Cytotoxicity and Anti-Hepatitis B Virus Activities of Saikosaponins from Bupleurum Species

• Abstract
• Introduction
• Materials and Methods
• Cell culture
• Treatment of saikosaponins and 3TC
• Cell proliferation inhibition assay
• Determination of HBsAg and HBeAg
• Detection of HBV DNA
• LDH release assay of cytotoxicity
• DNA fragmentation analysis
• Western blot
• Statistical analysis
• Results
• Effect of saikosaponins on 2.2.15 cell viability
• Effect of saikosaponins on HBsAg, HBeAg and HBV DNA expression
• Cytotoxic effect of saikosaponin d on human hepatoma HepG2 cells
• Discussion
• References

Abstract

Saikosaponins, the main active constituents of Bupleurum spp., have been shown to possess immunomodulatory, hepatoprotective, anti-tumor and anti-viral activities. In this study, saikosaponins a, c and d were evaluated for cytotoxicity and anti-hepatitis B virus (HBV) activities. Results showed that, with the exception of saikosaponins a and d, HBV-transfected human hepatoma cells (2.2.15 cells) cultured with saikosaponin c showed a significantly lower level of HBeAg in culture medium. Saikosaponin c also possessed activity in inhibiting HBV DNA replication; this inhibitory effect was not due to the cytotoxicity of saikosaponin c or its effect on 2.2.15 cell proliferation. Although saikosaponin d exhibited cytotoxicity on 2.2.15 cells, it failed to inhibit HBV multiplication. The cytotoxicity of saikosaponin d against HepG2 human hepatocellular carcinoma cells was due to the induction of apoptosis through the activation of caspases 3 and 7, which subsequently resulted in poly-ADP-ribose-polymerase (PARP) cleavage. DNA fragmentation was clearly noted at more than 6 h after HepG2 cells exposure to saikosaponin d. The present study concludes that saikosaponin c exhibits anti-HBV activity and saikosaponin d possesses potent cytotoxicity against human hepatocellular carcinoma cells.

Introduction

Saikosaponins (a, c and d) are the major active components present in Bupleurum spp., particularly B. chinese, B. scorzoneraefolium, B. falcatum L. var. komarowi koso-polj and B. kaoi Liu, Chao et Chuan [1]. Although studies found that B. kaoi contains the highest amount of saikosaponins, roots of these plant materials are interchangeably used as the ingredient of a popular traditional Chinese medicine known as ”Chai-Hu”. It is frequently used for treating liver diseases in China, Taiwan and Japan. In mainland China, Chai-Hu is considered to be the most highly recommended and widely accepted crude drug for treating hepatitis B. Previous studies have shown that B. chinese and B. falcatum possess anti-HTLV III [2], anti-HIV [3] and anti-HBV activities [4]. Chai-Hu was also postulated to contain ingredient(s) that might act as a reverse transcriptase inhibitor(s). Saikosaponins were reported to exhibit immunomodulatory [5], hepatoprotective [1], anti-tumor [6] and anti-viral [7] activities.

Hepatitis B virus (HBV) is well known as the major causative agent of human hepatitis. Its infection often leads to chronic hepatitis, cirrhosis, liver failure and hepatocellular carcinoma (HCC) [8]. Vaccination of HBsAg in infants has proved to be a successful strategy to prevent HBV infection [9] and further progression to HCC [10]. However, HBV carriers with immune systems unresponding to HBV infection or vaccination often require an alternative treatment. Alpha-interferon has been shown to be effective against HBV [11], but its effectiveness in achieving HBeAg seroconversion was only found in about 40 % of the patients, and severe side effects were also noted [12]. [-]-β-L-2′,3′-dideoxy-3′-thiacytidine (3TC, lamivudine), a nucleoside analogue which was originally developed as an anti-HIV agent [13], is effective in inhibiting HBV DNA replication [14] and has been approved for treatment of chronic hepatitis caused by HBV in many countries. HBV viremia relapse has been noted in patients shortly after the cessation of 3TC therapy [15]. Using 3TC for long-term treatment may result in the emergence of 3TC-resistant mutants [16]. Since there is no satisfactory therapeutic strategy available, it is necessary to find new alternative anti-HBV agents with less side-effects.

In this study, we evaluated the cytotoxicity and inhibitory activity of saikosaponins a, c, and d against secretion of hepatitis B virus soluble antigens, as well as their effect on HBV DNA replication.

