雷公藤红素增强多柔比星抑制肝癌细胞系Huh7细胞活性的实验研究

Celastrol Enhances the Cytotoxicity of Doxorubicin via Inhibiting the Glucose Metabolism in Hepatocellular Carcinoma Cell Line Huh7

目的观察雷公藤红素增强多柔比星对肝癌细胞的杀伤活性及机制。方法设0.5、1、2μmol/L浓度的雷公藤红素为雷公藤红素1、2、3组,设0.1、1g/m L浓度的多柔比星为多柔比星1、2组;MTT法检测多柔比星单独治疗及联合雷公藤红素治疗对肝癌细胞系Huh7的杀伤活性。荧光定量PCR方法检测人正常胎肝细胞系L-O2及肝癌细胞系Huh7、Hep G2和PLC的PKM2表达水平。MTT法检测PKM2表达载体转染对雷公藤红素联合多柔比星杀伤Huh7细胞疗效的影响。结果与对照组Huh7细胞活力零抑制率比较,雷公藤红素1、2、3组分别为(2.6±0.9)%、(4.7±1.3)%(P均<0.05)、(8.8±1.9)%(P<0.05);多柔比星1、2组分别为(7.5±1.9)%、(61.4±4.2)%(P均<0.05);1μmol/L雷公藤红素联合0.1、1g/m L多柔比星较多柔比星1、2组,Huh7细胞活力抑制率明显上升[(58.7±3.8)%比(7.5±1.9)%,P<0.05;(89.7±6.7)%比(61.4±4.2)%,P<0.05]。PKM2相对表达水平,相对人正常肝细胞系L-O2细胞系的(1.0±0.05),Huh7细胞系为(15.2±0.6)(P<0.05),Hep G2细胞系为(11.8±0.5)(P<0.05),PLC细胞系为(13.4±0.7)(P<0.05);雷公藤红素1、2、3组分别下调为(0.70±0.05)(P<0.05)、(0.42±0.04)(P<0.05)、(0.31±0.03)(P<0.05);多柔比星组无下调作用;1μmol/L雷公藤红素联合0.1、1g/m L多柔比星后PKM2相对表达水平较多柔比星1、2组明显下降[(0.44±0.04)比(0.98±0.07),P<0.05;(0.41±0.03)比(1.01±0.07),P<0.05]。转染PKM2表达载体后,1μmol/L雷公藤红素联合0.1、1g/m L多柔比星对Huh7细胞活力抑制率较未转染组下降[(60.5±4.3)%比(19.3±2.0)%,P<0.05;(91.6±6.9)%比(69.6±4.5)%,P<0.05]。结论雷公藤红素通过下调PKM2的表达抑制肿瘤细胞的糖代谢,增强多柔比星对肝癌细胞系Huh7的杀伤活性。

Objective To investigate the effect of celastrol on doxorubicin therapy in hepatocellular carcinoma（HCC） in vitro. Methods HCC cell line Huh7 cells were treated with celastrol alone at 0.5, 1, 2 μmol/L or doxorubicin alone at concentration of 0.1 and 1g/m L or the combination of celastrol（1μmol/L） and doxorubicin（0.1and 1g/m L）. MTT assay was performed to determine the viability inhibition of doxorubicin plus celastrolon Huh7 cells. RT-q PCR analysis was performed to detect the expression of PKM2 in normal liver cell line L-O2 and HCC cell lines Huh7, Hep G2, and PLC. PKM2 expression vector was constructed and then transfected into Huh7 cells treated with celastrol plus doxorubicin. The effect of PKM2 vectors on celastrol plus doxorubicin therapy was detected by MTT assay. Results The Huh7 cell viability inhibition rate was 2.6% ±0.9% in 0.5μmol/L celastrol group, 4.7% ±1.3% in 1μmol/L celastrol group, 8.8% ±1.9% in 2μmol/L celastrol group; 7.5% ±1.9% in 0.1μg/m L doxorubicin single group, 61.4%±4.2% in 1μg/m L doxorubicin single group, all with a significant difference to that of control group（all P 〈0.05）. Compared with doxorubicin single group, the cell viability inhibition rate increased to58.7% ±3.8% in 1μmol/L celastrol ＋0.1μg/m L doxorubicin group and 89.7% ±6.7% in 1μmol/L celastrol ＋1μg/m L doxorubicin group（P 〈0.05）. The relative expression of PKM2 was as follows： 1.0 ±0.05 on L-O2, 15.2 ±0.6 on Huh7, 11.8±0.5 on Hep G2, and 13.4±0.7 on PLC（P 〈0.05）. Celastrol decreased the expression of PKM2 on Huh7（0.70±0.05）, Hep G2（0.42±0.04）, and PLC（0.31±0.03） cells（P 〈0.05）. No effect was observed in doxorubicin single groups, but the expressions of PKM2 in 1μmol/L celastrol＋0.1μg/m L doxorubicin group and 1μmol/L celastrol＋1μg/m L doxorubicin group were lower than those of doxorubicin single group（0.44 ±0.04 vs 0.98 ±0.07; 0.41 ±0.03 vs1.01±0.07; all P 〈0.05）. The synergism of celastrol to doxorubicin was inhibited after the transfection of PKM2, and the cell viability inhibition rate was as follows： 60.5% ±4.3% in 1 μmol/L celastrol ＋0.1μg/m L doxorubicin group,91.6%±6.9% in 1μmol/L celastrol＋1μg/m L doxorubicin group, 19.3%±2.0% in 1μmol/L celastrol＋0.1μg/m L doxorubicin＋pc DNA3.1-PKM2 group, 69.6% ±4.5% in 1μmol/L celastrol＋1μg/m L doxorubicin ＋pc DNA3.1-PKM2 group（P 〈0.05）. Conclusion Celastrol enhances the cytotoxicity of doxorubicin via inhibiting the glucose metabolism in HCC cell line Huh7. This may relate to the downregulation of PKM2 in Huh7 cells.