PMC:3832756 / 18598-27374 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3832756","sourcedb":"PMC","sourceid":"3832756","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3832756","text":"Discussion\nIn this work, we found that sulforaphane efficiently decreases survival of phenotypically distinct breast cancer cell lines which differ in the expression pattern of growth factor or estrogen receptors and PTEN suppressor (Table 1). Although anti-proliferative activity of SFN against different breast cancer cell lines has already been reported, this is the first study exploring the effect of SFN on AktmTOR-S6K1 pro-survival pathway.\nPreviously, it has been reported that SFN at 15 μM concentration caused accumulation of MCF-7 and MDA MB 231 cells in G2/M phase of the cell cycle after 24 h treatment and longer incubation time resulted in apoptosis initiated by mitochondrial or death receptor pathway, respectively. Moreover, global HDAC activity, as determined with an in vitro activity assay, was inhibited by SFN in MCF-7, MDA MB 231 and, to a lesser extend, in MDA MB 468 and T47D cell lines [16]. Authors also reported that IC50 of SFN did not differ significantly between these cell lines when treated for 48 h [16]. Increased tubulin acetylation and suppression of microtubules dynamic instability was observed in MCF-7 exposed to 15, 25 or 50 μM SFN [17]. G2/M cell cycle arrest of MDA MB 231 cells was associated with increased p21 and p27 cdk-cyclin inhibitors and decreased levels of cyclin A, B1 and cdc2, and apoptosis was accompanied by decreased Bcl-2 and increased caspase-3 level [31]. The same authors showed that SFN induced autophagy in MDA MB 231 cells and this process played a protective role as its inhibition by bafilomycin A1 significantly enhanced SFN-induced apoptosis [31]. It was also reported that sensitivity of breast cancer cells to SFN is connected with upregulation of p38 MAP kinase and caspase -7 activation in MCF-7 cells [32], global changes in gene expression [33] or downregulation of ER, EGFR or HER2 mRNAs [16, 34]. Other studies indicate that SFN-induced apoptosis of MCF-7 and MDA MB 231 cells is initiated by reactive oxygen species due to p66Shc translocation to mitochondria and collapse of mitochondrial membrane potential [35]. Interestingly, non-tumorigenic human mammary epithelial MCF-10A cells were resistant to SFN-induced oxidative stress and cell death [35]. Thus, it seems that cytotoxic effect of SFN is specific for cancer cells. Other reports confirm it. For instance, human mammary epithelial cell line, MCF-12A, was significantly more resistant to 48-h treatment with SFN comparing to cancer cells, MCF-7 wt and MCF-7/Adr (IC50 = 40.5 μM for MCF-12A as compared to 27.9 μM for MCF-7 wt and 13.7 μM for MCF-7/Adr) [36]. Moreover, 30 μM SFN inhibited MCF-7 and ZR75-1 cancer cells proliferation by 80 % after 48-h exposure, while proliferation of non-tumorigenic mammary cells, MCF-10F, was inhibited by about 50 % as compared to the respective controls [34]. Dose- and time-dependent growth inhibition with SFN was observed in MCF-7 and MDA 231 cells, while MCF-10A epithelial cells were more resistant, even to higher (above 10 μM) SFN concentrations. Interestingly, authors found that SFN-induced apoptosis in breast cancer cells was mediated by epigenetic regulation of telomerase gene expression [37]. None of the above mentioned research explored connection of SFN activity with AktmTOR-S6K1 pathway in breast cancer cells.\nSignaling at the AktmTOR-S6K1 level is aberrantly activated in most human cancers. It is not surprising taking into account that all these kinases stimulate pro-survival processes. mTOR (the mammalian target of rapamycin) is a crucial regulator of translation and together with associated proteins, raptor, GβL, PRAS40 and DEPTOR, phosphorylates the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), an inhibitor of cap-dependent translation, and the S6 kinase 1 which positively regulates different stages of protein synthesis [38]. In addition to the role in translation, mTOR regulates the mRNA stability of some crucial cell cycle regulators, such as D cyclins or p27 cyclin-dependent kinase inhibitor as well as increases production of HIF1α, a transcription factor promoting expression of glycolytic genes [39].