Esther Raskopf

siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo.

BACKGROUND/AIMSnWe have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo.nnnMETHODSnThe influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining.nnnRESULTSnVEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors.nnnCONCLUSIONSnThe results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.

Role of hypoxia-inducible transcription factor 1α for progression and chemosensitivity of murine hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a hypervascularized tumor entity with association of arterial vessel density with poor prognosis. The hypoxia-inducible transcription factor HIF-1α represents a pivotal regulator of angiogenesis and is thought to determine the angiogenic nature of HCC. However, the precise role of HIF-1α during the pathogenesis of HCC remains elusive. We established a functional inactivation of HIF-1α in vitro and in vivo via RNAi and Cre/loxP-mediated recombination, respectively, to determine HIF-1α’s role for tumor growth and chemosensitivity in transgenic and orthotopic murine HCC models. HIF-1α-deficient HCC cells displayed significantly reduced anchorage-independent growth and enhanced sensitivity toward etoposide, while basic cellular proliferation was unaffected. Analysis of gross tumor growth failed to detect reduced growth of HIF-1α-deficient tumors in the orthotopic and the transgenic HCC model, respectively. In line with the in vitro data, treatment of HIF-1α-deficient tumors with etoposide resulted in greater antiproliferative efficacy when compared to wild-type mice. Taken together, our study does not support a pivotal role of HIF-1α for tumor growth and angiogenesis in two murine HCC models. However, our data point toward a significant function of HIF-1α in determining chemosensitivity of HCC and therefore warrant validation of HIF-1α-inhibitors as adjuvant therapeutic agents in clinical studies of human HCC.

Statins activate the canonical hedgehog-signaling and aggravate non-cirrhotic portal hypertension, but inhibit the non-canonical hedgehog signaling and cirrhotic portal hypertension.

Liver cirrhosis but also portal vein obstruction cause portal hypertension (PHT) and angiogenesis. This study investigated the differences of angiogenesis in cirrhotic and non-cirrhotic PHT with special emphasis on the canonical (Shh/Gli) and non-canonical (Shh/RhoA) hedgehog pathway. Cirrhotic (bile duct ligation/BDL; CCl4 intoxication) and non-cirrhotic (partial portal vein ligation/PPVL) rats received either atorvastatin (15u2009mg/kg; 7d) or control chow before sacrifice. Invasive hemodynamic measurement and Matrigel implantation assessed angiogenesis in vivo. Angiogenesis in vitro was analysed using migration and tube formation assay. In liver and vessel samples from animals and humans, transcript expression was analyzed using RT-PCR and protein expression using Western blot. Atorvastatin decreased portal pressure, shunt flow and angiogenesis in cirrhosis, whereas atorvastatin increased these parameters in PPVL rats. Non-canonical Hh was upregulated in experimental and human liver cirrhosis and was blunted by atorvastatin. Moreover, atorvastatin blocked the non-canonical Hh-pathway RhoA dependently in activated hepatic steallate cells (HSCs). Interestingly, hepatic and extrahepatic Hh-pathway was enhanced in PPVL rats, which resulted in increased angiogenesis. In summary, statins caused contrary effects in cirrhotic and non-cirrhotic portal hypertension. Atorvastatin inhibited the non-canonical Hh-pathway and angiogenesis in cirrhosis. In portal vein obstruction, statins enhanced the canonical Hh-pathway and aggravated PHT and angiogenesis.

Plasminogen fragment K1–5 improves survival in a murine hepatocellular carcinoma model

