Kalopanaxsaponin A induces apoptosis in human leukemia U937 cells through extracellular Ca2+ influx and caspase-8 dependent pathways

Food and Chemical Toxicology 46 (2008) 3486–3492

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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Kalopanaxsaponin A induces apoptosis in human leukemia U937 cells through extracellular Ca2+ influx and caspase-8 dependent pathways Jung-Hye Choi a, Heon-Woo Lee a, Hee-Juhn Park b, Sung-Hoon Kim c,*, Kyung-Tae Lee a,* a

Department of Biochemistry, College of Pharmacy, Kyung-Hee University, Dongdaemun-ku, 1 Hoegi-Dong, Seoul 130-701, South Korea Division of Applied Plant Sciences, Sang-Ji University, Woosan-Dong, Wonju 220-702, South Korea c Cancer Preventive Material Development Research Center (CPMDRC), College of Oriental Medicine, Kyunghee University, 1 Hoegi-Dong, Dongdaemun-ku, Seoul 130-7 01, South Korea b

a r t i c l e

i n f o

Article history: Received 10 March 2008 Accepted 26 August 2008

Keywords: Kalopanaxsaponin A Apoptosis Mitochondrial membrane potential Calcium Caspase-8

a b s t r a c t In the present study, we investigated the effect of KPS-A on the apoptotic activity and the molecular mechanism of the action in human leukemia. Treatment with KPS-A significantly increased apoptotic DNA fragmentation in human histiocytic lymphoma U937 cells as shown by DAPI staining, flow cytometry, and agarose gel electrophoresis. In addition, stimulation of U937 cell with KPS-A induced a series of intracellular events: (1) the activations of caspase-8, caspase-9, and caspase-3; (2) the translocations of Bid and Bax proteins to mitochondria; (3) the loss of mitochondrial membrane potential; and (4) the increased release of cytochrome c to the cytosol. Pretreatment with a specific caspases-8, -9 or -3 inhibitor, neutralized the pro-apoptotic activity of KPS-A in U937 cells. We further demonstrated that KPS-A markedly induced an increase in intracellular Ca2+ level, which was reversed by EGTA, a general calcium chelator, but not by TMB-8 and dantrolene, intracellular Ca2+ release blockers. Moreover, KPS-A-induced DNA fragmentation and caspase activation were substantially reduced in the presence of EGTA. Taken together, these results suggest that KPS-A may play therapeutic role for leukemia via the potent apoptotic activity through Ca2+/caspases-8/MPT/caspases-9/caspases-3 signaling pathway. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Although apoptotic cell death primarily plays role in normal cell development, tissue homeostasis, and the regulation of the immune system (Jacobson et al., 1997), aberrant apoptosis also acts to promote cancer development (Thompson, 1995). In this regard, many anticancer drugs have been developed to induce apoptotic cell death in various cancer cells (Friesen et al., 1996; Bhalla et al., 1993; Li and Yuan, 1999). It has been well known that the death circuitry in mammalian cells proceeds via two major apoptotic pathways. One is a receptor-mediated pathway involving Fas and other members of the tumor necrosis factor (TNF) receptor Abbreviations: ARC, apoptosis repressor with caspase recruitment domain; AIF, apoptosis-inducing factor; CCCP, carbonyl cyanide m-chlorophenylhydrazone; BAPTA, 1,2-bis(2-aminophenosy)ethane-N,N,N0 ,N0 -tetraacetic acid; DAPI, 40 ,6diamidino-2-phenylindole-dihydrochloride; DCFH-DA, dichlorodihydrofluorescein diacetate; ECL, enhanced chemiluminescence; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid; FBS, fetal bovine serum; KPS-A, kalopanaxsaponin A; KRB, Krebs–Ringer buffer; MMP, mitochondrial membrane potential; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PMSF, phenylmethanesulfonyl fluoride; tBid, truncated Bid; TMB-8, 3,4,5-trimethoxybenzoic acid 8diethylamino)octylester; TNF, tumor necrosis factor. * Corresponding authors. Tel.: +82 2 961 0860; fax: +82 2 962 0860 (K.-T. Lee), tel.: +82 2 961 9233; fax: +82 2 964 1054 (S.-H. Kim). E-mail addresses: [email protected] (S.-H. Kim), [email protected] (K.-T. Lee). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.08.026

