SEARCH

SEARCH BY CITATION

Keywords:

  • 17β-estradiol;
  • estrogen receptor;
  • hepatitis C virus;
  • sex difference

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh-7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β-estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E2, but not P4, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E2–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα-selective agonist 4, 4′, 4″- (4-propyl-[1H]-pyrazole-1, 3, 5-triyl)trisphenol (PPT), but not with the ERβ-selective agonist 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN) or the G protein-coupled receptor 30 (GPR30)-selective agonist 1-(4-[6-bromobenzo 1, 3 dioxol-5-yl]-3a, 4, 5, 9b-tetrahydro-3H-cyclopenta [c] quinolin-8-yl)-ethanone (G-1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E2, inhibits the production of HCV infectious particles in an ERα–dependent manner.

List of Abbreviations: 
DMEM

Dulbecco's modified Eagle's medium

DMSO

dimethyl sulfoxide

DPN

2, 3-bis (4-hydroxyphenyl)-propionitrile

E2

17β-estradiol

ER

estrogen receptor

G-1

1-(4-[6-bromobenzo 1, 3 dioxol-5-yl]-3a, 4, 5, 9b-tetrahydro-3H-cyclopenta [c] quinolin-8-yl)-ethanone

GPR30

G protein-coupled receptor 30

HCV

hepatitis C virus

P4

progesterone

PPT

4, 4′, 4″- (4-propyl-[1H]-pyrazole-1, 3, 5-triyl)trisphenol

SEM

standard error of the mean

HCV, an enveloped RNA virus which belongs to the genus Hepacivirus within the family Flaviviridae, prevails in most parts of the world with an estimated number of about 170 million carriers; hence HCV infection is a major global health-care problem (1). Persistent infection with HCV causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma (2, 3). In the USA, the prevalence of anti-HCV antibodies is twice as high in men as in women (4). The male gender is thought to be one of the critical factors in progression of hepatic fibrosis in chronic HCV infection (5, 6). It has also been reported that progression of hepatic fibrosis is faster in postmenopausal than in premenopausal women, and that hormone replacement therapy with estrogen and progesterone significantly delays progression of hepatic fibrosis in postmenopausal women (6, 7). This potential innate resistance of premenopausal women to hepatic fibrosis may be attributed to female hormones, such as estrogens and progesterone. In fact, E2, the most potent physiological estrogen, has been reported to suppress the progression of liver fibrosis and hepatocarcinogenesis (8, 9). Moreover, women are more likely than men to clear HCV in the acute phase of infection, even within a few months after infection (10). These observations imply the possibility that female hormones inhibit HCV infection, either at the level(s) of virus attachment/entry, virus RNA replication, virus protein synthesis or production of infectious virus particles (virions).

Estrogens utilize three kinds of ER; ERα, ERβ and GPR30 (11–15). Specific agonists and antagonists of ER are available and widely used to examine the roles of estrogens. In the present study, we examined the possible effects of female hormones, especially E2 and P4, on HCV RNA replication, protein synthesis and virion production in cultured cells.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Cell culture and virus infection

A human hepatoma-derived cell line, Huh-7.5, which is highly permissive to HCV RNA replication (16), was kindly provided by Dr. C. M. Rice (The Rockefeller University, New York, NY, USA). The cells were maintained in phenol red-free DMEM (Sigma–Aldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated and charcoal-stripped FBS (Israel Beit Haemek, Haemek, Israel), 0.1 mM non-essential amino acids (Invitrogen, Carlsbad, CA, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin (Invitrogen).

The pFL-J6/JFH1 plasmid that encodes the entire viral genome of a chimeric strain of HCV-2a, J6/JFH1 (17) was kindly provided by Dr. C. M. Rice. A cell culture-adapted mutant derived from J6/JFH1 (P-47 strain) (18, 19) was used for infection experiments. The virus was inoculated into Huh-7.5 cells at a multiplicity of infection of 1.0 and incubated for 2 hr. After the residual virus had been removed by washing, the cells were cultured in the presence or absence of female hormones, and agonists and an antagonist of estrogen receptors (see below). Culture supernatants were collected at 0, 1, 2 and 3 days postinfection and virus titers were determined, as described below.

