Redox status of thioredoxin-1 （TRX1） determines the sensitivity of human liver carcinoma cells （HepG2） to arsenic trioxide-induced cell death

lntracellular redox homeostasis plays a critical role in determining tumor cells＇ sensitivity to drug-induced apoptosis. Here we investigated the role of thioredoxin-1 （TRX1）, a key component of redox regulation, in arsenic trioxide （AS2O3）-induced apoptosis. Over-expression of wild-type TRX1 in HepG2 cells led to the inhibition of As2O3-induced cytochrome c （cyto c） release, caspase activation and apoptosis, and down-regulation of TRX1 expression by RNAi sensitized HepG2 cells to As2O3-induced apoptosis. Interestingly, mutation of the active site of TRX1 from Cys^32/35 to Ser^32/35 converted this molecule from an apoptotic protector to an apoptotic promoter. In an effort to understand the mechanisms of this conversion, we used isolated mitochondria from mouse liver and found that recombinant wild-type TRX1 could protect mitochondria from the apoptotic changes. In contrast, the mutant form of TRX1 alone elicited mitochondria-related apoptotic changes, including the mitochondrial permeability transition pore （mPTP） opening, loss of mitochondrial membrane potential, and cyto c release from mitochondria. These apoptotic effects were inhibited by cyclosporine A （CsA）, indicating that mutant TRX1 targeted to mPTP. Alteration of TRX1 from its reduced form to oxidized form in vivo by 2,4-dinitrochlorobenzene （DNCB）, a specific inhibitor ofTRX reductase, also sensitized HepG2 cells to As203-induced apoptosis. These data suggest that TRX1 plays a central role in regulating apoptosis by blocking cyto c release, and inactivation of TRX1 by either mutation or oxidization of the active site cysteines may sensitize tumor cells to As2O3-induced apoptosis.

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis,development and diseases.Autophagy is also described as a mechanism of death pathways,however,the precise mechanism of how autophagy links to cell death remains to be fully understood.Beclin 1 is a dual regulator for both autophagy and apoptosis.In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149,respectively.Furthermore,the autophagosome formation occurred,followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment.The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA.In addition,the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1,which could be blocked by z-VAD-fmk.Thus,our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.