family that activate caspase-8 (Ashkenazi and Dixit, 1998), while the other involves the mitochondrial pathway related to cytochrome c, Apaf-1, and caspase-9 (Hakem et al., 1998; Reed and Green, 2002; Nicholson and Thornberry, 2003). As caspases are activated in response to genotoxic stress they are often considered as a precondition for apoptosis. However, there is accumulating evidence that there are also caspase-independent pathways to induce apoptosis (Yuan et al., 2003). Candidate mechanisms that could mediate caspase-independent cell death involve the mitochondrial release of apoptosis-inducing factor (AIF) and/or endonuclease G (Susin et al., 1999; Li et al., 2001). Over the past few years, there is increasing evidence suggesting that intracellular Ca2+ ½Ca2þ i play a critical role in the programmed cell death. For example, hydrogen peroxide (H2O2)-induced apoptosis required the activation of ½Ca2þ i -dependent endonuclease leading to internucleosomal DNA fragmentation and poly (ADPribose) polymerase (PARP) activation (Li et al., 2000). It has been demonstrated that ½Ca2þ i induced apoptosis in cancer cells via the activation of the Ca2+-dependent protein kinases and phosphatases (Bonnefoy-Berard et al., 1994; Shibasaki et al., 1997). In addition, recent studies have suggested that ½Ca2þ i is involved in the apoptotic mechanism of several anti-cancer drugs including tamoxifen and tributyltin (McConkey and Orrenius, 1997; Kim et al., 1999; Stridh et al., 1999).

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after 3 passages. KPS-A was dissolved in DMSO, and DMSO concentrations in all assays did not exceed 0.1%. 2.3. DAPI staining DNA fragmentation was quantitated as previously reported (Ka et al., 2003). In brief, cells were lysed in a solution containing 5 mM Tris–HCl (pH 7.4), 1 mM EDTA, and 0.5% (w/v) Triton X-100 for 20 min on ice. Lysate and supernatant were centrifuged at 27,000g for 20 min and sonicated for 40 s. DNA levels in each fraction were measured using a DAPI based fluorometric method. The amount of fragmented DNA was expressed as the ratio of DNA in supernatant to that in lysate. Genomic DNA was prepared for gel electrophoresis as previously described (Ka et al., 2003). Electrophoresis was performed in a 1.5% (w/v) agarose gel in 40 mM Tris–acetate buffer (pH 7.4) at 50 V for 1 h. The fragmented DNA was visualized by staining with ethidium bromide after electrophoresis. 2.4. Cell fractionation and Western blotting assay

Fig. 1. Chemical structure of kalopanaxsaponin A isolated from K. pictus.

Plants have been found to be a rich source of unique compounds that can induce apoptosis in premalignant or malignant human cells (Tang et al., 2003). As a part of our screening program to identify a natural compound with potential chemopreventive/chemotherapeutic effect, we investigated the effect of kalopanaxsaponin A (KPS-A) (Fig. 1), which was isolated from the stem bark of Kalopanax pictus Nakai (Araliaceae). It is of interest that the stem bark of this plant has been traditionally used for the treatment of rheumatoid arthritis, neurotic pain, and diabetes mellitus (Choi et al., 2002; Kim et al., 2002). However, active compounds for the therapeutical function are poorly studied. We previously demonstrated the potent anti-tumor and anti-inflammatory effects of KPS-A in animal models (Park et al., 2001; Choi et al., 2002; Kim et al., 2002). Thus, the present study was designed to evaluate (1) the effect of KPS-A on apoptosis of U937 human leukemia cells, (2) the mechanisms of KPS-A-induced actions in the cell growth. 2. Materials and methods 2.1. Materials The kalopanaxaponin A used for this study was isolated from the stem bark of Kalopanax pictus Nakai (Araliaceae) as previously described (Park et al., 2001; Choi et al., 2002). This compound used for this study was checked by HPLC and were >98% pure. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies. Inc. (Grand Island, NY, USA). RNase, leupeptine, aprotinin, verapamil hydrochloride, phenylmethanesulfonyl fluoride (PMSF), ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), 3,4,5-trimethoxybenzoic acid 8-diethylamino)octylester (TMB-8), dantrolene, nickel chloride hexahydrate, nifedipine, carbonyl cyanide m-chlorophenylhydrazone (CCCP), propidium iodide (PI), and 40 ,6-diamidino-2-phenylindole-dihydrochloride (DAPI) were purchased from Sigma Chemical Co (St. Louis, MO, USA). Proteinase K was from Wako Pure Chemical Industries (Osaka, Japan). Antibodies for b-actin, Bcl-2, Bax, Bid, tBid, cytochrome c, caspase-8, caspase-9, and caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3,30 -dihexyloxacarbocyanine iodide (DIOC6(3)), Fluo-3 acetoxymethyl ester and 1,2-bis(2-Aminophenosy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA) acetoxymethyl ester were obtained from Molecular Probes Inc (Eugene, OR, USA). Calpain inhibitor I, calpain inhibitor II, z-VAD-fmk, z-IETD-fmk, z-DEVD-fmk were purchased from A.G. Scientific Inc. (San Diego, CA, USA). 2.2. Cell culture and kalopanaxsaponin A treatment Human promonocytic leukemia U937 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin sulfate (100 lg/mL) in a 95% air/5% CO2 atmosphere, and were seeded in to plates