Virus titration

Culture supernatants containing HCV were serially diluted 10-fold in DMEM and inoculated into Huh-7.5 cells (2 × 105 cells per well in a 24-well plate). After incubation at 37°C for 6 hr, the cells were fed with fresh DMEM. At 24 hr postinfection, the cells were fixed with ice-cold methanol, blocked with 5% goat serum in PBS and subjected to immunofluorescence analysis using mouse monoclonal antibody against the HCV core protein (2H9) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L, Molecular Probes, Eugene, OR, USA). Hoechst 33342 (Molecular Probes) was used for counterstaining of the nuclei. HCV-positive foci were counted under a fluorescent microscope (BX51; Olympus, Tokyo, Japan) and virus titers were expressed as focus-forming units per ml, as reported previously (18, 19).

Chemicals

E2 and P4 were purchased from Sigma–Aldrich (St Louis, MO, USA). ICI182780 (an antagonist of ERα and ERβ), PPT (an ERα-selective agonist) (20) and DPN (an ERβ-selective agonist) (21) were purchased from Tocris Bioscience (Bristol, UK). G-1 (a GPR30-selective agonist) (22) was purchased from Calbiochem (Darmstadt, Germany). DMSO, which was used as a solvent, was obtained from Wako Pure Chemical Industries (Osaka, Japan). The concentrations of E2 and P4 used in this study were 0.4 μM and 3 μM, respectively, which correspond to the estimated highest concentrations in the sera of pregnant women. ICI182780 was used at a concentration of 1 μM, PPT and DPN at 0.1, 1 and 10 μM, and G-1 at 0.1 and 1 μM. As G-1 has been reported to lose its GPR30-binding specificity at concentrations over 1 μM, a concentration of 10 μM for G-1 was not tested. The final concentration of DMSO as a control never exceeded 0.01%.

Cell viability assay

Cells plated on 96-well microtiter plates (2.0 × 104 cells/well) were inoculated with HCV and treated with E2, P4 or DMSO. The cell viability in each well was determined by WST-1 assay (Roche Diagnostics, Mannheim, Germany) until 3 days postinfection.

Real-time quantitative RT-PCR

Total cellular RNA was isolated using the RNAiso reagent (Takara Bio, Kyoto, Japan) and cDNA was generated using the QuantiTect Reverse Transcription system (Qiagen, Valencia, CA, USA). Real-time quantitative PCR was performed on a SYBR Premix Ex Taq (Takara Bio) using SYBR green chemistry in ABI PRISM 7000 (Applied Biosystems, Foster, CA, USA). Primer sets used in this study are shown below: HCV NS5B, 5′-ACCAAGCTCAAACTCACTCCA-3′ and 5′-AGCGGGGTCGGGCAC GAGACA-3′ (23); β-actin, 5′-GCGGGAAATCGTGCGTGACATT-3′ and 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′.

Immunoblotting

Cells were solubilized in lysis buffer as reported previously (18, 19). The cell lysates were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated with mouse monoclonal antibodies against HCV NS3 (Chemicon International, Temecula, CA, USA), followed by incubation with peroxidase-conjugated goat anti-mouse IgG (Medical & Biological Laboratories Co. Ltd., Nagoya, Japan). The positive bands were visualized by using ECL detection system (GE Healthcare UK, Buckinghamshire, UK).

Statistical analysis

Results were expressed as mean ± SEM. Statistical significance was evaluated by one-way analyses of variances.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

E2 inhibits HCV virion production, but not HCV RNA replication or HCV protein synthesis

