Small Interfering RNAs Induce Target-Independent Inhibition of Tumor Growth and Vasculature Remodeling in a Mouse Model of Hepatocellular Carcinoma
2010; Elsevier BV; Volume: 177; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.100157
ISSN1525-2191
AutoresMathieu Bergé, Philippe Bonnin, Eric Sulpice, José Vilar, David Allanic, Jean‐Sébastien Silvestre, Bernard Lévy, Gordon C. Tucker, G Tobelem, Tatyana Merkulova‐Rainon,
Tópico(s)MicroRNA in disease regulation
ResumoRNA interference mediated by small interfering RNAs (siRNAs) has emerged as a potential therapeutic approach to treat various diseases, including cancer. Recent studies with several animal models of posttraumatic revascularization demonstrated that synthetic siRNAs may produce therapeutic effects in a target-independent manner through the stimulation of the toll-like receptor-3 (TLR3)/interferon pathway and suppression of angiogenesis. To analyze the impact of siRNAs on tumor angiogenesis, we injected transgenic mice developing hepatocellular carcinoma (HCC) with either control siRNAs or siRNA targeting neuropilin-1. We found that treatment with these siRNAs led to a comparable reduction in tumor liver volume and to inhibition of tumor vasculature remodeling. We further determined that TLR3, which recognizes double-stranded siRNA, was up-regulated in mouse HCC. Treatment of HCC mice with polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 agonist, led to both a reduction of tumor liver enlargement and a decrease in hepatic arterial blood flow, indicating that TLR3 is functional and may mediate both anti-angiogenic and anti-tumor responses. We also demonstrated that siRNAs increased interferon-γ levels in the liver. In vitro, interferon-γ inhibited proliferation of endothelial cells. In addition, we found that siRNAs inhibited endothelial cell proliferation and morphogenesis in an interferon-γ–independent manner. Our results suggest that synthetic siRNAs inhibit target-independently HCC growth and angiogenesis through the activation of the innate interferon response and by directly inhibiting endothelial cell function. RNA interference mediated by small interfering RNAs (siRNAs) has emerged as a potential therapeutic approach to treat various diseases, including cancer. Recent studies with several animal models of posttraumatic revascularization demonstrated that synthetic siRNAs may produce therapeutic effects in a target-independent manner through the stimulation of the toll-like receptor-3 (TLR3)/interferon pathway and suppression of angiogenesis. To analyze the impact of siRNAs on tumor angiogenesis, we injected transgenic mice developing hepatocellular carcinoma (HCC) with either control siRNAs or siRNA targeting neuropilin-1. We found that treatment with these siRNAs led to a comparable reduction in tumor liver volume and to inhibition of tumor vasculature remodeling. We further determined that TLR3, which recognizes double-stranded siRNA, was up-regulated in mouse HCC. Treatment of HCC mice with polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 agonist, led to both a reduction of tumor liver enlargement and a decrease in hepatic arterial blood flow, indicating that TLR3 is functional and may mediate both anti-angiogenic and anti-tumor responses. We also demonstrated that siRNAs increased interferon-γ levels in the liver. In vitro, interferon-γ inhibited proliferation of endothelial cells. In addition, we found that siRNAs inhibited endothelial cell proliferation and morphogenesis in an interferon-γ–independent manner. Our results suggest that synthetic siRNAs inhibit target-independently HCC growth and angiogenesis through the activation of the innate interferon response and by directly inhibiting endothelial cell function. RNA interference (RNAi) is a naturally occurring mechanism of sequence-specific gene expression silencing that is mediated by double-stranded RNA and has been implicated in the control of the expression of endogenous and exogenous genes, including those of viruses and transposons.1Siomi H Siomi MC On the road to reading the RNA-interference code.Nature. 