U937 cells (2.5  107) were collected by centrifugation at 200g for 10 min at 4 °C. The cells were then washed twice with ice-cold PBS, pH 7.2, and centrifuged at 200g for 5 min. The cell pellet obtained was then resuspended in ice-cold cell extraction buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100 lM PMSF, protease inhibitor cocktail) for 30 min on ice. The cells were then homogenized with a glass dounce and a Btype pestle (80 strokes), homogenates were spun at 15,000g for 15 min at 4 °C, and the supernatant (cytosolic fraction) was removed whilst taking care to avoid the pellet. The resulting pellet (mitochondrial fraction) was resuspended in extraction buffer. Approximately 40 lg of cytosolic protein extracts was separated using 15% SDS–polyacrylamide gels and transferred to nitrocellulose. The blots were incubated with monoclonal b-actin, Bcl-2, Bax, anti-cytochrome c, and polyclonal anti-caspase-3, -8, and -9 antibodies and this was followed by enhanced chemiluminescence (ECL)-based detection (Amersham Bioscience, Uppsala, Sweden). 2.5. Determination of mitochondrial membrane potential Changes in mitochondrial transmembrane potential were monitored by flow cytometry. Briefly, U937 cells were exposed to KPS-A (15 lM), and the mitochondrial transmembrane potential was measured directly using 50 nM DiOC6(3). Fluorescence was measured after the cells had been stained for 30 min at 37 °C using a FACScater-plus Flow cytometer (Becton Dickinson Co., Germany). 2.6. Measurement of [Ca2+]i ½Ca2þ i was measured using the cell-permeable Ca2+-sensitive fluorescent dye Fluo-3 acetoxymethyl ester (Hail and Lotan, 2002). Where indicated, BAPTA acetoxymethyl ester (10 lM) was added to cell culture medium in 10 cm plastic tissue culture plates for a 1 h exposure prior to loading Fluo-3 acetoxymethyl ester. The medium then was removed from the tissue culture plates and replaced with 4 lM Fluo-3 acetoxymethyl ester diluted in Krebs-Ringer buffer (KRB) [10 mM D-glucose, 120 mM NaCl, 4.5 mM KCl, 0.7 mM Na2HPO4, 1.5 mM NaH2PO4, and 0.5 mM MgCl2 (pH 7.4 at 37 °C)] for 20 min. The dishes were then washed once with 5 mL KRB to remove residual dye. The cultures were then treated for the designated times (10, 20 and 30 min) with 15 lM of KPS-A or the vehicle Me2SO diluted in KRB alone. Cells were collected by centrifugation, washed in 5 mL of Ca2+-free PBS at 37 °C, pelleted by centrifugation, resuspended in 1 mL of Ca2+-free PBS at 37 °C and analyzed immediately for Fluo-3 fluorescence intensity by flow cytometry (Becton Dickinson Co., Germany). 2.7. Statistical analysis Data are reported as means ± S.D. All experiments were done at least three times, and three or more independent observations were made on each occasion. Statistically significant values were compared using Student’s t-test for single comparison and p-values less than 0.05 were considered statistically significant.

