PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T120	0-138	Sentence	denotes	Deficiency of C-C Chemokine Receptor 5 Suppresses Tumor Development via Inactivation of NF-κB and Upregulation of IL-1Ra in Melanoma Model
T1	0-138	Sentence	denotes	Deficiency of C-C Chemokine Receptor 5 Suppresses Tumor Development via Inactivation of NF-κB and Upregulation of IL-1Ra in Melanoma Model
T2	139-184	Sentence	denotes	Deficiency of CCR5 Prevents Tumor Development
T3	186-194	Sentence	denotes	Abstract
T121	195-384	Sentence	denotes	To evaluate the relevance of C-C chemokine receptor type 5 (CCR5) expression and tumor development, we compared melanoma growth in CCR5 knockout (CCR5−/−) mice and wild type (CCR5+/+) mice.
T4	195-384	Sentence	denotes	To evaluate the relevance of C-C chemokine receptor type 5 (CCR5) expression and tumor development, we compared melanoma growth in CCR5 knockout (CCR5−/−) mice and wild type (CCR5+/+) mice.
T122	385-499	Sentence	denotes	CCR5−/− mice showed reduced tumor volume, tumor weight, and increased survival rate when compared to CCR5+/+ mice.
T5	385-499	Sentence	denotes	CCR5−/− mice showed reduced tumor volume, tumor weight, and increased survival rate when compared to CCR5+/+ mice.
T123	500-665	Sentence	denotes	We investigated the activation of NF-κB since it is an implicated transcription factor in the regulation of genes involving cell growth, apoptosis, and tumor growth.
T6	500-665	Sentence	denotes	We investigated the activation of NF-κB since it is an implicated transcription factor in the regulation of genes involving cell growth, apoptosis, and tumor growth.
T124	666-917	Sentence	denotes	Significant inhibition of DNA binding activity of NF-κB, and translocation of p50 and p65 into the nucleus through the inhibition of phosphorylation of IκB was found in the melanoma tissues of CCR5−/− mice compared to melanoma tissues of CCR5+/+ mice.
T7	666-917	Sentence	denotes	Significant inhibition of DNA binding activity of NF-κB, and translocation of p50 and p65 into the nucleus through the inhibition of phosphorylation of IκB was found in the melanoma tissues of CCR5−/− mice compared to melanoma tissues of CCR5+/+ mice.
T125	918-1150	Sentence	denotes	NF-κB target apoptotic protein expression, such as cleaved caspase-3, cleaved PARP, and Bax, was elevated, whereas the survival protein expression levels, such as Bcl-2, C-IAP1, was decreased in the melanoma tissues of CCR5−/− mice.
T8	918-1150	Sentence	denotes	NF-κB target apoptotic protein expression, such as cleaved caspase-3, cleaved PARP, and Bax, was elevated, whereas the survival protein expression levels, such as Bcl-2, C-IAP1, was decreased in the melanoma tissues of CCR5−/− mice.
T126	1151-1346	Sentence	denotes	Interestingly, we found that the level of IL-1Ra, a tumor growth suppressive cytokine, was significantly elevated in tumor tissue and spleen of CCR5−/− mice compared to the level in CCR5+/+ mice.
T9	1151-1346	Sentence	denotes	Interestingly, we found that the level of IL-1Ra, a tumor growth suppressive cytokine, was significantly elevated in tumor tissue and spleen of CCR5−/− mice compared to the level in CCR5+/+ mice.
T127	1347-1539	Sentence	denotes	Moreover, infiltration of CD8+ cytotoxic T cell and CD57+ natural killer cells was significantly increased in melanoma tumor and spleen tissue of CCR5−/− mice compared to that of CCR5+/+ mice.
T10	1347-1539	Sentence	denotes	Moreover, infiltration of CD8+ cytotoxic T cell and CD57+ natural killer cells was significantly increased in melanoma tumor and spleen tissue of CCR5−/− mice compared to that of CCR5+/+ mice.
T128	1540-1688	Sentence	denotes	Therefore, these results showed that CCR5 deficiency caused apoptotic cell death of melanoma through inhibition of NF-κB and upregulation of IL-1Ra.
T11	1540-1688	Sentence	denotes	Therefore, these results showed that CCR5 deficiency caused apoptotic cell death of melanoma through inhibition of NF-κB and upregulation of IL-1Ra.
T12	1690-1702	Sentence	denotes	Introduction
T1404	1703-1832	Sentence	denotes	Chemokines are small soluble molecules that are best known for their potent ability to induce cancer cell growth by inflammation.
T13	1703-1832	Sentence	denotes	Chemokines are small soluble molecules that are best known for their potent ability to induce cancer cell growth by inflammation.
T1405	1833-1907	Sentence	denotes	Many types of cancer cells express chemokines and chemokine receptors [1].
T14	1833-1907	Sentence	denotes	Many types of cancer cells express chemokines and chemokine receptors [1].
T1406	1908-2171	Sentence	denotes	The accumulated evidence indicates that the chemokine (C-C motif) ligand 5 (CCL5) and C-C chemokine receptor (CCR5), which are potent chemotactic factors for inflammatory cells, may be significantly involved in the proliferation and metastasis of several cancers.
T15	1908-2171	Sentence	denotes	The accumulated evidence indicates that the chemokine (C-C motif) ligand 5 (CCL5) and C-C chemokine receptor (CCR5), which are potent chemotactic factors for inflammatory cells, may be significantly involved in the proliferation and metastasis of several cancers.
T1407	2172-2407	Sentence	denotes	Intermediate and strong CCR5 expression is significantly associated with nonmetastatic development of colorectal cancer [2], melanoma [3], hepatoma [4], glioblastoma, Hodgkin lymphoma [5], as well as oral and prostate cancer cells [6].
T16	2172-2407	Sentence	denotes	Intermediate and strong CCR5 expression is significantly associated with nonmetastatic development of colorectal cancer [2], melanoma [3], hepatoma [4], glioblastoma, Hodgkin lymphoma [5], as well as oral and prostate cancer cells [6].
T1408	2408-2540	Sentence	denotes	Local production of the CCL5 is also important in the progression of breast cancer [7], and also correlates with poor prognosis [8].
T17	2408-2540	Sentence	denotes	Local production of the CCL5 is also important in the progression of breast cancer [7], and also correlates with poor prognosis [8].
T1409	2541-2667	Sentence	denotes	Meanwhile, CCR5 disruption has been demonstrated to inhibit experimental tumor growth and metastasis of pancreatic cancer [9].
T18	2541-2667	Sentence	denotes	Meanwhile, CCR5 disruption has been demonstrated to inhibit experimental tumor growth and metastasis of pancreatic cancer [9].
T1410	2668-2882	Sentence	denotes	In addition, host absence of CCR5 potentiates the delay of tumor growth [10], and CCR5 inhibitors prevented cancer cell growth, such as prostate cancer [11], breast cancer, hepatoma cells [4], and lung cancer [12].
T19	2668-2882	Sentence	denotes	In addition, host absence of CCR5 potentiates the delay of tumor growth [10], and CCR5 inhibitors prevented cancer cell growth, such as prostate cancer [11], breast cancer, hepatoma cells [4], and lung cancer [12].
T1411	2883-3023	Sentence	denotes	These data suggest that the deficiency of inflammatory chemokine receptor CCR5 may function as a suppressive receptor in cancer progression.
T20	2883-3023	Sentence	denotes	These data suggest that the deficiency of inflammatory chemokine receptor CCR5 may function as a suppressive receptor in cancer progression.
T1412	3024-3135	Sentence	denotes	However, more studies are required to describe how CCR5 deficiency acts in the inhibition of tumor development.
T21	3024-3135	Sentence	denotes	However, more studies are required to describe how CCR5 deficiency acts in the inhibition of tumor development.
T1413	3136-3292	Sentence	denotes	Interleukin-1 receptor antagonist (IL-1Ra), a member of the IL-1 family, is a naturally occurring cytokine that competitively blocks the IL-1 receptor [13].
T22	3136-3292	Sentence	denotes	Interleukin-1 receptor antagonist (IL-1Ra), a member of the IL-1 family, is a naturally occurring cytokine that competitively blocks the IL-1 receptor [13].
T1414	3293-3519	Sentence	denotes	Because IL-1Ra has been shown to inhibit tumor progression by promoting antitumor immune responses and by enhancing the activity of chemotherapy, numerous studies have examined the ability of IL-1Ra to block tumor progression.
T23	3293-3519	Sentence	denotes	Because IL-1Ra has been shown to inhibit tumor progression by promoting antitumor immune responses and by enhancing the activity of chemotherapy, numerous studies have examined the ability of IL-1Ra to block tumor progression.
T1415	3520-3602	Sentence	denotes	IL-1Ra expression was reduced in prostate [14], breast [15], and skin cancer [16].
T24	3520-3602	Sentence	denotes	IL-1Ra expression was reduced in prostate [14], breast [15], and skin cancer [16].
T1416	3603-3729	Sentence	denotes	It was also reported that endogenous IL-1Ra deficient mice developed aggressive tumors following exposure to carcinogens [17].
T25	3603-3729	Sentence	denotes	It was also reported that endogenous IL-1Ra deficient mice developed aggressive tumors following exposure to carcinogens [17].
T1417	3730-3867	Sentence	denotes	Meanwhile, proliferation of the melanoma cells that were transduced with IL-1Ra gene was reduced and inhibited melanoma development [18].
T26	3730-3867	Sentence	denotes	Meanwhile, proliferation of the melanoma cells that were transduced with IL-1Ra gene was reduced and inhibited melanoma development [18].
T1418	3868-3922	Sentence	denotes	IL-1R blockade also reduced hepatic tumor growth [19].
T27	3868-3922	Sentence	denotes	IL-1R blockade also reduced hepatic tumor growth [19].
T1419	3923-4118	Sentence	denotes	Moreover, it has also been reported that IL-1Ra reduces fibrosarcoma development [20], B16 melanoma growth and metastasis [21], colon adenocarcinoma growth [22], and skin cancer development [23].
T28	3923-4118	Sentence	denotes	Moreover, it has also been reported that IL-1Ra reduces fibrosarcoma development [20], B16 melanoma growth and metastasis [21], colon adenocarcinoma growth [22], and skin cancer development [23].
T1420	4119-4197	Sentence	denotes	IL-1 knockout mice injected with melanoma failed to develop solid tumors [24].
T29	4119-4197	Sentence	denotes	IL-1 knockout mice injected with melanoma failed to develop solid tumors [24].
T1421	4198-4373	Sentence	denotes	It was also found that IL-1Ra, combined with temozolomide and docetaxel chemotherapy, demonstrated more significant antitumor activity against B16 melanoma cells in vivo [25].
T30	4198-4373	Sentence	denotes	It was also found that IL-1Ra, combined with temozolomide and docetaxel chemotherapy, demonstrated more significant antitumor activity against B16 melanoma cells in vivo [25].
T1422	4374-4458	Sentence	denotes	These data indicate that IL-1Ra is important as a tumor growth suppressing cytokine.
T31	4374-4458	Sentence	denotes	These data indicate that IL-1Ra is important as a tumor growth suppressing cytokine.
T1423	4459-4639	Sentence	denotes	NF-κB plays a crucial role in the suppression of apoptosis, as well as the induction of cell proliferation and inflammation, and is closely associated with cancer development [26].
T32	4459-4639	Sentence	denotes	NF-κB plays a crucial role in the suppression of apoptosis, as well as the induction of cell proliferation and inflammation, and is closely associated with cancer development [26].
T1424	4640-4884	Sentence	denotes	Constitutive activation of NF-κB has been described in a great number of cancers including colon cancers, prostate cancers and melanoma [27], and was found to up-regulate anti-apoptotic genes and/or down regulate apoptotic gene expression [28].
T33	4640-4884	Sentence	denotes	Constitutive activation of NF-κB has been described in a great number of cancers including colon cancers, prostate cancers and melanoma [27], and was found to up-regulate anti-apoptotic genes and/or down regulate apoptotic gene expression [28].
T1425	4885-4986	Sentence	denotes	Although NF-κB promotes tumor growth, it is required for the immune system to function normally [29].
T34	4885-4986	Sentence	denotes	Although NF-κB promotes tumor growth, it is required for the immune system to function normally [29].
T1426	4987-5122	Sentence	denotes	Studies on c-Rel-deficient mice demonstrated that c-Rel is essential for IL-2, IL-3, GM-CSF, and IFNγ expression in T lymphocytes [30].
T35	4987-5122	Sentence	denotes	Studies on c-Rel-deficient mice demonstrated that c-Rel is essential for IL-2, IL-3, GM-CSF, and IFNγ expression in T lymphocytes [30].
T1427	5123-5318	Sentence	denotes	C-Rel-deficient mice also have a tissue-specific deficiency of various cytokines and growth factors in T cells and macrophages affecting both innate and humoral immune responses in the host [31].
T36	5123-5318	Sentence	denotes	C-Rel-deficient mice also have a tissue-specific deficiency of various cytokines and growth factors in T cells and macrophages affecting both innate and humoral immune responses in the host [31].
T1428	5319-5564	Sentence	denotes	Therefore, NF-κB can be specially targeted to prevent cancer cell growth by (1) directly affecting apoptotic cancer cell death, (2) stimulating tumor killing lymphocytes infiltration or (3) increasing the production of tumor killing lymphokines.
T37	5319-5564	Sentence	denotes	Therefore, NF-κB can be specially targeted to prevent cancer cell growth by (1) directly affecting apoptotic cancer cell death, (2) stimulating tumor killing lymphocytes infiltration or (3) increasing the production of tumor killing lymphokines.
T1429	5565-5660	Sentence	denotes	Activation of NF-κB is important in the regulation of CCR-mediated tumor growth and metastasis.
T38	5565-5660	Sentence	denotes	Activation of NF-κB is important in the regulation of CCR-mediated tumor growth and metastasis.
T1430	5661-5831	Sentence	denotes	It was reported that inhibition of NF-κB by dehydroxymethyl-epoxyquinomicin (DHMEQ) induces cell death of primary effusion lymphoma by inhibition of CCR5 expression [32].
T39	5661-5831	Sentence	denotes	It was reported that inhibition of NF-κB by dehydroxymethyl-epoxyquinomicin (DHMEQ) induces cell death of primary effusion lymphoma by inhibition of CCR5 expression [32].
T1431	5832-5931	Sentence	denotes	Activation of NF-κB also increases CCL5-mediated oral cancer motility (5) and lung metastasis [33].
T40	5832-5931	Sentence	denotes	Activation of NF-κB also increases CCL5-mediated oral cancer motility (5) and lung metastasis [33].
T1432	5932-6031	Sentence	denotes	Thus, it is possible that inhibition of NF-κB could be involved in CCR5 mediated tumor development.
T41	5932-6031	Sentence	denotes	Thus, it is possible that inhibition of NF-κB could be involved in CCR5 mediated tumor development.
T1433	6032-6188	Sentence	denotes	NF-κB transcription factor binding sites are present in the promoter region of proinflammatory cytokine genes, such as IL-1alpha, IL-6, GM-CSF, and KC [34].
T42	6032-6188	Sentence	denotes	NF-κB transcription factor binding sites are present in the promoter region of proinflammatory cytokine genes, such as IL-1alpha, IL-6, GM-CSF, and KC [34].
T1434	6189-6272	Sentence	denotes	On the other hand, the IL-1Ra is a negative regulator of the inflammatory response.
T43	6189-6272	Sentence	denotes	On the other hand, the IL-1Ra is a negative regulator of the inflammatory response.
T1435	6273-6377	Sentence	denotes	Several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1.
T44	6273-6377	Sentence	denotes	Several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1.
T1436	6378-6583	Sentence	denotes	Smith et al. suggested that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors; NF-κB/PU.1/GA-binding protein binding site [35].
T45	6378-6583	Sentence	denotes	Smith et al. suggested that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors; NF-κB/PU.1/GA-binding protein binding site [35].
T1437	6584-6728	Sentence	denotes	Nuclear translocation of NF-κB (p65) was significantly enhanced and prolonged in IL-1Ra-deficient mice, compared to that in wild type mice [36].
T46	6584-6728	Sentence	denotes	Nuclear translocation of NF-κB (p65) was significantly enhanced and prolonged in IL-1Ra-deficient mice, compared to that in wild type mice [36].
T1438	6729-6865	Sentence	denotes	Intracellular IL-1Ra (icIL-1Ra) was found to inhibit IL-1 by blocking NF-κB signal transduction pathways in inflammatory responses [37].
T47	6729-6865	Sentence	denotes	Intracellular IL-1Ra (icIL-1Ra) was found to inhibit IL-1 by blocking NF-κB signal transduction pathways in inflammatory responses [37].
T1439	6866-7085	Sentence	denotes	Because IL-1 has been shown to be an inducer of NF-κB activation [38], we hypothesized that the expression of IL-1Ra could play an important role in the autocrine activation of transcription factors NF-κB or vice versa.
T48	6866-7085	Sentence	denotes	Because IL-1 has been shown to be an inducer of NF-κB activation [38], we hypothesized that the expression of IL-1Ra could play an important role in the autocrine activation of transcription factors NF-κB or vice versa.
T1440	7086-7233	Sentence	denotes	Thus, the aim of this study was to evaluate the roles of NF-κB and IL-1Ra in the chemokine receptor CCR5-mediated suppression of tumor development.
T49	7086-7233	Sentence	denotes	Thus, the aim of this study was to evaluate the roles of NF-κB and IL-1Ra in the chemokine receptor CCR5-mediated suppression of tumor development.
T50	7235-7242	Sentence	denotes	Results
T4493	7244-7291	Sentence	denotes	Inhibition of Tumor Development in CCR5−/− Mice
T51	7244-7291	Sentence	denotes	Inhibition of Tumor Development in CCR5−/− Mice
T4494	7292-7380	Sentence	denotes	The effect of CCR5 in tumor development was investigated using CCR5+/+ and CCR5−/− mice.
T52	7292-7380	Sentence	denotes	The effect of CCR5 in tumor development was investigated using CCR5+/+ and CCR5−/− mice.
T4495	7381-7473	Sentence	denotes	B16-F0 melanoma cells were inoculated subcutaneously into CCR5+/+ and CCR5−/− mice (n = 20).
T53	7381-7473	Sentence	denotes	B16-F0 melanoma cells were inoculated subcutaneously into CCR5+/+ and CCR5−/− mice (n = 20).
T4496	7474-7513	Sentence	denotes	Tumor growth was monitored for 31 days.
T54	7474-7513	Sentence	denotes	Tumor growth was monitored for 31 days.
T4497	7514-7601	Sentence	denotes	Tumor growth in the CCR5−/− mice was significantly lower than that in the CCR5+/+ mice.
T55	7514-7601	Sentence	denotes	Tumor growth in the CCR5−/− mice was significantly lower than that in the CCR5+/+ mice.
T4498	7602-7823	Sentence	denotes	The tumor growth in the CCR5−/− mice was reduced to 19.1%, whereas the tumor growth in the CCR5+/+ mice was only reduced to 24.3%, in both tumor weight (Figure 1A and 1B) and tumor volume, respectively (Figure 1A and 1C).
T56	7602-7823	Sentence	denotes	The tumor growth in the CCR5−/− mice was reduced to 19.1%, whereas the tumor growth in the CCR5+/+ mice was only reduced to 24.3%, in both tumor weight (Figure 1A and 1B) and tumor volume, respectively (Figure 1A and 1C).
T4499	7824-7874	Sentence	denotes	These results correlated with mice survival rates.
T57	7824-7874	Sentence	denotes	These results correlated with mice survival rates.
T4500	7875-8036	Sentence	denotes	There was a significant difference in survival rates between CCR5−/− mice (75% of starting time) and CCR5+/+ mice (15%) at the end of the experiment (Figure 1D).
T58	7875-8036	Sentence	denotes	There was a significant difference in survival rates between CCR5−/− mice (75% of starting time) and CCR5+/+ mice (15%) at the end of the experiment (Figure 1D).
T4501	8037-8128	Sentence	denotes	There was a significant difference in survival rates between CCR5−/− mice and CCR5+/+ mice.
T59	8037-8128	Sentence	denotes	There was a significant difference in survival rates between CCR5−/− mice and CCR5+/+ mice.
T4502	8129-8236	Sentence	denotes	At the end of the experiment, 75% of the CCR5−/− mice survived while only 15% of the CCR5+/+ mice survived.
T60	8129-8236	Sentence	denotes	At the end of the experiment, 75% of the CCR5−/− mice survived while only 15% of the CCR5+/+ mice survived.
T4503	8237-8323	Sentence	denotes	Subcutaneous tumors were harvested from the sacrificed mice 31 days after inoculation.
T61	8237-8323	Sentence	denotes	Subcutaneous tumors were harvested from the sacrificed mice 31 days after inoculation.
T4504	8324-8494	Sentence	denotes	The immunohistochemical analysis of tumor sections, stained with H&E and for proliferation antigen PCNA, revealed greater inhibition of tumor cell growth in CCR5−/− mice.
T62	8324-8494	Sentence	denotes	The immunohistochemical analysis of tumor sections, stained with H&E and for proliferation antigen PCNA, revealed greater inhibition of tumor cell growth in CCR5−/− mice.
T4505	8495-8661	Sentence	denotes	The histologic findings after H&E staining indicated that the tumor tissues of the CCR5−/− mice, but not those of the CCR5+/+ mice, contained large areas of necrosis.
T63	8495-8661	Sentence	denotes	The histologic findings after H&E staining indicated that the tumor tissues of the CCR5−/− mice, but not those of the CCR5+/+ mice, contained large areas of necrosis.
T4506	8662-8741	Sentence	denotes	PCNA reactive cells were significantly reduced in the CCR5−/− mice (Figure 1E).
T64	8662-8741	Sentence	denotes	PCNA reactive cells were significantly reduced in the CCR5−/− mice (Figure 1E).
T65	8742-8834	Sentence	denotes	10.1371/journal.pone.0033747.g001 Figure 1 Inhibition of tumor development in CCR5−/− mice.
T17206	8786-8834	Sentence	denotes	Inhibition of tumor development in CCR5−/− mice.
T17207	8835-8875	Sentence	denotes	A–C, Tumor images, volumes, and weights.
T66	8835-8875	Sentence	denotes	A–C, Tumor images, volumes, and weights.
T17208	8876-8980	Sentence	denotes	B16 melanoma cells (5×105 cells/mouse) were inoculated s.c. into CCR5+/+ mice and CCR5−/− mice (n = 20).
T67	8876-8980	Sentence	denotes	B16 melanoma cells (5×105 cells/mouse) were inoculated s.c. into CCR5+/+ mice and CCR5−/− mice (n = 20).
T17209	8981-9081	Sentence	denotes	Tumor volumes were measured every day and tumor weights were measured at study termination (Day 31).
T68	8981-9081	Sentence	denotes	Tumor volumes were measured every day and tumor weights were measured at study termination (Day 31).
T17210	9082-9182	Sentence	denotes	The CCR5−/− mice had a significant reduction in tumor growth and volume as compared to CCR5+/+ mice.
T69	9082-9182	Sentence	denotes	The CCR5−/− mice had a significant reduction in tumor growth and volume as compared to CCR5+/+ mice.
T17211	9183-9285	Sentence	denotes	The results are expressed as mean ± SD. * indicates significant difference from CCR5+/+ mice (P<0.05).
T70	9183-9285	Sentence	denotes	The results are expressed as mean ± SD. * indicates significant difference from CCR5+/+ mice (P<0.05).
T17212	9286-9399	Sentence	denotes	D, The CCR5−/− mice demonstrated significantly higher survival rates compared to CCR5+/+ mice (by Log-rank Test).
T71	9286-9399	Sentence	denotes	D, The CCR5−/− mice demonstrated significantly higher survival rates compared to CCR5+/+ mice (by Log-rank Test).
T72	9400-9402	Sentence	denotes	E.
T17213	9400-9529	Sentence	denotes	E. Tumor sections were analyzed by H&E stain and expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry.
T73	9403-9529	Sentence	denotes	Tumor sections were analyzed by H&E stain and expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry.
T17214	9530-9644	Sentence	denotes	Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (P<0.05).
T74	9530-9644	Sentence	denotes	Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (P<0.05).
T17215	9645-9670	Sentence	denotes	Scale bar indicates 50 m.
T75	9645-9670	Sentence	denotes	Scale bar indicates 50 m.
T5418	9672-9730	Sentence	denotes	Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice
T76	9672-9730	Sentence	denotes	Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice
T17744	9672-9858	Sentence	denotes	Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression.
T5419	9731-9858	Sentence	denotes	The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression.
T77	9731-9858	Sentence	denotes	The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression.
T5420	9859-10062	Sentence	denotes	To evaluate whether the tumor inhibition was related with the inactivation of NF-κB in melanoma, the DNA binding activity of NF-κB was determined by electromobility shift assay (EMSA) in melanoma tissue.
T78	9859-10062	Sentence	denotes	To evaluate whether the tumor inhibition was related with the inactivation of NF-κB in melanoma, the DNA binding activity of NF-κB was determined by electromobility shift assay (EMSA) in melanoma tissue.
T5421	10063-10170	Sentence	denotes	The constitutively activated DNA binding activity was reduced in the CCR5−/− mice tumor tissue (Figure 2A).
T79	10063-10170	Sentence	denotes	The constitutively activated DNA binding activity was reduced in the CCR5−/− mice tumor tissue (Figure 2A).
T5422	10171-10350	Sentence	denotes	Moreover, the translocation of p50 and p-p65 into the nucleus through the inhibition of phosphorylation of IκB was also prevented in the tumor tissues of CCR5−/− mice (Figure 2B).
T80	10171-10350	Sentence	denotes	Moreover, the translocation of p50 and p-p65 into the nucleus through the inhibition of phosphorylation of IκB was also prevented in the tumor tissues of CCR5−/− mice (Figure 2B).
T5423	10351-10504	Sentence	denotes	Immunofluorescence analysis also confirmed that the intensity of nuclear staining for p50 had decreased in the tumor tissues of CCR5−/− mice (Figure 2C).
T81	10351-10504	Sentence	denotes	Immunofluorescence analysis also confirmed that the intensity of nuclear staining for p50 had decreased in the tumor tissues of CCR5−/− mice (Figure 2C).
T82	10505-10608	Sentence	denotes	10.1371/journal.pone.0033747.g002 Figure 2 Decrease of NF-κB activity in tumor tissue of CCR5−/− mice.
T17745	10609-10778	Sentence	denotes	A, The DNA binding activity of NF-κB was determined in the nuclear extracts of the CCR5−/− mice and CCR5+/+ mice tumor tissues by EMSA described in material and methods.
T83	10609-10778	Sentence	denotes	A, The DNA binding activity of NF-κB was determined in the nuclear extracts of the CCR5−/− mice and CCR5+/+ mice tumor tissues by EMSA described in material and methods.
T17746	10779-10933	Sentence	denotes	B, Expression of p50 and p65 phosphorylation in nuclear extracts (NE), and IκB and IκB phosphorylation in the cytosol (CE) determined by Western blotting.
T84	10779-10933	Sentence	denotes	B, Expression of p50 and p65 phosphorylation in nuclear extracts (NE), and IκB and IκB phosphorylation in the cytosol (CE) determined by Western blotting.
T17747	10934-11084	Sentence	denotes	C, Immunolfluorescence analysis of p50 confirmed that the intensities of nuclear staining for p50 were decreased in the tumor tissues of CCR5−/− mice.