We first examined the effect of E2 or P4 treatment on HCV virion production. At 2 hr after virus inoculation, the HCV-infected Huh-7.5 cells were treated with E2 (0.4 μM) or P4 (3 μM) for 3 days. Culture supernatants were collected every day and titrated for viral infectivity. As shown in Figure 1a, E2 treatment significantly suppressed HCV virion production at 2 and 3 days postinfection, whereas treatment with P4 did not. The same treatment (E2 or P4) did not exert significant cytotoxicity (Fig. 1b). Next, we examined the effect of E2 on HCV RNA replication and HCV protein synthesis under the same experimental conditions. We found that HCV RNA replication and HCV protein synthesis in both HCV-infected cells and HCV RNA replicon-harboring cells (23) were all unaffected by treatment with E2 or P4 (Fig. 2a–c). Moreover, treatment of the cells with E2 either prior to, or during, virus inoculation did not significantly inhibit HCV virion production (Fig. 3a). These results collectively suggest that E2 inhibits HCV virion production, but not at the level of virus entry, RNA replication or protein synthesis. We also observed that E2–mediated inhibition of HCV virion production occurs in a dose-dependent manner (Fig. 3b).

image

Figure 1. Effects of E2 and P4 on HCV virion production and cell growth. (a) HCV virion production. Huh-7.5 cells were inoculated with HCV at a multiplicity of infection of 1.0, incubated for 2 hr, and cultured for 0, 1, 2 and 3 days after virus infection. The HCV-infected cells were treated with E2 (0.4 μM), P4 (3 μM) or DMSO (control) from 2 hr postinfection to sampling time (days 1, 2 and 3). The culture supernatants of HCV-infected cells were assayed for virus infectivity. Data are shown as mean ± SEM. (b) Cell growth. HCV-infected cells were treated with E2, P4 or DMSO (control) from 2 hr to 3 days postinfection. Cell growth in each culture was determined by WST-1 assay. Data are shown as mean ± SEM.

Download figure to PowerPoint

image

Figure 2. Effects of E2 and P4 on HCV RNA replication and HCV protein synthesis. (a) HCV RNA replication. Huh-7.5 cells were inoculated with HCV at a multiplicity of infection of 1.0, incubated for 2 hr, and cultured for 0, 1, 2 and 3 days after virus infection. The HCV-infected cells were treated with E2 (0.4 μM) or DMSO (control) from 2 hr to sampling time (days 1, 2 and 3). HCV RNA replication levels were determined by real-time quantitative RT-PCR and normalized with β-actin mRNA levels. Data are shown as mean ± SEM. (b) Huh-7.5 cells harboring a full-genomic HCV RNA replicon (23) were treated with E2 (0.4 μM) or DMSO, and HCV RNA replication levels determined as in (a). (c) HCV protein synthesis. HCV-infected cells were treated with E2 or DMSO as in (a) and the amount of HCV protein synthesis determined by immunoblot analysis using anti-NS3 antibody. The degree of β-actin expression as determined by anti-β-actin antibody served as a control. dpi, days postinfection.

Download figure to PowerPoint

image

Figure 3. Kinetic analysis of E2–mediated inhibition of HCV virion production. (a) Time-of-addition experiment. Huh-7.5 cells were inoculated with HCV at a multiplicity of infection of 1.0, incubated for 2 hr, and cultured up to 2 days after virus infection. Treatment of the cells with E2 (0.4 μM) was performed before or during virus inoculation for 2 hr, or after virus inoculation until sampling time (day 2). The culture supernatants of HCV-infected cells were assayed for viral infectivity. Data are shown as mean ± SEM. *P < 0.05, compared with DMSO control. (b) Dose–dependency experiment. Huh-7.5 cells were inoculated with HCV as in (a). The HCV-infected cells were treated with various concentrations of E2 (0.4 nM to 0.4 μM]) from 2 hr postinfection to sampling time (day 2). The culture supernatants of HCV-infected cells were assayed for viral infectivity. Data are shown as mean ± SEM. *P < 0.05, compared with DMSO control.

Download figure to PowerPoint

A nuclear estrogen receptor antagonist, ICI182780, abolishes E2-mediated inhibition of HCV virion production

We hypothesized that E2 signaling through nuclear ER (ERα and ERβ) was involved in the E2–mediated inhibition of HCV virion production. To test this possibility, we used ICI182780 (1 μM), an antagonist of ERα and ERβ. The results clearly demonstrated that treatment of cells with ICI182780 abolished E2–mediated inhibition of HCV virion production (Fig. 4).

image

Figure 4. Effects of ER antagonist, ICI182780, on HCV virion production. Huh-7.5 cells were inoculated with HCV at a multiplicity of infection of 1.0, incubated for 2 hr, and cultured for 0, 1, 2 and 3 days after virus infection. The HCV-infected cells were treated with E2 (0.4 μM) and/or ICI182780 (1 μM) or DMSO (control) from 2 hr postinfection to sampling time (days 1, 2 and 3). The culture supernatants of HCV-infected cells were assayed for virus infectivity. Data are shown as mean ± SEM. *P < 0.05, compared with DMSO control.

