Effect of Cx43 on acquired gefitinib-resistance mechanisms in human NSCLC HCC827 cells / 中国药理学通报

ABSTRACT

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.