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Fructus schisandrae (Wuweizi)-containing compound inhibits secretion of HBsAg and HBeAg in hepatocellular carcinoma cell line
Biomedicine & Pharmacotherapy 61 (2007) 606e610 www.elsevier.com/locate/biopha
Fructus schisandrae (Wuweizi)-containing compound inhibits secretion of HBsAg and HBeAg in hepatocellular carcinoma cell line Wings T.Y. Loo, Mary N.B. Cheung, Louis W.C. Chow* UNIMED Medical Institute, 10/F Luk Kwok Centre, 72 Gloucester Road, Wanchai, Hong Kong Available online 14 September 2007
Abstract Chronic hepatitis B is probably the major cause of cirrhosis and hepatocellular carcinoma. The detection of the hepatitis B virus surface antigen (HBsAg) and HBV core protein, the e antigen (HBeAg), indicates infection with the hepatitis B virus and replication activity, respectively. Fructus schisandrae containing compound (KY88) may affect the elimination of HBV, strengthen the immune system, as well as stimulate liver cell regeneration. The present study was conducted to demonstrate the ability of KY88 in inhibiting hepatocellular carcinoma cell proliferation and secretions of HBsAg and HBeAg. The hepatocellular carcinoma cell line HB-8064 was treated by KY88 followed by the measurement of cell proliferation rate and secretions of HBsAg and HBeAg on days 1, 3, 5, and 7. A semi-quantitative RTePCR method was used to quantify the expression of the mRNA. Seventy SpragueeDawley rats were fed for 28 days with purified KY88 for a toxicity test. The expression of surface and e antigens was lower when the cells were treated for a longer time with KY88 or when the doses were higher. Oneway ANOVA analysis confirmed the mRNA content of HBsAg to be significantly less than control. The body weight did not show a significant difference compared to the control group. Fructus schisandrae-containing compound (KY88) was potentially effective in suppressing the proliferation of hepatocellular carcinoma cells. The decreased secretion and gene expression of HBsAg and HBeAg might restrict the growth of tumour masses. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Fructus schisandrae; Hepatocellular carcinoma cells; HBsAg; HbeAg
1. Introduction On a worldwide scale, about 300 million people have chronic infection with hepatitis B virus (HBV). The prevalence of hepatitis B varies greatly in different parts of the world: it is higher in African and Asian (>75%) countries than in the Americas, Australia and Western Europe; in urban than in rural areas; and in men than in women [1]. In developed countries the risk of exposure to hepatitis B is high in certain categories of people [1]. Hepatitis B is caused by the hepatitis B virus (HBV), a DNA hepadnavirus (hepar liver þ DNA) which is structurally and immunologically complex [2]. HBV, being a hepatropic virus, will reside and multiply in hepatocytes after entering the body, and cause hepatic injury and inflammation
* Corresponding author. Tel.: þ852 2861 0286; fax: þ852 2861 1386. E-mail address: [email protected] (L.W.C. Chow). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.08.023
(hepatitis) to varying degrees. Chronic hepatitis B is probably the major cause of cirrhosis and hepatocellular carcinoma [2]. The detection of the hepatitis B virus surface antigen (HBsAg) indicates infection with the hepatitis B virus, while the detection of the HBV core protein, the e antigen (HBeAg), indicates replication activity [3]. Fructus schisandrae-containing compounds are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF), possess anti-inflammatory effects, and induce apoptosis, as reported by our previous study [4e6]. KY88 (Hon Ding International Ltd, Hong Kong, China) is one such compound, used by Chinese medicine practitioners in the treatment of acute and chronic hepatitis B virus infection. KY88 is a herbal extract and blend of Fructus schisandrae, Akebia, Bupleurum, Capillaris, Desmodium and some other Chinese herbs. It is believed to be able to affect the elimination of HBV, strengthen the immune system, as well as stimulate liver cell regeneration.
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 61 (2007) 606e610
The present study conducts to demonstrate the ability of a Fructus schisandrae-containing compound (KY88) in inhibiting hepatocellular carcinoma cell proliferation and secretions of HBsAg and HBeAg. 2. Methods and materials 2.1. Fructus schisandrae-containing compound (KY88) preparation KY88 was obtained from Hon Ding (HK) International Limited. The ingredients of KY88 are Schizandrae fructus, Bupleuri radix, Artemisiae capillaris, Desmodii herba, Poria sclerotium, Lithospermi radix, Paeoniae radix, Phellodendri cortex, Scutellariae radix, and Trichosanthis radix [4]. All Chinese herbs were purchased from Chengdu of China. Ten grams of each of the above ingredients were primarily washed. Through the process of extraction, concentration and purification, the essence of the herbal extractsdKY88dwas assembled. This capsule had been tested by SGS Hong Kong Ltd (Socie´te´ Ge´ne´rale de Surveillance) to be free of heavy metals and micro-organisms. Prior to conduction of experiments, KY88 (50 g) was extracted with methanol (500 ml 3) [7]. The solid residue of the crude extract was then dissolved in dimethyl sulphoxide to a concentration of 92 mg/ml and stored at 4 C until use. For use, the KY88 was diluted (10 mg/ml) in Hanks’ Balanced Salt Solution (HBSS) (Invitrogen, USA) and filtered using 0.22 MicroCellulose Acetate (Corning, USA) for sterilisation. 2.2. Animal study Female SpragueeDawley (SD) rats were bred and maintained in The University of Hong Kong Animal Unit. SD rats were housed under constant environmental conditions (photoperiod, temperature, air humidity, food) [8]. At a weight of 180e200 g, 40 rats were fed with 1 ml of 4 mg purified KY88 daily whereas 1 ml saline was fed for the other 30 rats in the control group. According to the description on the data sheet of KY88, 1.2 g should be taken by adults each day, assuming an average body weight of 60 kg. Hence, the amount to be fed for rats was calculated based on the body weight [9]. For the control group, 30 rats were given same amount of saline instead. Animals were monitored every day in terms of behaviour and excretions, and they were weighed once a week throughout a 28-day observation period. 