[Involvement of bcl-2 in multidrug resistance in human small cell lung cancer cell subline H446/DDP].

OBJECTIVE: To study the mechanism of bcl-2 involvement in multidrug resistance in human small cell lung cancer (SCLC) cell subline H446/DDP. METHODS: After the construction of pLXSN-bcl-2, in which the full length of bcl-2 cDNA amplified from total RNA of H446/DDP cells was integrated into the mammalian expression vector pLXSN, allowing transcription of a bicistronic mRNA, and the synthesis in vitro of 20-mer ODNs targeting the coding region of bcl-2 mRNA and the scrambled ODNs used as the control, the cationic lipid DOTAP was used to transfect them into H446 and H446/DDP cells, respectively. When H446 cells were transfected with mammalian expression vector pLXSN, the cells were divided into 3 groups, including cells transfected with the recombinant pLXSN-bcl-2, cells transfected with the vector pLXSN and cells untransfected (control); when H446 cells and H446/DDP cells were transfected with PS-ODNs, the cells were divided into 3 groups, including cells transfected with AS-PS-ODNs, cells transfected with NS-PS-ODNs and cells untransfected (control), respectively. After transfection, Western blot were carried out to detect the expression level of bcl-2 in the control cells and the transfected cells, respectively. Meanwhile flow cytometry (FCM) was used to detect cell apoptosis in the control cells and the transfected cells. RESULTS: (1) The data from Western blot showed that compared with the control H446 cells (gray-scale value 0.103 +/- 0.005), the expression level of bcl-2 in the pLXSN-bcl-2 transfected H446 cells (gray-scale value 0.854 +/- 0.016) was increased significantly (P < 0.01); and the apoptosis from DNA content analysis decreased significantly in the pLXSN-bcl-2 transfected H446 cells [(20.9 +/- 0.2)%] compared with that of the control H446 cells [(31.1 +/- 0.2)%] by DNA content analysis (P < 0.01). (2) The results from Western blot showed that bcl-2 expression in the AS-PS-ODNs transfected H446/DDP cells (gray-scale value 0.695 +/- 0.014) was significantly reduced compared with that of the control H446/DDP cells (gray-scale value 0.942 +/- 0.018), although not totally reduced to the level of the control H446 cells (P < 0.01); and the data from DNA content analysis indicated that the sensitivity of the AS-PS-ODNs transfected H446/DDP cells to 5 microg/ml DDP-induced apoptosis was strongly increased as compared with that of the control H446/DDP cells (P < 0.01). CONCLUSION: Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression may be one of the important therapeutic approaches to overcoming chemoresistance in human SCLC.