Study On The Molecular Mechanism Of Synergistic Inhibition By Co-treatment With PI3K Inhibitor And HDAC Inhibitor In Breast Cancer

Background:Breast cancer is one of the most common malignancies in women worldwide and the second breast cancer leading cause of cancer death among women. Although numerous advances in prevention, surgical resection, and adjuvant chemoradiotherapy have led to a decline in the overall mortality due to breast cancer, the survival rates for patients with metastatic disease have not significantly improved. Consequently, enhancing the understanding of the biology of breast cancer and the discovery of novel target therapy in individual patients is of great value.Recent years, the molecule targetedtherapy have achieved graveness progression and the curative effect. PI3K/mTOR dual inhibitor NVP-BEZ235 and histone deacetylase inhibitor TSA are effective drugs for some cancers, however, which were limited by the side effects and drug resistance after high-dose administration. Studies suggest that combination therapy may be more effective than single agents in some cancer cells. Co-treatment with BEZ235 and TSA was synergistially in non cell lung cancer and prostate cancer, while the role and mechanism in breast cancer is unclear. Our previous studies indicated that the acetylation of proteins and PI3K/Akt/mTOR pathway played important roles in development of breast cancer. Thus, deeply exploring the synergistic effect and potential mechanism of BEZ235 and TSA in breast cancer cells will of important meaning for clinical guideline.Objectives:To detect the effect on the biological behavior of breast cancer cells after co-treatment with PI3K/mTOR inhibitor BEZ235 and HDAC inhibitor TSA in vitro, explore the possible mechanism of synergistically joint inhibitory in breast cancers, and provide supporting evidence for clinical therapy.Materials and methods:MTT assay was used to detect the combination effect of TSA and BEZ235 on breast cancer cell lines T47D, SKBR3, MDA-MB-231, MCF7, MDA-MB-468, MDA-MB-453; the proliferation ability was tested by colony formation assay; the motility ability was measured by scratch experiment; the gene expression profiles of breast cancer cells treated by TSA and/or BEZ235 were compared using microarray analysis, and pathway enrichment analysis was also performed to clarify possible mechanism of combinational treatment. The flow cytometry and Hoechst33342 staining were used to detect the apoptosis of breast cancer cells after the treatment; Furthermore, western blot assay was used to detect the expression of PI3K/Akt signaling pathway, apoptosis and autophagy related proteins.Results:MTT assay showed synergistic inhibition of BEZ235 and TSA in proliferation of breast cancer MCF-7 and T47D cells; colony formation assays and scratch experiment showed that the cell motility and colony forming ability were inhibited by combined therapy. Microarray and Pathway Analyses showed different genes expression in combination group cells of MCF-7 with a wide range of biological functions, which focused on Pathway in Cancers, Cell Death and Survival, Cellular Development, and so on. Flow cytometry and Hoechst 33342 staining results showed that combined therapy enhanced apoptosis ability in MCF-7 and T47D cells; Western blot showed that combination of BEZ235 and TSA further reduced the protein levels related with PI3K/Akt/mTOR signaling pathway; up-regulate the level of PARP, down-regulate the ratio of Bcl-2/Bax, and enhanced the expression of caspase-3,8,9. Additionally, combined therapy resulted in a moderate increase in LC3B-Ⅱ and Beclinl expression, Furthermore, with the addition of 3-MA, we found that the expression of LC3B-Ⅱ in dual drug group was lower than in absence of 3-MA group, however, the expression of caspase-3 and PARP were increased significantly in MCF-7 and T47D cells.Conclusions:BEZ235 and TSA synergistically inhibited the proliferation, motility and colony capacity of breast cancer cells; the synergistic inhibition of combination with BEZ235 and TSA was related to further depression of PI3K/Akt/mTOR signaling pathway; co-treatment of BEZ235 and TSA enhanced the apoptosis of breast cancer cells via caspase-dependent way, and induced the autophagic cell death, which providing potential mechanisms of combined therapy jointly regulated the progression of breast cancer cells.