Potential molecular targets of statins in the prevention of hepatocarcinogenesis

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"documento" => "article" "crossmark" => 0 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Ann Hepatol. 2018;17:501-10" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 272 "formatos" => array:3 [ "EPUB" => 18 "HTML" => 178 "PDF" => 76 ] ] "en" => array:11 [ "idiomaDefecto" => true "titulo" => "Therapeutic Effect of HGF on NASH Mice Through HGF/c-Met and JAK2-STAT3 Signalling Pathway" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "501" "paginaFinal" => "510" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "f0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 2280 "Ancho" => 4442 "Tamanyo" => 1622685 ] ] "descripcion" => array:1 [ "en" => "Liver H&E staining (200x) after treatment. More severe steatohepatitis was observed in HFD mice when HGF, c-Met, or JAK2 was inhibited. A. Control group. B-F. Different intervention groups as shown in figure. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. group A (control);† P < 0.05, †† P < 0.01, ††† P < 0.001 vs. group B (HFD + normal saline); § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. group C (HFD + HGF)." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Ning Li, Zhangfeng Dou, Jinchun Liu, Bao Chai, Yue Li, Xiuqin An, Peiling Chu, Xiaolan Zhang" "autores" => array:8 [ 0 => array:2 [ "nombre" => "Ning" "apellidos" => "Li" ] 1 => array:2 [ "nombre" => "Zhangfeng" "apellidos" => "Dou" ] 2 => array:2 [ "nombre" => "Jinchun" "apellidos" => "Liu" ] 3 => array:2 [ "nombre" => "Bao" "apellidos" => "Chai" ] 4 => array:2 [ "nombre" => "Yue" "apellidos" => "Li" ] 5 => array:2 [ "nombre" => "Xiuqin" "apellidos" => "An" ] 6 => array:2 [ "nombre" => "Peiling" "apellidos" => "Chu" ] 7 => array:2 [ "nombre" => "Xiaolan" "apellidos" => "Zhang" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1665268119302066?idApp=UINPBA00004N" "url" => "/16652681/0000001700000003/v1_201905160920/S1665268119302066/v1_201905160920/en/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S1665268119302042" "issn" => "16652681" "doi" => "10.5604/01.3001.0011.7393" "estado" => "S300" "fechaPublicacion" => "2018-05-01" "aid" => "70061" "copyright" => "Fundación Clínica Médica Sur, A.C." "documento" => "article" "crossmark" => 0 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Ann Hepatol. 2018;17:482-9" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 199 "formatos" => array:3 [ "EPUB" => 10 "HTML" => 125 "PDF" => 64 ] ] "en" => array:11 [ "idiomaDefecto" => true "titulo" => "Modulatory Effect of Dietary Inclusion of Aegle marmelos Fruits against Cisplatin - Induced Hepatotoxicity In Wistar Rats" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "482" "paginaFinal" => "489" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "f0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 545 "Ancho" => 776 "Tamanyo" => 134086 ] ] "descripcion" => array:1 [ "en" => "Aegle marmelos. A. Tree. B. Leaf, flower. C. Inside the ripe fruit. D. Fruit pulp." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Saket S. Chandel, Mrunal Shirsat, Ram K. Sahu, Siva S. Nayak" "autores" => array:4 [ 0 => array:2 [ "nombre" => "Saket S." "apellidos" => "Chandel" ] 1 => array:2 [ "nombre" => "Mrunal" "apellidos" => "Shirsat" ] 2 => array:2 [ "nombre" => "Ram K." "apellidos" => "Sahu" ] 3 => array:2 [ "nombre" => "Siva S." "apellidos" => "Nayak" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1665268119302042?idApp=UINPBA00004N" "url" => "/16652681/0000001700000003/v1_201905160920/S1665268119302042/v1_201905160920/en/main.assets" ] "en" => array:17 [ "idiomaDefecto" => true "titulo" => "Potential molecular targets of statins in the prevention of hepatocarcinogenesis" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "490" "paginaFinal" => "500" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Ezequiel Ridruejo, Giselle Romero-Caími, María J. Obregón, Diana Kleiman de Pisarev, Laura Alvarez" "autores" => array:5 [ 0 => array:4 [ "nombre" => "Ezequiel" "apellidos" => "Ridruejo" "email" => array:1 [ 0 => "eridruejo@gmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "*" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "Giselle" "apellidos" => "Romero-Caími" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "**" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "María J." "apellidos" => "Obregón" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "***" "identificador" => "aff0015" ] ] ] 3 => array:3 [ "nombre" => "Diana Kleiman de" "apellidos" => "Pisarev" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "**" "identificador" => "aff0010" ] ] ] 4 => array:3 [ "nombre" => "Laura" "apellidos" => "Alvarez" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "**" "identificador" => "aff0010" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Hepatology Section, Department of Medicine. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno” (CEMIC). Ciudad Autónoma de Buenos Aires, Argentina" "etiqueta" => "*" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Laboratory of Biological Effects of Environmental Pollutants, Department of Human Biochemistry, School of Medicine. Universidad de Buenos Aires. Ciudad Autónoma de Buenos Aires, Argentina" "etiqueta" => "**" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Department of Molecular Pathophysiology, Instituto de Investigaciones Biomédicas (Centro mixto CSIC-UAM). Madrid, Spain" "etiqueta" => "***" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "*" "correspondencia" => "Corresponding author." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "f0040" "etiqueta" => "Figure 8" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr8.jpeg" "Alto" => 3455 "Ancho" => 2888 "Tamanyo" => 500907 ] ] "descripcion" => array:1 [ "en" => "Role of TGF-β1 in statins-induced antiproliferative effect. Lane1: Hep-G2 cells treated with ETOH, lane 2: Hep-G2 cells treated with HCB (5 μM), lane 3: Hep-G2 cells treated with conditioned medium (CM), lane 4: Hep-G2 cells pre-treated with AT (30 μM), lane 5: Hep-G2 cells pre-treated with AT + HCB, lane 6: Hep-G2 cells pre-treated with AT + CM, lane 7: Hep-G2 cells treated with ETOH, lane 8: Hep-G2 cells treated with HCB (5 μM), lane 9: Hep-G2 cells treated with TGF-β 1, lane 10: Hep-G2 cells treated with an exogenous TGF-β 1 inhibitor, lane 11: Hep-G2 cells pre-treated with TGF-β1 inhibitor + TGF-β 1, lane 12: Hep-G2 cells pre-treated with TGF-β 1 + AT (30 μM) and lane 13: Hep-G2 cells pre-treated with AT and TGF-β 1 inhibitor and after with HCB and TGF-β 1." ] ] ] "textoCompleto" => "IntroductionHepatocellular carcinoma (HCC), the most common type of liver cancer, is the fifth most common malignant tumor type worldwide and the second leading cause of cancer-related deaths.<a class="elsevierStyleCrossRef" href="#bib0005">1</a> Dysregulated cell proliferation is the main mechanism of hepatocarcinogenesis, leading to tumor growth and invasion. However, the mechanism of this proliferation is complex and poorly understood. Over the last few years, many molecular targets have been proposed as potential sites for advanced HCC treatment.<a class="elsevierStyleCrossRef" href="#bib0010">2</a>,<a class="elsevierStyleCrossRef" href="#bib0015">3</a> Limited clinical and epidemiologic data suggest that statins may improve HCC outcomes, a tumor associated with poor prognosis.<a class="elsevierStyleCrossRef" href="#bib0020">4</a>Statins are the most widely used therapeutic agents to treat hypercholesterolemia. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMGCoAR) is a key enzyme of the mevalonate pathway.<a class="elsevierStyleCrossRef" href="#bib0025">5</a> Seven HMG-CoAR inhibitors are available on the market: atorvastatin, rosuvastatin, simvastatin, fluvastatin, lovastatin, pitavastatin, and pravastatin. Inhibition of proliferation, migration and invasion, and induction of apoptosis are the proposed mechanisms of their antitumoral effect.<a class="elsevierStyleCrossRef" href="#bib0030">6</a>,<a class="elsevierStyleCrossRef" href="#bib0035">7</a> However, results of these studies are controversial and current evidence does not support their specific role in cancer chemoprevention and cancer related outcomes.<a class="elsevierStyleCrossRef" href="#bib0035">7</a> In recent years, several studies have demonstrated that statins have beneficial effect in patients with different liver diseases including chronic hepatitis C virus (HCV) infection, chronic hepatitis B virus (HBV) infection, and also decrease the risk of hepatocellular carcinoma (HCC) development.<a class="elsevierStyleCrossRefs" href="#bib0040">8</a>–<a class="elsevierStyleCrossRef" href="#bib0080">16</a> Several studies have shown that statins use in chronic liver disease and cirrhosis is safe, and even it was associated with lower mortality and lower rate of hepatic decompensation.<a class="elsevierStyleCrossRef" href="#bib0085">17</a> Statin use is also associated with a reduced risk of fibrosis progression in patients with chronic hepatitis C and a reduced risk of cirrhosis development.<a class="elsevierStyleCrossRef" href="#bib0090">18</a>–<a class="elsevierStyleCrossRef" href="#bib0095">19</a> So, there appears to be multiple benefits from statins use in patients with chronic liver diseases. So far, it has been proven that there are several mechanisms activated by statins in cancer cells, depending on the cell line, statins concentration, duration of exposure, and the type of statin being used. It has been shown that statins may inhibit cell-cycle progression and may induce apoptosis by both intrinsic and extrinsic pathways.<a class="elsevierStyleCrossRef" href="#bib0100">20</a>,<a class="elsevierStyleCrossRef" href="#bib0105">21</a>TGF-β1 is involved in the regulation of cell behavior in a variety of cellular contexts. Expression of TGF- β1 may be a critical step in the growth control of normal and proliferating rat hepatocytes. It has been reported that TGF-β1 has many roles in HCC development, including tumor growth, invasiveness, neo-angiogenesis and metastatic behavior, leading to cell survival promotion, proliferation, invasion and neoangiogenesis in many human cancers.Proto-oncogene tyrosine-protein kinase Src pathway activation, also known as cellular Src kinase or simply cSrc, has been observed in about 50% of tumors from various organs including the liver.<a class="elsevierStyleCrossRef" href="#bib0110">22</a> Sato, et al.<a class="elsevierStyleCrossRef" href="#bib0115">23</a> demonstrated that TGF-β1 can induce c-Src phosphorylation.Hexachlorobenzene (HCB), a persistent environmental pollutant, has been shown to induce cell growth imbalance in rat liver. HCB increases the development of rat liver preneoplastic lesions in a medium-term initiation/ promotion model.<a class="elsevierStyleCrossRef" href="#bib0120">24</a>The aim of our study was to investigate the effect of statins on cell proliferation in an initiation/promotion model in rat liver and in Hep-G2 cells. We also assessed the expression of TGF-β1, and its involvement in the potential key molecules involved in statins actions.Material and MethodsAnimals and TreatmentFemale Wistar rats (50 g at the onset of the experiment) were purchased from the Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. The rats were fed with Purine 3 rat chow (Cabeca S.C.A, Argentina) and water ad libitum. Environmental conditions consisted of a 12 hrs light-dark cycle, 19 ± 2 °C, and 45-75% humidity. After 7-day acclimation period, a total of 20 rats were divided into 4 equal groups. Rats in groups 1 and 2, received a single i.p. injection of saline vehicle. Animals in groups 3 and 4 were treated with a single i.p. injection of DEN (100 mg/kg body weight). One week later, the animals in group 2 and 4 received HCB (100 mg/kg body weight) administered 5 days a week, by gavage. HCB (40 mg/mL) was suspended in water containing Tween 20 (0.5 mL/100 mL) (<a class="elsevierStyleCrossRef" href="#f0005">Figure 1</a>). The doses of DEN and HCB were selected based on similar previous studies. At week 3 (day 21), a partial hepatectomy, which comprised approximately 70% of the total liver weight, was performed on all animals. Given the diurnal variation observed in liver DNA synthesis after partial hepatectomy in rodents, all rats underwent partial hepatectomy or sham hepatectomy between 11:00 and 15:00 h.<a class="elsevierStyleCrossRef" href="#bib0095">19</a> At the end ofthe eighth week, the animals were killed by exsanguinations.