C086,A Novel Heat Shock Protein 90 (HSP90) Inhibitor Induces Apoptosis And Autophagy In Multiple Myeloma Cells

Purpose:Heat Shock Protein 90(HSP90) as molecular chaperoneshas close relationship with occurrence and development, biological behaviour and prognosis of tumor. Hence,Hsp90 has attracted much attention recently and become a major therapeutic target for cancer. In this study, we aim to investigate the effects of novel HSP90 inhibitor C086 on proliferation, apoptosis and autophagy of human multiple myeloma U266 cells in vitro, and to investigate the role of autophagy in C086-induced apoptosis in U266 cells.Methods:Effects of C086 on cell proliferation were measured by CCK8 method.AO/EB and Annexin V-FITC/PI double-labeled cytometry were used to examine the apoptosis. The morphologic changes of autophagy was determined by fluorescencemicroscopy with monodansylcadaverine( MDC) staining and transmission electron microscope. The protein levels of PARP、LC3B、beclin1 were examined by western blot assay.Results:(1) The results showed that C086 simultaneously inhibitite the proliferation of multiple myeloma U266 cells in a time-and dose-dependent manner. The IC50(48h) was 0.95μg/ml.(2) 1.0μg/ml C086 can induce apoptosis with AO/EB staining showed thetypical morphological changes of apoptosis. Annexin V-FITC/PI assay showed a significantly increased apoptosis rate in the C086 group(28.12±0.481) than in the control group(p<0.01).Meanwhile, the expression of PARP and cleaved PRA was incresaed in a dose-dependent manner.(3)MDC staining showed that the fluorescent density was higher in U266 cells in C086 treatment group than in control group and electron microscopy further demonstrated formation of abundant autophagic vacuoles in U266 cells after C086 treatment.Western blot analysis showed that exposure of U266 cells to C086 resulted in the expression of LC3B-Ⅱand beclin1 increasing in a time and dose-dependent manner.(4) Inhibition of autophagy by 3-MA enchancced the sensitivity of C086 inhibititing the proliferation of U266 cells and promoted C086-induced apoptosis compared with C086 alone treatment groups(p<0.01), meanwhile the expression of LC3 B and beclin1 expression decreased more significantly.Conclusions: Taken together, these findings indicate that C086 is a potent antitumor agent for multiple myeloma. Both apoptosis and autophagy were activated during the C086-induced death of multiple myeloma U266 cells,and apoptosis is the main way to promote the death of U266 cells by C086.C086-induced autophagy is an adaptive response and functions as a protective mechanism in U266 cells, and blockade of autophagy increase C086-induced apoptosis. Therefore,inhibition of autophagy may be an attractive strategy for enhancing the antitumor potential of C086 in multiple myeloma.