The Antitumor Activity And Molecular Mechanism Of A Novel Multi-targets Inhibitor CUDC-101 And Docetaxel Combination On Prostate Cancer

Background:Prostate cancer(PCa)is one of the most common diagnosed malignancies in men,which is the third leading cause of cancer-related death among the world.The establishment of docetaxel(DTX)-based chemotherapeutic treatments has improved the survival of PCa patients.However,the incidence of complication,tumor malignant and poor selectivity of drugs led to chemotherapy failure and tumor recurrence.So finding a new efficient therapeutic agent remains a major task.As a novel multi-function small molecular,CUDC-101 simultaneously inhibits histone deacetylase(HDAC),EGFR,and HER2.Currently,the pharmacological characteristics,therapeutic effects and mechanisms of CUDC-101 have not been fully elucidated,and the synergistically efficacy of CUDC-101 combined with chemotherapeutic drug DTX did not reveal in PCa.Objectives:To investigate the anti-tumor effects and the molecular mechanism of combination of CUDC-101 and DTX,in vitro and in vivo.Materials and methods:1)Clinical specimens and database analysis:IHC staining was performed to evaluate the expression level of HD AC 4 and 6 in PCa and benign prostate tissues.And the expression of HDACs in protein and mRNA levels on PCa tissues were collected from HPA and Oncomine databases.2)Experiments in vitro:MTT,colony formation and CFSE cell proliferation assay were used to confirm the synergistic inhibitory effect of CUDC-101 and DTX treatment on human PCa cells.FACS analysis was used to detect the apoptosis and cell-cycle progression induced by CUDC-101 and DTX in both PCa cells.Moreover,western blot assay were used to detect the apoptosis-and cell cycle-associated proteins expression in PCa cells by the agents treatment.To determine the role of the co-treatment of CUDC-101 and DTX in regulation of EMT in PCa cells,wound healing assay,cell migration and invasion assay,and tube formation assay were conducted after the treatment for 48 h.Western blot assay was also used to detect the expression of PI3K,AKT,ERK and the activation status of proteins in PCa cells after CUDC-101 and/or DTX treatment.3)Experiments in vivo:PC-3 cells were injected subcutaneously into the nude mice.After the developing tumors were measured,mice were randomly divided into four groups(five mice per group)as follow:control,CUDC-101,DTX,or CUDC-101 + DTX.Control group was received the vehicle alone(saline),while other groups were treated with CUDC-101(60 mg/kg/d)and/or DTX(5 mg/kg/d),and given intraperitonally(i.p.)every 3 days for a total of five times.At the end of the experiments,mice were weighted,killed and tumors were subjected.To detect the synergistic effect of CUDC-101 and DTX in vivo,HDAC4,anti-proliferation cell nuclear antigen(ki-67),apoptosis-related marker(survivin)and EMT-associated marker(E-cadherin and vimentin)were analyzed by IHC.Results:1)IHC staining showed that HD AC 4 and 6 expressions were strong positive in PCa,while negative or weak in benign and PIN tissues.Moreover,HPA and Oncomine Data showed that HDACs protein and mRNA expression levels were both higher in PCa tissues rather than in normal tissues.2)MTT analysis showed that CUDC-101 and DTX suppressed PCa cell growth both in PC-3 and DU 145 cell lines.And combined treatment synergistically suppressed cell proliferation.3)Colony formation assay and CFSE cell proliferation assay indicated that the combination of CUDC-101 and DTX effectively increased the proliferation of PC-3 and DU145 cells.4)Cell cycle percentage was detected by flow cytometry(FACS)analysis,and the results showed that CUDC-101 as well as DTX induced accumulation of cells in G2/M phase.Additionally,FACS analysis also showed that CUDC-101 caused cell death through inducing apoptosis,and co-treated with DTX showed more prominent pro-apoptosis effect compared to the single agent.Furthermore,western blot assay determined that the expression levels of cleaved-PARP,cleaved caspase-3,-8,-9,Bax and p21 proteins were increased in combination treatment,while Bcl-2 and Cyclin B1 expression were decreased.The ratio of Bax/Bcl-2 was synergistically increased following the co-treatment of CUDC-101 and DTX.5)Co-treatment of agents showed lower number of cells migrating across the wound than the single agent.Additionally,combined treatment prominently prevented the migration,invasion and tube formation in PCa cells.Furthermore,western blot analysis indicated that the expression levels of epithelia cell-related proteins(E-cadherin and ZO-1)were enhanced;in contrast the levels of mesenchymal cell-related proteins,including vimentin,Snail,Slug and MMP-9 were decreased.The EMT-associated proteins expressions were also detected using IF staining.6)Western blot analysis indicated that PI3K/AKT/mTOR and ERK signaling pathways induced by CUDC-101 and DTX involved in anti-tumor activities.7)In vivo,combination therapy observably inhibited tumor growth in PC-3 subcutaneous xenograft mouse models.And compared with either control or agents alone,the combined treatment increased the expression level of E-cadherin and decreased the expression levels of HDAC4,vimentin,ki-67 and survivin.Conclusions:1)Co-treatment of CUDC-101 and DTX synergistically decreased the proliferation of PC-3 and DU145 cells compared to the single agent.2)Combined treatment of agents could significantly induce cell apoptosis,and prevented EMT in PCa cell lines,in vitro and in vivo.3)PI3KIAKT/mTOR and ERK signaling pathways involved in the anti-tumor activity of PCa induced by the combined treatment of CUDC-101 and DTX.