Baicalin induces apoptosis via mitochondrial pathway as prooxidant

Molecular Immunology 38 (2001) 781–791

Baicalin induces apoptosis via mitochondrial pathway as prooxidant Shugo Ueda a,b , Hajime Nakamura a , Hiroshi Masutani a , Tetsuro Sasada a,b , Arimichi Takabayashi b , Yoshio Yamaoka b , Junji Yodoi a,∗ a

Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan b Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan Received 13 August 2001; accepted 26 September 2001

Abstract Baicalin is a flavonoid and a major component of a herbal medicine, Sho-saiko-to, which is commonly used for treatment of chronic hepatitis in Japan and China. Flavonoids including baicalin have been reported to not only function as anti-oxidants but also cause cytotoxic effect. We investigated the mechanism of baicalin-induced cytotoxicity in leukemia-derived T cell line, Jurkat cells. When cells were cultured with 50–200 ␮g/ml baicalin for 6 h, caspase-3 was activated and then cells fell into apoptosis. Induction of apoptosis by baicalin was accompanied with the marginal generation of intracellular reactive oxygen species (ROS), the increase of the cytosolic fractions of cytochrome c, and the disruption of mitochondrial transmembrane potential (Ψ m ) prior to the activation of caspase-3. The pre-culture with 5 mM of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, facilitated baicalin-induced disruption of Ψ m and induction of apoptosis. The pre-culture with N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone (Z-VAD-fmk), a pan-caspase inhibitor, partially suppressed the induction of apoptosis. On the other hand, baicalin showed little toxic effect on peripheral blood mononuclear cells (PBMCs) from healthy volunteers. These results indicate that baicalin acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway. © 2002 Published by Elsevier Science Ltd. Keywords: Baicalin; Flavonoid; Apoptosis; Reactive oxygen species (ROS); Mitochondria

1. Introduction Flavonoids are used for the treatment of various types of chronic disease in Japan and China. Sho-saiko-to (TJ-9) is a herbal medicine, which is commonly used to treat chronic hepatitis in Japan (Hirayama et al., 1989; Kakumu et al., 1991; Oka et al., 1995; Tajiri et al., 1991). Baicalin (Fig. 1A) is a flavonoid, which is a major component of Sho-saiko-to (Liu et al., 1998; Shimizu et al., 1999a). Flavonoids have been reported to function as anti-oxidants (Gao et al., 1999; Middleton and Kandaswami, 1992; Robak and Gryglewski, 1988; Thompson et al., 1976), to cause cytotoxic effect (Csokay et al., 1997; Dickancaite et al., 1998; Ding et al., 1999; Hirano et al., 1995; Liu et al., 1998; Matsuzaki et al., 1996; Plaumann et al., 1996; Richter et al., 1999; Russo et al., 1999; Wu et al., 1995; Yano et al., 1994; Zi and Agarwal, 1999), and to have anti-viral effect by the inhibition of reverse transcriptase (Baylor et al., 1992; Li et al., 1993; Ono et al., 1989). Baicalin is a glucuronic compound of baicalein which is also a component of Sho-saiko-to. Both baicalin and baicalein have been reported to induce cell death, apoptosis ∗

Corresponding author. Tel.: +81-75-751-4024; fax: +81-75-761-5766. E-mail address: [email protected] (J. Yodoi).

0161-5890/02/$ – see front matter © 2002 Published by Elsevier Science Ltd. PII: S 0 1 6 1 - 5 8 9 0 ( 0 1 ) 0 0 1 1 5 - 8

or necrosis (Ding et al., 1999; Matsuzaki et al., 1996; Wu et al., 1995). Similarly, other flavonoids have been reported to induce cell death or apoptosis (Ahmad et al., 1998; Csokay et al., 1997; Dickancaite et al., 1998; Hirano et al., 1995; Plaumann et al., 1996; Richter et al., 1999; Russo et al., 1999; Zi and Agarwal, 1999). However, the precise mechanism of flavonoid-induced cell death has not been elucidated. Oxidative stress induces a variety of cellular responses including apoptosis (Nakamura et al., 1997; Nakamura et al., 1993; Yodoi and Uchiyama, 1992). Recent studies have demonstrated that mitochondria and caspases, cysteine proteases, have critical roles in the apoptosis signal (Kroemer et al., 1997; Nicholson and Thornberry, 1997; Reed, 1997; Susin et al., 1999). We have shown that diamide, a thioloxidant, induces apoptosis in leukemia-derived cell line, Jurkat cells, via mitochondrial pathway and that the intracellular reduction/oxidation (redox) state is important for the caspase activity and the induction of apoptosis (Sato et al., 1995; Ueda et al., 1998). According to previous reports, anti-oxidants or reducing factors have been believed to have cytoprotective function against oxidative stress (Chiba et al., 1996; Matsuda et al., 1991; Nakamura et al., 1994; Sasada et al., 1996). Thus, it should be clarified how a flavonoid with anti-oxidant activity induces cell death or apoptosis.

