Inhibition Effect Of HS-1200 On Hepatocarcinogenesis In Diethylnitrosamine-exposed Rats And HepG2 Transplanted Tumor In Nude Mice

Background and Aim:Liver cancer is one of the most common malignant tumors and the leading cause of cancer deaths worldwide.An estimated 782,500 new cases of liver cancer and 745,500 liver cancer deaths occurred worldwide in year 2012.Hepatocellular carcinoma(HCC),which accounts for a majority of primary liver cancers,is generally associated with chronic liver diseases such as hepatitis and cirrhosis.Despite considerable advances in the diagnosis and treatment of primary HCC,the associated mortality rate is still very high due to high metastasis and recurrence rates.At present,the number of drugs used for HCC prevention is quite limited and many of the agents are still undergoing clinical trials.Development of novel treatment options for liver cancer is a key imperative.Accumulated evidence suggests an anticarcinogenic effect of hydrophilic bile acids such as ursodeoxycholic acid(UDCA).In in vitro cultured HCC cells and in diethylnitrosamine(DEN)-induced rat HCC model,administration of UDCA appeared to suppress hepatocarcinogenesis by inhibiting cancer cell proliferation.Further,treatment with UDCA was shown to reduce the risk of liver cancer in patients with hepatitis C virus-associated liver cirrhosis,and to improve patient prognosis in primary biliary cirrhosis.Chenodeoxycholic acid(CDCA)is the most abundant primary bile acid that is known to decrease dietary cholesterol absorption.HS-1200{N-[(3a,5b,7a)-3,7-dihydroxy-24-oxocholan-24-yl]b-alanine benzyl ester},a synthetic CDCA derivative,has shown anti-cancer activity in several human cancers.For instance,HS-1200 was shown to sensitize breast cancer MCF-7 cell to'radiation-induced apoptosis.Further,HS-1200 was shown to inhibit proliferation and promote apoptosis of both prostate cancer cells and bone sarcoma cells.Similarly,pro-apoptotic effect of HS-1200 on human HCC cell lines BEL7402 and HepG2 cells has also been demonstrated.However,the potential in vivo anti-cancer effect of HS-1200 against HCC has not been elucidated.For this purpose,we established 2 kinds of hepatic carcinoma model,gave them HS-1200 by oral administration and intraperitoneal injection respectively,and observed in vivo anti-cancer effect of HS-1200.Part oneInhibition effect of HS-1200 on hepatocarcinogenesis in diethylnitrosamine-exposed ratsAim:Although the molecular mechanism of HCC development remains largely unknown,oxidative DNA damage is thought to be involved in human hepatocarcinogenesis and progression.Oxidative stress due to excessive production of free radicals such as reactive oxygen species(ROS)is thought to induce genetic instability during carcinogenesis.8-hydroxy-2'-deoxy-guanosine(8-OHdG)is a well-accepted biomarker of ROS-triggered oxidative DNA damage,and is also identified as a risk factor for developing HCC in patients with chronic hepatitis C virus infection.mutT homologue gene 1(MTH1),a hepatocyte DNA repair gene,encodes MTH1 protein which favors the repair of oxidation-induced DNA damage by inhibiting the incorporation of oxidized dNTPs.Oxidative stress upregulates expression of MTH1 which leads to removal of excess of 8-OHdG.Increased expression of MTH1 has been documented in several human cancers such as kidney,breast,and colorectal cancers.Moreover,increased expression of MTH1 in cancerous tissue as compared to that in adjacent non-cancerous tissues has also been demonstrated.However,its precise role in hepatocarcinogenesis is not well understood.We sought to investigate the potential ability of HS-1200 in inhibiting hepatocarcinogenesis and examine the involvement of MTH1 in carcinogenesis.For this purpose,a rat HCC model was established by injecting a chemical carcinogen DEN,which induces liver carcinogenesis by disrupting the antioxidant defense pathway.Our findings indicate that HS-1200 suppressed tumorigenesis and improved liver function in DEN-exposed rats.In addition,the anti-tumor activity of HS-1200 appears to be associated with its ability to downregulate the mRNA level of MTH1.Our findings suggest a potential role of HS-1200 in reducing oxidative DNA damage and,thereby,preventing HCC.Methods:145 male Wistar rats were needed in the experiment,50 for the evaluation of the safety of HS-1200,20 for establishment of the rat model,and 75 for next step.First,we made the evaluation of the hepatotoxicity and nephrotoxicity of HS-1200,and we established the rat model of primary hepatic carcinoma successfully.Then we conduct the following experiment:Rats were randomly assigned into five groups:Control(N=15),HS-1200(N=15),HCC(N=15),HCC+low dose HS-1200(N=15)and HCC+high dose HS-1200(N=15)groups.Rat HCC model was established by intraperitoneal injection of DEN.And rats were administered HS-1200 by daily oral gavage.After 20 weeks,we examined animal body weight,liver weight,liver pathological changes,serum levels of AST,ALT and AFP,mutT homologue gene 1(MTH1)in liver tissue.Result:1,At the end of the experiment,the DEN group,HS-1200 low dose group and high dose group established hepatoma model successfully,and the percentage of tumorigenesis was respectively 93%(14/15)?73%(11/15)?53%(8/15).The body weight,liver weight and the liver coefficient of the normal control group and HS-1200 control group had no difference,but the body weight of the DEN group and HS-1200 intervention group is significantly lower than the normal conrol group(P<0.05).When compared to HCC group,animals receiving low dose or high dose HS-1200 therapy showed increased