Materials and Methods

Cell culture

The 2.2.15 cell line is a clonal derivative from Hep G2 (ATCC HB8065) human hepatocellular carcinoma cell. It was obtained by stable transfection with a plasmid containing head-to-tail hepatitis B virus dimer and selected in the medium containing G418. The 2.2.15 cell line was kindly provided by Professor George Acs, Mount Sinai Medical Center, New York, USA. The cell line and Hep G2 were routinely cultured with RPMI 1640 medium supplemented with 10 % fetal calf serum and antibiotics (100 units/mL of penicillin G, 100 μg/mL of streptomycin and 0.25 μg/mL amphotericin B) at 37 °C in an atmosphere of 5 % CO₂ and 100 % humidity.

Treatment of saikosaponins and 3TC

For anti-HBV marker activity and LDH release assays, a density of 4 × 10⁵ 2.2.15 cells per well were seeded in a 6-well tissue culture plate. The cells were allowed to attach to the bottom of the plate for 24 h. Medium containing various concentrations of saikosaponins a, c and d (Wako Pure Chemical Industries, Ltd. Japan) and 3TC (Moravek Biochemical, California, USA) were renewed every 2 days for 6 days.

Cell proliferation inhibition assay

The cell proliferation inhibition assay was performed in a 96-well format with 4 × 10⁴ 2.2.15 cells per well. Cell viability of 2.2.15 and HepG2 cells treated with saikosaponins a, c and d was assayed on day 2 using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Germany). In brief, cells were washed with PBS (containing 137 mM NaCl, 4.5 mM KCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄ and 0.5 mM EDTA, pH 7.4), 100 μL culture medium and 50 μL assay reagent (containing 1 mg/mL sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate and 3.83 μg/mL N-methyldibenzopyrazine methyl sulfate) were then added to each well. After incubation at 37 °C for 4 h, samples were measured at OD₄₉₂ and OD₆₉₀ using an ELISA reader (Multiskan EX, Labsystems). The viability of treated cells was calculated as OD_T/OD_C. OD_T was obtained by subtracting OD₄₉₂ from OD₆₉₀ of the treated cells and OD_C was that of the 1 % DMSO control.

Determination of HBsAg and HBeAg

At the end of the incubation, culture medium was collected and clarified by centrifugation at 400 × g at 4 °C for 15 min. HBsAg and HBeAg were determined by ELISA kits and preformed according to the protocol provided together with the kit (General Biological Corp., Taiwan). The percentage of HBsAg and HBeAg present in the treated medium was calculated by comparing OD₄₉₂ of saikosaponin-treated medium to that of DMSO-treated medium. The concentration of DMSO present in the medium was 1 % (v/v).

Detection of HBV DNA

After treatment with saikosaponin c and 3TC, the media of day 6 in triplicate were collected and clarified as described above. Dane particles secreted by 2.2.15 cells were precipitated using the polyethylene glycol precipitation method [14]. In brief, an equal volume of 20 % polyethylene glycol-8000 was added to the medium, followed by incubating overnight at 4 °C. On the following day, the HBV particles were collected by centrifugation at 3,500 × g at 4 °C for 30 min. The pellet was suspended in 500 μL PBS and further incubated at 37 °C for 2 h, 25 μL proteinase K (20 mg/mL) and 100 μL 10 % SDS were added at the end of the incubation. The mixture was extracted with phenol and then with chloroform twice. DNA was precipitated with 2 volumes of absolute ethanol and 1/10 volume of sodium acetate (pH 5.2) with the aid of 10 μL tRNA (10 mg/mL) as carrier. HBV DNA isolated from the triplicate media were pooled and analyzed by slot blotting onto positively charged nylon membranes (Sartorius, Germany). Hybridization was performed using a PCR-generated Dig-labeled 469 bp fragment of HBV pol gene derived from a HBV genome-containing plasmid. HBV DNA was detected by Dig labeling and Detection System (Roche Molecular Biochemicals, Germany). The primers were adwpol370 5′-TCGCTGGATGTGTCTGCGGCGTTTTAT-3′ and adwpol838 5′-GCCCCATCTTTTTGTTTTCTGAGG-3′. Quantitative densitometry measurements of autoradiographs were performed by using BIO-1D software (Vilber Lourmat, France).

LDH release assay of cytotoxicity

The cytotoxicity of saikosaponin c against 2.2.15 cells was examined according to the changes of LDH level, which reflects cytolytic activity in the test. The measurement of LDH level in the medium was conducted according to the protocol provided by the manufacturer (Promega Corp. USA.)