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Fig. 1. (A) Chemical structure of baicalin. (B) % viability of Jurkat cells cultured with baicalin. Jurkat cells cultured with 0, 50, 100, 200, or 400 ␮g/ml baicalin for indicated hours were examined by trypan blue exclusion test. This result is a representative of two experiments. The data are mean ± S.D. of four samples.

In this paper, we report baicalin induces apoptosis in Jurkat cells, and examine the mechanism of apoptosis signal induced by baicalin and the involvement of the intracellular redox state. Our results indicate that baicalin acts as prooxidant and induces apoptosis and caspase-3 activation via mitochondrial pathway.

tively. Carbonylcyanide m-chlorophenylhydrazone (CCCP), diamide, and dl-buthionine-[S,R]-sulfoximine (BSO) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were purchased from Nacalai Tesque Inc. (Kyoto, Japan) unless otherwise stated. 2.2. Cells

2. Materials and methods 2.1. Reagents Baicalin was purchased from Wako (Osaka, Japan) and 100 mg/ml solution dissolved with DMSO was stored at −20 ◦ C until use. Anti-cytochrome c monoclonal antibody (mAb) and anti-caspase-3 polyclonal antibody (Ab) were purchased from PharMingen (San Diego, CA). A fluorogenic substrate for caspase-3, acetyl-l-aspartyl-l-glutamyll-valyl-l-aspartic acid ␣-(4-methyl coumaryl-7-amide) (Ac-DEVD-MCA) and a pan-caspase inhibitor, N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone (ZVAD-fmk), were purchased from Peptide Institute (Osaka, Japan) and Calbiochem (La Jolla, CA), respectively. Dihydroethidium and 3,3 -dihexyloxacarbocyanine iodide (DiOC6 (3)) were purchased from Peptide Institute (Osaka, Japan) and Molecular Probes Inc. (Eugene, OR), respec-

Jurkat cells were maintained in RPMI1640 (GIBCO Inc., Grand Island, NY) with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (100 IU/ml penicillin and 100 ␮g/ml streptomycin) at 37 ◦ C in humidified atmosphere containing 5% CO2 . Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood of healthy volunteers by Ficoll–Paque density gradient centrifugation (Kanof et al., 1999). Washed with PBS(−) twice, PBMCs were cultured in RPMI1640 with 10% FCS at 37 ◦ C in humidified atmosphere containing 5% CO2 . After the incubation on plastic plate for 6 h, non-adherent cells were collected and used for cytotoxicity assay. 2.3. Assay for caspase-3(-like) protease activity Caspase-3(-like) protease activity was measured using the method described previously (Ueda et al., 1998).

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2.4. Flow cytometric estimation of intracellular redox state and mitochondrial transmembrane potential Intracellular redox state was estimated by levels of intracellular reactive oxygen species (ROS), which were monitored by flow cytometric analysis with dihydroethidium as described previously (Kobzik et al., 1990; Rothe and Valet, 1990; Ueda et al., 1998). Mitochondrial transmembrane potential (Ψ m ) was measured by using DiOC6 (3), which incorporates into mitochondria in strict nonlinear dependence of Ψ m and emits exclusively within the spectrum of green light (Petit et al., 1990; Zamzami et al., 1995). Jurkat cells cultured with the indicated concentration of baicalin for 3 h, and 2 ␮M dihydroethidium or 40 ␮M DiOC6 (3) was added for the last 20 or 15 min at 37 ◦ C, respectively. Some samples of cells were pre-cultured with BSO for 24 h, and after wash with medium without FCS, cells were cultured with the indicated concentration of baicalin, followed by the treatment of dihydroethidium or DiOC6 (3). After wash with PBS(−) containing 1% BSA and 0.1% NaN3 , the cells were analyzed on a flow cytometer (FACSCalilbur, Beckton Dickinson, Mountain View, CA) measuring the fluorescence at 585 ± 21 nm (dihydroethidium) or 530 ± 15 nm (DiOC6 (3)) with an excitation wavelength of 488 nm.

