Connexin 43对非小细胞肺癌细胞吉非替尼原发性耐药的影响

Effect of connexin 43 on the primary resistance to gefitinib in non-small-cell lung cancer cells

目的:探讨缝隙连接蛋白43(connexin 43,Cx43)对非小细胞肺癌(non-small-cell lung cancer,NSCLC)吉非替尼原发性耐药的影响。方法:MTT法测定吉非替尼对3株非突变型EGFR(WT-EGFR)NSCLC细胞系A549、H1299、Calu3细胞的半数抑制浓度(IC50);Western blot观察Cx43、磷酸化Akt(phospho-Akt,p-Akt)蛋白在上述细胞系中的表达水平;"Parachute"法检测细胞缝隙连接功能(gap junction intercellular communication,GJIC);免疫荧光法检测Cx43蛋白在细胞中的定位。结果:吉非替尼作用于Calu3、A549、H1299细胞的IC50分别为(0.064±0.011)μmol/L、(13.64±0.015)μmol/L、(20.054±0.012)μmol/L,即A549、H1299细胞对吉非替尼原发性耐药;A549、H1299细胞中Cx43、p-Akt蛋白水平显著高于Calu3细胞(P<0.01);A549、H1299细胞有荧光传递现象,且Cx43蛋白定位在A549、H1299细胞膜上,而Calu3细胞无荧光传递现象,且Cx43主要表达于细胞质上。结论:NSCLC细胞膜上调的Cx43及其组成的GJIC可促进细胞对吉非替尼的原发性耐药,其机制可能与其激活PI3K/Akt信号通路有关。

Objective ：To investigate relationship between eonnexin 43 （Cx43） expression and non-small-cell lung cancer（NSCLC） cells with primary resistance to gefitinib. Methods：Gefitinib efficacy in A549,H1299 cells and Calu3 cells was detected by MTr as- say. The protein expression of Cx43 and phospho-Akt（p-Akt） was detected by Western blot. The functional gap junction intercellular eommunication（GJIC） was measured by ＇.parachute＇ assay. The cellular localization of Cx43 protein was evaluated by immunotluores- eenee staining. Results：MTr assay showed that gefitinib eytotoxieity in Calu3 cells is significantly more than that in A549 and H1299 cells with IC50 of （0.064 ±0.011） μmol/L versus（13.64 ± 0.015） μmol/L and （20.054 ± 0.012） μmol/L,indicating that A549 and H1299 cells were primary resistance to gefitinib. Expression of Cx43 in Calu3 cells was significantly lower than that in A549 and H1299 cells（P〈0.01）. Moreover,p-Akt protein in Calu3 cells was obviously lower than that in A549 and H1299 cells（P〈0.01）. Fur- thermore, functional GJIC was found in A549 and H1299 with or without RA（a well-defined GJIC enhancer） treatment, but not in Calu3 cells. Immunofluorescence staining clearly showed that Cx43 protein deferentially accumulates at＇the plasma membrane of A549 cells, the plasma membrane and cytoplasm of H1299 cells while the cytoplasm of Calu3 cells. Conclusion：Up-regulation of Cx43 at the plasma membrane and their derived GJIC in A549 and H1299 cells may contribute to the primary resistance to gefitinib by activation of PI3K/Akt signaling pathway.