<elsevierMultimedia ident="f0005"></elsevierMultimedia>Chemicals and antibodiesHexachlorobenzene (HCB, > 99% purity commercial grade), anti- -actin, and anti-proliferating cell nuclear antigen (PCNA) were purchased from Sigma (SigmaAldrich Co). TRI-Reagent used for mRNA extraction was purchased from MRC, Molecular Research Center, Inc. The reagents used for cDNA synthesis, were purchased from SRL Biodynamics. Polyvinyl difluoride membranes (PVDF), for immuno-blotting were purchased from Bio-Rad (Bio-Rad Laboratories). CP-BU plates were purchased from New Afghanistan (Afghanistan, Gevaerl ArgentinaSA). Monoclonal anti-TGF-β1 was obtained from Abcam Inc. Specific primers for transforming growth factor β1 (TGF-β1) and (L-19) were purchased from Invitrogen Life Technology. The compound for (RT) and PCR were purchased from Promega Corporation. All other reagents used were of analytical grade.Cell culture and treatmentHep-G2 cell line was supplied by the American Type Culture Collection (ATCC). The cells were cultured in Modified Eagle’s Medium (MEM) (Sigma) supplemented with 10% Fetal Bovine Serum (FBS) and 50 μg/ mL gentamicin and penicillin. Cells were maintained under standard conditions at 37 °C in 5% CO2 and 100% relative humidity. Cells in exponential growth with 80% confluence were washed with phosphate solution (PBS) and then treated with a solution of 0.025% trypsin-0.03% EDTA for 1-5 minutes at 37 °C. Cells were seeded in 12-well plates and pre-incubated for 24 h to allow cell adherence. For dose-response studies, cells were treated for 24 h with HCB (0.05, 0.5 and 5 ^M). Control cells were treated with the same volumes of ETOH. We used a conditioned medium (CM) obtained from Hep-G2 cells treated with HCB 5 μM for 24 h to obtain medium enriched in growth factors released by the cells.Western BlotttingTotal cellular protein lysates were electrophoresed in 10% SDS-polyacrylamide gel (SDS-PAGE) prior to transfer to polivinilidene difluoride membranes (PVDF), in a semidry transfer cell at 18 V for 1.5 h. Membranes were blocked overnight at 4 °C with 5% nonfat dry milk- 2.5% BSA in TBST buffer (10 mM Tris-HCl, pH 8.0, 0.5% Tween 20, 150 mM NaCl). Membranes were incubated with rabbit polyclonal specific antibodies (PCNA, c-Src and TGF-β1) (1:500), and anti- -actin at (1:500) dilution, and incubated overnight at 4 °C. After incubation, membranes were washed five times with TBST, and the suitable peroxidase- conjugated antispecies specific antibodies were used for protein detection. After washing, blots were reacted using an enzyme-linked enhanced chemoluminescence (ECL) detection kit (Amer- sham Biosciences, Inc., UK) and quantified by scanning laser densitometry in a Fotodyne (Foto/Analyst), Gel-Pro Analyzer 3.1.RT-PCR analysisTotal RNA was extracted from with a phenol-isotiocianate of guanidine (TRIZOL) solution, by a modification of Chomczynski’s method.<a class="elsevierStyleCrossRef" href="#bib0125">25</a>An aliquot of 2 μg of total RNA was used to synthesize first-strand cDNA with the random primers, deoxynucleotide triphosphates, transcriptase reverse (RT), and transcriptase reverse buffer. Reaction mixture for TGF-β1, and L-19 amplification contained GoTaq reaction buffer (1.5 mM MgCl2, 0.2 mM dNTP’s mix, 1.25 U GoTaq DNA polymerase, 0.5 μM of each forward and reverse primers, and 1 μL of RT products). PCR was performed as follows: reaction mixture for TGF-β1 amplification was incubated at 94 °C for 5 min and then amplified at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min. Reactions were repeated for 27 cycles. Reaction mixture for L-19 amplification was incubated at 94 °C for 5 min and then amplified at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. Reactions were repeated for 27 cycles. The sequences of forward and reverse primers were as follows:TGF-β1: 5’-CTGCTGGCAATAGCTTCCTA-3, 5-CGAGCCTTAGTTGGACAGGAT-3’. L-19: 5’-TGAACGGGAAGCTCACTGG-3, 5-TCCACCACCCTGTTGCTGTA-3’.L-19 cDNA was used as a loading control. PCR products were detected as a single band on 2% agarose gel, containing 0.05% (v/v) ethidium bromide. Bands were detected, and intensity was quantified by scanning laser densitometry in a Fotodyne (Foto/Analyst), Gel-Pro Analyzer 3.1.Statistical analysisData are expressed as means ± SEM. Differences between treated and control groups were analyzed by oneway ANOVA, at a 95% confidence interval, followed by Tukey post hoc test to identify significant differences between the samples and controls means, after testing homogeneity of variance using Barlett’s procedure. Differences between control and treated animals were considered significant when p values were < 0.05.ResultsEffect of HCB on HMGCoAR mRNA levels and serum cholesterol levels in an initiation-promotion model of HCCWe have previously demonstrated that HCB contributes to the development of preneoplastic foci by enhancing the proliferation of initiated cells in rat liver.<a class="elsevierStyleCrossRef" href="#bib0130">26</a> We focused on the effect of HCB on HMGCoAR mRNA levels in rat liver, in an initiation-promotion model. As shown in <a class="elsevierStyleCrossRef" href="#f0010">figure 2</a>, HMGCoAR mRNA levels were significantly increased in liver from DEN + HCB group compared to DEN group (31%) (<a class="elsevierStyleCrossRef" href="#f0010">Figure 2</a>A and 2B). The above mentioned increase in HMGCoAR activity, correlates with high cholesterol serum levels. Our results showed that serum cholesterol levels were significantly increased (28%) in DEN + HCB group compared with DEN group (<a class="elsevierStyleCrossRef" href="#f0010">Figure 2</a>C).<elsevierMultimedia ident="f0010"></elsevierMultimedia>Effect of statins treatment on Hep-G2 cells viabilityIn order to investigate the effect of statins on cell viability, Hep-G2 cells were treated with different doses of AT (10, 20 and 30 μΜ) and SM (5, 10 and 20 μΜ) for three hours. MTT assay shows that statins did not alter Hep-G2 cells viability (<a class="elsevierStyleCrossRef" href="#f0015">Figure 3</a>).<elsevierMultimedia ident="f0015"></elsevierMultimedia>Effect of HCB on HMGCoAR mRNA levels in Hep-G2 cellsHep-G2 is a well-differentiated human hepatocellular carcinoma cell line that preserves numerous liver-specific functions and thus can serve as an in vitro model.Consistent with our previous results showing that HCB induces cell proliferation in a dose-dependent manner in Hep-G2 cells,<a class="elsevierStyleCrossRef" href="#bib0130">26</a> we evaluated HCB treatment effect (0.05, 0.5 and 5 μΜ) on HMGCoAR mRNA levels. As shown in <a class="elsevierStyleCrossRef" href="#f0015">figure 3</a>, HMGCoAR mRNA levels significantly increased (32% to 54%) in total cell lysates of 0.5 and 5 μΜ HCB-treated cells, respectively, when compared to ETOH (<a class="elsevierStyleCrossRef" href="#f0020">Figure 4</a>).<elsevierMultimedia ident="f0020"></elsevierMultimedia>Effect of statins on HMGCoAR mRNA levels in HCB-treated Hep-G2 cellTo evaluate the effect of statins on HMGCoAR mRNA expression, Hep-G2 cells were pretreated with increasing doses of AT (10, 20 and 30 μΜ) or SM (5, 10 and 20 μΜ) for 3 h and then treated with HCB (5 μΜ) for 24 h. As shown in <a class="elsevierStyleCrossRef" href="#f0025">figure 5</a>, HMGCoAR mRNA levels significantly increased due to HCB effects. Nevertheless, HMGCoAR mRNA levels significantly decreased by 29% and 38% when pretreated with AT 10 and 20 μM (respectively), reaching baseline values with AT 30 μM, when compared to HCB-treated cells without AT pretreatment. Similarly, pretreatment with SM at 5 and 10 μM decreased HMG-CoAR mRNA levels by 21% and 31% (respectively), reaching baseline values with SM 20 μM, when compared to HCB-treated cells without SM pretreatment (<a class="elsevierStyleCrossRef" href="#f0025">Figure 5</a>).<elsevierMultimedia ident="f0025"></elsevierMultimedia>Statins effect on HCB-induced proliferation in Hep-G2 cellsSince we have previously demonstrated that 5 μM HCB induced PCNA-expression in Hep-G2 cells,<a class="elsevierStyleCrossRef" href="#bib0130">26</a> we evaluated the dose-response effects of statins on cell proliferation. Western blots analysis showed that AT pretreatment (10, 20 and 30 μΜ) significantly reduced HCB-induced PCNA levels by 24%, 52% and reaching baseline values at maximum (respectively) when compared to HCB group (<a class="elsevierStyleCrossRef" href="#f0030">Figure 6</a>A). In a similar manner SM pretreatment (5, 10 and 20 μΜ), significantly reduced HCB-induced PCNA levels (30%, 58% until reaching basal values at maximum doses, respectively), when compared to HCB group (<a class="elsevierStyleCrossRef" href="#f0030">Figure 6</a>B). Altogether, the above results demonstrate that statins antagonize HCB-induced cell proliferation in Hep-G2 cells.<elsevierMultimedia ident="f0030"></elsevierMultimedia>Statins effect on TGF-β1 levels in HCB treated Hep-G2 cellsGiven that TGF-β1 is one of the most relevant cytokines that intervene in the apoptotic and/or proliferative processes in hepatocytes, we evaluated the statins effect on TGF-β1 levels in HCB-treated Hep-G2 cells. Hep-G2 cells were pretreated with AT (10, 20 and 30 μΜ) or SM (5, 10 and 20 μΜ) or ETOH for 3 h, and then treated with HCB (5 μΜ) for 24 h. Western blot analysis showed a significant reduction in TGF-β1 levels, when Hep-G2 cells were treated with both statins: AT (23%, 29% and 45%), and SM (11%, 24% and 40%) respectively, until reaching baseline values at maximum doses, when compared to HCB group (<a class="elsevierStyleCrossRef" href="#f0035">Figure 7</a>).<elsevierMultimedia ident="f0035"></elsevierMultimedia>Role of TGF-β1 in statins-induced anti-proliferative effectWe further investigated whether the statins anti-proliferative effect in HCB-treated Hep-G2 cells was mediated by TGF-β1 When Hep-G2 cells were treated with HCB 5 μΜ (lane 2) or CM (lane 3), a significant increase in PCNA levels (42% and 38%) was observed when compared to ETOH, respectively.Our results show that AT pretreated cells did not show increases in PCNA levels with HCB or MC treatment (lanes 5 and 6, respectively). On the other hand, Hep-G2 cells treated with TGF-β1 showed a significant increase in PCNA levels (lanes 9) and this effect was blocked when these cells were pretreated with an exogenous TGF-β1 inhibitor (lane 11). Similarly, Hep-G2 cells pre-treated with AT and TGF-β1 showed no changes in PCNA levels (lane 12). Finally, Hep-G2 cells pre-treated with AT and I/TGF-1 and then with an exogenous TGF-β1 and HCB, showed no changes on PCNA levels (lane 13) (<a class="elsevierStyleCrossRef" href="#f0040">Figure 8</a>A and 8B).<elsevierMultimedia ident="f0040"></elsevierMultimedia>Statins effect on c-Src levels in HCB treated Hep-G2 cellsGiven that TGF-β1 activates c-Src kinase, and c-Src pathway activation has been observed in about 50% of tumors from various organs including the liver,<a class="elsevierStyleCrossRef" href="#bib0110">22</a> we evaluated the effect of statins on c-Src protein levels in HCB-treated cells. Our results showed that HCB significantly increased phosphorylated c-Src protein levels (136%). Pre-treatment with AT (30 μM) and SM (20 μM) decreased this HCB-induced elevation in phosphorylated c-Src levels (41% and 40%, respectively). However, it does not return to baseline values when compared to HCB 5 μM treated cells (<a class="elsevierStyleCrossRef" href="#f0045">Figure 9</a>).<elsevierMultimedia ident="f0045"></elsevierMultimedia>Statins effect on DI levels in HCB treated Hep-G2 cellsDisruptive hormone plays a critical role in the development of pre-neoplastic lesions. T3 is one of the major cell cycle regulators and its levels are regulated by the activity of the enzyme DI. In HCB treated cells, mRNA DI levels were significantly reduced by 45% (p ≤ 0.01). When both statins were added to treated cells, they reversed this effect and mRNA DI levels returned to their baseline value (<a class="elsevierStyleCrossRef" href="#f0050">Figure 10</a>).<elsevierMultimedia ident="f0050"></elsevierMultimedia>DiscussionDysregulated cell proliferation is the main mechanism of hepatocarcinogenesis, leading to tumor growth and invasion. However, the mechanism of this proliferation is complex and poorly understood. Over the last few years, many molecular targets have been proposed as potential sites for advanced HCC treatment.<a class="elsevierStyleCrossRef" href="#bib0010">2</a>,<a class="elsevierStyleCrossRef" href="#bib0015">3</a> Limited clinical and epidemiologic data suggest that statins may improve HCC outcomes, a tumor associated with poor prognosis.<a class="elsevierStyleCrossRef" href="#bib0020">4</a>In this study, we have investigated the cellular effects of statins (atorvastatin and simvastatin) on HCB-induced proliferation in Hep-G2 cells. We found that incubation with atorvastatin or simvastatin inhibits HCB-induced cell proliferation in cultured Hep-G2 cells. We have demonstrated for the first time that, statins induced inhibition of proliferation might at least be partially explained by a decrease in TGF-β1 expression and by a downstream decrease in c-Src phosphorylation.Other authors observed that simvastatin prevents proliferation and favors cellular apoptosis in a rat osteosarcoma line, UMR-106.