Background: The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, and new alternative treatments are needed. Aims: To comparatively test the angiostatic and antitumour effects of adenoviral gene transfer of angiostatin (PlgK1–4, amino acids 1–440) and full kringles 1–5 (PlgK1–5, amino acids 1–546) in a model of subcutaneously transferred HCC in mice. Methods: PlgK1–4 and PlgK1–5 were generated from human WtPlg cDNA and used for adenovirus construction. Vector function and angiostatic effects were confirmed in vitro and in vivo. Antitumoral efficacies of intratumoral vector injections were studied in a model of subcutaneously transferred HCC model. Results: Cell supernatants containing PlgK1–4 and PlgK1–5 reduced endothelial tube formation in vitro by about 30%, whereas WtPlg exerted no inhibitory effect. Endothelial cell infiltration in vivo was decreased by about 60%, but not in AdWtPlg-treated animals. Intratumoral treatment of subcutaneous HCC tumours inhibited growth by 40% for AdPlgK1–4 and 63% for AdPlgK1–5 in surviving mice 12 days after initiation of treatment, whereas treatment with AdWtPlg even led to accelerated growth. Although PlgK1–4 and PlgK1–5 have similar inhibitory effects on intratumoral microvessels, PlgK1–5 markedly improved the survival time compared with PlgK1–4. Conclusion: PlgK1–5 and PlgK1–4 effectively inhibited HCC growth. As PlgK1–5 could also prolong the survival time, inducing complete tumour elimination in half of the AdPlgK1–5-treated mice, PlgK1–5 might be the most potential plasminogen fragment for treatment of experimental HCC.

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-formulated, immune-stimulatory vascular endothelial growth factor a small interfering RNA (siRNA) increases antitumoral efficacy in murine orthotopic hepatocellular carcinoma with liver fibrosis.

Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy and nonfibrotic. The majority of hepatocellular carcinoma (HCC), however, develops in patients suffering from preexisting liver fibrosis. We investigated the efficacy of a standard experimental therapeutic approach to interrupt the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) cascade via VEGF-A silencing, with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; cationic lipid) formulation, in HCC mice with preexisting liver fibrosis. The data show that intraperitoneal treatment with naked VEGF-A small interfering RNA (siRNA; 200 µg/kg) was inefficient to treat HCC implanted into fibrotic livers. VEGF-A siRNA containing an immunostimulatory motif in combination with DOTAP formulation significantly reduced hepatic VEGF-A expression and additionally activated the innate and adapted immune system as shown by an increased intrahepatic interferon type 1 response (68-fold increased β-interferon expression). DOTAP-formulated VEGF-A siRNA markedly improved VEGF-A siRNA uptake and enhanced the antitumor response. This study shows for the first time the therapeutic feasibility of using synergistic effects (gene silencing and activation of the immune system) united in one siRNA sequence to reduce HCC growth and metastasis in mice with preexisting liver fibrosis. We expect that these results will help to direct and improve future experimental gene-silencing approaches and establish more efficient antitumoral therapies against HCC.

Improving immunotherapy of hepatocellular carcinoma (HCC) using dendritic cells (DC) engineered to express IL-12 in vivo

Interleukin 12 (IL‐12), one of the most potent Th1‐cytokines, has been used to improve dendritic cells (DC)‐based immunotherapy of cancer. However, it failed to achieve clinical response in patients with hepatocellular carcinoma (HCC). In this study, improved conditions of immunotherapy with DC engineered to express IL‐12 were studied in murine subcutaneous HCC.

Toxic Damage Increases Angiogenesis and Metastasis in Fibrotic Livers via PECAM-1

Excessive ethanol consumption is one of the main causes of liver fibrosis. However, direct effects of ethanol exposure on endothelial cells and their contribution to fibrogenesis and metastasis were not investigated. Therefore we analysed whether ethanol directly affects endothelial cells and if this plays a role during fibrogenesis and metastasis in the liver. Murine and human endothelial cells were exposed to ethanol for up to 72 hours. In vitro, effects on VEGF, HIF-1alpha, PECAM-1, and endothelial cell functions were analysed. In vivo, effects of continuous liver damage on blood vessel formation and metastasis were analysed by PECAM-1 immunohistochemistry. Ethanol increased HIF-1alpha and VEGF levels in murine and human endothelial cells. This resulted in enhanced intracellular signal transduction, and PECAM-1 expression as well as tube formation and wound healing. In vivo, toxic liver damage increased angiogenesis during fibrogenesis. Metastasis was also enhanced in fibrotic livers and located to PECAM-1 positive blood vessels compared to nonfibrotic mice. In conclusion, ethanol had strong effects on endothelial cells, which—at least in part—led to a profibrotic and prometastatic environment mediated by PECAM-1. Blockade of increased PECAM-1 expression could be a promising tool to inhibit fibrogenesis and metastasis in the liver.