T85	10934-11084	Sentence	denotes	C, Immunolfluorescence analysis of p50 confirmed that the intensities of nuclear staining for p50 were decreased in the tumor tissues of CCR5−/− mice.
T17748	11085-11199	Sentence	denotes	Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).
T86	11085-11199	Sentence	denotes	Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).
T17749	11200-11226	Sentence	denotes	Scale bar indicates 50 µm.
T87	11200-11226	Sentence	denotes	Scale bar indicates 50 µm.
T5957	11228-11337	Sentence	denotes	Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice
T88	11228-11337	Sentence	denotes	Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice
T18203	11228-12481	Sentence	denotes	Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed. Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells. TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A). Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C). Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D). 10.1371/journal.pone.0033747.g003 Figure 3 Induction of apoptotic cell death in tumor tissues of CCR5 −/− mice.
T5958	11338-11611	Sentence	denotes	To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed.
T89	11338-11611	Sentence	denotes	To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed.
T5959	11612-11743	Sentence	denotes	Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells.
T90	11612-11743	Sentence	denotes	Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells.
T5960	11744-11894	Sentence	denotes	TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A).
T91	11744-11894	Sentence	denotes	TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A).
T5961	11895-12082	Sentence	denotes	Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C).
T92	11895-12082	Sentence	denotes	Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C).
T5962	12083-12368	Sentence	denotes	Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D).
T93	12083-12368	Sentence	denotes	Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D).
T94	12369-12481	Sentence	denotes	10.1371/journal.pone.0033747.g003 Figure 3 Induction of apoptotic cell death in tumor tissues of CCR5 −/− mice.
T18204	12482-12627	Sentence	denotes	A, Apoptotic cells in tumor sections were examined by fluorescence microscopy after DAPI and TUNEL staining as described in material and methods.
T95	12482-12627	Sentence	denotes	A, Apoptotic cells in tumor sections were examined by fluorescence microscopy after DAPI and TUNEL staining as described in material and methods.
T18205	12628-12733	Sentence	denotes	The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells that were counted.
T96	12628-12733	Sentence	denotes	The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells that were counted.
T18206	12734-12859	Sentence	denotes	Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.
T97	12734-12859	Sentence	denotes	Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.
T18207	12860-12886	Sentence	denotes	Scale bar indicates 50 µm.
T98	12860-12886	Sentence	denotes	Scale bar indicates 50 µm.
T18208	12887-13002	Sentence	denotes	B and C, Apoptotic protein (cleaved caspase-3 and Bax) in tumor sections were detected by immunofluorescence assay.
T99	12887-13002	Sentence	denotes	B and C, Apoptotic protein (cleaved caspase-3 and Bax) in tumor sections were detected by immunofluorescence assay.
T18209	13003-13153	Sentence	denotes	The reactive cell number was determined as the number of DAPI-stained, Specific antibody (cleaved caspase-3 and Bax)-positive cells that were counted.
T100	13003-13153	Sentence	denotes	The reactive cell number was determined as the number of DAPI-stained, Specific antibody (cleaved caspase-3 and Bax)-positive cells that were counted.
T18210	13154-13279	Sentence	denotes	Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.
T101	13154-13279	Sentence	denotes	Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.
T18211	13280-13306	Sentence	denotes	Scale bar indicates 50 µm.
T102	13280-13306	Sentence	denotes	Scale bar indicates 50 µm.
T18212	13307-13502	Sentence	denotes	D, The expression of apoptotic proteins was detected by Western blotting using specific antibodies; cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bax, Bcl-2 and c-IAP1 in the tumor tissues.
T103	13307-13502	Sentence	denotes	D, The expression of apoptotic proteins was detected by Western blotting using specific antibodies; cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bax, Bcl-2 and c-IAP1 in the tumor tissues.
T18213	13503-13555	Sentence	denotes	The β-actin protein was used as an internal control.
T104	13503-13555	Sentence	denotes	The β-actin protein was used as an internal control.
T18214	13556-13626	Sentence	denotes	Each band is representative of three independent experimental results.
T105	13556-13626	Sentence	denotes	Each band is representative of three independent experimental results.
T18215	13627-13742	Sentence	denotes	Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).
T106	13627-13742	Sentence	denotes	Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).
T6636	13744-13806	Sentence	denotes	Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice
T107	13744-13806	Sentence	denotes	Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice
T18867	13744-14007	Sentence	denotes	Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A).
T6637	13807-14007	Sentence	denotes	To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A).
T108	13807-14007	Sentence	denotes	To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A).
T6638	14008-14159	Sentence	denotes	Among 40 tested cytokines, the levels of IL-1Ra, MIG, MIP-1α increased about 686, 9 and 25-fold respectively, in CCR5−/− mice compared to CCR5+/+ mice.
T109	14008-14159	Sentence	denotes	Among 40 tested cytokines, the levels of IL-1Ra, MIG, MIP-1α increased about 686, 9 and 25-fold respectively, in CCR5−/− mice compared to CCR5+/+ mice.
T6639	14160-14286	Sentence	denotes	Using this technique, no other cytokines were found to be different between the CCR5−/− mice and the CCR5+/+ mice (Figure 4B).
T110	14160-14286	Sentence	denotes	Using this technique, no other cytokines were found to be different between the CCR5−/− mice and the CCR5+/+ mice (Figure 4B).
T6640	14287-14373	Sentence	denotes	Similar altered cytokine levels were found in the spleens of CCR5−/− mice (Figure 4C).
T111	14287-14373	Sentence	denotes	Similar altered cytokine levels were found in the spleens of CCR5−/− mice (Figure 4C).
T6641	14374-14543	Sentence	denotes	Since the IL-1Ra level was the most significantly elevated, the expression of IL-1Ra in tumor and spleen tissues was analyzed by immunofluorescence and western blotting.
T112	14374-14543	Sentence	denotes	Since the IL-1Ra level was the most significantly elevated, the expression of IL-1Ra in tumor and spleen tissues was analyzed by immunofluorescence and western blotting.
T6642	14544-14647	Sentence	denotes	The expression of IL-1Ra was upregulated in tumor and spleen tissues of the CCR5−/− mice (Figure 4D-G).
T113	14544-14647	Sentence	denotes	The expression of IL-1Ra was upregulated in tumor and spleen tissues of the CCR5−/− mice (Figure 4D-G).
T114	14648-14755	Sentence	denotes	10.1371/journal.pone.0033747.g004 Figure 4 Upregulation of IL-1Ra in the tumor and spleen of CCR5−/− mice.
T18868	14756-14818	Sentence	denotes	A, Figure 4A indicates mouse cytokine array panel coordinates.
T115	14756-14818	Sentence	denotes	A, Figure 4A indicates mouse cytokine array panel coordinates.
T18869	14819-14911	Sentence	denotes	Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate.
T116	14819-14911	Sentence	denotes	Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate.
T18870	14912-15055	Sentence	denotes	B, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.
T117	14912-15055	Sentence	denotes	B, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.
T18871	15056-15120	Sentence	denotes	Representative blot from three independent experiments is shown.
T118	15056-15120	Sentence	denotes	Representative blot from three independent experiments is shown.
T18872	15121-15213	Sentence	denotes	Positive controls show the manufacturer’s internal positive control samples on the membrane.
T119	15121-15213	Sentence	denotes	Positive controls show the manufacturer’s internal positive control samples on the membrane.
T18873	15214-15299	Sentence	denotes	C, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues.
T120	15214-15299	Sentence	denotes	C, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues.
T18874	15300-15393	Sentence	denotes	There were differences in cytokines between CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.
T121	15300-15393	Sentence	denotes	There were differences in cytokines between CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.
T18875	15394-15458	Sentence	denotes	Representative blot from three independent experiments is shown.
T122	15394-15458	Sentence	denotes	Representative blot from three independent experiments is shown.
T18876	15459-15551	Sentence	denotes	Positive controls show the manufacturer’s internal positive control samples on the membrane.
T123	15459-15551	Sentence	denotes	Positive controls show the manufacturer’s internal positive control samples on the membrane.
T18877	15552-15672	Sentence	denotes	D and E, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues.
T124	15552-15672	Sentence	denotes	D and E, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues.
T18878	15673-15793	Sentence	denotes	The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted.
T125	15673-15793	Sentence	denotes	The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted.
T18879	15794-15919	Sentence	denotes	Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.
T126	15794-15919	Sentence	denotes	Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.
T18880	15920-15946	Sentence	denotes	Scale bar indicates 50 µm.
T127	15920-15946	Sentence	denotes	Scale bar indicates 50 µm.
T18881	15947-16038	Sentence	denotes	F and G, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues.
T128	15947-16038	Sentence	denotes	F and G, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues.
T18882	16039-16109	Sentence	denotes	Each band is representative of three independent experimental results.
T129	16039-16109	Sentence	denotes	Each band is representative of three independent experimental results.
T18883	16110-16225	Sentence	denotes	Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).
T130	16110-16225	Sentence	denotes	Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).
T7342	16227-16314	Sentence	denotes	Induction of CD8 T cells and NK Cell Infiltration into Tumor and Spleen of CCR5−/− Mice
T131	16227-16314	Sentence	denotes	Induction of CD8 T cells and NK Cell Infiltration into Tumor and Spleen of CCR5−/− Mice
T7343	16315-16554	Sentence	denotes	To investigate whether the inhibition of tumor growth in CCR5−/− mice is related to tumor-specific immune responses, we analyzed the distribution patterns of CD8+ cytotoxic T cell and CD57+ Natural Killer cells in tumor and spleen tissues.
T132	16315-16554	Sentence	denotes	To investigate whether the inhibition of tumor growth in CCR5−/− mice is related to tumor-specific immune responses, we analyzed the distribution patterns of CD8+ cytotoxic T cell and CD57+ Natural Killer cells in tumor and spleen tissues.
T7344	16555-16754	Sentence	denotes	By staining the tumor sections from CCR5−/− mice, we found that there was a significant influx of CD8+ cells into the tumor, as well as an increase in the number of infiltrating NK cells (Figure 5A).
T133	16555-16754	Sentence	denotes	By staining the tumor sections from CCR5−/− mice, we found that there was a significant influx of CD8+ cells into the tumor, as well as an increase in the number of infiltrating NK cells (Figure 5A).
T7345	16755-16840	Sentence	denotes	CD8+ T cells and CD57+ NK cells were spread diffusely throughout the entire sections.
T134	16755-16840	Sentence	denotes	CD8+ T cells and CD57+ NK cells were spread diffusely throughout the entire sections.
T7346	16841-17068	Sentence	denotes	To further investigate the differences in the numbers of CD8+ T cells and CD57+ NK cells between CCR5+/+ mice and CCR5−/− mice in immunity-related organ, we also analyzed the CD8 and CD57 reactive cell number in spleen tissues.
T135	16841-17068	Sentence	denotes	To further investigate the differences in the numbers of CD8+ T cells and CD57+ NK cells between CCR5+/+ mice and CCR5−/− mice in immunity-related organ, we also analyzed the CD8 and CD57 reactive cell number in spleen tissues.
T7347	17069-17212	Sentence	denotes	The number of CD8+ T cells and CD57+ NK cells were higher in the spleen of the CCR5−/− mice than in the spleen of the CCR5+/+ mice (Figure 5B).
T136	17069-17212	Sentence	denotes	The number of CD8+ T cells and CD57+ NK cells were higher in the spleen of the CCR5−/− mice than in the spleen of the CCR5+/+ mice (Figure 5B).
T7348	17213-17403	Sentence	denotes	These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in an anti-tumor effect in CCR5−/− mice.
T137	17213-17403	Sentence	denotes	These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in an anti-tumor effect in CCR5−/− mice.
T138	17404-17558	Sentence	denotes	10.1371/journal.pone.0033747.g005 Figure 5 Induction of the infiltration of CD8+ T cells and NK cells into tumor, and increase in spleen of CCR5−/− mice.
T19587	17448-17558	Sentence	denotes	Induction of the infiltration of CD8+ T cells and NK cells into tumor, and increase in spleen of CCR5−/− mice.
T19588	17559-17742	Sentence	denotes	A and B, Immunolfluorescence analysis was used to determine the expression levels of CD8 (CD8+ cytotoxic T-cell surface marker) and CD57 (NK cell marker) in tumor and spleen sections.
T139	17559-17742	Sentence	denotes	A and B, Immunolfluorescence analysis was used to determine the expression levels of CD8 (CD8+ cytotoxic T-cell surface marker) and CD57 (NK cell marker) in tumor and spleen sections.
T19589	17743-17833	Sentence	denotes	The images shown are representative of three separate experiments performed in triplicate.
T140	17743-17833	Sentence	denotes	The images shown are representative of three separate experiments performed in triplicate.
T19590	17834-17879	Sentence	denotes	Scale bars indicate 50 µm (A) and 100 µm (B).
T141	17834-17879	Sentence	denotes	Scale bars indicate 50 µm (A) and 100 µm (B).
T19591	17880-17923	Sentence	denotes	C and D, Analysis of lymphocyte phenotypes.
T142	17880-17923	Sentence	denotes	C and D, Analysis of lymphocyte phenotypes.
T19592	17924-17998	Sentence	denotes	The tumor and spleen tissues were separated at study termination (Day 31).
T143	17924-17998	Sentence	denotes	The tumor and spleen tissues were separated at study termination (Day 31).
T19593	17999-18098	Sentence	denotes	Flow cytometry analysis was performed using FACSAria flow cytometry, and represent data were shown.
T144	17999-18098	Sentence	denotes	Flow cytometry analysis was performed using FACSAria flow cytometry, and represent data were shown.
T19594	18099-18150	Sentence	denotes	Data are means ± S.D. of four experimental animals.
T145	18099-18150	Sentence	denotes	Data are means ± S.D. of four experimental animals.
T7349	18151-18318	Sentence	denotes	To analyze the cell phenotype change in CCR5−/− mice and CCR5+/+ mice by inoculation of the melanoma cell, lymphocytes were isolated from the tumor and spleen tissues.
T146	18151-18318	Sentence	denotes	To analyze the cell phenotype change in CCR5−/− mice and CCR5+/+ mice by inoculation of the melanoma cell, lymphocytes were isolated from the tumor and spleen tissues.
T7350	18319-18524	Sentence	denotes	The B, CD3 T, CD4 T, CD 8 T and NK cell portion of CCR5−/− mice tumor tissue were 7.2%, 48.2%, 16.9%, 32.1% and 7.8%, respectively, compared to 2.9%, 11.1%, 2.3%, 0.6% and 3.1% of CCR5+/+ mice (Figure 5C).
T147	18319-18524	Sentence	denotes	The B, CD3 T, CD4 T, CD 8 T and NK cell portion of CCR5−/− mice tumor tissue were 7.2%, 48.2%, 16.9%, 32.1% and 7.8%, respectively, compared to 2.9%, 11.1%, 2.3%, 0.6% and 3.1% of CCR5+/+ mice (Figure 5C).
T7351	18525-18684	Sentence	denotes	The lymphocytes that infiltrated the tumor tissue of the CCR5−/− mice also had higher levels of CD19, CD3, CD4, CD8 and CD57 when compared to the CCR5+/+ mice.
T148	18525-18684	Sentence	denotes	The lymphocytes that infiltrated the tumor tissue of the CCR5−/− mice also had higher levels of CD19, CD3, CD4, CD8 and CD57 when compared to the CCR5+/+ mice.
T7352	18685-18891	Sentence	denotes	The B, CD3 T, CD4 T, CD 8 T and NK cell portion of the CCR5−/− mice spleen were 61.3%, 33.4%, 17.6%, 9.2% and 0.6%, respectively compared to 38.3%, 5.9%, 4.0%, 1.0% and 3.9% of the CCR5+/+ mice (Figure 5D).
T149	18685-18891	Sentence	denotes	The B, CD3 T, CD4 T, CD 8 T and NK cell portion of the CCR5−/− mice spleen were 61.3%, 33.4%, 17.6%, 9.2% and 0.6%, respectively compared to 38.3%, 5.9%, 4.0%, 1.0% and 3.9% of the CCR5+/+ mice (Figure 5D).
T7353	18892-19092	Sentence	denotes	These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in the inhibition of tumor growth in CCR5−/− mice.
T150	18892-19092	Sentence	denotes	These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in the inhibition of tumor growth in CCR5−/− mice.
T151	19094-19104	Sentence	denotes	Discussion
T9010	19105-19286	Sentence	denotes	In the present study, we found that tumor weight and volume were much smaller in the CCR5−/− mice compared to the CCR5+/+ mice inoculated with B16 melanoma cells (Figure 1A and 1B).
T152	19105-19286	Sentence	denotes	In the present study, we found that tumor weight and volume were much smaller in the CCR5−/− mice compared to the CCR5+/+ mice inoculated with B16 melanoma cells (Figure 1A and 1B).
T9011	19287-19448	Sentence	denotes	The tumor growth inhibition in the CCR5−/− mice was associated with the inhibition of constitutively activated NF-κB and elevation of IL-1Ra in the tumor tissue.
T153	19287-19448	Sentence	denotes	The tumor growth inhibition in the CCR5−/− mice was associated with the inhibition of constitutively activated NF-κB and elevation of IL-1Ra in the tumor tissue.
T9012	19449-19727	Sentence	denotes	This anti-tumor activity was also associated with decreased expression of NF-κB target anti-apoptotic, cell proliferative, and tumor promoting genes (Bcl-2 and C-IAP1) but with increased expression of their target apoptotic genes; cleaved caspase-3 and 9, and Bax (Figure 3A-D).
T154	19449-19727	Sentence	denotes	This anti-tumor activity was also associated with decreased expression of NF-κB target anti-apoptotic, cell proliferative, and tumor promoting genes (Bcl-2 and C-IAP1) but with increased expression of their target apoptotic genes; cleaved caspase-3 and 9, and Bax (Figure 3A-D).
T9013	19728-19880	Sentence	denotes	In the tumor and the spleen, the number of cytotoxic CD8+ T cells, CD57+ natural killer cells, and the levels of IL-1Ra were increased (Figure 4 and 5).
T155	19728-19880	Sentence	denotes	In the tumor and the spleen, the number of cytotoxic CD8+ T cells, CD57+ natural killer cells, and the levels of IL-1Ra were increased (Figure 4 and 5).
T9014	19881-20096	Sentence	denotes	These findings suggest that melanoma growth was inhibited in the CCR5−/− mice, and that the elevation of IL-1Ra, accompanied with decreased NF-κB, is significant in the inhibition of melanoma growth in CCR5−/− mice.
T156	19881-20096	Sentence	denotes	These findings suggest that melanoma growth was inhibited in the CCR5−/− mice, and that the elevation of IL-1Ra, accompanied with decreased NF-κB, is significant in the inhibition of melanoma growth in CCR5−/− mice.
T9015	20097-20219	Sentence	denotes	NF-κB regulates the expression of over 200 genes that control the immune system, cancer cell growth and inflammation [39].
T157	20097-20219	Sentence	denotes	NF-κB regulates the expression of over 200 genes that control the immune system, cancer cell growth and inflammation [39].
T9016	20220-20508	Sentence	denotes	Because of its abilities to induce the expression of a large array of inflammatory mediators and its roles as core transcription factors in diverse immune responses, NF-κB has been recognized as a major factor responsible for cytokine-associated cancer development or anti-tumor immunity.
T158	20220-20508	Sentence	denotes	Because of its abilities to induce the expression of a large array of inflammatory mediators and its roles as core transcription factors in diverse immune responses, NF-κB has been recognized as a major factor responsible for cytokine-associated cancer development or anti-tumor immunity.
T9017	20509-20801	Sentence	denotes	In the melanoma tumor tissues of the CCR5−/− mice, the expression of NF-κB target cell death genes (Bax, caspase-3 and 9) and the DNA binding activity of NF-κB, were significantly inhibited, but the expression of cell death inhibitory NF-κB target genes, such as Bcl-2 and cIAP were enhanced.
T159	20509-20801	Sentence	denotes	In the melanoma tumor tissues of the CCR5−/− mice, the expression of NF-κB target cell death genes (Bax, caspase-3 and 9) and the DNA binding activity of NF-κB, were significantly inhibited, but the expression of cell death inhibitory NF-κB target genes, such as Bcl-2 and cIAP were enhanced.
T9018	20802-20957	Sentence	denotes	In addition, CCR5−/− prevented the phosphorylation of IκB, accompanied with the inhibition of the p50 and p65 translocation into the nucleus (Figure 2A-C).
T160	20802-20957	Sentence	denotes	In addition, CCR5−/− prevented the phosphorylation of IκB, accompanied with the inhibition of the p50 and p65 translocation into the nucleus (Figure 2A-C).
T9019	20958-21092	Sentence	denotes	Many tumors, including melanoma, have increased levels of NF-κB [40], which is likely acting as a survival factor for melanoma growth.
T161	20958-21092	Sentence	denotes	Many tumors, including melanoma, have increased levels of NF-κB [40], which is likely acting as a survival factor for melanoma growth.
T9020	21093-21231	Sentence	denotes	Thus, the inhibitions of NF-κB activity and the expression of target genes are critical in the inhibition of tumor growth in CCR5−/− mice.
T162	21093-21231	Sentence	denotes	Thus, the inhibitions of NF-κB activity and the expression of target genes are critical in the inhibition of tumor growth in CCR5−/− mice.
T9021	21232-21381	Sentence	denotes	Although the mechanism is not clear as to how CCR5−/− downregulates NF-κB, it is noteworthy that NF-κB is activated or inactivated by many cytokines.
T163	21232-21381	Sentence	denotes	Although the mechanism is not clear as to how CCR5−/− downregulates NF-κB, it is noteworthy that NF-κB is activated or inactivated by many cytokines.
T9022	21382-21460	Sentence	denotes	Numerous studies showed that cytokine affect tumor growth by regulating NF-κB.
T164	21382-21460	Sentence	denotes	Numerous studies showed that cytokine affect tumor growth by regulating NF-κB.
T9023	21461-21560	Sentence	denotes	Inflammatory cytokines, including IL-1, IL-6 and IL-8, activate NF-κB pathways in tumor cells [41].
T165	21461-21560	Sentence	denotes	Inflammatory cytokines, including IL-1, IL-6 and IL-8, activate NF-κB pathways in tumor cells [41].
T9024	21561-21676	Sentence	denotes	The interleukin 10 (IL-10) is well known as an anti-tumor factor as well as an anti-inflammatory factor [42], [43].
T166	21561-21676	Sentence	denotes	The interleukin 10 (IL-10) is well known as an anti-tumor factor as well as an anti-inflammatory factor [42], [43].
T9025	21677-21759	Sentence	denotes	IL-10 inhibits constitutively activated NF-κB both in vitro [44] and in vivo [45].
T167	21677-21759	Sentence	denotes	IL-10 inhibits constitutively activated NF-κB both in vitro [44] and in vivo [45].
T9026	21760-21910	Sentence	denotes	Inhibition of NF-κB by the IL-10 contributes to the anti-tumor effects through the activation of caspase-3 and apoptosis in Colon26-bearing mice [43].
T168	21760-21910	Sentence	denotes	Inhibition of NF-κB by the IL-10 contributes to the anti-tumor effects through the activation of caspase-3 and apoptosis in Colon26-bearing mice [43].
T9027	21911-22031	Sentence	denotes	Similarly, several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1 [46].
T169	21911-22031	Sentence	denotes	Similarly, several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1 [46].
T9028	22032-22224	Sentence	denotes	It has been reported that IL-1 induces the activation of NF-κB in several cancers, and increases cell cycle progression, and the suppression of apoptotic cell death leading to tumor promotion.
T170	22032-22224	Sentence	denotes	It has been reported that IL-1 induces the activation of NF-κB in several cancers, and increases cell cycle progression, and the suppression of apoptotic cell death leading to tumor promotion.
T9029	22225-22414	Sentence	denotes	The results of various studies over the past 10 years indicate that the major function of IL-1Ra is to regulate the pleiotropic effects of IL-1 by competitively blocking its functions [47].
T171	22225-22414	Sentence	denotes	The results of various studies over the past 10 years indicate that the major function of IL-1Ra is to regulate the pleiotropic effects of IL-1 by competitively blocking its functions [47].
T9030	22415-22577	Sentence	denotes	The ability of IL-1 to induce its own antagonist is of interest in understanding the regulation and dysregulation of IL-1 signaling in normal and diseased tissue.
T172	22415-22577	Sentence	denotes	The ability of IL-1 to induce its own antagonist is of interest in understanding the regulation and dysregulation of IL-1 signaling in normal and diseased tissue.
T9031	22578-22722	Sentence	denotes	Up-regulation of the receptor antagonist represents a mechanism for turning off IL-1 signaling and creating a temporary state of refractoriness.
T173	22578-22722	Sentence	denotes	Up-regulation of the receptor antagonist represents a mechanism for turning off IL-1 signaling and creating a temporary state of refractoriness.
T9032	22723-22876	Sentence	denotes	The elevated expression of IL-1Ra, without any change of IL-1 in tumors, suggests that the IL-1-signaling system may be dysregulated by IL-1Ra in tumors.
T174	22723-22876	Sentence	denotes	The elevated expression of IL-1Ra, without any change of IL-1 in tumors, suggests that the IL-1-signaling system may be dysregulated by IL-1Ra in tumors.
T9033	22877-22982	Sentence	denotes	IL-1Ra has been shown to inhibit tumor progression in prostate, breast, and skin cancer [14], [15], [16].
T175	22877-22982	Sentence	denotes	IL-1Ra has been shown to inhibit tumor progression in prostate, breast, and skin cancer [14], [15], [16].
T9034	22983-23097	Sentence	denotes	Intracellular IL-1Ra (icIL-1Ra) inhibits IL-6 and IL-8 production in Caco-2 cells by blocking NF-κB pathways [37].