<a class="elsevierStyleCrossRef" href="#bib0135">27</a> It is interesting to note that they use doses of 3-10 μM; however, we observed that 20 μM might be an optimal dose for proliferative effect reversion. Atorvastatin inhibits in vitro proliferation at dose of 0.4 nM in hepatic myofibroblasts.<a class="elsevierStyleCrossRef" href="#bib0140">28</a> In this study, although both statins present a complete reversion of the HCB proliferative effect, atorvastatin appears to be more effective at the highest dose (30 μΜ).In a previous study, we have demonstrated that HCB deregulates cell growth and alters growth factors such as EGF and TGF-β1 expression levels, as well as theirs signaling pathways, both being involved in developmental and tumorigenic processes.<a class="elsevierStyleCrossRef" href="#bib0130">26</a>,<a class="elsevierStyleCrossRef" href="#bib0145">29</a> Here, in the in vivo induction/ promotion HCC model and in the in vitro model, we use HCB to induce cell proliferation. This increase in cell proliferation (supported by an increased PCNA levels) was accompanied by increases in HMGCoAR and TGF-β1 mRNA levels.Previously, we had demonstrated that HCB-induced proliferation in Hep-G2 cell is mediated by an increase in TGF-β1 expression. In this study, we evaluated atorvastatin and simvastatin effect on HCB-induced proliferation. Likewise, we analyzed the potential role of TGF-β1 in the statins mechanism of action in the HCB-induced proliferation model. TGF-β1 involvement in the statins mechanism of action to reduce cell proliferation had been previously described.<a class="elsevierStyleCrossRef" href="#bib0150">30</a>,<a class="elsevierStyleCrossRef" href="#bib0155">31</a> However, it is unclear which molecules are involved in the downstream pathway of this cytokine.One of the potential molecules involved could be c-Src. Its pathway activation has been observed in about 50% of tumors from various organs including the liver,<a class="elsevierStyleCrossRef" href="#bib0110">22</a> c-Src can cooperate with kinases receptors to signal through downstream molecules, such as PI3K/PTEN/Akt, Ras/Raf/ Mek1/2/Erk1/2 and STATs. In many cancer cell lines, tumors signal transducers and transcription activator proteins (STAT) are often deregulated with persistent activation in tyrosine phosphorylation. STAT1 is generally considered a tumour suppressor but there is growing evidence that it can also act as a tumour promoter. Therefore, the JAK/STAT signaling pathway is one of the most promising targets in the cancer therapy. Our hypothesis about the role of cSrc in the inhibition of TGF-β1-induced proliferation is consistent with the observations of Sato, et al., who demonstrated that TGF-β1 can induce c-Src phosphorylation.<a class="elsevierStyleCrossRef" href="#bib0115">23</a>Here, we have demonstrated for the first time, the downstream role of c-Src in Hep-G2 cells proliferation. We have also demonstrated for the first time that TGF-β1/ c-Src pathway may be inhibited by both statins in a dose dependent manner.The role of thyroid hormones in carcinogenesis has been postulated for many years. However, there is not enough data for a complete understanding of this process. Triiodothyronine (T3) acts through its nuclear receptors, the thyroid hormone receptors (THR). T3 is produced by monodeiodination catalyzed by type 1 iodothyronine deiodinase (DI). This enzyme is present in almost all tissues, but the highest concentrations were found in the thyroid, the liver, and the kidneys.<a class="elsevierStyleCrossRef" href="#bib0160">32</a> Both its enzymatic activity and its gene expression have been examined in some neoplastic tissues. Significant decreases in gene expression and enzyme activity were found in several of them.<a class="elsevierStyleCrossRef" href="#bib0165">33</a> HCB is a hormone disruptor and induces hypothyroxinemia, and it is well known that T3 has a negative regulatory effect on the cell cycle.<a class="elsevierStyleCrossRef" href="#bib0145">29</a> A possible explanation could be that thyroid hormones deregulation may play an important role in HCC development as well as in the statins mechanism key on cell proliferation inhibition.In our previous study, we demonstrated that HCB increases cell proliferation, alters mRNA DIII and TH concentration in induction/promotion rat liver model.<a class="elsevierStyleCrossRef" href="#bib0145">29</a> In this study, our results showed that HCB increased cell proliferation and decreased mRNA DI levels in Hep-G2 cells. It is possible that DI decreased expression is partly responsible for the decrease in tissue T3 and this event contributes to the deregulation of the cell cycle. More experiments are needed to explore this finding.We showed here that the increase in cell proliferation as well as the decrease in mRNA DI levels are mediated by TGF-β1 We also demonstrated that statins inhibit cell proliferation by decreasing TGF-β1 levels.It is known that there is a regulation between TGF-β1 and DI enzyme. It is possible that statins mediated TGF-β1 reduction counteracts the effects on DI and T3 levels, resulting in normal T3 tissue levels.In preliminary experiments (data not shown) performed in our laboratory, we observed that exogenously administered thyroid hormones reduce HCB-induced cell proliferation in the Hep-G2 cell line. Won Park, et al. showed that THR could down regulate the activity of cSrc in the presence of T3 via phosphorylation, suggesting that low T3 levels generate a c-Src related pro-proliferative process.<a class="elsevierStyleCrossRef" href="#bib0170">34</a> More experiments are needed to validate these results in our experimental model.Summarizing, our study showed for the first time that atorvastatin and simvastatin can revert HCB-induced proliferative effects on Hep-G2 cells. In addition, this effect is mediated by the inhibition of TGF-β1 and the downstream c-Src phosphorylation pathway. The TH homeostasis deregulation could be potentially involved. TGF-β1 and c-Src may be the statins molecular targets in early events of hepatocarcinogenesis.Abbreviations<ul class="elsevierStyleList" id="l0005"><li class="elsevierStyleListItem" id="u0005">•AT: Atorvastatin.</li><li class="elsevierStyleListItem" id="u0010">•CM: conditioned medium.</li><li class="elsevierStyleListItem" id="u0015">•c-Src: cellular Src kinase, Proto-oncogene tyrosineprotein kinase Src.</li><li class="elsevierStyleListItem" id="u0020">•DEN: Diethylnitrosamine.</li><li class="elsevierStyleListItem" id="u0025">•DI: deiodinase I.</li><li class="elsevierStyleListItem" id="u0030">•HBV: hepatitis B virus.</li><li class="elsevierStyleListItem" id="u0035">•HCB: hexachlorobenzene.</li><li class="elsevierStyleListItem" id="u0040">•HCC: Hepatocellular carcinoma.</li><li class="elsevierStyleListItem" id="u0045">•HCV: hepatitis C virus.</li><li class="elsevierStyleListItem" id="u0050">•HMGCoAR: 3 -hydroxy-3-methylglutaryl coenzyme-A reductase.</li><li class="elsevierStyleListItem" id="u0055">•HSCs: hepatic stellate cells.</li><li class="elsevierStyleListItem" id="u0060">•HVPG: hepatic venous pressure gradient.</li><li class="elsevierStyleListItem" id="u0065">•i.p.: intra peritoneal.</li><li class="elsevierStyleListItem" id="u0070">•PCNA: Proliferating cell nuclear antigen.</li><li class="elsevierStyleListItem" id="u0075">•pc-Src: phosphorylated c-SRC.</li><li class="elsevierStyleListItem" id="u0080">•SM: Simvastatin.</li><li class="elsevierStyleListItem" id="u0085">•STAT: signal transducer and activator of transcription.</li><li class="elsevierStyleListItem" id="u0090">•T3: triiodothyronine.</li><li class="elsevierStyleListItem" id="u0095">•TGF-β1: transforming growth factor-β1.</li><li class="elsevierStyleListItem" id="u0100">•TH: thyroid hormones.</li><li class="elsevierStyleListItem" id="u0105">•THR: thyroid hormone receptor.</li></ul>Conflict of InterestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.Financial SupportThis work was supported by grant SAF2012-32491 from Plan Nacional (MINECO) and grant S2010/BDM-2423 (MOIR) from Comunidad de Madrid, Spain, and by a grant UBACYT 20020120200258 /EXP-UBA N° 17064/ 2012 from Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina." "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres1190773" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abs0010" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1110050" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "s0005" "titulo" => "Introduction" ] 3 => array:3 [ "identificador" => "s0010" "titulo" => "Material and Methods" "secciones" => array:6 [ 0 => array:2 [ "identificador" => "s0015" "titulo" => "Animals and Treatment" ] 1 => array:2 [ "identificador" => "s0020" "titulo" => "Chemicals and antibodies" ] 2 => array:2 [ "identificador" => "s0025" "titulo" => "Cell culture and treatment" ] 3 => array:2 [ "identificador" => "s0030" "titulo" => "Western Blottting" ] 4 => array:2 [ "identificador" => "s0035" "titulo" => "RT-PCR analysis" ] 5 => array:2 [ "identificador" => "s0040" "titulo" => "Statistical analysis" ] ] ] 4 => array:3 [ "identificador" => "s0045" "titulo" => "Results" "secciones" => array:9 [ 0 => array:2 [ "identificador" => "s0050" "titulo" => "Effect of HCB on HMGCoAR mRNA levels and serum cholesterol levels in an initiation-promotion model of HCC" ] 1 => array:2 [ "identificador" => "s0055" "titulo" => "Effect of statins treatment on Hep-G2 cells viability" ] 2 => array:2 [ "identificador" => "s0060" "titulo" => "Effect of HCB on HMGCoAR mRNA levels in Hep-G2 cells" ] 3 => array:2 [ "identificador" => "s0065" "titulo" => "Effect of statins on HMGCoAR mRNA levels in HCB-treated Hep-G2 cell" ] 4 => array:2 [ "identificador" => "s0070" "titulo" => "Statins effect on HCB-induced proliferation in Hep-G2 cells" ] 5 => array:2 [ "identificador" => "s0075" "titulo" => "Statins effect on TGF-β1 levels in HCB treated Hep-G2 cells" ] 6 => array:2 [ "identificador" => "s0080" "titulo" => "Role of TGF-β1 in statins-induced anti-proliferative effect" ] 7 => array:2 [ "identificador" => "s0085" "titulo" => "Statins effect on c-Src levels in HCB treated Hep-G2 cells" ] 8 => array:2 [ "identificador" => "s0090" "titulo" => "Statins effect on DI levels in HCB treated Hep-G2 cells" ] ] ] 5 => array:2 [ "identificador" => "s0095" "titulo" => "Discussion" ] 6 => array:2 [ "identificador" => "s0100" "titulo" => "Abbreviations" ] 7 => array:2 [ "identificador" => "s0105" "titulo" => "Conflict of Interest" ] 8 => array:2 [ "identificador" => "s0110" "titulo" => "Financial Support" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2017-06-07" "fechaAceptado" => "2017-08-19" "PalabrasClave" => array:1 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1110050" "palabras" => array:4 [ 0 => "Hepatocellular carcinoma" 1 => "TGF-βΙ" 2 => "HMGCoAR" 3 => "Statins" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "Introduction and alm. Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-βΙ) and thyroid hormones (TH) regulation. Aim. The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an in vivo and in vitro HCC model.Materials and methods. We used two models: in vivo (in rats) using diethylnitrosamine (DEN) and hexachlorobenzene (HCB) to develop HCC. We analyzed cell proliferation parameters (proliferating cell nuclear antigen, PCNA) and cholesterol metabolism (hydroxy-methylglutaryl-CoA reductase, HMGCoAR). In vitro (Hep-G2 cells) we evaluated the effects of different doses of Atorvastatin (AT) and Simvastatin (SM) on HCB induced proliferation and analyzed proliferative parameters, cholesterol metabolism, TGF-βΙ mRNA, c-Src and TH levels.Results.In vivo, we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB. In vitro, we observed the same results on PCNA as in vivo. The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB, reaching levels similar to controls at maximum doses: AT (30 μM), and SM (20 μM). Increases in PCNA, TGF-βΙ, and pc-Src, and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses.Conclusion. Statins can prevent the proliferative HCB effects on Hep-G2 cells. TGF-βΙ, c-Src and TH may be the statins molecular targets in hepatocarcinogenesis." ] ] "multimedia" => array:10 [ 0 => array:7 [ "identificador" => "f0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1854 "Ancho" => 2921 "Tamanyo" => 218291 ] ] "descripcion" => array:1 [ "en" => "Experimental design for the initiation/promotion study. The initiation agent, DEN, was administered i.p. (100 mg/kg) on Day 0. Saline solution or HCB was delivered by gavage starting on day 45 at a dose of 100 mg/kg b.w. per day, five days/week. A two-thirds partial hepatectomy was performed on all animals on day 52. Liver tissues were collected 100 days following initial DEN dosing." ] ] 1 => array:7 [ "identificador" => "f0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 3216 "Ancho" => 2133 "Tamanyo" => 373053 ] ] "descripcion" => array:1 [ "en" => "Analysis of HMGCoAR mRNA expression in rat liver and serum cholesterol levels in an initiation-promotion