Study On The Effect And Mechnism Of Polypeptide Extract From Scorpion Venom On Inhibition Angiogenesis Of H₂₂ Hepatoma

ObiectiveTo observe the effects of polypeptide extracted from scorpion venom (PESV) on tumorangiogenesis by evaluating the microvessel density(MVD) and the expression of PTEN、PI3K、P-Akt、COX-2、HIF-1α and VEGF-A in tumor tissues in order to investigate themolecular mechanisms through which PESV inhibit tumor angiogenesis. Provide theexperimental basis for the treatment of HCC patients.Methods1. The H22hepatoma tumor model was established by subcutaneously implanting H22hepatoma cells into mice. The tumor-bearing mice were randomly divided into4groups: thecontrol group, the high-dose(40mg/kg) PESV group and the low-dose(10mg/kg) PESVgroup and the5-Fluorouracil (5-FU)(20mg/kg) group,10mice in each group. The effect ofPESV on tumor growth was observed by recording tumor growth curve and calculating theinhibition rate.2. Pathological changes of the tumors were observed by H&E staining.3. Immunohistochemistry was applied to detect MVD，the levels of PTEN、PI3K、P-Akt、COX-2、HIF-1α and VEGF-A.4. Western blot was applied to detect the expressions of PTEN、PI3K、P-Akt、Akt、COX-2、HIF-1α and VEGF-A.5. ELISA was applied to detect the serum levels of PTEN、PI3K、P-Akt、COX-2、 HIF-1α and VEGF-A.Results1．Effects of PESV on H22hepatoma tumor growthWith the growth of the intervention period, the volume of each group of mice xenograftsincreasing,5-FU group, PESV high-and low-dose groups were15,17,19days starting fromthe control group were statistically significant difference in tumor volume (P<0.05).Compared to controls, treatments with5-FU alone, PESV high-dose and low-dose groupswere found to markedly inhibit the tumor growth(P<0.05，P<0.01). The inhibitory rate was64.8%、43.7%and32.4%, respectively. PESV high-and low-dose group between tumorweight and tumor volume were statistically significant (P<0.05, P<0.01).2．Pathological, morphometric of H22hepatocarcinoma after PESV treatment.H&E staining showed that H22hepatocarcinoma cells demonstrated flaky or nestedirregular growth. In the control group, tumor angiogenesis richness, rare nuclear pyknosis,nuclear karyorrhexis and other morphological changes of apoptosis were shown. PESV high-and low-dose groups showed multiple large patchy necrosis areas. Most of the tumor cellsexhibited morphological changes characteristic of apoptotic processes such as nuclearpyknosis and karyorrhexis.3．MVD analysis of H22hepatocarcinoma after PESV treatmentCD34were expressed in the cytoplasm or membrane of endothelial cells of microvessels.Cells positive for CD34were stained brown. The MVD of the control group,5-FU, high-doseand low-dose PESV were7.7±1.4、4.4±1.8、5.3±1.6、5.4±1.5，respectively. The high-andlow-dose PESV demonstrated inhibition of MVD in comparison to the control group (P<0.05for each comparison), which suggests that PESV inhibits angiogenesis.4．Expression of PTEN, PI3K, P-Akt, COX-2, HIF-1α and VEGF-A in H22hepatocarcinomatumors.4.1Detected by immunohistochemistry method resultsBased on immunohistochemical staining, PTEN, PI3K, P-Akt, COX-2, VEGF-A andCD34were expressed in the cytoplasm or membrane of tumor cells. HIF-1α was expressed inthe nucleus and cytoplasm of tumor cells. Cells positive for PTEN, PI3K, P-Akt, COX-2, HIF-1α、VEGF-A and CD34were stained brown. Gray scale intensity variants of PTEN,PI3K, P-Akt, COX-2, HIF-1α and VEGF-A immunoreactivity were evaluated by Leica QwinV3software. The expression of PI3K, P-Akt, COX-2, HIF-1and VEGF-A in the controlgroup was markedly higher than that in the high-and low-dose groups. In each comparison,there was a significant difference (P<0.05or P<0.01). Furthermore, PESV at eachconcentration was significantly different between groups (P<0.05). PI3K and P-Akt, COX-2,HIF-1α, VEGF-A expression were positively correlated(r=0.912，P<0.01；r=0.892，P<0.01；r=0.883, P<0.01；r=0.893，P<0.01). P-Akt and COX-2, HIF-1α, VEGF-A expression werepositively correlated(r=0.890，P<0.01；r=0.895，P<0.01；r=0.895，P<0.01). COX-2and HIF-1α,VEGF-A expression were positively correlated (r=0.969，P<0.01；r=0.965，P<0.01). HIF-1αand VEGF-A expression were positively correlated(r=0.973，P<0.01). The expression ofPTEN in the high-and low-dose PESV groups was higher than that in the control group. Ineach comparison, there was a significant difference (P<0.05). Furthermore, at each of thePESV concentrations there was a significant difference between groups (P<0.05). PTEN andPI3K, P-Akt, COX-2, HIF-1α, VEGF-A expression were negatively correlated(r=-0.895，P<0.01；r=-0.914，P<0.01；r=-0.963，P<0.01；r=-0.972，P<0.01；r=-0.958，P<0.01).4.2Detected by western blot method resultsPI3K、P-Akt、COX-2、HIF-1α and VEGF-A expression were decreased significantly intreatment groups when compared to the control group. PTEN expression was increasedsignificantly in treatment groups when compared to the control group. Band intensities wereanalyzed by ImageJ software. PI3K, P-Akt, COX-2, HIF-1α and VEGF-A expressiondecreased significantly in the H22hepatocarcinoma tissue treated with high-and low-dosePESV groups. Furthermore, PESV at each concentration showed a significant differencebetween groups (P<0.05). Furthermore, PI3K and P-Akt, COX-2, HIF-1α, VEGF-Aexpression were positively correlated (r=0.980，P<0.01；r=0.935，P<0.01；r=0.971，P<0.01；r=0.955，P<0.01). P-Akt and COX-2, HIF-1α, VEGF-A expressionwere positively correlated(r=0.931，P<0.01；r=0.950，P<0.01；r=0.961，P<0.01). COX-2and HIF-1α, VEGF-Aexpression were positively correlated (r=0.945，P<0.01；r=0.969，P<0.01). HIF-1α andVEGF-A expression were positively correlated (r=0.948，P<0.01). PTEN expression wasincreased significantly in high-and low-dose PESV groups. Furthermore, PTEN and PI3K, P-Akt, COX-2, HIF-1α, VEGF-A expression were negatively correlated (r=-0.972，P<0.01；r=-0.975，P<0.01；r=-0.962，P<0.01；r=-0.968，P<0.01；r=-0.989，P<0.01).4.3Detected by ELISA method resultsAfter PESV intervention, the levels of PI3K, P-Akt, COX-2, HIF-1α and VEGF-A in theserum of mice treated with PESV high-and low-dose groups were significantly lower than thetumor-bearing control group(P<0.05，P<0.01). In the treatment groups, the levels of PTEN inserum were significantly higher than those of the tumor-bearing control group(P<0.05). Inaddition, PESV at each concentration was significantly different between groups (P<0.05).Conclusion1．PESV can inhibit the growth of H22hepatocarcinoma and reduce tumor volume and weight.2．PESV can influence the expression of angiogenic factors PTEN、PI3K、P-Akt、COX-2、HIF-1α and VEGF-A in H22hepatocarcinoma which maybe the anti-angiogenic mechanismsof it.