Smac/DIABLO induces apoptosis in pancreatic cancer cells and increases the sensitivity of TRAIL and gemcitabine chemotherapy

Abstract

Objective To explore the effect of ectopic overexpression of Smac/DIABLO on the proliferation of pancreatic cancer cell line 5W1990, and the sensitization to TRAIL and Gemcitabine induced apoptosis. Methods The Smac/DIABLO gene was transfeeted into the pancreatic cancer cell line SW1990 with the participation of Lipofectamine 2000 （SW1990/Smac）. The cell line transfected with empty vector served as controls （SW1990/neo）. The SW1990/neo and SW1990/Smac cells were assigned into the following treatment groups： TRAIL group, Gemcitabine group, TRAIL plus Gemcitabine group, and the control group. The SW1990 cells were treated with TRAIL and Gemcitabine in different concentrations and time. The cell growth inhibition rate （CGIR） was detected by MTF,thc rate of apoptosis was measured by flow eytometry, the apoptosis morphous was observed by Heochst 33342 staining. The expressions of apoptosis-associated proteins such as Smas/DIABLO, XIAP, cytochrome C and caspase-3 were detected by Western blot. Results The cell growth of SW1990/Smac was significantly lower than growth of SW1990/ neo. The concentration of TRAIL were 200, 500, 1000 and 2500 ng/ml respectively. After 24 hours, the CGIR of SW1990/neo and SW1990/Smac were 11.11% , 46. 03% , 67.08% , 76. 19% and 22. 11% ,42.67%, 56. 63%, 67.6% respectively （P 〈 0. 05 ）. The concentration of Gemcitabine were 10, 20, 40 and 60 p.mol/L respectively. After 24 hours, the CGIR of SW1990/neo and SW1990/Smac were 15.2%, 34. 6%, 55.16%, 76.4% and 22. 65%, 36. 85%, 55.11%, 79.99% respectively （P〈0.05）. The cells of SW1990/neo and SW1990/Smac were treated by TRAIL（ 500 ng/ml） , Gemcitabine （20 μmol/L） and combination group. The apoptosis rate were 5.64% , 15.30% , 27.27% and 20. 37% , 23.27% , 67.30% （ P 〈 0. 05 ） respectively. In combination group, the expressions of activators of caspase such as Smas/DIABLO, cytochrome C and caspase-3 increased significantly, while the expressions of inhibitor of apoptosis protein XIAP decreased. Conclusions Ectopic expression of Smac/DIABLO could induce the apoptosis of SW1990 cell, inhibit the cell proliferation, and enhence the sensitivity of SW1990 cell to TRAIL and Gemeitabine. The mechanism of apoptosis sensitization effect by Smac/DIABLO was associated with significant up-regulation of Smae/DIABLO, cytochrome C, down-regulation of XIAP, and the activation of caspase-3. Key words: Pancreatic neoplasms; TNF related apoptosis-inducing ligand; Apoptosis; Gemcitabine