TNF? Induces Ca²⁺ Influx To Accelerate Extrinsic Apoptosis In Hepatocellular Carcinoma Cells

Tumor necrosis factor-?has been proven an effective anticancer agent in preclinical studies.However,the translation of TNF?from research to clinic has been blocked by significant systemic toxicity and limited efficacy at maximal tolerated dose,which need urgently to be solved.How to reduce the toxic effects induced by TNF?and to improve the efficiency of TNF?need to be solved.Our group firstly found that TNF?induced extracellular Ca²⁺influx in a dose-dependent manner in HCC cells.Numberous studies have reported that changes in the levels of intracellular calcium(Ca²⁺)provide dynamic and highly versatile signals that control a plethora of cellular processes,although their importance is perhaps most strikingly exemplified by their role in life-and-death decisions.Our results further showed that TNF?-mediated Ca²⁺influx was closely correlated with the cell apoptosis in HCC cells.Based on these previous results,we hypothesized that TNF?-mediated Ca²⁺influx promoted cell apoptosis and altering the levels of cytosolic Ca²⁺minght be a novel stragety to amplify the TNF?-induced cell apoptosis.?Objective?Our research aimed to study the roles of TNF?in mediating the extracellular Ca²⁺influx,and to study the underlying mechanism of TNF?-mediated Ca²⁺influx in promoting the cell apoptosis in HCC cells.?Methods?1.Fura-2/AM was used to assay the levels of cytosolic Ca²⁺;2.Annexin-?/PI assay and TUNEL assay were used to detect the cell apoptosis;3.Co-IP assay was used to study the underlying mechanism of TNF?in regulating the TRPM7 channel;4.The effects of TNF?on the cell growth of apoptosis were evaluated in a subcutaneous mice model;5.The mRNA and protein levels of apoptotic related molecules were measured by qPCR and western Blot to study the underlying mechanism of TNF?-mediated Ca²⁺flux in promoting the cell apoptosis;6.The mitochondrial membranal potential was assayed by TMRM assay;7.The subcellular location of Cyto c was detected by immunofluorescence.?Results?First part:TNF?induced extracellular Ca²⁺influx,which resulted in the significantly elevated of the levels of cytosolic Ca²⁺in HCC cells.TNF?induced extracellular Ca²⁺influx in a dose-dependent manner.The obvious changes of the levels of the cytosolic Ca²⁺was not observed after TNF?treatment when HCC cells were cultured in calcium-free medium.Second part:TNFR1 knockdown had no effect on the TNF?-mediated Ca²⁺influx,suggesting that TNF?mediated Ca²⁺influx was independent on the TNFR1.Moreover,the inhibitor of VGCC treatment also had no effect on the TNF?-mediated Ca²⁺influx,suggesting that TNF?-mediated Ca²⁺influx was not related with VGCC.However,the inhibitor of TRP channel significantly inhibited TNF?-mediated Ca²⁺influx,and TRPM7knockdown remarkably decreased the TNF?-mediated Ca²⁺influx in HCC cells.Furthermore,our results showed that TNF?didn't interact with TRPM7 channel directly,suggesting that TNF?activated TRPM7 channel in an indirect manner.Third part:Our results demonstrated that decreased cytosolic Ca²⁺level attenuated TNF?-induced apoptosis.In contrast,increased cytosolic Ca²⁺sensitized HCC cells to TNF?-induced apoptosis.In vivo assay,our results showed that overexpression of parvalbumin,which functioned as a calcium scavenger protein,significantly inhibited the anti-cancer activity of TNF?.However,TNF?combined with ionomycin significantly enhanced the pro-apoptotic activity of TNF?in a subcutaneous mice model.Fourth part:Our results showed that TNF?induced cell apoptosis through activating Calpain/IAP/Caspase 3 signal pathway.In addition,TNF?-mediated Ca²⁺influx had no effect on mitochondria-dependent intrinsic apoptosis.?Conclusion?In summary,our data provides the strong evidence to support the notion that extracellular Ca²⁺influx induced by TNF?facilitates the TNF?-mediated extrinsic apoptosis through activating calpain/IAPs/caspase3 signaling pathway,and the combination of ionomycin with TNF?remarkably increases the pro-apoptotic activity of TNF?.