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Research Article
Anti-viral effect of a compound isolated from Liriope platyphylla against
hepatitis B virus in vitro
Tsurng-Juhn Huanga*, Yu-Chi Tsai h, Shang-Yu Chiang h, Guei-Jane Wang b, c, d,
Yu-Cheng Kuoe, f, Yi-Chih Changg, Yi-Ying Wug, Yang-Chang Wuh, i, j, k*
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Department of Biological Science and Technology and Research Center for
Biodiversity, China Medical University, Taichung 404, Taiwan
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Graduate Institute of Clinical Medical Science, China Medical University, Taichung
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404, Taiwan
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Department of Medical Research, China Medical University Hospital, Taichung 404,
Taiwan
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University, Taichung 404, Taiwan
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Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung
807, Taiwan
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School of Pharmacy, College of Pharmacy, China Medical University, Taichung
404,Taiwan
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Chinese Medicine Research and Development Center, China Medical University
Hospital, Taichung 404, Taiwan
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Center for Molecular Medicine, China Medical University Hospital, Taichung 404,
Taiwan
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Correspondence should be addressed to:
Tsurng-Juhn Huang, Ph.D
TEL: +886 4 22053366 ext. 8105
FAX: +886 4 22071507
E-mail: tjhuang@mail.cmu.edu.tw
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413,
Taiwan
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Department of Radiation Oncology, China Medical University Hospital, Taichung,
Taiwan
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Department of Biomedical Imaging and Radiological Science, China Medical
University, Taichung 404, Taiwan
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Department of Medical Laboratory Science and Biotechnology, China Medical
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Co-correspondence should be addressed to:
Yang-Chang Wu, Ph.D
TEL: +886 4 22053366 ext. 1012
FAX: +886 4 22060248
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E-mail: yachwu@mail.cmu.edu.tw
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Abstract
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The compound LPRP-Et-97543 was isolated from Liriope platyphylla roots and was
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observed to have potential anti-viral effects in HepG2.2.15 cells against hepatitis B
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virus (HBV). The antiviral mode was further clarified, and the HBV-transfected Huh7
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cells were used as the platform. During viral gene expression, LPRP-Et-97543
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treatment had apparent effects on the viral precore/pregenomic and S/preS RNA.
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Promoter activity analysis demonstrated that LPRP-Et-97543 significantly reduced
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Core, S, and preS but not X promoter activities. Further examination showed that
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putative signaling pathways were involved in this inhibitory effect, indicating that
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NF-B may serve a putative mediator of HBV gene regulation with LPRP-Et-97543.
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In addition, the nuclear expression of p65/p50 NF-B member proteins was
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attenuated with LPRP-Et-97543 and augmented cytoplasmic IB protein levels but
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without affecting the expression of these proteins in HBV non-transfected cells during
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treatment. Moreover, LPRP-Et-97543 reduced the binding activity of NF-B protein
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to CS1 element of HBV surface gene in a gel retardation analysis and inhibited CS1
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containing promoter activity in HBV expressed cells. However, HBV transfection
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significantly enhances CS1 containing promoter activity without compound treatment
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in cells. Finally, transfection of the p65 expression plasmid significantly reversed the
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inhibitory effect of LPRP-Et-97543 on the replicated HBV DNA level in HBV
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positive cells. In conclusion, this study suggests that the mechanism of HBV
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inhibition by LPRP-Et-97543 may involve the feedback regulation of viral gene
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expression and viral DNA replication by HBV viral proteins, which interferes with the
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NF-B signaling pathway.
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Keywords: Liriope platyphylla, hepatitis B virus, NF-B, HepG2.2.15, Huh7
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1. Introduction
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Hepatitis B virus (HBV) is a causative agent that often leads to acute and chronic
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infections in humans (Gitlin, 1997). Liver damage occurs in approximately 80%
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chronic HBV carriers, which may lead to liver cirrhosis and hepatocellular carcinoma
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(HCC) (Park et al., 2006). Despite the development of a recombinant vaccine, which
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provides immunity against the HBV and the use of a specific Hepatitis B
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immunoglobulin in susceptible populations, there are still more than 400 million
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chronic HBV carriers (Gish, 2005).While interferons and nucleotide analogs have
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been widely used to treat chronically infected patients, the rapid development of
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resistance and undesirable side effects associated with this treatment makes alternate
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treatments necessary. Recent studies have been performed to determine how the viral
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gene is regulated and controlled to ameliorate HBV. This is a crucial issue for
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antiviral strategies (Huang et al., 2014a) because the focus is not on viral genome
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replication but on curing hepatitis B. Thus, continued development of new antiviral
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agents with novel targets and mechanisms are urgently required to eradicate HBV in
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chronic carriers.
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The large repertoire of herbal compounds may be the essential ingredients
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required for developing new drugs that relieve the threat of HBV and its associated
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liver diseases (Bent, 2008; Bent and Ko, 2004). At present, more that 80% of the
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population in developing countries uses alternative or traditional medical resources.
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Systematic methodologies have been developed to determine the active compounds in
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traditional Chinese herbal medicine because some of the herbal plants exhibit potent
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anti-viral effects (Li and Peng, 2013).