model. A. Representative pattern of RT-PCR amplification of HMGCoAR cDNA from liver of DEN and DEN+HCB treated rats, synthesized from total RNA. B. L-19 was used as a loading control. Quantification of HMGCoAR/L-19 cDNA ratio is shown in the lower panel. C. Serum cholesterol levels. Values are means ± SEM of three independent experiments of four rats per group. Significantly different (*p < 0.05) compared to DEN group." ] ] 2 => array:7 [ "identificador" => "f0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 3084 "Ancho" => 2130 "Tamanyo" => 348318 ] ] "descripcion" => array:1 [ "en" => "Effect of statins on Hep-G2 viable cell number. The viable cell number was evaluated using the MTT assay. The absorbance was measured at 570 nm, and the results were expressed as percentage of ETOH-treated cells. A. Cells were treated with AT (10, 20 and 30 μM) or (B) SM (5, 10 and 20 μM) for 24 h. Values represent means ± SEM of the three independent studies." ] ] 3 => array:7 [ "identificador" => "f0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 2316 "Ancho" => 2130 "Tamanyo" => 240313 ] ] "descripcion" => array:1 [ "en" => "HCB induces HMGCoAR mRNA expression. Hep-G2 cells were treated with HCB (0.05, 0.5 and 5 μM). A. Representative pattern of HMGCoAR cDNA from ETOH and HCB-treated cells, synthesized from total RNA. L-19 was used as a loading control. B. Quantification of cDNAs, after correction with L-19 cDNA. Values are means ± SEM of three independent experiments. Significantly different (*p < 0.05) compared to ETOH group." ] ] 4 => array:7 [ "identificador" => "f0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 4734 "Ancho" => 2133 "Tamanyo" => 549458 ] ] "descripcion" => array:1 [ "en" => "Analysis of statins effect on HMGCoA R mRNA expression in HCB-treated Hep-G2 cells. A. Hep-G2 cells were pretreated with AT (10, 20 and 30 μM) or SM (5, 10 and 20 μM) for 3 h and then treated with HCB (5 μM) for 24 h. B. L-19 was used as a loading control. Representative patterns of RT-PCR amplification of HMGCoAR cDNA from ETOH and statinstreated cells, synthesized from total RNA are shown in the upper panel. Quantification of HMGCoAR mRNA, after correction with L-19 is shown in the lower panel. Values are means ± SEM of three independent experiments. Significantly different (*p < 0.05) compared to ETOH group." ] ] 5 => array:7 [ "identificador" => "f0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 4620 "Ancho" => 2125 "Tamanyo" => 501993 ] ] "descripcion" => array:1 [ "en" => "Statins effect on HCB-induced cell proliferation. A. Western blot analysis of PCNA in total lysate of Hep-G2 cells treated with 5 βM HCB, and pretreated with AT (10, 20, and 30 βM) for 3 h. Quantification of PCNA protein levels by densitometric scanning of the immunoblots is shown in the lower panel. B. Western blot analysis of PCNA in total lysate of Hep-G2 cells treated with 5 μΜ HCB, and pretreated with SM (5, 10 and 20 μΜ). Quantification of PCNA protein levels by densitometric scanning of the immunoblots is shown in the lower panel. Values are means ± SEM of three independent experiments. Significantly different (*p < 0.05 and **p < 0.01) compared to HCB-treated cells." ] ] 6 => array:7 [ "identificador" => "f0035" "etiqueta" => "Figure 7" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr7.jpeg" "Alto" => 3066 "Ancho" => 2092 "Tamanyo" => 320000 ] ] "descripcion" => array:1 [ "en" => "Effect of statins on TGF-β 1 protein levels, in Hep-G2 cells treated with HCB. A. Western blot analysis of TGF-β1 protein levels in Hep-G2 cells pretreated with AT (10, 20 and 30 μM) or SM (5, 10 and 20 μM) for 3 h and then treated with HCB (5 μM) for 24 h. Quantification of TGF-β 1 L-19 ratio to ETOH is shown in the lower panel. B. Values are means ± SEM of three independent experiments. Significantly different (*p < 0.05 and **p < 0.05) compared to HCB-treated cells." ] ] 7 => array:7 [ "identificador" => "f0040" "etiqueta" => "Figure 8" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr8.jpeg" "Alto" => 3455 "Ancho" => 2888 "Tamanyo" => 500907 ] ] "descripcion" => array:1 [ "en" => "Role of TGF-β1 in statins-induced antiproliferative effect. Lane1: Hep-G2 cells treated with ETOH, lane 2: Hep-G2 cells treated with HCB (5 μM), lane 3: Hep-G2 cells treated with conditioned medium (CM), lane 4: Hep-G2 cells pre-treated with AT (30 μM), lane 5: Hep-G2 cells pre-treated with AT + HCB, lane 6: Hep-G2 cells pre-treated with AT + CM, lane 7: Hep-G2 cells treated with ETOH, lane 8: Hep-G2 cells treated with HCB (5 μM), lane 9: Hep-G2 cells treated with TGF-β 1, lane 10: Hep-G2 cells treated with an exogenous TGF-β 1 inhibitor, lane 11: Hep-G2 cells pre-treated with TGF-β1 inhibitor + TGF-β 1, lane 12: Hep-G2 cells pre-treated with TGF-β 1 + AT (30 μM) and lane 13: Hep-G2 cells pre-treated with AT and TGF-β 1 inhibitor and after with HCB and TGF-β 1." ] ] 8 => array:7 [ "identificador" => "f0045" "etiqueta" => "Figure 9" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr9.jpeg" "Alto" => 1513 "Ancho" => 2130 "Tamanyo" => 171631 ] ] "descripcion" => array:1 [ "en" => "Analysis of the expression of c-Src by Western blot. Hep-G2 cells were pretreated with AT (30 μM) or SM (20 μM) for 3 h and then HCB (5 μM) for 24 h. β-actin was used as charge control. The results of the bands optical analysis are shown in the bottom panel *p ≤ 0.05, ***p ≤ 0.01 respect HCB." ] ] 9 => array:7 [ "identificador" => "f0050" "etiqueta" => "Figure 10" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr10.jpeg" "Alto" => 1513 "Ancho" => 2130 "Tamanyo" => 159201 ] ] "descripcion" => array:1 [ "en" => "Analysis of the expression of mRNA DI by RT-PCR. Hep-G2 cells were pretreated with AT(30 µM) or SM (20 µM) for 3 h and then HCB (5 µM) for 24 h. L-19 was used as charge control. 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Ezequiel Ridruejo

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eridruejo@gmail.com

Corresponding author.

, Giselle Romero-Caími**, María J. Obregón***, Diana Kleiman de Pisarev**, Laura Alvarez**

* Hepatology Section, Department of Medicine. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno” (CEMIC). Ciudad Autónoma de Buenos Aires, Argentina

** Laboratory of Biological Effects of Environmental Pollutants, Department of Human Biochemistry, School of Medicine. Universidad de Buenos Aires. Ciudad Autónoma de Buenos Aires, Argentina

*** Department of Molecular Pathophysiology, Instituto de Investigaciones Biomédicas (Centro mixto CSIC-UAM). Madrid, Spain

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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="s0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0015">Introduction</span><p id="p0005" class="elsevierStylePara elsevierViewall">Hepatocellular carcinoma &#40;HCC&#41;&#44; the most common type of liver cancer&#44; is the fifth most common malignant tumor type worldwide and the second leading cause of cancer-related deaths&#46;<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> Dysregulated cell proliferation is the main mechanism of hepatocarcinogenesis&#44; leading to tumor growth and invasion&#46; However&#44; the mechanism of this proliferation is complex and poorly understood&#46; Over the last few years&#44; many molecular targets have been proposed as potential sites for advanced HCC treatment&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a>&#44;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> Limited clinical and epidemiologic data suggest that statins may improve HCC outcomes&#44; a tumor associated with poor prognosis&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a></p><p id="p0010" class="elsevierStylePara elsevierViewall">Statins are the most widely used therapeutic agents to treat hypercholesterolemia&#46; Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme&#46; A reductase &#40;HMGCoAR&#41; is a key enzyme of the mevalonate pathway&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> Seven HMG-CoAR inhibitors are available on the market&#58; atorvastatin&#44; rosuvastatin&#44; simvastatin&#44; fluvastatin&#44; lovastatin&#44; pitavastatin&#44; and pravastatin&#46; Inhibition of proliferation&#44; migration and invasion&#44; and induction of apoptosis are the proposed mechanisms of their antitumoral effect&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a>&#44;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a> However&#44; results of these studies are controversial and current evidence does not support their specific role in cancer chemoprevention and cancer related outcomes&#46;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a> In recent years&#44; several studies have demonstrated that statins have beneficial effect in patients with different liver diseases including chronic hepatitis C virus &#40;HCV&#41; infection&#44; chronic hepatitis B virus &#40;HBV&#41; infection&#44; and also decrease the risk of hepatocellular carcinoma &#40;HCC&#41; development&#46;<a class="elsevierStyleCrossRefs" href="#bib0040"><span class="elsevierStyleSup">8</span></a><span class="elsevierStyleSup">&#8211;</span><a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">16</span></a> Several studies have shown that statins use in chronic liver disease and cirrhosis is safe&#44; and even it was associated with lower mortality and lower rate of hepatic decompensation&#46;<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">17</span></a> Statin use is also associated with a reduced risk of fibrosis progression in patients with chronic hepatitis C and a reduced risk of cirrhosis development&#46;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">18</span></a>&#8211;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">19</span></a> So&#44; there appears to be multiple benefits from statins use in patients with chronic liver diseases&#46; So far&#44; it has been proven that there are several mechanisms activated by statins in cancer cells&#44; depending on the cell line&#44; statins concentration&#44; duration of exposure&#44; and the type of statin being used&#46; It has been shown that statins may inhibit cell-cycle progression and may induce apoptosis by both intrinsic and extrinsic pathways&#46;<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">20</span></a>&#44;<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">21</span></a></p><p id="p0015" class="elsevierStylePara elsevierViewall">TGF-&#946;1 is involved in the regulation of cell behavior in a variety of cellular contexts&#46; Expression of TGF- &#946;1 may be a critical step in the growth control of normal and proliferating rat hepatocytes&#46; It has been reported that TGF-&#946;1 has many roles in HCC development&#44; including tumor growth&#44; invasiveness&#44; neo-angiogenesis and metastatic behavior&#44; leading to cell survival promotion&#44; proliferation&#44; invasion and neoangiogenesis in many human cancers&#46;</p><p id="p0020" class="elsevierStylePara elsevierViewall">Proto-oncogene tyrosine-protein kinase Src pathway activation&#44; also known as cellular Src kinase or simply cSrc&#44; has been observed in about 50&#37; of tumors from various organs including the liver&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> Sato&#44; <span class="elsevierStyleItalic">et al&#46;</span><a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">23</span></a> demonstrated that TGF-&#946;1 can induce c-Src phosphorylation&#46;</p><p id="p0025" class="elsevierStylePara elsevierViewall">Hexachlorobenzene &#40;HCB&#41;&#44; a persistent environmental pollutant&#44; has been shown to induce cell growth imbalance in rat liver&#46; HCB increases the development of rat liver preneoplastic lesions in a medium-term initiation&#47; promotion model&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a></p><p id="p0030" class="elsevierStylePara elsevierViewall">The aim of our study was to investigate the effect of statins on cell proliferation in an initiation&#47;promotion model in rat liver and in Hep-G2 cells&#46; We also assessed the expression of TGF-&#946;1&#44; and its involvement in the potential key molecules involved in statins actions&#46;</p></span><span id="s0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0020">Material and Methods</span><span id="s0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0025">Animals and Treatment</span><p id="p0035" class="elsevierStylePara elsevierViewall">Female Wistar rats &#40;50 g at the onset of the experiment&#41; were purchased from the Facultad de Farmacia y Bioqu&#237;mica&#44; Universidad de Buenos Aires&#46; The rats were fed with Purine 3 rat chow &#40;Cabeca S&#46;C&#46;A&#44; Argentina&#41; and water ad libitum&#46; Environmental conditions consisted of a 12 hrs light-dark cycle&#44; 19 &#177; 2 &#176;C&#44; and 45-75&#37; humidity&#46; After 7-day acclimation period&#44; a total of 20 rats were divided into 4 equal groups&#46; Rats in groups 1 and 