CORD-19-Sentences  

CORD-19:f4f0d1b957340175fd951c8fa9c27815dd589f26 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-135 Sentence denotes The inhibitory effect of 7,7"-dimethoxyagastisflavone on the metastasis of melanoma cells via the suppression of F-actin polymerization
TextSentencer_T1 0-135 Sentence denotes The inhibitory effect of 7,7"-dimethoxyagastisflavone on the metastasis of melanoma cells via the suppression of F-actin polymerization
TextSentencer_T2 137-145 Sentence denotes Abstract
TextSentencer_T2 137-145 Sentence denotes Abstract
TextSentencer_T3 146-228 Sentence denotes 7,7"-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from Taxus x media cv.
TextSentencer_T3 146-228 Sentence denotes 7,7"-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from Taxus x media cv.
TextSentencer_T4 229-282 Sentence denotes Hicksii, induces apoptotic and autophagic cell death.
TextSentencer_T4 229-282 Sentence denotes Hicksii, induces apoptotic and autophagic cell death.
TextSentencer_T5 283-344 Sentence denotes However, whether DMGF suppresses tumor metastasis is unclear.
TextSentencer_T5 283-344 Sentence denotes However, whether DMGF suppresses tumor metastasis is unclear.
TextSentencer_T6 345-492 Sentence denotes The aim of this study was to investigate the anti-metastatic activities of DMGF on the metastatic processes of melanoma cells in vivo and in vitro.
TextSentencer_T6 345-492 Sentence denotes The aim of this study was to investigate the anti-metastatic activities of DMGF on the metastatic processes of melanoma cells in vivo and in vitro.
TextSentencer_T7 493-708 Sentence denotes A transwell assay showed that DMGF could effectively attenuate the motility of B16F10 cells, and the results of real-time PCR revealed that DMGF also suppressed the expressions of matrix metalloproteinase-2 (MMP-2).
TextSentencer_T7 493-708 Sentence denotes A transwell assay showed that DMGF could effectively attenuate the motility of B16F10 cells, and the results of real-time PCR revealed that DMGF also suppressed the expressions of matrix metalloproteinase-2 (MMP-2).
TextSentencer_T8 709-806 Sentence denotes Moreover, DMGF did not influence tube formation but inhibited the migration of endothelial cells.
TextSentencer_T8 709-806 Sentence denotes Moreover, DMGF did not influence tube formation but inhibited the migration of endothelial cells.
TextSentencer_T9 807-1069 Sentence denotes Furthermore, animal models were used to monitor the effects of DMGF on tumor metastasis, and all models showed that DMGF significantly suppressed the metastatic behaviors of B16F10 cells, including intravasation, colonization, and invasion of the lymphatic duct.
TextSentencer_T9 807-1069 Sentence denotes Furthermore, animal models were used to monitor the effects of DMGF on tumor metastasis, and all models showed that DMGF significantly suppressed the metastatic behaviors of B16F10 cells, including intravasation, colonization, and invasion of the lymphatic duct.
TextSentencer_T10 1070-1167 Sentence denotes In addition, DMGF could also reduce the densities of the blood vessels in the tumor area in vivo.
TextSentencer_T10 1070-1167 Sentence denotes In addition, DMGF could also reduce the densities of the blood vessels in the tumor area in vivo.
TextSentencer_T11 1168-1460 Sentence denotes Further investigation of the molecular mechanisms of anti-metastatic activity revealed that DMGF can down-regulate the levels of key modulators of the Cdc42/Rac1 pathway to interfere in F-actin polymerization and suppress the formation of lamellipodia by reducing the phosphorylation of CREB.
TextSentencer_T11 1168-1460 Sentence denotes Further investigation of the molecular mechanisms of anti-metastatic activity revealed that DMGF can down-regulate the levels of key modulators of the Cdc42/Rac1 pathway to interfere in F-actin polymerization and suppress the formation of lamellipodia by reducing the phosphorylation of CREB.
TextSentencer_T12 1461-1553 Sentence denotes These data suggested that DMGF presents anti-metastatic activities in B16F10 melanoma cells.
TextSentencer_T12 1461-1553 Sentence denotes These data suggested that DMGF presents anti-metastatic activities in B16F10 melanoma cells.
TextSentencer_T13 1554-1708 Sentence denotes Here, we demonstrated that DMGF can inhibit the metastasis of highly invasive melanoma cancer cells through the down-regulation of F-actin polymerization.
TextSentencer_T13 1554-1708 Sentence denotes Here, we demonstrated that DMGF can inhibit the metastasis of highly invasive melanoma cancer cells through the down-regulation of F-actin polymerization.
TextSentencer_T14 1709-1840 Sentence denotes Considering these findings, DMGF may be further developed to serve as a chemoprevention drug for patients with metastatic melanoma.
TextSentencer_T14 1709-1840 Sentence denotes Considering these findings, DMGF may be further developed to serve as a chemoprevention drug for patients with metastatic melanoma.
TextSentencer_T15 1842-1970 Sentence denotes A variety of polyphenolic compounds in dietary and medical plants have been reported to exhibit a variety of biological effects.
TextSentencer_T15 1842-1970 Sentence denotes A variety of polyphenolic compounds in dietary and medical plants have been reported to exhibit a variety of biological effects.
TextSentencer_T16 1971-2133 Sentence denotes Among polyphenolic substances, biflavonoids have potent anti-inflammatory, anti-cancer, anti-virus, anti-microbial, vasorelaxant and anticlotting properties [1] .
TextSentencer_T16 1971-2133 Sentence denotes Among polyphenolic substances, biflavonoids have potent anti-inflammatory, anti-cancer, anti-virus, anti-microbial, vasorelaxant and anticlotting properties [1] .
TextSentencer_T17 2134-2246 Sentence denotes Currently, many biflavonoids exhibit bioactive functions, and the mechanisms of such effects have been explored.
TextSentencer_T17 2134-2246 Sentence denotes Currently, many biflavonoids exhibit bioactive functions, and the mechanisms of such effects have been explored.
TextSentencer_T18 2247-2528 Sentence denotes For example, the amentoflavone derived from Selaginella tamariscina or Torreya nucifera has been shown to not only have potent anticandidal or anti-SARS-coV activities [2, 3] , but also exhibits anti-neovascularization by interfering in the interaction of VEGF and VEGFR-1 [4, 5] .
TextSentencer_T18 2247-2528 Sentence denotes For example, the amentoflavone derived from Selaginella tamariscina or Torreya nucifera has been shown to not only have potent anticandidal or anti-SARS-coV activities [2, 3] , but also exhibits anti-neovascularization by interfering in the interaction of VEGF and VEGFR-1 [4, 5] .
TextSentencer_T19 2529-2741 Sentence denotes In addition, 7,7"-dimethoxyagastisflavone (DMGF, the structure was shown as Figure 1A ) has been found to have anti-HSV-1 and -HSV-2 activity [6] , and it can cause the cell death via apoptosis or autophagy [7] .
TextSentencer_T19 2529-2741 Sentence denotes In addition, 7,7"-dimethoxyagastisflavone (DMGF, the structure was shown as Figure 1A ) has been found to have anti-HSV-1 and -HSV-2 activity [6] , and it can cause the cell death via apoptosis or autophagy [7] .
TextSentencer_T20 2742-2888 Sentence denotes Moreover, ginkgetin is a strong NF-κB inhibitor (IC50 = 7.5 μM) [8] , and it selectively inhibits the growth of human ovarian adenocarcinoma [9] .
TextSentencer_T20 2742-2888 Sentence denotes Moreover, ginkgetin is a strong NF-κB inhibitor (IC50 = 7.5 μM) [8] , and it selectively inhibits the growth of human ovarian adenocarcinoma [9] .
TextSentencer_T21 2889-3102 Sentence denotes Moreover, morelloflavone has anti-oxidative, antiviral, and anti-inflammatory properties [10] , and it was recently shown to inhibit tumor angiogenesis by targeting Rho GTPases and the ERK signaling pathway [11] .
TextSentencer_T21 2889-3102 Sentence denotes Moreover, morelloflavone has anti-oxidative, antiviral, and anti-inflammatory properties [10] , and it was recently shown to inhibit tumor angiogenesis by targeting Rho GTPases and the ERK signaling pathway [11] .
TextSentencer_T22 3103-3231 Sentence denotes Therefore, the biflavonoids in these traditional medical plants are a potential source of new therapies for many human diseases.
TextSentencer_T22 3103-3231 Sentence denotes Therefore, the biflavonoids in these traditional medical plants are a potential source of new therapies for many human diseases.
TextSentencer_T23 3232-3340 Sentence denotes Tumor cells metastasize from the original site to other organs, developing and growing to form a new nodule.
TextSentencer_T23 3232-3340 Sentence denotes Tumor cells metastasize from the original site to other organs, developing and growing to form a new nodule.
TextSentencer_T24 3341-3409 Sentence denotes This process results in dysfunction of the organ and eventual death.
TextSentencer_T24 3341-3409 Sentence denotes This process results in dysfunction of the organ and eventual death.
TextSentencer_T25 3410-3484 Sentence denotes Thus, the development of anti-metastasis drugs is important and necessary.
TextSentencer_T25 3410-3484 Sentence denotes Thus, the development of anti-metastasis drugs is important and necessary.
TextSentencer_T26 3485-3732 Sentence denotes However, tumor metastasis is a complex process and consists of a series of sequential steps [12] in which cell movement, extravasation [13] , and expressions of matrix metalloproteinases [14] are important processes that result in systemic spread.
TextSentencer_T26 3485-3732 Sentence denotes However, tumor metastasis is a complex process and consists of a series of sequential steps [12] in which cell movement, extravasation [13] , and expressions of matrix metalloproteinases [14] are important processes that result in systemic spread.
TextSentencer_T27 3733-3896 Sentence denotes In addition, angiogenesis is not only required for nourishment and the removal of metabolic wastes from the tumor region, but it also facilitates tumor metastasis.
TextSentencer_T27 3733-3896 Sentence denotes In addition, angiogenesis is not only required for nourishment and the removal of metabolic wastes from the tumor region, but it also facilitates tumor metastasis.
TextSentencer_T28 3897-4076 Sentence denotes Thus, chemoprotective drugs that can simultaneously inhibit the processes of metastasis and angiogenesis may provide better chemotherapeutic efficiency by inhibiting tumor spread.
TextSentencer_T28 3897-4076 Sentence denotes Thus, chemoprotective drugs that can simultaneously inhibit the processes of metastasis and angiogenesis may provide better chemotherapeutic efficiency by inhibiting tumor spread.
TextSentencer_T29 4077-4255 Sentence denotes In addition to the previous description of DMGF, only one study has described its activity, reporting that it can inhibit the production of aflatoxin by Aspergillus flavus [15] .
TextSentencer_T29 4077-4255 Sentence denotes In addition to the previous description of DMGF, only one study has described its activity, reporting that it can inhibit the production of aflatoxin by Aspergillus flavus [15] .
TextSentencer_T30 4256-4323 Sentence denotes Whether DMGF has other bioactivities in tumor migration is unclear.
