Ji Fu-yun, Qian Gui-sheng, Qian Pin, Chen Yan, Guo Rui-ling, Li Shu-ping, Huang Gui-jun
Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2006 Mar;29(3):156-60.
To study the mechanism of bcl-2 involvement in multidrug resistance in human small cell lung cancer (SCLC) cell subline H446/DDP.
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.
(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).
Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression may be one of the important therapeutic approaches to overcoming chemoresistance in human SCLC.
研究bcl-2参与人小细胞肺癌（SCLC）细胞亚系H446/DDP多药耐药的机制。
构建pLXSN-bcl-2，即将从H446/DDP细胞总RNA中扩增出的bcl-2 cDNA全长整合到哺乳动物表达载体pLXSN中，使其转录出双顺反子mRNA，同时体外合成靶向bcl-2 mRNA编码区的20聚体寡脱氧核苷酸（ODNs）及用作对照序列的乱序ODNs，然后用阳离子脂质体DOTAP分别将它们转染至H446和H446/DDP细胞。当用哺乳动物表达载体pLXSN转染H446细胞时，将细胞分为3组，包括转染重组pLXSN-bcl-2的细胞、转染载体pLXSN的细胞和未转染的细胞（对照）；当用PS-ODNs转染H446细胞和H446/DDP细胞时，将细胞分别分为3组，包括转染反义PS-ODNs的细胞、转染非特异性PS-ODNs的细胞和未转染的细胞（对照）。转染后，分别用蛋白质免疫印迹法检测对照细胞和转染细胞中bcl-2的表达水平。同时用流式细胞术（FCM）检测对照细胞和转染细胞的细胞凋亡情况。
（1）蛋白质免疫印迹数据显示，与对照H446细胞（灰度值0.103±0.005）相比，转染pLXSN-bcl-2的H446细胞中bcl-2的表达水平显著升高（灰度值0.854±0.016）（P<0.01）；通过DNA含量分析发现，转染pLXSN-bcl-2的H446细胞的凋亡率[(20.9±0.2)%]与对照H446细胞[(31.1±0.2)%]相比显著降低（P<0.01）。（2）蛋白质免疫印迹结果显示，与对照H446/DDP细胞（灰度值0.942±0.018）相比，转染反义PS-ODNs的H446/DDP细胞中bcl-2的表达显著降低（灰度值0.695±0.014），尽管未完全降至对照H446细胞的水平（P<0.01）；DNA含量分析数据表明，转染反义PS-ODNs的H446/DDP细胞对5μg/ml顺铂诱导凋亡的敏感性与对照H446/DDP细胞相比显著增加（P<0.01）。
靶向抑制抗凋亡线粒体基因Bcl-2的表达可能是克服人SCLC化疗耐药的重要治疗途径之一。
