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

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.

The Selenium Metabolite Methylselenol Regulates the Expression of Ligands That Trigger Immune Activation through the Lymphocyte Receptor NKG2D

“…Studies have shown that impairment of proteasomal activity can lead to increased autophagy (70,71), which would also explain the decreased amount of Ub-M-GFP accumulation in Fig. 5B, right panel.…”

Crosstalk between autophagy and proteasome protein degradation systems: possible implications for cancer therapy

“…Differently than in the thyroid cancer cells [78], both in SH-SY5Y cells and in the midbrain of the UPS-impaired mouse model of PD, treatment with lactacystin significantly increased the level of not only LC3-I/II but also Beclin-1 protein, and reduced the levels of p-mTOR and mTOR [85]. Moreover, Marchantin M, a novel proteasome inhibitor tested in prostate cancer cells, significantly induced expression of LC3-I and its further conversion to LC3-II [87].…”

“…Studies on Marchantin M-induced, PI3K/Vps34-dependent autophagy revealed that not the IRE1/JNK pathway but the PERK/eIF2a pathway seemed to be involved in autophagy induction by this proteasome inhibitor [87]. Although activation of the IRE1-mediated pathway of UPR was observed, a downstream effect of this activation was the increase in the spliced form of XBP-1 mRNA, however, the translated protein was found to be not involved in the autophagy induction [87]. In the same study, evaluation of ATF6-mediated pathway showed just a slight increase of ATF6 mRNA.…”

“…According to another report, treatment with resveratrol, also a covalent proteasome inhibitor, leads to blockade of autolysosome formation, as assessed by the absence of co-localization of LC3 and Lamp-2 [99]. Experimental studies designed to evaluate if non-covalent proteasome inhibitor Marchantin M has an influence on the fusion process of amphisome and lysosomes, did not show any blockade of autolysosome formation [87]. The main conclusion from those reports is that the effects of proteasome inhibition on mechanisms of autophagosome maturation and fusion are ambiguous, which probably results from the great variety of chemical structures and activities of various proteasome inhibitors.…”

“…Many studies showed that PERK/eIF2a canonical signalling pathway, which mediates autophagy during ER stress, may be triggered by proteasome inhibition [87,[108][109][110]. Studies on Marchantin M-induced, PI3K/Vps34-dependent autophagy revealed that not the IRE1/JNK pathway but the PERK/eIF2a pathway seemed to be involved in autophagy induction by this proteasome inhibitor [87].…”