TextSentencer_T30 4256-4323 Sentence denotes Whether DMGF has other bioactivities in tumor migration is unclear.
TextSentencer_T31 4324-4507 Sentence denotes In this study, the effects of DMGF on metastasis were determined, and the in vitro and in vivo results indicated that DMGF could decrease the activities of B16F10 cells in metastasis.
TextSentencer_T31 4324-4507 Sentence denotes In this study, the effects of DMGF on metastasis were determined, and the in vitro and in vivo results indicated that DMGF could decrease the activities of B16F10 cells in metastasis.
TextSentencer_T32 4508-4673 Sentence denotes In vivo, DMGF also showed that it could reduce new vessel formation in the tumor area and reduce the abilities of B16F10 cells to metastasize through the new vessel.
TextSentencer_T32 4508-4673 Sentence denotes In vivo, DMGF also showed that it could reduce new vessel formation in the tumor area and reduce the abilities of B16F10 cells to metastasize through the new vessel.
TextSentencer_T33 4674-4892 Sentence denotes The results showed that DMGF down-regulated the expressions of the genes associated with the Cdc42/Rac1 pathway to lower the formation of lamellipodia by reducing the phosphorylation of the CREB transcriptional factor.
TextSentencer_T33 4674-4892 Sentence denotes The results showed that DMGF down-regulated the expressions of the genes associated with the Cdc42/Rac1 pathway to lower the formation of lamellipodia by reducing the phosphorylation of the CREB transcriptional factor.
TextSentencer_T34 4893-4989 Sentence denotes Taken together, these results indicate that DMGF is a good candidate for an antimetastasis drug.
TextSentencer_T34 4893-4989 Sentence denotes Taken together, these results indicate that DMGF is a good candidate for an antimetastasis drug.
TextSentencer_T35 4990-5087 Sentence denotes Highly metastatic B16F10 melanoma cells were used to examine the anti-metastatic potency of DMGF.
TextSentencer_T35 4990-5087 Sentence denotes Highly metastatic B16F10 melanoma cells were used to examine the anti-metastatic potency of DMGF.
TextSentencer_T36 5088-5262 Sentence denotes First, the cytotoxicity of DMGF for B16F10 cells was determined, and it was shown to suppress the cell proliferation of B16F10 cells in a dose-dependent manner ( Figure 1B) .
TextSentencer_T36 5088-5262 Sentence denotes First, the cytotoxicity of DMGF for B16F10 cells was determined, and it was shown to suppress the cell proliferation of B16F10 cells in a dose-dependent manner ( Figure 1B) .
TextSentencer_T37 5263-5382 Sentence denotes Sequentially, low doses of DMGF were administered to determine whether DMGF could affect the migration of B16F10 cells.
TextSentencer_T37 5263-5382 Sentence denotes Sequentially, low doses of DMGF were administered to determine whether DMGF could affect the migration of B16F10 cells.
TextSentencer_T38 5383-5505 Sentence denotes Clearly, the migration rate of B16F10 cells was lowered to 35% compared with the untreated group within 6 h ( Figure 1C ).
TextSentencer_T38 5383-5505 Sentence denotes Clearly, the migration rate of B16F10 cells was lowered to 35% compared with the untreated group within 6 h ( Figure 1C ).
TextSentencer_T39 5506-5613 Sentence denotes Furthermore, the elevated levels of MMPs in tumor tissue were correlated with the invasion of cancer cells.
TextSentencer_T39 5506-5613 Sentence denotes Furthermore, the elevated levels of MMPs in tumor tissue were correlated with the invasion of cancer cells.
TextSentencer_T40 5614-5893 Sentence denotes Thus, the effects of DMGF on the expressions of MMP genes in tumors were further measured, and the results showed that treatments with DMGF for 12 or 24 h significantly inhibited the MMP-2 expression of B16F10 cells but not MMP-9 compared to untreated B16F10 cells ( Figure 1D ).
TextSentencer_T40 5614-5893 Sentence denotes Thus, the effects of DMGF on the expressions of MMP genes in tumors were further measured, and the results showed that treatments with DMGF for 12 or 24 h significantly inhibited the MMP-2 expression of B16F10 cells but not MMP-9 compared to untreated B16F10 cells ( Figure 1D ).
TextSentencer_T41 5894-6045 Sentence denotes Taking these in vitro results together revealed that DMGF might suppress the metastasis of tumor cells by suppressing migration and matrix degradation.
TextSentencer_T41 5894-6045 Sentence denotes Taking these in vitro results together revealed that DMGF might suppress the metastasis of tumor cells by suppressing migration and matrix degradation.
TextSentencer_T42 6046-6171 Sentence denotes Tumor metastasis is highly associated with angiogenesis; therefore, the effect of DMGF on angiogenesis was examined in vitro.
TextSentencer_T42 6046-6171 Sentence denotes Tumor metastasis is highly associated with angiogenesis; therefore, the effect of DMGF on angiogenesis was examined in vitro.
TextSentencer_T43 6172-6257 Sentence denotes Figure 2A showed that DMGF could inhibit the cell proliferation of endothelial cells.
TextSentencer_T43 6172-6257 Sentence denotes Figure 2A showed that DMGF could inhibit the cell proliferation of endothelial cells.
TextSentencer_T44 6258-6402 Sentence denotes In addition, a transwell assay indicated that low dosages of DMGF also significantly inhibited the migration of endothelial cells ( Figure 2B ).
TextSentencer_T44 6258-6402 Sentence denotes In addition, a transwell assay indicated that low dosages of DMGF also significantly inhibited the migration of endothelial cells ( Figure 2B ).
TextSentencer_T45 6403-6521 Sentence denotes However, the treatments of DMGF had no effect on the tube formation of mouse SVEC4-10 endothelial cells ( Figure 2C ).
TextSentencer_T45 6403-6521 Sentence denotes However, the treatments of DMGF had no effect on the tube formation of mouse SVEC4-10 endothelial cells ( Figure 2C ).
TextSentencer_T46 6522-6750 Sentence denotes These results indicated that DMGF could only inhibit the early stages of angiogenesis, including cell proliferation and migration for endothelial cells, but not influence the late stage of angiogenesis, including tube formation.
TextSentencer_T46 6522-6750 Sentence denotes These results indicated that DMGF could only inhibit the early stages of angiogenesis, including cell proliferation and migration for endothelial cells, but not influence the late stage of angiogenesis, including tube formation.
TextSentencer_T47 6751-6822 Sentence denotes Furthermore, the in vivo effect of DMGF on angiogenesis was determined.
TextSentencer_T47 6751-6822 Sentence denotes Furthermore, the in vivo effect of DMGF on angiogenesis was determined.
TextSentencer_T48 6823-7033 Sentence denotes When the average volumes of B16/F10 melanoma on the mouse backs were approximately 30 mm 3 , the tumor-bearing mice were treated with or without i.v. injections of DMGF to determine its effects on angiogenesis.
TextSentencer_T48 6823-7033 Sentence denotes When the average volumes of B16/F10 melanoma on the mouse backs were approximately 30 mm 3 , the tumor-bearing mice were treated with or without i.v. injections of DMGF to determine its effects on angiogenesis.
TextSentencer_T49 7034-7260 Sentence denotes Figure 3A revealed that larger new vessels formed near the tumor area in the untreated group, but there was less formation of new vessels near the tumor area in the DMGF-treated group www.impactjournals.com/oncotarget in vivo.
TextSentencer_T49 7034-7260 Sentence denotes Figure 3A revealed that larger new vessels formed near the tumor area in the untreated group, but there was less formation of new vessels near the tumor area in the DMGF-treated group www.impactjournals.com/oncotarget in vivo.
TextSentencer_T50 7261-7362 Sentence denotes The sizes of angiogenetic vessels in the dorsal skin were correlated with DMGF dosages ( Figure 3A ).
TextSentencer_T50 7261-7362 Sentence denotes The sizes of angiogenetic vessels in the dorsal skin were correlated with DMGF dosages ( Figure 3A ).
TextSentencer_T51 7363-7503 Sentence denotes H&E histological staining further indicated that DMGF treatments lowered the densities of the blood vessels in the tumor area ( Figure 3B ).
TextSentencer_T51 7363-7503 Sentence denotes H&E histological staining further indicated that DMGF treatments lowered the densities of the blood vessels in the tumor area ( Figure 3B ).
TextSentencer_T52 7504-7564 Sentence denotes Thus, DMGF treatment indeed suppressed angiogenesis in vivo.
TextSentencer_T52 7504-7564 Sentence denotes Thus, DMGF treatment indeed suppressed angiogenesis in vivo.
TextSentencer_T53 7565-7622 Sentence denotes Angiogenesis plays an important role in tumor metastasis.
TextSentencer_T53 7565-7622 Sentence denotes Angiogenesis plays an important role in tumor metastasis.
TextSentencer_T54 7623-7726 Sentence denotes Therefore, the suppression of angiogenesis is one of the mechanisms of metastatic suppression for DMGF.
TextSentencer_T54 7623-7726 Sentence denotes Therefore, the suppression of angiogenesis is one of the mechanisms of metastatic suppression for DMGF.
TextSentencer_T55 7727-7831 Sentence denotes With a high metastatic ability, B16F10 cells were used to determine the anti-metastasis ability of DMGF.
TextSentencer_T55 7727-7831 Sentence denotes With a high metastatic ability, B16F10 cells were used to determine the anti-metastasis ability of DMGF.
TextSentencer_T56 7832-7976 Sentence denotes The tumor-bearing mice were examined to determine whether the tumor cells could spontaneously metastasize from subcutaneous sites to the organs.
TextSentencer_T56 7832-7976 Sentence denotes The tumor-bearing mice were examined to determine whether the tumor cells could spontaneously metastasize from subcutaneous sites to the organs.
TextSentencer_T57 7977-8078 Sentence denotes All organs but the spleen were examined and the melanoma cells were undetectable (results not shown).
TextSentencer_T57 7977-8078 Sentence denotes All organs but the spleen were examined and the melanoma cells were undetectable (results not shown).
TextSentencer_T58 8079-8365 Sentence denotes In the spleen, B16F10 cells spontaneously metastasized from the subcutaneous inoculated sites to the spleen ( Figure 4A , right panel) whereas the administration of DMGF decreased the numbers of metastatic melanoma cells in a dose-dependent manner ( Figure 4A , middle and left panels).
TextSentencer_T58 8079-8365 Sentence denotes In the spleen, B16F10 cells spontaneously metastasized from the subcutaneous inoculated sites to the spleen ( Figure 4A , right panel) whereas the administration of DMGF decreased the numbers of metastatic melanoma cells in a dose-dependent manner ( Figure 4A , middle and left panels).
TextSentencer_T59 8366-8537 Sentence denotes Counting cells under a microscope, DMGF treatments lowered the number of spontaneously metastatic cells in the spleen 5-fold relative to the untreated group ( Figure 4B ).
TextSentencer_T59 8366-8537 Sentence denotes Counting cells under a microscope, DMGF treatments lowered the number of spontaneously metastatic cells in the spleen 5-fold relative to the untreated group ( Figure 4B ).
