Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells

Cancer Letters 163 (2001) 83±93

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Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells Eun-ok Im a,1, Yung Hyun Choi b,1, Kee-Joo Paik a, Hongsuk Suh c, Youngeup Jin c, Kyu-Won Kim d, Young Hyun Yoo e, Nam Deuk Kim a,* a Department of Pharmacy, Pusan Cancer Research Center, Pusan National University, Pusan 609-735, South Korea Department of Biochemistry, College of Oriental Medicine, Dong-Eui University and Research Center for Oriental Medicine, Pusan 614-054, South Korea c Department of Chemistry, Chemistry Institute for the Functional Materials, Pusan Cancer Research Center, Pusan National University, Pusan 609-735, South Korea d Department of Molecular Biology, Pusan Cancer Research Center, Pusan National University, Pusan 609-735, South Korea e Department of Anatomy and Cell Biology, Dong-A University College of Medicine, Pusan 602-103, South Korea b

Received 14 July 2000; received in revised form 18 October 2000; accepted 23 October 2000

Abstract We have compared the anti-proliferative effects of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and their derivatives, HS-1183, HS-1199 and HS-1200, on MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53) cells. While UDCA and CDCA exhibited no signi®cant effect, their novel derivatives inhibited the proliferation of both cell lines in a concentrationdependent manner, concomitant with apoptotic nuclear changes and the increase of a sub-G1 population and DNA fragmentation. Furthermore, we also observed an increase in the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 and cleavages of lamin B and poly(ADP-ribose) polymerase (PARP) in MCF-7 and MDA-MB-231 cells. Cell cycle related proteins, cyclin D1 and D3, as well as retinoblastoma protein (pRb) were down-regulated, while the level of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 was increased in both cancer cells after treatment with novel bile acids. These ®ndings suggest that these cytotoxic effects of novel bile acid derivatives on human breast carcinoma cells were mediated via apoptosis through a p53independent pathway. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Novel bile acids; Apoptosis; p53; p21 WAF1/CIP1; Bcl-2; Bax

1. Introduction Bile acids are polar derivatives of cholesterol essential for the absorption of dietary lipids and regulate the transcription of genes that control cholesterol * Corresponding author. Tel.: 182-51-5102801; fax: 182-515136754. E-mail address: [email protected] (N.D. Kim). 1 These authors contributed equally to this work.

homeostasis. However, depending on the nature of the chemical structures, different bile acids exhibit distinct biological effects [1]. Hydrophilic ursodeoxycholic acid (UDCA) and its taurine and glycine conjugates protect cells against apoptosis induced by several hydrophobic bile acids [2,3]. This protective effect is due to the direct prevention of mitochondrial membrane perturbation [4]. UDCA also functions as a chemopreventive agent in azoxymethane-treated rats [5] and an inhibitor of cell proliferation [1] and

0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00671-6

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initiation of SV40 DNA replication in vitro [6]. However, chenodeoxycholic acid (CDCA), a primary bile acid, has been shown to act as a tumor promoter in animal models and to enhance cell transformation in vitro and apoptosis in several different tumor cell lines [1,7,8]. We have previously reported that a novel glycine methyl ester conjugate of UDCA, HS-1030, induced apoptosis in HepG2 human hepatocellular carcinoma cells and MCF-7 human breast carcinoma cells [9± 11]. Moreover, UDCA and its novel derivatives, HS-1030 and HS-1183, inhibited SV40 DNA replication, and predominantly inhibited the initiation stage of DNA replication [6]. To ®nd a more effective agent than HS-1030 in apoptosis-inducing activity, we have synthesized and characterized a new series of synthetic derivatives of CDCA and examined their effects on apoptosis induction and the expression of apoptosis-related genes. We report here the structures and activities of the three most potent UDCA and CDCA derivatives.

