Lysosomal dysfunction and autophagy blockade contribute to IMB-6G-induced apoptosis in pancreatic cancer cells

Lu Liu,Chongwen Bi,Genxiang Mao,Yueying Dou,Na Zhang,Xiaojia Liu,Hongbin Deng,Danqing Song,Weiqiang Pang

doi:10.1038/srep41862

Abstract

Targeting the autophagic pathway is currently regarded as an attractive strategy for cancer drug discovery. Our previous work showed that IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells, yet the effect of IMB-6G on autophagy and pancreatic cancer cell death remains unknown. Here, we show that IMB-6G inhibits the growth of MiaPaCa-2 and HupT-3 pancreatic cancer cells and induces caspase-mediated apoptosis, which is correlated with an accumulation of autophagic vacuoles. IMB-6G promotes autophagosome accumulation from the early stage of treatment but blocks autophagic flux in the degradation stage, mainly through attenuation of lysosomal cathepsin activity in pancreatic cancer cells. Moreover, IMB-6G triggers lysosomal membrane permeabilization (LMP), followed by cathepsin B/CTSB and cathepsin D/CTSD release from lysosomes into the cytoplasm. Inhibition of autophagosome formation with siRNA against autophagy protein 5 (Atg5) attenuates IMB-6G-induced LMP and apoptosis. Furthermore, cathepsin inhibitors relieve IMB-6G-induced apoptosis as well. Altogether, our findings demonstrate that IMB-6G is a novel autophagy inhibitor, which induces autophagy-dependent apoptosis through autophagosomal-cathepsin axis in pancreatic cancer cells and indicate the potential value of IMB-6G as a novel antitumor drug candidate.

Highlights

Natural products provide a bountiful source of new chemotherapeutics
To examine whether cell apoptosis was involved in IMB-6G-induced pancreatic cancer cell death, the apoptotic cell death was investigated by immunoblotting
Data were the mean value of three independent experiments with each count of no less than 100 cells.*p < 0.05, **p < 0.01 compared with the untreated control group. (c) MiaPaCa-2 and HupT-3 cells were treated with IMB-6G at the indicated concentrations for 24 h, or treated with IMB-6G (5 μM) for indicated time points, the lipidation of LC3 and the levels of p62/SQSTM1 were detected by immunoblotting using corresponding antibodies

Summary

Introduction

Natural products provide a bountiful source of new chemotherapeutics. Sophoridine, one of the major bioactive components extracted from the traditional medicine herb Sophora alopecuroides L., has been approved by China FDA (CFDA) in 2005 to cure the cancer patients with malignant trophoblastic tumors[12,13]. Our results indicated that IMB-6G promoted autophagosome accumulation from the early stage of treatment but blocked autophagic flux through attenuation of lysosomal cathepsins activity in pancreatic cancer cells. This novel autophagy inhibitor eventually induced cathepsin releasing from lysosomes into the cytoplasm and a caspase-mediated apoptotic cell death. These findings have important clinical implications and provide a mechanistic rationale for the use of IMB-6G for the treatment of pancreatic cancer

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