2.3. Cell culture and growth studies The hepatocellular carcinoma cell line HB-8064 (ATCC, USA) was selected for this study. It possesses an integrated hepatitis B virus genome and produces HBsAg, HBeAg and the major plasma proteins [10]. The cells were seeded at a rate of 5000 per well in 24-well plates. After culture for 24 h, KY 88 was added into the MEM medium (Invitrogen, USA) with 10% foetal bovine serum and the cells were further cultured for 1, 3, 5 or 7 days. The cell viability and cell
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proliferation rate were measured on those days by Microplate Reader (Sunrise, TECAN, Austria) using MTT (Sigma, USA). A pilot test was carried out using various concentrations of KY88 to see which were most suited for the experiments. Concentrations of aqueous extracts of KY88 were tested in a range of 0.05e2 mg/ml. The final concentrations were confirmed to be 1, 0.5 and 0.1 mg/ml. 2.4. MTT assay MTT was dissolved at 5 mg/ml in PBS for use. In brief, 20 ml of MTT solution was added to each well of a 96-well microplate, and the microplate was further incubated at 37 C for 4 h. Two hundred microlitres of acidified isopropanol was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed using a Microplate Reader with 570 nm wavelength. Data were expressed as mean absorbance value (OD). 50% inhibition was calculated as: (OD of control group OD of KY test groups/OD of control group) 100%. 2.5. Cell viability measurement Samples of monolayer cells on the dish were detached using trypsin and ethylenediamine tetra-acetic acid (Invitrogen). Cells were mixed with 1:1 trypan blue and counted by a haemocytometer [11]. 2.6. HBeAg and HBsAg determination by ELISA The rate of 1.0 106 cells were seeded in a 100-mm tissue culture dish for each concentration of KY88, and the culture medium was collected on days 1, 3, 5, and 7 for measurement of secretions of HBsAg and HBeAg by ELISA kits (Equipar, Italy). The plates were incubated for 2 h and the substrate tetramethylbenizidine was added into each well for 20 min in the dark at room temperature. The optical density of the plates was read at 450 nm wavelength. Sensitivity was calculated as a minimum detectable concentration expressed in U PEI/ml. 2.7. HBeAg and HbsAg determination by semi-quantitative RTePCR RNA extraction of the cultured cells treated by different concentrations of KY88 was performed using Trizol (Invitrogen). Total RNA was transformed to cDNA by RTePCR by means of Superscript II (Invitrogen). The primer sequences used were as follows: HBVsAg: 50 -TTCCTATGGGAGTGG-30 (sense) 50 -CCCAATACCACATCATCC-30 (antisense) HBVeAg: 50 -ATGGACATTGACACGTATAAA-30 (sense) 50 -TTAAACAACAGTAGTTTC-30 (antisense) Platinum PCR Supermix (Invitrogen) was employed for running the PCR; the PCR products were analysed electrophoretically on a 1% agarose gel, followed by scanning
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 61 (2007) 606e610
densitometry (measuring optical density, OD), using Lab Works-Image Acquisition and Analysis Software (UVP, USA). 2.8. Statistical analysis Statistical significance of doseeresponse test (MTT), mRNA expression and secretion of HbeAg and HbsAg, and body weight of SD rats was analyzed by SPSS (14.0, USA) one-way ANOVA. 3. Results
.6
Mean of secretion of HbsAg (U PEI/ml)
608
.5
.4
.3
.2
.1
3.1. MTT assay and cell viability measurement 0.0
After serial trial, the range of the minimal concentrations was confirmed. The effect of KY88 on the potential inhibition of secretion of HBsAg and HBeAg was investigated using various techniques. In the MTT assay, a statistically significant suppression of hepatocellular carcinoma cell proliferation was observed in concentrations 0.5 mg/ml and 1 mg/ml KY88 on day 1, but this significance was seen in all three concentrations at other time points. Besides, the cell proliferation rate of the test groups was considerably lower than the control group (Fig. 1). The cell number in the control group increased up to day 5 when the cells were eventually deprived of the declining nutrients in culture medium on day 7. There was a remarkable suppression of cell proliferation in three concentrations from day 3 on. 3.2. HBeAg and HBsAg determination by ELISA The secretion of both HBeAg and HBsAg was directly proportional to the concentration of KY88. HBsAg showed a greater extent of inhibition compared to HBeAg (Figs. 2 and 3). The explanation for this pattern is likely that there is
control
1mg/ml
0.5mg/ml
0.1mg/ml
Groups Fig. 2. The mean level of HBsAg secreted by hepatocellular carcinoma cells pretreated with different concentrations of KY88.
a higher concentration of HBsAg produced by the hepatocellular carcinoma cell line HB-8064 than HBeAg. 3.3. HBeAg and HbsAg determination by semi-quantitative RTePCR A semi-quantitative RTePCR method was used to quantify the expression of the genes. It was found that the expression of surface antigens was lower when the cells were treated for a longer time with KY88 or when the doses were higher. The results are similar so only HBsAg is shown (Fig. 4). One-way ANOVA analysis confirmed the mRNA content of HBsAg to be statistically significant for 0.5 mg/ml and 1 mg/ml KY88. On the other hand, HBeAg showed a difference in all test groups compared with the control group although statistical significance was not achieved. .06
.8
Groups .6 0.1mg/ml
.4 0.5mg/ml
.2
1mg/ml
control
0.0 1
3
5
7
Day Fig. 1. Dose responsive cell proliferation of KY88 measured by MTT assay in terms of optical density on days 1, 3, 5 and 7.
