T7 Peptide Executes Its Anti-angiogenic Activity Via Downregulation Of Angiopoietin-2 And Combination With 3-MA

ObjectivesAccording to the statistics of the cancer incidence worldwide, liver cancer ranks sixth, and takes the second place in the mortality of malignant tumors. The incidence of hepatocellular carcinoma (HCC) accounts for 78% of liver cancer. HCC is considered as solid tumor enriched with blood supply characterized by hidden onset, rapid development, and enhanced invasion and metastasis ability. Angiogenesis of HCC is vital for the development and distant metastasis of HCC. Now the related mechanisms of drugs targeted to angiogenesis have been the hot study. Sorafenib is the only clinically recommended molecular target drug for the treatment of advanced HCC, and shows obvious anti-proliferation and anti-angiogenic activity in HCC. The median survival of HCC patients could be prolonged by 2-3 months with the treatment of sorafenib. However, the chemo-resistance of tumors to sorafenib has become one of the important factors to limit the clinical apply of sorafenib widely. So the exploration for new anti-angiogenic drugs supplies the new study direction for the replacement treatment of sorafenib or drugs combination with sorafenib.Tumstatin is a new endogenic inhibitor of angiogenesis derived from the type Ⅳ collagen α3 chain, which executes its anti-angiogenic activity via inhibition of endothelial cells (ECs) protein synthesis caused by downregulation of mTOR activity. T7 peptide, the 74-98 amino acid fragment of tumstatin, has an equivalent anti-angiogenic activity to that of full-length tumstatin, and exerts an anti-angiogenic activity through its L, V, and D amino acids. However, the anti-angiogenic mechanisms of T7 peptide are still unclear. So, we aim to study the signaling pathway and downstream target protein related to the anti-angiogenic activity of T7 peptide, which supplies experimental and theoretical basis for the future clinical medicine test and apply of T7 peptide.Angiopoietin (Ang) is another important pro-angiogenic factor in addition to vascular endothelial growth factor (VEGF), and its protein family consists of Ang-1, Ang-2, Ang-3, and Ang-4. Ang-2 is mainly stored in the Webel Paladebody (WPB) of ECs, which is released quickly by hypoxic stimulation, a hallmark of the intratumor environment. Ang-2 could improve either ECs disassociation from the extracellular matrix (ECM) or ECs migration and activation, meanwhile the sensitivity of ECs to VEGF is enhanced by Ang-2. It is also reported that the high expression of Ang-2 protein improves the angiogenesis, invasion, and metastasis of HCC. As an important modulation protein to tumor angiogenesis, the specific modulation mechanisms of Ang-2 and the role of Ang-2 in the anti-angiogenic process modulated by T7 peptide have important research significance. Autophagy is a process in which cellular proteins and cytoplasmic organelles are degraded by lysosome. The role of autophagy in angiogenesis have drawn people attention. And some inhibitors of angiogenesis could induce the increased autophagy of ECs, which results in the enhanced anti-apoptotic activity of ECs, and antagonism and even reverge the anti-angiogenic activity of angiogenesis-related inhibitors. To enhance the sensitivity of angiogenesis-related inhibitors combined with autophagy inhibitor such as 3-MA has drawn the study attention.3-MA executes its anti-autophagic ability by targeting to Ⅲ PI3K which is responsible for the phagocytosis of cellular components.Further study of molecular drugs target to HCC angiogenesis is vital for the prolonged survival and improved prognosis of HCC patients. We mainly explored the possible related signaling pathway and downstream target protein modulated by T7 peptide, and the role of Ang-2 in the anti-angiogenic process modulated by T7 peptide. We further studied the correlation of T7 peptide and increased autophagy of ECs in the angiogenesis.Methods(1) The xenogeneic nude mouse tumor model derived from human HCC line HepG2 was divided into control group and experiment group randomly (n=15), and dealt with NS and T7 peptide (4.4 mg/Kg) respectively. After 22 days, nude mouse were killed and the tumor specimen were embedded in paraffin. Immunohistochemistry was used to detect the expression of VE-cadherin and CD31 and analyze the potential anti-angiogenic activity of T7 peptide.(2) HUVECs were incubated in hypoxia chamber (37℃,1% O2,5% CO2,94% N2), and grouped into:normoxia group, hypoxia group, hypoxia+T7 peptide (1.0μM) group. The tube formation assay was applied to analyze the influence of T7 peptide on angiogenesis under hypoxia.(3) To determine the related mechanisms of T7 peptide, we incubated HUVECs with various concentrations of the T7 peptide (0.25,0.5,1.0 and 2.0μM), and T7 peptide of 1.0μM was used for subsequent experiments. Meanwhile we dealt with HUVECs with T7 peptide (1.0μM) for 12 h or 24 h. Cell Counting Kit-8 was applied to observe the cell viability.(4) We grouped HUVECs into:normoxia group, hypoxia group, hypoxia+T7 peptide group, and incubated for 24 h. The apoptosis rate was detected with Annexin V-FITC. Then we extracted the cell protein, and the expression levels of pro-apoptotic protein BAX and anti-apoptotic protein Bcl-2 were observed with western blot.(5) To determine whether Ang-2 is the target protein and signaling pathway modulated by T7 peptide, hypoxia+MK2206 (5.0μM) group was set up based on (4). MK2206 is a specific inhibitor of Akt, which acted as the positive control to T7 peptide. After 24h, western blot was used to detect the protein expression levels of Ang-2, Akt, and p-Akt.(6) The exogenous rhAng-2 (15 ng) was used to deal with HUVECs under hypoxia combined with T7 peptide. Scratch and transwell assays were applied to observe the migration ability of ECs.