3. Results 3.1. Induction of apoptosis by kalopanaxsaponin A Treatment of U937 human leukemia cells with KPS-A resulted in DNA fragmentation, DNA ladder formation, and an increase in the sub-G1 phase. As shown in Fig. 2A, DAPI staining assay revealed that KPS-A induce DNA fragmentation in a time- and dose-dependent manner in U937 cells; DNA fragmentation increased on increasing of KPS-A concentrations up to 15 M following 1, 2, 4 and 8 h treatment. In addition, we found that

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Fig. 2. Effects of kalopanaxsaponin A (KPS-A) on apoptotic induction and DNA fragmentation in U937 cells. (A) U937 cells were treated with 7.5 or 15 lM KPS-A for the indicated times. The extent (%) of fragmentation was determined using DAPI, as described in Section 2. Data are presented with the means ± S.D. of the results from three independent experiments. (d) 15 lM, (j) 7.5 lM, (s) Control. (B) U937 cells were treated with 15 lM KPS-A for the indicated times, and DNA fragmentation was analyzed by agarose gel electrophoresis. (C) Determination of sub-G1 cells in KPS-A-treated U937 cells by flow cytometry. U937 cells were treated with 15 lM KPS-A for the indicated times.

treatment with KPS-A at 15 lM for these times induced DNA ladder formation and an increase in the sub-G1 phase, suggesting the apoptotic activity of KPS-A in human leukemia cells (Figs. 2B and C). 3.2. Caspase activities during kalopanaxsaponin A-induced apoptosis Since it has been suggested that apoptosis requires the activation of caspases in many cases (Ashkenazi and Dixit, 1998; Hakem et al., 1998), we investigated the involvement of caspases activation in KPS-A-induced apoptosis in U937 cells using Western blot analysis. As illustrated in Fig. 3A, treatment with KPS-A stimulated a time-dependent cleavage of procaspase-3 and PARP, indicating

the activation of caspase-3. To elucidate the mechanism by which KPS-A activates caspase-3, we examined changes in the activity of caspase-8 and caspase-9 in KPS-A-treated U937 cells. The activations of both caspases-8 and -9 were evidenced by the degradation of their proenzymes (Fig. 3A). It is noteworthy that caspase-8 was activated by KPS-A faster and more significantly than caspase-9 or caspase-3. To validate whether the activations of caspase-8 and caspase-9 are required for KPS-A-stimulated apoptosis, we used various caspase inhibitors such as z-VAD-fmk (a broad caspase inhibitor), z-IETD-fmk (a caspase-8 inhibitor), and z-LEHD-fmk (a caspase-9 inhibitor). As shown in Fig. 3B all the three inhibitors markedly attenuated KPS-A-stimulated DNA fragmentation, suggesting that KPS-A-induced apoptotic cell death is largely

Fig. 3. Effect of KPS-A on the activity of caspase-8, -9, and -3. KPS-A induced the cleavages of procaspases-8, -9 and -3 in the cytosol. U937 cells were treated with KPS-A (15 lM) for indicated times. After treatment, the cytosolic fractions were separated by SDS-PAGE, transferred onto cellulose membranes, and then blotted with caspase-8, -9, and -3 specific antibodies. b-actin was used as an internal control. (B) The effect of caspase-inhibitors (z-VAD-fmk, z-IETD-fmk and z-LEHD-fmk) on apoptosis in response to KPS-A-induced DNA fragmentation. U937 cells were pretreated with or without 50 lM z-VAD-fmk, 50 lM z-IETD-fmk, or 50 lM z-LEHD-fmk for 1 h, and then challenged with KPS-A at 15 lM for 4 h. DNA fragmentation was measured using DAPI as described in Section 2. (h) None, (j) KPS-A. The data presented are the means ± S.D. of the results of three independent experiments. *p < 0.01, significantly different from the KPS-A-treated group by Student’s t-test.

