Non-apoptotic Cell Death Induced By Opening The Large-conductance Mechanosensitivs Channel MseL In HepG2 Cells

Most anticancer therapies trigger apoptosis to eliminate malignant cells,however,drug resistance caused by gene mutations or resistance to apoptosis often inhibit the effective therapy.Therefore,it's vital to find new methods for cancer therapy.Cell death with obvious characteristics have been revealed.In addition to apoptosis,autophagy,necrosis and immune killing,cell death also includes paraptosis,oncosis and pyroptosis,and so on.Different death pathways are chose according to environmental stimuli.Studies show that cancer cells that are not sensitive to apoptosis,may respond to non-apoptotic inducing factors.Mechanosensitive channel of large conductance,Msc L,exists ubiquitously in bacteria,which can be activated by membrane tension and achieves an open pore diameter of?30?,obtaining the exclusive large conductance of?3 n S.Msc L is the Emergency Release Valve in bacteria,and prevents cell lysis because of excessive dilation under hypotonic stress.Due to its small molecular weight of 15 k Da,stable function and various mutants,although no Msc L homologs have been identified in mammalian cells,reconstitution of Msc L in Chinese hamster ovary,human embryonic kidney,and renal erythropoietin-producing cell lines as well as primary cultured neurons has been implemented.Previous studies demonstrated the long-term activation of Msc L decreased cell viaility in both of the prokaryotes and eukaryotes.WT-Msc L,gain of function mutant V23A-Msc L and MTSET inducible mutant G26C-Msc L were expressed in hepatocellular carcinoma Hep G2 cells in our study.Compared with WT-Msc L,V23A-Msc L has a lower gating threshold and shows partial spontaneous openings,while MTSET-treated G26C-Msc L can maintain the opening state.We then estimated cell viability,cell membrane system,and nucleus,and Ca²⁺homeostasis after Msc L opening.MTSET-induced G26C channels caused membrane blebbing and rapid changes in cell morphology,leading to necrosis.In contrast,GOF mutant V23A-Msc L expressed Hep G2 cells showed cytoplasmic vacuolization,which mainly derived from swelling of endoplasmic reticulum,leading to cytoplasmic vacuolization cell death.Moreover,we employed the real time cellular analysis system to track the process of cell death.Acquired results indicate that Hep G2 cells with cytoplasmic vacuolization experienced similar process to that MTSET-activated G26C-Msc L expressed cells died soon after membrane blebbing,suggesting that the correlation between cell death and the extent of Msc L opening.Our study demonstrated the non-apoptotic cell death caused by opening Msc L in Hep G2 cells,and the lethal effect in a mouse model in vivo,which have never been seen before.The study suggests that we can regulate cancer cells by modulating Msc L,and Msc L-based tools may add to our arsenal as a new strategy for cancer therapy.