左旋紫草素对顺铂耐药人宫颈癌HeLa细胞的逆转作用研究

Study on Reversal Effects of Levoshikonin on Cisplatin Resistance of Human Cervical Carcinoma HeLa Cells

目的:探讨左旋紫草素(L-SHK)对顺铂(DDP)耐药人宫颈癌HeLa细胞(HeLa/DDP)的逆转作用及其可能机制。方法:以人宫颈癌HeLa细胞株为研究对象,以DDP诱导获得HeLa/DDP耐药细胞;采用CCK-8法测定HeLa/DDP细胞的耐药指数以及不同剂量L-SHK(0.125、0.25、0.5、1、2、4、8、16μmol/L)对该细胞的抑制率、半数抑制浓度(IC50)和逆转倍数;采用流式细胞术检测低、中、高剂量L-SHK(0.3、0.6、1.2μmol/L)联合DDP对HeLa/DDP细胞周期及凋亡率的影响,采用Western blotting法检测低、中、高剂量L-SHK(0.3、0.6、1.2μmol/L)联合DDP对HeLa/DDP细胞凋亡相关蛋白[剪切型胱天蛋白酶3(Cleaved caspase-3)、Bcl-2、Bax]表达的影响。结果:所得HeLa/DDP细胞的耐药指数为11.8。L-SHK对HeLa/DDP细胞的抑制率有随剂量增加而逐渐升高的趋势。与单用DDP比较,DDP+低、中、高剂量L-SHK组细胞的IC50值均显著降低,且呈剂量依赖性(P<0.05);DDP+低、中、高剂量L-SHK组的逆转倍数分别为1.38、2.80、6.71。与空白对照组比较,各药物组G0/G1期、S期细胞比例,各剂量L-SHK联用组细胞的早期、晚期凋亡率及总凋亡率以及Bax、Cleaved caspase-3蛋白的表达量均显著升高;各药物组G2/M期细胞比例以及各剂量L-SHK联用组Bcl-2蛋白的表达量均显著降低(P<0.05)。与DDP组比较,各剂量L-SHK联用组S期、G2/M期细胞比例和Bcl-2蛋白的表达量均显著降低;G0/G1期细胞比例,早期、晚期凋亡率及总凋亡率和Bax、Cleaved caspase-3蛋白的表达量均显著升高(P<0.05)。结论:HeLa/DDP细胞对DDP具有一定的耐药性,L-SHK可逆转这种耐药性。L-SHK和DDP联用可促进HeLa/DDP细胞凋亡,且作用强于DDP单用;这种作用可能与影响细胞周期、调控凋亡相关蛋白的表达有关。

OBJECTIVE:To investigate the reversal effects and potential mechanism of levoshikonin(L-SHK)on cisplatin(DDP) resistance of human cervical carcinoma HeLa cells. METHODS:Human cervical carcinoma HeLa cells were used as research objects,and drug-resistant HeLa/DDP cells were induced by DDP. CCK-8 assay was used to determine drug resistance index of HeLa/DDP cells,the inhibition rate of different doses of L-SHK(0.125,0.25,0.5,1,2,4,8,16 μ mol/L) on cell proliferation,IC50 and the reversion index of L-SHK on HeLa/DDP cells. Effects of low,medium and high doses of L-SHK(0.3,0.6,1.2 μmol/L)combined with DDP on cell cycle and apoptosis rate were determined by flow cytometry. Western blotting assay was used to detect the effects of low,medium and high doses of L-SHK(0.3,0.6,1.2 μ mol/L) combined with DDP on the expression of apoptosis-related protein(Cleaved caspase-3,Bcl-2 and Bax). RESULTS:The drug resistance index of He La/DDP cells was 11.8. The inhibition rate of L-SHK on HeLa/DDP cells increased with the increase of dose. Compared with DDP alone,IC50 of DDP + low-dose,medium-dose and high-dose L-SHK groups were decreased significantly,with a dose-dependent manner(P<0.05). The reversion indexes were 1.38,2.80,6.71 in DDP+low-dose,medium-dose and high-dose L-SHK groups. Compared with blank control group,the proportion of cells at phase G0/G1 and phase S in administration groups,as well as early and late apoptosis rate and total apoptosis rate of cells,the protein expression of Bax and Cleaved caspase-3 in L-SHK combination groups were increased significantly;the proportion of cells at phase G2/M in administration group as well as the protein expression of Bcl-2 in L-SHK combination groups were decreased significantly(P<0.05). Compared with DDP group,the proportion of cells at phase S and G2/M and the protein expression of Bcl-2 in L-SHK combination groups were significantly decreased;the proportion of cells at phase G0/G1,early and late apoptosis rate and total apoptosis rate,the protein expression of Bax and Cleaved caspase-3 in L-SHK combination groups were significantly increased(P<0.05). CONCLUSIONS:HeLa/DDP cells are resistant to DDP,and L-SHK can reverse the drug resistance. L-SHK combined with DDP can promote the apoptosis of HeLa/DDP cells,which is better than DDP alone. Its mechanism may be related to the influence of cell cycle and the regulation of apoptosis-related protein expression.