PAK1在康莱特联合5-氟脲嘧啶对Hepal-6肝癌细胞化疗增效中的作用

The Role of PAK1 in the Chemotherapy Efficiency of Hepal-6 Cell Induced by Kanglaite and 5-Fluorouracil

目的观察康莱特对化疗药物5-氟脲嘧啶(5-Fu)的最佳增效时间和最佳增效浓度,并探讨其发挥化疗增效作用的机制。方法采用MTT比色法观察不同浓度康莱特联合5-Fu对Hepal-6肝癌细胞的生长抑制作用,康莱特作用浓度分别设10、30、50mL/L,作用时间分别设5-Fu作用前0、8、16、24h和作用后8、16、24h,采用RT-PCR法检测PAK1mRNA的表达,细胞划痕愈合实验测定细胞扩散运动能力。结果不同浓度康莱特联合5-Fu给药组细胞生长抑制率明显高于5-Fu单药组(P<0.05),且康莱特的增效作用随其剂量的增加而增加(P<0.05),50mL/L康莱特于5-Fu作用前8h加入增效作用最佳(P<0.01)。正常肝细胞仅表达少量PAK1mRNA,康莱特8h-5-Fu给药组较肝癌细胞组、5-Fu单药组能明显下调Hepal-6肝癌细胞PAK1mRNA的表达(P<0.01),但仍较正常肝细胞组高。康莱特8h-5-Fu给药组较肝癌细胞组、5-Fu单药组能显著减弱细胞移行运动能力。结论康莱特以50mL/L于5-Fu作用前8h加入,化疗增效作用最佳,康莱特主要通过减少PAK1基因的表达,减弱细胞移行运动能力,从而发挥增加5-Fu化疗效果的作用。

Objective To observe the best enhancement time and concentration of kanglaite on chemotherapeutic response of 5-fluorouracil（5-Fu） and to explore the mechanism of chemosensitization.Methods MTT method was used to test the growth inhibitory rate of hepatocellular carcinoma cell Hepal-6 acted by kanglaite and 5-Fu.The concentration of kanglaite was 10,30,50 mL/L and the time point was 0,8,16,24 h before 5-Fu,and 8,16,24 h after 5-Fu.The expressions of PAK1 mRNA were detected by RT-PCR,monolayer wound healing assay was used to detect Hepal-6 cell motility.Results The inhibitory rates of cell growth in the kanglaite combination groups were significantly higher than that in the 5-Fu alone group（P 0.05）,and the enhancement increased with kanglaite dosage augment.50 mL/L kanglaite could exert the best enhancement on chemotherapeutic response of 5-Fu when it was added 8 h before 5-Fu（P 0.01）.The expression of PAK1 mRNA in the normal group was poor,the relative level of PAK1 mRNA in the kanglaite 8 h-5-Fu group was significantly decreased compared with the hepatocellular carcinoma group,the 5-Fu alone group（P 0.01）,but still higher than that in the normal group.The kanglaite 8 h-5-Fu group can weaken the cell migration ability significantly compared with the hepatocellular carcinoma group,the 5-Fu alone group.Conclusion Kanglaite could exert the best enhancement with 50 mL/L when added 8 h before 5-Fu.PAK1 plays an important role in the enhancement of kanglaite on the chemotherapeutic response of 5-Fu.