Wang Qilong, Li Min, Hu Chengping
Department of Respiratory Medicine, Xiangya Hospital of Central South University, Changsha 410008, China.
Zhongguo Fei Ai Za Zhi. 2015 Apr;18(4):193-8. doi: 10.3779/j.issn.1009-3419.2015.04.04.
Nowadays the secondary resistance of gefitinib in the treatment of lung adenocarcinoma is an outstanding problem. This research is to explore whether the gefitinib secondary resistance can be induced by gefitinib, to explore whether epidermal growth factor receptor (EGFR) promotor methylation correlate with the gefitinib-resistance in PC9/GR cell lines and to find a new therapeutic target to overcome the gefitinib secondary resistance in lung adenocarcinoma.
In vitro cultivation of lung adenocarcinoma PC9 cell lines, apply gefitinib on lung adenocarcinoma PC9 cell lines, and improve drug concentration. MTT for test of gefitinib resistance index in PC9 cell and PC9/GR cell. Bisulfite sequencing polymerase chain reaction (BSP) and Reverse transcription-polymerase chain reaction (RT-PCR) for detection of EGFR promoter methylation status and mRNA expression. In vitro cultivation of lung adenocarcinoma PC9 cell lines, apply 1 μmol/L 5-Aza-dc on lung adenocarcinoma PC9/GR cell lines for 72 h. MTT method for test of gefitinib resistance index in PC9/GR cell.
After improving the gefitinib concentration, MTT results showed that half maximal inhibitory concentration (IC50) of PC9 cell lines increase from (0.01 ± 0.002) μmol/L to (3.95 ± 0.23) μmol/L (P<0.05). BSP results showed that abnormal methylation sites compared the degree of methylation change: PC9: 59%; PC9/GR: 74% (P<0.05). RT-PCR results showed in PC9/GR cell lines, EGFR mRNA expression quantity increased (P<0.05). After applying 5-Aza-dc on PC9 cell lines, IC50 of PC9/GR decrease from (3.87 ± 0.034) μmol/L to (2.55 ± 0.14) μmol/L.
The PC9 cell line which is induced by improving gefitinib concentration will be resistant to gefitinib, and the gefitinib-resistant cell line PC9/GR could be built. EGFR gene promoter methylation may be one of the mechanisms for the secondary resistance to gefitinib.
目前，吉非替尼治疗肺腺癌的继发性耐药是一个突出问题。本研究旨在探讨吉非替尼是否能诱导其自身继发性耐药，探究表皮生长因子受体（EGFR）启动子甲基化与PC9/GR细胞系中吉非替尼耐药性的相关性，并寻找克服肺腺癌吉非替尼继发性耐药的新治疗靶点。
体外培养肺腺癌PC9细胞系，对肺腺癌PC9细胞系应用吉非替尼，并提高药物浓度。采用MTT法检测PC9细胞和PC9/GR细胞的吉非替尼耐药指数。采用亚硫酸氢盐测序聚合酶链反应（BSP）和逆转录聚合酶链反应（RT-PCR）检测EGFR启动子甲基化状态和mRNA表达。体外培养肺腺癌PC9细胞系，对肺腺癌PC9/GR细胞系应用1μmol/L 5-氮杂-2'-脱氧胞苷（5-Aza-dc）处理72小时。采用MTT法检测PC9/GR细胞的吉非替尼耐药指数。
提高吉非替尼浓度后，MTT结果显示PC9细胞系的半数最大抑制浓度（IC50）从（0.01±0.002）μmol/L增加至（3.95±0.23）μmol/L（P<0.05）。BSP结果显示，异常甲基化位点比较甲基化变化程度：PC9为59%；PC9/GR为74%（P<0.05）。RT-PCR结果显示，PC9/GR细胞系中EGFR mRNA表达量增加（P<0.05）。对PC9细胞系应用5-Aza-dc后，PC9/GR的IC50从（3.87±0.034）μmol/L降至（2.55±0.14）μmol/L。
通过提高吉非替尼浓度诱导的PC9细胞系对吉非替尼产生耐药，可构建吉非替尼耐药细胞系PC9/GR。EGFR基因启动子甲基化可能是吉非替尼继发性耐药的机制之一。