2009; 457: 396-404Crossref PubMed Scopus (508) Google Scholar RNAi, induced by artificial synthetic small interfering RNA (siRNAs) or plasmid or viral vectors encoding for short hairpin RNAs, has become a powerful tool to analyze gene function and has emerged as a potential therapeutic strategy to treat various pathologies including cancers.2Kim DH Rossi JJ Strategies for silencing human disease using RNA interference.Nat Rev Genet. 2007; 8: 173-184Crossref PubMed Scopus (926) Google Scholar Numerous siRNAs and short hairpin RNAs have been tested in preclinical cancer models and have provided encouraging results including an efficient inhibition of tumor angiogenesis, tumor cell proliferation and invasiveness, promotion of apoptosis, uphold of differentiation, and sensitization to chemotherapy.3Huang C Li M Chen C Yao Q Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications.Expert Opin Ther Targets. 2008; 12: 637-645Crossref PubMed Scopus (114) Google Scholar Several RNAi-based approaches have recently entered clinical trials.4Castanotto D Rossi JJ The promises and pitfalls of RNA-interference-based therapeutics.Nature. 2009; 457: 426-433Crossref PubMed Scopus (1046) Google Scholar Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, with a continuously increasing incidence, and is the third cause of cancer-related deaths.5Llovet JM Bruix J Novel advancements in the management of hepatocellular carcinoma in 2008.J Hepatol. 2008; 48: S20-S37Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar The principal treatment modalities for HCC include surgical rejection, liver transplantation, and local ablation, whereas HCC is relatively resistant to systemic therapy.6Yau T Chan P Epstein R Poon RT Evolution of systemic therapy of advanced hepatocellular carcinoma.World J Gastroenterol. 2008; 14: 6437-6441Crossref PubMed Scopus (58) Google Scholar RNAi-based approaches are currently being considered as potential new therapeutics to both prevent HCC by treating the underlying liver diseases and to cure established HCC.7Arbuthnot P Thompson LJ Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma.World J Gastroenterol. 2008; 14: 1670-1681Crossref PubMed Scopus (32) Google Scholar In animal models of HCC or of precancerous liver diseases, synthetic siRNA-mediated gene silencing targeting hepatitis B8Chen Y Cheng G Mahato RI RNAi for treating hepatitis B viral infection.Pharm Res. 2008; 25: 72-86Crossref PubMed Scopus (106) Google Scholar or hepatitis C9Watanabe T Umehara T Kohara M Therapeutic application of RNA interference for hepatitis C virus.Adv Drug Deliv Rev. 2007; 59: 1263-1276Crossref PubMed Scopus (39) Google Scholar viral RNAs and replication intermediates or endogenous liver proteins including the Fas receptor,10Song E Lee SK Wang J Ince N Ouyang N Min J Chen J Shankar P Lieberman J RNA interference targeting Fas protects mice from fulminant hepatitis.Nat Med. 2003; 9: 347-351Crossref PubMed Scopus (1028) Google Scholar vascular endothelial growth factor-A (VEGF-A),11Kornek M Lukacs-Kornek V Limmer A Raskopf E Becker U Klockner M Sauerbruch T Schmitz V 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.Mol Med. 2008; 14: 365-373Crossref PubMed Scopus (22) Google Scholar, 12Raskopf E Vogt A Sauerbruch T Schmitz V siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo.J Hepatol. 2008; 49: 977-984Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar cell-cycle-activating phosphatase CDC25B,13Yan X Chua MS He J So SK Small interfering RNA targeting CDC25B inhibits liver tumor growth in vitro and in vivo.Mol Cancer. 2008; 7: 1-9Crossref PubMed Scopus (23) Google Scholar and heme oxygenase 1,14Sass G Leukel P Schmitz V Raskopf E Ocker M Neureiter D Meissnitzer M Tasika E Tannapfel A Tiegs G Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice.Int J Cancer. 