The Effects And Molecular Mechanisms Of JWA Combined With SP-141 On Proliferation,apoptosis And Migration In Melanoma And Gastric Cancer Cells

The incidence and mortality of cancer have still been increasing and becoming a major public health problem worldwide.Gastric cancer is one of the most common malignant tumors of alimentary system in China,and it has become the second common cancer and the third leading cause of cancer death.For melanoma,although the incidence is relative low,the significant upward trend was observed in recent years.Despite great progress has been made in clinical multimodal treatments,such as surgery in early stage,chemotherapy and radiotherapy,the long-term outcome of patients with gastric cancer and melanoma are still unsatisfactory due to the metastasis of cancer.Cancer metastasis is a complex multi-step process,mainly including tumor invasion,dissemination and survival in the circulation.To dissemination,tumor cells fall off from the original site,and have to acquire the capabilities through epithelial-mesenchymal transformation(EMT)to break down the basement membrane,then they invade extracellular matrix,penetrate into the circulatory system,and manage to survive before they can arrest at a distant organs and gradually form clinically detectable metastases because of continuous proliferation.This complex process is driven by tumor heterogeneity,which mainly results from the genomic instability involving in oncogene activation and tumor suppressor gene inactivation.Therefore,in this study,we tried to observe whether the combined regulation of an oncogene MDM2 and a tumor suppressor gene JWA would synergistically exhibit the effects on proliferation,apoptosis and migration in melanoma and gastric cancer cells.Mouse double minute-2 homolog(MDM2)is one of the well known oncogenes,which exerts its biology function mainly through the p53 dependent or independent signal pathways.The Nutlin-3,a small molecule antagonist of MDM2,has been found and shown activities based on the biological characteristics of MDM2-p5 3 interaction.However,the biology function of Nutlin-3 depends on the expression of wild-type p53,unfortunately it is reported commonly mutated or deleted in half of cases of cancer.As a result,its clinical application is greatly limited.Fortunately,Wang W et al has successfully designed and synthesized another small molecule inhibitor of MDM2,SP-141,which could directly bind to MDM2 protein and induce its autoubiquitination and proteasomal degradation through a p53 independent signal pathway.JWA,also known as ARL6ip5,is one of the environmental response genes.It was firstly discovered and cloned by ourselves in 1996.JWA could inhibit oxidative stress and repair DNA damage,as well as suppress the angiogenesis and metastasis of gastric cancer and melanoma by regulating MAPK signaling pathways.The expressions of JWA in both melanoma and gastric cancer tissues are significantly lower than those in the normal adjacent tissues.In addition,patients with lower JWA expression have significantly poor survival time.Therefore,JWA is a potential tumor suppressor gene in melanoma and gastric cancer.In this study,firstly we detected the effects of SP-141 on proliferation,apoptosis and migration and their relative molecular mechanisms in melanoma and gastric cancer cells.Next,JWA protein was overexpressed by plasmid transfection to determine the combined effects of SP-141 and JWA on proliferation,apoptosis and migration and related molecular mechanisms in these cells.Objectives:To investigate the effects and molecular mechanisms of the SP-141 alone,and combined treatment of JWA and SP-141 on cell proliferation,apoptosis and migration in melanoma cell lines A375 and B16F10,as well as gastric cancer cell lines MGC803 and BGC823.Methods:Melanoma cell lines A375 and B16F10,gastric cancer cell lines MGC803 and BGC823 were treated with different concerntrations of SP-1415 or transfected with Flag-JWA for 24 h,followed by treatment of SP-141 for another 24 h.The cell viability,proliferation,cell cycle distribution,apoptosis and migration were determined by the CCK-8 assay,colony formation,flow cytometry,Hoechst staining and wound-healing,respectively.Western