小白菊内酯增强人肺腺癌A549细胞对顺铂敏感性实验研究

Experimental investigation of parthenolide enhancing the sensitivity of lung adenocarcinoma A549 cells to cisplatin

目的肺恶性肿瘤无论是发病还是致死率都居高不下,传统化学疗法通常会随着治疗的推移而产生耐药,出现化疗后毒副作用,因此寻找增强顺铂敏感性的药物,对于减少顺铂的使用剂量从而降低顺铂的毒副作用具有重要的临床价值。本研究拟从分子生物水平,探讨小白菊内酯(Parthenolide,PTL)是否具有增强肺腺癌A549细胞对顺铂(DDP)的敏感性。方法 A549细胞经PTL和(或)DDP处理后,以噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法检测细胞活力,流式细胞术(flow cytometry,FCM)法检测细胞凋亡及细胞周期,实时荧光定量PCR(reverse transcription PCR,RT-PCR)法检测Bcl-2和Bax的mRNA表达情况。结果 MTT检测结果显示,小剂量PTL(<10μmol/L)有促A549细胞增殖作用(P<0.05),大剂量PTL(>10μmol/L)可抑制A549细胞增殖(P<0.05);且大剂量PTL(15和20μmol/L)与DDP(2mg/L)联合能够明显抑制人肺腺癌A549细胞的增殖,与单独用药组相比有明显的协同抑制作用,q值分别为1.181和1.206,P<0.05。FCM法检测结果显示,PTL(15μmol/L)与DDP(2mg/L)联合作用于A549细胞,A549细胞凋亡率为24.45%,较对照组的0.91%及单独用药组的2.8%和3.07%增加;PTL(15μmol/L)和DDP(2 mg/L)联合组G1+S期细胞比例为81.10%,较对照组的60.75%及单独用药组的77.91%和70.83%增加。RT-PCR检测结果显示,PTL(15μmol/L)和DDP(2mg/L)联合后,A549细胞的抗凋亡基因Bcl-2的相对表达为0.015±0.002,较对照组及单独用药组的1、23.873±0.76和2.925±0.146显著下调,P<0.05;凋亡基因Bax的表达为32.175±1.651,较对照组及单独用药组的1、0.284±0.009和0.732±0.022显著上调,P<0.05。结论大剂量PTL具有抗肿瘤作用,可增强人肺腺癌A549细胞对DDP敏感性,且PTL(10、15和20μmol/L)与DDP(2mg/L)联合后具有协同作用,其作用机制可能与增加G1+S期细胞比例、诱导细胞凋亡及降低Bcl-2/Bax mRNA比例有关。

OBJECTIVE This study was to evaluate the coordination of parthenolide （PTL） with cisplatln（DDP） on lung adenocarcinoma A549 ceils from the level of molecular biology. METHODS After treated by parthenolide and （or） cisplatin,the viability of cell lines was detected through microscope and MTT. The apoptosis of cell lines and cell cycle was detected by flow cytometry（FCM）. The expressions of Bcl-2 and Bax mRNAs were measured by reverse transcription-PCR. RESULTS Determined by MTT/FCM/PCR, the results showed that small doses（~10μmol/L） of parthenol-ide had an effect on promoting proliferation of A549 cells, high dose（〉10μmol/L） of parthenolide could inhibit proliferation of A549 cells compared to control group or single medication group （P〈0. 05）. The apoptotic rate （24. 45%）was significantly increased in A549 cells treated with PTL in combination with cisplatin, compared with that in cisplatin-treated（3.07%） or PTL-treated（2.8%） A549 cells, and the cell cycle（8l. 10%） was arrested at phase G1＋S vs cisplatin-treated（77.91%） or PTL-treated（70.83）. The expressionlevel of Bcl-2 protein was decreased（P〈0.05） and the expression level of Bax was increased （P〈0.05） in a concentration-dependent manner in A549 cells treated with different drugs, and these effects（0. 015±0. 002, 32. 175±1. 651） were significantly enhanced in A549 cells treated with PTL in combination with cisplatin compared with cells treated with eisplatin（0. 284±0. 009, 23. 873±0. 76） or PTL（0. 732±0.022, 2.92±0. 146） alone （P〈0.05）. CONCLUSION Parthenolide （10, 15, 20μmol/L） and eisplatin（2 mg/L） have synergistic effects on inhibiting cellular proliferation and inducing cellular apoptosis of lung adenocarcinoma A549 cells,which may be associated with the increased proportion of G1＋S phase cells, inducing apoptosis and decreasing the ratio of Bcl-2/Bax mRNAs.