E. Raskopf

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.

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.

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