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Table 1 Percentage viability of PBMCs cultured with baicalin for 5.5 daysa Baicalin (␮g/ml)

Viability (%) (mean ± S.D.)

0 50 100 200 400

99.5 94.5 96.3 95.1 96.5

± ± ± ± ±

1.1 2.4 3.6 3.9 3.6

a PBMCs from two healthy volunteers were cultured with 0, 50, 100, 200, or 400 ␮g/ml baicalin for 5.5 days. The viability of cells was analyzed by trypan blue exclusion test. The data are mean ± S.D. of four samples.

however, baicalin showed little cytotoxicity on PBMCs even after 5.5 days of culture (Table 1). To investigate the regulatory mechanism of baicalininduced cell death, we next examined caspase-3(-like) protease activity in Jurkat cells cultured with baicalin. As shown in Fig. 2A, 50 or 100 ␮g/ml baicalin strongly induced activation of caspase-3(-like) protease. However, 400 ␮g/ml baicalin did not activate caspase-3(-like) protease and induced necrosis (data not shown). Large subunit of caspase-3 was able to be detected as early as 6 h after the treatment with 100 ␮g/ml baicalin (Fig. 2B). This

2.5. Immunoblot analysis For cytochrome c assay, soluble cytosolic fraction was prepared as described (Liu et al., 1996; Ueda et al., 1998). For caspase-3 immunoblotting, cells were collected and lysed with 50 mM Tris HCl (pH 7.4), 1 mM EDTA, and 10 mM EGTA containing 50 ␮M digitonin. Lysates were centrifuged at 15,000 rpm for 3 min, and supernatants were collected (Ueda et al., 1998). Immunoblot analyses were performed as previously described (Ueda et al., 1998). 2.6. Analysis of nuclear DNA content For the detection of hypodiploid cells, cells were permeabilized with ethanol, followed by propidium iodide (PI) staining, and analyzed on a flow cytometer, as previously described (Nicoletti et al., 1991).

3. Results 3.1. Induction of apoptosis and caspase-3 activation in Jurkat cells but not in normal lymphocytes by baicalin First, we examined whether baicalin induced cell toxicity. As shown in Fig. 1B, cell death was induced by baicalin dose-dependently. Jurkat cells cultured with 50–200 ␮g/ml baicalin showed the characteristic morphologic change of apoptosis (data not shown). When PBMCs from healthy volunteers were cultured with various concentration of baicalin,

Fig. 2. (A) Caspase-3(-like) protease activity in Jurkat cells cultured with baicalin. This result is a representative of at least four experiments. The data are mean ± S.D. of four samples. (B) Immunoblot analysis for processing of caspase-3 in Jurkat cells cultured with 100 ␮g/ml baicalin or 200 ␮M diamide for indicated hours. This result is a representative of three experiments. (C) Cell cycle distribution of Jurkat cells cultured with 0, 50, 100, or 200 ␮g/ml baicalin or 200 ␮M diamide for 6 h. Cells were analyzed for DNA content by PI staining. Each bar indicates hypodiploid cells. This result is a representative of at least three independent experiments.

