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.
ABSTRACT
Aim To investigate effects of Cx26 on pro-liferation,apoptosis,migration of human highly meta-static hepatocellular carcinoma HCCLM3 cells.Meth-ods The HCCLM3 cells were infected with lentiviral vector targeting interference of Cx26 and the stable transfectants were selected by puromycin.The interfer-ence efficiency of Cx26 was detected by Real-time PCR and Western blot.Gap junction function was assessed by “parachute”dye-coupling assay. The effects of Cx26 interference on proliferation,apoptosis,migra-tion of HCCLM3 cells were determined by MTT assay, flow cytometry,transwell migration assay,respective-ly.Results The mRNA and protein expression of Cx26 in LV-Cx26 group was significantly lower than the LV-NC group and wide group (P<0.01),and GJ function in LV-Cx26 group also significantly reduced compared with LV-NC group and wild group (P <0.01 ).The Cx26 interference significantly inhibited the proliferation (P<0.01)),promoted the apoptosis (P <0.01 ),and decreased migration of HCCLM3 cells in vitro (P <0.01 ).Conclusion Lentiviral vector targeting interference Cx26 expression of HC-CLM3 cells can significantly reduce its GJ function,in-hibit the proliferation and migration,promote apopto-sis,and reduce its malignant properties.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the effect of lentivirus-mediated shRNA interference of Cx26 on epithelial-mesenchymal transition (EMT) and invasion of highly invasive human hepatocellular carcinoma cells in vitro.</p><p><b>METHODS</b>SK-Hep-1 cells were infected with the lentivirus for delivering Cx26 shRNA, and the stably transfected cells were selected by puromycin. The interference efficiency of shRNA-Cx26 was assessed with real-time PCR and Western blotting. The morphological changes of the transfected SK-Hep-1 cells were observed microscopically, and the protein expressions of E-cadherin and vimentin were detected using Western blotting. The effect of Cx26 interference on the invasiveness of SK-Hep-1 cells was determined by Transwell invasion assay.</p><p><b>RESULTS</b>Compared with SK-Hep-1 cells infected with empty EGFP vector and uninfected cells, the cells transfected with shRNA-Cx26 showed significantly reduced mRNA and protein expressions of Cx26 (P<0.01), which resulted in obvious morphological conversion from mesenchymal cells to epithelial cells. shRNA-Cx26-transfected cells showed significantly increased E-cadherin protein expression (P<0.01) but decreased vimentin expression (P<0.01) with obviously attenuated invasive ability in vitro (P<0.01).</p><p><b>CONCLUSION</b>Targeted down-regulation of Cx26 expression can inhibit the EMT and invasion of SK-Hep-1 cells in vitro.</p>