The growth and metastasis of solid tumors depends on angiogenesis. Anti-angiogenesis therapy may represent a promising therapeutic option. Vasostatin, the N-terminal domain of calreticulin, is a very potent endogenous inhibitor of angiogenesis and tumor growth. In this study, we attempted to investigate whether plasmid-encoding vasostatin complexed with cationic liposome could suppress the growth and metastasis of hepatocellular carcinoma in vivo and discover its possible mechanism of action. Apoptosis induction of pSecTag2B-vasostatin plasmid on murine endothelial cells (MS1) was examined by flow cytometric analysis in vitro. Nude mice bearing HCCLM3 tumor received pSecTag2B-vasostatin, pSecTag2B-Null, and 0.9 % NaCl solution, respectively. Tumor net weight was measured and survival time was observed. Microvessel density within tumor tissues was determined by CD31 immunohistochemistry. H&E staining of lungs and TUNEL assay of primary tumor tissues were also conducted. The results displayed that pSecTag2B-vasostatin could inhibit the growth and metastasis of hepatocellular carcinoma xenografts and prolong survival time compared with the controls in vivo. Moreover, histologic analysis revealed that pSecTag2B-vasostatin treatment increased apoptosis and inhibited angiogenesis. The present data may be of importance to the further exploration of this new anti-angiogenesis approach in the treatment of hepatocellular cancer.

Liver resection and liver transplantation are the treatment modalities with the greatest potential for curing hepatocellular carcinoma (HCC). Tumor recurrence after resection for HCC is, however, a major problem, and an increased rate of recurrence after living donor transplantation versus cadaveric whole liver transplantation has been suggested. Factors involved in liver regeneration may stimulate the growth of occult tumors. The aim of this project was to test the hypothesis that a microscopic HCC tumor in the setting of partial hepatectomy would show enhanced growth and signs of increased invasiveness corresponding to the size of the liver resection. Hepatectomy was performed to various degrees in groups of Buffalo rats with the concomitant implantation of a fixed number of hepatoma cells in the remnant liver; a control group underwent only resection. After 21 days, the sizes and numbers of the tumors and the expression of alpha-fetoprotein (AFP), cyclin D1, calpain small subunit 1 (CAPNS1), CD34 (a microvessel density marker), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2) were evaluated and compared between the groups. The tumor volume and number increased significantly with the size of the partial hepatectomy (P < 0.05). The largest resections were also associated with increased hepatoma cell infiltration in the lungs and significant up-regulation of cyclin D1, AFP, CAPNS1, CD34, VEGF, and VEGFR2. The results suggest that liver regeneration after partial hepatectomy facilitates the growth and malignant transformation of microscopic HCC, and this could be significant for liver resection and partial liver transplantation strategies for HCC.

Hepatocellular carcinoma (HCC) is naturally resistant to radiotherapy and cytotoxic chemotherapy, leaving surgery as the mainstream therapeutic approach. However, the 5-year recurrence rate after curative resection is as high as 61.5%. The background hepatitis B- or C-induced cirrhosis and the presence of micrometastases at the time of surgery have been regarded as two main causes of recurrence. Recently, accumulating evidence suggests that growth factors and cytokines released during the physiological process of post-surgical liver regeneration could induce the activation of dormant micrometastatic lesions. The establishment of neovasculature to support either liver regeneration or HCC growth involves multiple cell types including liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and circulating endothelial progenitors. The crosstalks among these cells are driven by multiple molecules and signaling pathways, including vascular endothelial growth factors and their receptors, platelet-derived growth factor, the angiopoietin/Tie family, hepatocyte growth factor/c-Met signaling, and others. Anti-angiogenic agent targeting liver cancer vasculature has been reported to be able to generate limited survival benefit of the patients. In this review, discussions are focused on various angiogenic mechanisms of HCC and liver regeneration, as well as the prevailing anti-angiogenic strategies.

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death, and conventional treatments offer unsatisfactory response. We have previously reported that kallistatin gene therapy suppressed the growth of HCC tumors by its anti-angiogenic activity, and meloxicam, a selective COX-2 inhibitor, inhibited proliferation and induced apoptosis of human HCC cells in vitro. The aim of this study was to determine whether combining kallistatin gene therapy and meloxicam could offer a better therapeutic effect to combat HCC in mice. A kallistatin expression plasmid was constructed and its expression was detected after intratumoral gene transfer. Both kallistatin gene therapy and meloxicam suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, and the combinational therapy showed a stronger effect in suppressing tumor growth, tumor angiogenesis and cell proliferation, and increasing cell apoptosis, than the respective monotherapies. Gene transfer of kallistatin inhibited tumor angiogenesis, and slightly inhibited cell proliferation and increased cell apoptosis in situ, but had no effect on expression of vascular endothelial growth factor, basic fibroblast growth factor, proliferating cell nuclear antigen, Bcl-2, Bax, or activation of caspase-3. Meloxicam therapy inhibited cell proliferation, induced cell apoptosis, reduced expression of proliferating cell nuclear antigen, increased activation of caspase-3, and upregulated Bax. Meloxicam also slightly inhibited tumor angiogenesis with no effect on the expression of vascular endothelial growth factor or basic fibroblast growth factor. Combining two novel anticancer agents, kallistatin targeting tumoral vascularization and meloxicam targeting cell proliferation and apoptosis, warrants investigation as a therapeutic strategy to combat HCC.

The current treatments for hepatocellular carcinoma (HCC) are poor, particularly for metastatic HCC. Intraportal transfusion of adeno-associated virus (AAV) leads to long-term and persistent transgenic expression in livers. Kallistatin, a novel angiogenesis inhibitor, exhibits anti-tumor activity. The aim of the study was to investigate whether intraportal injection of AAV-kallistatin could suppress local and metastatic HCC in mice.