DNA fragmentation analysis

HepG2 cells treated with saikosaponin d were collected by centrifugation and cellular DNA was isolated as described by David et al. [17]. DNA was separated by electropheresis in a 2 % TAE agarose gel. Gel was stained in ethidium bromide solution (0.5 μg/mL) and then photographed.

Western blot

Total protein of HepG2 cells treated with saikosaponin d was extracted and subjected to SDS-PAGE. Protein was then electro-blotted onto nitrocellulose paper. Specific proteins of interest were detected by anti-caspase-3 (clone 556 425, BD Biosciences, USA), anti-caspase-7 (clone 556 541, BD Biosciences, USA) and anti-PARP (clone 556 494, BD Biosciences, USA) antibodies. They were then visualized by a relevant secondary antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Lab, USA) using the ECL system (Perkin Elmer Life Sciences, USA).

Statistical analysis

Data were expressed as means ± standard errors. The difference between the control and treated groups was evaluated using Student’s t-test. P value less than 0.05 was considered as statistically significant.

Results

Effect of saikosaponins on 2.2.15 cell viability

The results from the XTT method showed that saikosaponin d, but not a and c, exhibited a strong cytotoxicity on 2.2.15 cells (Fig. [1] A). Saikosaponin d significantly inhibited the growth of 2.2.15 cells and a dose-dependent fashion of response was observed at concentrations between 5 μg/mL and 20 μg/mL, with the IC₅₀ of 12.1 ± 1.9 μg/mL. Based on the results obtained from the LDH releasing assay, saikosaponin c was also shown to be non-toxic (Fig. [1] B).

Effect of saikosaponins on HBsAg, HBeAg and HBV DNA expression

The results showed that saikosaponin c significantly (p < 0.05) inhibited the expression of HBeAg and HBV DNA in the 2.2.15 cell line, whereas those viral markers were not affected by saikosaponins a and d (Fig. [2]). Saikosaponin c exhibited an anti-HBV activity against secretion of HBsAg (IC₅₀ = 11 μg/mL) and expression of HBV DNA (IC₅₀ = 13.4 μg/mL). Its inhibition on HBeAg was greater than on HBsAg. The inhibition of HBV DNA expression by saikosaponin c was more potent than 3TC.

Cytotoxic effect of saikosaponin d on human hepatoma HepG2 cells

Results showed that DNA fragmentation was clearly observed when HepG2 cells were treated with saikosaponin d at concentrations of 10 μg/mL or higher (Fig. [3] a). The appearance of an increasing level of internucleosomal DNA fragments showed a dose-dependent effect of saikosaponin d-induced apoptosis of HepG2 cells. Fig. [3] b and Fig. [3] c show that the activation of caspases 3 and 7 participated in the apoptosis of HepG2 cells. The levels of procaspases 3 and 7 were noted to decrease with increasing concentrations of saikosaponin d. Active caspase 7 was observed as a degraded form (lower molecular weight than procaspase 7). The activation of caspases 3 and 7 has resulted in the cleavage of PARP (113 kD) as shown by the presence of cleaved form of PARP (89 kD) (Fig. [3] d).

Discussion

Hepatitis B virus infection is an important health issue worldwide and usually results in chronic hepatitis B (CHB), liver failure, cirrhosis, and HCC [8]. According to the WHO’s report, there are about 2 billion people infected by HBV and about 350 million people known to be chronic carriers around the world [18]. HBsAg-positive patients were reported to have a relative risk of progression to HCC as compared to HBsAg-negative people [8]. In Taiwan, 15 - 20 % of HBV-infected people will develop to CHB and consequently die from cirrhosis or HCC [19].

Alpha-interferon is the drug of choice for treatment of CHB patients, but its effectiveness is unsatisfactory [11]. It was reported that less than 30 % of patients responded to the therapy and HBV-viremia relapsed in 50 % of patients at the end of the alpha-interferon treatment [20]. HBV belongs to the hepadnaviridae family and is a DNA virus with a partial double-stranded DNA genome, which replicates through a pregenomic RNA template by an RNA-dependent DNA polymerase. Lamivudine (3TC) was originally synthesized to inhibit reverse transcriptase of human immunodeficiency virus type 1 [13] and was demonstrated to effectively inhibit HBV DNA replication both in vitro and in vivo [14]. Treatment of 3TC completely eradicates HBV DNA replication in about 20 % of HBeAg-positive and HBeAg-negative patients but long-term treatment might confer to selection of a drug-resistant HBV strain [21]. Thus, there is a need for other more effective anti-HBV agents.

