Dioleoyl



Keywords: dioleoyl
Description: 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),

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.

In 1978, the World Health Organization defined cirrhosis as “a diffuse process characterized by fibrosis and the conversion of normal liver architecture into structurally abnormal nodules.” One of the most common causes of hepatic fibrosis is chronic alcohol abuse; other factors also have the potential to trigger hepatic fibrogenesis (1 ).

Liver fibrosis is of utmost relevance for hepatocellular carcinogenesis (HCC). Malignant hepatocellular transformation is characterized by a shortened half-life and increased proliferation and regeneration of hepatocytes secondary to ongoing inflammation (2 ). This leads to accumulation of genomic mutations and instability, alterations that sometimes accumulate in a neoplastic phenotype (3 ). For that reason,

HCC is strongly associated with chronic liver diseases, including chronic hepatitis and cirrhosis. In fact most cases of HCC, approximately 80%, occur in combination with underlying cirrhosis (4 ,5 ); <10% are observed in noncirrhotic livers, rarely without hepatitis (6 ). Notably, once cirrhosis is established there is no proven effective HCC prevention, yet (7 ). Recently, we showed that hepatic fibrosis relevantly accelerates orthotopic HCC tumor growth and metastasis in fibrotic C3H/He mice (8 ). Kuriyama et al. (9 ) reported that fibrosis seemed to affect metastasis. However, there is still the need for a robust murine liver fibrosis model to investigate antitumor efficacy.

Angiogenesis plays a major role in a wide range of biological processes, such as wound healing, organ regeneration, and the female reproduction cycle. Under normal conditions, angiogenesis otherwise does not occur in an adult organism but is needed for further tumor growth. Vascular endothelial growth factor (VEGF) is a major player in tumor angiogenesis, and the VEGF/VEGF receptor (VEGFR) pathway is a major focus of interest in basic cancer research (10 ). Several studies have used gene silencing targeted against VEGF-A mRNA in distinct tumor models (11 –14 ).

There are no data on functional VEGF-A knockdown in HCC with preexisting hepatic fibrosis. Here, we applied 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-formulated small interfering RNA (siRNA) targeted against VEGF-A to control HCC in mice with preexisting hepatic fibrosis.

C3H/He female mice (age matched) were obtained from Charles River (Sulzfeld, Germany) and housed under SPF conditions in the central animal facility of the University Hospital Bonn. Animal procedures were performed in accordance with approved protocols of the responsible local governmental administration and followed recommendations for proper care and use of laboratory animals.

Hepa129 cells (hepatoma 129 originating from C3H/He mice, obtained from NCI-Frederick Cancer Research and Development Center [DCT Tumor Repository; Frederick, MD, USA]) were maintained in RPMI 1640 (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (Invitrogen), 200 mM glutamine, and penicillin/streptomycin.

Before tumor cell implantation, induction of fibrosis was studied in female C3H/He mice using i.p. injections of thioacetamide (TAA) (Sigma-Aldrich, Taufkirchen, Germany; 0.15 mg/g body weight) three times per wk for up to 24 wks in combination with alcohol feeding in sweetened drinking water (10% vol/vol) according to a published protocol (8 ,15 ,16 ).

To minimize postoperational mortality, TAA administration was paused for up to 7 d after tumor cell implantation. At d 7 and 9, all mice received TAA i.p. again. EtOH feeding via drinking water was maintained.

DOTAP was purchased from Roth (Karlsruhe, Germany). The DOTAP siRNA complex was formulated according to the manufacturer’s protocol and adapted to the in vivo application. Fibrotic mice received 200 μg siRNA/kg body weight (5 μg siRNA for a 27-g mouse) according to published studies (18 –20 ). The DOTAP siRNA ratio was 2:1 (vol/wt). Therefore, 5 μg siRNA were incubated with 10 μL DOTAP for 20 min at room temperature and diluted in NaCl (0.9%) buffer for i.p. injections. siRNA was applied i.p. as described (21 ). siRNA with or without DOTAP formulation was injected at day –1, day 0 (HCC implantation), and every second d until d 10 ( Figure 2A ).




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