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


RESULTS
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.


CONCLUSIONS
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.
Lee
Yong J
YJ
Department of Surgery and Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. leeyj@msx.upmc.edu
Froelich
Christopher J
CJ
Fujita
Naoya
N
Tsuruo
Takashi
T
Kim
Jin H
JH
D001943:Breast Neoplasms; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000172:deficiency / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000494:pharmacology; D011471:Prostatic Neoplasms; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D005947:Glucose