\nS6K1 plays a pleiotropic role in translation. It participates in ribosomal biogenesis, initiation of mRNA translation through phosphorylation of eukaryotic translation initiation factor 4B (eIF4B) and regulates translation elongation through phosphorylation of eukaryotic elongation factor 2 kinase (eEF2K) [40–42]. Besides, S6K1 plays crucial role in cell growth, progression of cell cycle and cell survival inactivating pro-apoptotic proteins and stimulating synthesis of anti-apoptotic survivin [43]. S6K1 gene is amplified in 9 % of primary breast cancers which is associated with aggressive disease and poor prognosis of patients [44]. Moreover, S6K1 phosphorylates ERα leading to its transcriptional activation, which may contribute to breast cancer progression [45].\nBoth mTOR and S6K1 activities may be stimulated by Akt kinase. It inactivates the tuberous sclerosis TSC1/2 inhibitors of mTOR. Importantly, Akt activates other pro-survival pathways. For instance, it inhibits pro-apoptotic proteins such as Bad or transcription factors of FOXO family, promotes p53 degradation and indirectly activates pro-survival NF-κβ transcription factor [46]. AktmTOR-S6K1 are located downstream of PI3K, which is activated by membrane receptors, including the family of EGF receptors and the estrogen receptor. Hyperactivation of Akt due to activating mutations, gene amplification or enhanced signaling from receptors seems to be genetically selected during tumorigenesis and was found in many human cancers including carcinomas, glioblastoma multiforme and various hematological malignancies (reviewed in [47]). Akt not only stimulates cancer cell growth and viability but also leads to resistance to chemotherapeutics, particularly these targeting only one element of the pathway. For instance, drugs targeting mTOR-S6K1 at the same time may stimulate Akt due to inhibition of negative feedback loop where active S6K1 blocks growth factor-PI3K-Akt signaling [48]. Moreover, cancer cells treated with inhibitors of growth factor or estrogen receptors often become resistant to therapy which is caused by changes in downstream signaling components, such as activating mutations of PI3K or Akt genes and loss of suppressors, such as PTEN. Thus, agents targeting this pathway at multiple levels might be a promising alternative. Indeed, preclinical studies have shown that the mTOR antagonists can restore endocrine sensitivity in breast cancer cells [6].\nHere, we show that SFN, isothiocyanate present in edible plants, targets the pro-survival pathway in breast cancer cells in at least two levels: Akt activation, determined as a drop in phosphorylation in the position crucial for the kinase activity, and mTOR-S6K1 signaling, determined as a decrease in S6K1 phosphorylation in the position recognized by mTOR (Fig. 4). Inhibition of protein synthesis (Fig. 5 and [30]) and induction of autophagy (Figs. 2, 3), both controlled by mTOR, confirm that SFN inhibits this pathway. We did not explore here the role of autophagy in the response of breast cancer cells to SFN. Even if it plays protective role such as in MDA MB 231 [31] or prostate PC-3 cells [27], it delays but not suppresses cell death.\nThe argument for parallel, rather than linear inhibition of Akt and mTOR by SFN, is different dephosphorylation pattern of Akt and S6K1 kinases upon SFN treatment shown on immunoblotting (Fig. 4). Moreover, Akt activation is initiated by translocation to the plasma membrane mediated by docking of the PH domain in the N-terminal region of AKT to PI(3,4,5) P3 on the membrane, which results in a conformational change in Akt, exposing two critical amino acid residues (serine 473 and threonine 308) for phosphorylation [49]. On the other hand, generation of PI(3,4,5) P3 by PI3K is inhibited by mTOR-S6K1 signaling [48]. Thus, drop in p-S6K1 should rather stimulate Akt activation if SFN targets only one signal transducer. Interestingly, previous studies on sensitivity of the panel of breast cancer cell lines to mTOR inhibitor, CCI-779, revealed that cells sensitive to CCI-779 were estrogen receptor positive, overexpressed ErbB2 and/or had lost the tumor suppressor gene product PTEN. On the other hand, resistant cell lines (such as MDA MB 231) shared none of these properties [50]. In our model, SFN inhibits viability of all tested cell lines, including MDA MB 231, which also might indicate that it acts in at least two levels.\nTaken together, our data indicate that SFN might be a good therapeutics for breast cancers with different alterations of the PI3K-Ak-tmTOR-S6K1 pathway as it targets downstream elements of this 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