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Plant species belonging to the Liriope genus were reported to have a broad
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spectrum of biological activities, such as anti-inflammatory (Hu et al., 2011),
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antibacterial (Kim et al., 2002), anticancer (Sun et al., 2010; Wang et al., 2013; Wang
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et al., 2011), antidiabetic (Bai et al., 2009b), neuroprotective (Hur et al., 2004),
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oestrogenic, anti-platelet (Tsai et al., 2013), and hepatoprotective (Wu et al., 2001)
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functions. Recent studies showed the root extract from Liriope platyphylla reduces
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inflammation and alleviates symptoms from metabolic and neurodegenerative
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disorders, such as diabetes and pathological obesity (Bai et al., 2009a; Chen et al.,
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2009; Choi et al., 2011; Lee et al., 2005). Bioactivity-guided fractionation and
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purification of L. platyphylla can be used to examine its antiviral effects. In this study,
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an active compound of L. platyphylla root part extract (LPRP), named
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LPRP-Et-97543, and its anti-hepatitis B virus activities was examined. Further
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evaluation of LPRP-Et-97543 was performed by transfecting it into the HBV genome
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using a hepatoma cell.
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2. Materials and Methods
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2.1. Materials
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The anti-preSSclonesurface sc-23944, anti-HBcAg (13A9 clone) (sc-23946),
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anti-NF-B (p65/p50)(sc-109/sc-7178) and anti-IB (sc-371) antibodies were used
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as the HB antibodies and purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
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USA). Proliferating cell nuclear antigen (PCNA)(PC10 clone)(#2586) antibody was
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purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). The DIG high
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prime DNA labeling and detection starter kit was obtained from Roche (Mannheim,
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Germany) while the Power SYBR Green PCR master mix was purchased from
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Applied Biosystem (Foster City, CA, USA). The QIAquick Gel extraction kit was
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purchased from Qiagen (Valencia, CA, USA) and the dual-Luciferase reporter assay
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kit was purchased from Promega (Madison, WI, USA). The Trizoltotal RNA
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isolation solution and the Lipofectamine 2000 transfection reagent were from
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Invitrogen (Carlsbad, CA, USA). Lamivudine (2′, 3′-dideoxy-3′-thiacytidine,
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commonly known as 3TC) and an anti--tubulin antibody were purchased from
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Sigma (St Louis, MO, USA). pNF-B-Luc, p-activator protein (AP)-1-Luc,
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pAP2-Luc and p-interferon-sensitive response element (ISRE)-Luc
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promoter-luciferase reporter constructs from Stratagene (La Jolla, CA, USA) were
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provided by Dr. Cheng-Wen Lin (Department of Medical Laboratory Science and
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Biotechnology, China Medical University, Taichung, Taiwan). The pCMV-p65
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expression plasmid was provided by Dr. Chen-Kung Chou (Department of
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Biomedical Sciences, Chang Gung University, Tao-Yuan, Taiwan)
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2.2.Plant material
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In August 2009, Liriope platyphylla was collected from Taichung County, Taiwan,
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and the plant was identified by Dr. Ming-Hong Yen from the Graduate Institute of
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Natural Products (GINP), Kaohsiung Medical University, Kaohsiung, Taiwan. A
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voucher specimen (LP002) was deposited at GINP. The roots were air-dried at room
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temperature and crushed using a mill (Rong Tsong, Taichung, Taiwan) before
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extraction.
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2.3.Extraction and isolation of bioactive compound
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The roots of L. platyphylla (3.5 kg) were extracted with 95% ethanol (EtOH) at room
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temperature for 24 h; this process was repeated two more times so that the roots were
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extracted at room temperature for a total of 72 h. The ethanolic extract (654.6 g) was
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concentrated in vacuo, suspended in water and partitioned into n-hexane, 70% EtOH
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in an aqueous solution and n-butanol (BuOH), respectively. The aqueous EtOH layer
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(28.5 g) was subjected to silica gel column chromatography (CC) and successively
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eluted with n-hexane:CH2Cl2 (90:10 – 0:100) and CH2Cl2:MeOH (100:0 – 80:20)
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mobile phase system to afford 19 fractions. Fraction nine (1140.6 mg) was subjected
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to silica gel CC and eluted with CH2Cl2:MeOH (98:2) to yield nine subfractions.
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Compound LPRP-Et-97543 (6.8 mg) was purified using a HPLC with MeOH:H2O
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(75:25) solvent system. The structure of LPRP-Et-97543 was established as
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(3R)-3-(4'-hydroxybenzyl)-5,7-dihydroxy-6-methyl-chroman-4-one by spectroscopic
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analysis and was compared with the reported physical data (Nguyen et al., 2006; Tsai
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et al., 2013). Prior to treatment, LPRP-Et-97543 was dissolved in dimethyl sulfoxide
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(DMSO). The final DMSO concentration did not exceed 0.5%, and it did not cause
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any detectable effects during treatment.
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2.4.Cell culture
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HBV stably expressed cells were generated (in 1987) by transfecting HepG2 cells
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with the head-to-tail dimer of HBV to generate a stable cell line (HepG2.2.15) (Sells
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et al., 1987) and were maintained in MEM with heat-inactivated 10% fetal bovine
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serum (FBS) , 1% antibiotics and G418 (300 g/ml). In parallel experiments, human
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Huh7 hepatoma cells were maintained in DMEM supplemented with heat-inactivated
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10% FBS and 1% antibiotics. HepG2.2.15 and Huh7 cells were both grown at 37C in
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a humidified atmosphere of 5% CO2 and 95% air.
