Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells

V Kw Wong,K Yc Lam,B Yk Law,C Km Law,E Dl Ma,N C Yip,L Liu,M M Zhang,P L Chan,T Li,F Michelangeli

doi:10.1038/cddis.2013.217

Abstract

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump, leading to autophagy induction through the activation of the Ca2+/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

Highlights

Emerging evidence supports the role of autophagy in tumor suppression,[5,6] as increased levels of autophagy might lead to autophagic cell death in cancers.[7]
Autophagy is directly involved in reducing the growth of tumor cells,[20] chemical autophagy inducers could have the potential of inhibiting tumor cells.[21]
Results indicated that Ssd could time dependently induce autophagy as shown by the increased number of fluorescence green fluorescent protein light-chain 3 (GFP-LC3) puncta observed within the cells (Supplementary Figures S1a and b)

Summary

Introduction

Emerging evidence supports the role of autophagy in tumor suppression,[5,6] as increased levels of autophagy might lead to autophagic cell death (type II programmed cell death) in cancers.[7]. Mechanistic studies revealed that Ssd-induced autophagy occurred by direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2 þ ATPase (SERCA), leading to the increase of intracellular calcium ion levels and activating the Ca2 þ /calmodulin-dependent kinase kinase-b (CaMKKb)–AMP-activated protein kinase (AMPK)– mammalian target of rapamycin (mTOR) signaling cascade, endoplasmic reticulum (ER) stress and unfolded protein responses (UPR). The activation of these pathways leads to apoptotic and autophagic cell death in both cancer cells and, more importantly, apoptosis-resistant cancer cells

Methods

Results

Conclusion