Reconstitution of caspase-3 confers low glucose-enhanced tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity and Akt cleavage.

We have previously observed that glucose deprivation enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death as well as caspase activation (caspase-3, -9, and -8) in human prostate adenocarcinoma DU-145 cells. In this study, we used caspase-3-deficient MCF-7 breast cancer cells to examine the possible role of caspase-3 in glucose deprivation-enhanced TRAIL cytotoxicity. Combined glucose deprivation and 200 ng/ml TRAIL treatment markedly induced cytotoxicity in caspase-3 cDNA transfected cells (MCF-7/casp-3) but not in control vector transfected cells (MCF-7/vector). We also observed that the level of Akt, an antiapoptotic protein, was reduced by treatment with TRAIL in MCF-7/casp-3 cells but not in MCF-7/vector cells. The reduction of Akt by TRAIL was promoted in the absence of glucose in MCF-7/casp-3 cells. However, pretreatment with 20 micro M Z-LEHD-FMK, a caspase-9 inhibitor, protected MCF-7/casp-3 cells from the combinatorial treatment of TRAIL and glucose deprivation-induced cytotoxicity. This compound also prevented the reduction of Akt level during the combinatorial treatment. Moreover, this Akt reduction was not inhibited by treatment with MG-132, a proteosome inhibitor. Data from site-directed mutagenesis show that Akt was cleaved at amino acid 108, but not 119, during treatment with TRAIL and glucose deprivation. Our results suggest that caspase-3 is involved in the reduction of Akt level, and its involvement is mediated through caspase-9 activation. The reduction of Akt level is also due to cleavage of Akt rather than degradation of Akt.