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2.5.Cell viability assay
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The cytotoxic effects of LPRP-Et-97543 was determined by a CellTiter 96 AQueous
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one solution cell proliferation assay kit (MTS) (Promega, Madison, WI, USA) to
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pinpoint the non-toxic test compound concentration in HepG2.2.15 and Huh7 cells. In
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brief, HepG2.2.15 or HBV-transfected Huh7 cells were plated into 96-well plates at a
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density of 4  104cells /ml for 24 h. The cells were then treated with serial dilutions of
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LPRP-Et-97543, which ranged from 2.5–320 g/ml, and the mixture was incubated
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for 3 days. Cell toxicity was measured according to the manufacturer’s protocol. All
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measurements were performed in four replicates and the results are presented as
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relative percentages over that of the control group.
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2.6.Determination of HBsAg and HBeAg
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After treating the HepG2.2.15 cells or HBV-transfected Huh7 cells, the levels of the
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viral surface (HBsAg) and e (HBeAg) antigens were measured in the culture media
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using an enzyme immunoassay (EIA) kit (Johnson and Johnson, Skillman, NJ, USA)
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according to the manufacturer’s instructions.
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2.7.Plasmid construction
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All of the constructs were produced using standard recombinant DNA techniques
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(Sambrook et al., 1989). The pHBV1.2 plasmid containing the 1.2-fold of HBV adw2
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serotype genome (nt 2186-1986) was cloned into the DraI and BglII site of
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pGEM-7Zf (+) (Promega, Madison, WI, USA) (Blum et al., 1991) which was kindly
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provided by Dr. Cheng-Chan Lu (Department of Pathology, National Cheng-Kung
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University). The four viral promoter-reporter plasmids were constructed by
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amplifying the HBV genomic fragments that corresponded to the Core (nt1636~1851),
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S (nt3114~220), PreS (nt2438–2855) and X (nt1071–1357) gene promoter regions
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with a polymerase chain reaction (PCR) that used the pHBV1.2 plasmid as a template
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and was subsequently inserted into the SacI/XhoI sites of the pGL4.17
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luciferase-reporter expression vector (Promega) as previously described (Huang et al.,
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2014a). In addition, the reporter plasmid, p5CS1-Luc, which contains five copies of
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the CS1 site (nt 207-216) within HBV surface gene coding sequence (adw2 serotype),
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was constructed from the pGL4.17 expression vector. All of the DNA sequences were
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verified with the appropriate restriction enzyme digestion and direct sequencing.
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2.8.Promoter-reporter activity assay
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Cells were plated into 24-well culture plates, transfected with promoter-reporter
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constructs (1 g/well) and pRL-SV40 in serum-free DMEM for 24 h, washed with 1
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PBS, incubated in DMEM supplemented with 2% FBS, and treated with or without
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LPRP-Et-97543 (10 g/ml) for 2 days. The luciferase assay was performed according
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to the manufacturer’s instructions (Promega, Madison, WI, USA). The
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promoter-reporter construct transfection consisted of co-transfecting the pRL-SV40
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Renilla luciferase expression plasmid (0.02 g/well) (Promega, Madison, WI, USA)
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and using it to normalize the basal level luciferase activity.
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2.9.Transfection
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Huh7 cells were seeded in 60-mm dishes at the same density (as described in the cell
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viability assay) for 24 h. The cells were then transfected with the pHBV1.2 plasmid
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using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in DMEM which
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contained 10% FBS for 2 days which followed the manufacturer’s protocol. The cells
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were then treated with the LPRP-Et-97543 compound for another 2 days.
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2.10.Analysis of intracellular HBV-RNA by Northern blotting
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Total RNA from the treated cells was extracted using the Trizolisolation buffer
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(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Five
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micrograms of total RNA was denatured, separated on a 1.0% agarose gel using a
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commercial kit (Amresco, Solon, OH, USA), and transferred to a positively charged
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Hybond-N+ nylon membrane (Amersham, GE Healthcare, Buckinghamshire, UK).
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After UV cross-linking, the membrane was hybridized with a DIG-labeled full-length
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HBV genome probe, washed, and exposed to X-ray film. In brief, the full-length HBV
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probe was produced by the restriction enzymatic digestion of the pHBV2 plasmid
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with EcoRI (Will et al., 1985), purified and labeled with a DIG high prime DNA
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labeling kit (Roche, Mannheim, Germany). The total RNA was normalized by
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hybridizing the membrane with a glyceraldehyde-3-phosphate dehydrogenase
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(GAPDH) probe which was produced by the Pst I digestion of the pGAPDH plasmid
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(provided by Dr. Hsiao-Sheng Liu from the Graduate Institute of Microbiology and
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Immunology, National Cheng-Kung University, Tainan, Taiwan). The variations in
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transfection efficiency were controlled by co-transfecting the Huh 7 cells with the
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pCMV- (Clontech, Palo Alto, CA, USA), which contains the CMV immediate-early
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promoter driving the -galactosidase (-gal) gene.
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2.11.Southern blot and Real-time PCR analysis of intracellular HBV-DNA synthesis
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Encapsidated HBV DNA in cells was extracted from the viral core particles and
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fractionated on 1.0 % agarose gels and transferred onto the Hybond N+ membrane
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(GE Healthcare, Buckinghamshire, UK) as described by Pugh et al. (Pugh et al.,
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1988). HBV DNA was detected by Southern blot analysis using the DIG-labeled
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full-length HBV probe. In addition, HBV DNA in the virion from conditioned media
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that were untreated or treated with LPRP-Et-97543 or Lamivudine (3TC) was isolated
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and subjected to Southern blot analysis as described above. Real-time PCR analysis of
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HBV DNA used 5- AGGAGGCTGTAGGCATAAATTGG -3 as the forward primer
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and 5- CAGCTTGGAGGCTTGAACAGT-3 as the reverse primer (Feng et al.,
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2009). The PCR reactions were performed using SYBR Green PCR master mix and
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the primer pair followed the protocol described below: initial denaturation at 50C for
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2 min and 95C for 10 min, followed by 45 cycles of amplification at 95C for 15s
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and annealing/extending at 58C for 1 min.