Download figure to PowerPoint

Estrogen receptor-α-selective agonist 4, 4′, 4″- (4-propyl-[1H]-pyrazole-1, 3, 5-triyl) trisphenol inhibits HCV virion production

To determine which estrogen receptor(s) is/are involved in the E2–mediated down-regulation of HCV virion production, we used receptor-specific agonists, such as PPT (an ERα-selective agonist) (20), DPN (an ERβ-selective agonist) (21) and G-1 (a GPR30-selective agonist) (22). Treatment of cells with PPT (10 μM), but not with DPN (10 μM) or G-1 (1 μM), significantly inhibited HCV virion production (Fig. 5). PPT treatment at a concentration of 1 μM also brought about a weak, but significant, inhibition of HCV virion production at 2 days postinfection. On the other hand, PPT did not mediate significant cytotoxicity at the concentrations tested (data not shown).

image

Figure 5. Effects of ER-specific agonists on HCV virion production. Huh-7.5 cells were inoculated with HCV at a multiplicity of infection of 1.0, incubated for 2 hr, and cultured for 0, 1, 2 and 3 days after virus infection. The HCV-infected cells were treated with PPT (ERα-selective agonist; 1 and 10 μM), DPN (ERβ-selective agonist; 10 μM) or G-1 (GPR30-selective agonist; 1 μM) from 2 hr postinfection to sampling time (days 1, 2 and 3). The culture supernatants of HCV-infected cells were assayed for viral infectivity. Data are shown as mean ± SEM. *P < 0.05, compared with DMSO control.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We have demonstrated in the present study that treatment of Huh-7.5 cells with E2 inhibits HCV virion production, but not HCV RNA replication or HCV protein synthesis (Figs 1 and 2). Treatment of the cells with E2 either prior to, or during, virus inoculation did not significantly suppress HCV virion production (Fig. 3a). These results collectively suggest that E2 inhibits HCV infection at the virion assembly/secretion level, but not at the level of virus attachment/entry, virus RNA replication or virus protein synthesis. E2 has been reported to possess antioxidant and anti-apoptotic activities in fibrotic liver and cultured hepatocytes (24, 25). It should be noted, however, that E2 did not exert anti-apoptotic or cytotoxic (pro-apoptotic) effect under our experimental conditions (Fig. 1b). In contrast to E2, another female hormone, P4, did not significantly affect HCV virion production (Fig. 1a).

E2-mediated inhibition of HCV virion production was abolished by a nuclear ER (ERα and ERβ) antagonist, ICI182780 (Fig. 4), this result suggesting that suppression of HCV virion production may be induced by ER signal transduction. Three types of ER have been reported so far; ERα, ERβ and GPR30 (11–15). To determine which ER is involved in the suppression of HCV virion production, we used ER-specific agonists, PPT (for ERα) (20), DPN (for ERβ) (21) and G-1 (for GPR30) (22). We found that PPT, but not DPN or G-1, inhibits the production of HCV infectious particles (Fig. 5), suggesting that ERα plays an important role in the inhibition of HCV virion production. It has been reported that, in hepatocytes, ERα constitutes a minor proportion of the total ER, and that an estrogen-mediated anti-apoptotic effect is mediated principally through ERβ (26). However, the importance of ERα–mediated signal transduction should not be ignored. The rationale for this assertion is that ERα is known to be involved in lipid metabolism (27), that certain lipid metabolism disorder(s) possibly result(s) in abnormal accumulation of lipid droplets, and that such an accumulation is required for HCV virion maturation in virus-infected cells (27), that certain lipid metabolism disorder(s) possibly result(s) in abnormal accumulation of lipid droplets, and that such an accumulation is required for HCV virion maturation in virus-infected cells (28). Also, we should not yet exclude the possible importance of ERβ and GPR30, because they may not be expressed at a sufficient level in the Huh7.5 cell line maintained in our laboratory.