T176	22983-23097	Sentence	denotes	Intracellular IL-1Ra (icIL-1Ra) inhibits IL-6 and IL-8 production in Caco-2 cells by blocking NF-κB pathways [37].
T9035	23098-23233	Sentence	denotes	We found that the levels of IL-1Ra were significantly elevated in the tumor tissues of CCR5−/− mice inoculated with B16 melanoma cells.
T177	23098-23233	Sentence	denotes	We found that the levels of IL-1Ra were significantly elevated in the tumor tissues of CCR5−/− mice inoculated with B16 melanoma cells.
T9036	23234-23408	Sentence	denotes	Taken together, these data indicate IL-1Ra could be critically involved in the tumor growth inhibition of CCR5−/− mice through the inactivation of NF-κB in the present study.
T178	23234-23408	Sentence	denotes	Taken together, these data indicate IL-1Ra could be critically involved in the tumor growth inhibition of CCR5−/− mice through the inactivation of NF-κB in the present study.
T9037	23409-23555	Sentence	denotes	CCR5 increases tumor growth in a local model and inhibits the efficacy of a dendrite cell vaccine in CCR5+/+ mice compared with CCR5−/− mice [10].
T179	23409-23555	Sentence	denotes	CCR5 increases tumor growth in a local model and inhibits the efficacy of a dendrite cell vaccine in CCR5+/+ mice compared with CCR5−/− mice [10].
T9038	23556-23697	Sentence	denotes	Cheng J and Sung RS examined the effect of CCR5 on the immune response to adenovirus vectors and graft function in an islet transplant model.
T180	23556-23697	Sentence	denotes	Cheng J and Sung RS examined the effect of CCR5 on the immune response to adenovirus vectors and graft function in an islet transplant model.
T9039	23698-23802	Sentence	denotes	They found that CCR5 absence does not prevent the local immune response to adenovirus transduction [48].
T181	23698-23802	Sentence	denotes	They found that CCR5 absence does not prevent the local immune response to adenovirus transduction [48].
T9040	23803-24093	Sentence	denotes	Additionally, Tan MC et al. suggested that disruption of CCR5/CCL5 signaling, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (TAK-779), reduced Treg (regulatory T cells) migration to tumors so that tumors are smaller in control mice [9].
T182	23803-24093	Sentence	denotes	Additionally, Tan MC et al. suggested that disruption of CCR5/CCL5 signaling, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (TAK-779), reduced Treg (regulatory T cells) migration to tumors so that tumors are smaller in control mice [9].
T9041	24094-24237	Sentence	denotes	These data support the important premise that inhibition of tumor growth in CCR5−/− mice could be related with the inhibition of immune escape.
T183	24094-24237	Sentence	denotes	These data support the important premise that inhibition of tumor growth in CCR5−/− mice could be related with the inhibition of immune escape.
T9042	24238-24547	Sentence	denotes	Similar to this finding, we found that the CCR5+/+ mice injected with melanoma cells apparently have a lower number of cytotoxic T cells (CD8+positive T Lymphocytes cells) and natural killer cells (CD57 positive NK cells), as well as dendrite cells, in spleen tissues compared to those without melanoma cells.
T184	24238-24547	Sentence	denotes	Similar to this finding, we found that the CCR5+/+ mice injected with melanoma cells apparently have a lower number of cytotoxic T cells (CD8+positive T Lymphocytes cells) and natural killer cells (CD57 positive NK cells), as well as dendrite cells, in spleen tissues compared to those without melanoma cells.
T9043	24548-24639	Sentence	denotes	But the lymphocytes of the CCR5−/− mice did not decrease when injected with melanoma cells.
T185	24548-24639	Sentence	denotes	But the lymphocytes of the CCR5−/− mice did not decrease when injected with melanoma cells.
T9044	24640-24821	Sentence	denotes	These differences of the immune response between the CCR5+/+ mice and the CCR5−/− mice, involving tumor immune tolerance, may be connected with the inhibition of cancer progression.
T186	24640-24821	Sentence	denotes	These differences of the immune response between the CCR5+/+ mice and the CCR5−/− mice, involving tumor immune tolerance, may be connected with the inhibition of cancer progression.
T9045	24822-25027	Sentence	denotes	Taken together, in the cytokine array experiment, the level of MIP-1α (which has a potent activity of T cell and B cell migration) was increased in the CCR5−/− mice compared with those in the CCR5+/+ mice.
T187	24822-25027	Sentence	denotes	Taken together, in the cytokine array experiment, the level of MIP-1α (which has a potent activity of T cell and B cell migration) was increased in the CCR5−/− mice compared with those in the CCR5+/+ mice.
T9046	25028-25170	Sentence	denotes	Thus, relatively increased tumor associated cytotoxic lymphocytes could play an important role in inhibiting the tumor growth in CCR5−/− mice.
T188	25028-25170	Sentence	denotes	Thus, relatively increased tumor associated cytotoxic lymphocytes could play an important role in inhibiting the tumor growth in CCR5−/− mice.
T9047	25171-25265	Sentence	denotes	Activation of NF-κB could modulate subcellular localization of NK cells and pathogenesis [49].
T189	25171-25265	Sentence	denotes	Activation of NF-κB could modulate subcellular localization of NK cells and pathogenesis [49].
T9048	25266-25398	Sentence	denotes	Inhibition of NF-κB activity prevented NK cell depletion, and thus increased anti-tumor activity by decreasing IL-6 production [50].
T190	25266-25398	Sentence	denotes	Inhibition of NF-κB activity prevented NK cell depletion, and thus increased anti-tumor activity by decreasing IL-6 production [50].
T9049	25399-25564	Sentence	denotes	Thus, the decreased NF-κB and elevated IL-1Ra could also be associated in the infiltration of cytotoxic lymphocytes into tumor, resulting in tumor growth inhibition.
T191	25399-25564	Sentence	denotes	Thus, the decreased NF-κB and elevated IL-1Ra could also be associated in the infiltration of cytotoxic lymphocytes into tumor, resulting in tumor growth inhibition.
T9050	25565-25684	Sentence	denotes	In contrast, IL-23, a tumor growth promoting cytokine, increased NF-κB and immune cell infiltration in oral tumor [51].
T192	25565-25684	Sentence	denotes	In contrast, IL-23, a tumor growth promoting cytokine, increased NF-κB and immune cell infiltration in oral tumor [51].
T9051	25685-25797	Sentence	denotes	These data indicate that NF-κB could be involved in the cytokine mediated anti-tumor activities of immune cells.
T193	25685-25797	Sentence	denotes	These data indicate that NF-κB could be involved in the cytokine mediated anti-tumor activities of immune cells.
T9052	25798-25931	Sentence	denotes	It is also noteworthy that IL-1Ra inhibited melanoma tumor growth by increasing the number of myeloid suppressor cells in tumor [25].
T194	25798-25931	Sentence	denotes	It is also noteworthy that IL-1Ra inhibited melanoma tumor growth by increasing the number of myeloid suppressor cells in tumor [25].
T9053	25932-26113	Sentence	denotes	The 3-methylcholatrene-induced tumor incidence was reduced in IL-1α knockout mice, but increased in IL-1Ra mice with concomitant maturation of NK cells and anti-tumor immunity [52].
T195	25932-26113	Sentence	denotes	The 3-methylcholatrene-induced tumor incidence was reduced in IL-1α knockout mice, but increased in IL-1Ra mice with concomitant maturation of NK cells and anti-tumor immunity [52].
T9054	26114-26254	Sentence	denotes	These data indicate that the decrease of NF-κB, and thus increase of IL-1Ra could be significant in tumor growth inhibition of CCR5−/− mice.
T196	26114-26254	Sentence	denotes	These data indicate that the decrease of NF-κB, and thus increase of IL-1Ra could be significant in tumor growth inhibition of CCR5−/− mice.
T9055	26255-26435	Sentence	denotes	Our data thus show significant clinical implications of CCR5, because CCR5 is a G protein–coupled receptor and it is amenable to use as a small molecule for the inhibition of CCR5.
T197	26255-26435	Sentence	denotes	Our data thus show significant clinical implications of CCR5, because CCR5 is a G protein–coupled receptor and it is amenable to use as a small molecule for the inhibition of CCR5.
T9056	26436-26558	Sentence	denotes	Several such compounds have already been shown to be safe in clinical trials for use in certain diseases such as HIV [53].
T198	26436-26558	Sentence	denotes	Several such compounds have already been shown to be safe in clinical trials for use in certain diseases such as HIV [53].
T9057	26559-26638	Sentence	denotes	Thus, we believe CCR5 is a cancer target that warrants continued investigation.
T199	26559-26638	Sentence	denotes	Thus, we believe CCR5 is a cancer target that warrants continued investigation.
T9058	26639-26766	Sentence	denotes	In summary, this study showed that melanoma cells growth was inhibited via modulation of NF-κB/IL-1Ra pathways in CCR5−/− mice.
T200	26639-26766	Sentence	denotes	In summary, this study showed that melanoma cells growth was inhibited via modulation of NF-κB/IL-1Ra pathways in CCR5−/− mice.
T201	26768-26789	Sentence	denotes	Materials and Methods
T13206	26791-26807	Sentence	denotes	Ethics Statement
T202	26791-26807	Sentence	denotes	Ethics Statement
T13207	26808-26992	Sentence	denotes	All experiments were approved and carried out according to the Guide for the Care and Use of Animals [Animal Care Committee of Chungbuk National University, Korea (CBNUA-045-0902-01)].
T203	26808-26992	Sentence	denotes	All experiments were approved and carried out according to the Guide for the Care and Use of Animals [Animal Care Committee of Chungbuk National University, Korea (CBNUA-045-0902-01)].
T13309	26994-27001	Sentence	denotes	Animals
T204	26994-27001	Sentence	denotes	Animals
T13310	27002-27137	Sentence	denotes	The CCR5 wild type (CCR5+/+) and CCR5 knockout (CCR5−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine 04609 USA).
T205	27002-27137	Sentence	denotes	The CCR5 wild type (CCR5+/+) and CCR5 knockout (CCR5−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine 04609 USA).
T206	27138-27288	Sentence	denotes	The mice were housed and bred under specific pathogen free conditions at the Laboratory Animal Research Center of Chungbuk National University, Korea.
T13311	27138-27512	Sentence	denotes	The mice were housed and bred under specific pathogen free conditions at the Laboratory Animal Research Center of Chungbuk National University, Korea. The mice (n = 4/cage) were maintained in a room with a constant temperature of 22±1°C, relative humidity of 55±10%, and 12-h light/dark cycle, and fed standard rodent chow (Samyang, Korea) and purified tap water ad libitum.
T207	27289-27512	Sentence	denotes	The mice (n = 4/cage) were maintained in a room with a constant temperature of 22±1°C, relative humidity of 55±10%, and 12-h light/dark cycle, and fed standard rodent chow (Samyang, Korea) and purified tap water ad libitum.
T13312	27513-27639	Sentence	denotes	The CCR5+/+ mice (n = 20) and CCR5−/− mice (n = 20), that were used, had matched ages (10–11 weeks old) and weights (16–19 g).
T208	27513-27639	Sentence	denotes	The CCR5+/+ mice (n = 20) and CCR5−/− mice (n = 20), that were used, had matched ages (10–11 weeks old) and weights (16–19 g).
T13313	27640-27738	Sentence	denotes	Control mice (B6129PF2/J) were F2 hybrid mice from the C57BL/6J-AW-J and 129P3/J parental strains.
T209	27640-27738	Sentence	denotes	Control mice (B6129PF2/J) were F2 hybrid mice from the C57BL/6J-AW-J and 129P3/J parental strains.
T13314	27739-27902	Sentence	denotes	CCR5 mice (B6, 129P2-Ccr5tm1Kuz/J) have the entire coding region of the CCR5 gene deleted from the parental strains, B6;129P2-Ccr5tm1kuz and B6;129P2-Cmkbr5tm1Kuz.
T210	27739-27902	Sentence	denotes	CCR5 mice (B6, 129P2-Ccr5tm1Kuz/J) have the entire coding region of the CCR5 gene deleted from the parental strains, B6;129P2-Ccr5tm1kuz and B6;129P2-Cmkbr5tm1Kuz.
T13806	27904-27942	Sentence	denotes	Tumor Inoculation and Tumor Monitoring
T211	27904-27942	Sentence	denotes	Tumor Inoculation and Tumor Monitoring
T13807	27943-28156	Sentence	denotes	B16 melanoma cells were injected subcutaneously [5×105 tumor cells in 0.1 ml Phosphate buffered saline(PBS)/animal] using a 27 G needle into the right-lower flanks of the carrier mice as previously described [54].
T212	27943-28156	Sentence	denotes	B16 melanoma cells were injected subcutaneously [5×105 tumor cells in 0.1 ml Phosphate buffered saline(PBS)/animal] using a 27 G needle into the right-lower flanks of the carrier mice as previously described [54].
T13808	28157-28235	Sentence	denotes	The body weights and tumor volumes of the animals were monitored twice weekly.
T213	28157-28235	Sentence	denotes	The body weights and tumor volumes of the animals were monitored twice weekly.
T13809	28236-28409	Sentence	denotes	The tumor volumes were measured with Vernier calipers and calculated using the following formula: (A×B2)/2, where A is the larger and B is the smaller of the two dimensions.
T214	28236-28409	Sentence	denotes	The tumor volumes were measured with Vernier calipers and calculated using the following formula: (A×B2)/2, where A is the larger and B is the smaller of the two dimensions.
T13810	28410-28468	Sentence	denotes	At the end of the experiment, the animals were sacrificed.
T215	28410-28468	Sentence	denotes	At the end of the experiment, the animals were sacrificed.
T13811	28469-28535	Sentence	denotes	The tumors were separated from the surrounding muscles and dermis.
T216	28469-28535	Sentence	denotes	The tumors were separated from the surrounding muscles and dermis.
T14071	28537-28553	Sentence	denotes	Western Blotting
T217	28537-28553	Sentence	denotes	Western Blotting
T14072	28554-28706	Sentence	denotes	Tumor tissues were homogenized with protein extraction solution (PRO-PREPTM, Intron Biotechnology, Seoul, Korea), and lysed by 60 min incubation on ice.
T218	28554-28706	Sentence	denotes	Tumor tissues were homogenized with protein extraction solution (PRO-PREPTM, Intron Biotechnology, Seoul, Korea), and lysed by 60 min incubation on ice.
T219	28707-28769	Sentence	denotes	The tissue lysate centrifuged at 15,000 rpm for 15 min at 4°C.
T14073	28707-28974	Sentence	denotes	The tissue lysate centrifuged at 15,000 rpm for 15 min at 4°C. Equal amounts of proteins (40 µg) were separated on a SDS/12%-polyacrylamide gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Water and Process technologies, Trevose, PA, USA).
T220	28770-28974	Sentence	denotes	Equal amounts of proteins (40 µg) were separated on a SDS/12%-polyacrylamide gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Water and Process technologies, Trevose, PA, USA).
T14074	28975-29094	Sentence	denotes	Blots were blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-Buffered Saline Tween-20 [TBST:
T221	28975-29094	Sentence	denotes	Blots were blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-Buffered Saline Tween-20 [TBST:
T14075	29095-29167	Sentence	denotes	10 mM Tris (pH 8.0) and 150 mM NaCl solution containing 0.05% Tween-20].
T222	29095-29167	Sentence	denotes	10 mM Tris (pH 8.0) and 150 mM NaCl solution containing 0.05% Tween-20].
T14076	29168-29604	Sentence	denotes	After a short washing in TBST, the membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against p65 and p50 (1∶500 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies directed against Bax and PARP (1∶500 dilutions; Santa Cruz Biotechnology), and against caspase-3, caspase-9, Bcl-2 and c-IAP-1 (1∶1000 dilutions; Cell Signaling Technology, Beverly, MA).
T223	29168-29604	Sentence	denotes	After a short washing in TBST, the membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against p65 and p50 (1∶500 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies directed against Bax and PARP (1∶500 dilutions; Santa Cruz Biotechnology), and against caspase-3, caspase-9, Bcl-2 and c-IAP-1 (1∶1000 dilutions; Cell Signaling Technology, Beverly, MA).
T14077	29605-29759	Sentence	denotes	The blots were incubated with the respective horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1∶4000 dilutions; Santa Cruz Biotechnology).
T224	29605-29759	Sentence	denotes	The blots were incubated with the respective horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1∶4000 dilutions; Santa Cruz Biotechnology).
T14078	29760-29828	Sentence	denotes	Immunoreactive proteins were detected with the ECL detection system.
T225	29760-29828	Sentence	denotes	Immunoreactive proteins were detected with the ECL detection system.
T14660	29830-29850	Sentence	denotes	Immunohistochemistry
T226	29830-29850	Sentence	denotes	Immunohistochemistry
T14661	29851-29929	Sentence	denotes	All specimens were fixed in formalin and embedded in paraffin for examination.
T227	29851-29929	Sentence	denotes	All specimens were fixed in formalin and embedded in paraffin for examination.
T14662	29930-30011	Sentence	denotes	Sections (4 µm thick) were stained with H&E and analyzed by immunohistochemistry.
T228	29930-30011	Sentence	denotes	Sections (4 µm thick) were stained with H&E and analyzed by immunohistochemistry.
T14663	30012-30160	Sentence	denotes	The paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval.
T229	30012-30160	Sentence	denotes	The paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval.
T14664	30161-30305	Sentence	denotes	Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 15 min, followed by clearing in PBS for 5 min.
T230	30161-30305	Sentence	denotes	Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 15 min, followed by clearing in PBS for 5 min.
T14665	30306-30565	Sentence	denotes	The sections were blocked for 30 min with 3% normal horse serum diluted in PBS, blotted, and incubated with primary mouse anti-mouse proliferating cell nuclear antigen (PCNA, 1∶200 dilution) monoclonal antibodies in blocking serum for 4 h at room temperature.
T231	30306-30565	Sentence	denotes	The sections were blocked for 30 min with 3% normal horse serum diluted in PBS, blotted, and incubated with primary mouse anti-mouse proliferating cell nuclear antigen (PCNA, 1∶200 dilution) monoclonal antibodies in blocking serum for 4 h at room temperature.
T14666	30566-30713	Sentence	denotes	Thereafter, the slides were washed three times for 5 min each in PBS and incubated with biotinylated anti-mouse and anti-rabbit antibodies for 2 h.
T232	30566-30713	Sentence	denotes	Thereafter, the slides were washed three times for 5 min each in PBS and incubated with biotinylated anti-mouse and anti-rabbit antibodies for 2 h.
T14667	30714-30862	Sentence	denotes	The slides were washed in PBS, followed by the addition of the avidin biotin peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA).
T233	30714-30862	Sentence	denotes	The slides were washed in PBS, followed by the addition of the avidin biotin peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA).
T14668	30863-31123	Sentence	denotes	The slides were washed and the peroxidase reaction was developed with diaminobenzidine (DAB) and peroxide, followed by counterstaining with hematoxylin, mounting in aqua-mount, and evaluation under a light microscope (magnification×200; Olympus, Tokyo, Japan).
T234	30863-31123	Sentence	denotes	The slides were washed and the peroxidase reaction was developed with diaminobenzidine (DAB) and peroxide, followed by counterstaining with hematoxylin, mounting in aqua-mount, and evaluation under a light microscope (magnification×200; Olympus, Tokyo, Japan).
T14669	31124-31208	Sentence	denotes	A negative control was included in all experiments by omitting the primary antibody.
T235	31124-31208	Sentence	denotes	A negative control was included in all experiments by omitting the primary antibody.
T14670	31209-31513	Sentence	denotes	For the detection of apoptotic cell death in the tumor tissues, the paraffin-embedded sections were incubated in a mixture of the labeling solution (540 µl) and enzyme solution (60 µl) for 1 h at 37°C, and then washed three times in 0.1 M PBS for 5 min each, according to the manufacturer’s instructions.
T236	31209-31513	Sentence	denotes	For the detection of apoptotic cell death in the tumor tissues, the paraffin-embedded sections were incubated in a mixture of the labeling solution (540 µl) and enzyme solution (60 µl) for 1 h at 37°C, and then washed three times in 0.1 M PBS for 5 min each, according to the manufacturer’s instructions.
T237	31514-31609	Sentence	denotes	Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C.
T14671	31514-31727	Sentence	denotes	Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C. Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy (DAS microscope).
T238	31610-31727	Sentence	denotes	Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy (DAS microscope).
T15427	31729-31747	Sentence	denotes	Immunofluorescence
T239	31729-31747	Sentence	denotes	Immunofluorescence
T15428	31748-32313	Sentence	denotes	Sections were treated with 10% bovine serum albumin in PBS for 1 h at room temperature, incubated overnight at 4°C with p50 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), p65 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), Bax (1∶200 dilution), cleaved caspase-3 (1∶100), CD8 (1∶10), CD57 (1∶50) and IL-1Ra (1∶500) antibodies, followed by incubation in anti-mouse IgG conjugated with Alexa 488 (1∶100 dilution, Molecular Probes, Eugene, OR, USA) and anti-rabbit IgG conjugated with Alexa 568 (1∶100 dilution, Molecular Probes) for 40 min at room temperature.
T240	31748-32313	Sentence	denotes	Sections were treated with 10% bovine serum albumin in PBS for 1 h at room temperature, incubated overnight at 4°C with p50 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), p65 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), Bax (1∶200 dilution), cleaved caspase-3 (1∶100), CD8 (1∶10), CD57 (1∶50) and IL-1Ra (1∶500) antibodies, followed by incubation in anti-mouse IgG conjugated with Alexa 488 (1∶100 dilution, Molecular Probes, Eugene, OR, USA) and anti-rabbit IgG conjugated with Alexa 568 (1∶100 dilution, Molecular Probes) for 40 min at room temperature.
T15429	32314-32488	Sentence	denotes	Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy and photography using ApoTome microscopy (Carl Zeiss, Thornwood, NY, USA).
T241	32314-32488	Sentence	denotes	Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy and photography using ApoTome microscopy (Carl Zeiss, Thornwood, NY, USA).
T15688	32490-32521	Sentence	denotes	Gel Electromobility Shift Assay
T242	32490-32521	Sentence	denotes	Gel Electromobility Shift Assay
T15689	32522-32648	Sentence	denotes	A gel electromobility shift assay (EMSA) was performed according to the manufacturer’s recommendations (Promega, Madison, WI).
T243	32522-32648	Sentence	denotes	A gel electromobility shift assay (EMSA) was performed according to the manufacturer’s recommendations (Promega, Madison, WI).
T15690	32649-32920	Sentence	denotes	The tumor tissues were briefly homogenized in 200 µl of solution A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride), vortexed vigorously, incubated on ice for 10 min, and then centrifuged at 15000 rpm for 15 min.
T244	32649-32920	Sentence	denotes	The tumor tissues were briefly homogenized in 200 µl of solution A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride), vortexed vigorously, incubated on ice for 10 min, and then centrifuged at 15000 rpm for 15 min.
T15691	32921-33065	Sentence	denotes	The pelleted nuclei were resuspended in solution C (solution A supplemented with 420 mM NaCl and 20% glycerol), and incubated on ice for 20 min.
T245	32921-33065	Sentence	denotes	The pelleted nuclei were resuspended in solution C (solution A supplemented with 420 mM NaCl and 20% glycerol), and incubated on ice for 20 min.
T15692	33066-33220	Sentence	denotes	The resuspended pellet was centrifuged at 15000 rpm for 15 min, and the resulting nuclear extracts supernatant were collected in a chilled Eppendorf tube.
T246	33066-33220	Sentence	denotes	The resuspended pellet was centrifuged at 15000 rpm for 15 min, and the resulting nuclear extracts supernatant were collected in a chilled Eppendorf tube.
T247	33221-33329	Sentence	denotes	Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and [P32]-ATP for 10 min at 37°C.
T15693	33221-33403	Sentence	denotes	Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and [P32]-ATP for 10 min at 37°C. The gel shift reactions were assembled and incubated at room temperature.
T248	33330-33403	Sentence	denotes	The gel shift reactions were assembled and incubated at room temperature.
T15694	33404-33514	Sentence	denotes	Subsequently, 1 ml of gel loading buffer was added to each reaction and loaded onto a 6% non-denaturating gel.
T249	33404-33514	Sentence	denotes	Subsequently, 1 ml of gel loading buffer was added to each reaction and loaded onto a 6% non-denaturating gel.
T15695	33515-33610	Sentence	denotes	The gel was subjected to electrophoresis until the dye was four-fifths of the way down the gel.
T250	33515-33610	Sentence	denotes	The gel was subjected to electrophoresis until the dye was four-fifths of the way down the gel.
T15696	33611-33684	Sentence	denotes	The gel was dried for 1 h at 80°C and exposed to film overnight at -70°C.
T251	33611-33684	Sentence	denotes	The gel was dried for 1 h at 80°C and exposed to film overnight at -70°C.
T16197	33686-33710	Sentence	denotes	Flow Cytometric Analysis
T252	33686-33710	Sentence	denotes	Flow Cytometric Analysis
T16198	33711-33790	Sentence	denotes	Lymphocytes were obtained from tumor and spleen tissues for phenotype analysis.
T253	33711-33790	Sentence	denotes	Lymphocytes were obtained from tumor and spleen tissues for phenotype analysis.
T16199	33791-33906	Sentence	denotes	Spleen and tumor lymphocytes were isolated from fresh spleen and tumor biopsies obtained from three mice per group.
T254	33791-33906	Sentence	denotes	Spleen and tumor lymphocytes were isolated from fresh spleen and tumor biopsies obtained from three mice per group.
T16200	33907-34111	Sentence	denotes	Spleen and tumor biopsies were briefly homogenized mechanically in PBS, filtered (100 µm cell strainer; BD PharMingen) and then placed in lysis buffer (ACK lysing buffer, LONZA) to remove red blood cells.
T255	33907-34111	Sentence	denotes	Spleen and tumor biopsies were briefly homogenized mechanically in PBS, filtered (100 µm cell strainer; BD PharMingen) and then placed in lysis buffer (ACK lysing buffer, LONZA) to remove red blood cells.
T16201	34112-34246	Sentence	denotes	One million splenocytes were washed once in PBS containing 1% bovine serum albumin (BSA) and re-suspended in 100 µl of PBS/BSA buffer.