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2.12.Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and
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Western blot analyses of intracellular viral antigens and cellular signaling proteins
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Whole cell proteins of Huh7 cells were extracted by RIPA buffer with protease
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inhibitors. For another set of experiments, the nuclear and cytoplasmic proteins of
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cells were prepared by NE-PER extraction reagents (Thermo Fisher Scientific,
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Rockford, IL, USA) according to the manufacturer’s protocol. The BCA protein assay
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kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to determine the cellular
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protein concentration and 25 g of protein from each sample was used for the sodium
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dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis. Separated proteins
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were transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham, GE
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Healthcare, Buckinghamshire, UK) by electroblotting and then the proteins were
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blocked for 1 h with PBST (PBS buffer with 0.05% Tween-20) that contained 4% of
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nonfat milk. Immunoblots were incubated with the primary antibody followed by a
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HRP-conjugated secondary antibody. The antibody-bound proteins were detected
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using an ECL reagent (Amersham, GE Healthcare, Buckinghamshire, UK) and then
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the same membranes were stripped and reprobed using the anti--tubulin antibody
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(Sigma, St Louis, MO, USA) or anti-PCNA antibody as the loading control.
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2.13.Nuclear extract preparation and electrophoretic mobility shift assay (EMSA)
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Nuclear extracts were prepared from Huh7 cells that were transfected with or without
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pHBV1.2 for 2 days and treated with or without LPRP-Et-97543 (10 g/ml) for
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another 2 days to detect the DNA/protein-binding activity. For the EMSA, nuclear
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extracts (8 g) for every reaction was pre-incubated with or without a competitor and
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concomitantly mixed with 1 g poly[dI-dC] (Sigma) and 1 g of salmon sperm DNA
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(Sigma) which was placed on ice for 20 min and then incubated with a P-labeled
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double-stranded DNA probe for 20 min at 30 C. The mixture was separated on a 5%
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nondenatured polyacrylamide gel in 0.5 TBE (Tris-borate-EDTA) buffer at 150 V for
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90 min and the binding shift was detected from an autoradiograph. The
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oligonucleotide-probe for the DNA binding assay was the NF-B CS1- binding site
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which corresponded to the HBV adw serotype (nt 207-216 ),
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5′-TACAGGCGGGGTTTTTCTTGTTG-3′ (Lin et al.,2009).
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2.14.Statistical Analysis and Quantification of data
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The data were expressed as means and standard deviations (SD) of the mean for three
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independent experiments that used GraphPad Prism (GraphPad Software Inc., San
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Diego, CA, USA). Variance analysis and student t-tests were used for data analysis.
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Significant differences occurred when P < 0.05. Quantitative data from Northern and
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Western blot analysis were obtained using the computing densitometer and TotalLab
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Quant software (Nonlinear Dynamics Ltd.).
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3. Results
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3.1.Effect of LPRP-Et-97543 on cell viability
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To exclude the possibility of HBV production being suppressed by LPRP-Et-97543
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due to its cytotoxicity, cell viability of LPRP-Et-97543 on HepG2.2.15 and Huh7 cells
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were tested using the MTS assay. The results indicated that LPRP-Et-97543 caused
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cytotoxic effects at doses >10 g/ml in HepG2.2.15 and HBV-transfected Huh7 cells
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(Fig. 1B). Thus, a non-cytotoxic dose (< 10 g/ml) was used for the antiviral
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treatment of HepG2.2.15 or HBV-transfected Huh7 cells.
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3.2.Effects of LPRP-Et-97543 on the HBV virions and antigen secretion in
HepG2.2.15 cells
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LPRP-Et-97543 effects on the expression of viral antigens were examined by
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measuring the secreted levels of HBsAg and HBeAg by HepG2.2.15 cells using the
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EIA. Cells were treated with various LPRP-Et-97543 concentrations (2.5, 5 and 10
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g/ml) and the HBsAg and HBeAg secretions were significantly suppressed in a
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dose-dependent manner compared to controls. The HBeAg inhibition rate was higher
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than the HBsAg inhibition rate (Fig. 2A). LPRP-Et-97543 function on virion secretion
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was further examined by isolating the viral DNA from the conditioned medium.
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Real-time PCR was performed to detect the extracellular viral DNA level. The results
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showed that LPRP-Et-97543 treatment significantly reduced the secreted viral DNA
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level in a dose-dependent manner (Fig. 2B).
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3.3.Effect of LPRP-Et-97543 on the HBV gene expression
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The molecular regulatory effect of LPRP-Et-97543 on the HBV gene expression was
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determined by transiently transfecting the Huh 7 cells with the pHBV1.2 HBV
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genome-containing plasmid (Blum et al., 1991) and treating the cells with three doses
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(2.5, 5 and 10 g/ml) of LPRP-Et-97543 for 2 days. A Northern blot was performed to
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analyze the HBV viral RNA expression levels. LPRP-Et-97543 treatment significantly
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reduced both precore/pregenomic and major S/preS RNA with the LPRP-Et-97543
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inhibition potential higher on surface RNA than on the precore/ pregenomic RNA (Fig.
