丝裂霉素C与表达Smac/DIABLO的肿瘤靶向腺相关病毒联用对抗恶性黑色素瘤的效果

目的探讨重组腺相关病毒(rAAV)介导促凋亡因子Smac/DIABLO是否能提高恶性黑色素瘤细胞对丝裂霉素(MMC)的敏感性。方法将携有目的基因的rAAV加入到培养液中转染肿瘤细胞,荧光显微镜观察绿色荧光蛋白(EGFP)的表达,RT-PCR检测Smac/DIABLO基因的表达,MTT法测定肿瘤细胞增殖,流式细胞术测定肿瘤细胞的凋亡;动物在体实验测定抗肿瘤效果。结果 rAAV转染96h后几乎所有细胞表达明亮的黄绿色荧光。RT-PCR检测发现转染48h后目的基因Smac/DIABLO稳定表达。MTT检测显示rAAV-hTERT/Smac/DIABLO+MMC对肿瘤细胞的抑制率最高(P<0.05)。流式细胞检测结果显示,rAAV-hTERT/Smac/DIABLO+MMC组肿瘤细胞的凋亡率明显高于生理盐水(HBSS)组、MMC组和rAAV-hTERT/Smac/DIABLO组(P<0.01)。动物实验结果表明,rAAV-hTERT/Smac/DIABLO+MMC有非常强的抑制肿瘤作用,其抗癌效果优于HBSS、MMC、rAAV-hTERT/EGFP和rAAV-hTERT/Smac/DIABL组。结论 rAAV-hTERT/Smac/DIABLO在体内、体外均能提高肿瘤细胞对MMC的敏感性。此作用与Smac/DIABLO的促凋亡作用有关。

Potent anti-melanoma effect by combination of mytomycin C with recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO

Objective To investigate whether recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO can improve sensitivity of melanoma to mitomycin C both in vitro and in vivo.Methods Tumor-targeting expressed Smac/DIABLO or green fluorescence protein(EGFP) in recombinant adeno-associated virus vectors(rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP) was constructed.The culture cells were transfected with rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP.The expression of EGFP in culture cells was observed with fluorescence microscope.Smac/DIABLO expression was detected by RT-PCR method.Proliferation of tumor cells was measured by MTT method.Apoptosis of tumor cells at different drug concentrations was examined by flow cytometry.Synergistic anti-tumor activity of mitomycin C combined with rAAV-hTERT/Smac/DIABLO was measured by MTT in vitro and animal experiment in vivo.Data was evaluated by SPSS statistics software analysis.Results Green fluorescence could be observed in tumor cells but not in normal cells 48 h after rAAV-hTERT/EGFP transfection.Almost all tumor cells displayed bright yellow-green fluorescence after 96 h.The expression of Smac/DIABLO in rAAV-hTERT/Smac/DIABLO transfected tumor cells showed Smac/DIABLO mRNA band 24 h after transfection and stabilized 48 h after transfection.Tumor cell inhibition rate was increased obviously higher in group of combination of mitomycin C with rAAV-hTERT/Smac/DIABLO than mitomycin C alone(P<0.01).Flow cytometry results indicated that mitomycin C combined with rAAV-hTERT/Smac/DIABLO group had the highest apoptosis-induced effect in groups of negative,mitomycin C,and rAAV-hTERT/Smac/DIABLO(P<0.01).Animal experiment result indicated that tumor growth was inhibited and survival rate was improved significantly in mitomycin C combined with rAAV-hTERT/Smac/DIABLO group compared to rAAV-hTERT/Smac/DIABLO or mitomycin C alone. Conclusion Tumor-targeting rAAV-hTERT/Smac/DIABLO can improve sensitivity of tumor cells to mitomycin C treatment both in vitro and in vivo.This synergistic anti-tumor activity is related to the sensitization of tumor cells to apoptosis.