V. Schmitz

Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice

Endogenous overexpression of the antiapoptotic protein heme oxygenase 1 (HO‐1) has been shown to occur in various cancer diseases and might contribute to cancer progression. We compared the expression levels of HO‐1 in human liver to expression levels in hepatocellular carcinoma (HCC), as well as the effect of HO‐1 inhibition by small interfering RNA (siRNA) on cellular survival and apoptosis in the mouse hepatoma cell lines Hepa129 and Hepa1‐6 and on orthotopic tumor growth in immune‐competent C3H/HeN mice. Our results show that HO‐1 is frequently overexpressed in human HCC. Downmodulation of HO‐1 by siRNA resulted in increased cellular damage and apoptosis, reduced proliferation, reduced growth of orthotopic HCC and reduced angiogenesis. Livers and kidneys of treated animals did not reveal signs of damage by this treatment. In conclusion, a specific knockdown of HO‐1 might represent a novel therapeutic approach in HCC therapy.

Targeting heat‐shock protein 90 improves efficacy of rapamycin in a model of hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) remains associated with a poor prognosis, but novel targeted therapies in combination with anti‐angiogenic substances may offer new perspectives. We hypothesized that simultaneous targeting of tumor cells, endothelial cells, and pericytes would reduce growth and angiogenesis of HCC, which represents a highly vascularized tumor entity. Recently, because of their anti‐angiogenic properties, inhibitors of mammalian target of rapamycin (mTOR) have entered clinical trials for therapy of HCC. However, treatment with mTOR inhibitors may lead to paradoxical activation of Akt signaling in tumor cells via insulin‐like growth factor‐I receptor (IGF‐IR)–dependent and IGF‐IR–independent mechanisms. Because we have recently identified heat shock protein 90 (Hsp90) antagonists to impair both oncogenic and angiogenic signaling cascades in tumor cells, including Akt and IGF‐IR, we sought to investigate whether Hsp90 blockade could improve growth‐inhibitory and anti‐angiogenic effects of the mTOR inhibitor rapamycin. Human HCC cells, a murine hepatoma cell line, endothelial cells (ECs), and vascular smooth muscle cells (VSMC) were employed in experiments. Results show that dual inhibition of mTOR and Hsp90 leads to effective disruption of oncogenic signaling cascades and substantially improves growth‐inhibitory effects in vivo. Importantly, blocking Hsp90 abrogated the rapamycin‐induced activation of Akt and of the downstream effector nuclear factor kappa‐B (NF‐κB) in HCC tumors. Furthermore, Hsp90 inhibition reduced the expression of platelet‐derived growth factor‐receptor‐β (PDGF‐Rβ) on VSMCs, and diminished vascular endothelial growth factor‐receptor 2 (VEGFR‐2) expression on ECs, which further improves the anti‐angiogenic capacity of this regimen. Conclusion: Blocking Hsp90 disrupts rapamycin‐induced activation of alternative signaling pathways in HCCs and substantially improves the growth‐inhibitory effects of mTOR inhibition in vivo. Hence, the concept of targeting tumor cells, ECs, and VSMCs by blocking Hsp90/mTOR could prove valuable for treatment of HCC. (HEPATOLOGY 2008.)

Effective angiostatic treatment in a murine metastatic and orthotopic hepatoma model

Vascular endothelial growth factor (VEGF) activity is correlated with a progressive tumor disease in patients with hepatocellular carcinoma (HCC). In spite of the well‐recognized role of VEGF in HCC, there are few data available regarding therapeutic strategies to block VEGF activity. Therefore, we employed a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGF receptor 2 (Flk‐1), AdsFlk‐1, to control pre‐established murine orthotopic and metastatic hepatomas. Vector function was confirmed via reverse‐transcriptase polymerase chain reaction and ELISA, and angiostatic effects were analyzed in vitro and in vivo. Antitumoral effects of systemic AdsFlk‐1 application were studied in a subcutaneous and orthotopic Hepa129 HCC model. Cell supernatant containing the truncated form of Flk‐1 had no direct effect on cell proliferation of Hepa129 cells in vitro but reduced endothelial tube formation on matrigel matrix by approximately 80% in vitro. Endothelial‐like cell infiltration into matrigel plugs in vivo was also decreased by 80%. Systemic treatment of tumor‐bearing mice inhibited tumor growth by 84% compared with the corresponding control group within 16 days after vector application. Likewise, the survival rate was significantly improved in the AdsFlk‐1 group compared with control. Orthotopic tumor growth was reduced by 82%, and development of malignant ascites was also retarded. In conclusion, systemic adenoviral‐mediated gene transfer of an Flk‐1 fragment significantly inhibited tumor growth in orthotopic and metastatic murine HCC. The data support the value of VEGF blockade as an effective target for HCC treatment. (HEPATOLOGY 2005;41:1233–1240)

