苯胺类毒物在非小细胞肺癌细胞中诱导肺耐药蛋白表达和抗肿瘤药物耐受  

Aniline Poison Induce Lung Resistance Protein Expression and Antitumor Drug Resistance in Non-small Cell Lung Cancer Cells

作　　者：车向前[1] 马菲菲[1] 张云[1] 林育红[1] 

机构地区：[1]沈阳军区总医院呼吸与重症医学科,沈阳110016

出　　处：《科学技术与工程》2018年第5期185-189,共5页Science Technology and Engineering

摘　　要：苯胺类毒物(aniline poison)是一类常用化工原料和环境污染物,通过食物或饮水污染进入体内后能够干扰人体正常内分泌系统(endocrinium)稳态,影响细胞遗传物质的稳定最终导致细胞DNA突变增加癌变或畸变的风险。最近的研究表明,苯胺类毒物还有可能影响恶性肿瘤的治疗效果。选取代表性的三个苯胺类毒物:对苯二胺(p-phenylene diamine,p-PDA)、间苯二胺(m-phenylene diamine,m-PDA)、邻苯二胺(o-phenylene diamine,o-PDA),考察其在非小细胞肺癌(non-small cell lung cancer,NSCLC)中诱导肺耐药蛋白(human lung resistance protein,LRP)表达及其可能的分子机制。培养NSCLC细胞系A549,使用系列浓度梯度的对苯二胺(p-phenylene diamine,p-PDA)、间苯二胺(m-phenylene diamine,m-PDA)、邻苯二胺(o-phenylene diamine,o-PDA)处理细胞后,使用实时荧光定量PCR(q PCR)检测LRP的mRNA表达;蛋白印记实验(Western bolt,WB)检测LRP的蛋白表达;进一步使用芳香烃受体(aryl hydrocarbon receptor,AhR)的小干扰RNA(siRNA)抑制A549细胞中AHR的表达,确定对苯二胺、间苯二胺、邻苯二胺对LRP的诱导作用是否依赖于AhR;在此基础上,使用对苯二胺、间苯二胺、邻苯二胺预处理A549细胞后,再分别使用抗肿瘤药物吉非替尼(Gefitinib)、吉西他滨(Gemcitabine)处理A549细胞,利用MTT实验检测上述抗肿瘤药物对A549细胞存活的抑制率(inhibitory rate,IR),并计算药物作用的半数抑制率(IC50值)。q PCR实验与WB实验结果显示,对苯二胺、间苯二胺、邻苯二胺刺激A549细胞能够剂量依赖地诱导LRP的mRNA、蛋白表达水平。干扰AhR表达后,该三种苯胺类毒物不能诱导LRP的mRNA与蛋白表达。MTT实验结果表明,对苯二胺、间苯二胺、邻苯二胺能够下调抗肿瘤药物吉非替尼、吉西他滨对A549细胞的杀伤作用:吉非替尼作用于A549细胞的IC50值从(1.20±0.25)μmol/L上调为(5.67±0.44)μmol/L、(6.07±0.30)μmol/L与(7.51±0.28)μmol/L,其耐�Aniline poison is a kind of common chemical raw materials and environmental pollutants. It can interfere with the homeostasis of endocrine system,affecting the stability of the genetic material and eventually lead to DNA mutation and increase the risk of cancer or distortion. Recent studies have shown that aniline poison may also affect the treatment of malignant tumors. Three representative aniline poison： p-PDA,m-PDA and o-PDA were selected to investigate how they induce the expression of human lung resistance protein（ LRP） in non-small cell lung cancer（ NSCLC） and its possible molecular mechanism. NSCLC cell line A549 was cultured and treated with a series of concentrations of p-PDA,m-PDA and o-PDA. The q PCR-assay was used to detect the mRNA expression of LRP and western blot（ WB） to detect the protein level of LRP. Then the expression of AhR in A549 cells was knocked down by siRNA of AhR to determine whether the induction of LRP by p-PDA,m-PDA and o-PDA was depended on AhR. In this background,A549 cells were pretreated with p-PDA,m-PDA and o-PDA,and then treated with anti-tumor agents Gefitinib and Gemcitabine,respectively. The inhibitory rate（ IR） and IC_（50） value of the drug was calculated. The results from q PCR and WB showed that p-PDA,m-PDA and o-PDA stimulate A549 cells to induce LRP mRNA and protein expression in a dose-dependent manner. After interfering the expression of AhR,these three kinds of aniline poison cannot induce LRP mRNA and protein expression. Results from MTT-assay showed that p-PDA,m-PDA and o-PDA could down-regulate the anti-tumor effect of Gefitinib and Gemcitabine on A549 cells＇ survival： the IC50 values of Gefitinib on A549 cells was up-regulated from（ 1. 20 ± 0. 25） μmol/L to（ 5. 67 ± 0. 44） μmol/L,（ 6. 07 ± 0. 30） μmol/L or（ 7. 51 ± 0. 28） μmol/L. The resistance folds were 4. 73,5. 06 or 6. 26,respectively. The IC50 value of Gemcitabine on A549 cells was up-regulated from（ 0. 44 ± 0. 10）μmol/L to（ 1. 55 ± 0. 25） μmol

关 键 词：非小细胞肺癌 苯胺类毒物 抗肿瘤药物 化疗药耐药 

分 类 号：R996.1[医药卫生—毒理学] R73-34[医药卫生—药学]