| Tumor metastasis is not only the malignant marker and characteristics of lung cancer, but also the key cause of failure to cure and lose their life of the patients with lung cancer. Illuminating the molecular mechanism of tumor invasion, metastasis and signal transduction related to tumor metastasis will provide a new targeting molecules and pathway for reversion of lung cancer and developing anticancer metastastic targeting drugs. It was well known that nm23-H1 gene was a tumor metastasis suppressor gene, and the abnormality of its structure and function was closely related to the invasive and metastatic ability of lung cancer. It has been proved that low expression and hetero-deletion in nm23- H1 gene was closely related to the high metastatic ability and poor prognosis for patient with lung cancer. Lung cancer combined with nm23-H1 gene deletion always has abnormal expression of some metastatic relative gene. Therefore, Zhou QingHua hypothesize that nm23-H1 may be an upstream regulatory gene of metastasis-relating genes and control downstream regulatory genes to inhibit tumor metastasis .The metastatic phenotype of lung cancer is reversed by transfecting wild type nm23-H1 cDNA into cancer cells. It is rasied by professor Zhou Qinghua that the role of nm23-Hl gene inhibiting tumor metastasis by controlingdownstream regulatory genes can be called "lung cancer metastatic suppressive cascade".As an important signal transduction pathway which mediates extra-ellular signals into intra-cellular response, the MAP Kinase (mitogen-activated protein kinase) pathway regulates a number of cellular fates including growth, differentiation, division, death and functional synchronization of cells. The ERK pathway (also called Ras-to-MAPK pathway), which is a key and classic pathway of the MAPK systems, mainly transduce mitogen signals, regulate cell cycle, and increase cell proliferation and differentiation. In recent years, It has been found that MAPK play an important role in cell canceration and tumor invasion and metastasis, hi order to explore the molecular mechanism and signal transduction pathway of nm23-Hl gene for reversing lung cancer invasion and metastasis, and regulating MAPK signal transduction in lung cancer suppressive cascade based on our previous research work, we carried out the following experimental studies: (1) The phospho-ERKl/2, total-ERKl/2 expression and relative activity of ERK 1/2 was detected in human high metastatic large cell lung cancer cell line L9981, L9981-nm23-Hl (transfected with nm23-Hl gene ) and L9981- pLXSN (transfected with vector) by Western-blot, immuno-precipitation; (2)The proliferative and invasive abilities among the above three lung cancer cell lines was determined by MTT and Boyden chamber; (3)The changes of expressive level and kinase activity of phospho-ERKl/2, total-ERKl/2, relative activities of ERKl/2 were detected in the three cell lines before and after treating with U0126 by Western blot and immunoprecipitation technique; (4)The tumorigenicity and lung metastastic rate of transplantation tumor in nude mice were detected in the three lung cancer cell lines before and after treatment with U0126 by animal model. The results in this study first showed in the world as follows:1 . The phosphorylated ERKl/2 expression level, phosphorylated ERKl/2 relative content and ERKl/2 relative activity in L9981-nm23-Hllung cancer cell line is remarkably lower than those in L9981 and L9981-pLXSN lung cancer cell lines (P<0.00i),but there is no significant difference between L9981 and L9981-pLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed in the three lung cancer cell lines (P > 0.05).2. The phosphorylated ERK1/2 expression level, ERK1/2 relative content and ERK1/2 relative activity of the three lung cancer cell lines was gradually decreased accompanied with increase of U0126 after treating with different concentration of U0126, and a highly significant differences was existed among the different concentration groups of U0126 and the three cancer cell lines (P<0.0\)., but significant difference of total ERK1/2 expression was found among the three cancer cell lines.3. After treatment with same concentration of U0126 for different time, the phosphorylated ERK1/2 expression level, phosphorylated ERK1/2 relative content and ERK1/2 relative activity of the three lung cancer cell lines was gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference was observed among groups of different treatment time and the three cell lines (PO.01). But no significant of total ERK1/2 expression level was found among different time groups in all the three lung cancer cell lines before and after the treatement with different treatement time of U0126 was found^ > 0.05).4. Utilization of combining the two independent factors of U0126 (40HM, for 20 minutes) and nm23-Hl gene transfection showed good interaction to downregulate the phosphorylated