2&#44; received a single i&#46;p&#46; injection of saline vehicle&#46; Animals in groups 3 and 4 were treated with a single i&#46;p&#46; injection of DEN &#40;100 mg&#47;kg body weight&#41;&#46; One week later&#44; the animals in group 2 and 4 received HCB &#40;100 mg&#47;kg body weight&#41; administered 5 days a week&#44; by gavage&#46; HCB &#40;40 mg&#47;mL&#41; was suspended in water containing Tween 20 &#40;0&#46;5 mL&#47;100 mL&#41; &#40;<a class="elsevierStyleCrossRef" href="#f0005">Figure 1</a>&#41;&#46; The doses of DEN and HCB were selected based on similar previous studies&#46; At week 3 &#40;day 21&#41;&#44; a partial hepatectomy&#44; which comprised approximately 70&#37; of the total liver weight&#44; was performed on all animals&#46; Given the diurnal variation observed in liver DNA synthesis after partial hepatectomy in rodents&#44; all rats underwent partial hepatectomy or sham hepatectomy between 11&#58;00 and 15&#58;00 h&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">19</span></a> At the end ofthe eighth week&#44; the animals were killed by exsanguinations&#46;</p><elsevierMultimedia ident="f0005"></elsevierMultimedia></span><span id="s0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0030">Chemicals and antibodies</span><p id="p0040" class="elsevierStylePara elsevierViewall">Hexachlorobenzene &#40;HCB&#44; &#62; 99&#37; purity commercial grade&#41;&#44; anti- -actin&#44; and anti-proliferating cell nuclear antigen &#40;PCNA&#41; were purchased from Sigma &#40;SigmaAldrich Co&#41;&#46; TRI-Reagent used for mRNA extraction was purchased from MRC&#44; Molecular Research Center&#44; Inc&#46; The reagents used for cDNA synthesis&#44; were purchased from SRL Biodynamics&#46; Polyvinyl difluoride membranes &#40;PVDF&#41;&#44; for immuno-blotting were purchased from Bio-Rad &#40;Bio-Rad Laboratories&#41;&#46; CP-BU plates were purchased from New Afghanistan &#40;Afghanistan&#44; Gevaerl ArgentinaSA&#41;&#46; Monoclonal anti-TGF-&#946;1 was obtained from Abcam Inc&#46; Specific primers for transforming growth factor &#946;1 &#40;TGF-&#946;1&#41; and &#40;L-19&#41; were purchased from Invitrogen Life Technology&#46; The compound for &#40;RT&#41; and PCR were purchased from Promega Corporation&#46; All other reagents used were of analytical grade&#46;</p></span><span id="s0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0035">Cell culture and treatment</span><p id="p0045" class="elsevierStylePara elsevierViewall">Hep-G2 cell line was supplied by the American Type Culture Collection &#40;ATCC&#41;&#46; The cells were cultured in Modified Eagle&#8217;s Medium &#40;MEM&#41; &#40;Sigma&#41; supplemented with 10&#37; Fetal Bovine Serum &#40;FBS&#41; and 50 <span class="elsevierStyleItalic">&#956;</span>g&#47; mL gentamicin and penicillin&#46; Cells were maintained under standard conditions at 37 &#176;C in 5&#37; CO2 and 100&#37; relative humidity&#46; Cells in exponential growth with 80&#37; confluence were washed with phosphate solution &#40;PBS&#41; and then treated with a solution of 0&#46;025&#37; trypsin-0&#46;03&#37; EDTA for 1-5 minutes at 37 &#176;C&#46; Cells were seeded in 12-well plates and pre-incubated for 24 h to allow cell adherence&#46; For dose-response studies&#44; cells were treated for 24 h with HCB &#40;0&#46;05&#44; 0&#46;5 and 5 &#94;M&#41;&#46; Control cells were treated with the same volumes of ETOH&#46; We used a conditioned medium &#40;CM&#41; obtained from Hep-G2 cells treated with HCB 5 <span class="elsevierStyleItalic">&#956;</span>M for 24 h to obtain medium enriched in growth factors released by the cells&#46;</p></span><span id="s0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0040">Western Blottting</span><p id="p0050" class="elsevierStylePara elsevierViewall">Total cellular protein lysates were electrophoresed in 10&#37; SDS-polyacrylamide gel &#40;SDS-PAGE&#41; prior to transfer to polivinilidene difluoride membranes &#40;PVDF&#41;&#44; in a semidry transfer cell at 18 V for 1&#46;5 h&#46; Membranes were blocked overnight at 4 &#176;C with 5&#37; nonfat dry milk- 2&#46;5&#37; BSA in TBST buffer &#40;10 mM Tris-HCl&#44; pH 8&#46;0&#44; 0&#46;5&#37; Tween 20&#44; 150 mM NaCl&#41;&#46; Membranes were incubated with rabbit polyclonal specific antibodies &#40;PCNA&#44; c-Src and TGF-&#946;1&#41; &#40;1&#58;500&#41;&#44; and anti- -actin at &#40;1&#58;500&#41; dilution&#44; and incubated overnight at 4 &#176;C&#46; After incubation&#44; membranes were washed five times with TBST&#44; and the suitable peroxidase- conjugated antispecies specific antibodies were used for protein detection&#46; After washing&#44; blots were reacted using an enzyme-linked enhanced chemoluminescence &#40;ECL&#41; detection kit &#40;Amer- sham Biosciences&#44; Inc&#46;&#44; UK&#41; and quantified by scanning laser densitometry in a Fotodyne &#40;Foto&#47;Analyst&#41;&#44; Gel-Pro Analyzer 3&#46;1&#46;</p></span><span id="s0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0045">RT-PCR analysis</span><p id="p0055" class="elsevierStylePara elsevierViewall">Total RNA was extracted from with a phenol-isotiocianate of guanidine &#40;TRIZOL&#41; solution&#44; by a modification of Chomczynski&#8217;s method&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">25</span></a></p><p id="p0060" class="elsevierStylePara elsevierViewall">An aliquot of 2 <span class="elsevierStyleItalic">&#956;</span>g of total RNA was used to synthesize first-strand cDNA with the random primers&#44; deoxynucleotide triphosphates&#44; transcriptase reverse &#40;RT&#41;&#44; and transcriptase reverse buffer&#46; Reaction mixture for TGF-&#946;1&#44; and L-19 amplification contained GoTaq reaction buffer &#40;1&#46;5 mM MgCl2&#44; 0&#46;2 mM dNTP&#8217;s mix&#44; 1&#46;25 U GoTaq DNA polymerase&#44; 0&#46;5 <span class="elsevierStyleItalic">&#956;</span>M of each forward and reverse primers&#44; and 1 <span class="elsevierStyleItalic">&#956;</span>L of RT products&#41;&#46; PCR was performed as follows&#58; reaction mixture for TGF-&#946;1 amplification was incubated at 94 &#176;C for 5 min and then amplified at 94 &#176;C for 1 min&#44; 60 &#176;C for 1 min&#44; and 72 &#176;C for 1 min&#46; Reactions were repeated for 27 cycles&#46; Reaction mixture for L-19 amplification was incubated at 94 &#176;C for 5 min and then amplified at 94 &#176;C for 1 min&#44; 55 &#176;C for 1 min&#44; and 72 &#176;C for 1 min&#46; Reactions were repeated for 27 cycles&#46; The sequences of forward and reverse primers were as follows&#58;</p><p id="p0065" class="elsevierStylePara elsevierViewall">TGF-&#946;1&#58; 5&#8217;-CTGCTGGCAATAGCTTCCTA-3&#44; 5-CGAGCCTTAGTTGGACAGGAT-3&#8217;&#46; L-19&#58; 5&#8217;-TGAACGGGAAGCTCACTGG-3&#44; 5-TCCACCACCCTGTTGCTGTA-3&#8217;&#46;</p><p id="p0070" class="elsevierStylePara elsevierViewall">L-19 cDNA was used as a loading control&#46; PCR products were detected as a single band on 2&#37; agarose gel&#44; containing 0&#46;05&#37; &#40;v&#47;v&#41; ethidium bromide&#46; Bands were detected&#44; and intensity was quantified by scanning laser densitometry in a Fotodyne &#40;Foto&#47;Analyst&#41;&#44; Gel-Pro Analyzer 3&#46;1&#46;</p></span><span id="s0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0050">Statistical analysis</span><p id="p0075" class="elsevierStylePara elsevierViewall">Data are expressed as means &#177; SEM&#46; Differences between treated and control groups were analyzed by oneway ANOVA&#44; at a 95&#37; confidence interval&#44; followed by Tukey post hoc test to identify significant differences between the samples and controls means&#44; after testing homogeneity of variance using Barlett&#8217;s procedure&#46; Differences between control and treated animals were considered significant when p values were &#60; 0&#46;05&#46;</p></span></span><span id="s0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0055">Results</span><span id="s0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0060">Effect of HCB on HMGCoAR mRNA levels and serum cholesterol levels in an initiation-promotion model of HCC</span><p id="p0080" class="elsevierStylePara elsevierViewall">We have previously demonstrated that HCB contributes to the development of preneoplastic foci by enhancing the proliferation of initiated cells in rat liver&#46;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> We focused on the effect of HCB on HMGCoAR mRNA levels in rat liver&#44; in an initiation-promotion model&#46; As shown in <a class="elsevierStyleCrossRef" href="#f0010">figure 2</a>&#44; HMGCoAR mRNA levels were significantly increased in liver from DEN &#43; HCB group compared to DEN group &#40;31&#37;&#41; &#40;<a class="elsevierStyleCrossRef" href="#f0010">Figure 2</a>A and 2B&#41;&#46; The above mentioned increase in HMGCoAR activity&#44; correlates with high cholesterol serum levels&#46; Our results showed that serum cholesterol levels were significantly increased &#40;28&#37;&#41; in DEN &#43; HCB group compared with DEN group &#40;<a class="elsevierStyleCrossRef" href="#f0010">Figure 2</a>C&#41;&#46;</p><elsevierMultimedia ident="f0010"></elsevierMultimedia></span><span id="s0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0065">Effect of statins treatment on Hep-G2 cells viability</span><p id="p0085" class="elsevierStylePara elsevierViewall">In order to investigate the effect of statins on cell viability&#44; Hep-G2 cells were treated with different doses of AT &#40;10&#44; 20 and 30 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; and SM &#40;5&#44; 10 and 20 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; for three hours&#46; MTT assay shows that statins did not alter Hep-G2 cells viability &#40;<a class="elsevierStyleCrossRef" href="#f0015">Figure 3</a>&#41;&#46;</p><elsevierMultimedia ident="f0015"></elsevierMultimedia></span><span id="s0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0070">Effect of HCB on HMGCoAR mRNA levels in Hep-G2 cells</span><p id="p0090" class="elsevierStylePara elsevierViewall">Hep-G2 is a well-differentiated human hepatocellular carcinoma cell line that preserves numerous liver-specific functions and thus can serve as an <span class="elsevierStyleItalic">in vitro</span> model&#46;</p><p id="p0095" class="elsevierStylePara elsevierViewall">Consistent with our previous results showing that HCB induces cell proliferation in a dose-dependent manner in Hep-G2 cells&#44;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> we evaluated HCB treatment effect &#40;0&#46;05&#44; 0&#46;5 and 5 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; on HMGCoAR mRNA levels&#46; As shown in <a class="elsevierStyleCrossRef" href="#f0015">figure 3</a>&#44; HMGCoAR mRNA levels significantly increased &#40;32&#37; to 54&#37;&#41; in total cell lysates of 0&#46;5 and 5 <span class="elsevierStyleItalic">&#956;</span>&#924; HCB-treated cells&#44; respectively&#44; when compared to ETOH &#40;<a class="elsevierStyleCrossRef" href="#f0020">Figure 4</a>&#41;&#46;</p><elsevierMultimedia ident="f0020"></elsevierMultimedia></span><span id="s0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0075">Effect of statins on HMGCoAR mRNA levels in HCB-treated Hep-G2 cell</span><p id="p0100" class="elsevierStylePara elsevierViewall">To evaluate the effect of statins on HMGCoAR mRNA expression&#44; Hep-G2 cells were pretreated with increasing doses of AT &#40;10&#44; 20 and 30 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; or SM &#40;5&#44; 10 and 20 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; for 3 h and then treated with HCB &#40;5 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; for 24 h&#46; As shown in <a class="elsevierStyleCrossRef" href="#f0025">figure 5</a>&#44; HMGCoAR mRNA levels significantly increased due to HCB effects&#46; Nevertheless&#44; HMGCoAR mRNA levels significantly decreased by 29&#37; and 38&#37; when pretreated with AT 10 and 20 <span class="elsevierStyleItalic">&#956;</span>M &#40;respectively&#41;&#44; reaching baseline values with AT 30 <span class="elsevierStyleItalic">&#956;</span>M&#44; when compared to HCB-treated cells without AT pretreatment&#46; Similarly&#44; pretreatment with SM at 5 and 10 <span class="elsevierStyleItalic">&#956;</span>M decreased HMG-CoAR mRNA levels by 21&#37; and 31&#37; &#40;respectively&#41;&#44; reaching baseline values with SM 20 <span class="elsevierStyleItalic">&#956;</span>M&#44; when compared to HCB-treated cells without SM pretreatment &#40;<a class="elsevierStyleCrossRef" href="#f0025">Figure 5</a>&#41;&#46;</p><elsevierMultimedia ident="f0025"></elsevierMultimedia></span><span id="s0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0080">Statins effect on HCB-induced proliferation in Hep-G2 cells</span><p id="p0105" class="elsevierStylePara elsevierViewall">Since we have previously demonstrated that 5 <span class="elsevierStyleItalic">&#956;</span>M HCB induced PCNA-expression in Hep-G2 cells&#44;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> we