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(Ueda et al., 1998). We also showed that intracellular reducing environment is important for the activation of caspase-3 and the induction of apoptosis (Ueda et al., 1998). Therefore, we analyzed the amount of ROS in cells cultured with various concentration of baicalin. As shown in Fig. 3, ROS were slightly generated in cells cultured with 50, 100, 200, or 400 ␮g/ml baicalin for 3 h. This result is similar to that in cells cultured with 200 ␮M diamide. After 6 h of culture with baicalin, intracellular reducing condition was maintained only in cells cultured with 50 or 100 ␮g/ml baicalin. When cells were cultured with 200 or 400 ␮g/ml baicalin for 6 h, a considerable increase of ROS was detected in cells in addition to cells with the maintenance of an intracellular reducing environment. 3.3. Immunoblot analysis for the release of cytochrome c into cytosol and flow cytometric analysis for mitochondrial transmembrane potential (∆Ψ m ) As shown in Fig. 4A, immunoblot analysis demonstrated that cytochrome c was released from mitochondria into cytosol in cells cultured with 100 ␮g/ml baicalin for 2 h. Treatment with CCCP, a protonophore, was used for negative control of DiOC6 (3) staining. As shown in Fig. 4B, Ψ m was disrupted in Jurkat cells cultured with 50, 100, 200, 400 ␮g/ml baicalin or 200 ␮M diamide. The disruption of Ψ m was detected as early as 2 h of culture with baicalin, which was earlier than the activation of caspase-3(-like) protease. These results indicate that baicalin-induced caspase-3 activation is mediated by mitochondria. 3.4. Effect of pre-culture with BSO

Fig. 2. (Continued ).

processing of procaspase-3 well correlated with the time course of the caspase-3(-like) protease activity. Since the increase of hypodiploid cells is known to be characteristic of apoptotic cell death, we examined the effect of baicalin or 200 ␮M diamide that induced apoptosis (Ueda et al., 1998) using PI staining method. Hypodiploid cells were increased after the culture with 50, 100, 200 ␮g/ml baicalin as well as 200 ␮M diamide for 6 h (Fig. 2C), further confirming that baicalin induces apoptosis. 3.2. Generation of ROS and redox state estimated by flow cytometry Only a slight increase of generated ROS was detected in Jurkat cells cultured with 200 ␮M diamide that induced apoptosis, whereas a considerable increase was detected in cells cultured with 500 ␮M diamide that induced necrosis

We previously showed that apoptosis is induced by much lower concentration of diamide in glutathione (GSH)-depleted Jurkat cells pre-cultured with 5 mM BSO for 24 h (Sato et al., 1995; Ueda et al., 1998). The pre-culture with 5 mM BSO for 24 h itself did not induce apoptosis nor activate caspase-3(-like) protease in Jurkat cells (Ueda et al., 1998). As shown in Fig. 5A, caspase-3(-like) protease was highly activated in BSO-pre-treated cells cultured with 50 ␮g/ml baicalin for 6 h. The activity was not detected in BSO-pre-treated cells cultured with more than 100 ␮g/ml baicalin. Morphologic observation showed that GSH-depleted cells with 50 ␮g/ml baicalin were apoptotic and those with more than 100 ␮g/ml baicalin were necrotic (data not shown). As compared with BSO-untreated cells, lower concentration of baicalin could induce the activation of caspase-3(-like) protease as well as apoptosis in GSH-depleted cells by the pre-culture with BSO. These results were very similar to diamide-induced apoptosis, as previously reported (Sato et al., 1995; Ueda et al., 1998). As shown in Fig. 5B, Ψ m was not disrupted only by the pre-culture with 5 mM BSO for 24 h. In BSO-treated cells, marked disruption of Ψ m was detected by the culture

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Fig. 3. Flow cytometric analysis of intracellular generation of ROS. Jurkat cells were cultured with the indicated concentration of baicalin for 3 or 6 h, and 2 ␮M dihydroethidium was added for the last 20 min at 37 ◦ C. The fluorescence intensity was measured by flow cytometer. Dark histograms indicate the fluorescence in treated cells, and clear ones indicate the fluorescence in untreated cells. This result is a representative of at least three independent experiments.

with 50, 100, or 200 ␮g/ml baicalin for 3 h. Baicalin-induced disruption of Ψ m was facilitated by the GSH-depletion. 3.5. Effect of pre-culture with caspase inhibitor When Jurkat cells were pre-cultured with 100 ␮M Z-VAD-fmk, pan-caspase inhibitor, for 1 h, baicalin-induced

activation of caspase-3(-like) protease was inhibited (data not shown). Culture with 100 ␮M Z-VAD-fmk by itself hardly decreased Ψ m in Jurkat cells. Even after cells were pre-cultured with 100 ␮M Z-VAD-fmk for 1 h, Ψ m was decreased by the following culture with 100 ␮g/ml baicalin for 6 h (Fig. 6A).