Mean of secretion of HbeAg (U PEI/ml)
Standard error of mean optical densities
1.0
.05
.04
.03
.02 control
1mg/ml
0.5mg/ml
0.1mg/ml
Groups Fig. 3. The mean level of HBsAg secreted by hepatocellular carcinoma cells pretreated with different concentrations of KY88.
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 61 (2007) 606e610 8
Standard error of mean mRNA content of HBsAg
7 6 Group
5 4
control
3 0.1mg/ml
2 0.5mg/ml
1 1mg/ml
0 1
3
5
7
Day Fig. 4. The quantification of HBsAg analysed by PCR product density in terms of optical density.
4. Discussion The three main experimental tools currently used in clinical or laboratory studies of HBV are a drug called lamivudine and interferons, vaccines with Chinese medicine being the adjuvant therapy. Their antiviral effects are achieved by lowering the secretions of HBsAg and HBeAg into the serum in humans. Lai’s study showed that lamivudine in doses of 25 mg, 100 mg, and 300 mg were effective in suppressing HBV DNA in Chinese HBsAg carriers [12]. The results of So et al. demonstrated that recombinant interferon alpha-2a (rIFN) is effective in the management of patients who are HBsAg and/or HBeAg positive [13]. Senturk et al. concluded that a pre-S2-containing vaccine (Genhevac-B) decreased serum HBV DNA levels in the majority of patients with chronic hepatitis B and sustained clearance was achieved in some patients [14]. Similarly, Yu et al. tested YGAPM, which is an effective drug in treating chronic hepatitis B, which can effectively negatively convert the HBV marker [15]. With regard to the antiviral effect of the drug Phyllanthus, the first current reports by two Indian groups, Thyagarayan et al. and Blumberg et al., indicated that P. amarus extract reduced detectable hepatitis B surface antigen (HBsAg) to negative in 59% of HBV-positive patients compared with 4% in the controls [16,17]. Herein, we describe a herbal compound called KY88 used among Chinese communities. Each individual ingredient of this herbal mixture confers advantages in treating or protecting liver disorders. Desmodium was employed for its ability to interfere with histamine-induced contractions, therefore it is widely used in India in treating asthma attacks as an anaphylactic drug [18]. Lithospermum has been used for treating tumours and inflammation in China and also inhibited proliferation and migration of endothelial cells in culture and network formation by endothelial cells on Matrigel in vitro [19]. Yamamoto
609
et al. stated that Capillaris has been used for treatment of various liver disorders and it exhibited potent anti-apoptotic activity [20]. Hoelen has been shown in conjunction with other herbs to reduce elevated levels of lipid peroxide in serum [21]. Lian et al. on the other hand proved its potency in suppressing cell viability and telomerase activity in hormonerefractory and chemo-resistant cancer cell lines [22]. Peony was used in strengthening the viability and improving the nutritional state [23]. Treatment with phellodendron was shown to have a significant hepatic protective effect [24]. Leung et al. reported that trichosanthin could inhibit the growth of a murine malignant tumour (MBL-2), both in vivo and in vitro [25]. A combination of the above ingredients was effective in inducing lymphocyte proliferation and the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), which was linked to the host inflammatory response against pathogens as reported by us earlier [4,5]. Not only was this effect seen in humans, another study in SD rats, although not shown here, also revealed similar results. When the peritoneal injection of 5-flurouracil at a dosage of 250 mg/kg was performed preceded by orally fed 1 mg/ml KY88 for 7 days, the total white blood cell count was comparable with that of the control group. In the present study, KY88 at the designated concentrations was able to suppress cancer cell proliferation throughout the 7-day period. We found that a higher percentage of stained blue cells were present at higher concentrations of KY88. By referring to a similar study in which we performed DNA fragmentation, we concluded that the above findings were due to apoptosis [6]. The production of the viral antigen (HBsAg) is attributed to the integration of the viral genome into the DNA of the hepatocytes. The presence of HBsAg indicates that the person is a carrier and potentially infective. This state can persist for months until recovery, or for years in chronic carrier states [2]. HBV carriers demonstrate the significance of hepatitis B surface antigen (HBsAg) and hepatitis B ‘‘e’’ antigen (HBeAg) in their sera, these being indicative of active disease or high infectivity. Infectivity of this particle is so high that even 0.0001 ml serum containing infective virus could transmit the disease. Prolonged persistence of the e antigen in serum indicates that the patient will eventually develop chronic liver damage [2]. The herbal mixture described herein was able to lower the secretions of HBsAg using the designated kits. For secretion of HBsAg, the mean value was very similar for all three concentrations whereas the control was almost triple these values (Fig. 3). This was consistent with other studies carried out which also demonstrated a decrease of 60e70% after osthole (another Chinese herbal medicine) treatment, without any detectable cytotoxic effects [26]. Results for the control group illustrated that it was almost double that of other concentrations for the secretion of HBeAg (Fig. 4). Another experiment revealed that the maximum inhibitory rates were higher for HBsAg than HBeAg [27]. KY88 was proved to have induced apoptosis and production of interleukin-4 and tumour necrosis factor-a in liver cancer cells in vitro in our former study [6].