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3A). In addition, immunoblot analysis was used to detect the intracellular levels of
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HBV large surface (LHBsAg) and core (HBcAg) proteins and the results indicated
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that LPRP-Et-97543 potently reduced LHBsAg and HBcAg protein levels compared
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to vehicle controls (Fig. 3B). Furthermore, Southern blotting was used to assess the
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intracellular HBV DNA level and the results indicated that LPRP-Et-97543 treatment
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potently inhibits the replication HBV DNA level in HBV transfected Huh7 cells (Fig.
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3C).
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3.4.Effect of LPRP-Et-97543 on the HBV gene and cellular signaling pathways
related to the responsive element that contains the promoter activity
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The regulatory effect of LPRP-Et-97543 on the HBV gene expression was examined
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by isolating and cloning four promoters that corresponded to the viral gene into a
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pGL4.17 luciferase reporter vector (Huang et al., 2014a) and the LPRP-Et-97543
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effect on the promoter’s activity level was tested. In this study, pCore-Luc, pS-Luc (S
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promoter), pPreS-Luc (preS promoter) or pX-Luc constructs were transfected into
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Huh7 cells for 24 h and then the cells were treated with the maximum inhibitory
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LPRP-Et-97543 dose prior to performing the luciferase assay. The results showed that
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LPRP-Et-97543 treatment significantly reduced the Core, S and preS promoter
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activity whereas it had no effects on the X promoter activity (Fig. 4A). Meanwhile,
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four luciferase reporters that contained the NF-B, AP-1, AP-2 or the ISRE binding
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elements were co-transfected with pHBV1.2 into Huh7 cells to determine if
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LPRP-Et-97543 inhibits the HBV gene expression in the host’s mediated cellular
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signaling pathways. After LPRP-Et-97543 treatment (10 g/ml), cells were harvested
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and subjected to a promoter activity assay. Figure 4B shows that LPRP-Et-97543
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significantly inhibited NF-B-containing promoter activity but did not affect the AP-1,
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AP-2 and ISRE-containing promoter activity (p < 0.05).
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3.5.Effects of LPRP-Et-97543 on NF-B and IB protein expressions in Huh7 cells
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During LPRP-Et-97543 treatment, NF-B had a regulatory role on the HBV gene
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expression; thus Huh7 cells were transfected with pHBV1.2 and then treated with
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LPRP-Et-97543. Western blot was conducted to determine the effect of this
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compound on the NF-B signaling pathway relative protein expression. The results
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showed that LPRP-Et-97543 treatment reduced nuclear p65/p50 NF-B protein
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expression and reduced phosphorylated NF-B p65 (Fig. 5), whereas cytoplasmic
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IB expression was significantly enhanced in HBV-expressed cells. To our surprise,
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when the LPRP-Et-97543 was used to treat Huh7cells that without HBV expression,
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the expression levels of nuclear NF-B p65/p50, phosphor-p65 was not impaired
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during treatment but significantly increased by HBV expression (Fig. 5). However,
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IB protein slightly decreased by LPRP-Et-97543 in HBV-nontransfected cells.
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3.6.NF-B is essential for the inhibitory effects of LPRP-Et-97543 on HBV
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Several putative NF-B binding sites were identified in the HBV genome (Lin et al.,
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2009). Among them, a specific NF-B-binding site was proposed that have a positive
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regulatory role in HBV gene expression (Huang et al., 2014b). This site was located in
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the HBV genome at nt207 to 216 (CS1) which corresponds to HBV surface gene
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coding region. To determine whether LPRP-Et-97543 regulated viral gene expression
379
by NF-B as mediated through the CS1 site, nuclear extracts were prepared from
380
Huh7 cells with or without HBV transfection and tested for the NF-B binding
381
activity to CS1 site by EMSA. The results showed that LPRP-Et-97543 significantly
382
reduced the binding activity of NF-B to the CS1 DNA sequence in HBV-expressing
19
383
or HBV non-transfected nuclear extracts (Fig. 6A). Moreover, this binding is
384
mediated through NF-B p65 protein because of occurring supershift band when p65
385
antibody was added in EMSA binding reaction. The results also showed that
386
transfection of HBV genome significantly enhanced the binding activity of NF-B to
387
the CS1 site (Fig. 6A). To determine if LPRP-Et-97543 could affect CS1 containing
388
promoter activity, p5CS1-Luc reporter was constructed and co-transfected into Huh7
389
cells with or without HBV expression. The transfections were then treated with or
390
without LPRP-Et-97543 (10 g/ml) for 2 days and tested for luciferase assay. Results
391
revealed that CS1- containing promoter activity increased in HBV-expressing cells,
392
but not in HBV non-transfected cells. Furthermore, treatment with LPRP-Et-97543
393
significantly decreased CS1-containing promoter activity in HBV-expressed cells, but
394
not in HBV-nontransfected cells (Fig. 6B). To further examine whether the regulatory
395
effect of LPRP-Et-97543 on the HBV is mediated through the inhibition of the NF-B
396
signaling pathway, the p65 expression plasmid was co-transfected with pHBV1.2 in
397
Huh7 cells and treated with LPRP-Et-97543 (10 g/ml). The HBV DNA from the
398
cultural medium was extracted for Real-time PCR analysis and the results showed that
399
an increase in p65 expression dose (0.5 and 2.5 g) significantly reversed the
400
inhibitory effect of LPRP-Et-97543 on the HBV DNA level as compared to the empty
401
vector transfection (Fig. 6C).