ERK1/2 expression level, phosphorylated ERK1/2 relative content and ERK1/2 relative activity of the L9981 lung cancer cell line (P<0.001). However, no significant difference of total ERK1/2 expression level was observed after the treatement with ( 40HM ) of U0126 for 20 minutes (P > 0.05). The phosphorylated ERK1/2 expression level, phosphorylated ERK1/2 relative content and ERK1/2 relative activity in L9981-nm23-Hl lung cancer cell line was significantlower than those in L9981 and L9981-pLXSN lung cancer cell lines both before and after treatment with 40HM of U0126 for 20 minutes (p<0.01), but no significent difference was found between L9981 and L9981-pLXSN cell lines (P>0.05).5. Results of cell culture and animal model experiment on the effects of nm23-Hl gene transfection and Ras-to-MAPK pathway inhibitor for the biological behaviour of human high metastatic large cell lung cancer cell lines showed that:(1) After treatment with differene concentration of U0126, the proliferation of the three lung cancer cell lines was gradually decreased accompanied with increase of concentration and a highly significant differences was existed among different groups of U0126 concentration (P<0.001). Treating with the same concentration of U0126, the proliferation of 19981-nm23-Hl lung cancer cell line was significent lower than those of L9981 and L9981-pLXSN lung cancer cell lines (PO.000). But no significant difference was found between L9981 and L9981-pLXSN lung cancer cell lines (P > 0.05). Result of two-factor analyses showed that there was good interaction to downregulate the proliferation of the L9981 lung cancer cell line when combined with the two factors of U0126 (60MM) and nm23-Hl gene transfection (PO.01).(2) The in vitro invasion of the three cell lines was decreased gradually accompanied with increase of U0126 after treatement with different concentration of U0126. No significent difference of in vitro invasion of L9981 and L9981-pLXSN lung cancer cell lines was found when compared with OMM, 10MM and 20HM of U0126(P > 0.05), but a highly significent difference was observed among other groups treated with different concentration of U0126(P<0.001) . There was significent difference of in vitro invasion of L9981-nm23-Hl lung cancer cell line between group treated with 60HM of U0126 and groups treated with 0MM , 10MM, 20HM and 40MM of U0126 (PO.001), but no significent difference was found among othergroups treated with different concentration of U0126 (P > 0.05). After treating with the same concentration of U0126 , the in vitro invasion of L9981-nm-23Hl lung cancer cell line was remarkably lower than those of L9981 and L9981-pLXSN lung cancer cell lines (PO.001), but significant difference was found between L9981 and L9981-pLXSN lung cancer cell lines (P > 0.05). Results of two-factor analyses showed that there was good interaction to decrease the invasion of the L9981 cell line when combining the two factors of U0126 (60"M) and nm23-Hl gene transfection (PO.001).(3) The in vivo experiment of the human high metastatic large cell lung cancer cell lines in nude mices were divided into five groups: nm23-Hl transfection group, U0126 treatment group, empty control group, solvent control group and empty vector transfection group. The results showed that: (pThe tumorgenesis in the U0126 treatment group (51.6%) and nm23-Hl tranfection group (49.1%) was significent higher than those in other groups^O.Ol), but no significant difference was found among empty control group, solvent control group and empty vector transfection group (P > 0.05). There was significant difference of average numbers of metastasis lesion in lung among the five groups (PO.01). ?The average lung metastasis numbers in nude mices of nm23-Hl tranfectant group was significant lower than those in the other four groups (P<0.0l), but no significant difference was found among the other groups.Conclusion: (1) Transfection of wild type nm23-Hl gene can significantly downregulate the Ras-to-MAPK signal pathway of the human high metastatic large cell lung cancer cell lines; (3) The inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high metastatic large cell lung cancer cell lines is dose-dependence and time-dependence; (2) Transfectant nm23-Hl gene or/and U0126 can significantly downregulate the Ras-to-MAPK signal pathway of the human high metastatic large cell lung cancer cell lines and has good interaction when both of them are used . (4)Both in vitro and in vivo experiment showed that suppression or block of Ras-to MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high metastatic large cell lung cancer cell lines, suggesting that the molecular mechanism which nm23-Hl gene reverse invasion and metastasis in the human high metastatic large cell lung cancer cell line, may be related to its effects to inhibit Ras-to MAPK signal transduction pathway.
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