Proteasome inhibition attenuates rasfonin-induced autophagy concurring with the upregulation of caspase-dependent apoptosis

Siyuan Yan,Yongsheng Che,Xuejun Jiang

doi:10.1080/21501203.2016.1147091

Abstract

ABSTRACTTwo major protein quality control mechanisms exist in eukaryotic cells, the ubiquitin-proteasome system (UPS) and the autophagy–lysosome system. Generally, the inhibition of UPS is believed to enhance autophagic pathway; nevertheless, the crosstalk between these two degradation systems may be much more complicated. Rasfonin, a 2-pyrone derivative of fungal secondary metabolites, is demonstrated to have the antitumor effect and can function as an autophagy inducer. Here, we reported that rasfonin activated multiple cell death pathways, including caspase-dependent apoptosis. Using electroscopy and microscopy, we observed rasfonin increased the formation of autophagosome. In immunoblotting assay, rasfonin enhanced autophagic flux concomitant with the upregulation of ubiquitination. MG132, an inhibitor of proteasome, attenuated rasfonin-dependent autophagy, whereas its presentation stimulated rasfonin-induced cleavage of poly (ADP-ribose) polymerase, a marker of caspase-dependent apoptosis. Together, we demonstrated that rasfonin induced the activation of both UPS and autophagic pathway, and the inhibition of UPS attenuated rasfonin-induced autophagy and enhanced the cytotoxicity of rasonin by upregulation of caspase-dependent apoptosis.

Highlights

Rasfonin, a natural product isolated from the fermented mycelium of Talaromyces sp. 3656-A1, is a derivative of a class of fungal secondary metabolites known as 2-pyrones (Tomikawa et al 2000). 2-Pyrones have been previously reported to have a broad range of biological activities including antimicrobial, antiviral, antitumor and anti-inflammatory effects (Hilgeroth 2002; Marrison et al 2002; McGlacken & Fairlamb 2005)
We demonstrated that MG132 attenuate rasfonin-induced autophagy, accompanied with enhancing rasfonin-activated caspase-dependent apoptosis
We found that rasfonin significantly reduced the cell viability of ACHN cells in a time-dependent manner (Figure 1A). poly (ADP-ribose) polymerase (PARP-1) is one of the main cleavage targets of caspase-3 in vivo, and cleavage of PARP-1 serves as a marker of cells undergoing apoptosis (Amé et al 2004; Andrabi et al 2006)

Summary

Introduction

A natural product isolated from the fermented mycelium of Talaromyces sp. 3656-A1, is a derivative of a class of fungal secondary metabolites known as 2-pyrones (Tomikawa et al 2000). 2-Pyrones have been previously reported to have a broad range of biological activities including antimicrobial, antiviral, antitumor and anti-inflammatory effects (Hilgeroth 2002; Marrison et al 2002; McGlacken & Fairlamb 2005). Important sort of 2-pyrones derivative, target a number of pathways in cancer such as kinase inhibition, cell cycle arrest, angiogenesis inhibition and antimitotic activity (Thakur et al 2015). Rasfonin was shown to induce the death of ras-mutated pancreatic tumour (Panc-1) cells (Xiao et al 2014). There exist two major protein quality control mechanisms, the ubiquitin-proteasome system (UPS) and the autophagy–lysosome system. The UPS is a major cellular mechanism that regulates intracellular protein levels and eliminates damaged, misfolded and mutant proteins (Glickman & Ciechanover 2002). UPS substrates are covalently conjugated with ubiquitin (Ub), a highly conserved 76-residue protein, and polyubiquitinated proteins are mostly targeted to 26S proteasomes for proteolysis into small peptides of 3–24 amino acids (Pickart & Cohen 2004). MG132 is a potent, membranepermeable proteasome inhibitor, which can inhibit proteasome activity and block the degradation of polyubiquitinated proteins (Lee & Goldberg 1998)

Methods

Results

Conclusion