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This study provides insight into the mechanism of the action of KY88 on hepatocellular carcinoma cells. Knowledge on this subject is limited although KY88 is believed to be effective against HBV. While KY88 may act on the virus directly, this no doubt has given rise to the lower level of secretions of both HBsAg and HBeAg detected. Activation of the host inflammatory response is certainly an important means of combating HBV infection. Further clinical evaluation of this herbal compound would hence appear worthwhile. In conclusion, our experiments managed to show that Fructus schisandrae-containing compound (KY88) has a higher impact in suppressing the secretion of HBsAg than HBeAg. However, the mechanism of action has yet to be explained. References [1] Mertens T, Tondorf G, Siebolds M, Kruppenbacher JP, Shrestha SM, Mauff G, et al. Epidemiology of HIV and hepatitis B virus (HBV) in selected African and Asian populations. Infection 1989;17:4e7. [2] Samaranayake LP. Essential microbiology for dentistry. New York: Churchill Livingstone; 2002. p. 187e195. [3] Kamisango K, Kamogawa C, Sumi M, Goto S, Hirao A, Gonzales F, et al. Quantitative detection of hepatitis B virus by transcription-mediated amplification and hybridization protection assay. J Clin Microbiol 1999;37:310e4. [4] Chow LWC, Loo WTY, Sham JST. Effects of a herbal compound containing bupleurum on human lymphocytes. H K Med J 2001;7:408e13. [5] Chow LWC, Loo TY, Sham STJ. Cytokine production by human lymphocytes stimulated by a herbal compound containing Bupleurum (KY88 LIVER LIVO). Acta Pharmacol Sin 2003;24:140e4. [6] Chow LWC, Loo WTY, Cheung MNB. Radix bupleuri containing compound (KY88 Liver-Livo) induces apoptosis and production of interleukin-4 and tumor necrosis factor-a in liver cancer cells in vitro. Am J Chin Med 2004;32:185e93. [7] Lin MC, Lin JH, Wen KC. Detection and determination of phenformin in Chinese medicinal capsules by GC-MS and HPLC. J Food Drug Anal 2001;9:139e44. [8] Macejova D, Brtko J. Chemically induced carcinogenesis: a comparison of 1-methyl-1-nitrosourea, 7,12-dimethylbenzanthracene, diethylnitrosoamine and azoxymethan models (minireview). Endocrine Regul 2001;35: 53e9. [9] Shih CK, Chiang W, Kuo ML. Effects of adlay on azoxymethane-induced colon carcinogenesis in rats. Food Chem Toxicol 2004;42:1339e47. [10] Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980;209:497e9. [11] Phillips HJ. Dye exclusion tests for cell viability. In: Kruse Jr PF, Patterson Jr MK, editors. Tissue culture methods and applications. New York: Academic Press; 1973. p. 406e8.
[12] Lai CL, Chine CK, Tung AKM, Li E, Young J, Hill A, et al. Lamivudine is effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface antigen carriers: A placebo-controlled trial. Hepatology 1997;25:241e4. [13] Ola SO, Anomneze EE, Chukwuani CM, Ojo OS, Ndububa DA, Onyenekwe B, et al. Interferon alpha-2a (Roferon-A) in the management of chronic hepatitis B infection: results of an open prospective study in Nigerian patients. West Afr J Med 2000;19:259e64. [14] Senturk H, Tabak F, Akdogan M, Erdem L, Mert A, Ozaras R, et al. Therapeutic vaccination in chronic hepatitis B. J Gastroenterol Hepatol 2002;17:72e6. [15] Liu KZ. Relation between traditional Chinese medicine differentiation of syndromes and HBV antigen, HBV DNA in serum and liver tissues and pathological changes in chronic hepatitis B. Zhongguo Zhong Xi Yi Jie He Za Zhi 1992;12:11e33. [16] Thyagarajan SP, Subramanian S, Thirunalasundari T, Venkateswaran PS, Blumberg BS. Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus. Lancet 1988;2:764e6. [17] Blumberg BS, Millman I, Venkateswaran PS, Thyagarajan SP. Hepatitis B virus and primary hepatocellular carcinoma: Treatment of HBV carriers with Phyllanthus amarus. Vaccine 1990;8:S86e91. [18] Addy ME, Awumey EM. Effects of the extracts of Desmodium adscendens on anaphylaxis. J Ethnopharmacol 1984;11:283e92. [19] Hisa T, Kimura Y, Takada K, Suzuki F, Takigawa M. Shikonin, an ingredient of Lithospermum erythrorhizon, inhibits angiogenesis in vivo and in vitro. Anticancer Res 1998;18:783e90. [20] Yamamoto M, Ogawa K, Morita M, Fukuda K, Komatsu Y. The herbal medicine Inchin-ko-to inhibits liver cell apoptosis induced by transforming growth factor beta 1. Hepatology 1996;23:552e9. [21] Nishiyama N, Zhou Y, Saito H. Ameliorative effects of chronic treatment using DX-9386, a traditional Chinese prescription, on learning performance and lipid peroxide content in senescence accelerated mouse. Biol Pharm Bull 1994;17:1481e4. [22] Lian Z, Fujimoto J, Yokoyama Y, Niwa K, Tamaya T. Herbal complex suppresses telomerase activity in chemo-endocrine resistant cancer cell lines. Eur J Gynaecol Oncol 2001;22:347e9. [23] Wei L, Chen B, Ye R, Li H. Treatment of complications due to peritoneal dialysis for chronic renal failure with traditional Chinese medicine. J Trad Chin Med 1999;19:3e9. [24] Chiu HF, Lin CC, Yang CC, Yang F. The pharmacological and pathological studies on several hepatic protective crude drugs from Taiwan (I). Am J Chin Med 1988;16:127e33. [25] Leung KN, Yeung HW, Leung SO. The immunomodulatory and antitumor activities of trichosanthin-an abortifacient protein isolated from tian-hua-fen (Trichosanthes kirilowii). Asian Pacific J Allergy Immunol 1986;4:111e20. [26] Huang RL, Chen CC, Huang YL, Hsieh DJ, Hu CP, Chen CF, et al. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro. Hepatology 1996;24:508e15. [27] Zhang B, Chen J, Li H, Xu X. Study on the Rheum palmatum volatile oil against HBV in cell culture in vitro. Zhong Yao Cai 1998;21:524e6.