Other relevant observations are that ERα interacts with HCV NS5B, the viral RNA polymerase, and promotes association of NS5B with the replication complex in human hepatoma-derived Huh-7 cells, and that tamoxifen, a competitive inhibitor of estrogens, suppresses the ERα–mediated association of NS5B with the replication complex, thereby inhibiting HCV RNA replication (29). Similarly, E2 binding to ERα may abrogate its interaction with NS5B. However, in our experiments we did not observe E2–mediated inhibition of HCV RNA replication (Fig. 2a,b). We therefore assume that E2 inhibits HCV virion production through a mechanism other than E2– ERα–NS5B interactions. Further study is needed to elucidate this issue.

In conclusion, the most potent physiological estrogen, E2, inhibits production of HCV infectious particles in Huh-7.5 cell cultures in an ERα–dependent manner. This may explain, at least in part, why the incidence of HCV-associated liver disease is lower in premenopausal women than in postmenopausal women and men.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The authors are grateful to Dr. C. M. Rice for providing Huh7.5 cells and pFL-J6/JFH1. Thanks are also due to Dr. T. Adachi for his technical advice. This study was supported in part by Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare, Japan, and the Japan Science and Technology/Japan International Cooperation Agencies’ Science and Technology Research Partnership for Sustainable Development. This study was also carried out as part of the Japan Initiative for Global Research Network on Infectious Diseases, Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Global Center of Excellence Program at Kobe University Graduate School of Medicine.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  • 1
    Shepard C.W., Finelli L., Alter M.J. (2005) Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 5: 55867.
  • 2
    Alberti A., Benvegnù L., Boccato S., Ferrari A., Sebastiani G. (2004) Natural history of initially mild chronic hepatitis C. Dig Liver Dis 36: 64654.
  • 3
    Davis G.L., Alter M.J., El-Serag H., Poynard T., Jennings L.W. (2010) Aging of hepatitis C virus (HCV)-infected persons in the United States: a multiple cohort model of HCV prevalence and disease progression. Gastroenterology 138: 51321.
  • 4
    Armstrong G.L., Wasley A., Simard E.P., McQuillan G.M., Kuhnert W.L., Alter M.J. (2006) The prevalence of hepatitis C virus infection in the United States, 1999 through 2002. Ann Intern Med 144: 70514.
  • 5
    Poynard T., Ratziu V., Charlotte F., Goodman Z., McHutchison J., Albrecht J. (2001) Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J Hepatol 34: 7309.
  • 6
    Massard J., Ratziu V., Thabut D., Moussalli J., Lebray P., Benhamou Y., Poynard T. (2006) Natural history and predictors of disease severity in chronic hepatitis C. J Hepatol 44: S1924.
  • 7
    Di Martino V., Lebray P., Myers R.P., Pannier E., Paradis V., Charlotte F., Moussalli J., Thabut D., Buffet C., Poynard T. (2004) Progression of liver fibrosis in women infected with hepatitis C: long-term benefit of estrogen exposure. Hepatology 40: 142633.
  • 8
    Shimizu I., Yasuda M., Mizobuchi Y., Ma Y.R., Liu F., Shiba M., Horie T., Ito S. (1998) Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats. Gut 42: 1129.
  • 9
    Yasuda M., Shimizu I., Shiba M., Ito S. (1999) Suppressive effects of estradiol on dimethylnitrosamine-induced fibrosis of the liver in rats. Hepatology 29: 71927.
  • 10
    Wang C.C., Krantz E., Klarquist J., Krows M., McBride L., Scott E.P., Shaw-Stiffel T., Weston S.J., Thiede H., Wald A., Rosen H.R. (2007) Acute hepatitis C in a contemporary US cohort: modes of acquisition and factors influencing viral clearance. J Infect Dis 196: 147482.
