The Mechanism For The Synergistic Induction Of Apoptosis Of Tumor Cells By BH3-mimetic Gossypol In Combination With Valproic Acid

AimCancer is a major disease in humans. Although cancer research has made a great effort in the past years, and many important progressions have been made, the cure of cancer remains a difficult problem in the clinic; particularly, the chemoresistance of cancer is one crucial obstacle to the treatment of human cancer. Combination of different anticancer agents with distinct properties holds promise to overcome such resistance. Gossypol(GOS), a natural BH3 mimetic, possesses both anti-proliferative and pro-apoptotic activities but has limited effectiveness in inducing cell death in many cancer cells. The histone deacetylase(HDAC) inhibitors are a new type of anti-cancer agent. HDAC inhibitors suppress cell proliferation and induce apoptosis in tumor cells through different mechanisms. The combination of HDAC inhibitors with conventional chemotherapeutic agents is believed to enhance anti-tumor capacity of the latters. Previously we reported that valproic acid(VPA), a short-chain fatty acid HDAC inhibitor, acts in synergy with GOS to enhance cell death in human DU145 cells. However, the underlying mechanism remains elusive. In addition, it is unknown that whether this synergistic effect has universality, whether the other HDAC inhibitors have the same synergistic effect, and whether there is synergistic effect in vivo. In this study, by using human melanoma A375, cervical cancer HeLa and prostate cancer PC-3 cells as a model, we aimed to explore the mechanism by which of GOS and VPA co-treatment synergistically inhibits cell proliferation in various human cancers, and validate such synergistic effect in vivo in a xenografted mouse tumor model. MethodsFirstly, the effect of GOS and/or VPA treatment on the viability of A375, HeLa and PC-3 cells was evaluated by WST-1 assay. Secondly, western blotting was used to analyze the effect of various treatment on the related protein expression, including apoptosis, autophagy, and cell signaling pathways. Thridly, following the drug treatment, immunofluorescence microscopy was used to eva luate the fusion of autophagosomes with lysosomes by observing the colocalization of LC3 and LAMP2, indicating the on- going autophagy. Fourthly, specific inhibitor and small interfering RNAs were used to verify the effect of related proteins and cell signa ling pathways on cell death induced by GOS plus VPA treatment. The effect of GOS and/or VPA on cell cycle distribution was detected by flow cytometry assay. In addition, quantitative RT-PCR(qPCR) was used to detect mRNA levels of Bim, FasL and so on after drug treatment. Lastly, human melanoma A375 cells were subcutaneous injected into nude mice, and the in vivo antitumor effect of GOS and VPA co-treatment was evaluated. ResultsCell proliferation assay indicated that the noncytotoxic dose of VPA synergistically enhanced the cytotoxic effect of GOS on A375, HeLa and PC-3 cells. Immunoblotting and microscopic observation revealed that GOS and VPA co-treatment robustly induced caspase-3 activation, PARP cleavage and nuclear fragmentation in all three cell lines, suggesting GOS and VPA act in a synergistic manner to induce apoptosis. While other HDAC inhibitors, including SAHA, tubacin and nicotinamide(N ico), could not significantly enhance the cytotoxicity of GOS. However, autophagy seemed not involved in GOS+VPA-induced cytotoxicity as the blockade of autophagy had no influence on cell death. Such synergistic effects of GOS+VPA seemed unrelated to the acetylation levels of histones but strongly correlated with decreased levels of cyclin A2 and downregulation of Akt signaling. Immunoblotting indicated that owing to the reduction of cyclin A2, Akt signaling was suppressed, leading to dephosphorylation of FOXO3 a. Consequently, FOXO3 a was activated and the expression of its target genes, including pro-apoptotic FasL, Trail and Bim, was upregulated. GOS+VPA co-treatment obviously downregulated the expression of anti-apoptotic proteins Bcl-2 and survivin, and induced caspase-8/-9 activation, suggesting GOS and VPA synergistically induce apoptosis by activating both intrinsic and extrinsic apoptotic pathways. In addition, FOXO3 a knockdown attenuated FasL and Bim upregulation and apoptosis induction in GOS+VPA-treated cells. Furthermore, blocking proteasome activity by MG132 prevented the downregulation of cyclin A2, dephosphorylation of Akt and FOXO3 a, and induction of apoptosis in cells co-treated with GOS and VPA. In mouse model, administration with GOS or VPA alone slightly decreased the growth of xenografted tumors, whereas GOS and VPA combination significantly inhibited the growth of A375 melanoma xenografts, suggesting that GOS and VPA co-treatment also has a synergistic antitumor effect in vivo. ConclusionsOur data indicate that GOS and VPA co-treatment synergistically induces apoptosis in human A375, He La and PC-3 cells without overt cell-type preference, whereas other HDAC inhibitors in combination with GOS had no obvious synergistic antitumor effect. GOS+VPA co-treatment obviously suppressed the cyclin- A2/Akt/FOXO3 a signaling pathway and synergistically induced apoptosis by activating both intrinsic and extrinsic apoptotic pathways. GOS+VPA co-treatment dramatically induced cyclin A2 degradation, which was at least partly mediated by the ubiquitin-proteasome pathway. Moreover, GOS and VPA combination had synergistic antitumor effect in vivo. These data suggest that combination of GOS and VPA represents one promising therapeutic regimen for certain human cancers with chemo-resistance.