The Experiment Study Of The Inhibition Effects Of Tyroservatide(YSV) On Angiogenesis Of Human Hepatocellular Carcinoma

Primary liver cancer,particularly hepatocellular carcinoma(HCC) is one of the most common human tumors worldwide,with the sixth incidence rate and the third leading global cause of cancer-related deaths,and 82%new cases in developing coutries.There are more than 600,000 new cases in the world,with over 55%occur in China per year,especially in Southeast Asia and South Saharan coutries.The incidence rate of HCC in Western countries is also increasing in recent years.HCC remains a high prevanlent disease associated with the third incidence rate in all malignant tumors in China.New cases in China amount to approximately 55%of all new cases world wide.Surgical resection is the mainstay of curative treatment for HCC,but prognosis after resection remains unsatisfactory due to a high incidence of postoperative recurrence.Moreover,most patients are at advanced stage when was diagnosed,only about 30%patients have the chance of surgical intervention. Therefore,it is essential to develop new drugs with high efficacy,low toxicity,and better specificity for hepatocellular carcinoma therapy.The spleen contains many immune cells such as macrophages,reticuloendothelial cells,lymphocytes and dentritic cells.These cells as well as their produced compounds are known to modify the tumor cell signal transduction and tumor cell killing.Indeed,a number of such peptides possessing anticancer or immune-activating activities have been identified from splenic tissues.This observation provided us with the initial rationale to explore the spleen organ for peptides that could target cancer.The great potential for identifying anticancer peptides,derived from the spleen, one of these peptides,tyroservatide(YSV),showed notable antitumor effects against hepatocarcinoma.YSV is a small tripeptide compound that composed of three amino acids,L-tyrosine,L-serine,and L-valine,and its molecular formula is C₁₇H₂₅N₃O₆;Its molecular weight is 367.40.We obverse the inhibition effects of YSV on the proliferation of human hepatocaricoma cell SMMC-7721 in vitro and in vivo, respectively.We found that YSV could significantly inhibit the proliferation of tumor cell in vitro and in vivo through downregulation of proangiogenic factors such as VEGF and IL-8.Administer YSV can inhibit the growth of human hepatocarcinoma cell,and may bring about new hope to the heaptocarcinoma patients especially the advanced stage patients.1.YSV inhibit the proliferation of human hepatocarcinoma cell SMMC-7721 in vitro and the growth of hepatocarcinoma transplant tumor in nude mice.The inhibitory effects of YSV on SMMC-7721 were examined by MTT assay. SMMC-7721 cells(2.0×10³) were plated in 96-well cell culture plate at 200μL per wall.After 24h incubation,YSV was added at different concentrations to culture plates,the final concentrations of YSV were 0.01 mg/L,0.1mg/L,1.0mg/L,10mg/L,100mg/L,and the control group was add the same volume RPMI-1640 media instead of YSV.The tumor cells proliferation were measured after 24h,48h and 72h by the MTT array.After 72h incubation,YSV showed notable inhibitory effects,the inhibition ratio was 42.34%at 10mg/L YSV.In order to further investigate the inhibitory effects of YSV tumor cell in vivo,animal models of human HCC was established by orthotopic implantation of histological intact SMMC-7721 HCC tissue into the liver of nude(nu/nu) mice.Mice bearing human SMMC-7721 HCC tissue were randomized into the YSV groups(160,320 and 640μg/kg/day),or the saline group(0.2ml/day).Solutions were administered from the second day after tumor implantation by intraperitoneal injection,once per day for 35days.At the dose of 160, 320,or 640μg/kg/day,inhibitory ratio was 42.62%,60.66%,and 27.59%,respectively, which differ significantly from the inhibition in saline group(p＜0.05).While tumor volume of YSV group(320μg/kg/day) and the control group were 0.271±0.067 and 1.510±0.441 cm³(p＜0.05).These results suggest that YSV inhibit the proliferation of SMMC-7721 in vivo and in vitro.2.YSV inhibited angiogenesis of tumor tissueIn order to investigate the effects of YSV on angiogenesis of replanted tumor, immunohistochemical study of microvessels in different tissues using anti-CD31 antibody was used to display the extent of angiogenesis in tumor tissue.The staining of microvessle in YSV groups was weak,especially at the dose of 320μg/kg/day, compared with the control group.The mean value of tumor MVD in YSV (320μg/kg/day) group was 36(SD,11.6),and this value in control group was 114.5(SD,26.5),the difference between two groups had the statistical significance (P＜0.05).3.Changes in angiogenesis related gene expression profile after the administration of YSVIn order to explore the mechanism of alteration in angiogenesis by using YSV, angiogenesis related cDNA microarray was used to analysis the change of gene expression.The results showed that a among the total 113 angiogenesis genes,and 18 genes were down-regulated in expression level by twofold or more in YSV group compared with the control,VEGF,IL-8 were the leading ones.To the research in vitro, the gene chip showed the same tendency.The gene-chip results showed that YSV could exert its antitumor activity may through antiangiogenic routes.4.Validation of gene chip results by RT-PCR,Realtime-PCR,Western blot and ELISATo verify the above-mentioned changes in VEGF and IL-8 from the gene chip analysis,we tested the effect of YSV on their mRNA level by RT-PCR for the tumor cell cultured in vitro.Compared with the control,VEGF and IL-8 mRNA expression in YSV group(10mg/L,100mg/L) were significantly decreased.Realtime-PCR was used to examine the mRNA expression of VEGF and IL-8 in replanted tumor tissue, mRNA expression in VEGF(P＜0.05) and IL-8(P＜0.05) in YSV group were decreased.The expression of VEGF protein in tumor tissue of nude mice and tumor cell cultured in vitro was meseasured by Western blot.We also got the same tendency as the gene chip.ELISA was used to detect the concentration of VEGF and IL-8 in mice blood serum,the results showed that the targeted factors' concentrations were at the lower level compared with the control.Conclusions1.YSV inhibits the growth of replanted tumor in vivo and suppresses the proliferation of SMMC-7721 in vitro.2.YSV exhibit a significant antitumor activity by decreasing the expression of angiogenic factors such as VEGF and IL-8.