In this study, saikosaponin c was found to inhibit HBeAg expression and HBV DNA replication, while no significant cytotoxicity was observed (Fig. [1]). To exclude the possibility that inhibition of HBV markers was the result of 2.2.15 cell death, the LDH releasing assay was conducted on the same culture medium used in the experiments described above. The results showed that prolonged treatment (6 days) of 2.2.15 with saikosaponin c did not cause significant cell death (Fig. [1] B). Thus, the inhibition of HBsAg, HBeAg and HBV DNA expression in culture medium was not due to cytotoxicity of saikosaponin c against 2.2.15 cells. Since test compounds might inhibit the ELISA reaction and result in a false interpretation of the data, we performed an ELISA-inhibition test. The results confirmed that the inhibition of HBsAg and HBeAg by saikosaponin c was not due to the ELISA-inhibition effect (data not shown).

To identify whether the HepG2 cell viability inhibition of saikosaponin d was due to the induction of apoptosis, treated cells were subjected to DNA fragmentation analysis. After treatment with saikosaponin d for more than 6 h, DNA fragmentation was clearly observed (Fig. [3] a), suggesting HepG2 cell death. The cell apoptosis was demonstrated by internucleosomal cleavage of genomic DNA, a hallmark of apoptosis. Similarly, saikosaponin d was also noted to induce apoptosis in 2.2.15 cells, a derivative cell line of HepG2, at the concentrations used in this study (data not shown). This reveals that the mechanism of saikosaponin d-induced apoptosis might be independent of the existence of hepatitis B virus genome; it is possible that the HBV encoded protein (e. g., X protein) might act as a regulator for apoptosis signaling proteins (e. g., p53).

In investigating the role of caspase in saikosaponin d-induced apoptosis, we found that activation of caspases 3 and 7 was involved in saikosaponin d-induced apoptosis (Figs. [3] b and c). Activation of caspases 3 and 7 further resulted in PARP cleavage (Fig. [3] d). This finding was in contrast to a recent report by Hsu et al. [6], who demonstrated that saikosaponin d-induced apoptosis of human CEM lymphocytes was not involved in the activation of caspase. This discrepancy in results might probably due to the nature of cell line used. Among the three saikosaponins tested, the trans form of the hydroxy group on the D ring exhibited cytotoxic activity, whereas the anti-HBV activity was dependent on the type of sugar group attached on the A ring (Fig. [4]).

In conclusion, saikosaponin c exhibits effective anti-HBV activity and saikosaponin d possesses potent cytotoxic activity against hepatoma cells. With a potency that is greater than that of 3TC, saikosaponin c is therefore worthy to be further investigated as a potential new anti-HBV agent in vivo.

References

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Professor Chun-Ching Lin

Graduate Institute of Pharmaceutical Sciences

College of Pharmacy

Kaohsiung Medical University

100 Shih-Chuan 1st Road

Kaohsiung 807

Taiwan

Republic of China

Phone: +886-7-3121101 ext. 2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net