T120

Sentence

denotes

Deficiency of C-C Chemokine Receptor 5 Suppresses Tumor Development via Inactivation of NF-κB and Upregulation of IL-1Ra in Melanoma Model

T1

0-138

Sentence

denotes

Deficiency of C-C Chemokine Receptor 5 Suppresses Tumor Development via Inactivation of NF-κB and Upregulation of IL-1Ra in Melanoma Model

T2

139-184

Sentence

denotes

Deficiency of CCR5 Prevents Tumor Development

T3

186-194

Sentence

denotes

Abstract

T121

195-384

Sentence

denotes

To evaluate the relevance of C-C chemokine receptor type 5 (CCR5) expression and tumor development, we compared melanoma growth in CCR5 knockout (CCR5−/−) mice and wild type (CCR5+/+) mice.

T4

195-384

Sentence

denotes

To evaluate the relevance of C-C chemokine receptor type 5 (CCR5) expression and tumor development, we compared melanoma growth in CCR5 knockout (CCR5−/−) mice and wild type (CCR5+/+) mice.

T122

385-499

Sentence

denotes

CCR5−/− mice showed reduced tumor volume, tumor weight, and increased survival rate when compared to CCR5+/+ mice.

T5

385-499

Sentence

denotes

CCR5−/− mice showed reduced tumor volume, tumor weight, and increased survival rate when compared to CCR5+/+ mice.

T123

500-665

Sentence

denotes

We investigated the activation of NF-κB since it is an implicated transcription factor in the regulation of genes involving cell growth, apoptosis, and tumor growth.

T6

500-665

Sentence

denotes

We investigated the activation of NF-κB since it is an implicated transcription factor in the regulation of genes involving cell growth, apoptosis, and tumor growth.

T124

666-917

Sentence

denotes

Significant inhibition of DNA binding activity of NF-κB, and translocation of p50 and p65 into the nucleus through the inhibition of phosphorylation of IκB was found in the melanoma tissues of CCR5−/− mice compared to melanoma tissues of CCR5+/+ mice.

T7

666-917

Sentence

denotes

Significant inhibition of DNA binding activity of NF-κB, and translocation of p50 and p65 into the nucleus through the inhibition of phosphorylation of IκB was found in the melanoma tissues of CCR5−/− mice compared to melanoma tissues of CCR5+/+ mice.

T125

918-1150

Sentence

denotes

NF-κB target apoptotic protein expression, such as cleaved caspase-3, cleaved PARP, and Bax, was elevated, whereas the survival protein expression levels, such as Bcl-2, C-IAP1, was decreased in the melanoma tissues of CCR5−/− mice.

T8

918-1150

Sentence

denotes

NF-κB target apoptotic protein expression, such as cleaved caspase-3, cleaved PARP, and Bax, was elevated, whereas the survival protein expression levels, such as Bcl-2, C-IAP1, was decreased in the melanoma tissues of CCR5−/− mice.

T126

1151-1346

Sentence

denotes

Interestingly, we found that the level of IL-1Ra, a tumor growth suppressive cytokine, was significantly elevated in tumor tissue and spleen of CCR5−/− mice compared to the level in CCR5+/+ mice.