evaluated the dose-response effects of statins on cell proliferation&#46; Western blots analysis showed that AT pretreatment &#40;10&#44; 20 and 30 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; significantly reduced HCB-induced PCNA levels by 24&#37;&#44; 52&#37; and reaching baseline values at maximum &#40;respectively&#41; when compared to HCB group &#40;<a class="elsevierStyleCrossRef" href="#f0030">Figure 6</a>A&#41;&#46; In a similar manner SM pretreatment &#40;5&#44; 10 and 20 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41;&#44; significantly reduced HCB-induced PCNA levels &#40;30&#37;&#44; 58&#37; until reaching basal values at maximum doses&#44; respectively&#41;&#44; when compared to HCB group &#40;<a class="elsevierStyleCrossRef" href="#f0030">Figure 6</a>B&#41;&#46; Altogether&#44; the above results demonstrate that statins antagonize HCB-induced cell proliferation in Hep-G2 cells&#46;</p><elsevierMultimedia ident="f0030"></elsevierMultimedia></span><span id="s0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0085">Statins effect on TGF-&#946;1 levels in HCB treated Hep-G2 cells</span><p id="p0110" class="elsevierStylePara elsevierViewall">Given that TGF-&#946;1 is one of the most relevant cytokines that intervene in the apoptotic and&#47;or proliferative processes in hepatocytes&#44; we evaluated the statins effect on TGF-&#946;1 levels in HCB-treated Hep-G2 cells&#46; Hep-G2 cells were pretreated with AT &#40;10&#44; 20 and 30 &#956;&#924;&#41; or SM &#40;5&#44; 10 and 20 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; or ETOH for 3 h&#44; and then treated with HCB &#40;5 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41; for 24 h&#46; Western blot analysis showed a significant reduction in TGF-&#946;1 levels&#44; when Hep-G2 cells were treated with both statins&#58; AT &#40;23&#37;&#44; 29&#37; and 45&#37;&#41;&#44; and SM &#40;11&#37;&#44; 24&#37; and 40&#37;&#41; respectively&#44; until reaching baseline values at maximum doses&#44; when compared to HCB group &#40;<a class="elsevierStyleCrossRef" href="#f0035">Figure 7</a>&#41;&#46;</p><elsevierMultimedia ident="f0035"></elsevierMultimedia></span><span id="s0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0090">Role of TGF-&#946;1 in statins-induced anti-proliferative effect</span><p id="p0115" class="elsevierStylePara elsevierViewall">We further investigated whether the statins anti-proliferative effect in HCB-treated Hep-G2 cells was mediated by TGF-&#946;1 When Hep-G2 cells were treated with HCB 5 <span class="elsevierStyleItalic">&#956;</span>&#924; &#40;lane 2&#41; or CM &#40;lane 3&#41;&#44; a significant increase in PCNA levels &#40;42&#37; and 38&#37;&#41; was observed when compared to ETOH&#44; respectively&#46;</p><p id="p0120" class="elsevierStylePara elsevierViewall">Our results show that AT pretreated cells did not show increases in PCNA levels with HCB or MC treatment &#40;lanes 5 and 6&#44; respectively&#41;&#46; On the other hand&#44; Hep-G2 cells treated with TGF-&#946;1 showed a significant increase in PCNA levels &#40;lanes 9&#41; and this effect was blocked when these cells were pretreated with an exogenous TGF-&#946;1 inhibitor &#40;lane 11&#41;&#46; Similarly&#44; Hep-G2 cells pre-treated with AT and TGF-&#946;1 showed no changes in PCNA levels &#40;lane 12&#41;&#46; Finally&#44; Hep-G2 cells pre-treated with AT and I&#47;TGF-1 and then with an exogenous TGF<span class="elsevierStyleBold"><span class="elsevierStyleItalic">-</span></span>&#946;1 and HCB&#44; showed no changes on PCNA levels &#40;lane 13&#41; &#40;<a class="elsevierStyleCrossRef" href="#f0040">Figure 8</a>A and 8B&#41;&#46;</p><elsevierMultimedia ident="f0040"></elsevierMultimedia></span><span id="s0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0095">Statins effect on c-Src levels in HCB treated Hep-G2 cells</span><p id="p0125" class="elsevierStylePara elsevierViewall">Given that TGF-&#946;1 activates c-Src kinase&#44; and c-Src pathway activation has been observed in about 50&#37; of tumors from various organs including the liver&#44;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> we evaluated the effect of statins on c-Src protein levels in HCB-treated cells&#46; Our results showed that HCB significantly increased phosphorylated c-Src protein levels &#40;136&#37;&#41;&#46; Pre-treatment with AT &#40;30 <span class="elsevierStyleItalic">&#956;</span>M&#41; and SM &#40;20 <span class="elsevierStyleItalic">&#956;</span>M&#41; decreased this HCB-induced elevation in phosphorylated c-Src levels &#40;41&#37; and 40&#37;&#44; respectively&#41;&#46; However&#44; it does not return to baseline values when compared to HCB 5 <span class="elsevierStyleItalic">&#956;</span>M treated cells &#40;<a class="elsevierStyleCrossRef" href="#f0045">Figure 9</a>&#41;&#46;</p><elsevierMultimedia ident="f0045"></elsevierMultimedia></span><span id="s0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0100">Statins effect on DI levels in HCB treated Hep-G2 cells</span><p id="p0130" class="elsevierStylePara elsevierViewall">Disruptive hormone plays a critical role in the development of pre-neoplastic lesions&#46; T3 is one of the major cell cycle regulators and its levels are regulated by the activity of the enzyme DI&#46; In HCB treated cells&#44; mRNA DI levels were significantly reduced by 45&#37; &#40;p &#8804; 0&#46;01&#41;&#46; When both statins were added to treated cells&#44; they reversed this effect and mRNA DI levels returned to their baseline value &#40;<a class="elsevierStyleCrossRef" href="#f0050">Figure 10</a>&#41;&#46;</p><elsevierMultimedia ident="f0050"></elsevierMultimedia></span></span><span id="s0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0105">Discussion</span><p id="p0135" class="elsevierStylePara elsevierViewall">Dysregulated cell proliferation is the main mechanism of hepatocarcinogenesis&#44; leading to tumor growth and invasion&#46; However&#44; the mechanism of this proliferation is complex and poorly understood&#46; Over the last few years&#44; many molecular targets have been proposed as potential sites for advanced HCC treatment&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a>&#44;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> Limited clinical and epidemiologic data suggest that statins may improve HCC outcomes&#44; a tumor associated with poor prognosis&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a></p><p id="p0140" class="elsevierStylePara elsevierViewall">In this study&#44; we have investigated the cellular effects of statins &#40;atorvastatin and simvastatin&#41; on HCB-induced proliferation in Hep-G2 cells&#46; We found that incubation with atorvastatin or simvastatin inhibits HCB-induced cell proliferation in cultured Hep-G2 cells&#46; We have demonstrated for the first time that&#44; statins induced inhibition of proliferation might at least be partially explained by a decrease in TGF-&#946;1 expression and by a downstream decrease in c-Src phosphorylation&#46;</p><p id="p0145" class="elsevierStylePara elsevierViewall">Other authors observed that simvastatin prevents proliferation and favors cellular apoptosis in a rat osteosarcoma line&#44; UMR-106&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">27</span></a> It is interesting to note that they use doses of 3-10 <span class="elsevierStyleItalic">&#956;</span>M&#59; however&#44; we observed that 20 <span class="elsevierStyleItalic">&#956;</span>M might be an optimal dose for proliferative effect reversion&#46; Atorvastatin inhibits <span class="elsevierStyleItalic">in vitro</span> proliferation at dose of 0&#46;4 nM in hepatic myofibroblasts&#46;<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">28</span></a> In this study&#44; although both statins present a complete reversion of the HCB proliferative effect&#44; atorvastatin appears to be more effective at the highest dose &#40;30 <span class="elsevierStyleItalic">&#956;</span>&#924;&#41;&#46;</p><p id="p0150" class="elsevierStylePara elsevierViewall">In a previous study&#44; we have demonstrated that HCB deregulates cell growth and alters growth factors such as EGF and TGF-&#946;1 expression levels&#44; as well as theirs signaling pathways&#44; both being involved in developmental and tumorigenic processes&#46;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a>&#44;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> Here&#44; in the <span class="elsevierStyleItalic">in vivo</span> induction&#47; promotion HCC model and in the <span class="elsevierStyleItalic">in vitro</span> model&#44; we use HCB to induce cell proliferation&#46; This increase in cell proliferation &#40;supported by an increased PCNA levels&#41; was accompanied by increases in HMGCoAR and TGF-&#946;1 mRNA levels&#46;</p><p id="p0155" class="elsevierStylePara elsevierViewall">Previously&#44; we had demonstrated that HCB-induced proliferation in Hep-G2 cell is mediated by an increase in TGF-&#946;1 expression&#46; In this study&#44; we evaluated atorvastatin and simvastatin effect on HCB-induced proliferation&#46; Likewise&#44; we analyzed the potential role of TGF-&#946;1 in the statins mechanism of action in the HCB-induced proliferation model&#46; TGF-&#946;1 involvement in the statins mechanism of action to reduce cell proliferation had been previously described&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">30</span></a>&#44;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">31</span></a> However&#44; it is unclear which molecules are involved in the downstream pathway of this cytokine&#46;</p><p id="p0160" class="elsevierStylePara elsevierViewall">One of the potential molecules involved could be c-Src&#46; Its pathway activation has been observed in about 50&#37; of tumors from various organs including the liver&#44;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> c-Src can cooperate with kinases receptors to signal through downstream molecules&#44; such as PI3K&#47;PTEN&#47;Akt&#44; Ras&#47;Raf&#47; Mek1&#47;2&#47;Erk1&#47;2 and STATs&#46; In many cancer cell lines&#44; tumors signal transducers and transcription activator proteins &#40;STAT&#41; are often deregulated with persistent activation in tyrosine phosphorylation&#46; STAT1 is generally considered a tumour suppressor but there is growing evidence that it can also act as a tumour promoter&#46; Therefore&#44; the JAK&#47;STAT signaling pathway is one of the most promising targets in the cancer therapy&#46; Our hypothesis about the role of cSrc in the inhibition of TGF-&#946;1-induced proliferation is consistent with the observations of Sato&#44; <span class="elsevierStyleItalic">et al&#46;</span>&#44; who demonstrated that TGF-&#946;1 can induce c-Src phosphorylation&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">23</span></a></p><p id="p0165" class="elsevierStylePara elsevierViewall">Here&#44; we have demonstrated for the first time&#44; the downstream role of c-Src in Hep-G2 cells proliferation&#46; We have also demonstrated for the first time that TGF-&#946;1&#47; c-Src pathway may be inhibited by both statins in a dose dependent manner&#46;</p><p id="p0170" class="elsevierStylePara elsevierViewall">The role of thyroid hormones in carcinogenesis has been postulated for many years&#46; However&#44; there is not enough data for a complete understanding of this process&#46; Triiodothyronine &#40;T3&#41; acts through its nuclear receptors&#44; the thyroid hormone receptors &#40;THR&#41;&#46; T3 is produced by monodeiodination catalyzed by type 1 iodothyronine deiodinase &#40;DI&#41;&#46; This enzyme is present in almost all tissues&#44; but the highest concentrations were found in the thyroid&#44; the liver&#44; and the kidneys&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">32</span></a> Both its enzymatic activity and its gene expression have been examined in some neoplastic tissues&#46; Significant decreases in gene expression and enzyme activity were found in several of them&#46;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">33</span></a> HCB is a hormone disruptor and induces hypothyroxinemia&#44; and it is well known that T3 has a negative regulatory effect on the cell cycle&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> A possible explanation could be that thyroid hormones deregulation may play an important role in HCC development as well as in the statins mechanism key on cell proliferation inhibition&#46;</p><p id="p0175" class="elsevierStylePara elsevierViewall">In our previous study&#44; we demonstrated that HCB increases cell proliferation&#44; alters mRNA DIII and TH concentration in induction&#47;promotion rat liver model&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> In this study&#44; our results showed that HCB increased cell proliferation and decreased mRNA DI levels in Hep-G2 cells&#46; It is possible that DI decreased expression is partly responsible for the decrease in