塑化剂双酚A在肺癌细胞中诱导肺耐药蛋白的表达和耐药作用的体外实验

Bisphenol A induces expression of LRP and MDR process in lung cancer cells in vitro

目的探讨塑化剂双酚A(bisphenol A,BPA)对肺癌细胞中人肺耐药蛋白(lung resistance protein,LRP)和化疗药多药耐药作用(multi-drug resistance,MDR)的影响。方法利用系列浓度梯度的BPA处理肺癌细胞系A549,酶联免疫印迹实验(ELISA)检测BPA对LRP表达的影响,计算其作用的EC_(50)值。使用EC_(50)浓度的BPA预处理A549细胞后,配置系列浓度梯度的抗肿瘤药物处理A549细胞,计算其杀伤细胞的IC50值。蛋白印迹实验(Western blot)验证EC_(50)浓度的BPA对LRP蛋白表达的诱导作用,以及LRP在耐药细胞株A549/ADR中的表达水平。进一步实验使用脂质体转染LRP的小干扰RNA(siRNA)降低LRP蛋白的表达,讨论LRP在肺癌细胞MDR中的意义。结果 ELISA实验结果显示,BPA诱导A549细胞中LRP蛋白表达的EC_(50)值为(0.55±0.12)μmol/L;EC_(50)值的BPA能够下调抗肿瘤药物紫杉醇(Paclitaxel)、吉西他滨(Gemcitabine)和吉非替尼(Gefitinib)对A549细胞的杀伤作用,其IC50值从(0.08±0.01)μmol/L、(0.45±0.05)μmol/L和(1.36±0.22)μmol/L上调为(0.67±0.11)μmol/L、(2.75±0.33)μmol/L和(5.82±0.41)μmol/L,诱导的耐药倍数为7.12、6.11和4.28;Western blot实验的结果显示,A549/ADR细胞中LRP蛋白的表达水平显著高于A549。利用siRNA降低LRP的表达能够诱导A549/ADR细胞对Paclitaxel、Gemcitabine和Gefitinib的敏感性,IC50值从(0.59±0.07)μmol/L、(3.95±0.66)μmol/L和(8.72±0.71)μmol/L下调至(0.15±0.06)μmol/L、(0.86±0.14)μmol/L和(1.99±0.54)μmol/L。结论 BPA能够在肺癌细胞中诱导LRP蛋白的表达,是肺癌MDR的潜在诱导因素。

Objective To declare whether BPA（Bisphenol A） induces the expression of LRP（lung resistance protein） and MDR（multi-drug resistance） process in vitro. Methods Series concentration gradient of BPA was used in dose-effect experiments inducing LRP expression. Then, ELISA assay was used to identify the effect of BPA on LRP and the EC50 value（50% effective concentration） was calculated. The inhibition rate of anti-tumor drugs on A549 or A549/ADR was calculated by A490 nm from MTT assays and IC50 values（50% inhibitory concentration） were calculated. Protein level of LRP induced by BPA with EC50 concentration and the expression of LRP in A549/ADR were detected by western blot analysis. The small interfering RNA（siR NA） of LRP was transfected into A549/ADR cells to downregulate protein level of LRP, and its effect on MDR was investigated. Results Results from ELISA showed that BPA induced the expression of LRP in a dose-dependent manner（EC50=0.55±0.12 μmol/L, R^2=0.96, P=0.001 1）, and BPA（EC50 concentration） could downregulate the effects of Paclitaxel, Gemcitabine and Gefitinib to A549 with significant increase of IC50 value [（0.08±0.01） μmol/L vs（0.67±0.11） μmol/L, P〈0.05;（0.45±0.05） μmol/L vs（2.75±0.33） μmol/L, P〈0.05;（1.36±0.22） μmol/L vs（5.82±0.41） μmol/L, P〈0.05] and drug resistance of 7.12, 6.11, 4.28 times. In addition, Western blot analysis showed that high level of LRP was detected in A549/ADR cells compared with A549 cells. Transfection of LRP siR NA downregulated LRP expression and enhanced the sensitivity of A549/ADR cells to Paclitaxel [（0.59±0.07） μmol/L vs（0.15±0.06） μmol/L, P〈0.05], Gemcitabine [（3.95±0.66） μmol/L vs（0.86±0.14） μmol/L, P〈0.05] or Gefitinib [（8.72±0.71） μmol/L vs（1.99±0.54） μmol/L, P〈0.05]. Conclusion BPA can induce the expression of LRP and will participate in the MDR process in human lung cancer cells.