T9

1151-1346

Sentence

denotes

Interestingly, we found that the level of IL-1Ra, a tumor growth suppressive cytokine, was significantly elevated in tumor tissue and spleen of CCR5−/− mice compared to the level in CCR5+/+ mice.

T127

1347-1539

Sentence

denotes

Moreover, infiltration of CD8+ cytotoxic T cell and CD57+ natural killer cells was significantly increased in melanoma tumor and spleen tissue of CCR5−/− mice compared to that of CCR5+/+ mice.

T10

1347-1539

Sentence

denotes

Moreover, infiltration of CD8+ cytotoxic T cell and CD57+ natural killer cells was significantly increased in melanoma tumor and spleen tissue of CCR5−/− mice compared to that of CCR5+/+ mice.

T128

1540-1688

Sentence

denotes

Therefore, these results showed that CCR5 deficiency caused apoptotic cell death of melanoma through inhibition of NF-κB and upregulation of IL-1Ra.

T11

1540-1688

Sentence

denotes

Therefore, these results showed that CCR5 deficiency caused apoptotic cell death of melanoma through inhibition of NF-κB and upregulation of IL-1Ra.

T12

1690-1702

Sentence

denotes

Introduction

T1404

1703-1832

Sentence

denotes

Chemokines are small soluble molecules that are best known for their potent ability to induce cancer cell growth by inflammation.

T13

1703-1832

Sentence

denotes

Chemokines are small soluble molecules that are best known for their potent ability to induce cancer cell growth by inflammation.

T1405

1833-1907

Sentence

denotes

Many types of cancer cells express chemokines and chemokine receptors [1].

T14

1833-1907

Sentence

denotes

Many types of cancer cells express chemokines and chemokine receptors [1].

T1406

1908-2171

Sentence

denotes

The accumulated evidence indicates that the chemokine (C-C motif) ligand 5 (CCL5) and C-C chemokine receptor (CCR5), which are potent chemotactic factors for inflammatory cells, may be significantly involved in the proliferation and metastasis of several cancers.

T15

1908-2171

Sentence

denotes

The accumulated evidence indicates that the chemokine (C-C motif) ligand 5 (CCL5) and C-C chemokine receptor (CCR5), which are potent chemotactic factors for inflammatory cells, may be significantly involved in the proliferation and metastasis of several cancers.

T1407

2172-2407

Sentence

denotes

Intermediate and strong CCR5 expression is significantly associated with nonmetastatic development of colorectal cancer [2], melanoma [3], hepatoma [4], glioblastoma, Hodgkin lymphoma [5], as well as oral and prostate cancer cells [6].

T16

2172-2407

Sentence

denotes

Intermediate and strong CCR5 expression is significantly associated with nonmetastatic development of colorectal cancer [2], melanoma [3], hepatoma [4], glioblastoma, Hodgkin lymphoma [5], as well as oral and prostate cancer cells [6].

T1408

2408-2540

Sentence

denotes

Local production of the CCL5 is also important in the progression of breast cancer [7], and also correlates with poor prognosis [8].

T17

2408-2540

Sentence

denotes

Local production of the CCL5 is also important in the progression of breast cancer [7], and also correlates with poor prognosis [8].

T1409

2541-2667

Sentence

denotes

Meanwhile, CCR5 disruption has been demonstrated to inhibit experimental tumor growth and metastasis of pancreatic cancer [9].

T18

2541-2667

Sentence

denotes

Meanwhile, CCR5 disruption has been demonstrated to inhibit experimental tumor growth and metastasis of pancreatic cancer [9].

T1410

2668-2882

Sentence

denotes

In addition, host absence of CCR5 potentiates the delay of tumor growth [10], and CCR5 inhibitors prevented cancer cell growth, such as prostate cancer [11], breast cancer, hepatoma cells [4], and lung cancer [12].

T19

2668-2882

Sentence

denotes

In addition, host absence of CCR5 potentiates the delay of tumor growth [10], and CCR5 inhibitors prevented cancer cell growth, such as prostate cancer [11], breast cancer, hepatoma cells [4], and lung cancer [12].

T1411

2883-3023

Sentence

denotes

These data suggest that the deficiency of inflammatory chemokine receptor CCR5 may function as a suppressive receptor in cancer progression.

T20

2883-3023

Sentence

denotes

These data suggest that the deficiency of inflammatory chemokine receptor CCR5 may function as a suppressive receptor in cancer progression.

T1412

3024-3135

Sentence

denotes

However, more studies are required to describe how CCR5 deficiency acts in the inhibition of tumor development.

T21

3024-3135

Sentence

denotes

However, more studies are required to describe how CCR5 deficiency acts in the inhibition of tumor development.

T1413

3136-3292

Sentence

denotes

Interleukin-1 receptor antagonist (IL-1Ra), a member of the IL-1 family, is a naturally occurring cytokine that competitively blocks the IL-1 receptor [13].

T22

3136-3292

Sentence

denotes

Interleukin-1 receptor antagonist (IL-1Ra), a member of the IL-1 family, is a naturally occurring cytokine that competitively blocks the IL-1 receptor [13].

T1414

3293-3519

Sentence

denotes

Because IL-1Ra has been shown to inhibit tumor progression by promoting antitumor immune responses and by enhancing the activity of chemotherapy, numerous studies have examined the ability of IL-1Ra to block tumor progression.

T23

3293-3519

Sentence

denotes

Because IL-1Ra has been shown to inhibit tumor progression by promoting antitumor immune responses and by enhancing the activity of chemotherapy, numerous studies have examined the ability of IL-1Ra to block tumor progression.

T1415

3520-3602

Sentence

denotes

IL-1Ra expression was reduced in prostate [14], breast [15], and skin cancer [16].

T24

3520-3602

Sentence

denotes

IL-1Ra expression was reduced in prostate [14], breast [15], and skin cancer [16].

T1416

3603-3729

Sentence

denotes

It was also reported that endogenous IL-1Ra deficient mice developed aggressive tumors following exposure to carcinogens [17].

T25

3603-3729

Sentence

denotes

It was also reported that endogenous IL-1Ra deficient mice developed aggressive tumors following exposure to carcinogens [17].

T1417

3730-3867

Sentence

denotes

Meanwhile, proliferation of the melanoma cells that were transduced with IL-1Ra gene was reduced and inhibited melanoma development [18].

T26

3730-3867

Sentence

denotes

Meanwhile, proliferation of the melanoma cells that were transduced with IL-1Ra gene was reduced and inhibited melanoma development [18].

T1418

3868-3922

Sentence

denotes

IL-1R blockade also reduced hepatic tumor growth [19].

T27

3868-3922

Sentence

denotes

IL-1R blockade also reduced hepatic tumor growth [19].

T1419

3923-4118

Sentence

denotes

Moreover, it has also been reported that IL-1Ra reduces fibrosarcoma development [20], B16 melanoma growth and metastasis [21], colon adenocarcinoma growth [22], and skin cancer development [23].

T28

3923-4118

Sentence

denotes

Moreover, it has also been reported that IL-1Ra reduces fibrosarcoma development [20], B16 melanoma growth and metastasis [21], colon adenocarcinoma growth [22], and skin cancer development [23].

T1420

4119-4197

Sentence

denotes

IL-1 knockout mice injected with melanoma failed to develop solid tumors [24].

T29

4119-4197

Sentence

denotes

IL-1 knockout mice injected with melanoma failed to develop solid tumors [24].

T1421

4198-4373

Sentence

denotes

It was also found that IL-1Ra, combined with temozolomide and docetaxel chemotherapy, demonstrated more significant antitumor activity against B16 melanoma cells in vivo [25].

T30

4198-4373

Sentence

denotes

It was also found that IL-1Ra, combined with temozolomide and docetaxel chemotherapy, demonstrated more significant antitumor activity against B16 melanoma cells in vivo [25].

T1422

4374-4458

Sentence

denotes

These data indicate that IL-1Ra is important as a tumor growth suppressing cytokine.

T31

4374-4458

Sentence

denotes

These data indicate that IL-1Ra is important as a tumor growth suppressing cytokine.

T1423

4459-4639

Sentence

denotes

NF-κB plays a crucial role in the suppression of apoptosis, as well as the induction of cell proliferation and inflammation, and is closely associated with cancer development [26].

T32

4459-4639

Sentence

denotes

NF-κB plays a crucial role in the suppression of apoptosis, as well as the induction of cell proliferation and inflammation, and is closely associated with cancer development [26].

T1424

4640-4884

Sentence

denotes

Constitutive activation of NF-κB has been described in a great number of cancers including colon cancers, prostate cancers and melanoma [27], and was found to up-regulate anti-apoptotic genes and/or down regulate apoptotic gene expression [28].

T33

4640-4884

Sentence

denotes

Constitutive activation of NF-κB has been described in a great number of cancers including colon cancers, prostate cancers and melanoma [27], and was found to up-regulate anti-apoptotic genes and/or down regulate apoptotic gene expression [28].

T1425

4885-4986

Sentence

denotes

Although NF-κB promotes tumor growth, it is required for the immune system to function normally [29].

T34

4885-4986

Sentence

denotes

Although NF-κB promotes tumor growth, it is required for the immune system to function normally [29].

T1426

4987-5122

Sentence

denotes

Studies on c-Rel-deficient mice demonstrated that c-Rel is essential for IL-2, IL-3, GM-CSF, and IFNγ expression in T lymphocytes [30].

T35

4987-5122

Sentence

denotes

Studies on c-Rel-deficient mice demonstrated that c-Rel is essential for IL-2, IL-3, GM-CSF, and IFNγ expression in T lymphocytes [30].

T1427

5123-5318

Sentence

denotes

C-Rel-deficient mice also have a tissue-specific deficiency of various cytokines and growth factors in T cells and macrophages affecting both innate and humoral immune responses in the host [31].

T36

5123-5318

Sentence

denotes

C-Rel-deficient mice also have a tissue-specific deficiency of various cytokines and growth factors in T cells and macrophages affecting both innate and humoral immune responses in the host [31].

T1428

5319-5564

Sentence

denotes

Therefore, NF-κB can be specially targeted to prevent cancer cell growth by (1) directly affecting apoptotic cancer cell death, (2) stimulating tumor killing lymphocytes infiltration or (3) increasing the production of tumor killing lymphokines.

T37

5319-5564

Sentence

denotes

Therefore, NF-κB can be specially targeted to prevent cancer cell growth by (1) directly affecting apoptotic cancer cell death, (2) stimulating tumor killing lymphocytes infiltration or (3) increasing the production of tumor killing lymphokines.

T1429

5565-5660

Sentence

denotes

Activation of NF-κB is important in the regulation of CCR-mediated tumor growth and metastasis.

T38

5565-5660

Sentence

denotes

Activation of NF-κB is important in the regulation of CCR-mediated tumor growth and metastasis.

T1430

5661-5831

Sentence

denotes

It was reported that inhibition of NF-κB by dehydroxymethyl-epoxyquinomicin (DHMEQ) induces cell death of primary effusion lymphoma by inhibition of CCR5 expression [32].

T39

5661-5831

Sentence

denotes

It was reported that inhibition of NF-κB by dehydroxymethyl-epoxyquinomicin (DHMEQ) induces cell death of primary effusion lymphoma by inhibition of CCR5 expression [32].

T1431

5832-5931

Sentence

denotes

Activation of NF-κB also increases CCL5-mediated oral cancer motility (5) and lung metastasis [33].

T40

5832-5931

Sentence

denotes

Activation of NF-κB also increases CCL5-mediated oral cancer motility (5) and lung metastasis [33].

T1432

5932-6031

Sentence

denotes

Thus, it is possible that inhibition of NF-κB could be involved in CCR5 mediated tumor development.

T41

5932-6031

Sentence

denotes

Thus, it is possible that inhibition of NF-κB could be involved in CCR5 mediated tumor development.

T1433

6032-6188

Sentence

denotes

NF-κB transcription factor binding sites are present in the promoter region of proinflammatory cytokine genes, such as IL-1alpha, IL-6, GM-CSF, and KC [34].

T42

6032-6188

Sentence

denotes

NF-κB transcription factor binding sites are present in the promoter region of proinflammatory cytokine genes, such as IL-1alpha, IL-6, GM-CSF, and KC [34].

T1434

6189-6272

Sentence

denotes

On the other hand, the IL-1Ra is a negative regulator of the inflammatory response.

T43

6189-6272

Sentence

denotes

On the other hand, the IL-1Ra is a negative regulator of the inflammatory response.

T1435

6273-6377

Sentence

denotes

Several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1.

T44

6273-6377

Sentence

denotes

Several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1.

T1436

6378-6583

Sentence

denotes

Smith et al. suggested that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors; NF-κB/PU.1/GA-binding protein binding site [35].

T45

6378-6583

Sentence

denotes

Smith et al. suggested that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors; NF-κB/PU.1/GA-binding protein binding site [35].

T1437

6584-6728

Sentence

denotes

Nuclear translocation of NF-κB (p65) was significantly enhanced and prolonged in IL-1Ra-deficient mice, compared to that in wild type mice [36].

T46

6584-6728

Sentence

denotes

Nuclear translocation of NF-κB (p65) was significantly enhanced and prolonged in IL-1Ra-deficient mice, compared to that in wild type mice [36].

T1438

6729-6865

Sentence

denotes

Intracellular IL-1Ra (icIL-1Ra) was found to inhibit IL-1 by blocking NF-κB signal transduction pathways in inflammatory responses [37].

T47

6729-6865

Sentence

denotes

Intracellular IL-1Ra (icIL-1Ra) was found to inhibit IL-1 by blocking NF-κB signal transduction pathways in inflammatory responses [37].

T1439

6866-7085

Sentence

denotes

Because IL-1 has been shown to be an inducer of NF-κB activation [38], we hypothesized that the expression of IL-1Ra could play an important role in the autocrine activation of transcription factors NF-κB or vice versa.

T48

6866-7085

Sentence

denotes

Because IL-1 has been shown to be an inducer of NF-κB activation [38], we hypothesized that the expression of IL-1Ra could play an important role in the autocrine activation of transcription factors NF-κB or vice versa.

T1440

7086-7233

Sentence

denotes

Thus, the aim of this study was to evaluate the roles of NF-κB and IL-1Ra in the chemokine receptor CCR5-mediated suppression of tumor development.

T49

7086-7233

Sentence

denotes

Thus, the aim of this study was to evaluate the roles of NF-κB and IL-1Ra in the chemokine receptor CCR5-mediated suppression of tumor development.

T50

7235-7242

Sentence

denotes

Results

T4493

7244-7291

Sentence

denotes

Inhibition of Tumor Development in CCR5−/− Mice

T51

7244-7291

Sentence

denotes

Inhibition of Tumor Development in CCR5−/− Mice

T4494

7292-7380

Sentence

denotes

The effect of CCR5 in tumor development was investigated using CCR5+/+ and CCR5−/− mice.

T52

7292-7380

Sentence

denotes

The effect of CCR5 in tumor development was investigated using CCR5+/+ and CCR5−/− mice.

T4495

7381-7473

Sentence

denotes

B16-F0 melanoma cells were inoculated subcutaneously into CCR5+/+ and CCR5−/− mice (n = 20).

T53

7381-7473

Sentence

denotes

B16-F0 melanoma cells were inoculated subcutaneously into CCR5+/+ and CCR5−/− mice (n = 20).

T4496

7474-7513

Sentence

denotes

Tumor growth was monitored for 31 days.

T54

7474-7513

Sentence

denotes

Tumor growth was monitored for 31 days.

T4497

7514-7601

Sentence

denotes

Tumor growth in the CCR5−/− mice was significantly lower than that in the CCR5+/+ mice.

T55

7514-7601

Sentence

denotes

Tumor growth in the CCR5−/− mice was significantly lower than that in the CCR5+/+ mice.

T4498

7602-7823

Sentence

denotes

The tumor growth in the CCR5−/− mice was reduced to 19.1%, whereas the tumor growth in the CCR5+/+ mice was only reduced to 24.3%, in both tumor weight (Figure 1A and 1B) and tumor volume, respectively (Figure 1A and 1C).

T56

7602-7823

Sentence

denotes

The tumor growth in the CCR5−/− mice was reduced to 19.1%, whereas the tumor growth in the CCR5+/+ mice was only reduced to 24.3%, in both tumor weight (Figure 1A and 1B) and tumor volume, respectively (Figure 1A and 1C).

T4499

7824-7874

Sentence

denotes

These results correlated with mice survival rates.

T57

7824-7874

Sentence

denotes

These results correlated with mice survival rates.

T4500

7875-8036

Sentence

denotes

There was a significant difference in survival rates between CCR5−/− mice (75% of starting time) and CCR5+/+ mice (15%) at the end of the experiment (Figure 1D).

T58

7875-8036

Sentence

denotes

There was a significant difference in survival rates between CCR5−/− mice (75% of starting time) and CCR5+/+ mice (15%) at the end of the experiment (Figure 1D).

T4501

8037-8128

Sentence

denotes

There was a significant difference in survival rates between CCR5−/− mice and CCR5+/+ mice.

T59

8037-8128

Sentence

denotes

There was a significant difference in survival rates between CCR5−/− mice and CCR5+/+ mice.

T4502

8129-8236

Sentence

denotes

At the end of the experiment, 75% of the CCR5−/− mice survived while only 15% of the CCR5+/+ mice survived.

T60

8129-8236

Sentence

denotes

At the end of the experiment, 75% of the CCR5−/− mice survived while only 15% of the CCR5+/+ mice survived.

T4503

8237-8323

Sentence

denotes

Subcutaneous tumors were harvested from the sacrificed mice 31 days after inoculation.

T61

8237-8323

Sentence

denotes

Subcutaneous tumors were harvested from the sacrificed mice 31 days after inoculation.

T4504

8324-8494

Sentence

denotes

The immunohistochemical analysis of tumor sections, stained with H&E and for proliferation antigen PCNA, revealed greater inhibition of tumor cell growth in CCR5−/− mice.

T62

8324-8494

Sentence

denotes

The immunohistochemical analysis of tumor sections, stained with H&E and for proliferation antigen PCNA, revealed greater inhibition of tumor cell growth in CCR5−/− mice.

T4505

8495-8661

Sentence

denotes

The histologic findings after H&E staining indicated that the tumor tissues of the CCR5−/− mice, but not those of the CCR5+/+ mice, contained large areas of necrosis.

T63

8495-8661

Sentence

denotes

The histologic findings after H&E staining indicated that the tumor tissues of the CCR5−/− mice, but not those of the CCR5+/+ mice, contained large areas of necrosis.

T4506

8662-8741

Sentence

denotes

PCNA reactive cells were significantly reduced in the CCR5−/− mice (Figure 1E).

T64

8662-8741

Sentence

denotes

PCNA reactive cells were significantly reduced in the CCR5−/− mice (Figure 1E).

T65

8742-8834

Sentence

denotes

10.1371/journal.pone.0033747.g001 Figure 1 Inhibition of tumor development in CCR5−/− mice.

T17206

8786-8834

Sentence

denotes

Inhibition of tumor development in CCR5−/− mice.

T17207

8835-8875

Sentence

denotes

A–C, Tumor images, volumes, and weights.

T66

8835-8875

Sentence

denotes

A–C, Tumor images, volumes, and weights.

T17208

8876-8980

Sentence

denotes

B16 melanoma cells (5×105 cells/mouse) were inoculated s.c. into CCR5+/+ mice and CCR5−/− mice (n = 20).

T67

8876-8980

Sentence

denotes

B16 melanoma cells (5×105 cells/mouse) were inoculated s.c. into CCR5+/+ mice and CCR5−/− mice (n = 20).

T17209

8981-9081

Sentence

denotes

Tumor volumes were measured every day and tumor weights were measured at study termination (Day 31).

T68

8981-9081

Sentence

denotes

Tumor volumes were measured every day and tumor weights were measured at study termination (Day 31).

T17210

9082-9182

Sentence

denotes

The CCR5−/− mice had a significant reduction in tumor growth and volume as compared to CCR5+/+ mice.

T69

9082-9182

Sentence

denotes

The CCR5−/− mice had a significant reduction in tumor growth and volume as compared to CCR5+/+ mice.

T17211

9183-9285

Sentence

denotes

The results are expressed as mean ± SD. * indicates significant difference from CCR5+/+ mice (P<0.05).

T70

9183-9285

Sentence

denotes

The results are expressed as mean ± SD. * indicates significant difference from CCR5+/+ mice (P<0.05).

T17212

9286-9399

Sentence

denotes

D, The CCR5−/− mice demonstrated significantly higher survival rates compared to CCR5+/+ mice (by Log-rank Test).

T71

9286-9399

Sentence

denotes

D, The CCR5−/− mice demonstrated significantly higher survival rates compared to CCR5+/+ mice (by Log-rank Test).

