The Effect Of Liver-targeting Peptide CSP I-plus Modified Recombinant Human Endostatin On Human Hepatocellular Carcinoma

IntroductionThe occurrence of Hepatocellular carcinoma(HCC) is a complex process of multi-factor and multi-stage. So far there is no effective cure for this disease. Looking for new treatment and new therapeutic molecular target for the treatment of hepatocellular carcinoma is fitting for the urgent demands. Angiogenesis inhibitors, particularly polypeptides or endogenous peptides, may become the safest and least toxic therapy for disease associated with abnormal angiogenesis. At present, the domestic and foreign research has been committed by preventing the tumors from creating blood vessels. Endostatin, a 20 k D C-terminal fragment of collagen XVIII, is one of the most effective anti-angiogenesis agents availabe. Endostatin could inhibit endothelial cell proliferation, migration, tube formation and eventually interrupting angiogenesis and tumor growth. In addition to its antiangiogenic activity, r Endostatin exerts a direct anticancer action in certain tumor cell lines such as breast cancer and colon cancer cells. But like many angiogenesis inhibitors, the signal administration of Endostatin can’t get significant curative effects, in addition to the existing problem of protein preparation thus the USA terminate the clinical development of Endostatin in 2003. In recent years, targeting drug research has become one of the hot spot. CSP I-plus peptide screened from Plasmodium falciparum Circumsprozoite protein(CSP) can specifically targeting liver. Thus, we have successfully synthesized liver targeting r Endostatin by using genetic engineering technology and prokaryotic expression system. At present, the inhibitory effect of the fusion protein on endothelial cells has been confirmed, But on HCC cells has not been reported, which need to be further discussed. In this study, the liver-targeting function of r ES-CSP will be futher verified in the models with nude mice orthotopic transplantation tumor in human hepatocellular carcinoma, and than Hep G2 and nude mice orthotopic transplantation tumor in human hepatocellular carcinoma were used to evaluate anti-HCC effect of r ES-CSP. The molecular mechanism of anti-HCC effect of r ES-CSP was also investigated.Objective The objectives of the present study are to investigate the direct inhibition of liver targeting r Endostatin(r ES-CSP) on human hepatocellular carcinoma cell line Hep G2, which supplemented the inhibitory target of r ES-CSP on hepatocellular carcinoma. Thus provide new scientific evidence and drug development ideas in targeting therapy of HCC.Methods 1. To study the Liver-target function of r ES-CSP 1.1 The establishment of models with nude mice orthotopic transplantation tumor in human hepatocellular carcinoma: nude mice subcutaneous transplantation tumor model was established, and serially planted the subcutaneous transplantation tumor into liver. 1.2 Animal in vivo distribution of r ES-CSP: Nude mice orthotopic transplantation tumor in human hepatocellular carcinoma were randomly divided into blank control(saline) group, r Endostatin group and r ES-CSP group. One month after signal tail vein administration of r ES-CSP and r Endostatin, the heart, liver, spleen, lung, kidney, blood, tumor tissues of nude mice were homogenized on 5、30 and 60 min. The endostatin concentration in each sample was measured by ELISA method. 2. In vitro anti-HCC and apoptosis related molecular mechanism of r ES-CSP 2.1 Cytotoxicity test: The HCC cell line Hep G2, human liver cell Chang’s and lung carcinoma cell A549 were added r Endostatin, r ES-CSP and CSP I-plus in equimolar concentrations(12 μM, 6 μM, 1.2 μM, 0.6 μM, 0.06 μM), the cytotoxicity was determined by MTS method. Thus the appropriate drug concertration was selected for subsequent functional studies. Than we analyzed the migration、cell cycle and apoptosis of r ES-CSP and r Endostatin on hepatocellular carcinoma cells. 2.2 Western blot assay detected apoptosis related molecular mechanism: Hep G2 cells were added r Endostatin, r ES-CSP in equimolar concentrations(6 μM). After 24 hours, protein was extracted from cells and incubated with anti-Caspase 8、anti-Bcl-2 antibody for analysis of intracellular expression of apoptosis related proteins by western blot assay. 3. Anti-HCC effect of r ES-CSP in vivo 3.1 Nude mice orthotopic transplantation tumor in human hepatocellular carcinoma was used as animal model, randomly divided into 3 groups, each with 10 mices. ①blank group(saline), ② r Endostatin(6 μM), ③ r ES-CSP(6 μM). Two weeks after intravenous administration once two days for 28 days. After the last administration, dissected the liver cancer and observed the tumor growth status, finally calculated the inhibition rate. 3.2 Nude mice tissue paraffin sections were stained with HE staining and observed pathological changes. The expression changes of microvessel density CD31 was detected by immunohistochemistry. 