The Mechanism Of Ang-(1-7) On Inhibiting Proliferation And Migration Of Tumor Cells And Its Application On Cancer Gene Therapy

Background and objectives:Angiotensin 1-7(Ang-(1-7)),as an endogenous heptapeptide in renin angiotensin system(RAS),has antitumor effect in a wide variety of tumors.Its low toxicity makes it a preeminent anticancer agent,but the intrinsic mechanism is still not established.However,the efficacy of this peptide in vivo is severely hampered due to rapid degradation by peptidases.In the study,we aim to illustrate the antitumor mechanisms of Ang-(1-7)and evaluate the feasibility of AAV-mediated Ang-(1-7)Over-expression in vivo and its antitumor potential.Methods and Results1.The mechanisms of Ang-(1-7)on the inhibition of proliferation in non-small cell lung cancer cells.Lentiviral vector LV-Ang-(1-7)or LV-eGFP were used to infect lung cancer cells,the result showed that LV-Ang-(1-7)can mediate Ang-(1-7)expression efficiency in vitro.The Ang-(1-7)over-expression can markedly inhibit the proliferation of lung cancer cells.The results in BrdU incorporation assays show Ang-(1-7)reduces DNA synthesis of the cells.To the mechanism study suggest that,Ang-(1-7)markedly decreases the level of DNA pre-replication protein Cdc6 and reduces the chromatin-binding fraction of Mcm2,Mcm6,Mcm7,suggests that Ang-(1-7)impairs the assemble of pre-replication complex,thereby inhibits the DNA replication.2.The mechanisms of Ang-(1-7)suppresses EMT in tumor cells.At first,our results show the migration ability of lung cancer cells and nasopharyngeal carcinoma cells declined after Ang-(1-7)treatment,detected by the transwell migration assay.Second,Ang-(1-7)elevated the protein level of the epithelial marker E-cadherin,with no effect on the mesenchymal marker vimentin;in nasopharyngeal carcinoma cells,Ang-(1-7)down-regulates Slug and Snail,these results show that Ang-(1-7)can inhibit epithelial-mesenchymal transition.Further study show that Ang-(1-7)inhibits the phosphorylation of Smad2 and Smad3;besides,Ang-(1-7)can reduce the phosphorylation of p38.The results suggest that Ang-(1-7)can inhibit the migration of cancer cell by suppresses EMT through Slug/Snail-Smad and p38 pathway.3.Effects of AAV8-mediated Ang-(1-7)over-expression on cell proliferation of lung cancer cells in vivoWe constructed subcutaneous cancer models in nude mice by inoculation of lung adenocarcinoma cell line Spc-Al.Then mice were treated with recombination vectors AAV8(Y733F)-Ang-(1-7)and AAV8(Y733F)-eGFP by a single intravenous injection into the tail.The results show the level of Ang-(1-7)in liver and serum in AAV8-Ang-(1-7)group was significantly higher than in control group.Ang-(1-7)over-expression can inhibit the transplanted lung tumor growth.The results of immunohistochemistry showed that the expression of Ki67,Cdc6 and VEGF in tumor tissues in AAV8-Ang-(1-7)group were down-regulated.Moreover the vascular density were markedly reduced compared with the control groups.4.Effects of AAV8(Y703F)-based Ang-(1-7)over-expression on cell proliferation of hepatoma carcinoma cells in immune-intact mice.Gene therapy based on Ang-(1-7)for treating the hepatoma carcinoma was validated furthermore.We used a more efficient liver-targeting recombinant adeno-associated viral vector mutant AAV8(Y703F)mediated Ang-(1-7)over-expression in Balb/c mice,showed inhibition of the growth of transplanted hepatocellular carcinoma significantly.Accordingly,the VEGF/VEGFR and PIGF in tumor tissue reduced in AAV8(Y703F)-Ang-(1-7)group.ConclusionAng-(1-7)inhibits proliferation of tumor cells by reducing Cdc6 expression and impairing Pre-RC assemble.Ang-(1-7)can suppress migration and EMT by Slug and Snail down-regulation and inhibition of Smad and p38 phosphorylation.Ang-(1-7)overexpression vector based on AAV8 can induce to consistent and steady Ang-(1-7)expression both in immune-defective nude mouse and in immune-intact Balb/c mouse and significantly attenuated tumor growth by inhibiting proliferation and angiogenesis.