20
402
403
4. Discussion
In this study, the LPRP-Et-97543 compound was used to evaluate the potential
404
antiviral effects against HBV. The results demonstrated that LPRP-Et-97543 inhibited
405
viral HBsAg and HBeAg secretion and supernatant viral DNA levels in HepG2.2.15
406
cells (Fig. 2A and 2B). To elucidate the inhibitory mode of LPRP-Et-97543 on HBV,
407
the HBV-transfected Huh7 cells were used as a platform to determine how this
408
compound regulates viral gene expression and viral propagation, which was
409
facilitated by the low sensitivity of determination for the viral gene products in
410
HepG2.2.15cells (Huang et al., 2014b). LPRP-Et-97543 significantly down-regulated
411
viral S/preS relative to precore/pregenomic RNA levels (Fig. 3A). LPRP-Et-97543
412
treatment significantly inhibited the synthesized viral protein expression level of a
413
large form of viral surface protein (LHBsAg) and core protein (HBcAg) (Fig. 3B).
414
Southern blot analysis was used to detect the isolated viral DNA in cells and the
415
results revealed that intracellular HBV DNA was decreased by LPRP-Et-97543 (Fig.
416
3C). In view of our results demonstrated that LPRP-Et-97543 could regulate the HBV
417
gene at the transcription level, therefore, viral promoter activity was further examined
418
and the results showed that HBV core, S, and preS promoters were attenuated by
419
LPRP-Et-97543 but the X promoter activity in the HBV-expressed Huh7 cells were
420
not affected (Fig. 4A). These results suggest that LPRP-Et-97543 could potentially
21
421
inhibit the HBV gene expression by regulating its promoter activity. In addition, It is
422
important to figure out if viral covalently closed circular DNA (cccDNA) was affected
423
during treatment in HBV transfected cells to distinguish the exact transcription
424
potential of produced viral genome than the transfected plasmid. However, there is no
425
detectable level of cccDNA was observed that when we conducted the experiments
426
for cccDNA isolation and for Southern blot analysis (data not shown). This may due
427
to the very low level of cccDNA during treatment or poor detecting efficiency in
428
Southern blot.
429
To examine the possible mechanism by which LPRP-Et-97543 affects HBV
430
function is mediated through host’s cellular signaling pathways; transcriptional
431
activities of four major signaling pathways, AP-1, AP-2, NF-B and ISRE were
432
analyzed using a promoter-reporter assay. After transfection and treatment,
433
LPRP-Et-97543 specifically decreased NF-B promoter activity, while there were no
434
effects on AP-1, AP-2 and ISRE promoters in the HBV-transfected Huh7cells. This
435
result indicates that LPRP-Et-97543 may regulate the NF-B signaling pathway in
436
HBV transfected host cells (Fig. 4B). Moreover, immunoblots revealed that the
437
nuclear expression of NF-B p65/p50 proteins and the phosphorylated p65 protein
438
level were decreased by LPRP-Et-97543 while the cytoplasmic IB protein level
439
was augmented by the same treatment in a dose-dependent manner (Fig. 5). These
22
440
results suggest that LPRP-Et-97543 could potentially enhance IB expression in
441
cytoplasm and inhibits the p65/p50 NF-B nuclear translocation and then attenuates
442
the p65 phosphorylation in subsequent activation. Surprisingly, the immunoblot also
443
reveals that without compound treatment, HBV transfection could enhance NF-B
444
nuclear translocation of NF-B protein and subsequent activation of p65 in Huh7 cells,
445
while the expression of NF-B member proteins was not changed in HBV
446
non-transfected cells, suggests that HBV infection might be required for the actions of
447
NF-B factors on gene promoter activity and LPRP-Et-97543 could interfere this
448
regulation. Previous reports have shown that several binding elements for NF-B are
449
essential for HBV viral gene regulation. Among them, a study by Kwon and Rho
450
(2002) clearly demonstrated that the HBV core protein (HBcAg) upregulates the HBV
451
enhancer II/ pregenomic promoter through the NF-B binding site located upstream
452
of the promoter. In addition, our recent studies also indicated that several natural
453
occurring compounds could inhibit HBV gene expression and genome replication
454
base on the interfering NF-B factor on viral surface promoter activity (Huang et al.,
455
2014b). Therefore, the importance of NF-B in HBV gene regulation is the main issue
456
we further addressed. On the other hand, to determine the modulatory role of
457
LPRP-Et-97543 on HBV gene regulation probably raised the possible mechanisms
458
that HBV genes could be regulated by HBV viral protein. It makes sense since
23
459
previous studies showed that transcription activators like HBx (Doria et al., 1995),
460
HBcAg (Kwon and Rho, 2002), a C-terminally truncated form of HBV middle surface
461
antigen (MHBst)(Meyer et al., 1992), and HB large surface antigens (LHBs) (Hildt et
462
al., 1996) are capable of NF-B activators. Thus, the effects of LPRP-Et-97543 on
463
HBV gene regulation may suggest that link to cellular NF-B signaling pathway.
