Vector-mediated Tum-5 expression in neovascular endothelial cells for treating hepatocellular carcinoma

Abstract

Hypervascular hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality. Angiogenesis is an important contributor to HCC progression and metastasis; therefore, inhibiting angiogenesis may be an effective method of treating HCC. Tumstatin is a novel type of efficient endogenous vascular endothelial cell growth inhibiting factor. The anti-angiogenic activity of tumstatin is localized to the 54-132 amino acid region (Tum-5). In a previous study performed by our group, the gene fragment encoding Tum-5 was cloned and inserted into a pLXSN retroviral vector. In the present study, the anti-angiogenic effects of Tum-5 and the antitumor effects exerted by the pLXSN-Tum-5 vector in vivo were investigated. The results demonstrated that pLXSN-Tum-5 significantly inhibited the growth of human umbilical vein endothelial cells compared with pLXSN, but had no obvious effect on HepG2 cell growth. Moreover, the antitumor and anti-angiogenic activity of Tum-5 was examined in vivo using a xenograft of H22 HCC cells. The results indicated that pLXSN-Tum-5 significantly inhibited tumor growth following 5 injections over 10 days. The size and weight of tumors in the pLXSN-Tum-5 group were lower than those in the saline and pLXSN groups. Furthermore, immunohistochemical analysis with CD31 antibodies indicated that the average microvessel density in the pLXSN-Tum-5 group were significantly lower than that in the saline and pLXSN groups. These results suggested that Tum-5 exerts its antitumor activity by suppressing vascular endothelial cells. The gene fragment of Tum-5 may be developed as an effective inhibitor of angiogenesis and used to treat patients with HCC.

Keywords: hepatocellular carcinoma; retroviral vector; tumor angiogenesis; tumstatin.

Figure 1.

Effect of Tum-5 on cell growth in vitro. The proliferation rate of (A) human umbilical vein endothelial cells and (B) HepG2 cells was assessed following pLXSN-Tum-5 and pLXSN virus transfection at MOIs of 0, 1, 5, 10, 25 and 50 for 72 h. *P<0.05 and **P<0.01, compared with the pLXSN group. Tum, tumstatin; MOI, multiplicity of infection.

Figure 2.

Antitumor efficacy of Tum-5 in vivo. (A) Tumor volume of H22 tumor-bearing mice treated with saline, pLXSN and pLXSN-Tum-5 measured over 10 days. Mice received injections on days 0, 2, 4, 6 and 8. (B) Images of excised tumors on day 10. (C) Weight of excised tumors. (D) Body weight of H22 tumor-bearing mice treated with saline, pLXSN and pLXSN-Tum-5 over 10 days. *P<0.05 for the pLXSN-Tum-5 group vs. the pLXSN group and **P<0.01 for the pLXSN-Tum-5 group vs. the saline group. Tum, tumstatin.

Figure 3.

Immunostaining for CD31 in tumor tissues from H22 xenograft mice in (A) the saline group, (B) the pLXSN group and (C) the pLXSN-Tum-5 group (magnification, ×400). (D) Graph of average microvessel density in CD31-stained sections. *P<0.05 compared with saline and pLXSN groups. HPF, high-power field; Tum, tumstatin.