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dependent on caspase activation and both caspases-8 and -9 as well as caspases-3 are required for the apoptosis. 3.3. Release of cytochrome c into cytosol and loss of mitochondrial membrane potential induced by kalopanaxsaponin A Considering the crucial role of the mitochondrial pathway in apoptosis, we examined changes in cytochrome c release into the cytosol and in mitochondrial membrane potential in KPS-A-treated U937 cells. Fig. 4A shows that KPS-A significantly increased the level of cytosolic cytochrome c. It is known that the release of cytochrome c into the cytosol is preceded or accompanied by a decrease in the mitochondrial membrane potential (DWm). To evaluate DWm changes in U937 cells exposed to KPS-A, we used DiOC6(3), a mitochondria-specific and voltage-dependent dye. Following treatment of U937 cells with KPS-A (15 lM), DWm was significantly reduced at 1.5 and 3 h (Fig. 4B). Interestingly, as with KPS-A, CCCP, an uncoupling agent for oxidative phosphorylation, also induced DWm depolarization. Because it has been demonstrated that there is a causal relationship between ROS production and a loss of DWm (Ka et al., 2003), we attempted to measure changes in ROS levels in KPS-A-treated cells using flow cytometry technique with dichlorodihydrofluorescein diacetate (DCFH-DA), a ROS-specific dye. However, no significant increase was observed in the levels of intracellular oxidants, e.g., hydrogen peroxide and superoxide (data not shown). Considering that Bid cleavage mediate the release of cytochrome c from mitochondria in CD95-induced apoptosis (Luo et al., 1998), we further examined the kinetics of Bid cleavage in U937 cells following treatment with KPS-A. As shown in Fig. 4A, KPS-A stimulated Bid cleavage in a time-dependent manner, and that this coincided with changes in caspase-8 activation, suggesting that Bid is involved in cytochrome c release induced by KPS-

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A treatment. Since it has been shown that tBid, which translocates to mitochondria during apoptosis, is able to trigger a change in Bax conformation (Eskes et al., 2000), cytochrome c release (Hengartner, 2000), and subsequent caspase activation (McConkey and Orrenius, 1996), we evaluated the abundance of cytoplasmic and mitochondrial Bax in U937 cells treated with KPS-A (15 lM). Treatment with KPS-A dramatically decreased the cytosolic levels of Bax and increased its mitochondrial levels. In contrast, no significant change in the levels of anti-apoptotic Bcl-2 and Bcl-xL was observed. Caspase-8 cleaves Bid and then the truncated Bid (tBid) triggers the release of mitochondrial cytochrome c into the cytosol and activates caspase-9 followed by caspase-3 activation (McConkey and Orrenius, 1996). We employed caspases-8 specific inhibitor z-IETD-fmk to test whether the translocation of tBid and Bax occurs in a caspase-8-dependent manner. Pretreatment of U937 cells with the z-IETD-fmk significantly prevented KPS-A-induced intracellular Bid and Bax translocation, cytochrome c release to the cytosol, and caspase-3 activation (Fig. 4C). These results suggest that KPS-A induces the translocations of Bid and Bax to mitochondria via the activation of caspase-8. 3.4. Kalopanaxsaponin A elevates [Ca2+]i MacConkey and Orrenius have proposed that the abnormal elevation of ½Ca2þ i is one of the major features of apoptosis (Carafoli and Molinari, 1998). To examine whether KPS-A-induced apoptosis is associated with an increase in ½Ca2þ i , we evaluated the intensity of intracellular fluo-3 fluorescence, an indicator of ½Ca2þ i , in U937 cells treated with or without KPS-A. Following treatment with KPSA (15 lM) for 10 min, ½Ca2þ i was found to be 2.3 times higher than that of the controls, and this elevated ½Ca2þ i was sustained for up to 30 min (Fig. 5A). Since ½Ca2þ i elevation is due to either Ca2+

Fig. 4. Determination of the release of cytochrome c into the cytosol, Bcl-2 family protein levels, and mitochondrial membrane potentials in kalopanaxsaponin A (KPS-A)induced apoptosis. (A) Immunoblotting was performed to determine cytochrome c release from mitochondria into the cytosol and the translocation of Bid and Bax from the cytosol to mitochondria in KPS-A-treated U937 cells. This experiment was repeated three times with similar results. (B) U937 cells were treated with 15 lM KPS-A for 1.5 and 3 h and then DWm was determined using DiOC6(3) by flow cytometry as described in Section 2. (C) Effect of caspase-8 inhibitor on KPS-A-induced apoptotic signals. U937 cells were pretreated for 1 h with z-IETD-fmk (a caspase-8 inhibitor) at 50 lM and then treated with 15 lM KPS-A for 4 h. Cytosolic protein extracts were separated by SDS– PAGE and immunoblotted as described in Section 2.