TextSentencer_T60 8538-8824 Sentence denotes Metastatic tumor cells have the ability to colonize at new sites through the circulatory system; therefore, B16F10 cells were i.v. injected to examine the effect of were treated with various concentrations of DMGF (5, 4, 3, 2 or 1 μg/ml) for 48 h; cell growth was measured by MTT assay.
TextSentencer_T60 8538-8824 Sentence denotes Metastatic tumor cells have the ability to colonize at new sites through the circulatory system; therefore, B16F10 cells were i.v. injected to examine the effect of were treated with various concentrations of DMGF (5, 4, 3, 2 or 1 μg/ml) for 48 h; cell growth was measured by MTT assay.
TextSentencer_T61 8825-9026 Sentence denotes The readings from controls were normalized to 100%, and readings from DMGF-treated cells were expressed as a percentage of controls. (C) DMGF (1 μg/ml) inhibited the migration of B16F10 melanoma cells.
TextSentencer_T61 8825-9026 Sentence denotes The readings from controls were normalized to 100%, and readings from DMGF-treated cells were expressed as a percentage of controls. (C) DMGF (1 μg/ml) inhibited the migration of B16F10 melanoma cells.
TextSentencer_T62 9027-9157 Sentence denotes Migration was quantitated by counting the cells that migrated to the lower side of the filter after fixation and staining (n = 3).
TextSentencer_T62 9027-9157 Sentence denotes Migration was quantitated by counting the cells that migrated to the lower side of the filter after fixation and staining (n = 3).
TextSentencer_T63 9158-9240 Sentence denotes Migrated cells were counted by an inverted light microscope at 100× magnification.
TextSentencer_T63 9158-9240 Sentence denotes Migrated cells were counted by an inverted light microscope at 100× magnification.
TextSentencer_T64 9241-9557 Sentence denotes The relative percent of migrated cells was counted and summarized in the bottom panel, and ten random fields were chosen for each count. (D) The expression level of MMP2 and MMP9 relative to β-actin was compared between the untreated group and DMGF group in B16F10 melanoma cells using quantitative PCR. (*p < 0.05).
TextSentencer_T64 9241-9557 Sentence denotes The relative percent of migrated cells was counted and summarized in the bottom panel, and ten random fields were chosen for each count. (D) The expression level of MMP2 and MMP9 relative to β-actin was compared between the untreated group and DMGF group in B16F10 melanoma cells using quantitative PCR. (*p < 0.05).
TextSentencer_T65 9558-9636 Sentence denotes Oncotarget 60049 www.impactjournals.com/oncotarget DMGF on tumor colonization.
TextSentencer_T65 9558-9636 Sentence denotes Oncotarget 60049 www.impactjournals.com/oncotarget DMGF on tumor colonization.
TextSentencer_T66 9637-9821 Sentence denotes At 19 days after tumor injection, B16F10 cells developed serious lung metastasis, and the numbers of metastatic nodules in the lungs of some mice were too large to count ( Figure 5A ).
TextSentencer_T66 9637-9821 Sentence denotes At 19 days after tumor injection, B16F10 cells developed serious lung metastasis, and the numbers of metastatic nodules in the lungs of some mice were too large to count ( Figure 5A ).
TextSentencer_T67 9822-9978 Sentence denotes Consistent with previous results, administration of 10 mg/kg DMGF significantly decreased the colonization of B16F10 cells from the circulation to the lung.
TextSentencer_T67 9822-9978 Sentence denotes Consistent with previous results, administration of 10 mg/kg DMGF significantly decreased the colonization of B16F10 cells from the circulation to the lung.
TextSentencer_T68 9979-10153 Sentence denotes Furthermore, 50 mg/kg DMGF displayed stronger effects to inhibit tumor colonization, and even abolish the colonization of metastatic cells in some treated mice ( Figure 5A ).
TextSentencer_T68 9979-10153 Sentence denotes Furthermore, 50 mg/kg DMGF displayed stronger effects to inhibit tumor colonization, and even abolish the colonization of metastatic cells in some treated mice ( Figure 5A ).
TextSentencer_T69 10154-10252 Sentence denotes The nodules of melanoma cells in the lungs in each group were counted, and the lungs were weighed.
TextSentencer_T69 10154-10252 Sentence denotes The nodules of melanoma cells in the lungs in each group were counted, and the lungs were weighed.
TextSentencer_T70 10253-10362 Sentence denotes The results indicated that the DMGF treatments reduced the colonization more than 3-fold ( Figure 5B and 5C).
TextSentencer_T70 10253-10362 Sentence denotes The results indicated that the DMGF treatments reduced the colonization more than 3-fold ( Figure 5B and 5C).
TextSentencer_T71 10363-10430 Sentence denotes Another common pathway for metastatic cells is the lymphoid system.
TextSentencer_T71 10363-10430 Sentence denotes Another common pathway for metastatic cells is the lymphoid system.
TextSentencer_T72 10431-10683 Sentence denotes Figure 6A shows that the EGFPexpressing B16F10 cells filled the cortex of the lymph node and invaded the medulla at day 9 after inoculation 1 cm from the inguinal lymph node; DMGF treatments slowed down the migration of tumor cells into the lymph node.
TextSentencer_T72 10431-10683 Sentence denotes Figure 6A shows that the EGFPexpressing B16F10 cells filled the cortex of the lymph node and invaded the medulla at day 9 after inoculation 1 cm from the inguinal lymph node; DMGF treatments slowed down the migration of tumor cells into the lymph node.
TextSentencer_T73 10684-10753 Sentence denotes After counting, the fluorescent cells occupied 80% of the lymph node.
TextSentencer_T73 10684-10753 Sentence denotes After counting, the fluorescent cells occupied 80% of the lymph node.
TextSentencer_T74 10754-10835 Sentence denotes However, DMGF suppressed metastasis to the lymph node in a dose-dependent manner:
TextSentencer_T74 10754-10835 Sentence denotes However, DMGF suppressed metastasis to the lymph node in a dose-dependent manner:
TextSentencer_T75 10836-10950 Sentence denotes 10 mg/Kg DMGF lowered lymphoid metastasis from 80% to 16%, and 50 mg/kg DMGF lowered it to below 5% ( Figure 6B ).
TextSentencer_T75 10836-10950 Sentence denotes 10 mg/Kg DMGF lowered lymphoid metastasis from 80% to 16%, and 50 mg/kg DMGF lowered it to below 5% ( Figure 6B ).
TextSentencer_T76 10951-11074 Sentence denotes These studies clearly revealed that treatment with DMGF efficiently inhibited the B16F10 cells' metastatic ability in vivo.
TextSentencer_T76 10951-11074 Sentence denotes These studies clearly revealed that treatment with DMGF efficiently inhibited the B16F10 cells' metastatic ability in vivo.
TextSentencer_T77 11075-11229 Sentence denotes Metastatic invasion is directly correlated with the formation of lamellipodia, which is caused by the polymerization and depolymerization of actin fibers.
TextSentencer_T77 11075-11229 Sentence denotes Metastatic invasion is directly correlated with the formation of lamellipodia, which is caused by the polymerization and depolymerization of actin fibers.
TextSentencer_T78 11230-11352 Sentence denotes Figure 7A revealed that 2.5 mg/ml of DMGF treatments suppressed the formation of lamellipodia ( Figure 7A , upper panels).
TextSentencer_T78 11230-11352 Sentence denotes Figure 7A revealed that 2.5 mg/ml of DMGF treatments suppressed the formation of lamellipodia ( Figure 7A , upper panels).
TextSentencer_T79 11353-11501 Sentence denotes In addition, intracellular actin fiber staining clearly showed that DMGF treatments caused the fragmentation of F-actin ( Figure 7A , lower panels).
TextSentencer_T79 11353-11501 Sentence denotes In addition, intracellular actin fiber staining clearly showed that DMGF treatments caused the fragmentation of F-actin ( Figure 7A , lower panels).
TextSentencer_T80 11502-11509 Sentence denotes It is .
TextSentencer_T80 11502-11509 Sentence denotes It is .
TextSentencer_T81 11510-11592 Sentence denotes Migrated cells were counted by an inverted light microscope at 100× magnification.
TextSentencer_T81 11510-11592 Sentence denotes Migrated cells were counted by an inverted light microscope at 100× magnification.
TextSentencer_T82 11593-11799 Sentence denotes The relative percent of migrated cells was counted and summarized in the bottom panel, and ten random fields were chosen for each count. (C) DMGF (1 μg/ml) had no effect on tube formation of SVEC4-10 cells.
TextSentencer_T82 11593-11799 Sentence denotes The relative percent of migrated cells was counted and summarized in the bottom panel, and ten random fields were chosen for each count. (C) DMGF (1 μg/ml) had no effect on tube formation of SVEC4-10 cells.
TextSentencer_T83 11800-11845 Sentence denotes Micrographs were taken at 100× magnification.
TextSentencer_T83 11800-11845 Sentence denotes Micrographs were taken at 100× magnification.
TextSentencer_T84 11846-11926 Sentence denotes Experiments were repeated three times, and values were the means of triplicates.
TextSentencer_T84 11846-11926 Sentence denotes Experiments were repeated three times, and values were the means of triplicates.
TextSentencer_T85 11927-12067 Sentence denotes The relative percent of tubes was counted and summarized in the bottom panel, and ten random fields were chosen for each count. (*p < 0.05).
TextSentencer_T85 11927-12067 Sentence denotes The relative percent of tubes was counted and summarized in the bottom panel, and ten random fields were chosen for each count. (*p < 0.05).
TextSentencer_T86 12068-12258 Sentence denotes Oncotarget 60050 www.impactjournals.com/oncotarget well known that the Cdc42 and Rac pathway can regulate actin polymerization or depolymerization to influence the formation of lamellipodia.
TextSentencer_T86 12068-12258 Sentence denotes Oncotarget 60050 www.impactjournals.com/oncotarget well known that the Cdc42 and Rac pathway can regulate actin polymerization or depolymerization to influence the formation of lamellipodia.
TextSentencer_T87 12259-12411 Sentence denotes Figure 7B shows that DMGF treatments suppressed the expression of genes associated with the formation of lamellipodia, including Cdc42, PAK1 and N-WASP.
TextSentencer_T87 12259-12411 Sentence denotes Figure 7B shows that DMGF treatments suppressed the expression of genes associated with the formation of lamellipodia, including Cdc42, PAK1 and N-WASP.
TextSentencer_T88 12412-12535 Sentence denotes For actin depolymerization, DMGF dramatically suppressed the expression of LIMK, and also decreased the expression of PAK1.
TextSentencer_T88 12412-12535 Sentence denotes For actin depolymerization, DMGF dramatically suppressed the expression of LIMK, and also decreased the expression of PAK1.
TextSentencer_T89 12536-12715 Sentence denotes Taken together, these results indicated that DMGF could enhance actin depolymerization and suppress the formation of lamellipodia by lowering the expression of the relative genes.