464
In view of the potential inhibitory effect of LPRP-Et-97543 on HBV surface
465
transcripts from Northern blot analysis (Fig. 3A) and a putative CS1 site was
466
identified available for NF-B on HBV surface gene upregulation (Lin et al.,
467
2009)(Huang et al., 2014b). We further examine the possible role of LPRP-ET-97543
468
on the binding activity of NF-B to DNA elements and CS1 NF-B binding sequence
469
was synthesized and used for an EMSA analysis. As seen in result Fig. 6A,
470
LPRP-Et-97543 treatment significantly decreased the binding activity of NF-B to the
471
CS1 site and this binding was specific because the protein/DNA shift band can be
472
supershift when p65 NF-B antibody was used in the binding reaction and the similar
473
result has been reported previously (Huang et al., 2014b). In addition, Lin et al. (2009)
474
have proposed that CS1 site of HB surface gene is capable for p65 homodimer
475
binding. In view of important role of CS1 NF-B binding sequence for the action of
476
LPRP-Et-97543 on HBV surface gene expression, we constructed CS1 site containing
477
promoter-reporter (luciferase) and examine whether LPRP-Et-97543 could regulate
24
478
CS1 containing gene expression. The result indicates that LPRP-Et-97543 compound
479
could inhibit CS1-containing promoter activity and can be enhanced by HBV
480
co-transfection (Fig. 6B). These results clearly indicate that HBV viral proteins might
481
be involved in LPRP-Et-97543 regulated viral gene expression and showed that host
482
NF-B signaling pathways inhibited by LPRP-Et-97543 were investigated in an
483
HBV-dependent manner.
484
To determine if LPRP-Et-97543 effects HBV gene expression and viral
485
propagation is indeed correlated with NF-B regulation, the pCMV-p65 NF-B
486
expression plasmid was co-transfected with pHBV1.2 to Huh7cells. The secreted
487
HBV viral particles were isolated and Southern blot analysis detected the viral DNA
488
level by viral DNA real-time PCR. As seen in Fig.6C, when cells were treated with a
489
maximum dose (10 g/ml) of LPRP-Et-97543, the secretion of the HBV viral particle
490
was inhibited about 41% compared to the vehicle treated controls. However, the
491
inhibition effect of LPRP-Et-97543 on the HBV viral particle secretion was
492
significantly reversed when cells were co-transfected with an increasing dose (0.5 and
493
2.5 g) of pCMV-p65 plasmid using the same treatment. There was no difference in
494
the secreted levels of HBV DNA in a maximum transfection dose of pCMV-p65
495
plasmid combined with LPRP-Et-97543 treatment compared to non-treated controls.
496
This result suggests the LPRP-Et-97543 effects on HBV gene expression and viral
25
497
498
DNA replication might be mediated through the NF-B inhibition.
For medicinal applications, controlling the HBV well enough to ameliorate the
499
disease is the most crucial issue in affecting the antiviral therapeutic strategies, as well
500
as preventing the viral resistance commonly caused by viral reverse transcriptase
501
inhibitors. In this study, LPRP-Et-97543 regulated the HBV gene expression and viral
502
DNA replication by interfering with the host cell’s NF-B signaling pathway. In
503
addition, previous studies have proposed that NF-B activation in cells that were
504
infected with HBV (Doria et al., 1995; Hildt et al., 1996; Kwon and Rho, 2002;
505
Meyer et al., 1992) might be a possible treatment for the HBV and that activation of
506
the NF-B pathway is mediated by viral proteins (Huang et al., 2014b). However, the
507
discrepancy was raised that the inhibitory effect of tumor necrosis factor alpha
508
(TNF-) or interleukin-1 (IL-1) on HBV replication was mediated through of NF-B
509
activation in previous studies (Lin et al., 2009). In contrast, elevated expression of
510
NF-B in HBV-infected hepatocytes seemed to be critical to HBV gene upregulation
511
and viral replication in our study. A possible explanation is that the inhibition of HBV
512
by NF-B activation is mediated through indirect protein interaction between NF-B
513
and other factor such as SP-1 (Lin et al., 2009) and do not bind directly to putative
514
NF-B site in HBV genome. However, our study clearly indicated that the possible
515
upregulatory role of viral protein on HBV gene expression and via through direct
26
516
binding of NF-B to its element and this regulation can be interfered by drug during
517
treatment. Thus, the activation of NF-B by which to inhibit or activate HBV gene
518
expression and viral replication seems to have distinct pathways. On the other hand,
519
several issues remain to be elucidated in the future, including how viral factor (i.e.,
520
Core and X proteins) involved in LPRP-Et-97543 action on HBV gene regulation;
521
how NF-B is involved in regulating viral gene promoter activity other than the
522
surface gene and how HBV switches the regulatory machinery and takes over cellular
523
signaling pathways in the chronic-infected phase of HBV in response to drug
524
treatment. In conclusion, the mechanism underlying the anti-HBV effects has been
525
elucidated and a novel drug, LPRP-Et-97543, for treating HBV infected liver disease
526
was discovered.
527
528
529
Acknowledgements
This work was funded by grants from the Committee on Chinese Medicine and
530
Pharmacy, Department of Health, Executive Yuan, Taiwan (CCMP102-RD-116),
531
China Medical University Hospital (DMR-103-042). The authors express heartfelt
532
thanks to Dr. Cheng-Chan Lu for the pHBV1.2 and pHBV2 plasmids, to Dr.
533
Hsiao-Sheng Liu for the pGAPDH plasmid, and to Dr. Cheng-Wen Lin for pAP1-Luc,
534
pAP2-Luc, pNF-kB-Luc, and pISRE-Luc promoter–reporter constructs.
