Bisindolylmaleimide alkaloid BMA-155CI induces autophagy and apoptosis in human hepatocarcinoma HepG-2 cells through the NF-KB p65 pathway

摘要

Bisindolylmaleimides,a series of derivatives of a PKC inhibitor staurosporine,exhibit potential as anti-cancer drugs and have received considerable attention in clinical trials.This study aims to investigate the effects of a bisindolylmaleimide alkaloid 155CI (BMA-155CI) with a novel structure on autophagy and apoptosis in human hepatocarcinoma HepG-2 cells in vitro and in vivo.The cell poliferation was assessed with a MTT assay.Autophagy was evaluated by MDC staining and TEM analysis.Apoptosis was investigated using Annexin V-FITC/PI and DAPI staining.The antitumor effects were further evaluated in nude mice bearing HepG-2 xenografts,which received BMA-155CI (10,20 mg/kg,ip) for 18 days.AUtophagy-and apoptosis-associated proteins and their mRNA levels were examined with Western blotting,immunohistochemistry,and RT-PCR.BMA-155CI (2.5-20 μmol/L) inhibited the growth of HepG-2 cells with IC50 values of 16.62±1.34,12.21±0.83,and 8.44±1.82 μmol/L at 24,48,and 72 h,respectively.Furthermore,BMA-155CI (5-20 μmol/L) dose-dependently induced autophagy and apoptosis in HepG-2 cells.The formation of autophagic vacuoles was induced by BMA-155CI (10 μmol/L) at approximately 6 h and peaked at approximately 15 h.Pretreatment with 3-MA potentiated BMA-155CI-mediated apoptotic cell death.This compound dose-dependently increased the mRNA and protein levels of Beclin-1,NF-κB p65,p53,and Bax,but decreased the expression of IκB and Bcl-2.Pretreatment with BAY 11-7082,a specific inhibitor of NF-κB p65,blocked BMA-155CI-induced expression of autophagy-and apoptosis-associated proteins.BMA-155CI administration effectively suppressed the growth of HepG-2 xenografts in vivo,and increased the protein expression levels of LC3B,Beclin-1,NF-κB p65,and Bax in vivo.We conclude that the NF-κB p65 pathway is involved in BMA-155CI-triggered autophagy,followed by apoptosis in HepG-2 cells in vitro and in vivo.Hence,BMA-155CI could be a promising pro-apoptotic candidate for developing as a novel anti-cancer drug.