Browsing by Author "Pan, Kao-Lu"

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Development of a Whole-Cell Screening System for Evaluation of the Human CYP1A2-Mediated Metabolism
(2011-12-01)
Chu, Chih-Chieh
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Pan, Kao-Lu
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Yao, Hsien-Tsung
;
Hsu, John Tsu-An
;
生物科技學系
Cytochrome P450 1A2 (CYP1A2) is an important member of cytochrome P450 involved in drug metabolism. In this study, a cell line, Huh7-1A2-I-E, with high expression level of CYP1A2 is established based on Huh7 cells. To achieve this, we constructed a recombinant lentiviral vector, pLenti-1A2-I-E, containing a single promoter encoding CYP1A2 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP1A2 and EGFP proteins would be based on a single bi-cistronic mRNA. The Huh7-1A2-I-E cells were evaluated as a cell-based model for identification of CYP1A2 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of Huh7-1A2-I-E cells and the Huh7-E control cells with aflatoxin B1 showed that cells with CYP1A2 expression are much more sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in Huh7-1A2-I-E cells could be prevented by furafylline, a CYP1A2 inhibitor. A collection of approximately 200 drugs were screened using this system and results indicate that for most drugs the metabolism by CYP1A2 is unlikely to have made a major contribution to the in vitro cytotoxicity except for thimerosal and evoxine. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study. Biotechnol. Bioeng. 2011; 108: 2932-2940. (C) 2011 Wiley Periodicals, Inc.
Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection
(2009-11-01)
Pan, Kao-Lu
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Lee, Jin-Ching
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Sung, Hsing-Wen
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Chang, Teng-Yuang
;
Hsu, John T. -A.
A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Delta 4B5A) SEAP, that carries a dual reporter, EG(Delta 4B5A) SEAP. The EG(Delta 4B5A) SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Delta 4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Delta 4B5A) SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Delta 4B5A) SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Delta 4B5A) SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z'-factorof this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.
Diosgenin, a Plant-Derived Sapogenin, Exhibits Antiviral Activity in Vitro against Hepatitis C Virus
(2011-04-01)
Wang, Ya-Jean
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Pan, Kao-Lu
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Hsieh, Tsung-Chih
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Chang, Teng-Yuan
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Lin, Wen-Hsing
Diosgenirr (3 beta-hydroxy-5-spirostene, 1); a plant-derived sapogenin, is used as a dietary: supplement However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 mu M No cellular toxicity was observed at this concentration.,Diosgenin, (1) also significantly reduced the levels of viral,RNA and viral proteins as evaluated by quantitative real-time reverse :transcriptase PCR and Western blot analysis, respectively. In addition, in an HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 mu M. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1:and interferon-alpha exerted an additive effect on the resultant anti-HCV activity.