Idetification Of JWA-activating Small Molecular Compounds And Its Potential Function Of Tumor Suppression

In recent years,the mortality of cancer has become the first death cause in many countries.Although the surgery and chemotherapy technology has made significant progress in the past ten years,the cancer prognosis is not ideal due to no significant effect,toxic side effects and so on.The prognosis of tumor is closely related to its metastasis;and metastasis is responsible for approximately 90%of all cancer-related deaths,therefore,the inhibition of tumor metastasis is an extremely important topic of medicine.Since 1996,we have focused on the relationships between environmental response gene-JWA and tumor development.Our previous data showed the melanoma cells with reduced expression of JWA indicated significant higher migration,invasion,adhesion abilities than that with vector control.Retrospective cohort study on melanoma and gastric cancer showed that JWA expression is significantly down-regulated in cancer tissues compared with their adjacent normal tissues.The lower expression of JWA in cancer tissues,the more risk of the lymph node metastasis and distant metastasis.Therefore,low expression of JWA in cancer tissue is an unfavourable factor for the prognosis of melanoma and gastric cancer patients.Small molecular compounds have been widely used in the experimental study of tumor suppression.In the present study,based on the small molecular compound library,we used the luciferase reporter gene assay system to screen small molecular compounds which can significantly activate the expression of JWA gene not only in HBE cells,but in B16/HGC-27 cancer cells.We then used these compounds to further investigate their effects on the biological behaviors of cancer cells such as migration and proliferation in vitro and mestatasis in vivo animal models.Objective:To screen small molecular compounds which can activate the expression of JWA gene and explore the function of these small molecular compounds in inhibiting of cancer cells migration and metastasis.Result:1.We successfully constructed JWA promoter reporter gene plasmid with the neomycin resistance,and HBE cells were stably transfected with the recombinant plasmid pGL3-JWA-neo.Finally we established a reporter-gene HBE cell culture model,which was used for screening of the compounds that can activate expression of JWA gene.2.We used the Iuciferase assay system to screen about 40000 compounds from small molecular compound library.Based on the molecular weights,lipid water partition coefficients,structures of the compounds,8 of 65 compounds were further used to in vitro and in vivo functional studies.3.Western Blot showed the synthesized compounds could significantly activate the expression of JWA protein in HBE,B16F10 and HGC-27cells,rspectively.4.In vitro studies found that the small molecular compounds without obvious cytotoxicity.The CCK8 assay was used to detect cell proliferation in compounds treated cells and data showed that these compounds didi not show significant effects on cellular proliferation after treatment for 12,24,48 or 72h,respectively.5.JWA-activating small molecular compounds can inhibit the migration of gastric cancer cells in vitro.The migration of gastric cancer cells was decreased after treated with the compounds,and meanwhile,the cells with increased expression of JWA while decreased expression of FAK.6.In vivo mouse models indicated that,JWA-activating compounds effectively inhibited melanoma metastasis in experimental mouse.Metastatic foci in lung tissue was significantly reduced in JAC-6 corupound treated mouse compared with the control group.In the mouse melanoma survival model,JAC-4 compound showed signifieant role in increase mouse survival compared with the mouse in control group(P<0.01);and with an 10.34%life extension rate.Conclusion:In the present study,we used a comprehensive strategy to screen JWA activating compounds and then determined its anti-cancer effects on melanoma and gastric cancer cells in vitro and in vivo models.These findings provided a new direction for the study on the treatment of tumor metastasis by using JWA as a target and with potential application.