T72

9400-9402

Sentence

denotes

E.

T17213

9400-9529

Sentence

denotes

E. Tumor sections were analyzed by H&E stain and expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry.

T73

9403-9529

Sentence

denotes

Tumor sections were analyzed by H&E stain and expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry.

T17214

9530-9644

Sentence

denotes

Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (P<0.05).

T74

9530-9644

Sentence

denotes

Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (P<0.05).

T17215

9645-9670

Sentence

denotes

Scale bar indicates 50 m.

T75

9645-9670

Sentence

denotes

Scale bar indicates 50 m.

T5418

9672-9730

Sentence

denotes

Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice

T76

9672-9730

Sentence

denotes

Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice

T17744

9672-9858

Sentence

denotes

Decrease of NF-κB Activity in Tumor Tissue of CCR5−/− Mice The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression.

T5419

9731-9858

Sentence

denotes

The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression.

T77

9731-9858

Sentence

denotes

The activation of NF-κB plays a critical role in cancer cell survival, especially melanoma cancer cells and in CCR5 expression.

T5420

9859-10062

Sentence

denotes

To evaluate whether the tumor inhibition was related with the inactivation of NF-κB in melanoma, the DNA binding activity of NF-κB was determined by electromobility shift assay (EMSA) in melanoma tissue.

T78

9859-10062

Sentence

denotes

To evaluate whether the tumor inhibition was related with the inactivation of NF-κB in melanoma, the DNA binding activity of NF-κB was determined by electromobility shift assay (EMSA) in melanoma tissue.

T5421

10063-10170

Sentence

denotes

The constitutively activated DNA binding activity was reduced in the CCR5−/− mice tumor tissue (Figure 2A).

T79

10063-10170

Sentence

denotes

The constitutively activated DNA binding activity was reduced in the CCR5−/− mice tumor tissue (Figure 2A).

T5422

10171-10350

Sentence

denotes

Moreover, the translocation of p50 and p-p65 into the nucleus through the inhibition of phosphorylation of IκB was also prevented in the tumor tissues of CCR5−/− mice (Figure 2B).

T80

10171-10350

Sentence

denotes

Moreover, the translocation of p50 and p-p65 into the nucleus through the inhibition of phosphorylation of IκB was also prevented in the tumor tissues of CCR5−/− mice (Figure 2B).

T5423

10351-10504

Sentence

denotes

Immunofluorescence analysis also confirmed that the intensity of nuclear staining for p50 had decreased in the tumor tissues of CCR5−/− mice (Figure 2C).

T81

10351-10504

Sentence

denotes

Immunofluorescence analysis also confirmed that the intensity of nuclear staining for p50 had decreased in the tumor tissues of CCR5−/− mice (Figure 2C).

T82

10505-10608

Sentence

denotes

10.1371/journal.pone.0033747.g002 Figure 2 Decrease of NF-κB activity in tumor tissue of CCR5−/− mice.

T17745

10609-10778

Sentence

denotes

A, The DNA binding activity of NF-κB was determined in the nuclear extracts of the CCR5−/− mice and CCR5+/+ mice tumor tissues by EMSA described in material and methods.

T83

10609-10778

Sentence

denotes

A, The DNA binding activity of NF-κB was determined in the nuclear extracts of the CCR5−/− mice and CCR5+/+ mice tumor tissues by EMSA described in material and methods.

T17746

10779-10933

Sentence

denotes

B, Expression of p50 and p65 phosphorylation in nuclear extracts (NE), and IκB and IκB phosphorylation in the cytosol (CE) determined by Western blotting.

T84

10779-10933

Sentence

denotes

B, Expression of p50 and p65 phosphorylation in nuclear extracts (NE), and IκB and IκB phosphorylation in the cytosol (CE) determined by Western blotting.

T17747

10934-11084

Sentence

denotes

C, Immunolfluorescence analysis of p50 confirmed that the intensities of nuclear staining for p50 were decreased in the tumor tissues of CCR5−/− mice.

T85

10934-11084

Sentence

denotes

C, Immunolfluorescence analysis of p50 confirmed that the intensities of nuclear staining for p50 were decreased in the tumor tissues of CCR5−/− mice.

T17748

11085-11199

Sentence

denotes

Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

T86

11085-11199

Sentence

denotes

Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

T17749

11200-11226

Sentence

denotes

Scale bar indicates 50 µm.

T87

11200-11226

Sentence

denotes

Scale bar indicates 50 µm.

T5957

11228-11337

Sentence

denotes

Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice

T88

11228-11337

Sentence

denotes

Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice

T18203

11228-12481

Sentence

denotes

Induction of Apoptotic Cell Death and Inhibition of the Expression of Anti-apoptotic Proteins in CCR5−/− Mice To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed. Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells. TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A). Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C). Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D). 10.1371/journal.pone.0033747.g003 Figure 3 Induction of apoptotic cell death in tumor tissues of CCR5 −/− mice.

T5958

11338-11611

Sentence

denotes

To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed.

T89

11338-11611

Sentence

denotes

To see whether inhibition of NF-κB activity results in the induction of apoptotic cell death and expression of NF-κB target apoptotic cell death regulatory protein, the levels of apoptotic cell death and expression of apoptotic cell death regulatory proteins were assessed.

T5959

11612-11743

Sentence

denotes

Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells.

T90

11612-11743

Sentence

denotes

Apoptotic cell death in the tumor sections were evaluated in the CCR5+/+ and CCR5−/− mice that were inoculated with melanoma cells.

T5960

11744-11894

Sentence

denotes

TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A).

T91

11744-11894

Sentence

denotes

TUNEL staining revealed a higher frequency of apoptotic cell death in the tumor tissues of the CCR5−/− mice than that of the CCR5+/+ mice (Figure 3A).

T5961

11895-12082

Sentence

denotes

Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C).

T92

11895-12082

Sentence

denotes

Immunohistochemical and immunofluorescence showed that high levels of cleaved caspase-3 and Bax were found more frequently in the CCR5−/− mice than in the CCR5+/+ mice (Figure 3B and 3C).

T5962

12083-12368

Sentence

denotes

Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D).

T93

12083-12368

Sentence

denotes

Consistent with the immunohistochemical data, Western blot analysis of tumor proteins showed greater increased levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, whereas the levels of Bcl-2 and C-IAP1 were significantly decreased in CCR5−/− mice tissues (Figure 3D).

T94

12369-12481

Sentence

denotes

10.1371/journal.pone.0033747.g003 Figure 3 Induction of apoptotic cell death in tumor tissues of CCR5 −/− mice.

T18204

12482-12627

Sentence

denotes

A, Apoptotic cells in tumor sections were examined by fluorescence microscopy after DAPI and TUNEL staining as described in material and methods.

T95

12482-12627

Sentence

denotes

A, Apoptotic cells in tumor sections were examined by fluorescence microscopy after DAPI and TUNEL staining as described in material and methods.

T18205

12628-12733

Sentence

denotes

The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells that were counted.

T96

12628-12733

Sentence

denotes

The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells that were counted.

T18206

12734-12859

Sentence

denotes

Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.

T97

12734-12859

Sentence

denotes

Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.

T18207

12860-12886

Sentence

denotes

Scale bar indicates 50 µm.

T98

12860-12886

Sentence

denotes

Scale bar indicates 50 µm.

T18208

12887-13002

Sentence

denotes

B and C, Apoptotic protein (cleaved caspase-3 and Bax) in tumor sections were detected by immunofluorescence assay.

T99

12887-13002

Sentence

denotes

B and C, Apoptotic protein (cleaved caspase-3 and Bax) in tumor sections were detected by immunofluorescence assay.

T18209

13003-13153

Sentence

denotes

The reactive cell number was determined as the number of DAPI-stained, Specific antibody (cleaved caspase-3 and Bax)-positive cells that were counted.

T100

13003-13153

Sentence

denotes

The reactive cell number was determined as the number of DAPI-stained, Specific antibody (cleaved caspase-3 and Bax)-positive cells that were counted.

T18210

13154-13279

Sentence

denotes

Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.

T101

13154-13279

Sentence

denotes

Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.

T18211

13280-13306

Sentence

denotes

Scale bar indicates 50 µm.

T102

13280-13306

Sentence

denotes

Scale bar indicates 50 µm.

T18212

13307-13502

Sentence

denotes

D, The expression of apoptotic proteins was detected by Western blotting using specific antibodies; cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bax, Bcl-2 and c-IAP1 in the tumor tissues.

T103

13307-13502

Sentence

denotes

D, The expression of apoptotic proteins was detected by Western blotting using specific antibodies; cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bax, Bcl-2 and c-IAP1 in the tumor tissues.

T18213

13503-13555

Sentence

denotes

The β-actin protein was used as an internal control.

T104

13503-13555

Sentence

denotes

The β-actin protein was used as an internal control.

T18214

13556-13626

Sentence

denotes

Each band is representative of three independent experimental results.

T105

13556-13626

Sentence

denotes

Each band is representative of three independent experimental results.

T18215

13627-13742

Sentence

denotes

Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

T106

13627-13742

Sentence

denotes

Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

T6636

13744-13806

Sentence

denotes

Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice

T107

13744-13806

Sentence

denotes

Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice

T18867

13744-14007

Sentence

denotes

Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A).

T6637

13807-14007

Sentence

denotes

To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A).

T108

13807-14007

Sentence

denotes

To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A).

T6638

14008-14159

Sentence

denotes

Among 40 tested cytokines, the levels of IL-1Ra, MIG, MIP-1α increased about 686, 9 and 25-fold respectively, in CCR5−/− mice compared to CCR5+/+ mice.

T109

14008-14159

Sentence

denotes

Among 40 tested cytokines, the levels of IL-1Ra, MIG, MIP-1α increased about 686, 9 and 25-fold respectively, in CCR5−/− mice compared to CCR5+/+ mice.

T6639

14160-14286

Sentence

denotes

Using this technique, no other cytokines were found to be different between the CCR5−/− mice and the CCR5+/+ mice (Figure 4B).

T110

14160-14286

Sentence

denotes

Using this technique, no other cytokines were found to be different between the CCR5−/− mice and the CCR5+/+ mice (Figure 4B).

T6640

14287-14373

Sentence

denotes

Similar altered cytokine levels were found in the spleens of CCR5−/− mice (Figure 4C).

T111

14287-14373

Sentence

denotes

Similar altered cytokine levels were found in the spleens of CCR5−/− mice (Figure 4C).

T6641

14374-14543

Sentence

denotes

Since the IL-1Ra level was the most significantly elevated, the expression of IL-1Ra in tumor and spleen tissues was analyzed by immunofluorescence and western blotting.

T112

14374-14543

Sentence

denotes

Since the IL-1Ra level was the most significantly elevated, the expression of IL-1Ra in tumor and spleen tissues was analyzed by immunofluorescence and western blotting.

T6642

14544-14647

Sentence

denotes

The expression of IL-1Ra was upregulated in tumor and spleen tissues of the CCR5−/− mice (Figure 4D-G).

T113

14544-14647

Sentence

denotes

The expression of IL-1Ra was upregulated in tumor and spleen tissues of the CCR5−/− mice (Figure 4D-G).

T114

14648-14755

Sentence

denotes

10.1371/journal.pone.0033747.g004 Figure 4 Upregulation of IL-1Ra in the tumor and spleen of CCR5−/− mice.

T18868

14756-14818

Sentence

denotes

A, Figure 4A indicates mouse cytokine array panel coordinates.

T115

14756-14818

Sentence

denotes

A, Figure 4A indicates mouse cytokine array panel coordinates.

T18869

14819-14911

Sentence

denotes

Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate.

T116

14819-14911

Sentence

denotes

Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate.

T18870

14912-15055

Sentence

denotes

B, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.

T117

14912-15055

Sentence

denotes

B, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.

T18871

15056-15120

Sentence

denotes

Representative blot from three independent experiments is shown.

T118

15056-15120

Sentence

denotes

Representative blot from three independent experiments is shown.

T18872

15121-15213

Sentence

denotes

Positive controls show the manufacturer’s internal positive control samples on the membrane.

T119

15121-15213

Sentence

denotes

Positive controls show the manufacturer’s internal positive control samples on the membrane.

T18873

15214-15299

Sentence

denotes

C, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues.

T120

15214-15299

Sentence

denotes

C, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues.

T18874

15300-15393

Sentence

denotes

There were differences in cytokines between CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.

T121

15300-15393

Sentence

denotes

There were differences in cytokines between CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra.

T18875

15394-15458

Sentence

denotes

Representative blot from three independent experiments is shown.

T122

15394-15458

Sentence

denotes

Representative blot from three independent experiments is shown.

T18876

15459-15551

Sentence

denotes

Positive controls show the manufacturer’s internal positive control samples on the membrane.

T123

15459-15551

Sentence

denotes

Positive controls show the manufacturer’s internal positive control samples on the membrane.

T18877

15552-15672

Sentence

denotes

D and E, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues.

T124

15552-15672

Sentence

denotes

D and E, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues.

T18878

15673-15793

Sentence

denotes

The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted.

T125

15673-15793

Sentence

denotes

The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted.

T18879

15794-15919

Sentence

denotes

Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.

T126

15794-15919

Sentence

denotes

Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice.

T18880

15920-15946

Sentence

denotes

Scale bar indicates 50 µm.

T127

15920-15946

Sentence

denotes

Scale bar indicates 50 µm.

T18881

15947-16038

Sentence

denotes

F and G, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues.

T128

15947-16038

Sentence

denotes

F and G, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues.

T18882

16039-16109

Sentence

denotes

Each band is representative of three independent experimental results.

T129

16039-16109

Sentence

denotes

Each band is representative of three independent experimental results.

T18883

16110-16225

Sentence

denotes

Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

T130

16110-16225

Sentence

denotes

Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).

T7342

16227-16314

Sentence

denotes

Induction of CD8 T cells and NK Cell Infiltration into Tumor and Spleen of CCR5−/− Mice

T131

16227-16314

Sentence

denotes

Induction of CD8 T cells and NK Cell Infiltration into Tumor and Spleen of CCR5−/− Mice

T7343

16315-16554

Sentence

denotes

To investigate whether the inhibition of tumor growth in CCR5−/− mice is related to tumor-specific immune responses, we analyzed the distribution patterns of CD8+ cytotoxic T cell and CD57+ Natural Killer cells in tumor and spleen tissues.

T132

16315-16554

Sentence

denotes

To investigate whether the inhibition of tumor growth in CCR5−/− mice is related to tumor-specific immune responses, we analyzed the distribution patterns of CD8+ cytotoxic T cell and CD57+ Natural Killer cells in tumor and spleen tissues.

T7344

16555-16754

Sentence

denotes

By staining the tumor sections from CCR5−/− mice, we found that there was a significant influx of CD8+ cells into the tumor, as well as an increase in the number of infiltrating NK cells (Figure 5A).

T133

16555-16754

Sentence

denotes

By staining the tumor sections from CCR5−/− mice, we found that there was a significant influx of CD8+ cells into the tumor, as well as an increase in the number of infiltrating NK cells (Figure 5A).

T7345

16755-16840

Sentence

denotes

CD8+ T cells and CD57+ NK cells were spread diffusely throughout the entire sections.

T134

16755-16840

Sentence

denotes

CD8+ T cells and CD57+ NK cells were spread diffusely throughout the entire sections.

T7346

16841-17068

Sentence

denotes

To further investigate the differences in the numbers of CD8+ T cells and CD57+ NK cells between CCR5+/+ mice and CCR5−/− mice in immunity-related organ, we also analyzed the CD8 and CD57 reactive cell number in spleen tissues.

T135

16841-17068

Sentence

denotes

To further investigate the differences in the numbers of CD8+ T cells and CD57+ NK cells between CCR5+/+ mice and CCR5−/− mice in immunity-related organ, we also analyzed the CD8 and CD57 reactive cell number in spleen tissues.

T7347

17069-17212

Sentence

denotes

The number of CD8+ T cells and CD57+ NK cells were higher in the spleen of the CCR5−/− mice than in the spleen of the CCR5+/+ mice (Figure 5B).

T136

17069-17212

Sentence

denotes

The number of CD8+ T cells and CD57+ NK cells were higher in the spleen of the CCR5−/− mice than in the spleen of the CCR5+/+ mice (Figure 5B).

T7348

17213-17403

Sentence

denotes

These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in an anti-tumor effect in CCR5−/− mice.

T137

17213-17403

Sentence

denotes

These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in an anti-tumor effect in CCR5−/− mice.

T138

17404-17558

Sentence

denotes

10.1371/journal.pone.0033747.g005 Figure 5 Induction of the infiltration of CD8+ T cells and NK cells into tumor, and increase in spleen of CCR5−/− mice.

T19587

17448-17558

Sentence

denotes

Induction of the infiltration of CD8+ T cells and NK cells into tumor, and increase in spleen of CCR5−/− mice.

T19588

17559-17742

Sentence

denotes

A and B, Immunolfluorescence analysis was used to determine the expression levels of CD8 (CD8+ cytotoxic T-cell surface marker) and CD57 (NK cell marker) in tumor and spleen sections.

T139

17559-17742

Sentence

denotes

A and B, Immunolfluorescence analysis was used to determine the expression levels of CD8 (CD8+ cytotoxic T-cell surface marker) and CD57 (NK cell marker) in tumor and spleen sections.

T19589

17743-17833

Sentence

denotes

The images shown are representative of three separate experiments performed in triplicate.

T140

17743-17833

Sentence

denotes

The images shown are representative of three separate experiments performed in triplicate.

T19590

17834-17879

Sentence

denotes

Scale bars indicate 50 µm (A) and 100 µm (B).

T141

17834-17879

Sentence

denotes

Scale bars indicate 50 µm (A) and 100 µm (B).

T19591

17880-17923

Sentence

denotes

C and D, Analysis of lymphocyte phenotypes.

T142

17880-17923

Sentence

denotes

C and D, Analysis of lymphocyte phenotypes.

T19592

17924-17998

Sentence

denotes

The tumor and spleen tissues were separated at study termination (Day 31).

T143

17924-17998

Sentence

denotes

The tumor and spleen tissues were separated at study termination (Day 31).

T19593

17999-18098

Sentence

denotes

Flow cytometry analysis was performed using FACSAria flow cytometry, and represent data were shown.

T144

17999-18098

Sentence

denotes

Flow cytometry analysis was performed using FACSAria flow cytometry, and represent data were shown.

T19594

18099-18150

Sentence

denotes

Data are means ± S.D. of four experimental animals.

T145

18099-18150

Sentence

denotes

Data are means ± S.D. of four experimental animals.

T7349

18151-18318

Sentence

denotes

To analyze the cell phenotype change in CCR5−/− mice and CCR5+/+ mice by inoculation of the melanoma cell, lymphocytes were isolated from the tumor and spleen tissues.

T146

18151-18318

Sentence

denotes

To analyze the cell phenotype change in CCR5−/− mice and CCR5+/+ mice by inoculation of the melanoma cell, lymphocytes were isolated from the tumor and spleen tissues.

T7350

18319-18524

Sentence

denotes

The B, CD3 T, CD4 T, CD 8 T and NK cell portion of CCR5−/− mice tumor tissue were 7.2%, 48.2%, 16.9%, 32.1% and 7.8%, respectively, compared to 2.9%, 11.1%, 2.3%, 0.6% and 3.1% of CCR5+/+ mice (Figure 5C).

T147

18319-18524

Sentence

denotes

The B, CD3 T, CD4 T, CD 8 T and NK cell portion of CCR5−/− mice tumor tissue were 7.2%, 48.2%, 16.9%, 32.1% and 7.8%, respectively, compared to 2.9%, 11.1%, 2.3%, 0.6% and 3.1% of CCR5+/+ mice (Figure 5C).

T7351

18525-18684

Sentence

denotes

The lymphocytes that infiltrated the tumor tissue of the CCR5−/− mice also had higher levels of CD19, CD3, CD4, CD8 and CD57 when compared to the CCR5+/+ mice.

T148

18525-18684

Sentence

denotes

The lymphocytes that infiltrated the tumor tissue of the CCR5−/− mice also had higher levels of CD19, CD3, CD4, CD8 and CD57 when compared to the CCR5+/+ mice.

T7352

18685-18891

Sentence

denotes

The B, CD3 T, CD4 T, CD 8 T and NK cell portion of the CCR5−/− mice spleen were 61.3%, 33.4%, 17.6%, 9.2% and 0.6%, respectively compared to 38.3%, 5.9%, 4.0%, 1.0% and 3.9% of the CCR5+/+ mice (Figure 5D).

T149

18685-18891

Sentence

denotes

The B, CD3 T, CD4 T, CD 8 T and NK cell portion of the CCR5−/− mice spleen were 61.3%, 33.4%, 17.6%, 9.2% and 0.6%, respectively compared to 38.3%, 5.9%, 4.0%, 1.0% and 3.9% of the CCR5+/+ mice (Figure 5D).