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Fig. 5. EGTA attenuates kalopanaxsaponin A (KPS-A)-induced Ca2+ influx, apoptosis, and caspase activation. (A) KPS-A increases ½Ca2þ i in U937 cells. Cells were loaded with Fluo-3 acetoxymethylester (4 lM) diluted in KRB for 20 min, washed once with 5 mL KRB to remove residual dye, treated with 15 mM KPS-A in the presence (N) or absence (h) of EGTA for the indicated times, and analyzed using a FACSscan. Control (j) values were obtained in the absence of KPS-A or EGTA. The values are the means ± S.D. of three independent experiments. (B) Effect of EGTA on sub-G1 cells in KPS-A-treated U937 cells by flow cytometry. (C) Effect of EGTA on the cleavages of procaspase-3, -6, and -9 induced by KPS-A.

influx from the external medium or Ca2+ released from internal stores, EGTA was used to chelate extracellular free Ca2+ so to prevent its entry into the cells. Fig. 5A shows that pretreatment with 5 mM EGTA for 5 min significantly suppressed the KPS-A-induced ½Ca2þ I levels, the sub-G1 phase, and the activation of caspases-3, -8 and -9 (Fig. 5B and C). It is of note that the calcium ion is known to act on multiple targets to trigger apoptosis. For instance, calpain, a calcium-dependent protease is considered as a potential target through which elevated calcium triggers apoptosis (Kaufmann, 1989). However, either calpain inhibitors I or II did not show any significant change in KPS-A-induced actions in U937 cells (data not shown). These results indicate that an increase in [Ca2+] induce apoptosis via the activation of caspases rather than calpain. 4. Discussion Uncontrolled imbalance between cell proliferation, cell loss, physiologic cell death (apoptosis), and differentiation, leads to the development of the clones of malignant cells. From this understanding of tumor biology with respect to the kinetics of cell populations, two new strategies, namely, apoptosis and differentiation have recently emerged as fields of interest in studies on cancer chemoprevention and chemotherapy. Various compounds that are used in cancer chemotherapy, such as VP16, cisplatin, adriamycin, and taxol have apoptosis-inducing activity (Friesen et al., 1996; Bhalla et al., 1993; Li and Yuan, 1999). Therefore, chemical agents with strong apoptosis-inducing activity, but minimal toxicity, would be expected to have potential as anticancer drugs. The present study shows that KPS-A is able to induce apoptosis in U937 cells, and that this is mediated via the Ca2+-dependent and caspase-8-dependent apoptotic pathway. Apoptosis is a cell-suicide mechanism that requires specialized cellular machinery, and a central component of this machinery is a

proteolytic system involving caspases, a highly conserved family of cystein proteases with specific substrates. The caspase family is divided into two groups. The members of one group, derived from caspases with long predomains (caspases-2, -8, -9, and -10), are referred to as initiator or upstream caspases, and the members of the other, which are derived from precursor with short predomains are called the effector or down-stream caspases (caspases-3, -6, -7, and -14) (Muzio et al., 1998). In our study, z-VAD-fmk (a broad caspase inhibitor) was found to inhibit the DNA fragmentation induced by KPS-A, suggesting that caspase may be involved in the process of KPS-A-triggered apoptosis. Furthermore, caspase-8 or caspase-9 inhibitor also completely inhibited KPS-A-induced DNA fragmentation. According to a previous report, caspase-8 not only activates caspase-3, but also activates caspase-9 via cytochrome c release (Li et al., 1998). Therefore, we hypothesize that caspase-8 is likely to be the most apical caspase in KPS-A-induced apoptosis, and finally converges to caspase-3. According to our observations, the loss of mitochondrial membrane potential by KPS-A seems to support this hypothesis. Bid, a proapoptotic Bcl-2 family member, can be cleaved by caspase-8. The carboxyl-terminal fragment of Bid then translocates to mitochondria to induce cytochrome c release; moreover, this translocation is 500 times more numerous than Bax (Finucane et al., 1999). In the present study, a decrease in the pro-form of Bid indicates that it was cleaved during KPS-A-induced apoptosis; moreover, either broad caspases inhibitor or caspase-8 specific inhibitor attenuated this cleavage. These data suggest that the cleavage of Bid is caspase8-dependent. In addition to Bid, other Bcl-2 family members are also associated with the release of cytochrome c. For example, Bax promotes cytochrome c release, but Bcl-2 and Bcl-xL counteract the effect of Bax and inhibit cytochrome c release (Kluck et al., 1997; Kharbanda et al., 1997). Bcl-xL binds to cytochrome c or Apaf-1 to prevent