blotting was performed to detect the levels of MDM2 and other relative biomarkers of apoptosis and migration.Results:1.SP-141 inhibits the proliferation of melanoma and gastric cancer cells.The cell viability of A375,B16F10,MGC803 and BGC823 cells was decreased after SP-141 treatment,the IC50 values of the four cell lines were 0.41 ?M,0.52 ?M,0.62 ?M and 0.60 respectively.Colony formation assay showed that the numbers of colony in both melanoma and gastric cancer cells were significantly decreased after SP-141 treatment(P<0.01).2.SP-141 induces melanoma and gastric cancer cell cycle arrest at G2/M phase.Flow cytometry assay showed that 0.5 ?M concentration of SP-141 increased G2/M phase arrest to 82.38 ± 2.08%,49.72±4.77%,94.22 ± 0.03%and 93.00± 1.29%compared with their controls in A375,B16F10,MGC803 and BGC823 cells,respectively(all P<0.01).3.SP-141 promotes the apoptosis of melanoma and gastric cancer cells.Hoechst staining assay showed that SP-141 significantly increased the proportion of apoptosis compared with their controls in A375,B16F10,MGC803 and BGC823,respectively(all P<0.05).In the four cell lines,0.5 ?M concentration of SP-141 increased the apoptotic index 0.69± 0.003,0.62± 0.21,3.48± 0.13 and 1.54± 0.05 folds higher than those in their controls,respectively(all P<0.001).Similarly,Western blotting assay indicated that SP-141 down-regulated the protein level of Bcl-2 but up-regulated protein levels of Bax,Cleaved-Caspase-3 and Cleaved-PARP1 in the above cell lines.4.SP-141 suppresses migration of melanoma and gastric cancer cells.The wound healing assay showed that SP-141 significantly inhibited the migration of B16f10 cells when cells were treated with 0.1 or 0.2 ?M of SP-141 for 12 or24h.The similar result was observed in the gastric cancer MGC803 cells.Western blotting assay indicated that SP-141 decreased FAK protein expressions in A375,B16F10,MGC803 and BGC823 cells.5.The effects of JWA are independent to MDM2 in melanoma and gastric cancer cells.Western blotting assay showed that the expression of JWA was not altered when MDM2 expression was inhibited by SP-141 in A375 and MGC803 cells.Meanwhile,the protein expression of MDM2 was not altered either after intervention of JWA in both A375 and MGC803 cells.6.Overexpressing JWA and inhibition of MDM2 dose not show combined inhibitory effect on proliferation in melanoma and gastric cancer cells.Overexpressing JWA in B16F10,MGC803 and BGC823 cells followed by SP-141 treatment,the colony numbers were 10.33 ± 1.53,40.33 ± 4.40,and 6.00 ± 1.73,respectively,which did not show statistical differences in the number of clones formed by SP-141 treatment alone(all P>0.05).7.Overexpressing JWA and inhibition of MDM2 indicates partial synergistic effects on apoptosis in melanoma and gastric cancer cells.A375,B16F10,MGC803 and BGC823 were transfected with Flag-JWA plasmid,then treated with SP-141 for 24 h,Bcl-2 was down-regulated,but Bax and PARP1 were up-regulated in Western blotting assay.Notably,in MGC803 cells,the protein levels of Bax and Cleaved-PARP1 were 8.20-fold and 5.50-fold higher than that of their basic levels.8.JWA combined with SP-141 significantly inhibits the migration in melanoma and gastric cancer cells.B16F10 and BGC823 were transfected with Flag-JWA plasmid,followed by SP-141 treatment for 24 h,cell migration was greatly suppressed compared with the treatment with Flag-JWA or SP-141 alone.Similar result was observed in the Transwell migration assay.In the molecular mechanism,Western blotting assay exhibited that the protein expression of FAK in the combined treatment group was significantly lower than that in the group of Flag-JWA or SP-141 treatment alone.Conclusions:Our study confirmed that the small molecule inhibitor of MDM2,SP-141,effectively inhibited proliferation,migration and promoted their apoptosis in melanoma and gastric cancer cells for the first time.Meanwhile,our results indicated that JWA combined with SP-141 has a synergistic inhibitory effect on migration in melanoma and gastric cancer cells.We speculate that both JWA and MDM2 could be the useful biomarkers and potential targets for prevention and therapy in melanoma and gastric cancer metastasis.