TextSentencer_T89 12536-12715 Sentence denotes Taken together, these results indicated that DMGF could enhance actin depolymerization and suppress the formation of lamellipodia by lowering the expression of the relative genes.
TextSentencer_T90 12716-12787 Sentence denotes Gene expression is driven by the promoter located upstream of the gene.
TextSentencer_T90 12716-12787 Sentence denotes Gene expression is driven by the promoter located upstream of the gene.
TextSentencer_T91 12788-13073 Sentence denotes By analyzing the promoters of these DMGF-effective genes for the binding sites of transcriptional factors listed on GeneCards (http:// www.genecards.org), the results showed that only one transcriptional factor binding site is displayed on all 5 promoter sequences of these genes-CREB.
TextSentencer_T91 12788-13073 Sentence denotes By analyzing the promoters of these DMGF-effective genes for the binding sites of transcriptional factors listed on GeneCards (http:// www.genecards.org), the results showed that only one transcriptional factor binding site is displayed on all 5 promoter sequences of these genes-CREB.
TextSentencer_T92 13074-13260 Sentence denotes According to this information, we examined the interaction of DMGF and CREB by bioinformatics tool and the results predicted DMGF would interact with several residuals in CREB (PDB code:
TextSentencer_T92 13074-13260 Sentence denotes According to this information, we examined the interaction of DMGF and CREB by bioinformatics tool and the results predicted DMGF would interact with several residuals in CREB (PDB code:
TextSentencer_T93 13261-13357 Sentence denotes 2LXT) including Arg131, Pro132, Tyr134, Lys136, Ile137, Tyr658, Gln661 and Lys662 ( Figure 8A ).
TextSentencer_T93 13261-13357 Sentence denotes 2LXT) including Arg131, Pro132, Tyr134, Lys136, Ile137, Tyr658, Gln661 and Lys662 ( Figure 8A ).
TextSentencer_T94 13358-13433 Sentence denotes Therefore, the phosphorylation on Ser133 of CREB may be interfered by DMGF.
TextSentencer_T94 13358-13433 Sentence denotes Therefore, the phosphorylation on Ser133 of CREB may be interfered by DMGF.
TextSentencer_T95 13434-13548 Sentence denotes Consecutively, we further examined the CREB protein expression level and its phosphorylation after DMGF treatment.
TextSentencer_T95 13434-13548 Sentence denotes Consecutively, we further examined the CREB protein expression level and its phosphorylation after DMGF treatment.
TextSentencer_T96 13549-13697 Sentence denotes The results showed that DMGF did not notably decrease CREB protein expression, and CREB was only minorly reduced after DMGF treatment ( Figure 8B ).
TextSentencer_T96 13549-13697 Sentence denotes The results showed that DMGF did not notably decrease CREB protein expression, and CREB was only minorly reduced after DMGF treatment ( Figure 8B ).
TextSentencer_T97 13698-13815 Sentence denotes However, the results indicated that phosphorylation at Ser133 of CREB was suppressed by DMGF treatment ( Figure 8B ).
TextSentencer_T97 13698-13815 Sentence denotes However, the results indicated that phosphorylation at Ser133 of CREB was suppressed by DMGF treatment ( Figure 8B ).
TextSentencer_T98 13816-13955 Sentence denotes These results revealed that DMGF could interfere with phosphorylation on the Ser133 of CREB which involves in the transcriptional activity.
TextSentencer_T98 13816-13955 Sentence denotes These results revealed that DMGF could interfere with phosphorylation on the Ser133 of CREB which involves in the transcriptional activity.
TextSentencer_T99 13956-14101 Sentence denotes By this reaction, DMGF could decrease the activity of CREB to lower the expression of the genes that correspond to the formation of lamellipodia.
TextSentencer_T99 13956-14101 Sentence denotes By this reaction, DMGF could decrease the activity of CREB to lower the expression of the genes that correspond to the formation of lamellipodia.
TextSentencer_T100 14102-14181 Sentence denotes DMGF is a bioactive biflavonoid from Ouratea parviflora [6] or Taxus media var.
TextSentencer_T100 14102-14181 Sentence denotes DMGF is a bioactive biflavonoid from Ouratea parviflora [6] or Taxus media var.
TextSentencer_T101 14182-14400 Sentence denotes Hicksii [7] ; however, little is known about its functions in tumor angiogenesis, metastasis and tumor growth except for its effect on inducing tumor death through apoptosis or autophagy in different cancer cells [7] .
TextSentencer_T101 14182-14400 Sentence denotes Hicksii [7] ; however, little is known about its functions in tumor angiogenesis, metastasis and tumor growth except for its effect on inducing tumor death through apoptosis or autophagy in different cancer cells [7] .
TextSentencer_T102 14401-14563 Sentence denotes In this study, the DMGF was shown to dramatically suppress the spontaneous metastasis of B16F10 melanoma cells from subcutaneous sites to the spleen ( Figure 4 ).
TextSentencer_T102 14401-14563 Sentence denotes In this study, the DMGF was shown to dramatically suppress the spontaneous metastasis of B16F10 melanoma cells from subcutaneous sites to the spleen ( Figure 4 ).
TextSentencer_T103 14564-14841 Sentence denotes Another two in vivo animal models further indicated that DMGF could suppress the processes of metastasis, including colonization from blood Oncotarget 60051 www.impactjournals.com/oncotarget circulation ( Figure 5 ) or invasion/intravasation into the lymph system ( Figure 6 ).
TextSentencer_T103 14564-14841 Sentence denotes Another two in vivo animal models further indicated that DMGF could suppress the processes of metastasis, including colonization from blood Oncotarget 60051 www.impactjournals.com/oncotarget circulation ( Figure 5 ) or invasion/intravasation into the lymph system ( Figure 6 ).
TextSentencer_T104 14842-15206 Sentence denotes These results showed that the decrease in the metastasis of melanoma cells may result from low migratory ability ( Figure 1B ) because DMGF could reduce the formation of lamellipodia ( Figure 7A ) by interfering with F-actin polymerization by lowering the phosphorylation of CREB to suppress the expression of Cdc42/Rac pathway associated genes (Figures 7 and 8 ).
TextSentencer_T104 14842-15206 Sentence denotes These results showed that the decrease in the metastasis of melanoma cells may result from low migratory ability ( Figure 1B ) because DMGF could reduce the formation of lamellipodia ( Figure 7A ) by interfering with F-actin polymerization by lowering the phosphorylation of CREB to suppress the expression of Cdc42/Rac pathway associated genes (Figures 7 and 8 ).
TextSentencer_T105 15207-15306 Sentence denotes This study is the first to show the medical potency of DMGF to suppress the metastasis of melanoma.
TextSentencer_T105 15207-15306 Sentence denotes This study is the first to show the medical potency of DMGF to suppress the metastasis of melanoma.
TextSentencer_T106 15307-15357 Sentence denotes Cell migration is a key step for tumor metastasis.
TextSentencer_T106 15307-15357 Sentence denotes Cell migration is a key step for tumor metastasis.
TextSentencer_T107 15358-15687 Sentence denotes Some reports have demonstrated that certain flavonoids can suppress the migration of melanoma under different concentrations: quercetin at 60 µM (18 mg/ml) [16] , (-)-epigallocatechin-3-gallate (EGCG) at 21.8 µM (10 mg/ml) [17] , and genistein at 25 μM (6.8 mg/ml) [18] could cause similar effects on the inhibition of migration.
TextSentencer_T107 15358-15687 Sentence denotes Some reports have demonstrated that certain flavonoids can suppress the migration of melanoma under different concentrations: quercetin at 60 µM (18 mg/ml) [16] , (-)-epigallocatechin-3-gallate (EGCG) at 21.8 µM (10 mg/ml) [17] , and genistein at 25 μM (6.8 mg/ml) [18] could cause similar effects on the inhibition of migration.
TextSentencer_T108 15688-15835 Sentence denotes Comparing the above information, only 1.8 µM (1 mg/ml) DMGF could have such the inhibitory effect on the migration of melanoma cells ( Figure 1B) .
TextSentencer_T108 15688-15835 Sentence denotes Comparing the above information, only 1.8 µM (1 mg/ml) DMGF could have such the inhibitory effect on the migration of melanoma cells ( Figure 1B) .
TextSentencer_T109 15836-15944 Sentence denotes Thus, we thought that DMGF might have a higher potency than other flavonoids on the inhibition of migration.
TextSentencer_T109 15836-15944 Sentence denotes Thus, we thought that DMGF might have a higher potency than other flavonoids on the inhibition of migration.
TextSentencer_T110 15945-16136 Sentence denotes Highly metastatic cancer cells frequently produce high levels of matrix metalloproteinases (MMPs) to degrade the extracellular matrix (ECM) surrounding them and to invade other tissues [19] .
TextSentencer_T110 15945-16136 Sentence denotes Highly metastatic cancer cells frequently produce high levels of matrix metalloproteinases (MMPs) to degrade the extracellular matrix (ECM) surrounding them and to invade other tissues [19] .
TextSentencer_T111 16137-16317 Sentence denotes MMP-2 and MMP-9 are key enzymes for degrading type IV collagen, which is a major component of the basement membrane in ECM Oncotarget 60052 www.impactjournals.com/oncotarget [14] .
TextSentencer_T111 16137-16317 Sentence denotes MMP-2 and MMP-9 are key enzymes for degrading type IV collagen, which is a major component of the basement membrane in ECM Oncotarget 60052 www.impactjournals.com/oncotarget [14] .
TextSentencer_T112 16318-16490 Sentence denotes Figure 1C shows that DMGF has a stronger ability to suppress only MMP2 gene expression, but only a slight effect on MMP9 expression for B16F10 melanoma cells ( Figure 1C ).
TextSentencer_T112 16318-16490 Sentence denotes Figure 1C shows that DMGF has a stronger ability to suppress only MMP2 gene expression, but only a slight effect on MMP9 expression for B16F10 melanoma cells ( Figure 1C ).
TextSentencer_T113 16491-16661 Sentence denotes Flavonoids such as quercetin and EGCG have been shown to decrease the expression of both MMP2 and MMP9 in human melanoma cells [16] and breast cancer [20] , respectively.
TextSentencer_T113 16491-16661 Sentence denotes Flavonoids such as quercetin and EGCG have been shown to decrease the expression of both MMP2 and MMP9 in human melanoma cells [16] and breast cancer [20] , respectively.
TextSentencer_T114 16662-16724 Sentence denotes These flavonoids inhibit the activities of both MMP2 and MMP9.
TextSentencer_T114 16662-16724 Sentence denotes These flavonoids inhibit the activities of both MMP2 and MMP9.
TextSentencer_T115 16725-16825 Sentence denotes However, 1 mg/ml DMGF only significantly suppressed the expression of MMP-2, but not MMP-9, at 12 h.
TextSentencer_T115 16725-16825 Sentence denotes However, 1 mg/ml DMGF only significantly suppressed the expression of MMP-2, but not MMP-9, at 12 h.
TextSentencer_T116 16826-16911 Sentence denotes Perhaps the dosages needed to inhibit the expression of MMP-2 or MMP-9 are different.
