Apoptosis of Prostate Cancer by Inhibition of Bcl-xL Expression

Kwak, Cheol; Jeong, Hyeon; Kim, Jeong Hyun; Lee, Eun sik; Lee, Sang Eun

Abstract

PURPOSE: In this study, we evaluated in vitro whether the anti-sense transfection that was targeted against Bcl-xL would induce cytotoxicity via apoptosis in prostate cancer cells.
MATERIALS AND METHODS
cDNA of the human Bcl-xL gene was obtained by RT-PCR amplification, and the anti-sense Bcl-xL mRNA plasmid was generated using the pCR 3.1 TOPO plasmid vector. The function of the cloned anti-sense Bcl-xL plasmid vector (pCR3.1-AS-Bcl-xL) was evaluated by the Western blot analysis. Using the MTT assay, the efficacy of growth inhibition by transfection with pCR3.1-AS-Bcl-xL was tested in vitro on PC-3 and DU145 human prostate cancer cell lines. Immunoblot analyses of Bax and caspase-9 were also performed. To evaluate the apoptosis, DNA fragmentation and caspase-3 assay were performed.
RESULTS
Bcl-xL expression after transfection with pCR3.1-AS-Bcl-xL was gradually decreased in PC-3 cells and was continuously decreased in DU145 cells, compared to the parent cells. Bax protein was not expressed in DU145 cells, and the levels of Bax protein expression was not altered in the transfected PC-3 cells compared to the parent cells. The cytotoxicity of pCR3.1-AS-Bcl-xL on PC-3 and DU145 cells increased significantly compared to the empty vector, pCR3.1. This increased cytotoxicity was associated with enhanced apoptosis as assessed by the DNA fragmentation assay and the caspase-3 assay. The expression of the active caspase-9 was increased in the PC-3 cells but not in the DU145 cells after transfection with pCR3.1-AS-Bcl-xL.
CONCLUSIONS
Our results showed that the suppression of Bcl-xL by anti-sense transfection efficiently inhibited the growth of PC-3 and DU145 human prostate cancer cell lines. The inhibition of Bcl-xL expression can possibly be a novel therapeutic alternative in the treatment of hormone refractory prostate carcinoma.