2008; 123: 1269-1277Crossref PubMed Scopus (83) Google Scholar has been shown to efficiently reduce target protein expression and inhibit disease progression. Another promising RNAi-based therapeutic strategy consists in the administration of microRNAs, which are down-regulated in HCC.15Kota J Chivukula RR O'Donnell KA Wentzel EA Montgomery CL Hwang HW Chang TC Vivekanandan P Torbenson M Clark KR Mendell JR Mendell JT Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.Cell. 2009; 137: 1005-1017Abstract Full Text Full Text PDF PubMed Scopus (1497) Google Scholar Such a treatment has been found to effectively inhibit the proliferation of cancer cells and stimulate apoptosis and to strongly reduce liver tumor growth without inducing deleterious toxic effects. HCC is a highly vascularized tumor, and its progression and prognosis is associated with angiogenesis and vascular remodeling.16Fernandez M Semela D Bruix J Colle I Pinzani M Bosch J Angiogenesis in liver disease.J Hepatol. 2009; 50: 604-620Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar Arterialization of the blood supply is one of the main features of HCC and a part of the noninvasive criteria to establish an HCC diagnosis.16Fernandez M Semela D Bruix J Colle I Pinzani M Bosch J Angiogenesis in liver disease.J Hepatol. 2009; 50: 604-620Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 17Nascimento C Bottino A Nogueira C Pannain V Analysis of morphological variables and arterialization in the differential diagnosis of hepatic nodules in explanted cirrhotic livers.Diagn Pathol. 2007; 2: 51Crossref PubMed Scopus (13) Google Scholar, 18Yang ZF Poon RT Vascular changes in hepatocellular carcinoma.Anat Rec (Hoboken). 2008; 291: 721-734Crossref PubMed Scopus (146) Google Scholar Other vascular changes in HCC include increase in microvessel density, formation of immature vessels with increased permeability, sinusoidal capillarization, and development of intrahepatic shunts.16Fernandez M Semela D Bruix J Colle I Pinzani M Bosch J Angiogenesis in liver disease.J Hepatol. 2009; 50: 604-620Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 18Yang ZF Poon RT Vascular changes in hepatocellular carcinoma.Anat Rec (Hoboken). 2008; 291: 721-734Crossref PubMed Scopus (146) Google Scholar Several studies have demonstrated that VEGF-A, a key regulator of vessel growth and homeostasis, is overexpressed in HCC and plays a central role in vascular remodeling, angiogenesis and HCC progression.18Yang ZF Poon RT Vascular changes in hepatocellular carcinoma.Anat Rec (Hoboken). 2008; 291: 721-734Crossref PubMed Scopus (146) Google Scholar, 19Kaseb AO Hanbali A Cotant M Hassan MM Wollner I Philip PA Vascular endothelial growth factor in the management of hepatocellular carcinoma: a review of literature.Cancer. 2009; 115: 4895-4906Crossref PubMed Scopus (94) Google Scholar Targeting angiogenesis by use of RNAi-based strategies directed against different components of VEGF signaling system appears as a rational strategy for therapy of HCC. We have recently described the main steps of angiogenesis and vascular remodeling in a transgenic mouse model of HCC.20Dupuy E Hainaud P Villemain A Bodevin-Phedre E Brouland JP Briand P Tobelem G Tumoral angiogenesis and tissue factor expression during hepatocellular carcinoma progression in a transgenic mouse model.J Hepatol. 2003; 38: 793-802Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar The evolution of HCC in this model is reproducible and characterized by a classical sequence of events: hyperplasia, dysplasia (between the fourth and eighth week after birth), adenoma (from the 12th week), and carcinoma (at the 16th week). As in humans, angiogenesis precedes then accompanies hepatocarcinogenesis in this model, and the VEGF-Delta-like 4/Notch4-ephrin B2 pathway plays a major role in the regulation of tumor vessel growth and remodeling and HCC progression.21Hainaud P Contreres JO Villemain A Liu LX Plouet J Tobelem G Dupuy E The role of the vascular endothelial growth factor-Delta-like 4 ligand/Notch4-ephrin B2 cascade in tumor vessel remodeling and endothelial cell functions.Cancer Res. 2006; 66: 8501-8510Crossref PubMed Scopus (145) Google Scholar We demonstrated that the hepatocellular tumor growth and vascular remodeling can be followed noninvasively in this model by using ultrasound Doppler imaging.22Bonnin P Villemain A Vincent F Debbabi H Silvestre JS Contreres JO Levy BI Tobelem G Dupuy E Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar We used this model to evaluate the therapeutic potential of targeting angiogenesis in HCC by treatment the mice with synthetic siRNA specific for neuropilin-1 (NRP1), a nontyrosine kinase receptor for VEGF. Moreover, it has been shown recently that synthetic siRNAs inhibit angiogenesis in a sequence- and a target-independent manner in the mouse models of choroidal neovascularization, dermal wound healing,23Kleinman ME Yamada K Takeda A Chandrasekaran V Nozaki M Baffi JZ Albuquerque RJ Yamasaki S Itaya M Pan Y Appukuttan B Gibbs D Yang Z Kariko K Ambati BK Wilgus TA DiPietro LA Sakurai E Zhang K Smith JR Taylor EW Ambati J Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.Nature. 2008; 452: 591-597Crossref PubMed Scopus (799) Google Scholar corneal neovascularization, and hindlimb ischemia.24Cho WG Albuquerque RJ Kleinman ME Tarallo V Greco A Nozaki M Green MG Baffi JZ Ambati BK De Falco M Alexander JS Brunetti A De Falco S Ambati J Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth.Proc Natl Acad Sci USA. 2009; 106: 7137-7142Crossref PubMed Scopus (121) Google Scholar Therefore we treated HCC mice with two different control siRNAs to analyze whether tumor angiogenesis could be target-independently suppressed by siRNAs. Approval was obtained from the local ethical committee for studies involving mice. The generation of ASV-B transgenic mice (C57BL/6 background) as a model of HCC has been reported previously.25Dubois N Bennoun M Allemand I Molina T Grimber G Daudet-Monsac M Abelanet R Briand P Time-course development of differentiated hepatocarcinoma and lung metastasis in transgenic mice.J Hepatol. 1991; 13: 227-239Abstract Full Text PDF PubMed Scopus (82) Google Scholar Because of the integration of the transgene into chromosome Y, only male mice develop HCC. The female mice of the same lineage do not harbor the transgene and were used as wild-type controls. The absence of structural and functional differences in liver architecture and liver vasculature between wild-type C57BL/6 male and female mice has been demonstrated previously.20Dupuy E Hainaud P Villemain A Bodevin-Phedre E Brouland JP Briand P Tobelem G Tumoral angiogenesis and tissue factor expression during hepatocellular carcinoma progression in a transgenic mouse model.J Hepatol. 2003; 38: 793-802Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Silencer In vivo Ready Predesigned siRNA targeting mouse NRP1 (NRP1 siRNA) and nontargeting Negative Control # 1 siRNA (NT siRNA) were purchased from Ambion (ID # 155679 and 103860, respectively, Austin, TX). siSTABLE siRNA targeting the firefly enzyme luciferase (Luc siRNA, sense 5′-UAAGGCUAUGAAGAGAUACdTdT-3′ and antisense 5′-GUAUCUCUUCAUAGCCUUAdTdT-3′)23Kleinman ME Yamada K Takeda A Chandrasekaran V Nozaki M Baffi JZ Albuquerque RJ Yamasaki S Itaya M Pan Y Appukuttan B Gibbs D Yang Z Kariko K Ambati BK Wilgus TA DiPietro LA Sakurai E Zhang K Smith JR Taylor EW Ambati J Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.Nature. 2008; 452: 591-597Crossref PubMed Scopus (799) Google Scholar, 24Cho WG Albuquerque RJ Kleinman ME Tarallo V Greco A Nozaki M Green MG Baffi JZ Ambati BK De Falco M Alexander JS Brunetti A De Falco S Ambati J Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth.Proc Natl Acad Sci USA. 2009; 106: 7137-7142Crossref PubMed Scopus (121) Google Scholar was purchased from Dharmacon (Lafayette, CO). siRNAs were resuspended in sterile phosphate buffered saline (PBS) and injected intraperitoneally, twice a week, at 1.2 mg/kg into both wild-type and HCC mice. Polyinosinic-polycytidylic acid [poly(I:C)] was purchased from Sigma-Aldrich (St. Louis, MO). For injection, poly(I:C) was resuspended in PBS and administered intraperitoneally, twice a week, at 2 mg/kg. Treatments were started when mice reached the age of 8 weeks and continued until mice were 16 weeks old. Ultrasound imaging was performed every 2 weeks. Echo-derived liver volume and time-average mean blood flow velocities (BFV) were estimated using a Vivid 7 echocardiograph with a 12-MHz linear transducer (GE Medical Systems ultrasound, Horten, Norway) as