2. Materials and methods 2.1. Chemicals UDCA and CDCA were obtained from Dae-Woong Pharmaceutical Co. (Seoul, South Korea) or Aldrich (Milwaukee, MI). N,N-Dimethylformamide (DMF), dicyclohexylcarbodiimide (DCC), hydroxybenztriazole (HOBT), triethylamine (TEA), glycine methyl ester hydrochloride, l-leucine benzyl ester p-toluenesulfonate salt, and l-leucine methyl ester hydrochloride were purchased from Aldrich or Sigma (St. Louis, MO) and used without further puri®cation. All melting points were determined by using an electrothermal melting point apparatus. 1H NMR spectra were determined either by a Varian Gemini 300 MHz or a Varian Gemini 200 MHz spectrometer using tetramethylsilane or chloroform as internal standards. 13C NMR spectra were recorded on a Varian Gemini 200 MHz spectrometer operating at 50 MHz. IR spectra were obtained using the Mattson Polaris FT-IR for samples as KBr pellets or thin ®lms. Column chromatography was performed on silica gel (Merck, 70±230 mesh, ASTM).

2.2. Preparation of CDCA derivatives 2.2.1. Conjugate form of CDCA with l-phenyl alanine benzyl ester (N-((3a,5b,7a)-3,7-dihydroxy-24oxocholan24-yl) l-phenyl alanine benzyl ester; HS1199) To a solution of 1.0 g (2.76 mmol) of CDCA in 20 ml of DMF, 631 mg (3.06 mmol) of DCC and 400 mg (2.96 mmol) of HOBT were added at 48C. After stirring at 48C for 40 min, 1.9 g (4.44 mmol) of l-phenyl alanine benzyl ester p-toluenesulfonate salt in 10 ml of DMF and 590 ml of TEA (4.24 mmol) was added. After stirring overnight at room temperature, the reaction mixture was diluted with 200 ml of ethyl acetate, and washed with magnesium sulfate, and concentrated under reduced pressure. The residue was puri®ed by ¯ash chromatography to give 1.03 g (58%) of N((3a,5b,7a)-3,7-dihydroxy-24-oxocholan24-yl) lphenyl alanine benzyl ester (HS-1199) as a yellow foam: Rf 0.33 (SiO2, 30% ethyl acetate±hexane); 1H NMR (200 MHz, CDCl3) d 7.42±6.94 (m, 10H), 5.94 (d, 1H, J ˆ 7:69), 5.15 (dd, 2H, J ˆ 12:08, 3.64), 4.94 (q, 1H, J ˆ 7:33), 3.54±3.42 (m, 2H), 3.14 (d, 2H, J ˆ 5:51), 2.30±0.86 (m, 34H), 0.63 (s, 3H); 13C NMR (50 MHz, CDCl3) d 173.6, 171.5, 135.7, 134.9, 129.0, 128.5, 128.4, 128.2, 126.9, 126.5, 125.6, 117.8, 110.8, 71.6, 67.0, 56.1, 54.7, 52.9, 44.2, 42.5, 40.0, 39.3, 37.6, 37.1, 36.5, 35.2, 34.9, 33.9, 33.3, 31.2, 30.4, 28.7, 26.8, 23.4, 21.0, 18.3, 12.1. 2.2.2. Conjugate form of CDCA with b -alanine benzyl ester (N-((3a,5b,7a)-3,7-dihydroxy-24-oxocholan24yl) b-alanine benzyl ester; HS-1200) To a solution of 1.0 g (2.76 mmol) of CDCA in 20 ml of DMF, 631 mg (3.06 mmol) of DCC and 400 mg (2.96 mmol) of HOBT were added at 48C. After stirring at 48C for 40 min, 1.0 g (4.64 mmol) of b-alanine benzyl ester p-toluenesulfonate salt in 10 ml of DMF and 590 ml of TEA (4.24 mmol) was added. After stirring overnight at room temperature, the reaction mixture was diluted with 200 ml of ethyl acetate, and washed with magnesium sulfate, and concentrated under reduced pressure. The residue was puri®ed by ¯ash chromatography to give 1.05 g (67%) of N-((3a,5b,7a)-3,7-dihydroxy-24-oxocholan24-yl) balanine benzyl ester (HS-1200) as a yellow foam: Rf 0.33 (SiO2, 80% ethyl acetate±hexane); 1H NMR (200