References

• 1 Yen M H, Lin C C, Chuang C H, Liu S Y. Evaluation of root quality of Bupleurum species by TLC scanner and the liver protective effects of ”Xiao-Chai-Hu-Tang” prepared using three different Bupleurum species.  J Ethnopharmacol. 1991;  34 155-65
  CrossrefPubMedSearch in Google Scholar
• 2 Chang R S, Yeung H W. Inhibition of growth of human immunodeficiency virus in vitro by crude extracts of Chinese medicinal herbs.  Antiviral Res. 1988;  9 163-75
  CrossrefPubMedSearch in Google Scholar
• 3 Ono K, Nakane H, Fukushima M, Chermann J C, Barre-Sinoussi F. Differential inhibition of the activities of reverse transcriptase and various cellular DNA polymerases by a traditional Kampo drug, sho-saiko-to.  Biomed Pharmacother. 1990;  44 13-6
  CrossrefPubMedSearch in Google Scholar
• 4 Gato W, Kadota S, Namba T, Kurokawa M, Shiraki K. Suppression of hepatitis B virus surface antigen secretion by traditional plant medicines.  Phytother Res. 1996;  10 504-7
  CrossrefPubMedSearch in Google Scholar
• 5 Ushio Y, Abe H. The effects of saikosaponin on macrophage functions and lymphocyte proliferation.  Planta Med. 1991;  57 511-4
  PubMedSearch in Google Scholar
• 6 Hsu M J, Cheng J S, Huang H C. Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl-2 mRNA.  Br J Pharmacol. 2000;  131 1285-93
  CrossrefPubMedSearch in Google Scholar
• 7 Ushio Y, Abe H. Inactivation of measles virus and herpes simplex virus by saikosaponin d.  Planta Med. 1992;  58 171-3
  PubMedSearch in Google Scholar
• 8 Beasley R P, Hwang L Y, Lin C C, Chien C S. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan.  Lancet. 1981;  2 1129-33
  PubMedSearch in Google Scholar
• 9 Beasley R P, Hwang L Y, Lee G C, Lan C C, Roan C H, Huang F Y. et al . Prevention of perinatally transmitted hepatitis B virus infections with hepatitis B immune globulin and hepatitis B vaccine.  Lancet. 1983;  2 1099-102
  PubMedSearch in Google Scholar
• 10 Chang M H, Shau W Y, Chen C J, Wu T C, Kong M S, Liang D C. et al . Hepatitis B vaccination and hepatocellular carcinoma rates in boys and girls.  JAMA. 2000;  284 3040-2
  CrossrefPubMedSearch in Google Scholar
• 11 Hope R L, Weltman M, Dingley J, Fiatarone J, Hope A H, Craig P I. et al . Interferon alpha for chronic active hepatitis B. Long-term follow-up of 62 patients: outcomes and predictors of response.  Med J Aust. 1995;  162 8-11
  PubMedSearch in Google Scholar
• 12 Niederau C, Heintges T, Lange S, Goldmann G, Niederau C M, Mohr L. et al . Long-term follow-up of HBeAg-positive patients treated with interferon alfa for chronic hepatitis B.  N Engl J Med. 1996;  334 1422-7
  CrossrefPubMedSearch in Google Scholar
• 13 Soudeyns H, Yao X I, Gao Q, Belleau B, Kraus J L, Nguyen-Ba N. et al . Anti-human immunodeficiency virus type 1 activity and in vitro toxicity of 2′-deoxy-3′-thiacytidine (BCH-189), a novel heterocyclic nucleoside analog.  Antimicrob Agents Chemother. 1991;  35 1386-90
  CrossrefPubMedSearch in Google Scholar
• 14 Doong S L, Tsai C H, Schinazi R F, Liotta D C, Cheng Y C. Inhibition of the replication of hepatitis B virus in vitro by 2′,3′-dideoxy-3′-thiacytidine and related analogues.  Proc Natl Acad Sci (USA). 1991;  88 8495-9
  PubMedSearch in Google Scholar
• 15 Honkoop P, de Man R A, Heijtink R A, Schalm S W. Hepatitis B reactivation after lamivudine.  Lancet. 1995;  346 1156-7
  CrossrefPubMedSearch in Google Scholar
• 16 Honkoop P, Niesters H G, de Man R A, Osterhaus A D, Schalm S W. Lamivudine resistance in immunocompetent chronic hepatitis B. Incidence and patterns.  J Hepatol. 1997;  26 1393-5
  CrossrefPubMedSearch in Google Scholar
• 17 David L, Spector R DG, Leslie A. Leinwand Cell - A Laboratory Manual. CSHL Press 1998: pp. 1511
  Search in Google Scholar
• 18 Ayoola E A, Balayab M S, Deinhardt F, Gust I, Kureshi A W, Maynard J E. et al . Bull.  World Health Org. 1988;  66 443-55
  PubMedSearch in Google Scholar
• 19 Beasley R P, Hwang L Y, Lin C C, Leu M L, Stevens C E, Szmuness W, Chen K P. Incidence of hepatitis B virus infections in preschool children in Taiwan.  J Infect Dis. 1982;  146 198-204
  PubMedSearch in Google Scholar
• 20 Fattovich G, Brollo L, Alberti A, Pontisso P, Giustina G, Realdi G. Long-term follow-up of anti-HBe-positive chronic active hepatitis B.  Hepatol. 1988;  8 1651-4
  PubMedSearch in Google Scholar
• 21 Tipples G A, Ma M M, Fischer K P, Bain V G, Kneteman N M, Tyrrell D L. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo .  Hepatol. 1996;  24 714-7
  PubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Pharmaceutical Sciences

College of Pharmacy

Kaohsiung Medical University

100 Shih-Chuan 1st Road

Kaohsiung 807

Taiwan

Republic of China

Phone: +886-7-3121101 ext. 2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net