27
535
536
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637
638
Figure legends
639
Fig. 1. (A) Schematic illustration of the LPRP-Et-97543 structure. (B) Cytotoxicity of
640
LPRP-Et-97543 on HepG2.2.15 and Huh7 cells. To determine the cytotoxicity, cells
641
were plated in 96-well plates for 24 h and treated with serial dilutions (0.625~40
642
g/ml) of LPRP-Et-97543 for 3 days. After treatment, the cells were subjected to a
643
cytotoxic assay. The data are expressed as mean and the standard deviation of the
644
mean. (n = 4) (*P<0.05 vs untreated cells)
645
646
Fig. 2. The effects of LPRP-Et-97543 on (A) the secretion of HBV viral antigens and
647
(B) replicated viral DNA levels in HepG2.2.15 cells. Cells were treated with various
648
concentrations (2.5, 5 and 10 g/ml) of LPRP-Et-97543 for 3 days and the
33
649
conditioned media were collected for the EIA analysis of HBsAg and HBeAg. In
650
addition, the HBV DNA was isolated from virion particles and subjected to Real
651
time-PCR analysis. The data are expressed as the mean and the standard deviation of
652
the mean. (n = 3) (*P<0.05 vs untreated cells)
653
654
Fig. 3A. The effects of LPRP-Et-97543 on HBV gene expression and viral DNA
655
replication in Huh7 cells. Huh7 cells were transfected with pHBV1.2 plasmid for 2
656
days and treated with three concentrations (2.5, 5 and 10 g/ml) of LPRP-Et-97543
657
for another 2 days. Treated cells were harvested and subjected to total RNA, protein
658
and DNA isolation. (A) The total RNA from the transfected and treated Huh7cells
659
were subjected to Northern blot analysis using the HBV whole genome DNA as the
660
probe, as described in the Materials and Methods section. GAPDH was used as an
661
RNA loading control and -gal was used to detect the efficiency of each transfection.
662
(B) Total cellular proteins were extracted and immunoblotting was used to detect the
663
LHBsAg, HBcAg and -tubulin for comparison. (C) Intracellular HBV DNA were
664
isolated and subjected to Southern blot analysis. RC, relaxed circular; SS, single
665
stranded; RI, replicated intermediates; HBV standard, 2 ng of 3.2 kb linear HBV
666
genome marker. The intensity of each protein band was quantitated with a
667
densitometer and the relative amount was normalized with an internal control. Data
34
668
shown in (A), (B) and (C) are representative of three sets of experiments.
669
670
Fig. 4. The effects of LPRP-Et-97543 on the HBV gene promoter or the cellular
671
signaling pathway responsive to the promoter activity. Huh7 cells were seeded in
672
24-well plates for 24 h and co-transfected with pHBV1.2 and with either (A)
673
pCore-Luc, pS-Luc, pPreS-Luc, and pX-Luc viral gene promoter reporter constructs
674
or (B) pAP1-Luc, pAP2-Luc, pNF-B-Luc and pISRE-Luc cellular signaling pathway
675
responsive to the promoter reporter constructs together with pRL-SV40 for 24 h and
676
then treated with LPRP-Et-97543 (10 g/ml) for another 24 h. The cellular lysates
677
were prepared for the luciferase assay as described in the Materials and Methods
678
Section. The data are expressed as the mean and the standard deviation of the mean.
679
(n=3) (*p0.05 vs untreated cells)
680
681
Fig. 5. The effects of LPRP-Et-97543 on nuclear NF-B p65/p50, phospho- p65 and
682
cytoplasmic IB protein expressions in Huh7 cells that with or without HBV
683
genome expression. Huh7 cells were transfected with or without pHBV1.2 for 2 days
684
and treated with three concentrations (2.5, 5 and 10 g/ml) of LPRP-Et-97543 for
685
another 2 days. Nuclear fraction of cellular proteins was extracted and
686
immunoblotting was used to detect p65, p50, and phosphorylated p65 of NF-B
35
687
protein. The cytoplasmic fraction of cellular proteins was used to detect IB for
688
comparison. PCNA protein was detected in immunoblot as control for nuclear protein
689
expression. The data shown are representative of the three sets of experiments.
690
691
Fig. 6. Effect of the NF-B signaling pathway on LPRP-Et-97543 mediated HBV
692
gene inhibition. (A) For the DNA binding activity assay, Huh7 cells were transfected
693
with pHBV1.2 for 2 days, treated with or without LPRP-Et-97543 (10 g/ml) and
694
then the cellular nuclear proteins were extracted and subjected to EMSA analysis. (B)
695
For the CS1 site-containing promoter activity, Huh7 cells were transfected with either
696
the p5CS1-Luc promoter-reporter constructs together with or without pHBV1.2
697
plasmid for 24 h and treated with or without LPRP-Et-97543 for 2 days. (C) In the
698
other set of experiments, Huh7 cells were co-transfected with pHBV1.2 and with two
699
doses (0.5, and 2.5 g) of pCMV-p65 plasmid for 2 days and then treated with
700
LPRP-Et-97543 (10 g/ml) for another 2 days. The secreted viral particles from the
701
cells were isolated by the kit and subjected to Real-time PCR analysis of HBV DNA.
702
For comparison, total protein of transfected and treated cells were extracted for
703
NF-B p65 protein immunoblot.
(n=3) (*p0.05 vs untreated cells)
704
705
36
706
707
708
709
710
711
Figure
712
37
713
714
Fig. 1.
715
716
717
38
718
719
720
Fig. 2.
39
721
722
40
723
724
Fig. 3.
725
726
727
41
728
729
730
Fig. 4.
42
731
732
Fig. 5.
733
734
735
43
736
737
44
738
739
Fig. 6.
740
45