吉非替尼可能通过EGFR启动子甲基化诱导肺腺癌细胞继发性耐药

Relationship between EGFR Promoter Region Methylation and Secondary Resistance Which may be Induced by Gefitinib

背景与目的在肺腺癌靶向治疗中的吉非替尼继发性耐药是临床遇到的重要问题。本研究旨在探讨吉非替尼是否可诱导肺腺癌PC9细胞发生继发性耐药,以及表皮生长因子受体(epidermal growth factor receptor,EGFR)启动子甲基化在耐药过程中的作用,拟为肺腺癌耐药提供新的治疗靶点。方法体外培养肺腺癌吉非替尼敏感细胞株PC9,应用不同浓度吉非替尼进行干预。MTT法检测PC9细胞株对吉非替尼的敏感性。亚硫酸氢盐处理后测序法(bisulfite sequencing polymerase chain reaction,BSP)检测肺腺癌PC9细胞株EGFR启动子甲基化水平。使用5-氮杂-2’-脱氧胞苷(5-Aza-dc)对吉非替尼耐药细胞株PC9/GR细胞株进行干预。MTT法检测耐药株PC9/GR对吉非替尼敏感性的变化。结果经过从0.01μmol/L逐渐提升吉非替尼诱导浓度至3μmol/L之后,MTT显示耐药细胞株PC9/GR细胞株半抑制浓度(half maximal inhibitory concentration,IC50)[(3.95±0.23)μmol/L]较之前敏感株PC9[(0.01±0.002)μmol/L]明显升高(P<0.05),BSP显示发生异常甲基化的位点结果比较:PC9/GR甲基化水平明显升高(74%vs59%,P<0.05)。RT-PCR显示PC9/GR细胞株较PC9细胞株EGFR m RNA表达量升高(P<0.05)。使用5-Aza-dc处理PC9/GR细胞后,PC9/GR细胞株IC50较对照组降低[(2.55±0.14)μmol/L vs(3.87±0.034)μmol/L,P<0.05)]。结论吉非替尼体外浓度递增诱导法可诱导PC9细胞株产生吉非替尼继发性耐药,成功构建PC9/GR耐药细胞株。PC9细胞EGFR启动子甲基化异常可能参与了吉非替尼继发性耐药机制。

Background and objective 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 gefitinibresistance in PC9/GR cell lines and to find a new therapeutic target to overcome the gefitinib secondary resistance in lung adenocarcinoma. Methods In vitro cultivation of lung adenocarcinoma PC9 cell lines, apply gefitinib on lung adenocarcinoma PC9 cell lines, and improve drug concentration. MTF for test of gefitinib resistance index in PC9 cell and PC9/GR cell. Bisulrite 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 S-Aza-dc on lung adenocarcinoma PC9/GR cell lines for 72 h. MTT method for test of gefitinib resistance index in PC9/GR cell. Results After improving the gefitinib concentration, MTF 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） [anol/L. Conclusion 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.