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MHz, CDCl3) d 7.34 (br s, 5H), 6.00 (br s, 1H), 5.13 (s, 2H), 4.11 (q, 2H, J ˆ 7:33), 3.55±3.47 (m, 2H), 2.57 (t, 2H, J ˆ 6:59), 2.29±0.89 (m, 34H), 0.63 (s, 3H); 13C NMR (50 MHz, CDCl3) d 173.5, 172.3, 135.5, 128.4, 128.2, 128.0, 72.2, 71.7, 68.1, 66.3, 60.2, 55.7, 50.2, 42.5, 41.4, 39.6, 39.3, 35.3, 34.9, 34.7, 34.5, 34.0, 33.3, 32.6, 31.6, 30.5, 23.5, 22.6, 20.8, 20.4, 18.2, 14.0, 11.6. 2.3. Cell culture and growth study MCF-7 and MDA-MB-231 human breast carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD). They were routinely cultured in DMEM (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 50 mg/ml gentamicin and 135 mg/ml glutamine. The cell growth was measured by the MTT (3-(4,5-dimethylthiazol-2y1)-2,5-diphenyl tetrazolium bromide) colorimetric dye reduction method as described previously [11]. Bile acids were dissolved in 100% ethanol and stored at 2208C before the experiments.

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Monoclonal antibodies to Bcl-2, lamin B, poly(ADP-ribose) polymerase (PARP) and p53, and polyclonal antibodies to Bax, cyclin D1 and D3, cyclindependent kinase (Cdk) 2, Cdk4 and retinoblastoma protein (pRb) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-p21 WAF1/CIP1 antibody was obtained from Transduction Lab. (Lexington, KY). Peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidaselabeled sheep anti-mouse immunoglobulin were purchased from Amersham Life Science. 3. Results 3.1. Novel bile acids reduced the viability of cells The structures of UDCA and its conjugate form (HS-1183) and CDCA and its conjugate forms (HS-

2.4. Apoptosis assays DNA content analysis using ¯ow cytometry was performed as described previously [12]. For the DNA fragmentation assay, cell pellets were lysed in lysis buffer (5 mM Tris±HCl (pH 7.5), 5 mM EDTA, and 0.5% Triton X-100) at 48C for 30 min. After centrifugation at 27 000 £ g for 15 min, DNA from supernatant fractions was isolated by phenol/chloroform extraction, analyzed by electrophoresis through 1.5% agarose gels in TAE (40 mM Tris-acetate and 1 mM EDTA) buffer, and then visualized by ethidium bromide (EtBr, Sigma) staining. 2.5. Western immunoblot analysis Whole cell extracts were prepared in lysis buffer as described previously [12]. The protein concentration was determined with the BCA (Bicinchoninic acid) reagent (Pierce, Rockford, IL). Equal amounts of protein were subjected to electrophoresis on SDSpolyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by electroblotting. Proteins were visualized with the enhanced chemiluminescence (ECL) detection system (Amersham Life Science, Arlington Heights, IL).

Fig. 1. Structures of UDCA and its derivative, HS-1183, and CDCA and its derivatives, HS-1199 and HS-1200.

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Fig. 2. Effects of bile acids and their derivatives on the viability of breast carcinoma cells. (A), MCF-7; and (B), MDA-MB-231 cells were treated with different concentrations of UDCA, CDCA and their derivatives for 24 h, and then the percentage of cell survival was determined using the MTT assay. Each value is a mean calculated from at least three independent experiments. The values from each treatment were expressed as a percentage relative to the control (100%). Bars represent the mean ^ SEM.