tissue T3 and this event contributes to the deregulation of the cell cycle&#46; More experiments are needed to explore this finding&#46;</p><p id="p0180" class="elsevierStylePara elsevierViewall">We showed here that the increase in cell proliferation as well as the decrease in mRNA DI levels are mediated by TGF-&#946;1 We also demonstrated that statins inhibit cell proliferation by decreasing TGF-&#946;1 levels&#46;</p><p id="p0185" class="elsevierStylePara elsevierViewall">It is known that there is a regulation between TGF-&#946;1 and DI enzyme&#46; It is possible that statins mediated TGF-&#946;1 reduction counteracts the effects on DI and T3 levels&#44; resulting in normal T3 tissue levels&#46;</p><p id="p0190" class="elsevierStylePara elsevierViewall">In preliminary experiments &#40;data not shown&#41; performed in our laboratory&#44; we observed that exogenously administered thyroid hormones reduce HCB-induced cell proliferation in the Hep-G2 cell line&#46; Won Park&#44; <span class="elsevierStyleItalic">et al&#46;</span> showed that THR could down regulate the activity of cSrc in the presence of T3 via phosphorylation&#44; suggesting that low T3 levels generate a c-Src related pro-proliferative process&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">34</span></a> More experiments are needed to validate these results in our experimental model&#46;</p><p id="p0195" class="elsevierStylePara elsevierViewall">Summarizing&#44; our study showed for the first time that atorvastatin and simvastatin can revert HCB-induced proliferative effects on Hep-G2 cells&#46; In addition&#44; this effect is mediated by the inhibition of TGF-&#946;1 and the downstream c-Src phosphorylation pathway&#46; The TH homeostasis deregulation could be potentially involved&#46; TGF-&#946;1 and c-Src may be the statins molecular targets in early events of hepatocarcinogenesis&#46;</p></span><span id="s0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0110">Abbreviations</span><p id="p0200" class="elsevierStylePara elsevierViewall"><ul class="elsevierStyleList" id="l0005"><li class="elsevierStyleListItem" id="u0005"><span class="elsevierStyleLabel">&#8226;</span><p id="p0205" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">AT&#58;</span> Atorvastatin&#46;</p></li><li class="elsevierStyleListItem" id="u0010"><span class="elsevierStyleLabel">&#8226;</span><p id="p0210" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">CM&#58;</span> conditioned medium&#46;</p></li><li class="elsevierStyleListItem" id="u0015"><span class="elsevierStyleLabel">&#8226;</span><p id="p0215" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">c-Src&#58;</span> cellular Src kinase&#44; Proto-oncogene tyrosineprotein kinase Src&#46;</p></li><li class="elsevierStyleListItem" id="u0020"><span class="elsevierStyleLabel">&#8226;</span><p id="p0220" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">DEN&#58;</span> Diethylnitrosamine&#46;</p></li><li class="elsevierStyleListItem" id="u0025"><span class="elsevierStyleLabel">&#8226;</span><p id="p0225" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">DI&#58;</span> deiodinase I&#46;</p></li><li class="elsevierStyleListItem" id="u0030"><span class="elsevierStyleLabel">&#8226;</span><p id="p0230" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HBV&#58;</span> hepatitis B virus&#46;</p></li><li class="elsevierStyleListItem" id="u0035"><span class="elsevierStyleLabel">&#8226;</span><p id="p0235" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HCB&#58;</span> hexachlorobenzene&#46;</p></li><li class="elsevierStyleListItem" id="u0040"><span class="elsevierStyleLabel">&#8226;</span><p id="p0240" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HCC&#58;</span> Hepatocellular carcinoma&#46;</p></li><li class="elsevierStyleListItem" id="u0045"><span class="elsevierStyleLabel">&#8226;</span><p id="p0245" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HCV&#58;</span> hepatitis C virus&#46;</p></li><li class="elsevierStyleListItem" id="u0050"><span class="elsevierStyleLabel">&#8226;</span><p id="p0250" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HMGCoAR&#58;</span> 3 -hydroxy-3-methylglutaryl coenzyme-A reductase&#46;</p></li><li class="elsevierStyleListItem" id="u0055"><span class="elsevierStyleLabel">&#8226;</span><p id="p0255" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HSCs&#58;</span> hepatic stellate cells&#46;</p></li><li class="elsevierStyleListItem" id="u0060"><span class="elsevierStyleLabel">&#8226;</span><p id="p0260" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">HVPG&#58;</span> hepatic venous pressure gradient&#46;</p></li><li class="elsevierStyleListItem" id="u0065"><span class="elsevierStyleLabel">&#8226;</span><p id="p0265" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">i&#46;p&#46;&#58;</span> intra peritoneal&#46;</p></li><li class="elsevierStyleListItem" id="u0070"><span class="elsevierStyleLabel">&#8226;</span><p id="p0270" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">PCNA&#58;</span> Proliferating cell nuclear antigen&#46;</p></li><li class="elsevierStyleListItem" id="u0075"><span class="elsevierStyleLabel">&#8226;</span><p id="p0275" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">pc-Src&#58;</span> phosphorylated c-SRC&#46;</p></li><li class="elsevierStyleListItem" id="u0080"><span class="elsevierStyleLabel">&#8226;</span><p id="p0280" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">SM&#58;</span> Simvastatin&#46;</p></li><li class="elsevierStyleListItem" id="u0085"><span class="elsevierStyleLabel">&#8226;</span><p id="p0285" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">STAT&#58;</span> signal transducer and activator of transcription&#46;</p></li><li class="elsevierStyleListItem" id="u0090"><span class="elsevierStyleLabel">&#8226;</span><p id="p0290" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">T3&#58;</span> triiodothyronine&#46;</p></li><li class="elsevierStyleListItem" id="u0095"><span class="elsevierStyleLabel">&#8226;</span><p id="p0295" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">TGF-&#946;1&#58;</span> transforming growth factor<span class="elsevierStyleBold">-&#946;</span>1&#46;</p></li><li class="elsevierStyleListItem" id="u0100"><span class="elsevierStyleLabel">&#8226;</span><p id="p0300" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">TH&#58;</span> thyroid hormones&#46;</p></li><li class="elsevierStyleListItem" id="u0105"><span class="elsevierStyleLabel">&#8226;</span><p id="p0305" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">THR&#58;</span> thyroid hormone receptor&#46;</p></li></ul></p></span><span id="s0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0115">Conflict of Interest</span><p id="p0310" class="elsevierStylePara elsevierViewall">The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript&#46;</p></span><span id="s0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0120">Financial Support</span><p id="p0315" class="elsevierStylePara elsevierViewall">This work was supported by grant SAF2012-32491 from Plan Nacional &#40;MINECO&#41; and grant S2010&#47;BDM-2423 &#40;MOIR&#41; from Comunidad de Madrid&#44; Spain&#44; and by a grant UBACYT 20020120200258 &#47;EXP-UBA N&#176; 17064&#47; 2012 from Universidad de Buenos Aires&#44; Ciudad Aut&#243;noma de Buenos Aires&#44; Argentina&#46;</p></span></span>"
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          "identificador" => "xres1190773"
          "titulo" => "Abstract"
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            0 => array:1 [
              "identificador" => "abs0010"
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        1 => array:2 [
          "identificador" => "xpalclavsec1110050"
          "titulo" => "Keywords"
        ]
        2 => array:2 [
          "identificador" => "s0005"
          "titulo" => "Introduction"
        ]
        3 => array:3 [
          "identificador" => "s0010"
          "titulo" => "Material and Methods"
          "secciones" => array:6 [
            0 => array:2 [
              "identificador" => "s0015"
              "titulo" => "Animals and Treatment"
            ]
            1 => array:2 [
              "identificador" => "s0020"
              "titulo" => "Chemicals and antibodies"
            ]
            2 => array:2 [
              "identificador" => "s0025"
              "titulo" => "Cell culture and treatment"
            ]
            3 => array:2 [
              "identificador" => "s0030"
              "titulo" => "Western Blottting"
            ]
            4 => array:2 [
              "identificador" => "s0035"
              "titulo" => "RT-PCR analysis"
            ]
            5 => array:2 [
              "identificador" => "s0040"
              "titulo" => "Statistical analysis"
            ]
          ]
        ]
        4 => array:3 [
          "identificador" => "s0045"
          "titulo" => "Results"
          "secciones" => array:9 [
            0 => array:2 [
              "identificador" => "s0050"
              "titulo" => "Effect of HCB on HMGCoAR mRNA levels and serum cholesterol levels in an initiation-promotion model of HCC"
            ]
            1 => array:2 [
              "identificador" => "s0055"
              "titulo" => "Effect of statins treatment on Hep-G2 cells viability"
            ]
            2 => array:2 [
              "identificador" => "s0060"
              "titulo" => "Effect of HCB on HMGCoAR mRNA levels in Hep-G2 cells"
            ]
            3 => array:2 [
              "identificador" => "s0065"
              "titulo" => "Effect of statins on HMGCoAR mRNA levels in HCB-treated Hep-G2 cell"
            ]
            4 => array:2 [
              "identificador" => "s0070"
              "titulo" => "Statins effect on HCB-induced proliferation in Hep-G2 cells"
            ]
            5 => array:2 [
              "identificador" => "s0075"
              "titulo" => "Statins effect on TGF-&#946;1 levels in HCB treated Hep-G2 cells"
            ]
            6 => array:2 [
              "identificador" => "s0080"
              "titulo" => "Role of TGF-&#946;1 in statins-induced anti-proliferative effect"
            ]
            7 => array:2 [
              "identificador" => "s0085"
              "titulo" => "Statins effect on c-Src levels in HCB treated Hep-G2 cells"
            ]
            8 => array:2 [
              "identificador" => "s0090"
              "titulo" => "Statins effect on DI levels in HCB treated Hep-G2 cells"
            ]
          ]
        ]
        5 => array:2 [
          "identificador" => "s0095"
          "titulo" => "Discussion"
        ]
        6 => array:2 [
          "identificador" => "s0100"
          "titulo" => "Abbreviations"
        ]
        7 => array:2 [
          "identificador" => "s0105"
          "titulo" => "Conflict of Interest"
        ]
        8 => array:2 [
          "identificador" => "s0110"
          "titulo" => "Financial Support"
        ]
        9 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2017-06-07"
    "fechaAceptado" => "2017-08-19"
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        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1110050"
          "palabras" => array:4 [
            0 => "Hepatocellular carcinoma"
            1 => "TGF-&#946;&#921;"
            2 => "HMGCoAR"
            3 => "Statins"
          ]
        ]
      ]
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    "resumen" => array:1 [
      "en" => array:2 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abs0010" class="elsevierStyleSection elsevierViewall"><p id="sp0055" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleBold">Introduction and alm&#46;</span> Hepatocellular carcinoma &#40;HCC&#41; represents 90&#37; of liver tumors&#46; Statins&#44; may reduce the incidence of various tumors&#44; including HCC&#46; Antitumoral activities may be mediated by changes in transforming growth factor-beta &#40;TGF-&#946;&#921;&#41; and thyroid hormones &#40;TH&#41; regulation&#46; Aim&#46; The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an <span class="elsevierStyleItalic">in vivo</span> and <span class="elsevierStyleItalic">in vitro</span> HCC model&#46;</p><p id="sp0155" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleBold">Materials and methods&#46;</span> We used two models&#58; <span class="elsevierStyleItalic">in vivo</span> &#40;in rats&#41; using diethylnitrosamine &#40;DEN&#41; and hexachlorobenzene &#40;HCB&#41; to develop HCC&#46; We analyzed cell proliferation parameters &#40;proliferating cell nuclear antigen&#44; PCNA&#41; and cholesterol metabolism &#40;hydroxy-methylglutaryl-CoA reductase&#44; HMGCoAR&#41;&#46; <span class="elsevierStyleItalic">In vitro</span> &#40;Hep-G2 cells&#41; we evaluated the effects of different doses of Atorvastatin &#40;AT&#41; and Simvastatin &#40;SM&#41; on HCB induced proliferation and analyzed proliferative parameters&#44; cholesterol metabolism&#44; TGF-&#946;&#921; mRNA&#44; c-Src and TH levels&#46;</p><p id="sp0165" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleBold">Results&#46;</span><span class="elsevierStyleItalic">In vivo</span>&#44; we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB&#46; <span class="elsevierStyleItalic">In vitro</span>&#44; we observed the same results on PCNA as <span class="elsevierStyleItalic">in vivo&#46;</span> The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB&#44; reaching levels similar to controls at maximum doses&#58; AT &#40;30 &#956;M&#41;&#44; and SM &#40;20 &#956;M&#41;&#46; Increases in PCNA&#44; TGF-&#946;&#921;&#44; and pc-Src&#44; and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses&#46;</p><p id="sp0175" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleBold">Conclusion&#46;</span> Statins can prevent the proliferative HCB effects on Hep-G2 cells&#46; TGF-&#946;&#921;&#44; c-Src and TH may be the statins molecular targets in hepatocarcinogenesis&#46;</p></span>"
