CUDC-907 Inhibits Cell Proliferation And Enhances TRAIL-induced Apoptosis In Human Breast Cancer Cells

ObjectiveCUDC-907 is a novel dual-acting small molecule compound designed to inhibit the activity of phosphoinositide 3-kinase(PI3K)and histone deacetylase(HDAC).In this study,we mainly investigated the effect of CUDC-907 on the cell proliferation and apoptosis in MCF-7 breast cancer cells and MDA-MB-231 triple-negative breast cancer cells.In addition,we also examined the potential role of CUDC-907 on the tumor necrosis factor-related apoptosis inducing ligand(TRAIL)-induced apoptosis in vitro and explored the intrinsic molecular mechanism in human breast cancer cells,which provides a new strategy for the clinical treatment of human breast cancer.Methods1.MCF-7 and MDA-MB-231 cells were routinely cultured in vitro.The logarithmic growth phase cells were cultured 96-well or 6-well plates.After treatment with different concentrations of CUDC-907 for 24 h,CCK-8 assay was used to detect the changes of cell viability.Flow cytometry was used to analyze the cell cycle distribution of breast cancer.Cell apoptosis was determined by ANNEXIN V-FITC/PI double staining.Western blotting was used to detect the expression of cell cycle and apoptosis-related proteins in CUDC-907-treated breast cancer cells.2.Breast cancer cells were seeded in 6-well plates.The experiment was designed as DMSO treatment group(control group),CUDC-907 treatment group,TRAIL treatment group and CUDC-907 combined TRAIL treatment group.The change of cell apoptosis was determined by ANNEXIN V/PI double staining method.The cleavage of Caspase-8,Caspase-9 and PARP were detected by immune blotting assay.The DR5 double-stranded small interfering RNA(si RNA)was synthesized and transfected into breast cancer cells.After 48 h transfection,the breast cancer cells were treated with CUDC-907 and TRAIL for another 24 h,ant the expression of Caspase-8,-9 and PARP were detected by immunoblotting assay.In addition,breast cancer cells were treated with CUDC-907 for 24 h,and the levels of some key factors in the endoplasmic reticulum stress pathway and the phosphorylation of MAPK family members were detect by western blotting assay.At last,breast cancer cells were pretreated with JNK specific inhibitor SP600125 and p38 MAPK inhibitor SB203580 for 1h,and then treated with CUDC-907 for another 24 h,the expression of DR5 was detect by western blotting assay.Results1.CUDC-907 inhibits the cell proliferation and induces cell apoptosis in human breast cancer cells.Our study indicated that CUDC-907 significantly inhibited the phosphorylation of Akt and p70S6 K were both in MCF-7 and MDA-MB-231 cells.CCK-8assay showed that CUDC-907 inhibited the viability of breast cancer cells in a dosedependent manner.Flow cytometry results showed that lower concentrations of CUDC-907 induced breast cancer cells to arrest in G1 phase,while higher concentrations of CUDC-907 induced cell arrest in G2/M phase.When breast cancer cells were treated with CUDC-907 for 24 h,the expression levels of p21 and p27 were significantly up-regulated in MCF-7 cells.However,there was no significant change in p27 protein expression in CUDC-907-treated MDA-MB-231 cells.After the treatment with the CUDC-907 for 24 h,the levels of the Cdc2,p-Cdc2,cyclinD1 and cyclinB1 were decreased in breast cancer cells.The expression of p53 was unchanged in CUDC-907-treated breast cancer cells.Annexin V-FITC and propidium iodide(PI)double staining results revealed that treatment with CUDC-907 significantly triggered cell apoptosis both in MCF-7 and MDAMB-231 cells.Western blotting results showed that treatment with CUDC-907 for 24 h clearly induced the cleavages of caspase-8,-9 and PARP in MCF-7 cells,compared to untreated controls.Likewise,similar events also occurred in MDA-MB-231 cells.In addition,CUDC-907 increased foci formation and upregulated the protein expression of γ-H2 AX,a DNA damage marker molecule,both in MCF-7 and MDA-MB-231 human breast cancer cells.2.Upregulation of DR5 by CUDC-907 contributes to the TRAIL-induced cell death in breast cancer cells.Breast cancer cells were treated with CUDC-907 alone,TRAIL alone,and combined treatment with CUDC-907 and TRAIL.Annexin V-FITC and PI double staining showed that co-treatment with CUDC-907 and TRAIL synergistically increased the apoptotic population compared with either CUDC-907 or TRAIL alone.Cotreatment with CUDC-907 and TRAIL markedly induced the cleavage of caspase-8 and-9,which in turn led to increased PARP cleavage.CUDC-907 treatment upregulated the expression of DR5 both in MCF-7 and MDA-MB-231 cells.However,the levels of DR4 remained constant upon treatment of MCF-7 and MDA-MB-231 cells with CUDC-907.Knockdown the expression of DR5 by RNA interference antagonized the effect of CUDC-907 on TRAIL-induced apoptosis both in MCF-7 and MDA-MB-231 cells.The cleaved caspase-8,-9 and PARP protein level induced by CUDC-907 plus TRAIL treatment was significantly attenuated in DR5 knockdown cells.CUDC-907 up-regulated the expression of endoplasmic reticulum stress-related factors BIP and PERK,but had no significant effect on CHOP.CUDC-907 promoted the phosphorylation of JNK and p38 MAPK in breast cancer cells.However,the expression of p-ERK was decreased in CUDC-907 treated human breast cancer cells.Pretreatment with the JNK inhibitor SP600125 abrogate the CUDC-907-induced DR5 upregulation both in MCF-7 and MDA-MB-231 cells.However,p38 MAPK inhibitor treatment did not influence the DR5 expression Conclusions1.CUDC-907 inhibits PI3K/Akt signaling pathway in breast cancer cells,inhibits proliferation of breast cancer cells by regulating cell cycle progression;induces apoptosis of breast cancer cells by activating members of the casapse family.2.CUDC-907 enhances TRAIL-induced apoptosis in breast cancer cells by upregulating DR5 protein expression.3.CUDC-907 promotes DR5 protein expression in breast cancer cells by activating MAPK family member JNK.