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apoptosis (Pan et al., 1998). In KPS-A-induced apoptosis, no changes in the levels of Bcl-2 or Bcl-xL were observed while the ratio of mitochondrial and cytosolic Bax was markedly increased in a time-dependent manner following treatment with KPS-A. Taken together, these results indicate that Bid and Bax, but not Bcl-2 and Bcl-xL, are involved in the caspase-8-dependent pathway of KPS-A-induced apoptosis and that their involvements result in cytochrome c release. In this study, we observed that KPS-A activated caspase-8 as well as caspase-9 and -3 (Fig. 3A). At this point, however, we do not understand the precise underlying mechanisms by which KPS-A increase the activity of caspases-8. Because caspase-8 is a key component of the FAS death receptor-triggered apoptotic pathway, it is of interest to study whether KPS-A would elevate the expression of Fas ligand. In fact, many other apoptotic pathways initiated by quite distinct stimuli require Fas engagement. For instance, cell apoptosis triggered by anticancer drugs (Fulda et al., 1997a,b), irradiation (Caricchio et al., 1998), or ceramide (Belka et al., 1999) is mediated by the up-regulation of Fas-L and its interaction with Fas. It is also possible that KPS-A activates caspases-8 via an Lck-controlled pathway in a Fas-L-independent manner (Harman and Maxwell, 1995). Further study is necessary to elaborate the precise mechanism. In addition, we demonstrated that the prolonged activation of ½Ca2þ i may mediate the apoptotic action of KPS-A. ½Ca2þ i is known to act as a common mediator of chemical-induced cellular toxicity (Baek et al., 1997) and apoptotic cell death (McConkey and Orrenius, 1997). The major pathways of ½Ca2þ i increase are Ca2+ influx from extracellular space and Ca2+ release from internal Ca2+ stores. Numerous studies have demonstrated that these two pathways appear to be involved in the ½Ca2þ i increase associated with apoptosis (Baek et al., 1997). In particular, KPS-A seems to be selectively associated with a external Ca2+ influx mechanism, since the apoptotic activity of KPS-A were completely blocked by EGTA, a blocker of Ca2+ influx but not by TMB-8 or dantrolene, ½Ca2þ i blockers (data not shown). We also tested the effect of voltage-operated Ca2+ channel blockers such as Ni2+, nifedipine, or verapamil on KPS-A-induced apoptosis, but they had no effect in U937 cells (data not shown). These data indicate that KPS-A increases cell membrane permeability and induces the influx of extracellular Ca2+ through an undefined Ca2+ channel type in human leukemia cells. Apoptosis repressor with caspase recruitment domain (ARC) seems to be a calcium-binding protein and functions as a cytosolic Ca2+ buffer in cells. There is increasing evidence showing that ARC possesses the ability not only to block activation of caspase-8 but to modulate caspase-independent mitochondrial events associated with cell death (Jo et al., 2004). Moreover, it has been reported that expressional regulation of ARC interfered with various types of Ca2+-mediated cell death, including caspase-8 dependent and caspase-8 independent cell death. The mechanism by which KPS-A induced caspase-8 dependent apoptosis via modulation of ARC remains to be defined. In summary, our data proposes a model of kalopanaxsaponin Ainduced apoptosis in which ½Ca2þ i elevation via Ca2+ influx and caspase-8 activation play a crucial role. These two events may cause a release of cytochrome c from mitochondria and downstream caspase activation leading to apoptosis. The anti-tumor or chemopreventive effects of kalopanaxsaponin A described in this work will contribute to potential therapeutical strategies for leukeminogenesis.

Conflict of interest statement The authors declare that there are no conflicts of interest.

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Acknowledgment This work was supported by MRC Grant (R13-2007-019-000000) from MOST.

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