previously described.22Bonnin P Villemain A Vincent F Debbabi H Silvestre JS Contreres JO Levy BI Tobelem G Dupuy E Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar At the end of treatments, a subgroup of mice was subjected to microangiographic analysis of liver vascularization. Microangiography was performed as previously described with minor modifications.22Bonnin P Villemain A Vincent F Debbabi H Silvestre JS Contreres JO Levy BI Tobelem G Dupuy E Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Before perfusion with barium sulfate (1 g/ml), the mesenteric artery and aorta upstream from the celiac trunk were ligated. Image acquisition was performed with a digital Trophy X-ray transducer (Trophy Radiologie, Vincennes, France). Quantification of arterial vessel density was performed using the PRIMed Angio v0.9 software (Primed Microvision, France) and expressed as a percentage of pixels per image occupied by vessels in the total liver area. Another subgroup of treated mice was sacrificed, livers were excised, weighed, and processed for RNA and protein extraction and immunohistochemical analysis. All cell culture reagents were purchased from Invitrogen (Cergy Pontoise, France) unless otherwise specified. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described.26Ding S Merkulova-Rainon T Han ZC Tobelem G HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro.Blood. 2003; 101: 4816-4822Crossref PubMed Scopus (140) Google Scholar Lung-derived normal human microvascular endothelial cells (HMVECs) were purchased from Lonza (Walkersville, MD) and cultured according to the manufacturer's instructions. The human HCC cell lines Hep G2, SK-HEP-1, and PLC/PRF/5 were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI 1640 (Hep G2) or DMEM (SK-HEP-1 and PLC/PRF/5) basal medium supplemented with 10% fetal bovine serum (FBS, BioWest, Cholet, France), 50 U/ml penicillin, 50 μg/ml streptomycin, and 2.5 μg/ml amphotericin B. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors by density gradient centrifugation using Pancoll (Pan-Biotech GmbH, Aidenbach, Germany). Cells were washed twice with PBS, resuspended in RPMI 1640 medium supplemented with 5% FBS at 3 × 106 cells/ml, and incubated in 24-well plates (0.5 ml/well) in the presence or absence of 50 μg/ml of poly(I:C) or NT siRNA. After 24 hours, supernatants were harvested, centrifuged at 14000 × g for 10 minutes, and interferon-γ (INF-γ) levels were determined by ELISA. The rat anti-CD31 antibody was purchased from BD Pharmingen (San Diego, CA). The goat anti-neuropilin-1 antibody was purchased from R&D Systems (Minneapolis, MN). The rabbit anti-laminin antibody was purchased from Sigma-Aldrich (Saint Louis, MO). The rabbit anti-Ki-67 antibody was purchased from NeoMarkers (Fremont, CA). The goat anti-TLR3 and rabbit anti-tubulin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-phospho-TLR3 (Tyr 759) antibody was purchased from Imgenex (San Diego, CA). The rabbit anti-phospho-IRF-3 (Ser 396), anti-phospho-NF-κB p65 (Ser 536) and the mouse anti-caspase-8 antibody were purchased from Cell Signaling Technology (Danvers, MA). Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 555-conjugated donkey anti-goat IgG and Alexa Fluor 555-conjugated goat anti-rabbit antibody were purchased from Molecular Probes (Eugene, OR). Peroxidase-conjugated species-specific secondary IgGs were obtained from Jackson ImmunoResearch (West Grove, PA). Liver samples were homogenized in RIPA buffer using a MagNA Lyser Instrument (Roche, Rotkreuz, Switzerland). Protein extraction, Western blotting, band visualization, and quantification were performed as previously described.27Sulpice E Plouet J Berge M Allanic D Tobelem G Merkulova-Rainon T Neuropilin-1 and neuropilin-2 act as coreceptors, potentiating proangiogenic activity.Blood. 