Synergistic Effect Of Histone Deacetylase Inhibitor SBHA With Proteasome Inhibitors Bortezomib Or MG-132 On Breast Cancer Cells

Objectives:To elucidate effects and mechanisms of histone deasetylase inhibitor SBHA (bishydroxamate/Suberoyl bishydroxamic acid) alone or in combination bortezomib or MG-132 on the growth inhibition and induced apoptosis of breast cancer cells.Methods:The effect of growth inhibition and induced apoptosis were investigated in the breast cancer MCF-7 and MDA-MB-231 cells treated with SBHA alone at various concentrations and time points by WST and FITC-labeled Annexin V/PI and flow cytometer (FCM).Light microscopy was used to observe morphological change.The apoptosis morphology characters were observed by fluorescence microscope by hoechst33258 staining. For the analysis of impacts of the combined treatment on cell proliferation and apoptosis,cells were incubated with variable combination of drugs:â‘ conâ‘¡SBHAâ‘¢Bortezomibâ‘£SBHA + Bortezomib⑤MG-132â‘¥SBHA + MG-132.Synergistically effects on growth and apoptosis were evaluated in MCF-7 and MDA-MB-231 cells by WST and FITC-labeled Annexin V/PI and FCM.Cell cycle changes were analyzed by FCM method.DNA fragmentation was evaluated by agarose gel electrophoresis.Reverse transcriptase polymerase chain(RT-PCR) method was used to explore the change in the mRNA of p21,p27,p53,Bcl-2,Bax,smad2 and smad4. Western blotting was used to investigate expression of p21,p27,p53 and Bcl-2 family, including Bcl-2,Bcl-Xs,Bax,Bim and Bak.In addition,JNK/c-Jun pathway was also evaluated by Western blotting.Results:This study showed that SBHA alone inhibited growth and induced apoptosis in concentration and time dependently in breast cancer MCF-7 and MDA-MB-231 cells.Changes of cell morphology and typical apoptotic bodies were found.G₀-G₁ phase arrest was been observed in MCF-7 cells treated with SBHA alone.Interestingly,combination of SBHA with bortezomib or MG-132 synergistically inhibited proliferation and induced apoptosis in both MCF-7 and MDA-MB-231 cells.The combination indexes were less than 1 in groupâ‘£andâ‘¥in both MCF-7 and MDA-MB-231 cells.Combination of SBHA with bortezomib or MG-132 resulted in cell cycle G₂-M phase arrest in MCF-7 cells.DNA fragmentation was found.SBHA alone significantly increased the level of p21,p27 and p53,and in combination with Bortezomib or MG-132 further enhanced expression levels of p21,p53 but not p27. Treatments for 72 hours with SBHA alone or in combination with Bortezomib or MG-132 were effective at modifying the level of the members of Bcl-2 family.Western blotting analysis demonstrated that modest level of Bcl-2 is present in MCF-7 cells and that SBHA alone or in combination treatment further decreased the intensity of this band with elevated level ofBcl-Xs,Bax.In particular,SBHA alone or in combination with Bortezomib or MG-132 increased the level of the three members of Bim family.Smad 4 was increased in MCF-7 cells treatment with SBHA alone or in combination treatment with a little increase of smad 2.Combined treatment was effective in inducing a marked increase in the content of phospho-c-Jun and JNK.Conclusions:SBHA inhibited growth and induced apoptosis in breast cancer MCF-7 and MDA-MB-231 cells in concentration-dependent and time-dependent manner.SBHA exerted a synergistic effects on growth inhibition and apoptosis in MCF-7 and MDA-MB-231 cells in combination with Bortezomib or MG-132.SBHA alone or in combination treatment significantly changed the expression of cell cycle-related proteins,including increase of p21, p27 and p53 and regulating multiple apoptosis related genes or pathways including Bcl-2 family,smad pathway and JNK/c-Jun pathway.