白花蛇舌草通过调控Hedgehog通路增加大肠癌耐药细胞的药物蓄积研究

Hedyotis Diffusa Willd Increase Drugs Accumulation by Regulating Hedgehog Pathway in Drug Resistant Colorectal Cancer Cells

目的:探讨白花蛇舌草(HDW)对大肠癌耐药细胞药物外排功能、Hedgehog信号通路调控的影响及其逆转耐药作用机制。方法:采用白花蛇舌草乙醇提取物(EEHDW)对大肠癌5-氟尿嘧啶(5-FU)耐药细胞HCT-8/5-FU进行药物干预,用MTT法检测HCT-8/5-FU的细胞活力,计算EEHDW对细胞增殖的抑制率;倒置显微镜观察EEHDW对HCT-8/5-FU细胞形态的影响;HPLC检测细胞内5-FU蓄积;倒置荧光显微镜观察细胞内阿霉素(ADM)蓄积;Western Blot检测ABC转运蛋白家族多药耐药相关蛋白1(MRP1)、P-糖蛋白(P-gp)、肺耐药蛋白(LRP)、乳腺癌耐药蛋白(BCRP)的表达及Hedgehog通路相关蛋白(SHH、SMO、SUFU、Gli-1)的表达。结果:经EEHDW干预后,HCT-8/5-FU的增殖受到显著抑制,并呈剂量依赖性;细胞内5-FU、ADM蓄积明显增加,提示药物泵功能受到显著抑制(P<0.01);MRP1、P-gp、LRP、BCRP等膜转运蛋白的表达明显下降;耐药相关Hedgehog信号通路受到显著抑制。结论:HDW可通过调控抑制Hedgehog信号通路活化,抑制大肠癌耐药细胞的药物外排功能,进而增加细胞内化疗药物的蓄积,发挥逆转大肠癌细胞耐药的作用。

Objective ：To explore the effect and underlying mechanism of Hedyotis diffusa Willd（HDW） on drug efflux and regulation of Hedgehog（HH） signaling pathway in human colorectal cancer HCT-8/5-FU cells. Methods：HCT-8/5-FU Cells were treated with different concentrations of ethanol extract of HDW（EEHDW）. Cell viability was determined by MTT assay,then the in hibitory rate was calculated to evaluate the effect of EEHDW on cell proliferation. Cell morphology was observed under phase-contrast microscope. Intracellular 5-Fluorouracil（5-FU） and Doxorubicin（ADM） accumulation were analyzed using HPLC and observed by fluorescence microscope,respectively. Western Blot was used to determine the expression of multidrug resistance（MDR） related proteins,such as MRP1,P-gp,LRP,BCRP,and the core proteins of HH signaling pathway,including SHH,SMO,SUFU and Gli-1. Results：After treated with different concentration of EEHDW,proliferation of HCT-8/5-FU cells were inhibited in a dose dependent（P〈0.01）. Cell density decreased with the increase concentration of EEHDW. Accumulation of intracellular 5-FU and ADM significantly increased in HCT-8/5-FU cells. The expression of MRP1,P-gp,LRP and BCRP decreased markedly,and activation of Hedgehog signaling pathway was significantly inhibited. Conclusion：HDW could inhibit drug efflux of human colorectal cancer HCT-8/5-FU cells and increase the accumulation of 5-FU and ADM by inhibiting the activation of HH signaling pathway,which play a role in reversing the drug resistance of colorectal cancer cells.