Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

H Jiang,Q Xu,J Wei,H Lou,C Y F Young,J Sun,Y Liu,H Yuan

doi:10.1038/cddis.2013.285

Abstract

We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF2α), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF2α and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as a potential agent in cancer chemotherapy.

Highlights

Docetaxel, a natural product derivative, is approved to offer some extent of symptomatic and survival benefits for hormone-refractory prostate cancer (HRPC) patients
As to whether Mar is an active chemical to inhibit proteasome activity, we initiated our studies to examine the effects of Mar on the purified 20S proteasome by monitoring chymotrypsinlike (ChT-L), trypsin-like (Try-L) and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities in vitro
We investigated the alteration in proteasome activity in prostate cancer (PCa) cells exposed to Mar

Summary

Introduction

A natural product derivative, is approved to offer some extent of symptomatic and survival benefits for HRPC patients. Autophagic cell death appears to be responsible for the death of some cancer cells (especially when they defect essential apoptotic modulators) with increased autophagic flux.[10] Recent studies have demonstrated that autophagy, including autophagic cell death, is often activated in tumor cells in response to multiple chemotherapeutic agents or radiation.[11,12] For example, proteasome inhibitor activates cellular protective autophagy via a phospho-eukaryotic translation initiation factor 2a (eIF2a)-dependent mechanism required for endoplasmic reticulum (ER) stress signaling.[13] ER is essential for cell survival through modulating both the folding and trafficking of newly synthesized proteins as well as the balance of intracellular calcium concentration. Upon disruption of ER functions by excess loading of unfolded/misfolded proteins in the ER, cells trigger the unfolded protein response (UPR) to avert ER stress. It is the cell’s inherent nature that prolonged or excessive ER stress naturally leads to cell death through apoptosis. We report the novel finding that Mar induced autophagy-dependent cell death, which was accompanied with the induction of ER stress and the inhibition of proteasome activity

Methods

Results

Conclusion