T7353

18892-19092

Sentence

denotes

These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in the inhibition of tumor growth in CCR5−/− mice.

T150

18892-19092

Sentence

denotes

These data suggest that by promoting the infiltration of CD8 T cells and NK cells, which are potent cytotoxic effectors for tumors, could play a role in the inhibition of tumor growth in CCR5−/− mice.

T151

19094-19104

Sentence

denotes

Discussion

T9010

19105-19286

Sentence

denotes

In the present study, we found that tumor weight and volume were much smaller in the CCR5−/− mice compared to the CCR5+/+ mice inoculated with B16 melanoma cells (Figure 1A and 1B).

T152

19105-19286

Sentence

denotes

In the present study, we found that tumor weight and volume were much smaller in the CCR5−/− mice compared to the CCR5+/+ mice inoculated with B16 melanoma cells (Figure 1A and 1B).

T9011

19287-19448

Sentence

denotes

The tumor growth inhibition in the CCR5−/− mice was associated with the inhibition of constitutively activated NF-κB and elevation of IL-1Ra in the tumor tissue.

T153

19287-19448

Sentence

denotes

The tumor growth inhibition in the CCR5−/− mice was associated with the inhibition of constitutively activated NF-κB and elevation of IL-1Ra in the tumor tissue.

T9012

19449-19727

Sentence

denotes

This anti-tumor activity was also associated with decreased expression of NF-κB target anti-apoptotic, cell proliferative, and tumor promoting genes (Bcl-2 and C-IAP1) but with increased expression of their target apoptotic genes; cleaved caspase-3 and 9, and Bax (Figure 3A-D).

T154

19449-19727

Sentence

denotes

This anti-tumor activity was also associated with decreased expression of NF-κB target anti-apoptotic, cell proliferative, and tumor promoting genes (Bcl-2 and C-IAP1) but with increased expression of their target apoptotic genes; cleaved caspase-3 and 9, and Bax (Figure 3A-D).

T9013

19728-19880

Sentence

denotes

In the tumor and the spleen, the number of cytotoxic CD8+ T cells, CD57+ natural killer cells, and the levels of IL-1Ra were increased (Figure 4 and 5).

T155

19728-19880

Sentence

denotes

In the tumor and the spleen, the number of cytotoxic CD8+ T cells, CD57+ natural killer cells, and the levels of IL-1Ra were increased (Figure 4 and 5).

T9014

19881-20096

Sentence

denotes

These findings suggest that melanoma growth was inhibited in the CCR5−/− mice, and that the elevation of IL-1Ra, accompanied with decreased NF-κB, is significant in the inhibition of melanoma growth in CCR5−/− mice.

T156

19881-20096

Sentence

denotes

These findings suggest that melanoma growth was inhibited in the CCR5−/− mice, and that the elevation of IL-1Ra, accompanied with decreased NF-κB, is significant in the inhibition of melanoma growth in CCR5−/− mice.

T9015

20097-20219

Sentence

denotes

NF-κB regulates the expression of over 200 genes that control the immune system, cancer cell growth and inflammation [39].

T157

20097-20219

Sentence

denotes

NF-κB regulates the expression of over 200 genes that control the immune system, cancer cell growth and inflammation [39].

T9016

20220-20508

Sentence

denotes

Because of its abilities to induce the expression of a large array of inflammatory mediators and its roles as core transcription factors in diverse immune responses, NF-κB has been recognized as a major factor responsible for cytokine-associated cancer development or anti-tumor immunity.

T158

20220-20508

Sentence

denotes

Because of its abilities to induce the expression of a large array of inflammatory mediators and its roles as core transcription factors in diverse immune responses, NF-κB has been recognized as a major factor responsible for cytokine-associated cancer development or anti-tumor immunity.

T9017

20509-20801

Sentence

denotes

In the melanoma tumor tissues of the CCR5−/− mice, the expression of NF-κB target cell death genes (Bax, caspase-3 and 9) and the DNA binding activity of NF-κB, were significantly inhibited, but the expression of cell death inhibitory NF-κB target genes, such as Bcl-2 and cIAP were enhanced.

T159

20509-20801

Sentence

denotes

In the melanoma tumor tissues of the CCR5−/− mice, the expression of NF-κB target cell death genes (Bax, caspase-3 and 9) and the DNA binding activity of NF-κB, were significantly inhibited, but the expression of cell death inhibitory NF-κB target genes, such as Bcl-2 and cIAP were enhanced.

T9018

20802-20957

Sentence

denotes

In addition, CCR5−/− prevented the phosphorylation of IκB, accompanied with the inhibition of the p50 and p65 translocation into the nucleus (Figure 2A-C).

T160

20802-20957

Sentence

denotes

In addition, CCR5−/− prevented the phosphorylation of IκB, accompanied with the inhibition of the p50 and p65 translocation into the nucleus (Figure 2A-C).

T9019

20958-21092

Sentence

denotes

Many tumors, including melanoma, have increased levels of NF-κB [40], which is likely acting as a survival factor for melanoma growth.

T161

20958-21092

Sentence

denotes

Many tumors, including melanoma, have increased levels of NF-κB [40], which is likely acting as a survival factor for melanoma growth.

T9020

21093-21231

Sentence

denotes

Thus, the inhibitions of NF-κB activity and the expression of target genes are critical in the inhibition of tumor growth in CCR5−/− mice.

T162

21093-21231

Sentence

denotes

Thus, the inhibitions of NF-κB activity and the expression of target genes are critical in the inhibition of tumor growth in CCR5−/− mice.

T9021

21232-21381

Sentence

denotes

Although the mechanism is not clear as to how CCR5−/− downregulates NF-κB, it is noteworthy that NF-κB is activated or inactivated by many cytokines.

T163

21232-21381

Sentence

denotes

Although the mechanism is not clear as to how CCR5−/− downregulates NF-κB, it is noteworthy that NF-κB is activated or inactivated by many cytokines.

T9022

21382-21460

Sentence

denotes

Numerous studies showed that cytokine affect tumor growth by regulating NF-κB.

T164

21382-21460

Sentence

denotes

Numerous studies showed that cytokine affect tumor growth by regulating NF-κB.

T9023

21461-21560

Sentence

denotes

Inflammatory cytokines, including IL-1, IL-6 and IL-8, activate NF-κB pathways in tumor cells [41].

T165

21461-21560

Sentence

denotes

Inflammatory cytokines, including IL-1, IL-6 and IL-8, activate NF-κB pathways in tumor cells [41].

T9024

21561-21676

Sentence

denotes

The interleukin 10 (IL-10) is well known as an anti-tumor factor as well as an anti-inflammatory factor [42], [43].

T166

21561-21676

Sentence

denotes

The interleukin 10 (IL-10) is well known as an anti-tumor factor as well as an anti-inflammatory factor [42], [43].

T9025

21677-21759

Sentence

denotes

IL-10 inhibits constitutively activated NF-κB both in vitro [44] and in vivo [45].

T167

21677-21759

Sentence

denotes

IL-10 inhibits constitutively activated NF-κB both in vitro [44] and in vivo [45].

T9026

21760-21910

Sentence

denotes

Inhibition of NF-κB by the IL-10 contributes to the anti-tumor effects through the activation of caspase-3 and apoptosis in Colon26-bearing mice [43].

T168

21760-21910

Sentence

denotes

Inhibition of NF-κB by the IL-10 contributes to the anti-tumor effects through the activation of caspase-3 and apoptosis in Colon26-bearing mice [43].

T9027

21911-22031

Sentence

denotes

Similarly, several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1 [46].

T169

21911-22031

Sentence

denotes

Similarly, several studies have demonstrated the ability of IL-1Ra to abrogate the proinflammatory effects of IL-1 [46].

T9028

22032-22224

Sentence

denotes

It has been reported that IL-1 induces the activation of NF-κB in several cancers, and increases cell cycle progression, and the suppression of apoptotic cell death leading to tumor promotion.

T170

22032-22224

Sentence

denotes

It has been reported that IL-1 induces the activation of NF-κB in several cancers, and increases cell cycle progression, and the suppression of apoptotic cell death leading to tumor promotion.

T9029

22225-22414

Sentence

denotes

The results of various studies over the past 10 years indicate that the major function of IL-1Ra is to regulate the pleiotropic effects of IL-1 by competitively blocking its functions [47].

T171

22225-22414

Sentence

denotes

The results of various studies over the past 10 years indicate that the major function of IL-1Ra is to regulate the pleiotropic effects of IL-1 by competitively blocking its functions [47].

T9030

22415-22577

Sentence

denotes

The ability of IL-1 to induce its own antagonist is of interest in understanding the regulation and dysregulation of IL-1 signaling in normal and diseased tissue.

T172

22415-22577

Sentence

denotes

The ability of IL-1 to induce its own antagonist is of interest in understanding the regulation and dysregulation of IL-1 signaling in normal and diseased tissue.

T9031

22578-22722

Sentence

denotes

Up-regulation of the receptor antagonist represents a mechanism for turning off IL-1 signaling and creating a temporary state of refractoriness.

T173

22578-22722

Sentence

denotes

Up-regulation of the receptor antagonist represents a mechanism for turning off IL-1 signaling and creating a temporary state of refractoriness.

T9032

22723-22876

Sentence

denotes

The elevated expression of IL-1Ra, without any change of IL-1 in tumors, suggests that the IL-1-signaling system may be dysregulated by IL-1Ra in tumors.

T174

22723-22876

Sentence

denotes

The elevated expression of IL-1Ra, without any change of IL-1 in tumors, suggests that the IL-1-signaling system may be dysregulated by IL-1Ra in tumors.

T9033

22877-22982

Sentence

denotes

IL-1Ra has been shown to inhibit tumor progression in prostate, breast, and skin cancer [14], [15], [16].

T175

22877-22982

Sentence

denotes

IL-1Ra has been shown to inhibit tumor progression in prostate, breast, and skin cancer [14], [15], [16].

T9034

22983-23097

Sentence

denotes

Intracellular IL-1Ra (icIL-1Ra) inhibits IL-6 and IL-8 production in Caco-2 cells by blocking NF-κB pathways [37].

T176

22983-23097

Sentence

denotes

Intracellular IL-1Ra (icIL-1Ra) inhibits IL-6 and IL-8 production in Caco-2 cells by blocking NF-κB pathways [37].

T9035

23098-23233

Sentence

denotes

We found that the levels of IL-1Ra were significantly elevated in the tumor tissues of CCR5−/− mice inoculated with B16 melanoma cells.

T177

23098-23233

Sentence

denotes

We found that the levels of IL-1Ra were significantly elevated in the tumor tissues of CCR5−/− mice inoculated with B16 melanoma cells.

T9036

23234-23408

Sentence

denotes

Taken together, these data indicate IL-1Ra could be critically involved in the tumor growth inhibition of CCR5−/− mice through the inactivation of NF-κB in the present study.

T178

23234-23408

Sentence

denotes

Taken together, these data indicate IL-1Ra could be critically involved in the tumor growth inhibition of CCR5−/− mice through the inactivation of NF-κB in the present study.

T9037

23409-23555

Sentence

denotes

CCR5 increases tumor growth in a local model and inhibits the efficacy of a dendrite cell vaccine in CCR5+/+ mice compared with CCR5−/− mice [10].

T179

23409-23555

Sentence

denotes

CCR5 increases tumor growth in a local model and inhibits the efficacy of a dendrite cell vaccine in CCR5+/+ mice compared with CCR5−/− mice [10].

T9038

23556-23697

Sentence

denotes

Cheng J and Sung RS examined the effect of CCR5 on the immune response to adenovirus vectors and graft function in an islet transplant model.

T180

23556-23697

Sentence

denotes

Cheng J and Sung RS examined the effect of CCR5 on the immune response to adenovirus vectors and graft function in an islet transplant model.

T9039

23698-23802

Sentence

denotes

They found that CCR5 absence does not prevent the local immune response to adenovirus transduction [48].

T181

23698-23802

Sentence

denotes

They found that CCR5 absence does not prevent the local immune response to adenovirus transduction [48].

T9040

23803-24093

Sentence

denotes

Additionally, Tan MC et al. suggested that disruption of CCR5/CCL5 signaling, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (TAK-779), reduced Treg (regulatory T cells) migration to tumors so that tumors are smaller in control mice [9].

T182

23803-24093

Sentence

denotes

Additionally, Tan MC et al. suggested that disruption of CCR5/CCL5 signaling, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (TAK-779), reduced Treg (regulatory T cells) migration to tumors so that tumors are smaller in control mice [9].

T9041

24094-24237

Sentence

denotes

These data support the important premise that inhibition of tumor growth in CCR5−/− mice could be related with the inhibition of immune escape.

T183

24094-24237

Sentence

denotes

These data support the important premise that inhibition of tumor growth in CCR5−/− mice could be related with the inhibition of immune escape.

T9042

24238-24547

Sentence

denotes

Similar to this finding, we found that the CCR5+/+ mice injected with melanoma cells apparently have a lower number of cytotoxic T cells (CD8+positive T Lymphocytes cells) and natural killer cells (CD57 positive NK cells), as well as dendrite cells, in spleen tissues compared to those without melanoma cells.

T184

24238-24547

Sentence

denotes

Similar to this finding, we found that the CCR5+/+ mice injected with melanoma cells apparently have a lower number of cytotoxic T cells (CD8+positive T Lymphocytes cells) and natural killer cells (CD57 positive NK cells), as well as dendrite cells, in spleen tissues compared to those without melanoma cells.

T9043

24548-24639

Sentence

denotes

But the lymphocytes of the CCR5−/− mice did not decrease when injected with melanoma cells.

T185

24548-24639

Sentence

denotes

But the lymphocytes of the CCR5−/− mice did not decrease when injected with melanoma cells.

T9044

24640-24821

Sentence

denotes

These differences of the immune response between the CCR5+/+ mice and the CCR5−/− mice, involving tumor immune tolerance, may be connected with the inhibition of cancer progression.

T186

24640-24821

Sentence

denotes

These differences of the immune response between the CCR5+/+ mice and the CCR5−/− mice, involving tumor immune tolerance, may be connected with the inhibition of cancer progression.

T9045

24822-25027

Sentence

denotes

Taken together, in the cytokine array experiment, the level of MIP-1α (which has a potent activity of T cell and B cell migration) was increased in the CCR5−/− mice compared with those in the CCR5+/+ mice.

T187

24822-25027

Sentence

denotes

Taken together, in the cytokine array experiment, the level of MIP-1α (which has a potent activity of T cell and B cell migration) was increased in the CCR5−/− mice compared with those in the CCR5+/+ mice.

T9046

25028-25170

Sentence

denotes

Thus, relatively increased tumor associated cytotoxic lymphocytes could play an important role in inhibiting the tumor growth in CCR5−/− mice.

T188

25028-25170

Sentence

denotes

Thus, relatively increased tumor associated cytotoxic lymphocytes could play an important role in inhibiting the tumor growth in CCR5−/− mice.

T9047

25171-25265

Sentence

denotes

Activation of NF-κB could modulate subcellular localization of NK cells and pathogenesis [49].

T189

25171-25265

Sentence

denotes

Activation of NF-κB could modulate subcellular localization of NK cells and pathogenesis [49].

T9048

25266-25398

Sentence

denotes

Inhibition of NF-κB activity prevented NK cell depletion, and thus increased anti-tumor activity by decreasing IL-6 production [50].

T190

25266-25398

Sentence

denotes

Inhibition of NF-κB activity prevented NK cell depletion, and thus increased anti-tumor activity by decreasing IL-6 production [50].

T9049

25399-25564

Sentence

denotes

Thus, the decreased NF-κB and elevated IL-1Ra could also be associated in the infiltration of cytotoxic lymphocytes into tumor, resulting in tumor growth inhibition.

T191

25399-25564

Sentence

denotes

Thus, the decreased NF-κB and elevated IL-1Ra could also be associated in the infiltration of cytotoxic lymphocytes into tumor, resulting in tumor growth inhibition.

T9050

25565-25684

Sentence

denotes

In contrast, IL-23, a tumor growth promoting cytokine, increased NF-κB and immune cell infiltration in oral tumor [51].

T192

25565-25684

Sentence

denotes

In contrast, IL-23, a tumor growth promoting cytokine, increased NF-κB and immune cell infiltration in oral tumor [51].

T9051

25685-25797

Sentence

denotes

These data indicate that NF-κB could be involved in the cytokine mediated anti-tumor activities of immune cells.

T193

25685-25797

Sentence

denotes

These data indicate that NF-κB could be involved in the cytokine mediated anti-tumor activities of immune cells.

T9052

25798-25931

Sentence

denotes

It is also noteworthy that IL-1Ra inhibited melanoma tumor growth by increasing the number of myeloid suppressor cells in tumor [25].

T194

25798-25931

Sentence

denotes

It is also noteworthy that IL-1Ra inhibited melanoma tumor growth by increasing the number of myeloid suppressor cells in tumor [25].

T9053

25932-26113

Sentence

denotes

The 3-methylcholatrene-induced tumor incidence was reduced in IL-1α knockout mice, but increased in IL-1Ra mice with concomitant maturation of NK cells and anti-tumor immunity [52].

T195

25932-26113

Sentence

denotes

The 3-methylcholatrene-induced tumor incidence was reduced in IL-1α knockout mice, but increased in IL-1Ra mice with concomitant maturation of NK cells and anti-tumor immunity [52].

T9054

26114-26254

Sentence

denotes

These data indicate that the decrease of NF-κB, and thus increase of IL-1Ra could be significant in tumor growth inhibition of CCR5−/− mice.

T196

26114-26254

Sentence

denotes

These data indicate that the decrease of NF-κB, and thus increase of IL-1Ra could be significant in tumor growth inhibition of CCR5−/− mice.

T9055

26255-26435

Sentence

denotes

Our data thus show significant clinical implications of CCR5, because CCR5 is a G protein–coupled receptor and it is amenable to use as a small molecule for the inhibition of CCR5.

T197

26255-26435

Sentence

denotes

Our data thus show significant clinical implications of CCR5, because CCR5 is a G protein–coupled receptor and it is amenable to use as a small molecule for the inhibition of CCR5.

T9056

26436-26558

Sentence

denotes

Several such compounds have already been shown to be safe in clinical trials for use in certain diseases such as HIV [53].

T198

26436-26558

Sentence

denotes

Several such compounds have already been shown to be safe in clinical trials for use in certain diseases such as HIV [53].

T9057

26559-26638

Sentence

denotes

Thus, we believe CCR5 is a cancer target that warrants continued investigation.

T199

26559-26638

Sentence

denotes

Thus, we believe CCR5 is a cancer target that warrants continued investigation.

T9058

26639-26766

Sentence

denotes

In summary, this study showed that melanoma cells growth was inhibited via modulation of NF-κB/IL-1Ra pathways in CCR5−/− mice.

T200

26639-26766

Sentence

denotes

In summary, this study showed that melanoma cells growth was inhibited via modulation of NF-κB/IL-1Ra pathways in CCR5−/− mice.

T201

26768-26789

Sentence

denotes

Materials and Methods

T13206

26791-26807

Sentence

denotes

Ethics Statement

T202

26791-26807

Sentence

denotes

Ethics Statement

T13207

26808-26992

Sentence

denotes

All experiments were approved and carried out according to the Guide for the Care and Use of Animals [Animal Care Committee of Chungbuk National University, Korea (CBNUA-045-0902-01)].

T203

26808-26992

Sentence

denotes

All experiments were approved and carried out according to the Guide for the Care and Use of Animals [Animal Care Committee of Chungbuk National University, Korea (CBNUA-045-0902-01)].

T13309

26994-27001

Sentence

denotes

Animals

T204

26994-27001

Sentence

denotes

Animals

T13310

27002-27137

Sentence

denotes

The CCR5 wild type (CCR5+/+) and CCR5 knockout (CCR5−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine 04609 USA).

T205

27002-27137

Sentence

denotes

The CCR5 wild type (CCR5+/+) and CCR5 knockout (CCR5−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine 04609 USA).

T206

27138-27288

Sentence

denotes

The mice were housed and bred under specific pathogen free conditions at the Laboratory Animal Research Center of Chungbuk National University, Korea.

T13311

27138-27512

Sentence

denotes

The mice were housed and bred under specific pathogen free conditions at the Laboratory Animal Research Center of Chungbuk National University, Korea. The mice (n = 4/cage) were maintained in a room with a constant temperature of 22±1°C, relative humidity of 55±10%, and 12-h light/dark cycle, and fed standard rodent chow (Samyang, Korea) and purified tap water ad libitum.