1199 and HS-1200) are shown in Fig. 1. To investigate the effects of three bile acid derivatives on the viability of MCF-7 and MDA-MB-231 cells, we performed the MTT assay. Cell viability was signi®cantly decreased by treatment of the novel bile acid derivatives for 24 h in a concentration-dependent manner; their parent compounds, UDCA and CDCA, showed weak activities (Fig. 2). HS-1183 showed a moderate cytotoxic effect when compared with HS-1199 and HS-1200 in both cell lines. The doses required for half-maximal inhibition (IC50) of MCF-7 cell growth were about 30 mM for HS-1199 and HS-1200, and about 150 mM for HS-1183. These results show that HS-1199 and HS-1200 had stronger cytotoxic effects on MCF-7 cells than HS-1030, which had an IC50 value of about 150 mM [11]. For MDA-MB-231 cells, the IC50 values were about 30 mM for HS-1200, and about 45 mM for both HS1199 and HS-1183, whereas no effect was observed in control cells treated with the vehicle solvent (data not shown). 3.2. Novel bile acids induced apoptosis To study whether the cytotoxic effect of novel bile acid derivatives might be mediated by apoptosis, ¯ow

cytometry analysis and DNA fragmentation assays were performed. As shown in Fig. 3A, sub-G1 populations of cells were signi®cantly increased by treatment with novel bile acid derivatives at 50 mM for 24 h. Moreover, agarose gel electrophoresis of DNA extracted from cells treated with novel bile acid derivatives revealed ladders of DNA fragmentation (Fig. 3B), indicative of induced apoptosis in both MCF-7 and MDA-MB-231 cells. Since apoptosis might be regulated by the alteration of the ratio of Bcl-2/Bax family protein expression [13], we tested whether novel bile acid derivative-induced apoptosis was accompanied by the change of the expression of Bax and Bcl-2. The results from Western immunoblotting showed that treatment of cells with novel bile acids derivatives resulted in the up-regulation of Bax in both cell lines (Fig. 4A). HS-1183, HS-1199 and HS-1200 were also found to signi®cantly downregulate the Bcl-2 expression in MDA-MB-231 cells (Fig. 4B). However, no effect of these three compounds on Bcl-2 expression was found in MCF7 cells (Fig. 4B). Previously, we reported that a novel UDCA derivative, HS-1030, also showed a similar result in MCF-7 cells [11]. The degradation of polypeptides, such as lamin B and PARP, was used to further examine the possible involvement of apopto-

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Fig. 3. Apoptosis inducing effects of bile acids treated with 50 mM for 24 h on breast carcinoma cells. (A) With ¯ow cytometry analysis, HS1200, HS-1199 and HS-1183 increased the sub-G1 fraction of MCF-7 and MDA-MB-231 cells. Each bar represents the mean numbers of three independent experiments. Bars represent the mean ^ SEM. (B) HS-1200, HS-1199 and HS-1183 also induced DNA fragmentation in MCF-7 and MDA-MB-231 cells treated with 50 mM bile acids for 24 h. Marker represents a 1 kb ladder used as a molecular size marker. Control, without bile acid.

sis-associated proteases in the induction of apoptosis and growth inhibition of breast carcinoma cells. When both cell lines were treated with the three novel compounds (50 mM) and underwent apoptosis, the cleavages of lamin B and PARP proteins were clearly observed (Fig. 5A,B). Cleavage of PARP was evident by the appearance of the p85 PARP cleavage fragments in MDA-MB-231 cells treated with HS-1200.

3.3. Novel bile acids down-regulated D-type G1 cyclins To understand the mechanism of inhibition of cell growth, asynchronous MCF-7 and MDA-MB-231 cells were treated with or without 50 mM bile acids for 24 h, and the expressions of cell cycle regulating proteins (i.e. D-type cyclins, Cdk2 and Cdk4) were

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Fig. 4. Effect on Bcl-2 family in MCF-7 and MDA-MB-231 cells after treatment with 50 mM bile acids for 24 h. Alteration of ratio of Bax to Bcl-2 expression in both cell lines treated with bile acids was analyzed by Western blot analysis using: (A), anti-Bax; and (B), anti-Bcl-2 antibodies, and ECL detection.

compared by Western blot analysis. The intracellular protein levels of cyclin D1 and/or D3 (Fig. 6A,B) were down-regulated by HS-1199 and HS-1200, but not by HS-1183 in both cell lines. However, the levels of Cdks (Fig. 6C,D) were not changed in both cell lines. 3.4. Novel bile acids increased intracellular levels of p21 WAF1/CIP1 and inhibited phosphorylation of pRb It has been shown that the tumor suppressor, p53, regulates a DNA damage-triggered G1 checkpoint by up-regulation of p21 WAF1/CIP1 expression [14±16].

