An Experimental Study On The Regulation Effect Of Nm-23-H1 Gene For PKC Signal Pathway In Human High-metastasis Large Cell Lung Carcinoma Cell Line L9981 And Its Molecular Mechanism

At present, lung cancer has become the most frenquent malignant tumor in the world which its mortality and morbidity is increasing fastest. It becomes NO.l in leading malignant tumors, which is harmful to human health and life, also is the first killer in all cancers. Tumor metastasis is not only the malignant marker and charateristics of lung cancer, but also the key cause of failure to cure and high mortality in lung cancer. Studying and understanding the molecular mechanism of its invasion and metastasis provide a new targeting molecules and pathway for retro-transduction of the signal pathway. Our research group guided by Dr. Zhou QING Hua, keep on studying in this area for a long time. In order to explore the molecular mechanism of nm23-Hl gene for reversing metastasis phenotype in lung cancer, we have first established three cell lines in the world: L9981, L9981-pLXSN and L9981-nm23-Hl. A expression plasmid of pLXSN-nm23-Hl-EGFP with EGFP and wild nm23-Hl gene has been successfully constructed and transfected into the L9981 cell line. We haveproved that low expression and hetero-deletion of tumor metastasis suppress gene nm23-Hl to be correlated with the high metastasis ability and poor prognosis in patient with lung cancer. Nm23-H1 gene is a key and upstream regulative gene in "lung cancer metastatic suppressive cascade". Transfecting wild type nm23-Hl cDNA into lung cancer cell L9981, which has been proved to has nm23-Hl hetero-deletion, can regulate the expression of metastatic relative gene and reverse the metastatic phenotype of lung cancer. The regulative effection of nm23-Hl may be through some importent signaling pathways. On the basis of our previous research results, the changes of PKC located in subcellular areas and the change of [Ca²⁺] in cytosol and biological behavior were observed in L9981, L9981-pLXSN and L9981-nm23-Hl human high-metastic large cell lung cancer cell lines, and the molecular mechanisms of nm23-Hl for regulating PKC signal pathway before and after transfecting with nm23-Hl gene. The results first showed in the world as follows:1. The expression of PKC α , PKC β â…¡ in L9981 and L9981- pLXSN cytoplasm,which was in activate states, is remarkably higher than those in L9981-nm23-Hl cell line (P<0.001); while expression of PKC α , PKC β â…¡ in cytoslol in L9981 and L9981-pLXSN cell lines.which is in inactivate states, was lower than those in L9981-nm23-H1 cell line (P<0.001).It means that the PKC signal pathway was activated in L9981 and L9981-pLXSN cell lines.2. The expression of PKC α ,PKC β â…¡ in cytosol in L9981-nm23-H1 cell line was remarkably higher than those in L9981 and L9981-pLXSN cell lines (P<0.001), while expression of PKCα . PKCβ â…¡in cytosplasma in L9981-nm23-H1 cell line,which is in activate states, was significantly lower than those in L9981 and L9981- pLXSN cell lines (P<0.001).It means that the PKC signal pathway was inactivated in L9981-nm23-Hl cell lines.3. The expression of PKC α , PKC β â…¡ in cytoplasm in L9981, L9981-pLXSN and L9981-nm23-Hl was downregulated after treating with PKC inhibitor Calphostin C (P<0.00l)4. PKC a , PKC P II mainly located in nucleus and perinucleus in L9981 and L9981-pLXSN ,which was in active status;5. In L9981-nm23-Hl cell line, which was transfected nm23-Hl gene, PKC a ., PKC ^ II mainly located in soluble cytosolic section, which was in inactive status.6. PKC a ? PKC 3 II mainly located in cytosolic site in all the three cell lines after treatment with PKC inhibitor Calphostin C, which was mainly in inactive status7. In L9981 and L9981-pLXSN cell lines, which untransfected nm23-Hl gene, the Ca2+ concentration in cells was obviously higher than that in L9981-nm23-Hl cell line (PO.01).8. The Ca2+ concentration was found to decrease in all the three cell lines after treating with PKC inhibitor Calphostin C.Conclusion: (1) Transfection of nm23-Hl gene can remarkably suppress PKC signal transduction. It may be releated to the effect of nm23-Hl on translocation of PKC and decrease of Ca2+ concentration in human high-metastic large cell lung cancer cell line L9981. (2) Both nm23-Hl and Calphostin can suppress PKC signal transduction,which has add and cooperative effects in human high-metastic large cell lung cancer cell line L9981. (3) nm23-Hl gene can significantly inhibit the cell proliferation and invasion ability in L9981 lung cancer line. (4) The effect of nm23-Hl for reversing malignant phenotype might be correlated with downregulation of PKC signal transduction in human high-metastic large cell lung cancer cell line.