T207

27289-27512

Sentence

denotes

The mice (n = 4/cage) were maintained in a room with a constant temperature of 22±1°C, relative humidity of 55±10%, and 12-h light/dark cycle, and fed standard rodent chow (Samyang, Korea) and purified tap water ad libitum.

T13312

27513-27639

Sentence

denotes

The CCR5+/+ mice (n = 20) and CCR5−/− mice (n = 20), that were used, had matched ages (10–11 weeks old) and weights (16–19 g).

T208

27513-27639

Sentence

denotes

The CCR5+/+ mice (n = 20) and CCR5−/− mice (n = 20), that were used, had matched ages (10–11 weeks old) and weights (16–19 g).

T13313

27640-27738

Sentence

denotes

Control mice (B6129PF2/J) were F2 hybrid mice from the C57BL/6J-AW-J and 129P3/J parental strains.

T209

27640-27738

Sentence

denotes

Control mice (B6129PF2/J) were F2 hybrid mice from the C57BL/6J-AW-J and 129P3/J parental strains.

T13314

27739-27902

Sentence

denotes

CCR5 mice (B6, 129P2-Ccr5tm1Kuz/J) have the entire coding region of the CCR5 gene deleted from the parental strains, B6;129P2-Ccr5tm1kuz and B6;129P2-Cmkbr5tm1Kuz.

T210

27739-27902

Sentence

denotes

CCR5 mice (B6, 129P2-Ccr5tm1Kuz/J) have the entire coding region of the CCR5 gene deleted from the parental strains, B6;129P2-Ccr5tm1kuz and B6;129P2-Cmkbr5tm1Kuz.

T13806

27904-27942

Sentence

denotes

Tumor Inoculation and Tumor Monitoring

T211

27904-27942

Sentence

denotes

Tumor Inoculation and Tumor Monitoring

T13807

27943-28156

Sentence

denotes

B16 melanoma cells were injected subcutaneously [5×105 tumor cells in 0.1 ml Phosphate buffered saline(PBS)/animal] using a 27 G needle into the right-lower flanks of the carrier mice as previously described [54].

T212

27943-28156

Sentence

denotes

B16 melanoma cells were injected subcutaneously [5×105 tumor cells in 0.1 ml Phosphate buffered saline(PBS)/animal] using a 27 G needle into the right-lower flanks of the carrier mice as previously described [54].

T13808

28157-28235

Sentence

denotes

The body weights and tumor volumes of the animals were monitored twice weekly.

T213

28157-28235

Sentence

denotes

The body weights and tumor volumes of the animals were monitored twice weekly.

T13809

28236-28409

Sentence

denotes

The tumor volumes were measured with Vernier calipers and calculated using the following formula: (A×B2)/2, where A is the larger and B is the smaller of the two dimensions.

T214

28236-28409

Sentence

denotes

The tumor volumes were measured with Vernier calipers and calculated using the following formula: (A×B2)/2, where A is the larger and B is the smaller of the two dimensions.

T13810

28410-28468

Sentence

denotes

At the end of the experiment, the animals were sacrificed.

T215

28410-28468

Sentence

denotes

At the end of the experiment, the animals were sacrificed.

T13811

28469-28535

Sentence

denotes

The tumors were separated from the surrounding muscles and dermis.

T216

28469-28535

Sentence

denotes

The tumors were separated from the surrounding muscles and dermis.

T14071

28537-28553

Sentence

denotes

Western Blotting

T217

28537-28553

Sentence

denotes

Western Blotting

T14072

28554-28706

Sentence

denotes

Tumor tissues were homogenized with protein extraction solution (PRO-PREPTM, Intron Biotechnology, Seoul, Korea), and lysed by 60 min incubation on ice.

T218

28554-28706

Sentence

denotes

Tumor tissues were homogenized with protein extraction solution (PRO-PREPTM, Intron Biotechnology, Seoul, Korea), and lysed by 60 min incubation on ice.

T219

28707-28769

Sentence

denotes

The tissue lysate centrifuged at 15,000 rpm for 15 min at 4°C.

T14073

28707-28974

Sentence

denotes

The tissue lysate centrifuged at 15,000 rpm for 15 min at 4°C. Equal amounts of proteins (40 µg) were separated on a SDS/12%-polyacrylamide gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Water and Process technologies, Trevose, PA, USA).

T220

28770-28974

Sentence

denotes

Equal amounts of proteins (40 µg) were separated on a SDS/12%-polyacrylamide gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Water and Process technologies, Trevose, PA, USA).

T14074

28975-29094

Sentence

denotes

Blots were blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-Buffered Saline Tween-20 [TBST:

T221

28975-29094

Sentence

denotes

Blots were blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-Buffered Saline Tween-20 [TBST:

T14075

29095-29167

Sentence

denotes

10 mM Tris (pH 8.0) and 150 mM NaCl solution containing 0.05% Tween-20].

T222

29095-29167

Sentence

denotes

10 mM Tris (pH 8.0) and 150 mM NaCl solution containing 0.05% Tween-20].

T14076

29168-29604

Sentence

denotes

After a short washing in TBST, the membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against p65 and p50 (1∶500 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies directed against Bax and PARP (1∶500 dilutions; Santa Cruz Biotechnology), and against caspase-3, caspase-9, Bcl-2 and c-IAP-1 (1∶1000 dilutions; Cell Signaling Technology, Beverly, MA).

T223

29168-29604

Sentence

denotes

After a short washing in TBST, the membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against p65 and p50 (1∶500 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies directed against Bax and PARP (1∶500 dilutions; Santa Cruz Biotechnology), and against caspase-3, caspase-9, Bcl-2 and c-IAP-1 (1∶1000 dilutions; Cell Signaling Technology, Beverly, MA).

T14077

29605-29759

Sentence

denotes

The blots were incubated with the respective horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1∶4000 dilutions; Santa Cruz Biotechnology).

T224

29605-29759

Sentence

denotes

The blots were incubated with the respective horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1∶4000 dilutions; Santa Cruz Biotechnology).

T14078

29760-29828

Sentence

denotes

Immunoreactive proteins were detected with the ECL detection system.

T225

29760-29828

Sentence

denotes

Immunoreactive proteins were detected with the ECL detection system.

T14660

29830-29850

Sentence

denotes

Immunohistochemistry

T226

29830-29850

Sentence

denotes

Immunohistochemistry

T14661

29851-29929

Sentence

denotes

All specimens were fixed in formalin and embedded in paraffin for examination.

T227

29851-29929

Sentence

denotes

All specimens were fixed in formalin and embedded in paraffin for examination.

T14662

29930-30011

Sentence

denotes

Sections (4 µm thick) were stained with H&E and analyzed by immunohistochemistry.

T228

29930-30011

Sentence

denotes

Sections (4 µm thick) were stained with H&E and analyzed by immunohistochemistry.

T14663

30012-30160

Sentence

denotes

The paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval.

T229

30012-30160

Sentence

denotes

The paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval.

T14664

30161-30305

Sentence

denotes

Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 15 min, followed by clearing in PBS for 5 min.

T230

30161-30305

Sentence

denotes

Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 15 min, followed by clearing in PBS for 5 min.

T14665

30306-30565

Sentence

denotes

The sections were blocked for 30 min with 3% normal horse serum diluted in PBS, blotted, and incubated with primary mouse anti-mouse proliferating cell nuclear antigen (PCNA, 1∶200 dilution) monoclonal antibodies in blocking serum for 4 h at room temperature.

T231

30306-30565

Sentence

denotes

The sections were blocked for 30 min with 3% normal horse serum diluted in PBS, blotted, and incubated with primary mouse anti-mouse proliferating cell nuclear antigen (PCNA, 1∶200 dilution) monoclonal antibodies in blocking serum for 4 h at room temperature.

T14666

30566-30713

Sentence

denotes

Thereafter, the slides were washed three times for 5 min each in PBS and incubated with biotinylated anti-mouse and anti-rabbit antibodies for 2 h.

T232

30566-30713

Sentence

denotes

Thereafter, the slides were washed three times for 5 min each in PBS and incubated with biotinylated anti-mouse and anti-rabbit antibodies for 2 h.

T14667

30714-30862

Sentence

denotes

The slides were washed in PBS, followed by the addition of the avidin biotin peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA).

T233

30714-30862

Sentence

denotes

The slides were washed in PBS, followed by the addition of the avidin biotin peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA).

T14668

30863-31123

Sentence

denotes

The slides were washed and the peroxidase reaction was developed with diaminobenzidine (DAB) and peroxide, followed by counterstaining with hematoxylin, mounting in aqua-mount, and evaluation under a light microscope (magnification×200; Olympus, Tokyo, Japan).

T234

30863-31123

Sentence

denotes

The slides were washed and the peroxidase reaction was developed with diaminobenzidine (DAB) and peroxide, followed by counterstaining with hematoxylin, mounting in aqua-mount, and evaluation under a light microscope (magnification×200; Olympus, Tokyo, Japan).

T14669

31124-31208

Sentence

denotes

A negative control was included in all experiments by omitting the primary antibody.

T235

31124-31208

Sentence

denotes

A negative control was included in all experiments by omitting the primary antibody.

T14670

31209-31513

Sentence

denotes

For the detection of apoptotic cell death in the tumor tissues, the paraffin-embedded sections were incubated in a mixture of the labeling solution (540 µl) and enzyme solution (60 µl) for 1 h at 37°C, and then washed three times in 0.1 M PBS for 5 min each, according to the manufacturer’s instructions.

T236

31209-31513

Sentence

denotes

For the detection of apoptotic cell death in the tumor tissues, the paraffin-embedded sections were incubated in a mixture of the labeling solution (540 µl) and enzyme solution (60 µl) for 1 h at 37°C, and then washed three times in 0.1 M PBS for 5 min each, according to the manufacturer’s instructions.

T237

31514-31609

Sentence

denotes

Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C.

T14671

31514-31727

Sentence

denotes

Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C. Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy (DAS microscope).

T238

31610-31727

Sentence

denotes

Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy (DAS microscope).

T15427

31729-31747

Sentence

denotes

Immunofluorescence

T239

31729-31747

Sentence

denotes

Immunofluorescence

T15428

31748-32313

Sentence

denotes

Sections were treated with 10% bovine serum albumin in PBS for 1 h at room temperature, incubated overnight at 4°C with p50 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), p65 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), Bax (1∶200 dilution), cleaved caspase-3 (1∶100), CD8 (1∶10), CD57 (1∶50) and IL-1Ra (1∶500) antibodies, followed by incubation in anti-mouse IgG conjugated with Alexa 488 (1∶100 dilution, Molecular Probes, Eugene, OR, USA) and anti-rabbit IgG conjugated with Alexa 568 (1∶100 dilution, Molecular Probes) for 40 min at room temperature.

T240

31748-32313

Sentence

denotes

Sections were treated with 10% bovine serum albumin in PBS for 1 h at room temperature, incubated overnight at 4°C with p50 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), p65 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), Bax (1∶200 dilution), cleaved caspase-3 (1∶100), CD8 (1∶10), CD57 (1∶50) and IL-1Ra (1∶500) antibodies, followed by incubation in anti-mouse IgG conjugated with Alexa 488 (1∶100 dilution, Molecular Probes, Eugene, OR, USA) and anti-rabbit IgG conjugated with Alexa 568 (1∶100 dilution, Molecular Probes) for 40 min at room temperature.

T15429

32314-32488

Sentence

denotes

Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy and photography using ApoTome microscopy (Carl Zeiss, Thornwood, NY, USA).

T241

32314-32488

Sentence

denotes

Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy and photography using ApoTome microscopy (Carl Zeiss, Thornwood, NY, USA).

T15688

32490-32521

Sentence

denotes

Gel Electromobility Shift Assay

T242

32490-32521

Sentence

denotes

Gel Electromobility Shift Assay

T15689

32522-32648

Sentence

denotes

A gel electromobility shift assay (EMSA) was performed according to the manufacturer’s recommendations (Promega, Madison, WI).

T243

32522-32648

Sentence

denotes

A gel electromobility shift assay (EMSA) was performed according to the manufacturer’s recommendations (Promega, Madison, WI).

T15690

32649-32920

Sentence

denotes

The tumor tissues were briefly homogenized in 200 µl of solution A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride), vortexed vigorously, incubated on ice for 10 min, and then centrifuged at 15000 rpm for 15 min.

T244

32649-32920

Sentence

denotes

The tumor tissues were briefly homogenized in 200 µl of solution A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride), vortexed vigorously, incubated on ice for 10 min, and then centrifuged at 15000 rpm for 15 min.

T15691

32921-33065

Sentence

denotes

The pelleted nuclei were resuspended in solution C (solution A supplemented with 420 mM NaCl and 20% glycerol), and incubated on ice for 20 min.

T245

32921-33065

Sentence

denotes

The pelleted nuclei were resuspended in solution C (solution A supplemented with 420 mM NaCl and 20% glycerol), and incubated on ice for 20 min.

T15692

33066-33220

Sentence

denotes

The resuspended pellet was centrifuged at 15000 rpm for 15 min, and the resulting nuclear extracts supernatant were collected in a chilled Eppendorf tube.

T246

33066-33220

Sentence

denotes

The resuspended pellet was centrifuged at 15000 rpm for 15 min, and the resulting nuclear extracts supernatant were collected in a chilled Eppendorf tube.

T247

33221-33329

Sentence

denotes

Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and [P32]-ATP for 10 min at 37°C.

T15693

33221-33403

Sentence

denotes

Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and [P32]-ATP for 10 min at 37°C. The gel shift reactions were assembled and incubated at room temperature.

T248

33330-33403

Sentence

denotes

The gel shift reactions were assembled and incubated at room temperature.

T15694

33404-33514

Sentence

denotes

Subsequently, 1 ml of gel loading buffer was added to each reaction and loaded onto a 6% non-denaturating gel.

T249

33404-33514

Sentence

denotes

Subsequently, 1 ml of gel loading buffer was added to each reaction and loaded onto a 6% non-denaturating gel.

T15695

33515-33610

Sentence

denotes

The gel was subjected to electrophoresis until the dye was four-fifths of the way down the gel.

T250

33515-33610

Sentence

denotes

The gel was subjected to electrophoresis until the dye was four-fifths of the way down the gel.

T15696

33611-33684

Sentence

denotes

The gel was dried for 1 h at 80°C and exposed to film overnight at -70°C.

T251

33611-33684

Sentence

denotes

The gel was dried for 1 h at 80°C and exposed to film overnight at -70°C.

T16197

33686-33710

Sentence

denotes

Flow Cytometric Analysis

T252

33686-33710

Sentence

denotes

Flow Cytometric Analysis

T16198

33711-33790

Sentence

denotes

Lymphocytes were obtained from tumor and spleen tissues for phenotype analysis.

T253

33711-33790

Sentence

denotes

Lymphocytes were obtained from tumor and spleen tissues for phenotype analysis.

T16199

33791-33906

Sentence

denotes

Spleen and tumor lymphocytes were isolated from fresh spleen and tumor biopsies obtained from three mice per group.

T254

33791-33906

Sentence

denotes

Spleen and tumor lymphocytes were isolated from fresh spleen and tumor biopsies obtained from three mice per group.

T16200

33907-34111

Sentence

denotes

Spleen and tumor biopsies were briefly homogenized mechanically in PBS, filtered (100 µm cell strainer; BD PharMingen) and then placed in lysis buffer (ACK lysing buffer, LONZA) to remove red blood cells.

T255

33907-34111

Sentence

denotes

Spleen and tumor biopsies were briefly homogenized mechanically in PBS, filtered (100 µm cell strainer; BD PharMingen) and then placed in lysis buffer (ACK lysing buffer, LONZA) to remove red blood cells.

T16201

34112-34246

Sentence

denotes

One million splenocytes were washed once in PBS containing 1% bovine serum albumin (BSA) and re-suspended in 100 µl of PBS/BSA buffer.

T256

34112-34246

Sentence

denotes

One million splenocytes were washed once in PBS containing 1% bovine serum albumin (BSA) and re-suspended in 100 µl of PBS/BSA buffer.

T16202

34247-34440

Sentence

denotes

Meanwhile, one million cells obtained from tumor tissues were placed in RPMI medium, incubated to obtain suspension cell for 1hour at 37°C, centrifuged (1200 rpm, 3 min) and then washed in PBS.

T257

34247-34440

Sentence

denotes

Meanwhile, one million cells obtained from tumor tissues were placed in RPMI medium, incubated to obtain suspension cell for 1hour at 37°C, centrifuged (1200 rpm, 3 min) and then washed in PBS.

T16203

34441-34638

Sentence

denotes

Next, the lymphocytes were incubated with various conjugated monoclonal antibodies, including CD4-APC, CD8-FITC and CD19-PE for 20min at 4°C, washed twice in PBS, and re-suspended in 400 µl of PBS.

T258

34441-34638

Sentence

denotes

Next, the lymphocytes were incubated with various conjugated monoclonal antibodies, including CD4-APC, CD8-FITC and CD19-PE for 20min at 4°C, washed twice in PBS, and re-suspended in 400 µl of PBS.

T16204

34639-34821

Sentence

denotes

A flow cytometric analysis was performed on a FACSAria flow cytometry (BD Biosciences), and the data were analyzed using the WinMDI statistical software (Scripps, La Jolla, CA, USA).

T259

34639-34821

Sentence

denotes

A flow cytometric analysis was performed on a FACSAria flow cytometry (BD Biosciences), and the data were analyzed using the WinMDI statistical software (Scripps, La Jolla, CA, USA).

T16205

34822-34891

Sentence

denotes

Forward and side scatter parameters were used to gate on lymphocytes.

T260

34822-34891

Sentence

denotes

Forward and side scatter parameters were used to gate on lymphocytes.

T16728

34893-34914

Sentence

denotes

Protein Immuno-arrays

T261

34893-34914

Sentence

denotes

Protein Immuno-arrays

T16729

34915-35076

Sentence

denotes

The tumor and spleen tissues were excised and homogenized in PBS with protease inhibitor cocktail (Sigma-Aldrich, USA) and Triton X-100 (final concentration 1%).

T262

34915-35076

Sentence

denotes

The tumor and spleen tissues were excised and homogenized in PBS with protease inhibitor cocktail (Sigma-Aldrich, USA) and Triton X-100 (final concentration 1%).

T16730

35077-35183

Sentence

denotes

The samples were frozen at −70°C, thawed, and centrifuged at 10,000×g for 5 min to remove cellular debris.

T263

35077-35183

Sentence

denotes

The samples were frozen at −70°C, thawed, and centrifuged at 10,000×g for 5 min to remove cellular debris.

T16731

35184-35272

Sentence

denotes

The tissue lysates were performed protein assay in the same way as for western blotting.

T264

35184-35272

Sentence

denotes

The tissue lysates were performed protein assay in the same way as for western blotting.

T16732

35273-35453

Sentence

denotes

4.5 mg of proteins, collected from three samples per group, were used for Mouse cytokine array as the protocol provided by supplier (Proteome Profiler™, #ARY006, R&D Systems, USA).

T265

35273-35453

Sentence

denotes

4.5 mg of proteins, collected from three samples per group, were used for Mouse cytokine array as the protocol provided by supplier (Proteome Profiler™, #ARY006, R&D Systems, USA).

T16970

35455-35475

Sentence

denotes

Statistical Analysis

T266

35455-35475

Sentence

denotes

Statistical Analysis

T16971

35476-35530

Sentence

denotes

The data were analyzed using the GraphPad Prism 4 ver.

T267

35476-35530

Sentence

denotes

The data were analyzed using the GraphPad Prism 4 ver.

T16972

35531-35579

Sentence

denotes

4.03 software (GraphPad Software, La Jolla, CA).

T268

35531-35579

Sentence

denotes

4.03 software (GraphPad Software, La Jolla, CA).

T16973

35580-35612

Sentence

denotes

Data are presented as mean ± SD.

T269

35580-35612

Sentence

denotes

Data are presented as mean ± SD.

T16974

35613-35689

Sentence

denotes

Difference between CCR5−/− mice and CCR5+/+ mice were compared using t test.

T270

35613-35689

Sentence

denotes

Difference between CCR5−/− mice and CCR5+/+ mice were compared using t test.

T16975

35690-35755

Sentence

denotes

A value of p<0.05 was considered to be statistically significant.

T271

35690-35755

Sentence

denotes

A value of p<0.05 was considered to be statistically significant.

T16976

35756-35896

Sentence

denotes

Survival data were presented by Kaplan-Meier survival estimates and compared and calculated by Log-rank (Mantel-Cox) Test in GraphPad Prism.

T272

35756-35896

Sentence

denotes

Survival data were presented by Kaplan-Meier survival estimates and compared and calculated by Log-rank (Mantel-Cox) Test in GraphPad Prism.

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