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          "en" => "<p id="sp0005" class="elsevierStyleSimplePara elsevierViewall">Experimental design for the initiation&#47;promotion study&#46; The initiation agent&#44; DEN&#44; was administered i&#46;p&#46; &#40;100 mg&#47;kg&#41; on Day 0&#46; Saline solution or HCB was delivered by gavage starting on day 45 at a dose of 100 mg&#47;kg b&#46;w&#46; per day&#44; five days&#47;week&#46; A two-thirds partial hepatectomy was performed on all animals on day 52&#46; Liver tissues were collected 100 days following initial DEN dosing&#46;</p>"
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          "en" => "<p id="sp0010" class="elsevierStyleSimplePara elsevierViewall">Analysis of HMGCoAR mRNA expression in rat liver and serum cholesterol levels in an initiation-promotion model&#46; <span class="elsevierStyleBold">A&#46;</span> Representative pattern of RT-PCR amplification of HMGCoAR cDNA from liver of DEN and DEN&#43;HCB treated rats&#44; synthesized from total RNA&#46; <span class="elsevierStyleBold">B&#46;</span> L-19 was used as a loading control&#46; Quantification of HMGCoAR&#47;L-19 cDNA ratio is shown in the lower panel&#46; C&#46; Serum cholesterol levels&#46; Values are means &#177; SEM of three independent experiments of four rats per group&#46; Significantly different &#40;&#42;p &#60; 0&#46;05&#41; compared to DEN group&#46;</p>"
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          "en" => "<p id="sp0015" class="elsevierStyleSimplePara elsevierViewall">Effect of statins on Hep-G2 viable cell number&#46; The viable cell number was evaluated using the MTT assay&#46; The absorbance was measured at 570 nm&#44; and the results were expressed as percentage of ETOH-treated cells&#46; <span class="elsevierStyleBold">A&#46;</span> Cells were treated with AT &#40;10&#44; 20 and 30 &#956;M&#41; or <span class="elsevierStyleBold">&#40;B&#41;</span> SM &#40;5&#44; 10 and 20 &#956;M&#41; for 24 h&#46; Values represent means &#177; SEM of the three independent studies&#46;</p>"
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            "Alto" => 2316
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            "Tamanyo" => 240313
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        "descripcion" => array:1 [
          "en" => "<p id="sp0020" class="elsevierStyleSimplePara elsevierViewall">HCB induces HMGCoAR mRNA expression&#46; Hep-G2 cells were treated with HCB &#40;0&#46;05&#44; 0&#46;5 and 5 &#956;M&#41;&#46; <span class="elsevierStyleBold">A&#46;</span> Representative pattern of HMGCoAR cDNA from ETOH and HCB-treated cells&#44; synthesized from total RNA&#46; L-19 was used as a loading control&#46; <span class="elsevierStyleBold">B&#46;</span> Quantification of cDNAs&#44; after correction with L-19 cDNA&#46; Values are means &#177; SEM of three independent experiments&#46; Significantly different &#40;&#42;p &#60; 0&#46;05&#41; compared to ETOH group&#46;</p>"
        ]
      ]
      4 => array:7 [
        "identificador" => "f0025"
        "etiqueta" => "Figure 5"
        "tipo" => "MULTIMEDIAFIGURA"
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        "descripcion" => array:1 [
          "en" => "<p id="sp0025" class="elsevierStyleSimplePara elsevierViewall">Analysis of statins effect on HMGCoA R mRNA expression in HCB-treated Hep-G2 cells&#46; <span class="elsevierStyleBold">A&#46;</span> Hep-G2 cells were pretreated with AT &#40;10&#44; 20 and 30 &#956;M&#41; or SM &#40;5&#44; 10 and 20 &#956;M&#41; for 3 h and then treated with HCB &#40;5 &#956;M&#41; for 24 h&#46; <span class="elsevierStyleBold">B&#46;</span> L-19 was used as a loading control&#46; Representative patterns of RT-PCR amplification of HMGCoAR cDNA from ETOH and statinstreated cells&#44; synthesized from total RNA are shown in the upper panel&#46; Quantification of HMGCoAR mRNA&#44; after correction with L-19 is shown in the lower panel&#46; Values are means &#177; SEM of three independent experiments&#46; Significantly different &#40;&#42;p &#60; 0&#46;05&#41; compared to ETOH group&#46;</p>"
        ]
      ]
      5 => array:7 [
        "identificador" => "f0030"
        "etiqueta" => "Figure 6"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr6.jpeg"
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        "descripcion" => array:1 [
          "en" => "<p id="sp0030" class="elsevierStyleSimplePara elsevierViewall">Statins effect on HCB-induced cell proliferation&#46; <span class="elsevierStyleBold">A&#46;</span> Western blot analysis of PCNA in total lysate of Hep-G2 cells treated with 5 &#946;M HCB&#44; and pretreated with AT &#40;10&#44; 20&#44; and 30 &#946;M&#41; for 3 h&#46; Quantification of PCNA protein levels by densitometric scanning of the immunoblots is shown in the lower panel&#46; <span class="elsevierStyleBold">B&#46;</span> Western blot analysis of PCNA in total lysate of Hep-G2 cells treated with 5 &#956;&#924; HCB&#44; and pretreated with SM &#40;5&#44; 10 and 20 &#956;&#924;&#41;&#46; Quantification of PCNA protein levels by densitometric scanning of the immunoblots is shown in the lower panel&#46; Values are means &#177; SEM of three independent experiments&#46; Significantly different &#40;&#42;p &#60; 0&#46;05 and &#42;&#42;p &#60; 0&#46;01&#41; compared to HCB-treated cells&#46;</p>"
        ]
      ]
      6 => array:7 [
        "identificador" => "f0035"
        "etiqueta" => "Figure 7"
        "tipo" => "MULTIMEDIAFIGURA"
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          "en" => "<p id="sp0035" class="elsevierStyleSimplePara elsevierViewall">Effect of statins on TGF-&#946; 1 protein levels&#44; in Hep-G2 cells treated with HCB&#46; <span class="elsevierStyleBold">A&#46;</span> Western blot analysis of TGF-&#946;1 protein levels in Hep-G2 cells pretreated with AT &#40;10&#44; 20 and 30 &#956;M&#41; or SM &#40;5&#44; 10 and 20 &#956;M&#41; for 3 h and then treated with HCB &#40;5 &#956;M&#41; for 24 h&#46; Quantification of TGF-&#946; 1 L-19 ratio to ETOH is shown in the lower panel&#46; B&#46; Values are means &#177; SEM of three independent experiments&#46; Significantly different &#40;&#42;p &#60; 0&#46;05 and &#42;&#42;p &#60; 0&#46;05&#41; compared to HCB-treated cells&#46;</p>"
        ]
      ]
      7 => array:7 [
        "identificador" => "f0040"
        "etiqueta" => "Figure 8"
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            "Tamanyo" => 500907
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        "descripcion" => array:1 [
          "en" => "<p id="sp0040" class="elsevierStyleSimplePara elsevierViewall">Role of TGF-&#946;1 in statins-induced antiproliferative effect&#46; Lane1&#58; Hep-G2 cells treated with ETOH&#44; lane 2&#58; Hep-G2 cells treated with HCB &#40;5 &#956;M&#41;&#44; lane 3&#58; Hep-G2 cells treated with conditioned medium &#40;CM&#41;&#44; lane 4&#58; Hep-G2 cells pre-treated with AT &#40;30 &#956;M&#41;&#44; lane 5&#58; Hep-G2 cells pre-treated with AT &#43; HCB&#44; lane 6&#58; Hep-G2 cells pre-treated with AT &#43; CM&#44; lane 7&#58; Hep-G2 cells treated with ETOH&#44; lane 8&#58; Hep-G2 cells treated with HCB &#40;5 &#956;M&#41;&#44; lane 9&#58; Hep-G2 cells treated with TGF-&#946; 1&#44; lane 10&#58; Hep-G2 cells treated with an exogenous TGF-&#946; 1 inhibitor&#44; lane 11&#58; Hep-G2 cells pre-treated with TGF-&#946;1 inhibitor &#43; TGF-&#946; 1&#44; lane 12&#58; Hep-G2 cells pre-treated with TGF-&#946; 1 &#43; AT &#40;30 &#956;M&#41; and lane 13&#58; Hep-G2 cells pre-treated with AT and TGF-&#946; 1 inhibitor and after with HCB and TGF-&#946; 1&#46;</p>"
        ]
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      8 => array:7 [
        "identificador" => "f0045"
        "etiqueta" => "Figure 9"
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        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr9.jpeg"
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        "descripcion" => array:1 [
          "en" => "<p id="sp0045" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Analysis of the expression of c-Src by Western blot&#46; Hep-G2 cells were pretreated with AT &#40;30 &#956;M&#41; or SM &#40;20 &#956;M&#41; for 3 h and then HCB &#40;5 &#956;M&#41; for 24 h&#46; &#946;-actin was used as charge control&#46; The results of the bands optical analysis are shown in the bottom panel &#42;p</span> &#8804; <span class="elsevierStyleItalic">0&#46;05&#44; &#42;&#42;&#42;p</span> &#8804; <span class="elsevierStyleItalic">0&#46;01 respect HCB&#46;</span></p>"
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      9 => array:7 [
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        "etiqueta" => "Figure 10"
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            "imagen" => "gr10.jpeg"
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          "en" => "<p id="sp0050" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Analysis of the expression of mRNA DI by RT-PCR&#46; Hep-G2 cells were pretreated with AT&#40;30 &#181;M&#41; or SM &#40;20 &#181;M&#41; for 3 h and then HCB &#40;5 &#181;M&#41; for 24 h&#46; L-19 was used as charge control&#46; The results of the bands optica analysis are shown in the bottom panel &#42;&#42;p</span> &#8804; <span class="elsevierStyleItalic">0&#46;05 respect HCB&#46;</span></p>"
        ]
      ]
    ]
    "bibliografia" => array:2 [
      "titulo" => "References"
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                  "contribucion" => array:1 [
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                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
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              "identificador" => "bib0050"
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                0 => array:2 [
                  "contribucion" => array:1 [
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                      "titulo" => "Combination Therapy of Metformin and Statin May Decrease Hepatocellular Carcinoma Among Diabetic Patients in Asia"
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                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "Chen H&#46;H&#46;"
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                  "contribucion" => array:1 [
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Article information

ISSN: 16652681
Original language: English

DOI:10.5604/01.3001.0011.7394

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	4	0	4
2024 October	11	8	19
2024 September	13	2	15
2024 August	12	7	19
2024 July	11	0	11
2024 June	13	2	15
2024 May	39	2	41
2024 April	25	4	29
2024 March	19	3	22
2024 February	13	4	17
2024 January	9	5	14
2023 December	10	3	13
2023 November	12	3	15
2023 October	18	4	22
2023 September	9	2	11
2023 August	7	1	8
2023 July	13	4	17
2023 June	19	2	21
2023 May	49	1	50
2023 April	40	3	43
2023 March	34	2	36
2023 February	32	2	34
2023 January	29	6	35
2022 December	12	3	15
2022 November	20	7	27
2022 October	13	10	23
2022 September	24	11	35
2022 August	26	8	34
2022 July	25	8	33
2022 June	57	5	62
2022 May	26	11	37
2022 April	29	6	35
2022 March	28	11	39
2022 February	23	5	28
2022 January	44	5	49
2021 December	35	11	46
2021 November	27	7	34
2021 October	40	17	57
2021 September	13	7	20
2021 August	19	4	23
2021 July	44	10	54
2021 June	26	4	30
2021 May	17	9	26
2021 April	27	13	40
2021 March	10	5	15
2021 February	10	5	15
2021 January	25	5	30
2020 December	24	11	35
2020 November	18	6	24
2020 October	17	4	21
2020 September	15	10	25
2020 August	18	5	23
2020 July	30	10	40
2020 June	4	1	5
2020 May	7	6	13
2020 April	3	2	5
2020 March	7	2	9
2020 February	7	3	10
2020 January	5	4	9
2019 December	5	4	9
2019 November	3	1	4
2019 October	4	4	8
2019 September	8	1	9
2019 August	5	3	8
2019 July	2	4	6
2019 June	6	10	16
2019 May	11	9	20

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