TNFα induces Ca influx to accelerate extrinsic apoptosis in hepatocellular carcinoma cells.

Background: Tumor necrosis factor-α has been proven an effective anticancer agent in preclinical studies. However, the translation of TNFα from research to clinic has been blocked by significant systemic toxicity and limited efficacy at maximal tolerated dose, which need urgently to be solved.

Methods: The level of cytosolic Ca was assessed by Fura-2 in HCC cells. After changing cytosolic Ca level by using agonists or inhibitors, cell apoptosis was detected by flow cytometry. We also detected the effect of ionomycin or parvalbumin on the anti-tumor activity of TNFα in a mice model. Lastly, we studied the roles of cytosolic Ca in the mitochondrial-dependent intrinsic apoptosis pathway.

Results: Here, we demonstrated that TNFα induced extracellular Ca influx into cytoplasm through transient receptor potential channel in HCC cells. Both cytosolic Ca scavenger and Ca-binding protein PV effectively desensitized hepatocellular carcinoma cells to TNFα, whereas combination ionomycin or 1,4,5-inositol triphosphate significantly sensitized HCC cells to TNFα, indicating that the increased level of cytosolic Ca was positively correlated with the TNFα-induced cell apoptosis in vitro. In a nude mice xenograft model, our data revealed that TNFα combined with ionomycin remarkably synergized the anti-tumor effect of TNFα. Furthermore, we found that TNFα-mediated extracellular Ca influx accelerated TNFα-induced extrinsic apoptosis through activating calpain/IAP/caspase3 pathway.

Conclusions: Our study provides the evidence supporting a novel mechanism by which TNFα induces extracellular Ca influx to enhance cell apoptosis and suggests that increasing the level of cytosolic Ca might be an alternative strategy to improve the pro-apoptotic activity of TNFα in HCC cells, although suitable chemical or biological reagents need to be further tested.