Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines

CANCER LETTERS Cancer

ELSEVIER

Letters 86 (1994) 91-95

Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines Yoshiharu Motoo*, Norio Sawabu Department of Internal Medicine, Cancer Research Institute, Kanazawa University, 4-86 Yoneixmi.

Received

17 June 1994; revision

received 23 August

1994; accepted

24 August

Kanazawa 921, Japan

1994

Abstract

Antitumor effects of nine components of a herbal medicine, ‘Sho-saiko-to’, were investigated on human hepatoma cell lines (PLC/PRF/S, Hep-G2), human liver cells (Chang) and a human pancreatic cancer cell line (BxPC-3). The concentration of each component required for 50% inhibition of cell growth of PLC/PRF/S cells was as follows: saikosaponin-d, baicalin, 20 &ml; saikosaponin-a, baicalein, 50 &ml; saikosaponin-b,, -c, ginsenoside-Rb,, -Rg,, glycyrrhizin, > 1000 &ml. Saikosaponin-a in 50-pg/ml quantities inhibited the cell growth and DNA synthesis of all the cell lines tested. These results indicate that ‘Sho-saiko-to’ includes potent antitumor components such as saikosaponin-a, -d, baicalin and baicalein against human hepatoma cells as well as other human cell lines. Keywords: Sho-saiko-to; Hepatoma cells; Antitumor effects; Saikosaponins;

1. Introduction A herbal medicine, ‘Sho-saiko-to’ (TJ-9), is one of the most common drugs used for chronic liver diseases such as chronic hepatitis and cirrhosis [lo]. A clinical study showed that TJ-9 signilicantly decreased hepatoma development in cirrhotic patients [17]. In vivo experiments disclosed inhibitory effects of TJ-9 on chemical hepatocarcinogenesis in rats [ 11,141. Furthermore, recent in vitro studies have shown that TJ-9 and its ingredients inhibited the growth of human hepatoma cell lines [ 12,151. Therefore, in addition to immunomodulating actions [ 1,3,4,5], TJ-9 may have * Corresponding author. 0304-3835/94/$07.00 0 1994 Elsevier SSDI 0304-3835(94)03544-S

Baicalin; Baicalein

a direct antitumor activity on human hepatoma cells. However, it is still unknown what components are responsible for the antitumor activity of TJ-9, and it is also uncertain whether the effects of TJ-9 are especially pronounced in hepatoma cells. We attempted to verify the antitumor effects of nine purified components of TJ-9 on two human hepatoma cell lines (PLC/PRF/S and HepG2), comparing the effects on human liver cells (Chang) and a human pancreatic cancer cell line (BxPC-3).

2. Materials and methods 2.1. Cell lines Two human hepatoma cell lines (PLC/PRF/S

Science Ireland Ltd. All rights reserved

92

Y. Moroo. N. Sawabui

and Hep-G2), a human liver cell line (Chang) and a pancreatic cancer cell line (B,PC-3) were used. PLC/PRF/S and Chang cells were provided by the Japanese Cancer Research Resources Bank (Tokyo, Japan), Hep-G2 bells were from the RIKEN Cell Bank (Tsukuba, Japan), and B,PC-3 cells were from the American Type Culture Collection (Rockville, MD). Cells were cultured as monolayers in RPM1 1640 medium supplemented with 10% fetal calf serum (GIBCO-Life Technologies, Gaithersburg, MD), 0.1 mg/ml kanamycin sulfate (Meiji Seika, Co., Tokyo, Japan) and 0.2 mg/ml amphotericin B (Flow Laboratories, Irvine, UK). 2.2. Preparation of components All the nine purified components were purchased from Wako Pure Chemicals (Osaka, Japan). Saikosaponin-a, -bz, -d and baicalein were insoluble in RPM1 1640 medium, dimethyl sulphoxide (DMSO) or water. Therefore, these four components were dissolved in 100% ethanol, and then diluted in the RPM1 medium with a final dilution of 0.1% or less. Other components were easily dissolved in RPM1 1640 medium. Since glycyrrhizin and baicalin lowered the pH of the solution, the pH was adjusted to 7.4 using 1 N NaOH. 2.3. Assay for antitumor activity The antitumor activity of each component was evaluated with cell counting, as reported previously [7,8] or by the MTT assay [6]. The cells were seeded in 96-well microtiter plates (Nunc Inc., Napervill, IL) at a cell density of 3 x lo3 cells/O.1 ml/well. After a 24-h incubation, 0.1 ml of fresh medium containing different concentrations of each component was added. The cell numbers were counted every 24 h for 3 days with a hemacytometer using a Trypan Blue solution for cell viability evaluation. The MTT assay was performed using an MTT assay kit (Chemicon International Inc., Temecula, CA). Each experiment had control wells containing 0.1% ethanol, which showed no effects on the cell growth. Since baicalin and baicalein made the color of the solution yellowish purple, which interfered with the MTT assay, each experiment on baicalin and baicalein included control wells containing only these components without cells.