TextSentencer_T116 16826-16911 Sentence denotes Perhaps the dosages needed to inhibit the expression of MMP-2 or MMP-9 are different.
TextSentencer_T117 16912-17102 Sentence denotes The other possibility is that the mechanism by which DMGF suppresses MMP expression may be different from these flavonoids; it may suppress the signals needed by the MMP2 expression pathway.
TextSentencer_T117 16912-17102 Sentence denotes The other possibility is that the mechanism by which DMGF suppresses MMP expression may be different from these flavonoids; it may suppress the signals needed by the MMP2 expression pathway.
TextSentencer_T118 17103-17229 Sentence denotes The lamellipodium is a flat, sheet-like and F-actinrich membrane protrusion, and it is the main organelle for cell locomotion.
TextSentencer_T118 17103-17229 Sentence denotes The lamellipodium is a flat, sheet-like and F-actinrich membrane protrusion, and it is the main organelle for cell locomotion.
TextSentencer_T119 17230-17372 Sentence denotes Tumor cells utilize this organelle to explore and move into the surrounding environment, then form invasive matrix-degrading structures [21] .
TextSentencer_T119 17230-17372 Sentence denotes Tumor cells utilize this organelle to explore and move into the surrounding environment, then form invasive matrix-degrading structures [21] .
TextSentencer_T120 17373-17679 Sentence denotes In addition, the Rac1 or Cdc42 pathways can lead to the formation of membrane lamellipodia or filopodia, respectively, and have been shown to regulate a variety of biological functions, including changes in cell shape, adhesion, and motility, through the reorganization of the actin cytoskeleton [22, 23] .
TextSentencer_T120 17373-17679 Sentence denotes In addition, the Rac1 or Cdc42 pathways can lead to the formation of membrane lamellipodia or filopodia, respectively, and have been shown to regulate a variety of biological functions, including changes in cell shape, adhesion, and motility, through the reorganization of the actin cytoskeleton [22, 23] .
TextSentencer_T121 17680-17773 Sentence denotes Recently, the literature demonstrated that some flavonoids could suppress such cell activity.
TextSentencer_T121 17680-17773 Sentence denotes Recently, the literature demonstrated that some flavonoids could suppress such cell activity.
TextSentencer_T122 17774-17889 Sentence denotes For example, apigenin (50 µM) could influence F-actin cytoskeleton formation to suppress tumor cell motility [18] .
TextSentencer_T122 17774-17889 Sentence denotes For example, apigenin (50 µM) could influence F-actin cytoskeleton formation to suppress tumor cell motility [18] .
TextSentencer_T123 17890-18125 Sentence denotes Moreover, 30 µM luteolin Oncotarget 60053 www.impactjournals.com/oncotarget inhibits the migration of human glioblastoma U-87 MG and T98G cells through the down-regulation of Cdc42 gene expression and ERK protein phosphorylation [24] .
TextSentencer_T123 17890-18125 Sentence denotes Moreover, 30 µM luteolin Oncotarget 60053 www.impactjournals.com/oncotarget inhibits the migration of human glioblastoma U-87 MG and T98G cells through the down-regulation of Cdc42 gene expression and ERK protein phosphorylation [24] .
TextSentencer_T124 18126-18260 Sentence denotes Moreover, 60 µM wogonin could suppress cell migration and actin remodeling of B16F10 cells by inhibiting the expression of Rac1 [25] .
TextSentencer_T124 18126-18260 Sentence denotes Moreover, 60 µM wogonin could suppress cell migration and actin remodeling of B16F10 cells by inhibiting the expression of Rac1 [25] .
TextSentencer_T125 18261-18455 Sentence denotes Here, the F-actin of the DMGFtreated B16F10 cells was remarkably disintegrated (Figure 7A) , and the formation of lamellipodia was decreased to influence the cell's shape in vitro ( Figure 7A) .
TextSentencer_T125 18261-18455 Sentence denotes Here, the F-actin of the DMGFtreated B16F10 cells was remarkably disintegrated (Figure 7A) , and the formation of lamellipodia was decreased to influence the cell's shape in vitro ( Figure 7A) .
TextSentencer_T126 18456-18663 Sentence denotes Sequentially, the expression of the relative genes in Cdc42/ Rac1 pathways were examined, and the results showed that the expression of all relative genes was down-regulated by DMGF treatments ( Figure 7B ).
TextSentencer_T126 18456-18663 Sentence denotes Sequentially, the expression of the relative genes in Cdc42/ Rac1 pathways were examined, and the results showed that the expression of all relative genes was down-regulated by DMGF treatments ( Figure 7B ).
TextSentencer_T127 18664-18858 Sentence denotes Taken together, the decrease in the expressions of genes in Cdc42/Rac1 pathway may involve in the blockade of cell migration and invasion for DMGF's anti-metastatic activity as other flavonoids.
TextSentencer_T127 18664-18858 Sentence denotes Taken together, the decrease in the expressions of genes in Cdc42/Rac1 pathway may involve in the blockade of cell migration and invasion for DMGF's anti-metastatic activity as other flavonoids.
TextSentencer_T128 18859-19092 Sentence denotes To identify which factors are involved in the DMGF-induced down-regulation of the genes, the transcriptional factor binding sites on the promoter sequences of these relative genes were assayed on GeneCards (http://www.genecards.org).
TextSentencer_T128 18859-19092 Sentence denotes To identify which factors are involved in the DMGF-induced down-regulation of the genes, the transcriptional factor binding sites on the promoter sequences of these relative genes were assayed on GeneCards (http://www.genecards.org).
TextSentencer_T129 19093-19178 Sentence denotes Only one factor, CREB, was proposed to regulate the expression of all relative genes.
TextSentencer_T129 19093-19178 Sentence denotes Only one factor, CREB, was proposed to regulate the expression of all relative genes.
TextSentencer_T130 19179-19354 Sentence denotes Therefore, it was assumed that DMGF may reduce the phosphorylation or expression of CREB to down-regulate the expression levels of the genes related to the Cdc42/Rac1 pathway.
TextSentencer_T130 19179-19354 Sentence denotes Therefore, it was assumed that DMGF may reduce the phosphorylation or expression of CREB to down-regulate the expression levels of the genes related to the Cdc42/Rac1 pathway.
TextSentencer_T131 19355-19405 Sentence denotes Sequentially, our data fit with such suppositions.
TextSentencer_T131 19355-19405 Sentence denotes Sequentially, our data fit with such suppositions.
TextSentencer_T132 19406-19675 Sentence denotes Figure 8A showed that DMGF treatments reduced the phosphorylation at Ser133 of CREB, but it did not influence the expression of CREB protein, which is in accord with the bioinformatics result which DMGF interacted with the amino acids near Ser133 of CREB ( Figure 8B ).
TextSentencer_T132 19406-19675 Sentence denotes Figure 8A showed that DMGF treatments reduced the phosphorylation at Ser133 of CREB, but it did not influence the expression of CREB protein, which is in accord with the bioinformatics result which DMGF interacted with the amino acids near Ser133 of CREB ( Figure 8B ).
TextSentencer_T133 19676-19769 Sentence denotes These results indicated that the transcriptional activity of CREB would be decreased by DMGF.
TextSentencer_T133 19676-19769 Sentence denotes These results indicated that the transcriptional activity of CREB would be decreased by DMGF.
TextSentencer_T134 19770-19901 Sentence denotes As previously reported, the metastatic phenotype of melanoma could benefit from the expression of CREB transcription factors [26] .
TextSentencer_T134 19770-19901 Sentence denotes As previously reported, the metastatic phenotype of melanoma could benefit from the expression of CREB transcription factors [26] .
TextSentencer_T135 19902-20137 Sentence denotes Moreover, Xie et al. also demonstrated the decrease of CREB activity in metastatic melanoma cells by a dominant-negative form of CREB (KCREB), which led to a decrease in their tumorigenicity and metastatic potential in nude mice [27] .
TextSentencer_T135 19902-20137 Sentence denotes Moreover, Xie et al. also demonstrated the decrease of CREB activity in metastatic melanoma cells by a dominant-negative form of CREB (KCREB), which led to a decrease in their tumorigenicity and metastatic potential in nude mice [27] .
TextSentencer_T136 20138-20349 Sentence denotes Moreover, a p21-activated kinase 4 (PAK4) inhibitor, PF-3758309, Oncotarget 60055 www.impactjournals.com/oncotarget could suppress migration and invasion by reducing the phosphorylation of CREB and ERK1/2 [28] .
TextSentencer_T136 20138-20349 Sentence denotes Moreover, a p21-activated kinase 4 (PAK4) inhibitor, PF-3758309, Oncotarget 60055 www.impactjournals.com/oncotarget could suppress migration and invasion by reducing the phosphorylation of CREB and ERK1/2 [28] .
TextSentencer_T137 20350-20567 Sentence denotes Thus, DMGF down-regulates the phosphorylation of CREB to suppress the expression levels of these genes related to the Cdc42/ Rac1 pathway, resulting in the inhibition of lamellipodia formation to attenuate metastasis.
TextSentencer_T137 20350-20567 Sentence denotes Thus, DMGF down-regulates the phosphorylation of CREB to suppress the expression levels of these genes related to the Cdc42/ Rac1 pathway, resulting in the inhibition of lamellipodia formation to attenuate metastasis.
TextSentencer_T138 20568-20697 Sentence denotes In addition to lamellipodia formation, we proposed the other anti-metastatic mechanism for DMGF: the suppression of angiogenesis.
TextSentencer_T138 20568-20697 Sentence denotes In addition to lamellipodia formation, we proposed the other anti-metastatic mechanism for DMGF: the suppression of angiogenesis.
TextSentencer_T139 20698-20854 Sentence denotes Tumor angiogenesis could provide an escape route for those tumor cells that can loosen themselves from the primary tumor to new sites via new vessels [29] .
TextSentencer_T139 20698-20854 Sentence denotes Tumor angiogenesis could provide an escape route for those tumor cells that can loosen themselves from the primary tumor to new sites via new vessels [29] .
TextSentencer_T140 20855-20995 Sentence denotes Figure 3 shows in vivo DMGF treatment reduced the formation of new vessels and caused a decrease in the intensity of vessels in tumor areas.
TextSentencer_T140 20855-20995 Sentence denotes Figure 3 shows in vivo DMGF treatment reduced the formation of new vessels and caused a decrease in the intensity of vessels in tumor areas.
TextSentencer_T141 20996-21207 Sentence denotes In vitro, DMGF could inhibit neovascularization by suppressing the motility of SVEC4-10 endothelial cells but not influencing the capillary-like tube formation in endothelial cells in vitro ( Figure 2B and 2C) .
TextSentencer_T141 20996-21207 Sentence denotes In vitro, DMGF could inhibit neovascularization by suppressing the motility of SVEC4-10 endothelial cells but not influencing the capillary-like tube formation in endothelial cells in vitro ( Figure 2B and 2C) .
TextSentencer_T142 21208-21386 Sentence denotes Therefore, we assume that DMGF could inhibit angiogenesis at early stages, and its anti-angiogenesis activity may also result from the blocking movement of the endothelial cells.