2008; 111: 2036-2045Crossref PubMed Scopus (133) Google Scholar For immunofluorescence analysis, frozen liver sections (5 μm) or cell cultures were fixed in cold acetone (15 minutes), washed three times with PBS containing 0.1% Tween-20 (PBST), and incubated in PBST containing 0.1% BSA for 20 minutes. Samples were then incubated with primary antibody for 1 hour at room temperature followed by the appropriate secondary antibody (30 minutes). Negative controls were incubated with the secondary antibodies only. Sections were observed with an Axioskop 2 plus fluorescence microscope equipped with an Achroplan ×40/0.65 and a Plan-Neofluar ×5/0.16 Ph1 objectives (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Images were taken using a QICAM 12 bit Fast 1394 Cooled Color Camera (QImaging, Pleasanton, CA) and Archimed 5.6 software (Microvision Instruments, Evry, France), and quantified using Histolab 5.8 software (Microvision Instruments). Cell cultures were observed using an Observer.Z1 inverted microscope equipped with an EC Plan-Neofluar ×40/0.75 objective (Carl Zeiss). Images were taken with a KY-F75U digital camera (JVC, Tokyo, Japan). Total cellular RNA was extracted with RNAXEL reagent (Eurobio, Les Ulis, France) and purified using the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The first strand cDNA template was synthesized using the Transcriptor First Strand cDNA Synthesis Kit and random hexamer primers (Roche Applied Science, Mannheim, Germany). The cDNA product was amplified using LightCycler FastStart DNA Master SYBR Green I and a Light Cycler 1.5 (Roche). The primers used for PCR were: mouse TLR3 forward 5′-TTGTCTTCTGCACGAACCTG-3′, and mouse TLR3 reverse 5′-CGCAACGCAAGGATTTTATT-3′; mouse 18S forward 5′-CGCGGTTCTATTTTGTTGGT-3′, and mouse 18S reverse 5′-AGTCGGCATCGTTTATGGTC-3′. mRNA levels were normalized to the 18S ribosomal RNA. TLR3 mRNA was detected in human cell lines after cDNA amplification with FastStart TaqDNA Polymerase (Roche) and following primers: human TLR3 forward 5′ AGCCTTCAACGACTGATGCT-3′, and human TLR3 reverse 5′-TTTCCAGAGCCGTGCTAAGT-3′. Interferon-α, -β, and interleukin-12 concentrations in liver homogenates were estimated using the Mouse Interferon α ELISA Kit, Mouse Interferon β ELISA Kit, and Quantikine Mouse IL-12 p70 Immunoassay (R&D Systems), respectively, according to the manufacturer's instructions. The BD OptEIA mouse IFN-γ ELISA Set (BD Biosciences, San Diego, CA) was used to quantify interferon-γ. Data were normalized to total protein content estimated with the BCA Protein Assay Reagent (Pierce, Rockford, IL). Interferon-γ concentration in human PBMC supernatants was determined using Quantikine Human INF-γ Immunoassay (R&D Systems). Cells were plated in 96-well plates at a density of 104 cells per well (HUVEC and Hep G2) or 5 × 103 cells per well (SK-HEP-1 and PLC/PRF/5) and allowed to grow at 37°C with 5% CO2 for 48 hours. Cells were then incubated with various concentrations of INF-γ (Reliatech GmBH, Wolfenbüttel, Germany), poly(I:C), NRP1 siRNA or NT siRNA for a further 48 hours, followed by an incubation with 0.5 mg/ml of 3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich). After 4 hours, a water-insoluble formazan produced by viable cells was solubilized with DMSO and cell viability and growth over time was estimated by measuring the absorbance at 560 nm using a Victor3 spectrofluorimeter (Perkin Elmer, Turku, Finland). HUVECs were resuspended in M199 medium supplemented with 1% FBS and seeded at 105 cells per well on the top of a preformed collagen gel (BD Biosciences, Bedford, MA) in 24-well plate. Capillary-like tube formation was induced by the addition of 100 ng/ml of VEGF-A165 and HGF (R&D systems) in the presence or absence of 100 ng/ml of INF-γ, 50 μg/ml of poly(I:C), or 50 μg/ml of NT siRNA. After 24 hours of incubation, cultures were observed using an Observer.Z1 inverted microscope equipped with an EC Plan-Neofluar 10x/0.3 Ph1 objective (Carl Zeiss). Images were taken with a KY-F75U digital camera (JVC) and quantified using Histolab 5.8 software. Data are presented as the mean ± SEM. The echo-derived volume of the liver, heart rate, peak systolic, end-diastolic, and mean BFV in hepatic and mesenteric arteries were compared between HCC mice treated with PBS, NRP1 siRNA, or control siRNAs by analysis of variance for repeated measurements (with time as within subject factor and group as between subject factor) followed by