Fructus schisandrae (Wuweizi)-containing compound inhibits secretion of HBsAg and HBeAg in hepatocellular carcinoma cell line Wings T.Y. Loo, Mary N.B. Cheung, Louis W.C. Chow* UNIMED Medical Institute, 10/F Luk Kwok Centre, 72 Gloucester Road, Wanchai, Hong Kong Available online 14 September 2007
Abstract Chronic hepatitis B is probably the major cause of cirrhosis and hepatocellular carcinoma. The detection of the hepatitis B virus surface antigen (HBsAg) and HBV core protein, the e antigen (HBeAg), indicates infection with the hepatitis B virus and replication activity, respectively. Fructus schisandrae containing compound (KY88) may affect the elimination of HBV, strengthen the immune system, as well as stimulate liver cell regeneration. The present study was conducted to demonstrate the ability of KY88 in inhibiting hepatocellular carcinoma cell proliferation and secretions of HBsAg and HBeAg. The hepatocellular carcinoma cell line HB-8064 was treated by KY88 followed by the measurement of cell proliferation rate and secretions of HBsAg and HBeAg on days 1, 3, 5, and 7. A semi-quantitative RTePCR method was used to quantify the expression of the mRNA. Seventy SpragueeDawley rats were fed for 28 days with purified KY88 for a toxicity test. The expression of surface and e antigens was lower when the cells were treated for a longer time with KY88 or when the doses were higher. Oneway ANOVA analysis confirmed the mRNA content of HBsAg to be significantly less than control. The body weight did not show a significant difference compared to the control group. Fructus schisandrae-containing compound (KY88) was potentially effective in suppressing the proliferation of hepatocellular carcinoma cells. The decreased secretion and gene expression of HBsAg and HBeAg might restrict the growth of tumour masses. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Fructus schisandrae; Hepatocellular carcinoma cells; HBsAg; HbeAg
1. Introduction On a worldwide scale, about 300 million people have chronic infection with hepatitis B virus (HBV). The prevalence of hepatitis B varies greatly in different parts of the world: it is higher in African and Asian (>75%) countries than in the Americas, Australia and Western Europe; in urban than in rural areas; and in men than in women [1]. In developed countries the risk of exposure to hepatitis B is high in certain categories of people [1]. Hepatitis B is caused by the hepatitis B virus (HBV), a DNA hepadnavirus (hepar liver þ DNA) which is structurally and immunologically complex [2]. HBV, being a hepatropic virus, will reside and multiply in hepatocytes after entering the body, and cause hepatic injury and inflammation
* Corresponding author. Tel.: þ852 2861 0286; fax: þ852 2861 1386. E-mail address: [email protected] (L.W.C. Chow). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.08.023
(hepatitis) to varying degrees. Chronic hepatitis B is probably the major cause of cirrhosis and hepatocellular carcinoma [2]. The detection of the hepatitis B virus surface antigen (HBsAg) indicates infection with the hepatitis B virus, while the detection of the HBV core protein, the e antigen (HBeAg), indicates replication activity [3]. Fructus schisandrae-containing compounds are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF), possess anti-inflammatory effects, and induce apoptosis, as reported by our previous study [4e6]. KY88 (Hon Ding International Ltd, Hong Kong, China) is one such compound, used by Chinese medicine practitioners in the treatment of acute and chronic hepatitis B virus infection. KY88 is a herbal extract and blend of Fructus schisandrae, Akebia, Bupleurum, Capillaris, Desmodium and some other Chinese herbs. It is believed to be able to affect the elimination of HBV, strengthen the immune system, as well as stimulate liver cell regeneration.
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 61 (2007) 606e610
The present study conducts to demonstrate the ability of a Fructus schisandrae-containing compound (KY88) in inhibiting hepatocellular carcinoma cell proliferation and secretions of HBsAg and HBeAg. 2. Methods and materials 2.1. Fructus schisandrae-containing compound (KY88) preparation KY88 was obtained from Hon Ding (HK) International Limited. The ingredients of KY88 are Schizandrae fructus, Bupleuri radix, Artemisiae capillaris, Desmodii herba, Poria sclerotium, Lithospermi radix, Paeoniae radix, Phellodendri cortex, Scutellariae radix, and Trichosanthis radix [4]. All Chinese herbs were purchased from Chengdu of China. Ten grams of each of the above ingredients were primarily washed. Through the process of extraction, concentration and purification, the essence of the herbal extractsdKY88dwas assembled. This capsule had been tested by SGS Hong Kong Ltd (Socie´te´ Ge´ne´rale de Surveillance) to be free of heavy metals and micro-organisms. Prior to conduction of experiments, KY88 (50 g) was extracted with methanol (500 ml 3) [7]. The solid residue of the crude extract was then dissolved in dimethyl sulphoxide to a concentration of 92 mg/ml and stored at 4 C until use. For use, the KY88 was diluted (10 mg/ml) in Hanks’ Balanced Salt Solution (HBSS) (Invitrogen, USA) and filtered using 0.22 MicroCellulose Acetate (Corning, USA) for sterilisation. 2.2. Animal study Female SpragueeDawley (SD) rats were bred and maintained in The University of Hong Kong Animal Unit. SD rats were housed under constant environmental conditions (photoperiod, temperature, air humidity, food) [8]. At a weight of 180e200 g, 40 rats were fed with 1 ml of 4 mg purified KY88 daily whereas 1 ml saline was fed for the other 30 rats in the control group. According to the description on the data sheet of KY88, 1.2 g should be taken by adults each day, assuming an average body weight of 60 kg. Hence, the amount to be fed for rats was calculated based on the body weight [9]. For the control group, 30 rats were given same amount of saline instead. Animals were monitored every day in terms of behaviour and excretions, and they were weighed once a week throughout a 28-day observation period. 2.3. Cell culture and growth studies The hepatocellular carcinoma cell line HB-8064 (ATCC, USA) was selected for this study. It possesses an integrated hepatitis B virus genome and produces HBsAg, HBeAg and the major plasma proteins [10]. The cells were seeded at a rate of 5000 per well in 24-well plates. After culture for 24 h, KY 88 was added into the MEM medium (Invitrogen, USA) with 10% foetal bovine serum and the cells were further cultured for 1, 3, 5 or 7 days. The cell viability and cell