TextSentencer_T142 21208-21386 Sentence denotes Therefore, we assume that DMGF could inhibit angiogenesis at early stages, and its anti-angiogenesis activity may also result from the blocking movement of the endothelial cells.
TextSentencer_T143 21387-21537 Sentence denotes Interestingly, the published literature also shows that two biflavonoids, amentoflavone (AF) and morelloflavone (MF), have anti-angiogenic activities.
TextSentencer_T143 21387-21537 Sentence denotes Interestingly, the published literature also shows that two biflavonoids, amentoflavone (AF) and morelloflavone (MF), have anti-angiogenic activities.
TextSentencer_T144 21538-21668 Sentence denotes However, these reports only focus on the anti-angiogenic abilities of the two biflavonoids, but not their anti-metastatic effects.
TextSentencer_T144 21538-21668 Sentence denotes However, these reports only focus on the anti-angiogenic abilities of the two biflavonoids, but not their anti-metastatic effects.
TextSentencer_T145 21669-21946 Sentence denotes The anti-angiogenic activity of AF is due to its ability to bind VEGF-A and VEGF-B, preventing the interaction with VEGF receptors [5] ; however, our data indicated that DMGF did not impact VEGF-induced capillary-like tube formation in SVEC4-10 endothelial cells ( Figure 2B ).
TextSentencer_T145 21669-21946 Sentence denotes The anti-angiogenic activity of AF is due to its ability to bind VEGF-A and VEGF-B, preventing the interaction with VEGF receptors [5] ; however, our data indicated that DMGF did not impact VEGF-induced capillary-like tube formation in SVEC4-10 endothelial cells ( Figure 2B ).
TextSentencer_T146 21947-22087 Sentence denotes Based on this different effect, we thought that DMGF may not bind to VEGF to prevent interactions with VEGF receptors for anti-angiogenesis.
TextSentencer_T146 21947-22087 Sentence denotes Based on this different effect, we thought that DMGF may not bind to VEGF to prevent interactions with VEGF receptors for anti-angiogenesis.
TextSentencer_T147 22088-22217 Sentence denotes In addition, MF could inhibit the activation of Raf/MEK/ERK protein kinases and suppress the activation of AP-1 and p90RSK [11] .
TextSentencer_T147 22088-22217 Sentence denotes In addition, MF could inhibit the activation of Raf/MEK/ERK protein kinases and suppress the activation of AP-1 and p90RSK [11] .
TextSentencer_T148 22218-22319 Sentence denotes DMGF could suppress the phosphorylation of CREB, which is also an event downstream of ERK activation.
TextSentencer_T148 22218-22319 Sentence denotes DMGF could suppress the phosphorylation of CREB, which is also an event downstream of ERK activation.
TextSentencer_T149 22320-22443 Sentence denotes Therefore, we assume that DMGF may inhibit both tumor angiogenesis by blocking the ERK/pCREB pathway in the same way as MF.
TextSentencer_T149 22320-22443 Sentence denotes Therefore, we assume that DMGF may inhibit both tumor angiogenesis by blocking the ERK/pCREB pathway in the same way as MF.
TextSentencer_T150 22444-22636 Sentence denotes In the C57/BL6 mouse model, we found that DMGF at 10 mg/kg and 50 mg/kg significantly inhibited lung and lymph node metastasis, but did not affect the body weight of the mice (data not shown).
TextSentencer_T150 22444-22636 Sentence denotes In the C57/BL6 mouse model, we found that DMGF at 10 mg/kg and 50 mg/kg significantly inhibited lung and lymph node metastasis, but did not affect the body weight of the mice (data not shown).
TextSentencer_T151 22637-22705 Sentence denotes DMGF inhibited the cell proliferation of SVEC4-10 as well as B16F10.
TextSentencer_T151 22637-22705 Sentence denotes DMGF inhibited the cell proliferation of SVEC4-10 as well as B16F10.
TextSentencer_T152 22706-22815 Sentence denotes SVEC4-10 is a highly proliferative endothelial cell line derived by SV40 transformation of endothelial cells.
TextSentencer_T152 22706-22815 Sentence denotes SVEC4-10 is a highly proliferative endothelial cell line derived by SV40 transformation of endothelial cells.
TextSentencer_T153 22816-22865 Sentence denotes Therefore, SVEC4-10 is sensitive as B16F10 cells.
TextSentencer_T153 22816-22865 Sentence denotes Therefore, SVEC4-10 is sensitive as B16F10 cells.
TextSentencer_T154 22866-23039 Sentence denotes However, in our previous results, DMGF had different antiproliferative effects between cancer cell lines and primary cells, including human PBMCs and mouse splenocytes [7] .
TextSentencer_T154 22866-23039 Sentence denotes However, in our previous results, DMGF had different antiproliferative effects between cancer cell lines and primary cells, including human PBMCs and mouse splenocytes [7] .
TextSentencer_T155 23040-23129 Sentence denotes These results showed that DMGF has selective cytotoxicity between normal and tumor cells.
TextSentencer_T155 23040-23129 Sentence denotes These results showed that DMGF has selective cytotoxicity between normal and tumor cells.
TextSentencer_T156 23130-23228 Sentence denotes Thus, DMGF was assumed to be a novel chemoprevention drug with limited toxicity in normal tissues.
TextSentencer_T156 23130-23228 Sentence denotes Thus, DMGF was assumed to be a novel chemoprevention drug with limited toxicity in normal tissues.
TextSentencer_T157 23229-23461 Sentence denotes Here, we first reported that DMGF has could suppress metastasis, including angiogenesis, due to its ability to suppress F-action polymerization by inhibiting the relative gene expression through the suppression CREB phosphorylation.
TextSentencer_T157 23229-23461 Sentence denotes Here, we first reported that DMGF has could suppress metastasis, including angiogenesis, due to its ability to suppress F-action polymerization by inhibiting the relative gene expression through the suppression CREB phosphorylation.
TextSentencer_T158 23462-23590 Sentence denotes Therefore, we propose that DMGF may be considered to be a potential chemoprevention drug for the inhibition of tumor metastasis.
TextSentencer_T158 23462-23590 Sentence denotes Therefore, we propose that DMGF may be considered to be a potential chemoprevention drug for the inhibition of tumor metastasis.
TextSentencer_T159 23591-23702 Sentence denotes DMGF (98% purity by high performance liquid chromatography) isolated from the dried needles of Taxus media var.
TextSentencer_T159 23591-23702 Sentence denotes DMGF (98% purity by high performance liquid chromatography) isolated from the dried needles of Taxus media var.
TextSentencer_T160 23703-23802 Sentence denotes Hicksii was kindly provided by Yung-Shin Pharmaceutical Industry Co., Ltd. (Taichung, Taiwan) [7] .
TextSentencer_T160 23703-23802 Sentence denotes Hicksii was kindly provided by Yung-Shin Pharmaceutical Industry Co., Ltd. (Taichung, Taiwan) [7] .
TextSentencer_T161 23803-23849 Sentence denotes Briefly, the dried needles of Taxus media var.
TextSentencer_T161 23803-23849 Sentence denotes Briefly, the dried needles of Taxus media var.
TextSentencer_T162 23850-23984 Sentence denotes Hicksii (10 kg) were extracted with methanol (MeOH) at a preferred ratio of 1:1 (w/v) at room temperature to yield a methanol extract.
TextSentencer_T162 23850-23984 Sentence denotes Hicksii (10 kg) were extracted with methanol (MeOH) at a preferred ratio of 1:1 (w/v) at room temperature to yield a methanol extract.
TextSentencer_T163 23985-24252 Sentence denotes The crude methanol extract was partitioned with water/ethyl acetate (EtOAc) (1:1), mixed with 350 g silical gel 60 N (Timely, Japan) for packing in an open column, and eluted with different ratios of hexane/EtOAc to afford 5 fractions (4:1, 3:1, 7:3, 13:7, 3:2, A-E).
TextSentencer_T163 23985-24252 Sentence denotes The crude methanol extract was partitioned with water/ethyl acetate (EtOAc) (1:1), mixed with 350 g silical gel 60 N (Timely, Japan) for packing in an open column, and eluted with different ratios of hexane/EtOAc to afford 5 fractions (4:1, 3:1, 7:3, 13:7, 3:2, A-E).
TextSentencer_T164 24253-24426 Sentence denotes Fraction E was further purified by recrystallization from hexane/acetone (3:1) then re-chromatographed with the same elution condition to give 4.2 g (98.5 %) of DMGF sample.
TextSentencer_T164 24253-24426 Sentence denotes Fraction E was further purified by recrystallization from hexane/acetone (3:1) then re-chromatographed with the same elution condition to give 4.2 g (98.5 %) of DMGF sample.
TextSentencer_T165 24427-24806 Sentence denotes SVEC4-10 mouse vascular endothelial cells and B16F10 mouse melanoma cells were purchased from BCRC (Hsinchu, Taiwan, ROC) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco/ Invitrogen, Carlsbad, CA, USA) supplemented with heatinactivated 10% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin/ amphotericin in 5% CO 2 at 37°C.
TextSentencer_T165 24427-24806 Sentence denotes SVEC4-10 mouse vascular endothelial cells and B16F10 mouse melanoma cells were purchased from BCRC (Hsinchu, Taiwan, ROC) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco/ Invitrogen, Carlsbad, CA, USA) supplemented with heatinactivated 10% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin/ amphotericin in 5% CO 2 at 37°C.
TextSentencer_T166 24807-24880 Sentence denotes The cells (1 × 10 4 cells/well) were seeded overnight in a 96-well plate.
TextSentencer_T166 24807-24880 Sentence denotes The cells (1 × 10 4 cells/well) were seeded overnight in a 96-well plate.
TextSentencer_T167 24881-25041 Sentence denotes After treatment with serial concentrations of DMGF in dimethyl sulfoxide (DMSO, final concentration of DMSO is 0.1%), the cells were incubated at 37°C for 48 h.
TextSentencer_T167 24881-25041 Sentence denotes After treatment with serial concentrations of DMGF in dimethyl sulfoxide (DMSO, final concentration of DMSO is 0.1%), the cells were incubated at 37°C for 48 h.
TextSentencer_T168 25042-25101 Sentence denotes Subsequently, the cell viability was measured by MTT assay.
TextSentencer_T168 25042-25101 Sentence denotes Subsequently, the cell viability was measured by MTT assay.
TextSentencer_T169 25102-25245 Sentence denotes The cell viability ratio (%) was calculated using the following equation: % viability = absorbance of test sample/absorbance of control × 100%.
TextSentencer_T169 25102-25245 Sentence denotes The cell viability ratio (%) was calculated using the following equation: % viability = absorbance of test sample/absorbance of control × 100%.
TextSentencer_T170 25246-25324 Sentence denotes The results were expressed in duplicate and for three independent experiments.
TextSentencer_T170 25246-25324 Sentence denotes The results were expressed in duplicate and for three independent experiments.