Cancer Left. 86 (19941 91-95

2.4. Expression of proliferating cell nuclear antigen (PCNA)

Cells were cultured on the Chamber Slide (LabTek, Nunc, Napervill, IL) in the media containing different concentrations of each component for 3 days, then fixed with 4% paraformaldehyde at 4°C for 2 h. After washing with sucrose gradients, cells were stained with the ABC method [2] using the murine monoclonal antidody against human PCNA (PC-lo, DAKO, Kyoto, Japan) as a primary antibody. 2.5. Statistical analysis Student’s t test was used, and a two-tailed P value of co.05 was considered to indicate statistical significance. 3. Results The antitumor effects of each component on PLC/PRF/S cells are shown in Fig. 1. There were significant differences in the antitumor effect among the components tested. Saikosaponin-a, -d,

d'

\

Fig. I. Antitumor effects of nine components of a herbal medicine ‘Sho-saiko-to’ (TJ-9) on human hepatoma cells PLC/PRF/S. Mean f SEM (n = 6). *P < 0.05, **P < 0.00, ***p < 0.001.

Y. Mofoo, N. Sawabu/Cuncer

baicalin and baicaiein showed 100% inhibition of cell growth at a concentration of 100 &ml, whereas saikosaponin-bz, -c, ginsenoside-Rbt, -Rg, and glycyrrhizin showed only 30-50% inhibition even at a concentration of 1000 yglml. The concentration of each component necessary to obtain 5Oo/ inhibition was as follows: saikosaponin-d, baicalin, 20 pg/ml; saikosaponina, baicalein, 50 pg/ml; saikosaponin-b2, -c, ginsenoside-Rb,, -Rgi, glycyrrhizin, > 1000 c&ml. Serial changes in the growth of PLC/PRF/S cells treated with saikosaponin-a and baicalin were studied. Saikosaponin-a at a concentration of 50 &ml suppressed the cell growth on day 1, inducing a decrease in the number of viable cells. On the other hand, baicalin at 50 &ml did not show a dytotoxic effect, with the starting cell number maintained, although 100 &ml of baicalin induced complete disappearance of viable cells on day 3 (data not shown). The antitumor effects of saikosaponin-a on four cell lines were compared. At a concentration of 20 &ml of saikosaponin-a, the growth of hepatoma cells Hep-G2 and PLC/PRF/S was significantly inhibited, compared with Chang liver cells and pancreatic cancer cells B,PC-3 (P < 0.01). However, at a concentration of 50 pg/ml, almost complete inhibition of the growth of all the cell lines was observed. The PCNA labeling index (PCNA-LI) in PLCIPRFIS cells treated with different concentrations of saikosaponin-a was determined, At ~on~ntrations of 20 and 50 &ml, the expression of PCNA was significantly (P < 0.01 and P < 0.001, respectively) suppressed: PCNA-LI, 20.1 f 4.0% and 2.0 f 0.4%, respectively (mean f SD, n = 5). 4. Discussion This study showed the direct antitumor effects of the major components of a herbal medicine, Sho-saiko-to (TJ-9), which is one of the most commonly used drugs for the treatment of chronic liver diseases in Japan. Previously, the antitumor effects of TJ-9 were considered to be mainly immunomodulating actions such as an enhancement of the natural killer activity or an induction of

