目的：探讨缝隙连接蛋白43(connexin 43,Cx43)与非小细胞肺癌( NSCLC)细胞产生吉非替尼( Gefitinib)获得性耐药的关系。方法在Gefitinib敏感细胞株HCC827上,通过逐步递增 Gefitinib 浓度诱导获得 Gefitinib 耐药细胞株HCC827 GR；MTT 法检测 Gefitinib 对 HCC827/GR 细胞的IC50；RT-PCR 检测 Cx43的 mRNA 水平；Western blot 检测Cx43和磷酸化Akt(p-Akt)的蛋白水平；parachute荧光示踪法检测细胞缝隙连接功能( gap junction intercellular communi-cation, GJIC)；免疫荧光技术检测 Cx43的细胞定位。结果Gefitinib 作用于 HCC827和 HCC827 GR 的 IC50分别为(0．07±0．019)μmol·L-1、(10．84±0．021)μmol·L-1( P<0．01)；HCC827 GR 中 Cx43的 mRNA 和蛋白水平较HCC827明显降低( P<0．05),但 p-Akt蛋白水平明显升高(P<0．05)。在HCC827 GR细胞上加PI3K的特异性抑制剂LY294002(25μmol·L-1,24 h)后,p-Akt蛋白水平明显下降(P<0．01),且Cx43的蛋白水平明显升高(P<0．01)。HCC827及HCC827 GR均未检测到GJIC,用GJIC增强剂RA (retinoic acid)处理(10μmol·L-1,24 h)上述细胞,亦未检测到荧光传递,免疫荧光结果显示 Cx43表达在细胞胞质。结论胞质中Cx43的下调可能促进NSCLC细胞对Gefitinib的获得性耐药,其机制可能与Cx43非GJIC依赖的PI3K/Akt信号通路激活有关。%Aim To explore the effect of connexin 43 ( Cx 4 3 ) on acquired gefitinib-resistance in human non small cell lung cancer ( NSCLC ) . Methods HCC827 GR, a gefitinib-resistant ( GR) NSCLC cell lines from their parental cells was established by gradually in-creasing the concentration of gefitinib. Gefitinib effica-cy in HCC827 and HCC827 GR cells was detected by MTT assay. Expression of Cx43 mRNA in HCC827 and HCC827 GR cells was determined by RT-PCR. The protein expressions of Cx43 and phospho-Akt ( p-Akt) in these cells were detected by Western blot. The func-tional gap junction intercellular communication ( GJIC ) was measured by parachute assay. The cellular locali-zation of Cx43 protein was evaluated by immunofluores-cence staining. Results MTT assay showed less ge-fitinib cytotoxicity in HCC827 GR cells than that in their parental cells with IC50 of (10. 84 ± 0. 021) μmol ·L-1 versus (0. 07 ± 0. 019) μmol·L-1 , respective-ly. Moreover, both mRNA and protein expressions of Cx43 in HCC827 GR cells were significantly lower than those in HCC827 cells ( P<0. 05 ) . However, the p-Akt protein in HCC827 GR cells was obviously higher than that in HCC827 cells ( P<0. 05 ) . Furthermore, treatment with LY294002 caused a significant reduced p-Akt expression, but a significant increased Cx43 ex-pression in HCC827 GR cells. Moreover, no detecta-ble GJIC was found in HCC827 and their GR cells with or without RA ( a well-defined GJIC enhancer ) treat-ment. Immunofluorescence staining clearly showed that Cx43 protein accumulated in the cytoplasm of HCC827 and their GR cells. Conclusion The down-regulation of Cx43 expression in cytoplasm of HCC827 GR cells may contribute to the acquired gefitinib resistance in NSCLC cells by GJIC-independent activation of PI3 K/Akt signaling pathway.
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