TextSentencer_T171 25325-25578 Sentence denotes For one set of experiments, SVEC4-10 cells were pre-cultured with 1 μg/ml DMGF for 6 h and then plated Oncotarget 60056 www.impactjournals.com/oncotarget (3 × 10 4 cells/well) in the upper well of a transwell plate (8 μm pore; Costar, Corning, NY, USA).
TextSentencer_T171 25325-25578 Sentence denotes For one set of experiments, SVEC4-10 cells were pre-cultured with 1 μg/ml DMGF for 6 h and then plated Oncotarget 60056 www.impactjournals.com/oncotarget (3 × 10 4 cells/well) in the upper well of a transwell plate (8 μm pore; Costar, Corning, NY, USA).
TextSentencer_T172 25579-25740 Sentence denotes In addition, B16F10 cells were pre-cultured with 1 μg/ml DMGF for 4 h (37°C, 5% CO 2 ), then plated (3 × 10 4 cells/well) in the upper well of a transwell plate.
TextSentencer_T172 25579-25740 Sentence denotes In addition, B16F10 cells were pre-cultured with 1 μg/ml DMGF for 4 h (37°C, 5% CO 2 ), then plated (3 × 10 4 cells/well) in the upper well of a transwell plate.
TextSentencer_T173 25741-25848 Sentence denotes These cells were allowed to migrate toward the lower well with DMEM growth medium for 6 h (37°C, 5% CO 2 ).
TextSentencer_T173 25741-25848 Sentence denotes These cells were allowed to migrate toward the lower well with DMEM growth medium for 6 h (37°C, 5% CO 2 ).
TextSentencer_T174 25849-26071 Sentence denotes Then, the cells on the top of the filter were removed with a cotton swab and the migrated cells on the underside were fixed with methanol, stained with 50 μg/ml propidium iodide, and counted with a fluorescence microscope.
TextSentencer_T174 25849-26071 Sentence denotes Then, the cells on the top of the filter were removed with a cotton swab and the migrated cells on the underside were fixed with methanol, stained with 50 μg/ml propidium iodide, and counted with a fluorescence microscope.
TextSentencer_T175 26072-26233 Sentence denotes Fifty microliters of growth factor-reduced matrigel (BD Bioscience, San Jose, CA, USA) was added to each well of a 96-well plate and polymerized for 2 h at 37°C.
TextSentencer_T175 26072-26233 Sentence denotes Fifty microliters of growth factor-reduced matrigel (BD Bioscience, San Jose, CA, USA) was added to each well of a 96-well plate and polymerized for 2 h at 37°C.
TextSentencer_T176 26234-26386 Sentence denotes SVEC 4-10 cells (3 × 10 4 ) were treated with or without 1 μg/ml DMGF in growth medium containing 16 ng/ml of VEGF (Upstate Inc., Lake Placid, NY, USA).
TextSentencer_T176 26234-26386 Sentence denotes SVEC 4-10 cells (3 × 10 4 ) were treated with or without 1 μg/ml DMGF in growth medium containing 16 ng/ml of VEGF (Upstate Inc., Lake Placid, NY, USA).
TextSentencer_T177 26387-26499 Sentence denotes These cells were incubated for a further 4 h at 37°C and photographed under a microscope (100 × magnifications).
TextSentencer_T177 26387-26499 Sentence denotes These cells were incubated for a further 4 h at 37°C and photographed under a microscope (100 × magnifications).
TextSentencer_T178 26500-26551 Sentence denotes The total number of network formations was counted.
TextSentencer_T178 26500-26551 Sentence denotes The total number of network formations was counted.
TextSentencer_T179 26553-26684 Sentence denotes Total cellular RNA was extracted with TriZol reagent (Life Technologies, Glasgow, UK) according to the manufacturer's instructions.
TextSentencer_T179 26553-26684 Sentence denotes Total cellular RNA was extracted with TriZol reagent (Life Technologies, Glasgow, UK) according to the manufacturer's instructions.
TextSentencer_T180 26685-26777 Sentence denotes Total RNA was reverse-transcribed using the Superscript™-III kit (Invitrogen, Carlsbad, CA).
TextSentencer_T180 26685-26777 Sentence denotes Total RNA was reverse-transcribed using the Superscript™-III kit (Invitrogen, Carlsbad, CA).
TextSentencer_T181 26778-26868 Sentence denotes PCR analysis was performed on aliquots of the cDNA preparations to detect gene expression.
TextSentencer_T181 26778-26868 Sentence denotes PCR analysis was performed on aliquots of the cDNA preparations to detect gene expression.
TextSentencer_T182 26869-26927 Sentence denotes The amplified products were visualized on 2% agarose gels.
TextSentencer_T182 26869-26927 Sentence denotes The amplified products were visualized on 2% agarose gels.
TextSentencer_T183 26928-27049 Sentence denotes The PCR conditions were 4 min at 94°C followed by 30 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 1 min at 72°C.
TextSentencer_T183 26928-27049 Sentence denotes The PCR conditions were 4 min at 94°C followed by 30 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 1 min at 72°C.
TextSentencer_T184 27050-27113 Sentence denotes The following primers were used for the amplification reaction:
TextSentencer_T184 27050-27113 Sentence denotes The following primers were used for the amplification reaction:
TextSentencer_T185 27114-27860 Sentence denotes 5ʹ-GTG CCT GAG ATA ACT CAC CAC-3ʹ as the forward primer and 5ʹ-TTC TGC ACT TAC ACA GAA AGG-3ʹ as the reverse primer for Cdc42; 5ʹ-AAG ACA TCC AAC AGC CAG AA-3ʹ as the forward primer and 5ʹ-TGT AGC CAC GTC CCG AGT-3ʹ as the reverse primer for PAK1; 5ʹ-CCC TAT CCT ATC CGC AAA CA-3ʹ as the forward primer and 5ʹ-CGC ACC TCA GGA TAC CAC TT-3ʹ as the reverse primer for Rac1; 5ʹ-CCC CCA AAT GGT CCT AAT CT-3ʹ as the forward primer and 5ʹ-ACA TGT CCA ATG TCT GGA A-3ʹ as the reverse primer for N-WASP1; 5ʹ-GGG GCA TCA TCA AGA GCA-3ʹ as the forward primer and 5ʹ-GAG GAC TAG GGT GGT TCAG-3ʹ as the reverse primer for LIMK; and 5ʹ-CCA GCC GAG CCA CAT CGC TC-3ʹ as the forward primer and 5ʹ-ATG AGC CCC AGC CTT CTC CAT-3ʹ as the reverse primer for GAPDH.
TextSentencer_T185 27114-27860 Sentence denotes 5ʹ-GTG CCT GAG ATA ACT CAC CAC-3ʹ as the forward primer and 5ʹ-TTC TGC ACT TAC ACA GAA AGG-3ʹ as the reverse primer for Cdc42; 5ʹ-AAG ACA TCC AAC AGC CAG AA-3ʹ as the forward primer and 5ʹ-TGT AGC CAC GTC CCG AGT-3ʹ as the reverse primer for PAK1; 5ʹ-CCC TAT CCT ATC CGC AAA CA-3ʹ as the forward primer and 5ʹ-CGC ACC TCA GGA TAC CAC TT-3ʹ as the reverse primer for Rac1; 5ʹ-CCC CCA AAT GGT CCT AAT CT-3ʹ as the forward primer and 5ʹ-ACA TGT CCA ATG TCT GGA A-3ʹ as the reverse primer for N-WASP1; 5ʹ-GGG GCA TCA TCA AGA GCA-3ʹ as the forward primer and 5ʹ-GAG GAC TAG GGT GGT TCAG-3ʹ as the reverse primer for LIMK; and 5ʹ-CCA GCC GAG CCA CAT CGC TC-3ʹ as the forward primer and 5ʹ-ATG AGC CCC AGC CTT CTC CAT-3ʹ as the reverse primer for GAPDH.
TextSentencer_T186 27861-27961 Sentence denotes The amplification was performed in 30 cycles of 50-60°C for 30 s, 72°C for 1 min, and 94°C for 30 s.
TextSentencer_T186 27861-27961 Sentence denotes The amplification was performed in 30 cycles of 50-60°C for 30 s, 72°C for 1 min, and 94°C for 30 s.
TextSentencer_T187 27962-28044 Sentence denotes The cells were incubated for 12 h in the presence or absence of 2.5 μg/ml of DMGF.
TextSentencer_T187 27962-28044 Sentence denotes The cells were incubated for 12 h in the presence or absence of 2.5 μg/ml of DMGF.
TextSentencer_T188 28045-28116 Sentence denotes Then, the cells were collected and lysed in ice-cold RIPA lysis buffer.
TextSentencer_T188 28045-28116 Sentence denotes Then, the cells were collected and lysed in ice-cold RIPA lysis buffer.
TextSentencer_T189 28117-28234 Sentence denotes The protein concentration of the cell lysate was estimated with the Bradford protein assay using BSA as the standard.
TextSentencer_T189 28117-28234 Sentence denotes The protein concentration of the cell lysate was estimated with the Bradford protein assay using BSA as the standard.
TextSentencer_T190 28235-28371 Sentence denotes Total proteins (50−60 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane.
TextSentencer_T190 28235-28371 Sentence denotes Total proteins (50−60 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane.
TextSentencer_T191 28372-28502 Sentence denotes The membrane was blocked with 5% skim milk in phosphate buffered saline with Tween 20 (0.05% v/v Tween-20 in PBS, pH 7.2) for 1 h.
TextSentencer_T191 28372-28502 Sentence denotes The membrane was blocked with 5% skim milk in phosphate buffered saline with Tween 20 (0.05% v/v Tween-20 in PBS, pH 7.2) for 1 h.
TextSentencer_T192 28503-28788 Sentence denotes The membranes were then incubated with anti-CREB antibody (1:1,000; GeneTex, Irvine, CA, USA) and anti-pCREB antibody (1:1000; Abcam, Cambridge, CA, USA) at 4°C overnight, followed by incubation with a horseradish peroxide-linked secondary antibody (1:10000; GeneTex, Irvine, CA, USA).
TextSentencer_T192 28503-28788 Sentence denotes The membranes were then incubated with anti-CREB antibody (1:1,000; GeneTex, Irvine, CA, USA) and anti-pCREB antibody (1:1000; Abcam, Cambridge, CA, USA) at 4°C overnight, followed by incubation with a horseradish peroxide-linked secondary antibody (1:10000; GeneTex, Irvine, CA, USA).
TextSentencer_T193 28789-28959 Sentence denotes The protein bands were visualized using the ChemiLucent ECL Detection System (Millipore, Billerica, MA, USA) and the Biospectrum AC Imaging System (UVP, Upland, CA, USA).
TextSentencer_T193 28789-28959 Sentence denotes The protein bands were visualized using the ChemiLucent ECL Detection System (Millipore, Billerica, MA, USA) and the Biospectrum AC Imaging System (UVP, Upland, CA, USA).
TextSentencer_T194 28960-29116 Sentence denotes The intensities of the chemiluminescence signal were quantified using the UVP VisionWorks LS Image Acquisition and Analysis Software (UVP, Upland, CA, USA).