Fig. 4. (A) Immunoblot analysis for the release of cytochrome c into cytosol in Jurkat cells cultured with 100 ␮g/ml baicalin for indicated hours. Soluble cytosolic fractions (5 ␮g protein/lane) were electrophoresed and subjected to immunoblot analysis with anti-cytochrome c mAb. Cytochrome c from bovine heart, purchased from Sigma, was used as a positive control. (B) Flow cytometric analysis for mitochondrial transmembrane potential (Ψ m ). Jurkat cells were cultured with the indicated concentration of baicalin for 2, 4, or 6 h, and incubated in the presence of 40 nM DiOC6 (3) for 15 min at 37 ◦ C. The fluorescence intensity was measured by flow cytometer. Dark histograms indicate the fluorescence in treated cells, and clear ones indicate the fluorescence in untreated cells. This result is a representative of five independent experiments.

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Fig. 4. (Continued ).

Fig. 5. Effect of GSH depletion by the culture with 5 mM BSO for 24 h on the activity of caspase-3(-like) protease (A) or mitochondrial transmembrane potential (Ψ m ) (B). (A): Caspase-3(-like) protease activity in BSO-treated or untreated Jurkat cells cultured with 0, 20, 50, 100, 200, 300, or 400 ␮g/ml baicalin for 6 h. (B): Flow cytometric analysis of Ψ m in BSO-treated or untreated Jurkat cells cultured with the indicated concentration of baicalin for 3 h.

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Fig. 6. Effect of caspase inhibitor on mitochondrial transmembrane potential (Ψ m ) (A) or hypodiploid cells (B). (A): Flow cytometric analysis of Ψ m in Jurkat cells pre-treated with or without 100 ␮M Z-VAD-fmk, pan-caspase inhibitor, for 1 h or 100 ␮M CCCP for 15 min, followed by the culture with 100 ␮g/ml baicalin for 6 h. CCCP is an uncoupling agent that abolishes Ψ m . Each bar indicates low Ψ m cells. This result is a representative of at least three independent experiments. (B): Intracellular DNA contents were analyzed by PI staining, and hypodiploid cell were counted. After cells were pre-cultured with or without 100 ␮M Z-VAD-fmk for 1 h, cells were cultured with 100 ␮g/ml baicalin for 6 h (closed bars). Open bars indicate the percentage of hypodiploid cells cultured with or without 100 ␮M Z-VAD-fmk for 7 h. Data (mean ± S.D.) are representative of two independent experiments.

Hypodiploid cells, analyzed by PI staining, were not increased only by the culture with 100 ␮M Z-VAD-fmk for 7 h. Pre-culture with 100 ␮M Z-VAD-fmk for 1 h partially suppressed the increase in hypodiploid cells induced by baicalin (Fig. 6B).

4. Discussion According to previous reports, anti-oxidants or reducing factors have cytoprotective function against oxidative stress (Chiba et al., 1996; Matsuda et al., 1991; Nakamura

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et al., 1994; Sasada et al., 1996). It has been believed that flavonoids including baicalin suppress cytotoxicity with their anti-oxidant activity (Gao et al., 1999; Middleton and Kandaswami, 1992; Robak and Gryglewski, 1988). Recently, there are other reports about flavonoid-induced cytotoxicity with prooxidant character, however, the precise mechanism has not been clarified yet (Cao et al., 1997; Csokay et al., 1997; Dickancaite et al., 1998; Ding et al., 1999; Hirano et al., 1995; Liu et al., 1998; Matsuzaki et al., 1996; Plaumann et al., 1996; Richter et al., 1999; Russo et al., 1999; Wu et al., 1995; Yano et al., 1994; Zi and Agarwal, 1999). In this paper, we have proved that baicalin, which is one of major ingredients of Sho-saiko-to, induces apoptosis in Jurkat cells as prooxidant. An oxidoreductase acts as an oxidant against a target molecule with lower redox potential, and acts reversibly as an anti-oxidant against one with higher redox potential. Although further investigation is required, it is possible that flavonoids including baicalin, which have reducing activity against ROS, act as prooxidants against an intracellular unknown target molecule. We have shown that intracellular reducing environment is important for the activation of caspase-3 and the induction of apoptosis (Ueda et al., 1998). GSH is one of major component of intracellular reducing factor. Our results indicate that GSH-depleted Jurkat cells by the culture with BSO are more sensitive to oxidative stress (Fig. 5) (Ueda et al., 1998). Especially, we have shown in this paper that GSH-depletion facilitated the disruption of Ψ m as well as the release of cytochrome c induced by baicalin. Thus, intracellular redox is also involved in baicalin-induced apoptosis. Many investigators have demonstrated that mitochondria play an important role in the induction of apoptosis. We also have shown that diamide induced cytochrome c release from mitochondria and activation of caspase-3, followed by apoptosis (Ueda et al., 1998). In this paper, we demonstrated that baicalin induced cytochrome c release from mitochondria into cytosol and loss of Ψ m , followed by caspase activation and nuclear degradation in Jurkat cells, which is very similar pathway to diamide-induced apoptosis. Baicalin may attack thiols of some target molecules. It is important to investigate whether permeability transition pore complex in mitochondria, including voltage-dependent anion channel (VDAC) (Shimizu et al., 1999b) or adenine nucleotide translocator (Marzo et al., 1998), is involved in baicalin-induced apoptosis. Caspase inhibitor, Z-VAD-fmk, did not suppress the disruption of Ψ m , but partially inhibited the induction of apoptosis by baicalin (Fig. 6). This result suggests that the disruption of Ψ m precedes caspase activation (Jacotot et al., 2000), which is different from Fas-induced apoptosis, where mitochondria are involved in the downstream of caspase activation (Hirata et al., 1998; Li et al., 1998). Thus, our results indicate that baicalin acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway. Other possibilities involved in baicalin-induced activation of caspase-3(-like) protease or induction of apoptosis is