607
proliferation rate were measured on those days by Microplate Reader (Sunrise, TECAN, Austria) using MTT (Sigma, USA). A pilot test was carried out using various concentrations of KY88 to see which were most suited for the experiments. Concentrations of aqueous extracts of KY88 were tested in a range of 0.05e2 mg/ml. The final concentrations were confirmed to be 1, 0.5 and 0.1 mg/ml. 2.4. MTT assay MTT was dissolved at 5 mg/ml in PBS for use. In brief, 20 ml of MTT solution was added to each well of a 96-well microplate, and the microplate was further incubated at 37 C for 4 h. Two hundred microlitres of acidified isopropanol was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed using a Microplate Reader with 570 nm wavelength. Data were expressed as mean absorbance value (OD). 50% inhibition was calculated as: (OD of control group OD of KY test groups/OD of control group) 100%. 2.5. Cell viability measurement Samples of monolayer cells on the dish were detached using trypsin and ethylenediamine tetra-acetic acid (Invitrogen). Cells were mixed with 1:1 trypan blue and counted by a haemocytometer [11]. 2.6. HBeAg and HBsAg determination by ELISA The rate of 1.0 106 cells were seeded in a 100-mm tissue culture dish for each concentration of KY88, and the culture medium was collected on days 1, 3, 5, and 7 for measurement of secretions of HBsAg and HBeAg by ELISA kits (Equipar, Italy). The plates were incubated for 2 h and the substrate tetramethylbenizidine was added into each well for 20 min in the dark at room temperature. The optical density of the plates was read at 450 nm wavelength. Sensitivity was calculated as a minimum detectable concentration expressed in U PEI/ml. 2.7. HBeAg and HbsAg determination by semi-quantitative RTePCR RNA extraction of the cultured cells treated by different concentrations of KY88 was performed using Trizol (Invitrogen). Total RNA was transformed to cDNA by RTePCR by means of Superscript II (Invitrogen). The primer sequences used were as follows: HBVsAg: 50 -TTCCTATGGGAGTGG-30 (sense) 50 -CCCAATACCACATCATCC-30 (antisense) HBVeAg: 50 -ATGGACATTGACACGTATAAA-30 (sense) 50 -TTAAACAACAGTAGTTTC-30 (antisense) Platinum PCR Supermix (Invitrogen) was employed for running the PCR; the PCR products were analysed electrophoretically on a 1% agarose gel, followed by scanning
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 61 (2007) 606e610
densitometry (measuring optical density, OD), using Lab Works-Image Acquisition and Analysis Software (UVP, USA). 2.8. Statistical analysis Statistical significance of doseeresponse test (MTT), mRNA expression and secretion of HbeAg and HbsAg, and body weight of SD rats was analyzed by SPSS (14.0, USA) one-way ANOVA. 3. Results
.6
Mean of secretion of HbsAg (U PEI/ml)
608
.5
.4
.3
.2
.1
3.1. MTT assay and cell viability measurement 0.0
After serial trial, the range of the minimal concentrations was confirmed. The effect of KY88 on the potential inhibition of secretion of HBsAg and HBeAg was investigated using various techniques. In the MTT assay, a statistically significant suppression of hepatocellular carcinoma cell proliferation was observed in concentrations 0.5 mg/ml and 1 mg/ml KY88 on day 1, but this significance was seen in all three concentrations at other time points. Besides, the cell proliferation rate of the test groups was considerably lower than the control group (Fig. 1). The cell number in the control group increased up to day 5 when the cells were eventually deprived of the declining nutrients in culture medium on day 7. There was a remarkable suppression of cell proliferation in three concentrations from day 3 on. 3.2. HBeAg and HBsAg determination by ELISA The secretion of both HBeAg and HBsAg was directly proportional to the concentration of KY88. HBsAg showed a greater extent of inhibition compared to HBeAg (Figs. 2 and 3). The explanation for this pattern is likely that there is
control
1mg/ml
0.5mg/ml
0.1mg/ml
Groups Fig. 2. The mean level of HBsAg secreted by hepatocellular carcinoma cells pretreated with different concentrations of KY88.
a higher concentration of HBsAg produced by the hepatocellular carcinoma cell line HB-8064 than HBeAg. 3.3. HBeAg and HbsAg determination by semi-quantitative RTePCR A semi-quantitative RTePCR method was used to quantify the expression of the genes. It was found that the expression of surface antigens was lower when the cells were treated for a longer time with KY88 or when the doses were higher. The results are similar so only HBsAg is shown (Fig. 4). One-way ANOVA analysis confirmed the mRNA content of HBsAg to be statistically significant for 0.5 mg/ml and 1 mg/ml KY88. On the other hand, HBeAg showed a difference in all test groups compared with the control group although statistical significance was not achieved. .06
.8
Groups .6 0.1mg/ml
.4 0.5mg/ml
.2
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control
0.0 1
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Day Fig. 1. Dose responsive cell proliferation of KY88 measured by MTT assay in terms of optical density on days 1, 3, 5 and 7.