Lert. 86 (1994) 91-95

93

cytokine production [ 1,3-5,181. Recently, however, accumulating evidence for direct antitumor effects of TJ-9 has been reported [ 12,151. Okita et al. [12] reported the effects of the TJ-9 components on the growth of a human hepatoma cell line HUH-~. They found that baicalein, baicalin and saikosaponin-a showed an inhibitory action on the cell growth. In particular, saikosaponin-a was found to have a strong killing effect. In their work, DMSG was used as a solvent for all the components of TJ-9. However, we found that saikosaponin-a, -bz, -d and baicalein were insoluble in DMSO, RPM1 1640 medium or water. We used 100% ethanol as a solvent. These four components were completely dissolved in ethanol. In order to avoid a toxic effect of ethanol, we tested the effect of 0.1% ethanol, a final concentration as a solvent, on the cell growth. There was no significant effect of ethanol on the growth of the cell lines tested. In addition to the six components which were studied in the report of Okita et al. [12], we examined the effects of saikosaponin-by, -d and glycyrrhizin, which are also present in TJ-9 191.Among these three components, only saikosaponin-d showed a marked growth-inhibitory action. The antitumor effect of saikosaponin-d has been reported in vivo [ 131. During the preparation of this manuscript, Yano et al. [15] reported that TJ-9 inhibited the proliferation of a human hepatoma cell line (KIM1) and a cholangiocarcinoma cell line (KMC-1) in vitro. They used normal human peripheral blood lymphocytes and normal rat hepatocytes as negative controls. TJ-9 suppressed the cell growth significantly more strongly than did each of its major components. They evaluated the effect of TJ-9 referring to the blood level of glycyrrhizin as a measurable index, although glycyrrhizin did not show any antitumor effect. In their study, only water-soluble ingredients of TJ-9 were used, The flavonoids such as baicalin and baicalein are barely soluble in water, as described by Yano et al. [ 151, and even saikosaponins are water-insoluble, as we tested. Therefore, it is considered that in the work of Yano et al. 1151the effects of some unknown components of TJ-9, different from those we examined in this study or in the report by Okita et al. [12], were tested. There were striking differences in the antitumor

Y. Motoo. N. Suwabu / Cancer Lerr. 86

94

effects among the saikosaponins tested. Saikosaponin-a and -d showed a marked cytotoxic effect, whereas saikosaponin-b2 and -c had little antitumor effect. The reasons for these differences are unknown, but saikosaponin-a and -d are also stronger than saikosaponin-c in the antiinflammatory activity [16]. However, in the report of Yano et al. [15], even saikosaponin-a and -d showed no significant antitumor effect, while the work of Okita et al. [12] and our study clearly revealed a strong cytotoxic effect of saikosaponina and -d. As described above, there were differences in the pretreatment or the dissolving method of these components in the different studies, and therefore, further studies are needed to clarify the antitumor effect of saikosaponins under consistent conditions. Although we compared the antitumor effects of saikosaponin-a among four cell lines (two hepatoma cell lines, one liver cell line, and one pancreatic cancer cell line), there was no significant difference among the cell lines tested at a concentration of 50 &ml. Since TJ-9 is reported to show an antitumor activity against lung cancer cells [3], the tissue specificity of the antitumor effect of TJ-9 is not feasible. We used the labeling index of PCNA for the evaluation of DNA synthesis inhibition. It was confirmed that the components of TJ-9 exerted an antitumor effect on hepatoma cells by inhibiting the DNA synthesis of the cancer cells. Apoptosis is now considered to be the main growth-inhibiting mechanism of TJ-9 on hepatoma cells [ 151. Studying the direct antitumor effect of each component of TJ-9 provides a rationale for using this drug for patients with liver cirrhosis and hepatocellular carcinoma. Since TJ-9 has already been shown to possess antiinflammatory and immunomodulating actions including cytokine production [ 1,181, evidence of a direct antitumor activity strongly supports the idea of using TJ-9 as a potent chemopreventive agent for hepatocellular carcinoma or using TJ-9 in combination with other anticancer drugs to potentiate their effects and to avoid side-effects.

technical assistance and Tsumura & Co. (Tokyo, Japan) for their cooperation. References Haranaka,K., Satomi, N., Sakurai, A., Haranaka, R.. Okada. N. and Kobayashi, M. (1985) Antitumor activities and tumor necrosis factor producibility of traditional Cbinese medicines and crude drugs. Cancer Immunol. Immunother., 20. I-5. 121Hsu, M., Raine, L. and Fanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabelled (PAP) procedure. J. Histochem. Cytochem., 29. 557-580. 131 Itoh. H. and Shimura, K. (1986) Effects of a blended Chinese medicine, Xiao-chi-hu-tang, on Lewis lung carcinoma growth and inhibition of lung metastasis, with special reference to macrophage activation. Jpn. J. Pharmacol., 41, 307-314. Ill

S., Shibata. 141 Kitamura, M.. Mizoguchi, Y.. Yamamoto, Y. and Morisawa, S. (1985) Antitumor activities of the Syo-saiko-to-activated macrophages in mice. J. Med. Pharm. Sot. Wakan-Yaku, 2. 32-38. 151 Kumazawa, Y.. Takimoto. H., Mura, S., Nishimura, C..