body weight,reduced liver weight and decreased liver coefficient(P<0.05 vs.HCC;).Moreover,high dose HS-1200 treatment appeared to have a greater efficacy in inhibiting hepatocarcinogenesis.2.In the liver tissues obtained from the Control or HS-1200 groups,hepatocytes were arranged around the central vein in a radial configuration,and hepatic lobule structures were well preserved.In contrast,disorganized hepatocytes and hepatic lobules,pseudolobule formation,cancer cell nests and tumor cell atypia were observed in livers of HCC rats.In addition,HCC rats exhibited hepatocyte necrosis as well as severe hepatic inflammatory cell infiltration.In HCC rats receiving low dose of HS-1200 treatment,the cancer cell nest formation and cell necrosis appeared to be much attenuated.High dose of HS-1200 therapy was associated with much improved pathological findings in HCC rats as compared to that observed in rats in the low dose HS-1200 treatment group.3.Serum levels of ALT,AST and AFP were significantly elevated in HCC rats(P<0.05 vs.Control),which indicated liver dysfunction in these animals.Administration of low or high dose of HS-1200 appeared to reverse the upregulation of the three biochemical parameters in HCC rats(P<0.05 vs.HCC).Moreover,the serum levels of these parameters were lower in the HCC rats in the high dose HS-1200 group(P<0.05.vs.HCC+Iow dose HS-1200).4.We found significant upregulation of mRNA level of MTH1 in liver tissues of HCC rats(Control,1;HCC,16.23 ± 0.74;P<0.05;Fig.7).Further,upregulation of MTH1 mRNA was significantly reversed by low-or high-dose HS-1200 therapy;high dose appeared to have a higher efficacy in this respect(HCC+low dose HS-1200,9.48± 0.46;HCC+high dose HS-1200,6.13 ± 0.33;P<0.05 vs.HCC).Conclusion:1.No significant difference was observed in the liver function and renal function of any of the HS-1200 toxicity assessment groups.The results suggested that HS-1200 was relatively safe for rats in the dose range of our exoeriment.2.HS-1200 can reduce the rate of rat liver neoplasia,and lower serum AFP levels in rats,HS-1200 obviously inhibited hepatocarcinogenesis.3.HS-1200 remarkably reduced the level of AST,ALT and AFP in serum,HS-1200 significantly downregulated the expression of MTH1.The inhibitory effect of primary liver cancer in rats might relate to the inhibition of inflammation and oxidative stress.Part twoInhibition effect of HS-1200 on HepG2 transplanted tumor in nude mice Aim:In order to further study of HS-1200 for liver tumor angiogenesis and metastatic tumors of the liver,we investigated organizational structure,ultrastructure and angiogenesis of human hepatic carcinoma cell strarin HepG2 transplanted tumor in nude mice.We also observed expression of VEGF and bFGF of transplanted tumor.We hope to provide experimental evidence or HS-1200 in hepatic carcinoma clinical treatmeat.Methods:We adjusted the cell density of human liver cell line HepG2 to 1.5 × 107/ml,inoculated the cell suspension to axillary subcutaneous tissue of nude mice.When subcutaneous tumor grew to diameter of about 10 mm,we stripped tumors,took it to 2×2×2mm3 and inoculated it to axillary subcutaneous tissue of 60 nude mice.When the inoculation tumors grew to diameter of about 10 mm,rats were randomly assigned to three groups:Control(N=20),low dose HS-1200(N=15)and high dose HS-1200(N=15)groups.In low dose HS-1200 or high dose HS-1200 groups,rats were administered HS-1200 by intraperitoneal injectionat at dose of 20 and 60 mg/kg/d,respectively,for 2 weeks.Then mice were sacrificed,transplanted tumor were stripped completely.We measured the tumors,calculated volume of tumors,calculated inhibition ratios of the tumor weight.Tumor tissue structure and ultrastructure of each transplanted tumor were observed by light microscope and electron microscope.Expression of VEGF and bFGF were detected by immune histochemical method.Result:1.The volume and weight of low or high dose of the HS-1200 transplanted tumors were significantly less than the control group,inhibition ratios of the tumor were 38.23%and 47.05%,respectively.2.We compared the histological findings in liver among the three groups.Active growth of blood vessel,disorganized hepatocytes,pseudolobule formation,cancer cell nests and tumor cell atypia were observed in livers of the control group.In low dose of HS-1200 treatment,blood vessels are few or absent,the cancer cell nest formation and cell necrosis appeared to be much attenuated.High dose of HS-1200 therapy was associated with much improved pathological findings as compared to low dose HS-1200 group.3.Transmission electron microscope:Paramorphia of cells,nuclear atypia and karyokinesis were showed in the HepG2 transplanted tumor of control group,swelling mitochondrion and lipid droplet were appeared in the cytoplasm.Apoptosis and necrosis were showed in the HepG2 transplanted tumor of HS-1200 group,apoptosis body was appeared,and chromatin was broken into pieces in the stroma.4.Immunohistochemistry showed expression of VEGF and bFGF were reduced in low or high dose of HS-1200 groups.Conclusion:1.HS-1200 can inhibit human hepatic carcinoma cell strarin HepG2 transplanted tumor in nude mice.2.HS-1200 can make the transplanted tumor blood supply decreased significantly.3.Ultrastructure showed HS-1200 can induce apoptosis of transplanted tumor cell.4.Immunohistochemistry showed expression of VEGF and bFGF were reduced obviously in transplanted tumor of low or high dose of HS-1200 groups.HS-1200 can inhibit angiogenesis of transplanted tumor.So we speculate that HS-1200 have a performance of anti-hepatoma.