Plasminogen fragment K1–3 inhibits expression of adhesion molecules and experimental HCC recurrence in the liver

PurposeThere is no established adjuvant or neo-adjuvant treatment to curb tumor recurrence of hepatocellular carcinoma (HCC). Recent data showed that angiostatic factors can inhibit tumor cell adhesion to the endothelium and therefore recurrence/metastasis. We tested a potential preventive, pre-operative strategy using plasminogen kringles 1–3 (K1–3) to overcome this hurdle.Materials and methodsEffects of K1–3 on the intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) expression was analyzed in vitro and in vivo on RNA and protein levels. Influence of K1–3 on HCC recurrence in the liver was analyzed in an orthotopic tumor model.ResultsK1–3 decreased ICAM expression in Hepa129 tumor cells and VCAM expression in SVEC4-10 endothelial cells in vitro. In vivo, ICAM was reduced in histological tumor sections. Preventive treatment with AdK1–3 inhibited experimental HCC recurrence and tumor growth in the liver.ConclusionsWe were able to show that K1–3 inhibits intrahepatic tumor recurrence. This novel aspect elucidates a possible approach to prevent HCC recurrence.

Plasminogen Derivatives Encoding Kringles 1-4 and Kringles 1-5 Exert Indirect Antiangiogenic and Direct Antitumoral Effects in Experimental Lung Cancer

Recently, increasing evidence has been found demonstrating direct effects of angiostatin on tumor cells themselves. We have applied the plasminogen derivatives K1-4 and K1-5 to a lung cancer model to analyse indirect angiostatic effects against endothelial and direct effects against tumor cells. In accordance with preceding findings both derivatives inhibited endothelial cell functions in vitro. Additionally K1-4 and K1-5 have also shown substantial anti-proliferative and pro-apoptotic effects in tumor cells and have inhibited tumor growth. In addition our data supports the recent conclusion that plasminogen derivatives have a dual antitumor mechanism affecting both tumor angiogenesis and tumor cells.

Combination of Hypoxia and RNA-Interference Targeting VEGF Induces Apoptosis in Hepatoma Cells Via Autocrine Mechanisms

Control of VEGF signaling is an intense objective of pre-clinical and clinical studies in HCC disease with steadily increasing clinical application. Despite its emerging role, several aspects of anti-VEGF based treatments are poorly investigated, like the impact on tumor cells themselves, such as the effect on intracellular signaling and apoptosis induction in hepatoma cells. Effects of siRNA-VEGF on VEGF, VEGF-receptor expression and VEGF-A signaling such as AKT and JNK phosphorylation were determined under normoxic or hypoxic conditions in murine hepatoma cells. Apoptosis induction was analyzed by SubG1-fraction, JC1-staining and caspase-8 activation. VEGF receptor expression was analysed by semiquantitative real time PCR. Independent of oxygen status, siRNA-VEGF reduced VEGF levels resulting in decreased AKT and increased JNK phosphorylation in Hepa129 cells. The VEGF-receptors neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) were downregulated following siRNA-VEGF treatment or hypoxia induction respectively. Functionally, hypoxia significantly increased the apoptosis rate (as analyzed by SubG1-fraction, JC1-staining and JNKphosphorylation) which was further stimulated by siRNA-VEGF treatment. Our data indicate that antitumoral efficacy of an anti-VEGF based treatment with siRNA is partly based on negative autocrine feedback mechanisms which are even enhanced under hypoxic conditions. This observation helps to understand why antitumoral efficacy can be maintained despite of counteracting stimulation of tumoral VEGF secretion due to hypoxia. The direct impact on tumor cells further underscores the attractiveness of an anti-VEGF based siRNA treatment.