  • 11
    Hall J.M., Couse J.F., Korach K.S. (2001) The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276: 36,86972.
  • 12
    Gustafsson J.A. (2003) What pharmacologists can learn from recent advances in estrogen signaling. Trends Pharmacol Sci 24: 47985.
  • 13
    Revankar C.M., Cimino D.F., Sklar L.A., Arterburn J.B., Prossnitz E.R. (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307: 162530.
  • 14
    Thomas P., Pang Y., Filardo E.J., Dong J. (2005) Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146: 62432.
  • 15
    Maggiolini M., Picard D. (2010) The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol 204: 10514.
  • 16
    Blight K.J., McKeating J.A., Rice C.M. (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76: 1300114.
  • 17
    Lindenbach B.D., Evans M.J., Syder A.J., Wolk B., Tellinghuisen T.L., Liu C.C., Maruyama T., Hynes R.O., Burton D.R., McKeating J.A., Rice C.M. (2005) Complete replication of hepatitis C virus in cell culture. Science 309: 6236.
  • 18
    Bungyoku Y., Shoji I., Makine T., Adachi T., Hayashida K., Nagano-Fujii M., Ide Y.H., Deng L., Hotta H. (2009) Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells. J Gen Virol 90: 168191.
  • 19
    Deng L., Adachi T., Kitayama K., Bungyoku Y., Kitazawa S., Ishido S., Shoji I., Hotta H. (2008) Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway. J Virol 82: 10,37585.
  • 20
    Stauffer S.R., Coletta C.J., Tedesco R., Nishiguchi G., Carlson K., Sun J., Katzenellenbogen B.S., Katzenellenbogen J.A. (2000) Pyrazole ligands: structure-affinity/activity relationships and estrogen receptor-alpha-selective agonists. J Med Chem 43: 493447.
  • 21
    Meyers M.J., Sun J., Carlson K.E., Marriner G.A., Katzenellenbogen B.S., Katzenellenbogen J.A. (2001) Estrogen receptor-beta potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues. J Med Chem 44: 423051.
  • 22
    Bologa C.G., Revankar C.M., Young S.M., Edwards B.S., Arterburn J.B., Kiselyov A.S., Parker M.A., Tkachenko S.E., Savchuck N.P., Sklar L.A., Oprea T.I., Prossnitz E.R. (2006) Virtual and biomolecular screening converge on a selective agonist for GPR30. Nat Chem Biol 2: 20712.
  • 23
    Kasai D., Adachi T., Deng L., Nagano-Fujii M., Sada K., Ikeda M., Kato N., Ide Y.H., Shoji I., Hotta H. (2009) HCV replication suppresses cellular glucose uptake through down-regulation of cell surface expression of glucose transporters. J Hepatol 50: 88394.
  • 24
    Liu Y., Shimizu I., Omoya T., Ito S., Gu X.S., Zuo J. (2002) Protective effect of estradiol on hepatocytic oxidative damage. World J Gastroenterol 8: 3636.
  • 25
    Lu G., Shimizu I., Cui X., Itonaga M., Tamaki K., Fukuno H., Inoue H., Honda H., Ito S. (2004) Antioxidant and antiapoptotic activities of idoxifene and estradiol in hepatic fibrosis in rats. Life Sci 74: 897907.
  • 26
    Inoue H., Shimizu I., Lu G., Itonaga M., Cui X., Okamura Y., Shono M., Honda H., Inoue S., Muramatsu M., Ito S. (2003) Idoxifene and estradiol enhance antiapoptotic activity through estrogen receptor-beta in cultured rat hepatocytes. Dig Dis Sci 48: 57080.
  • 27
    Cooke P.S., Heine P.A., Taylor J.A., Lubahn D.B. (2001) The role of estrogen and estrogen receptor-alpha in male adipose tissue. Mol Cell Endocrinol 178: 14754.
  • 28
    Miyanari Y., Atsuzawa K., Usuda N., Watashi K., Hishiki T., Zayas M., Bartenschlager R., Wakita T., Hijikata M., Shimotohno K. (2007) The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9: 108997.
  • 29
    Watashi K., Inoue D., Hijikata M., Goto K., Aly H.H., Shimotohno K. (2007) Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B. J Biol Chem 282: 32,76572.