Accelerated orthotopic hepatocellular carcinomas growth is linked to increased expression of pro-angiogenic and prometastatic factors in murine liver fibrosis.

Background: Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy. We questioned whether a pre‐existing fibrosis affects tumour development of implanted syngenic hepatoma cells. To further investigate a selected panel of factors involved in tumour growth, tumour organ samples were characterized for gene expression of vascular endothelial growth factor (VEGF)‐A/‐C, VEGF receptors Flt1, Flk‐1, Flt‐4 and for VEGF‐A protein levels.

Gene Therapy of Hepatocellular Carcinoma and Gastrointestinal Tumors

Abstract: Primary liver cancer and liver metastases from gastrointestinal tumors lack effective therapy. Gene therapy is a promising therapeutic approach and is based on the introduction of genetic material into cells to generate a curative biological effect. Adenoviral vectors can very efficiently transduce a wide variety of malignant epithelial cells both in vitro and in vivo. A variety of gene therapy‐based anticancer strategies have been effective in animal tumor models, including replacement of tumor suppressor genes, selective activation of prodrugs, genetic immunotherapy, and antiangiogenic actions. Enzymes used for genetic activation include viral thymidine kinase (tk), which may activate nucleoside analogs such as ganciclovir. We and others have demonstrated the efficacy of the tk/ganciclovir system in the treatment of hepatocellular carcinoma and metastatic colorectal cancer in experimental models. Also, this strategy can be safely applied to patients with liver tumors. Interleukin‐12 (IL‐12) is among the most potent cytokines in stimulating antitumor immunity. In models of primary and metastatic liver cancer we showed that intratumoral administration of recombinant adenovirus encoding IL‐12 activates natural killer cells, induces specific antitumor immunity, and displays a powerful antiangiogenic effect, resulting in tumor regression. There is a synergistic effect with the gene transfer of the chemokine IP‐10. Also, intratumoral injection of either dendritic cells transfected ex vivo with recombinant adenovirus encoding IL‐12 (Ad.IL‐12) or an adenovirus coding for the CD40 ligand have shown an intense antitumor effect against experimental colorectal cancer. In summary, a variety of gene therapy strategies have been effective against animal models of gastrointestinal tumors. Clinical trials should determine whether human patients can be treated safely and effectively by such strategies.

Effective antitumour mono- and combination therapy by gene delivery of angiostatin-like molecule and interleukin-12 in a murine hepatoma model.

MethodsWe applied an experimental approach employing two recombinant adenoviral vectors (Ad) that express interleukin-12 (IL-12) and angiostatin-like molecule (K1-3) respectively to a subcutaneous hepatoma model in mice.ResultsInjection of AdK1-3 into tumour nodules established by subcutaneous (s.c.) implantation of Hepa129 hepatoma cells in C3H mice resulted in a significant dose-dependent reduction in tumour growth by 57% in the high dosage group (5×109xa0plaque-forming units [pfu], n=8) 10xa0days after treatment initiation. Similar antitumoural effects were found for the intratumoural mono-therapy with IL-12 (2.5×109xa0pfu, n=8) resulting in 60% tumour inhibition at the same time point. The survival rate was significantly (p=0.009) improved in the IL-12 but not in the K1-3 treatment group. A combination therapy of AdK1-3 and AdIL-12 was also effective, but did not further improve antitumour efficacy compared with the monotherapy.Conclusion In conclusion, both mono- and combination therapy of K1-3 and IL-12 significantly inhibited tumour progression in this experimental tumour model. The co-administration of both compounds did not result in additive antitumour effects. We hypothesise that the lack of additive antitumour effects of the combination treatment might be attributed to partially counteracting antitumour effects and further studies are needed to illustrate the interference of tumour angiogenesis and tumour inflammation in this tumour model.

