An Experimental Study On The Regulation Effect Of The Nm23-H1 Gene For PKA Signal Pathway Of Lung Cancer Cell And Its Molecular Mechanism

Lung cancer has become the most frenquent malignant tumor in the world which its mortality and morbidity is increasing fastest among all the cancers,and it also has become the No1 malignant tumors which is harmful to human health and life,and the tirst killer in all cancers.Meanwhile.tumor metastasis is not only the malignant marker and characteristics of lung cancer, but also the main cause of failure to cure and lose their life of the patients with lung cancer. It is a complex biological consquence that involved in many factors, genes, signal pathways and processes. The study of lung cancer cell signal transduction will illuminate the molecules mechanism of tumor invasion and metastasis, provide a new targeting molecules and pathway for retro-transduction of the signal pathway It has been proved that nm23-H1 is a putative tumor metastasis suppressor gene.there exists an close relationship between the abnormality of nm23-H1 structure and function and the tumor metastatic ability. The low expression and hetero-deletion of nm23-Hl has also been proved to be correlated with lung cancer, metastasis. Dr.Zhou Qinhua first hypothesized that nm23-H1 may be an upsteam key regulatory gene of "lung cancer metastatic suppressive cascade",and it can regulate thedownstream metastasis-relating gene to inhibit tumor metastasis and reverse the metastatic photype of lung cancer.Meanwhile,as showed in many studies ,up-regulation of cAMP-PKA signal pathway will inhibit growth and proliferation ,induce differtiation and lead to apoptosis in many malignant tumor,so it was deemed to be a negative-regulative factor in tumorigenesis and development in many tumors.And nm23-Hl was demonstrated to exhibit nucleoside diphosphate kinase(NDPK) activity,which can transfer a phosphate among tri- and diphosphate so as to improve the GTP level.GTP is an essential cofactor of adenylyl cyclase(AC) regulation.Thus,it can be estimated that a close relationship might be exsited between nm23-Hl and PKA signal pathway.In oder to explore the fundation and moleculor mechanism of nm23-Hl gene in "lung cancer metastatic suppressive cascade",three lung cancer cell lines including primary human high-metastatic large cell lung cancer cell line(L9981),vector transfection lung cancer cell line(L9981-pLXSN) and nm23-Hl gene transfectionlung cancer cell line (L9981-nm23-Hl-pLXSN) was studied in this research.PKA activity and expression level of relating gene such as CREB ,Raf ,MAPK and the bio-characteristic in vitro in the three lung cancer cell lines before and after treated with PKA pathway activator forskolin was observed by radioimmunoassay,Western blot , MTT .and Boyden chamber methods, respectively. The results first showed in the world as follows:l.The activity of PKA in L9981-nm23-Hl-pLXSN lung cnacer cell line [1.906±0.034umol/(min.g)] was significantly higher than that in L9981 [0.441±0.021umol/(min.g)] and L9981-pLXSN lung cnacer cell lines [0.443±0.059^mol/(min.gl] (P=0.000), but no significant difference of the PKA activity was observed between L9981 and L9981-pLXSN lung cnacer cell lines (P>0.05) .2.No significant difference of the expression level of CREB was observed among L998U L9981-pLXSN and L9981-nm23-Hl-pLXSN lung cnacer cell lines (P>0.05), but the expression level of p-CREB in L9981-nm23-Hl-pLXSN lung cnacer cell line was remarkably higher than that in L9981 and L9981-pLXSN lung cnacer cell lines (P=0.000).3.The expression level of p-Raf in L9981-nm23-Hl-pLXSN lung cnacer cell line was significantly higher than that in L9981 and L9981-pLXSN lung cnacer cell lines (iD=0.000),but no significant difference was observed between L9981 and L9981-pLXSN lung cnacer cell lines (P>0.05).4.The expression level of p-MAPK in L9981-nm23-Hl-pLXSN lung cnacer cell line was remarkably lower than that in L9981 and L9981 -pLXSN lung cnacer cell lines (P — 0.000),but no significant difference of the p-MAPK expression was observed between L9981 and L9981-pLXSN lung cnacer cell lines (P=0.622).5.The cell proliferation and invasion ability in L9981-nm23-Hl-pLXSN lung cnacer cell line was significantly lower than that in L9981 and L9981-pLXSN lung cnacer cell lines (P = 0.000), but no significant difference existed between L9981 and L9981-pLXSN lung cnacer cell lines (P>0.05).6.The PKA activity was remarkably increased in all the three lung cancer cell lines after treatment with different concentratin of forskolin for 30 minutesCP = 0.000). The PKA activity in L9981-nm23-Hl-pLXSN lung cnacer cell line was remarkably higher than that in L9981 and L9981-pLXSN lung cnacer cell lines after treatment with 0,l,10uM concentration groups of forskolin. But there was no significant difference among L9981-nm23-Hl-pLXSN, L9981 and L9981-pLXSN lung cnacer cell lines at lOOuM concentration of forsklin.7.The PKA activity in all the three lung cancer cell lines after treatment with lOOuM forskolin for 30 minutes.was remarkably higher than those for 0, 1CK 60 minutes (/?