Mean of secretion of HbeAg (U PEI/ml)
Standard error of mean optical densities
1.0
.05
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.02 control
1mg/ml
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Groups Fig. 3. The mean level of HBsAg secreted by hepatocellular carcinoma cells pretreated with different concentrations of KY88.
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Standard error of mean mRNA content of HBsAg
7 6 Group
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3 0.1mg/ml
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Day Fig. 4. The quantification of HBsAg analysed by PCR product density in terms of optical density.
4. Discussion The three main experimental tools currently used in clinical or laboratory studies of HBV are a drug called lamivudine and interferons, vaccines with Chinese medicine being the adjuvant therapy. Their antiviral effects are achieved by lowering the secretions of HBsAg and HBeAg into the serum in humans. Lai’s study showed that lamivudine in doses of 25 mg, 100 mg, and 300 mg were effective in suppressing HBV DNA in Chinese HBsAg carriers [12]. The results of So et al. demonstrated that recombinant interferon alpha-2a (rIFN) is effective in the management of patients who are HBsAg and/or HBeAg positive [13]. Senturk et al. concluded that a pre-S2-containing vaccine (Genhevac-B) decreased serum HBV DNA levels in the majority of patients with chronic hepatitis B and sustained clearance was achieved in some patients [14]. Similarly, Yu et al. tested YGAPM, which is an effective drug in treating chronic hepatitis B, which can effectively negatively convert the HBV marker [15]. With regard to the antiviral effect of the drug Phyllanthus, the first current reports by two Indian groups, Thyagarayan et al. and Blumberg et al., indicated that P. amarus extract reduced detectable hepatitis B surface antigen (HBsAg) to negative in 59% of HBV-positive patients compared with 4% in the controls [16,17]. Herein, we describe a herbal compound called KY88 used among Chinese communities. Each individual ingredient of this herbal mixture confers advantages in treating or protecting liver disorders. Desmodium was employed for its ability to interfere with histamine-induced contractions, therefore it is widely used in India in treating asthma attacks as an anaphylactic drug [18]. Lithospermum has been used for treating tumours and inflammation in China and also inhibited proliferation and migration of endothelial cells in culture and network formation by endothelial cells on Matrigel in vitro [19]. Yamamoto
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et al. stated that Capillaris has been used for treatment of various liver disorders and it exhibited potent anti-apoptotic activity [20]. Hoelen has been shown in conjunction with other herbs to reduce elevated levels of lipid peroxide in serum [21]. Lian et al. on the other hand proved its potency in suppressing cell viability and telomerase activity in hormonerefractory and chemo-resistant cancer cell lines [22]. Peony was used in strengthening the viability and improving the nutritional state [23]. Treatment with phellodendron was shown to have a significant hepatic protective effect [24]. Leung et al. reported that trichosanthin could inhibit the growth of a murine malignant tumour (MBL-2), both in vivo and in vitro [25]. A combination of the above ingredients was effective in inducing lymphocyte proliferation and the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), which was linked to the host inflammatory response against pathogens as reported by us earlier [4,5]. Not only was this effect seen in humans, another study in SD rats, although not shown here, also revealed similar results. When the peritoneal injection of 5-flurouracil at a dosage of 250 mg/kg was performed preceded by orally fed 1 mg/ml KY88 for 7 days, the total white blood cell count was comparable with that of the control group. In the present study, KY88 at the designated concentrations was able to suppress cancer cell proliferation throughout the 7-day period. We found that a higher percentage of stained blue cells were present at higher concentrations of KY88. By referring to a similar study in which we performed DNA fragmentation, we concluded that the above findings were due to apoptosis [6]. The production of the viral antigen (HBsAg) is attributed to the integration of the viral genome into the DNA of the hepatocytes. The presence of HBsAg indicates that the person is a carrier and potentially infective. This state can persist for months until recovery, or for years in chronic carrier states [2]. HBV carriers demonstrate the significance of hepatitis B surface antigen (HBsAg) and hepatitis B ‘‘e’’ antigen (HBeAg) in their sera, these being indicative of active disease or high infectivity. Infectivity of this particle is so high that even 0.0001 ml serum containing infective virus could transmit the disease. Prolonged persistence of the e antigen in serum indicates that the patient will eventually develop chronic liver damage [2]. The herbal mixture described herein was able to lower the secretions of HBsAg using the designated kits. For secretion of HBsAg, the mean value was very similar for all three concentrations whereas the control was almost triple these values (Fig. 3). This was consistent with other studies carried out which also demonstrated a decrease of 60e70% after osthole (another Chinese herbal medicine) treatment, without any detectable cytotoxic effects [26]. Results for the control group illustrated that it was almost double that of other concentrations for the secretion of HBeAg (Fig. 4). Another experiment revealed that the maximum inhibitory rates were higher for HBsAg than HBeAg [27]. KY88 was proved to have induced apoptosis and production of interleukin-4 and tumour necrosis factor-a in liver cancer cells in vitro in our former study [6].
