Molecular Mechanism Of Nodal Promoting Prostate Cancer Cell Growth

Prostate cancer(PCa)is one of the most common malignancies in men,with the second highest mortality rate in developed countries and regions(e.g.,Europe and the United States),and the incidence of PCa in Asia is lower than that in Europe,but has shown a rapid increase in recent years.Although significant progress has been made in the treatment of PCa,PCa remains an important medical problem affecting men ’s health.Nodal is a member of the transforming growth factor beta(TGF-β)superfamily whose re-expression and signaling has been implicated in a variety of cancers and has properties that promote tumor cell plasticity and tumorigenicity.Nodal signaling regulates different transcription by inducing a TGF-β family receptor signaling cascade at the plasma membrane and plays an important role in maintaining pluripotency as well as embryonic morphology of embryonic stem cells.Nodal has a decisive role in early embryonic development in vertebrates as well as maintaining the cellular function of human embryonic stem cells(HESCs).It has been shown that Nodal is highly expressed in a variety of tumor cells and is associated with proliferation,metastasis,and angiogenesis of tumor cells,but there are few reports on the association between Nodal and PCa.ObjectiveTo investigate the effect of Nodal on PCa cell proliferation,migration and vasculogenic mimicry and its possible mechanism.MethodsTwo kinds of human prostate cancer cells(DU145 cells and LNCaP cells)were selected as the study subjects,and the cultured DU145 cells and LNCa P cells were divided into three groups after corresponding treatment: the control group,the Nodal group,and the Nodal + SB431542(Nodal signaling pathway inhibitor)group.The cell viability of each group was detected by MTT assay;the proliferation ability of each group was detected by plate cloning assay;and the migration ability of each group was detected by Transwell migration assay.DU145 cells were divided into three groups:control group,Nodal group and Nodal + SB431542 group.The expression levels of VE-cadherin protein,a mimicry marker of angiogenesis,in each group of cells were detected by Western blot;the expression levels of ALK4 and VEGF in each group of cells were detected by Western blot.Upregulation of ALK4 expression in DU145 cells by cell transfection was performed to examine the effect of up-regulation of ALK4 on VEGF expression.Results1.MTT assay showed that in DU145 cells and LNCaP cells,the cell viability of Nodal group was higher than that of control group and Nodal + SB431542 group(P <0.05).2.The results of plate cloning assay showed that 12 colonies were randomly selected from DU145 cells and LNCa P cells.In DU145 cells,the mean cell density was74.3 ± 3.2 in the control group,141.3 ± 4.1 in the Nodal group,and 107 ± 4.2 in the Nodal + SB431542 group.In LNCa P cells,the mean cell density was 66.8 ± 2.3 in the control group,138.9 ± 4.2 in the Nodal group,and 83 ± 3.5 in the Nodal + SB431542 group.The results showed that in DU145 cells and LNCa P cells,the mean cell density in the Nodal group was significantly greater than that in the control and Nodal +SB431542 groups(P < 0.05).3.The results of Transwell migration assay showed that in DU145 cells,the number of migrated cells was 96 ± 5.6 in the control group,202 ± 10.2 in the Nodal group,and 102 ± 6.1 in the Nodal + SB431542 group.In LNCa P cells,the number of cells migrated was 65 ± 6.9 in the control group,193 ± 7.1 in the Nodal group,and 74 ±6.3 in the Nodal + SB431542 group.The results showed that in DU145 and LNCa P cells,the number of cell migration in the Nodal group was higher than that in both the control and Nodal + SB431542 groups(P < 0.05).4.The expression levels of VE-cadherin,a vasculogenic mimicry marker,in DU145 cells from the control,Nodal,and Nodal + SB431542 groups were detected by Western blot.The results showed that VE-cadherin protein expression levels in the Nodal group were significantly increased compared with the control and Nodal +SB431524 groups(P < 0.05).5.The expression levels of ALK4 and VEGF in DU145 cells of control group,Nodal group and Nodal + SB431542 group were detected by Western blot.The results showed that the protein expression levels of ALK4 and VEGF in the Nodal group were significantly increased compared with those in the control and Nodal + SB431524groups(P < 0.05).6.The effect of high ALK4 expression on VEGF expression in DU145 cells was detected by Western blot.The results showed that high ALK4 expression could promote the expression of VEGF(P < 0.05).ConclusionNodal promotes proliferation,migration,and VM formation in PCa cells.Nodal may promote proliferation and migration of PCa cells via ALK4,and promote VM formation in PCa cells via ALK4/VEGF signaling pathway.