Insulin pathway proteins are down-regulated by JFH1.

<p><b>A</b>: Western blot from Huh7.5 cells in the presence or absence of insulin (20 nM) and infected or not with JFH-1 particles. 40 µgrams of total protein were loaded per lane. A representative gel is shown for each protein. All experiments were performed in triplicate. β-Actin was used as a loading control. <b>B</b>: IRS1 and PTP1B abundance at 24 and 48 hours post-infection. Huh7.5 cells were infected with JFH1 particles and incubated at the indicated time. Performance of western-blot was as indicated for panel <b>A</b>.</p

Interferon effect on insulin pathway gene expression.

<p>Huh7.5 cultures were treated with 500 IU/ml and infected with JFH-1 particles. Experiments were performed in triplicate. Control experiment (non-infected cells and no insulin added) was used as a reference (fold induction = 1). (*) = p<0.05.</p

PTP1B is highly induced in the presence of metformin and HCV.

<p>Gene expression (fold induction) of insulin pathway-related genes in Huh7.5 cells treated with metformin (2 mM) was analyzed. Experiments were performed in triplicate. (*) = p<0.05.</p

Insulin effect on gene expression in vitro.

<p>Relative gene expression (fold induction [+] and fold inhibition [−]) in Huh7.5 cells grown in the presence of insulin (10 nM) and JFH-1 particles (one particle per cell). Experiments were performed in triplicate. Control experiment (non-infected cells and no insulin added) was used as a reference (fold induction = 1). (*) = p<0.05.</p

Metformin (2 mM) inhibits viral replication in vitro.

<p>Huh7.5 cells treated with interferon (500 IU/ml) were used as a positive control for inhibition of viral replication (100%). Cells were infected with JFH1 and treated with metformin for 48 hours and 7 days. Metformin significantly inhibited viral replication (p<0.05).</p

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy

<div><p>Hepatitis C virus (HCV) infection has been related to increased risk of development of hepatocellular carcinoma (HCC) while metformin (M) and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metformin and simvastatin (S) could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP) and phosphatase and tensin homolog (PTEN) proteins while M inhibited mammalian target of rapamycin (mTOR) and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII) were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection <i>in vitro</i>. In human hepatocytes, metformin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.</p></div

Metformin and simvastatin effects on HCV infection.

<p><b>a</b>. Intracellular levels of negative strand of HCVRNA (%) in Huh7.5 cells infected with JFH-1and treated with simvastatin (SIM; 2μM), metformin (MET; 2mM) or in combination (SIM-MET; S: 2μM, M: 2mM), relative to complete infection (100% without any treatment). α-Interferon (500 IU/ml) was used as a positive control. <b>b</b>: Extracellular level of HCVRNA (%) measure in supernatant from Huh7.5 culture cells treated with simvastatin and metformin quantified by COBAS^® Taqman^® HCV test v2.0. <b>c</b>: core protein expression in Huh7.5 cells treated with simvastatin (2μM) or metformin (2mM) quantified by western-blot. <b>d</b>: core protein quantification using ChemiDoc^™ MP Imaging System. * p<0.05, ** p<0.01, ***p<0.001.</p

Cell viability of Huh7.5 cells treated with simvastatin and metformin.

<p><b>A</b>: Percentage of cell kinetic in Huh7.5 treated with metformin (M), simvastatin (S) or combination (S: 2μM, M: 2mM). Cell number was quantified by Neubauer chamber. <b>B</b>: Percentage of Huh7.5 cell treated with these treatments in different phases of cell cycle measured by FACS analysis. * p<0.05, ** p<0.01, ***p<0.001.</p

Autophagy modification in Huh7.5 cell and in primary hepatocytes.

<p><b>A</b>: MAPLC3B gene expression in Huh7.5 cells infected with JFH1 particles (1 particle/cell) and treated 24 hours post-infection with metformin (M: 2mM) alone or in combination with simvastatin (SIM-MET; S:2μM, M: 2mM). <b>B</b>: Western-blot analysis of autophagy markers in control or JFH1 infected in Huh 7.5 cells treated with simvastatin (SIM; 2μM), metformin (MET; 2mM) or in combination (SIM-MET; S:2μM, M: 2mM) over 72 hours. All gels have been run under the same experimental conditions. <b>C</b>: Protein expression of autophagy markers in human primary hepatocytes treated with simvastatin (2μM) or metformin (2mM) quantified by western-blot of three different donors. <b>D</b>: Protein quantification of Huh7.5 using ChemiDoc^™ MP Imaging System. <b>E</b>: Protein quantification of human primary hepatocytes using ChemiDoc^™ MP Imaging System. <b>F</b>: p62 protein expressions in Huh7.5 cells treated with three different concentration of metformin (M2: 2mM, M5: 5mM and M10; 10mM). * p<0.05, ** p<0.01, ***p<0.001.</p

mTOR pathway modification in Huh7.5 cell and in primary hepatocytes.

<p><b>A</b>: PTP1B gene expression in Huh7.5 cells infected with JFH1 particles (1 particle/cell) and treated 24 hours post-infection with simvastatin (S: 2μM) alone or in combination with metformin (SIM-MET; S:2μM, M: 2mM). <b>B</b>: Gene expression of mTOR pathway in Huh7.5 cells infected with JFH1 particles and treated at 24 hours post-infection with metformin (M: 2mM) alone or in combination with simvastatin. <b>C</b>: Protein expression by western-blot of mTOR pathway in control or JFH1 infected in Huh 7.5 cells treated with simvastatin (SIM; 2μM), metformin (MET; 2mM) or in combination (SIM-MET; S:2μM, M: 2mM) over 72 hours. All gels have been run under the same experimental conditions. <b>D</b>: Protein quantification of Huh7.5 using ChemiDoc^™ MP Imaging System. <b>E</b>: Protein expression of the mTOR pathway in human primary hepatocytes treated with simvastatin (2μM) or metformin (2mM) quantified by western-blot of three different donors. <b>F</b>: Protein quantification of human primary hepatocytes using ChemiDoc^™ MP Imaging System. * p<0.05, ** p<0.01, ***p<0.001.</p