Inhibition Effects And Mechanisms Of Tautomycetin On Breast Cancer Cells

Tautomycetin (TMC) is polyketide natural product originally isolated as antifungal antibiotics from Streptomyces griseochromogens. TMC was later found to be able to specifically inhibit protein phosphatase type1(PP1). TMC has been also identified as a potent immunosuppressor and a potential drug for colorectal cancer. The anticancer activities of TMC are both thought to be related to its ability to inhibit protein phosphatases. Unfortunately, this hypothesis has not yet been fully clarified. Our studies showed that TMC could inhibit proliferation of different kinds of cancer cells in vitro.In this study, we presented the correlation between the anti-cancer activity of TMC and the inhibitory activity of PP1, with the focus on the signaling pathway of the anticancer activity of TMC. The main results and conclusions were listed as follows:(1) Phosphatase inhibition, immunosuppressive and anticancer activities of TMC and its analogues were determinded. The results showed that some analogues of TMC exhibited strong PP1-inhibiting activity rather than anti-tumor activity and immunosuppressive activity; some analogues exhibited cytotoxic activity rather than PP1-inhibiting activity. TMC (1μmol/L) and TMC-F1(50μmol/L) could both inhibited PPl activity in MCF-7cell lines by60%, but with90%and24%inbibiting percentages of MCF-7cell proliferation, respectively. Therefore, the anti-tumor activity and immunosuppressive activity were not correlated with the inhibitory activity of protein phosphatase PP1.(2) The effects of TMC on the MCF-7cell proliferation, invasion and apoptosis were examined by MTT, cell invasion assays and flow cytometry analysis, respectively. The estimated IC50value was0.36umol/L in MCF-7cell lines; the percentages of cell migration and invasion of MCF-7cell lines inhibiting by0.4μmol/L TMC were76%and79%, respectively; the percentages of MCF-7cell early and advanced apoptosis induced by0.5μmol/L TMC were21%and34%, respectively. Furthermore, we examined the effects of TMC on the apoptosis relative protein expression by western blotting. The results indicated that the expression level of Bcl-2and phosphorylation of Bad were both decreased, expression of Bax and mitochondria release of cytochrome-c were enhanced, caspase-9and caspase-7were activated by TMC treatment. Therefore, it was argued that TMC could activate caspase cascade pathway through the regulations of Bcl-2family and mitochondrial/cytochrome-c mediated apoptosis pathway, which could induce the apoptosis of MCF-7cells.(3) Detecting the influence of TMC on Adriamycin resistant human breast adenocarcinoma MCF-7/ADR cells by MTT and Western blotting. The results showed that TMC inhibited the proliferation of MCF-7/ADR cells with the IC50values being1.26μmol/L. The early and late apoptosis rate of MCF-7/ADR cells induced by1μmol/L TMC increased to17.2%and28.4%, respectively. Similarly. TMC could significantly decrease the phosphorylation of Akt in MCF-7/ADR cells and the expression of Bcl-2. while activate caspase-9and caspase-7. It indicated that TMC is sensitive to MCF-7/ADR cells and could activate caspase cascade pathway through the regulations of Bcl-2family and mitochondrial/cytochrome-c mediated apoptosis pathway, which could induce the apoptosis of MCF-7/ADR cells.(4) The effects of TMC on the relative protein phosphorylations and transcription factor activities of MAPK (ERK. p-38and JNK), Wnt. p53and Akt signal pathway were examined by Western blotting and dual luciferase assay system. The results showed that the phosphorylation of Akt and its downstream target FKHR were decreased significantly, and the transcription activity of CREB were also decreased by70%. Meanwhile, the phosphorylation of kinase PDK1was inhibited by TMC, but those of tyrosine kinase EGFR and HER2were not. Next, we examined the role of Akt in TMC-mediated inhibition. We found that TMC inhibited proliferation of MCF-7cells transfected with blank vector and myr-Akt plasmid by64%and31%, respectively. We demonstrated that TMC inhibited Akt and its downstream signaling, subsequently resulting in apoptosis of MCF-7cells.(5) The gene PP1was silenced by siRNA technology, then the influence on phosphorylation of Akt was detected and no significant change was observed. It was further indicated that the inhibitory of TMC on MCF-7was not clearly correlated with the inhibition of PP1.(6) To investigate whether there is synergistic effect between TMC and Leptomycin B (LMB), MTT, Western blotting and Cell transfection assays were conducted. The results showed that the synergistic effect was better by adding LMB first and TMC was added24h later, with a CI value0.38-0.49. In combination treatment, the required dosage of TMC and LMB for half-inhibition is reduced to23.8%and17.7%compared to single treatment. LMB could enhance the down-regulating effect of TMC on phosphorylation of Akt and FKHR. LMB could also enhance the gathering of nuclear induced by TMC. It was implied that combination treatment of TMC and LMB has notable synergistic effect on the inhibitory of MCF-7cell growth. The effect may be related to the function of LMB to promote the nuclear gathering which is induced by TMC.In summary, our results indicated that cytotoxicity of TMC was not associated with its PP1inhibition activity. Moreover, we provided a plausible mechanism of TMC inducing the MCF-7cell apoptosis and proved that there was a synergistic antitumor activity of Akt inhibitors combined with nuclear export inhibitors. The results of this study proposed a new paradigm for investigating the mechanism of the anti-tumor drugs which inhibit protein phosphatase, laid the foundation for further identifying the direct target of its anti-tumor activity. Lastly, our study provided a new design strategy for rational drug combination.