335 VEGF-A GENE SILENCING AND IMMUNOSTIMULATION COMBINED IN ONE SIRNA INCREASED ANTI TUMORAL THERAPY EFFICACY IN AN HCC ORTHOTOPIC FIBROTIC TUMOR MODEL

Four HBV associated-HCC cell lines, SNU-739, SNU-761, SNU-878 and SNU-886, which had been established from HCC patients with chronic HBV infection, were used to study the expression of PPARr and they were cultured in the 10 mmol/L to 100 mmol/L concentration of PPARr synthetic agonists, troglitzone, pioglitazone, rosiglitazone, and its natural ligand, 15deoxy-D12, 14-prostaglandin J2 (15d-PGJ2). Cell growth was determined via MTT assay in 24, 48 and 72 hour intervals. The cell cycle was analyzed by flow cytometry. DNA fragments were measured for apoptosis assay. Caspase 3, 8, and 9 activities were also determined by colorimetric protease assay. The amount of bcl-2 and bax mRNA was quantified by RT-PCR. The expression of PPARr was all found in the above HBV-associated HCC cell lines. Cell growth was inhibited by all the PPARr agonists doseand time-dependently. Especially, the effect of pioglitazone and 15d-PGJ2 was prominent. Cell cycle analysis revealed an increased proportion of cells in sub-G1 phase only by pioglitazone and 15d-PGJ2. In apoptosis assay, DNA fragments were also increased in all the HCC cell lines when treated with pioglitazone and 15d-PGJ2, but the effect of troglitazone and rosiglitazone was minimal. Pioglitazone and 15d-PGJ2 were found to increase caspase-3 and 9 activities but not caspase-8 and they decreased the level of expression of antiapoptotic factor, bcl-2, but not the proapoptotic factor, bax. Apoptosis induced by pioglitazone and 15d-PGJ2 was revealed caspasedependent. The pancaspase inhibitor and capase-3 inhibitor inhibited the apoptosis induced by various PPARr agonists dose-dependently. In conclusion, these findings suggest that pioglitazone and 15d-PGJ2 have antineoplastic effects on HBV-associated HCC cells by induction of apoptosis through a caspase dependent intrinsic pathway.

Antitumoral efficacy of adenoviral mediated gene transfer of angiostatin-like molecule in subcutaneously growing HEPA129 hepatomas in mice depends on the application modus

Introduction: Hepatocellular carcinomas (HCC) are regularly hypervascularized and therefore present a rationale aim for angiostatic antitumor therapy. We tested the effect of an intratumoral or systemic application of an angiostatic adenoviral vector encoding angiostatin-like molecule (AdKl-3) representing the first three kringle regions of plasminogen in subcutaneously growing HCC. Method: AdKl-3 had been constructed and tested for activity as described before. Systemic in viva activity was confirmed in Matrigel assay. HCC were established by S.C. injection of 1000 x lo3 Hepa cells in syngenie C3H-mice. When tumor sizes reached about 1.50 mm3 intratumoral treatment was initiated by injection of AdlacZ (0,5 x 10” pfu; n=13) or AdKl-3 (0,5 x 10” pfu; n=8), respectively. For systemic treatment vectors were administered intraperitoneally (10” pfu) when tumors reached about 50 mm3 in volume (AdlacZ, n=5; AdKl-3, n=6). Tumor growth and survival rate were assessed. Results: Intratumoral tumor growth was inhibited by AdKl-3 by about 60%. Although systemic administration of AdKl-3 reduced the endothelial cell-like infiltration into Matrigel plugs by about 50%, tumor growth was not inhibited in the AdKl-3 group (852 mm3 versus 757 mm3 in the control). Survival rates were neither improved by intratumoral nor by systemic administration of AdKl-3. Conclusion: Intratumoral but not systemic administration of gene transfer for angiostatin-like molecule resulted in significant inhibition of tumor growth in this tumor model. The lack of improvement in survival rate might be related to transient protein expression by first generation adenoviruses. I 743 INHIBITION OF TUMOR GROWTH BY USING AN ADENOVIRUS EXPRESSING THE DOMINANT NEGATIVE RECEPTOR FLK-1 IN A MURINE TUMOR MODELS