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the activation of apoptosis signal-regulating kinase (ASK) 1 (Ichijo et al., 1997), followed by the activation of c-Jun N-terminal kinase (JNK) or p38 MAP kinase. The p38 MAP kinase or JNK was indeed phosphorylated slightly after the culture with baicalin (S.U., unpublished observations). However, the phosphorylation of p38 MAP kinase or JNK was detected after 4 h of culture with baicalin (S.U., unpublished observations), which is later than other apoptosis inducer (Kawasaki et al., 1997). The activation of these kinases does not seem to be mainly involved in the activation of caspase-3(-like) protease or the induction of apoptosis induced by baicalin, although additional experiments are required. Although p53 activation is also important for induction of apoptosis (Miyashita and Reed, 1995; Plaumann et al., 1996), protein levels of p53 or Bax did not change after the treatment with baicalin (S.U., unpublished observations). Further study is required to clarify whether NO is involved in baicalin-induced cytotoxicity. Sho-saiko-to, a herbal medicine, has been used for the treatment of chronic hepatitis in Japan for a long time and possesses little side-effect. However, recently it was reported that this drug rarely induces interstitial pneumonia (Ohtake et al., 2000). As baicalin is a one of major ingredients of Sho-saiko-to, the cytotoxicity induced by baicalin may cause interstitial pneumonia. We have shown that baicalin is cytotoxic against Jurkat cells. Baicalin is also cytotoxic against other leukemia-derived cells such as U937 and HL-60, or hepatoma-derived cell lines (S.U., unpublished observations), but not against normal PBMCs (Table 1). As unstimulated PBMCs are not mitotic, it is possible that resting cells are more resistant to baicalin. There might be some unknown mechanism that cancer cells are more susceptible to baicalin. There are some other flavonoids including baicalein, which are reported to have cytotoxicity against tumor cells (Ahmad et al., 1998; Csokay et al., 1997; Ding et al., 1999; Hirano et al., 1995; Liu et al., 1998; Matsuzaki et al., 1996; Richter et al., 1999; Russo et al., 1999; Yano et al., 1994; Zi and Agarwal, 1999). Furthermore, both baicalin and baicalein have been reported to inhibit reverse transcriptase activity (Baylor et al., 1992; Li et al., 1993; Ono et al., 1989). It is possible that baicalin is clinically applied to new therapeutics of chemoprevention against malignancy including retrovirus-related leukemia (Ahmad et al., 1998; Oka et al., 1995). In conclusion, baicalin, a flavonoid, acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway in Jurkat cells. It is suggested that this induction of apoptosis by baicalin may be involved in the development of intersititial pneumonia, a rare side-effect of Sho-saiko-to.

Acknowledgements We thank Dr. Tetsuo Ohkuma for critical reading, Dr. Akira Yamauchi and Dr. Michiyuki Maeda for scien-

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