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To study the molecular mechanisms of nm23-H1 for regulating PKC signal pathway before and after transfection with nm23-H1 gene.Using Western-blot, Boyden-chamber, MTT and laser scanning confocal microscopy (LSCM) techniques to detect the distribution of PKC in cytosol and plasma membrane, changes of invasion and proliferation activity, PKC translocation status and changes of intracellular Ca(2+) concentration among different human pulmonary carcinoma cells with transfected or untransfected nm23-H1 gene, and changes of the three cell lines after treatment with Calphostin C, a PKC inhibitor.(1) The expression of PKCalpha, PKCbeta II on L9981 and L9981-pLXSN cell membrane, which was in activated status, was remarkably higher than those in L9981-nm23-H1 cell line (P0.001). The expression of PKCalpha, PKCbeta II in cytosol in L9981 and L9981-pLXSN cell lines, which was in inactivated status, was lower than those in L9981-nm23-H1 cell line (P0.001). It means that the PKC signal pathway was activated in L9981 and L9981-pLXSN cell lines. (2) PKCalpha and PKCbeta II mainly located in nuclei and perinuclear area in L9981 and L9981-pLXSN cells, which were in active status, and the Ca(2+) concentration in these cells was obviously higher than that in L9981-nm23-H1 cell line (P0.01). In L9981-nm23-H1 cell line, which was transfected with nm23-H1 gene, PKCalpha and PKCbeta II mainly located in soluble cytosolic section, in an inactive status. (3) The invasion and proliferation ability of L9981 and L9981-pLXSN lung cancer cells was higher than that of L9981-nm23-H1 cell line (P0.001). There was no statistically significant difference between L9981 and L9981-pLXSN cell lines (P0.05). (4) After treated with PKC inhibitor Calphstin C, the expression of PKC and PKCbeta II in membrane in L9981 and L9981-pLXSN cell lines was down-regulated (P0.001), PKCalpha and PKCbeta II were mainly located in cytosolic area, mainly in an inactive status, and the Ca(2+) concentration was found to be decreased in all the three cell lines. The invasion and proliferation ability of the three lung cancer cell lines were obviously decreasing (P0.001). However, the invasion and proliferation ability of L9981-nm23-H1 lung cancer cell line was still lower than that of L9981 and L9981-pLXSN lung cancer cell lines (P0.001). There was also no significant difference between L9981 and L9981-pLXSN cell lines (P0.05).The results of this study suggest that nm23-H1 gene might inhibit the invasion and metastasis of lung cancer cells by down-regulating PKC signaling pathway. The Ca(2+) in cells might be involved in this process. |
