防己诺林碱增强吉西他滨诱导的黑色素瘤细胞凋亡

Fangchinoline promote the effect of Gemcitabine on melanoma apoptosis

目的观察防己诺林碱对吉西他滨导致的细胞凋亡的影响,并初步探讨其作用机制。方法将人黑色素瘤细胞A375分为4组,分别用生理盐水(对照),吉西他滨、防己诺林碱以及两种药物联合处理,通过MTT的方法检测不同时间不同处理因素作用下细胞的光密度值(OD490),确定药物对细胞增殖的作用;用AV-PI染色的方法用流式细胞仪检测不同组凋亡细胞比例;Hoechest 33258染色的方法观察不同组别下细胞的凋亡情况;通过Western Blot以及Realtime PCR检测不同因素作用下凋亡相关蛋白的变化。结果对细胞作用24 h时,10μg/ml防己诺林碱,1μg/ml吉西他滨,以及两种药物联合处理组A375细胞增殖的抑制率分别为4.07%±1.95%,45.22%±2.25%和75.71%±1.01%。两种药物联用A375细胞增殖的抑制作用显著强于各单药组作用,各组数据间差异显著(P<0.05)。对细胞作用24 h时,不同处理组细胞凋亡的百分比分别为2.07%±0.27%,3.35%±0.20%,4.15%±0.07%和6.12%±0.51%。药物联用作用下细胞凋亡率增加。各组数据之间有显著差异(P<0.05)。防己诺林碱也可以显著增强吉西他滨对凋亡相关蛋白Bcl-2、BAX和Caspase 3的调控。结论低浓度防己诺林碱(10μg/ml)可以显著增强极低浓度的吉西他滨(0.01×PPC)对抑制黑色素瘤细胞A375细胞凋亡的促进作用,联合应用可达到更好的治疗效果。

Objective To observe the effect of fangchinoline （FAN） on cell apoptosis caused by gemeitabine （GEM） , and discuss its mechanism. Methods A375 cells were divided into four groups ： control group （ NACL）, GEM treatment group, FAN treatment group and GEM combined with FAN treatment group. The proliferation of A375 cells at different time points treated by different concentration of drugs were tested by MTT. Then observed the apoptosis of different groups by AV-PI and Hoeehest 33258 staining methods. At last the protein level and RNA level of proteins were detected by Western Blot and Reahime PCR. Results 10 μg/ml of FAN, 1 μg/ml of GEM or FAN combined with GEM were treated on A375 cells, the inhibition of cell proliferation were 4.07% ± 1.95% , 45.22% ±2.25% and 75.71% ±1.01%. In different treated groups, the percentages of apoptotie cells were 2.07% ±0.27% , 3.35% ±0.20% , 4.15% ±0.07% and 6.12% ±0.51%. At the same time, FAN can significantly enhance the affection of Bc1-2, BAX and Caspase 3 by GEM. Conclusion FAN combined with GEM have a synergistic effect on the promotion of the apoptosis of melanoma cells, which can decrease the side effects of GEM.