161

[71

[81

191

[lOI

1111

Acknowledgments We wish to thank Miss Mari Kurose for her

( 1994) 91-95

1121

Yamada, A., Kawakita, T. and Nomoto, K. (1988) Activation of murine peritoneal macrophages by intraperitoneal administration of a traditional Chinese herbal medicine, Xiao-chai-hu-tang (Japanese name: Sho-saiko-to). Int. J. Immunopharm., 10, 395-403. Mosmann, T. (1983) Rapid calorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63. Motoo, Y.. Hill, N.O. and Osther. K. (1986) Comparison of the antitumor effects of human natural and recombinant interferons (alpha and beta) on human cancer cell lines. Jpn. J. Exp. Med., 56, 13-18. Motoo, Y., Hill, N.O., Mahmoudi. M. and Osther, K. (1986) Antitumor effect of human tumor necrosis factor on human hepatoma cells PLC/PRF/S. Jpn. J. Exp. Med., 56, 151-154. Nishioka. Y., Kyoya, S., Miyamura. M. and Kususe, M. (1990) Studies on the quantification of the components of Sho-saiko-to. Med. Consult. New Remed., 27. 1996-2002. Oda. T. and Oka, H. (1988) Kampo preparations as biological response modifiers in the treatment of chronic viral hepatitis. In: Recent Advances in the Pharmacology of Kampo (Japanese Herbal) Medicine, pp. 369-377. Editors: E. Hosoya and Y. Yamamura. Excerpta Medica, Tokyo. Okita, K.. Kurokawa, F., Yamazaki, T.. Furukawa, T.. Li, Q., and Sasaki, K.( 1991) Chemoprevention of chemical hepatocarcinogenesis in rats with Sho-saiko-to (TJ9) and its possible pharmacological action. Prog. Med., II. 412-418. Okita, K., Li, Q., Murakami, T. and Takahashi, M.

Y. Moroo. N. Sawabu/Caneer (1993) Anti-growth

effects with components of Shosaiko-to (TJ-9) on cultured human hepatoma cells. Eur. J. Cancer Prevent., 2, 169-176. 1131 Sugiyama, K., Tauchi, Y. and Yokota, M. (1988) Antitumor activity of saikosaponin d. J. Med. Pharm. Sot. Wakan-Yaku, 5, 426-427. 1141 Tatsuta, M., I&hi, H., Baba, M., ~aka~zumi, A. and Uehara, H. (1991) Inhibition by Xiao-chai-hu-tang (TJ9) of development of hepatic foci induced by Nnitrosomorpholine in Sprague-Dawley rats. Jpn. J. Cancer Res., 82, 987-992. [IS] Yano, H., Mizoguchi, A., Fukuda, K., Haramaki. M., Ogasawara, S., Momosaki, S. and Kojiro. M. (1994) The herbal medicine Sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the GdG, phase. Cancer Res., 54. 448-454.

Lert. 86 (19941 91-95

9s

[l6] Yamamoto, M., Kumagai, A. and Yamamura, Y. (1975) Structure and actions of saikosaponins isolated from Bupleurum falcatum L. I. Anti-inflammatory action of saikosaponins. Arzneim. Forsch. (Drug Res.), 25, 1021-1023. 1171 Yamamoto, S., Oka, H., Kanno, T., Mizoguchi, Y and Kobayashi, K. (1989) ControlIed prospective trial to evaluate Sho-saiko-to for the prevention of hepatocellular carcinoma in patients with cirrhosis of the liver. Jpn. J. Cancer Chemother., 16, 1519-1524. [18] Yamashiki, M., Nishimura, A., Asakawa, M., Kayaba, Y. and Kosaka, Y. (1992) Herbal medicine ‘Sho-saikoto’ induces in vitro granuloeyte colony-stimulating factor production on peripheral blood mononuclear cells. J. Clin. Lab. Immunol., 37, 83-90.