4. Statistical analysis All datas were expressed as the mean±standard error of the mean( sx ±). All statistical analyses were performed by SPSS(version 13.0 for Windows) statistical software. Quantitative variables were analyzed by one-way analysis of variance. When the data are homogeneity, multiple comparison use LSD method. When the data are not homogeneity, multiple comparison use Dunnett T 3 method. The difference was statistically significant when P<0.05.Result 1. The Liver-target function of r ES-CSP 1.1 The subcutaneous tumor formation rate was 100.0%, tumors were grown by nine-tenths mice which were injected subcutaneous transplantation tumor, and all of the mice survived. 1.2 In-vivo distribution of r ES-CSP in the transplantation tumor bearing mice liver: to determine whether r ES-CSP was able to accumulate rapidly in liver for potential treatment of HCC, the concentration of r ES-CSP in liver and liver cancer as a functional of time were obtained after i.v. administration of r ES-CSP or r Endostatin to mice. The levels of endostatin in the liver and liver cancer at 5、30 and 60 min post-administration were analyzed by ELISA. The results showed that the concentrations of Endostatin in heart, liver, spleen, lung, kidney, blood and tumor between r ES-CSP and r Endostatin groups were significantly different(P<0.05). At the three time intervals post-administration, the liver and liver cancer Endostatin levels in r ES-CSP group higher than in the r Endostatin group(P<0.01). 2. In vitro anti-HCC and apoptosis related molecular mechanism of r ES-CSP 2.1 Cytotoxicity test: MTS results showed that the strong inhibitory effect of r ES-CSP on the proliferation of Hep G2 showed a concentration-dependent manner. However, CSP I-plus、r Endostatin can hardly affect the proliferation of hepatocellular carcinoma cells. At the dose of 6 μM, r ES-CSP showed the strongest inhibitory effect on the proliferation of Hep G2 with an survival ratio of 46.3±2.31%. However the A549 and Chang’s cells were 92.8±2.11% and 93.2±2.96% respectively. 2.2 Inhibition of r ES-CSP suppresses Hep G2 cells migration in vitro:Wound healing assay and Transwell assay showed that the rates of migration in the cells treated with r ES-CSP were significantly lower compared with r Endostatin. 2.3 Flow cytometry detected the cell cycle and apoptosis:At 24 and 48 h of treatment with r ES-CSP(6 μM), an increase in G2/M phase concomitant with a decrease in G0/G1 and S phase was observed when compared with r Endostatin group, and both early and late apoptosis rates in Hep G2 cells significantly higher than r Endostatin group(P<0.01). 2.4 Transmission electron microscope observe the Hep G2 cell apoptosis morphologic changes: the results showed that hepatocellular carcinoma cells almostly appeared the mid and late stage apoptosis, visible cell membrane budding and apoptotic bodies with degenerated organelles inside. 2.5 Western blotting assay detected the expression changes of apoptosis related protein: the results showed that r ES-CSP can significantly decrease the expression of Bcl-2 protein, while up regulate the expression of Caspase 8 protein compared with r Endostatin-treated group. 3. In vivo anti-HCC and related molecular mechanism of r ES-CSP 3.1 The inhibition of human hepatocellular carcinoma in vivo nude mice orthotopic transplantation tumor: compared with r Endostatin group and control group, the tumor weight and tumor volume in r ES-CSP group were significantly reduced. The tumor inhibition rates of r Endostatin and r ES-CSP groups were 5.30±1.23% and 29.6±4.39%. 3.2 HE staining observed the tissue pathological changes: the results showed the hepatocellular carcinoma cells were infiltrating growth, the tumor in r ES-CSP group occurred necrosis, and the pathological karyokinesis and interstitial blood vessel were fewer, and the inflammatory cell infiltration. That is to say the r ES-CSP has certain inhibitory effect on Hep G2 cell growth. 3.3 immunohistochemistry detected the expression changes of CD31 in tumor: the results showed that the tumor microvessel density in r Endostatin and r ES-CSP groups were significantly reduced, statistically significant differences between the two groups(P<0.05).ConclusionLiver-targeting peptide CSP I-plus modified recombinant human Endostatin(rES-CSP) could target binding to hepatocellular carcinoma cells surface molecular HSPG and improve liver homing. Anti-HCC effect of r ES-CSP in vitro assay confirmed that r ES-CSP can significantly inhibit the proliferation and migration of Hep G2 cells, inducing cell cycle arrest and increasing the apoptosis of hepatocellular carcinoma cells. Animal experiments confirmed that r ES-CSP can significantly inhibit the growth of HCC in nude mice orthotopic transplantation tumor, the down regulated-expression of CD31 protein was more abvious. In sum, liver target recombinant human endostatin has obvious liver-target potency, can accumulate in liver tissue, which can improve the specificity of r Endostatin in the treatment of HCC and provide new scientific evidence and drug development ideas for the targeted therapy of HCC.