An AAV vector encoding kallistatin was constructed, and its transduction efficiency by intraportal transfusion in livers was examined by RT-PCR, immunohistochemical and Western blotting analysis. The anti-tumor activity was tested in three HCC models including hepatic and subcutaneous human Hep3B HCC tumors in BALB/c athymic (nu/nu) mice, and subcutaneous mouse BNL HCC tumors in BALB/c mice. Tumor cell proliferation in situ was examined by anti-Ki-67 staining, and apoptosis by TUNEL.

Gene transfection by rAAV-kallistatin inhibited proliferation of human umbilical vein endothelial cells and HCC cells in vitro. Intraportal injection of rAAV-kallistatin resulted in persistent and specific expression of kallistatin in livers detected by RT-PCR and immunohistochemical analysis, and kallistatin protein in circulation detected by Western blotting analysis. Intraportal injection of rAAV-kallistatin significantly suppressed angiogenesis and growth of hepatic Hep3B tumors. The kallistatin released by hepatocytes into the circulation suppressed remote Hep3B and BNL tumors established subcutaneously. The rAAV-kallistatin gene therapy significantly inhibited tumor cell proliferation and induced apoptosis.

Intraportal injection of rAAV-kallistatin suppressed hepatic and subcutaneous HCC tumors, relying on its anti-angiogenic and anti-proliferative activities.

To investigate in vitro effects and mechanisms of silibinin on hepatocellular carcinoma (HCC) cell growth.

Human HCC cell lines were treated with different doses of silibinin. The effects of silibinin on HCC cell growth and proliferation, apoptosis, cell cycle progression, histone acetylation, and other related signal transductions were systematically examined.

We demonstrated that silibinin significantly reduced the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells. Silibinin-reduced HuH7 cell growth was associated with significantly up-regulated p21/CDK4 and p27/CDK4 complexes, down-regulated Rb-phosphorylation and E2F1/DP1 complex. Silibinin promoted apoptosis of HuH7 cells that was associated with down-regulated survivin and up-regulated activated caspase-3 and -9. Silibinin's anti-angiogenic effects were indicated by down-regulated metalloproteinase-2 (MMP2) and CD34. We found that silibinin-reduced growth of HuH7 cells was associated with increased activity of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and decreased p-Akt production, indicating the role of PTEN/PI(3)K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a possible role of altered histone acetylation in silibinin-reduced HCC cell proliferation.

Our results defined silibinin's in vitro anti-HCC effects and possible mechanisms, and provided a rationale to further test silibinin for HCC chemoprevention.

Integrin alphaVbeta3 plays a critical role in tumour angiogenesis and metastasis formation, and is recognized as a key therapeutic target in the treatment of cancer.

To investigate whether antisense alphaV and beta3 gene therapy has utility in the treatment of hepatocellular carcinomas.

Antisense expression plasmids targeting integrin alphaV or beta3 were constructed, and examined by immunohistochemistry and Western blot analyses for their ability to inhibit alphaV and beta3 expression. The antisense alphaV and beta3 expression vectors, either alone or in combination, were injected into HepG2 hepatomas established subcutaneously in nude mice and tumour growth, angiogenesis and apoptosis were monitored.

Antisense alphaV and beta3 downregulated the alphaV and beta3 subunits expressed by human umbilical vein endothelial cells, and the alphaV subunit expressed by HepG2 cells. Gene transfer of antisense alphaV and beta3 expression vectors downregulated alphaV and beta3 in HepG2 tumours established in nude mice, inhibited tumour vascularization and growth, and enhanced tumour cell apoptosis. Antisense alphaV suppressed tumour growth more strongly than antisense beta3; however antisense therapy that simultaneously targeted both integrin subunits was more effective than the respective monotherapies. Antisense alphaV and beta3 inhibited tumour angiogenesis to similar extents, by a process that is independent of vascular endothelial growth factor.

Antisense gene therapy targeting alphaV integrins warrants consideration as an approach to treat hepatocellular carcinomas.

B7H3 immunogene therapy is able to completely eradicate tumors when combined with an anti-vascular agent. The aim of this study was to determine whether vasostatin, a potent anti-angiogenic agent, could synergize with B7H3-mediated immunotherapy to combat hepatocellular carcinoma (HCC).

Vasostatin and B7H3 expression plasmids were constructed, and the in vitro and in vivo expression and anti-angiogenic activity of recombinant vasostatin were measured. The anti-tumor activities of B7H3 and vasostatin alone and in combination were assessed using single and multiple H22 tumor models.

Gene transfer of vasostatin inhibited the proliferation of aortic endothelial cells, and angiogenesis in the chorioallantoic membrane assay. Subcutaneous H22 tumors established in BALB/c mice were completely eradicated in response to intratumoral injection of B7H3-expressing plasmids followed 24h later by vasostatin-expressing plasmids. In contrast, neither vasostatin nor B7H3 monotherapy was effective. Gene transfer of vasostatin inhibited tumor angiogenesis and enhanced infiltration of NK cells, whereas B7H3 therapy activated CD8+ and NK cells and increased their infiltration into tumors, and enhanced the levels of circulating IFN-gamma. B7H3 and vasostatin combination gene therapy was effective in combating a systemic challenge of parental H22 cells, and caused the complete regression of multiple distant tumor nodules.

Combining vasostatin anti-angiogenic therapy with B7H3-mediated immunotherapy warrants investigation as a therapeutic strategy to combat HCC, and other malignancies.