TextSentencer_T194 28960-29116 Sentence denotes The intensities of the chemiluminescence signal were quantified using the UVP VisionWorks LS Image Acquisition and Analysis Software (UVP, Upland, CA, USA).
TextSentencer_T195 29117-29506 Sentence denotes To observe the structures of lamellipodia, B16F10 cells plated on cover slides were incubated at 37°C overnight and then were treated by DMGF for 12 h. www.impactjournals.com/oncotarget B16F10 cells were fixed with 4% formaldehyde for 10 min and were stained by phallotoxin (Invitrogen, Carlsbad, CA, USA) or ConA conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 60 min.
TextSentencer_T195 29117-29506 Sentence denotes To observe the structures of lamellipodia, B16F10 cells plated on cover slides were incubated at 37°C overnight and then were treated by DMGF for 12 h. www.impactjournals.com/oncotarget B16F10 cells were fixed with 4% formaldehyde for 10 min and were stained by phallotoxin (Invitrogen, Carlsbad, CA, USA) or ConA conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 60 min.
TextSentencer_T196 29507-29671 Sentence denotes The stained B16F10 cells were observed with 5 ml; of Hoest33342 mounting medium (Mounting Medium with Hoest33342, Abcam, Cambridge, CA, USA) by confocal microscopy.
TextSentencer_T196 29507-29671 Sentence denotes The stained B16F10 cells were observed with 5 ml; of Hoest33342 mounting medium (Mounting Medium with Hoest33342, Abcam, Cambridge, CA, USA) by confocal microscopy.
TextSentencer_T197 29672-29772 Sentence denotes Female C57BL/6JNarl mice (8 weeks of age) were purchased from the National Laboratory Animal Center.
TextSentencer_T197 29672-29772 Sentence denotes Female C57BL/6JNarl mice (8 weeks of age) were purchased from the National Laboratory Animal Center.
TextSentencer_T198 29773-29934 Sentence denotes All experiments with animals were performed in accordance with and approved by the Institutional Animal Care and Use Committee at National Chiao Tung University.
TextSentencer_T198 29773-29934 Sentence denotes All experiments with animals were performed in accordance with and approved by the Institutional Animal Care and Use Committee at National Chiao Tung University.
TextSentencer_T199 29935-30001 Sentence denotes Mice were inoculated subcutaneously with 1 × 10 6 of B16F10 cells.
TextSentencer_T199 29935-30001 Sentence denotes Mice were inoculated subcutaneously with 1 × 10 6 of B16F10 cells.
TextSentencer_T200 30002-30146 Sentence denotes Once a tumor mass of 30 mm 3 was established, mice were intravenously injected with PBS, 10 mg/ kg DMGF and 50 mg/kg DMGF once every three days.
TextSentencer_T200 30002-30146 Sentence denotes Once a tumor mass of 30 mm 3 was established, mice were intravenously injected with PBS, 10 mg/ kg DMGF and 50 mg/kg DMGF once every three days.
TextSentencer_T201 30147-30330 Sentence denotes Mice were sacrificed on the final day of the experiments; tumors were removed and fixed by 4% paraformaldehyde, then examined by H&E staining to calculate the number of blood vessels.
TextSentencer_T201 30147-30330 Sentence denotes Mice were sacrificed on the final day of the experiments; tumors were removed and fixed by 4% paraformaldehyde, then examined by H&E staining to calculate the number of blood vessels.
TextSentencer_T202 30331-30429 Sentence denotes The vessel counts were counted with randomly ten fields (200× magnification) in each tumor tissue.
TextSentencer_T202 30331-30429 Sentence denotes The vessel counts were counted with randomly ten fields (200× magnification) in each tumor tissue.
TextSentencer_T203 30430-30524 Sentence denotes The vessel numbers were considered as endothelial cells and countable microvessels morphology.
TextSentencer_T203 30430-30524 Sentence denotes The vessel numbers were considered as endothelial cells and countable microvessels morphology.
TextSentencer_T204 30525-30617 Sentence denotes The experimental metastasis model was established by the methods described previously [18] .
TextSentencer_T204 30525-30617 Sentence denotes The experimental metastasis model was established by the methods described previously [18] .
TextSentencer_T205 30618-30794 Sentence denotes B16F10 cells (1 × 10 6 cells in 0.2 ml per mouse) were injected into C57BL/6JNarl mice via the lateral tail vein, and the mice were then randomly divided into different groups.
TextSentencer_T205 30618-30794 Sentence denotes B16F10 cells (1 × 10 6 cells in 0.2 ml per mouse) were injected into C57BL/6JNarl mice via the lateral tail vein, and the mice were then randomly divided into different groups.
TextSentencer_T206 30795-30951 Sentence denotes Then, the mice were intravenously injected with DMGF at doses of 10 or 50 mg/kg, and the mice in the untreated group were injected with saline every 3 days.
TextSentencer_T206 30795-30951 Sentence denotes Then, the mice were intravenously injected with DMGF at doses of 10 or 50 mg/kg, and the mice in the untreated group were injected with saline every 3 days.
TextSentencer_T207 30952-31022 Sentence denotes All of the mice were sacrificed on the 19th day after tumor injection.
TextSentencer_T207 30952-31022 Sentence denotes All of the mice were sacrificed on the 19th day after tumor injection.
TextSentencer_T208 31023-31184 Sentence denotes Their lungs were then removed and fixed, the metastatic foci at the lung surfaces were photographed, and the number of tumor nodules on the lungs was calculated.
TextSentencer_T208 31023-31184 Sentence denotes Their lungs were then removed and fixed, the metastatic foci at the lung surfaces were photographed, and the number of tumor nodules on the lungs was calculated.
TextSentencer_T209 31185-31308 Sentence denotes All spleen tissues were collected and embedded in paraffin, and sections stained by H&E staining without melanin bleaching.
TextSentencer_T209 31185-31308 Sentence denotes All spleen tissues were collected and embedded in paraffin, and sections stained by H&E staining without melanin bleaching.
TextSentencer_T210 31309-31430 Sentence denotes The numbers of mtastatic melanoma cells were counted with randomly ten fields (400× magnification) in all spleen tissues.
TextSentencer_T210 31309-31430 Sentence denotes The numbers of mtastatic melanoma cells were counted with randomly ten fields (400× magnification) in all spleen tissues.
TextSentencer_T211 31431-31566 Sentence denotes The percentages of melanoma cells were calculated as (melanin positive cells/ total splenocytes and melanocytes)/ × 100% in each group.
TextSentencer_T211 31431-31566 Sentence denotes The percentages of melanoma cells were calculated as (melanin positive cells/ total splenocytes and melanocytes)/ × 100% in each group.
TextSentencer_T212 31567-31747 Sentence denotes C57BL/6JNarl mice were inoculated in situ with 5 × 10 5 B16F10-EGFP cells approximately 1 cm from the inguinal lymph node to directly establish experimental lymph nodes metastasis.
TextSentencer_T212 31567-31747 Sentence denotes C57BL/6JNarl mice were inoculated in situ with 5 × 10 5 B16F10-EGFP cells approximately 1 cm from the inguinal lymph node to directly establish experimental lymph nodes metastasis.
TextSentencer_T213 31748-31872 Sentence denotes Mice bearing metastatic cells were intravenously injected with saline, 10 mg/kg DMGF or 50 mg/kg DMGF once every three days.
TextSentencer_T213 31748-31872 Sentence denotes Mice bearing metastatic cells were intravenously injected with saline, 10 mg/kg DMGF or 50 mg/kg DMGF once every three days.
TextSentencer_T214 31873-31964 Sentence denotes At day 9, the mice were sacrificed; lymph nodes were removed and embedded in O.C.T reagent.
TextSentencer_T214 31873-31964 Sentence denotes At day 9, the mice were sacrificed; lymph nodes were removed and embedded in O.C.T reagent.
TextSentencer_T215 31965-32043 Sentence denotes The frozen sections of lymph nodules were examined by fluorescence microscopy.
TextSentencer_T215 31965-32043 Sentence denotes The frozen sections of lymph nodules were examined by fluorescence microscopy.
TextSentencer_T216 32044-32224 Sentence denotes Ten micrometer frozen sections of lymph nodes were stained with propidium iodide (PI) as a counter stain and analyzed with randomly ten fields (400× magnification) for each sample.
TextSentencer_T216 32044-32224 Sentence denotes Ten micrometer frozen sections of lymph nodes were stained with propidium iodide (PI) as a counter stain and analyzed with randomly ten fields (400× magnification) for each sample.
TextSentencer_T217 32225-32379 Sentence denotes The percentages of metastatic melanoma cells were calculated as [green fluorescence cells (GFP)/total red fluorescence cells (PI)] / × 100% in each group.
TextSentencer_T217 32225-32379 Sentence denotes The percentages of metastatic melanoma cells were calculated as [green fluorescence cells (GFP)/total red fluorescence cells (PI)] / × 100% in each group.
TextSentencer_T218 32380-32558 Sentence denotes The docking of DMGF into CREB was explored using iGEMDOCK software [30] [31] [32] [33] , which have been shown to be powerful tools for molecular recognition and screening [34] .
TextSentencer_T218 32380-32558 Sentence denotes The docking of DMGF into CREB was explored using iGEMDOCK software [30] [31] [32] [33] , which have been shown to be powerful tools for molecular recognition and screening [34] .
TextSentencer_T219 32559-32575 Sentence denotes CREB1 (PDB code:
TextSentencer_T219 32559-32575 Sentence denotes CREB1 (PDB code:
TextSentencer_T220 32576-32652 Sentence denotes 2LXT) was selected from Protein Data Bank (PDB) and DMGF was docked with it.
TextSentencer_T220 32576-32652 Sentence denotes 2LXT) was selected from Protein Data Bank (PDB) and DMGF was docked with it.
TextSentencer_T221 32653-32787 Sentence denotes The parameters of iGEMDOCK were setting such as population was set to 300, generation was set to 70 and solution was set to 100 [32] .
TextSentencer_T221 32653-32787 Sentence denotes The parameters of iGEMDOCK were setting such as population was set to 300, generation was set to 70 and solution was set to 100 [32] .
TextSentencer_T222 32788-32889 Sentence denotes The results were analyzed using the SAS statistical software package (SAS Institute Inc., Cary, USA).
TextSentencer_T222 32788-32889 Sentence denotes The results were analyzed using the SAS statistical software package (SAS Institute Inc., Cary, USA).
TextSentencer_T223 32890-32935 Sentence denotes The results were expressed as the means ± SD.
TextSentencer_T223 32890-32935 Sentence denotes The results were expressed as the means ± SD.
TextSentencer_T224 32936-33052 Sentence denotes Student's t-test was used when comparing two independent samples and ANOVA was used when comparing multiple samples.
TextSentencer_T224 32936-33052 Sentence denotes Student's t-test was used when comparing two independent samples and ANOVA was used when comparing multiple samples.
TextSentencer_T225 33053-33121 Sentence denotes Differences with p < 0.05 were considered statistically significant.
TextSentencer_T225 33053-33121 Sentence denotes Differences with p < 0.05 were considered statistically significant.