post hoc Student's t-test with Bonferroni correction (MedCalc Software, Mariakerke, Belgium). Statistical analyses of other data were performed in Excel using Student's t-test. Data were considered statistically significant if P < 0.05. To evaluate the effect of synthetic siRNAs on HCC growth and angiogenesis, we used an siRNA targeting the VEGF receptor NRP1 (NRP1 siRNA) and two control siRNAs, one non-targeting (NT siRNA), with limited sequence similarity to known mouse, human, and rat genes, and the other one targeting the firefly enzyme luciferase (Luc siRNA). The NRP1 siRNA was selected from preliminary in vitro experiments based on its capacity to effectively down-regulate (by 90%) NRP1 protein expression in a cultured mouse melanoma cell line B16-F10, which expresses high levels of NRP1 (data not shown). We found that twice a week intraperitoneal administration of this siRNA (at 1.2 mg/kg) reduced the expression of NRP1 protein by 25% in the liver of transgenic mice developing HCC (data not shown). In contrast, the control siRNAs, which had no effect on NRP1 expression in B16-F10 cells, did not significantly modulate the NRP1 protein level in the liver. We have previously established that between the fourth and 20th weeks, hepatocellular tumor growth is accompanied by an approximately sixfold increase in liver volume that can be assessed using ultrasound imaging.22Bonnin P Villemain A Vincent F Debbabi H Silvestre JS Contreres JO Levy BI Tobelem G Dupuy E Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar We used this approach to follow the rate of HCC growth in siRNA-treated mice. Ultrasonography demonstrated that similar to our previous observations, the echo-derived liver volume was increased almost threefold between the eighth and 16th week in HCC mice treated with vehicle alone (Figure 1A). Treatment with NRP1 siRNA inhibited liver volume growth in HCC mice (1.9-fold increase, P = 0.0053 versus PBS), whereas it did not affect liver volume in wild-type mice. The two control siRNAs decreased HCC growth to an extent similar to that observed with the NRP1 siRNA (Figure 1A). These results were confirmed by weighing the livers after sacrifice at the 16th week (Figure 1B). We showed previously that capillarization of blood supply in mouse HCC is accompanied by the up-regulation of laminin expression within tumor nodules.20Dupuy E Hainaud P Villemain A Bodevin-Phedre E Brouland JP Briand P Tobelem G Tumoral angiogenesis and tissue factor expression during hepatocellular carcinoma progression in a transgenic mouse model.J Hepatol. 2003; 38: 793-802Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar We used laminin immunostaining to visualize tumor nodules in liver sections of treated mice and determined their size with Histolab software. We found that treatment with each siRNA led to the reduction of tumor nodule size compared to PBS-treated animals (Figure 1C). Whereas the number of tumor nodules per section was higher in siRNA-treated mice (4.5 ± 0.75, 2.67 ± 0.82, 4.75 ± 0.87, for NRP1 siRNA, NT siRNA, and Lus NRP1 siRNA, respectively, against 1 nodule per section of PBS treated mice) the percentage of section area occupied by all tumor nodules was significantly lower in siRNA-treated mice compared to PBS-treated mice (Figure 1C). Thus, our results suggest that treatment with siRNAs does not affect the incidence of tumor nodules but limit their growth. Finally, to verify whether the decrease in HCC liver growth correlated with a decrease in tumor cell proliferation, we costained liver sections from sacrificed animals with 4′-6-diamidino-2-phenylindole (DAPI) and the proliferation marker Ki-67. We found that the proportion of Ki-67–positive nuclei was reduced by 30–40% in the liver of HCC mice treated with siRNAs compared to mice treated with PBS (Figure 1D). Thus, the siRNA-induced inhibition of HCC growth could at least be partially explained by the inhibition of proliferation of tumor hepatocytes. We next analyzed whether treatment with synthetic siRNAs affected tumor vasculature in transgenic mice developing
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