(7) To determine the role of Ang-2 and T7 peptide in invasion of HepG2 cells, HepG2 cells were incubated in upper chamber of a transwell plate while HUVECs were in lower chamber. HUVECs were divided into normoxia group, hypoxia group, hypoxia+T7 peptide group, hypoxia+T7 peptide+rhAng-2 group. ELISA was used to detect the levels of Ang-2 in culture supernatants of HUVECs, while the invasion ability of HepG2 was determined by transwell assay. HUVECs were incubated in 6-well plate and grouped as same as the above. The culture supernatants were used to incubate HepG2, and western blot was used to detect the expression of MMP-2 protein.(8) To observe the role of T7 peptide in autophagy, HUVECs were incubated with or without T7 peptide and/or 3-MA under hypoxia, acridine orange (AO) staining was used to observe autophagy-lysosome and western blot was used to detect the expression of LC-3Ⅱ of ECs.(9) To determine the role of 3-MA in the anti-angiogenic activity of T7 peptide, we grouped HUVECs as same as (8). Tube formation assay was applied to observe the influence of 3-MA (5.0 nM) and T7 peptide on angiogenesis in vitro, and the apoptosis rate was detected by Annexin V-FITC assay.Results(1) The xenogeneic nude mouse tumor model derived from human HCC line HepG2 was divided into two groups randomly, and dealt with NS and T7 peptide respectively. Immunohistochemistry indicated that the protein expression levels of hallmarks of angiogenesis, CD31 and VE-cadherin, were inhibited by T7 peptide. In vitro, T7 peptide reversed the capillary-like tube formation induced by hypoxia, which was consistent with the in vivo results.(2) T7 peptide inhibited ECs viability significantly in a concentration-dependent manner. The concentration of T7 peptide at 0.5μM showed lessened ability to decrease the ECs ability compared with that of T7 peptide at 1.0μM, however no statistical difference existed. The ECs viability was less than 20% while the concentration of T7 peptide at 2.0μM. The concentration of T7 peptide at 1.0μM was used in subsequent experiments. The ECs viability was inhibited more significant at 24 h than that at 12 h (P<0.05), and T7 peptide could induce ECs decreased viability at the same time check point.(3) Annexin V-FITC indicated that T7 peptide could increase the apoptosis rate significantly under hypoxia while hypoxia decreased the apoptosis rate (P<0.01). Western blot revealed that T7 peptide resulted in the downregulation of anti-apoptotic protein Bcl-2 and upregulation of pro-apoptotic protein Bax under hypoxia (P<0.01).(4) Hypoxia induced the up-regulation of Ang-2 expression and the phosphorylation of Akt (P<0.01). However, T7 peptide could inhibit Ang-2 expression and the phosphorylation of Akt (P<0.01). Our results revealed that MK2206, a specific inhibitor of Akt, decreased the expression of Ang-2 significantly (P<0.01). We demonstrated that T7 peptide downregulated the expression of Ang-2 via inhibition of phosphorylation of Akt indirectly.(5) The results of scratch assay indicated that T7 peptide decreased the migration ability compared with hypoxia (P<0.05). Combination T7 peptide and rhAng-2 enhanced the migration ability significantly compared with T7 peptide only (P<0.05). The results of transwell assay was consistent with scratch assay.(6) ELIS A indicated that hypoxia increased the Ang-2 level in cellular supernatants, and T7 peptide reversed the process (P<0.01). We also observed that the cellular supernatants of HUVECs with high concentration of Ang-2 increased the invasion ability of HepG2 (P<0.05) and the expression of MMP-2 (P<0.01). T7 peptide modulated the invasion ability of HepG2 via Ang-2.(7) AO staining revealed that T7 peptide induced ECs autophagy, and 3-MA reversed the process. The expression level of LC3-Ⅱ was increased by T7 peptide. The results of western blot was consistent with those of AO staining.3-MA induced ECs apoptosis under hypoxia, which resulted in decreased angiogenesis (P<0.01). Combination T7 peptide and 3-MA enhanced the apoptosis significantly compared with T7 peptide only (P<0.01), and tube formation assay revealed the result that combination T7 peptide and 3-MA showed stronger anti-angiogenic activity (P<0.05).Conclusions(1) T7 peptide inhibits angiogenesis significantly in vivo and in vitro under hypoxic conditions.(2) T7 peptide executes its anti-angiogenic activity via inhibiting cell viability and inducing cell apoptosis under hypoxic conditions.(3) T7 peptide reduces EC migration via downregulation of Ang2, and Akt phosphorylation acts as the bridge between T7 peptide and Ang2 expression.(4) T7 peptide inhibits the invasion of tumor cells via downregulation of Ang-2 expression.(5) 3-MA decreases the angiogenesis in vitro and enhances the anti-apoptotic and anti-angiogenic ability of T7 peptide. Autophagy may be a protective mechanism against the anti-angiogenic activity of the T7 peptide.Significance(1) We found that T7 peptide partially exerts its anti-angiogenic activity by inhibiting cell viability, migration and inducing the apoptosis of ECs。(2) We demonstrated that Ang-2 is one target protein of T7 peptide, and Akt phosphorylation acts as the key bridge. We supply the experimental and theoretical basis for the further research of T7 peptide and Ang-2.(3) We revealed that Ang-2 plays important role in modulating the invasion of tumor cells. T7 peptide was demonstrated to execute both anti-angiogenic activity and inhibition of invasion ability of tumor cells.(4) The inhibitor of autophagy enhances the anti-angiogenic activity of T7 peptide, which supports the further study for future combination drug therapy.(5) The study for related mechanisms of T7 peptide, an important angiogenesis-related inhibitor, is important for the research and development of the novel anti-angiogenic drug.