=().OOO). The PKA activity in L9981-nm23-Hl-pLXSN lung cnacer cell line was significantly higher than that in L9981 and L9981-pLXSN lung cnacer cell lines after treatment with lOOuM forskolin among 0> 10> 60 minutes groups (P=0.000). But no significant difference was observed among the different time groups between L9981 and L9981-pLXSN lung cnacer cell lines.8.No significant difference of the expression level of CREB was observed among L998K L9981-pLXSN and L9981-nm23-Hl-pLXSN lung cnacer cell lines (P>0.05) after treatment with forskolin, but the expression level of p-CREB was remarkably increased in all the three lung cancer cell lines(P=0.000).9.After treatment with forskolin,the expression level of p-Raf in L9981-nm23-Hl-pLXSN lung cnacer cell line was remarkably higher than that in L9981 and L998l-pLXSN lung cnacer cell lines (P=0.000),but no significant difference of the p-Raf was observed between L9981 and L9981-pLXSN lung cnacer cell lines (P=0.554). The expression level of p-Raf was remarkably increased in all the three lung cancer cell lines after treatment with forskolin^'^O.OOO).10.The expression level of p-MAPK was remarkably decreased in all the three lung cancer cell lines after treatment with forskolin(P=0.000),and the expression level of p-MAPK in L9981-nm23-Hl-pLXSN lung cancer cell line was significantly lower than that in L9981 and L9981-pLXSN lung cancer cell lines after treatment with lOOfiM forskolin for 30 minutes^=0.000).11.The expression level of p-MAPK in L9981-nm23-Hl-pLXSN lung cancer cell line was significantly lower than that in L9981 andL9981-pLXSN lung cancer cell lines after treatment with 50uM U0126 for 30 minutes (P=0.000).But no significant difference was observed between L9981 and L9981-pLXSN lung cancer cell lines.There was significant difference of expression level of p-MAPK in L9981-nm23-Hl-pLXSN lung cancer cell line between treatment with lOOuM forskolin for 60 minutes and with 50uM U0126 for 30 minutesCf^O.OSXbut the expression level of p-MAPK of both in L9981 and L9981-pLXSN lung cancer cell lines after treatment with lOOuM forskolin for 60 minutes was significantly higher than that after treatment with 50uM U0126(f>=0.000).12.After treatment with forskolin, the cell proliferation and invasion ability in vitro in L9981-nm23-Hl-pLXSN lung cnacer cell line was significantly lower than that in L9981 and L9981-pLXSN lung cnacer cell lines (P — 0.000).After treatment with PKA activator forskolin,the cell proliferation and invasion ability in vitro was significantly decreased in all the three lung cancer cell lines was (P=0.000), but there was no significant difference both proliferation and invasion ability in vitro compared between L9981 and L9981-pLXSN lung cancer cell lines(P>0.05).Conclusion (l)Transfection of nm23-Hl gene can significantly up-regulate the PKA signal transduction and down-regulated the MAPK signal transduction in human high-metastic large cell lung cancer cell line L9981 suggesting that a cross-talk relationship exist between PKA signal transduction and MAPK signal transduction;(2) Transfection of nm23-Hl gene can significantly reverse or/and suppress the cell proliferation and invasion of human high-metastic large cell lung cancer cell line L9981;(3)Forskolin also upregulated the PKA signal transduction, down-regulated the MAPK signal transduction and partly reversed the cell proliferation and invasion of the L9981 cancer cell line,which suggest that a add or corporative effect exist between nm23-Hl transfection and forskolinin the regulation of PKA and MAPK signal transduction in the human high-metastatic large cell lung cancer cell line L9981;(4)The molecular mechanisms of nm23-Hl gene for reversing the invasion and metastasis of human high-matastatic large cell lung cancer might be related to its effecfs of activating PKA signal pathway and suppressing MAPK pathway.