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This study provides insight into the mechanism of the action of KY88 on hepatocellular carcinoma cells. Knowledge on this subject is limited although KY88 is believed to be effective against HBV. While KY88 may act on the virus directly, this no doubt has given rise to the lower level of secretions of both HBsAg and HBeAg detected. Activation of the host inflammatory response is certainly an important means of combating HBV infection. Further clinical evaluation of this herbal compound would hence appear worthwhile. In conclusion, our experiments managed to show that Fructus schisandrae-containing compound (KY88) has a higher impact in suppressing the secretion of HBsAg than HBeAg. However, the mechanism of action has yet to be explained. References [1] Mertens T, Tondorf G, Siebolds M, Kruppenbacher JP, Shrestha SM, Mauff G, et al. Epidemiology of HIV and hepatitis B virus (HBV) in selected African and Asian populations. Infection 1989;17:4e7. [2] Samaranayake LP. Essential microbiology for dentistry. New York: Churchill Livingstone; 2002. p. 187e195. [3] Kamisango K, Kamogawa C, Sumi M, Goto S, Hirao A, Gonzales F, et al. Quantitative detection of hepatitis B virus by transcription-mediated amplification and hybridization protection assay. J Clin Microbiol 1999;37:310e4. [4] Chow LWC, Loo WTY, Sham JST. Effects of a herbal compound containing bupleurum on human lymphocytes. H K Med J 2001;7:408e13. [5] Chow LWC, Loo TY, Sham STJ. Cytokine production by human lymphocytes stimulated by a herbal compound containing Bupleurum (KY88 LIVER LIVO). Acta Pharmacol Sin 2003;24:140e4. [6] Chow LWC, Loo WTY, Cheung MNB. Radix bupleuri containing compound (KY88 Liver-Livo) induces apoptosis and production of interleukin-4 and tumor necrosis factor-a in liver cancer cells in vitro. Am J Chin Med 2004;32:185e93. [7] Lin MC, Lin JH, Wen KC. Detection and determination of phenformin in Chinese medicinal capsules by GC-MS and HPLC. J Food Drug Anal 2001;9:139e44. [8] Macejova D, Brtko J. Chemically induced carcinogenesis: a comparison of 1-methyl-1-nitrosourea, 7,12-dimethylbenzanthracene, diethylnitrosoamine and azoxymethan models (minireview). Endocrine Regul 2001;35: 53e9. [9] Shih CK, Chiang W, Kuo ML. Effects of adlay on azoxymethane-induced colon carcinogenesis in rats. Food Chem Toxicol 2004;42:1339e47. [10] Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980;209:497e9. [11] Phillips HJ. Dye exclusion tests for cell viability. In: Kruse Jr PF, Patterson Jr MK, editors. Tissue culture methods and applications. New York: Academic Press; 1973. p. 406e8.
[12] Lai CL, Chine CK, Tung AKM, Li E, Young J, Hill A, et al. Lamivudine is effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface antigen carriers: A placebo-controlled trial. Hepatology 1997;25:241e4. [13] Ola SO, Anomneze EE, Chukwuani CM, Ojo OS, Ndububa DA, Onyenekwe B, et al. Interferon alpha-2a (Roferon-A) in the management of chronic hepatitis B infection: results of an open prospective study in Nigerian patients. West Afr J Med 2000;19:259e64. [14] Senturk H, Tabak F, Akdogan M, Erdem L, Mert A, Ozaras R, et al. Therapeutic vaccination in chronic hepatitis B. J Gastroenterol Hepatol 2002;17:72e6. [15] Liu KZ. Relation between traditional Chinese medicine differentiation of syndromes and HBV antigen, HBV DNA in serum and liver tissues and pathological changes in chronic hepatitis B. Zhongguo Zhong Xi Yi Jie He Za Zhi 1992;12:11e33. [16] Thyagarajan SP, Subramanian S, Thirunalasundari T, Venkateswaran PS, Blumberg BS. Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus. Lancet 1988;2:764e6. [17] Blumberg BS, Millman I, Venkateswaran PS, Thyagarajan SP. Hepatitis B virus and primary hepatocellular carcinoma: Treatment of HBV carriers with Phyllanthus amarus. Vaccine 1990;8:S86e91. [18] Addy ME, Awumey EM. Effects of the extracts of Desmodium adscendens on anaphylaxis. J Ethnopharmacol 1984;11:283e92. [19] Hisa T, Kimura Y, Takada K, Suzuki F, Takigawa M. Shikonin, an ingredient of Lithospermum erythrorhizon, inhibits angiogenesis in vivo and in vitro. Anticancer Res 1998;18:783e90. [20] Yamamoto M, Ogawa K, Morita M, Fukuda K, Komatsu Y. The herbal medicine Inchin-ko-to inhibits liver cell apoptosis induced by transforming growth factor beta 1. Hepatology 1996;23:552e9. [21] Nishiyama N, Zhou Y, Saito H. Ameliorative effects of chronic treatment using DX-9386, a traditional Chinese prescription, on learning performance and lipid peroxide content in senescence accelerated mouse. Biol Pharm Bull 1994;17:1481e4. [22] Lian Z, Fujimoto J, Yokoyama Y, Niwa K, Tamaya T. Herbal complex suppresses telomerase activity in chemo-endocrine resistant cancer cell lines. Eur J Gynaecol Oncol 2001;22:347e9. [23] Wei L, Chen B, Ye R, Li H. Treatment of complications due to peritoneal dialysis for chronic renal failure with traditional Chinese medicine. J Trad Chin Med 1999;19:3e9. [24] Chiu HF, Lin CC, Yang CC, Yang F. The pharmacological and pathological studies on several hepatic protective crude drugs from Taiwan (I). Am J Chin Med 1988;16:127e33. [25] Leung KN, Yeung HW, Leung SO. The immunomodulatory and antitumor activities of trichosanthin-an abortifacient protein isolated from tian-hua-fen (Trichosanthes kirilowii). Asian Pacific J Allergy Immunol 1986;4:111e20. [26] Huang RL, Chen CC, Huang YL, Hsieh DJ, Hu CP, Chen CF, et al. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro. Hepatology 1996;24:508e15. [27] Zhang B, Chen J, Li H, Xu X. Study on the Rheum palmatum volatile oil against HBV in cell culture in vitro. Zhong Yao Cai 1998;21:524e6.