Fig. 5. Cleavages of lamin B and PARP during bile acid-induced apoptosis. MCF-7 and MDA-MB-231 cells were incubated with 50 mM bile acids for 24 h. Aliquots of cellular extracts were examined by Western blot analysis using monoclonal antibodies to: (A), lamin B; and (B), PARP. The full-length forms of lamin B (p69) and PARP (p116), and their cleaved fragments of lamin B (p32) and PARP (p85) are indicated.

Moreover, transcription of the p21 WAF1/CIP1 gene is also induced by other factors in p53-independent pathways [17,18]. Therefore, experiments were carried out to examine if the Cdk inhibitor would be induced by bile acids via a p53-dependent or a p53independent pathway. The results show that treatment with novel bile acid derivatives resulted in a marked increase in the level of p21 WAF1/CIP1 protein in both cancer cells (Fig. 7A). Even though there were slight increases in the expression levels of wild-type p53 protein in MCF-7 treated with HS-1199 and HS1200, the induction of p21 WAF1/CIP1 in MDA-MB-231 cells was independent of p53 (Fig. 7B). The Rb gene product pRb is an important checkpoint protein in the G1/S phase transition of the cell cycle. Furthermore, p21 WAF1/CIP1 inhibits the phosphorylation of pRb, which is mediated by cyclin D/Cdk [19±21]. We therefore examined whether the effect of decreased expression of D-type G1 cyclins by novel bile acids was associated with the decreased phosphorylation of pRb in cells treated with bile acids. As shown in Fig. 7C, the levels of both expression and phosphorylation of pRb were decreased slightly in MCF-7 cells treated with HS-1183, and remarkably decreased in MDAMB-231 cells treated with novel bile acid derivatives. 4. Discussion In the present study, using two different human breast carcinoma cell lines, MCF-7, which expresses the wild-type p53 gene, and MDA-MB-231, which

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Fig. 6. Effects of bile acids on the protein levels of D-type cyclins and Cdks. MCF-7 and MDA-MB-231 cells were treated with 50 mM bile acids for 24 h. Total cell lysates were prepared and immunoblotted. Western blots were detected with antibodies against: (A), cyclin D1; (B), cyclin D3; (C), Cdk2; and (D), Cdk4, and ECL detection.

contains the mutant p53 gene [22], we have demonstrated that the treatment of both cell lines with novel bile acid derivatives showed signi®cant cytotoxic effects in a dose-dependent manner compared with the parental compounds. However, in the human immortalized breast epithelial cell line, MCF-10A, there was little cytotoxic effects at concentrations of 50, 100 and 150 mM (data not shown). The treatment also induced apoptosis as demonstrated by an increase in the apoptotic sub-G1 population and the presence of DNA fragmentation and cleavages of lamin B and

PARP. Furthermore, we elucidated the mechanism of action of novel bile acid derivative-induced apoptosis in human breast carcinoma cells. DNA fragmentation has been observed in cells undergoing apoptosis, which can be induced by a variety of agents. This cleavage produces ladders of DNA fragments that are the size of integer multiples of a nucleosome length (180±200 bp) [23]. Due to their characteristic patterns revealed by agarose gel electrophoresis, these nucleosomal DNA ladders are widely used as biochemical markers of apoptosis. When cells

Fig. 7. Effects of bile acids treatment on the protein levels of p21 WAF1/CIP1, p53 and pRb in MCF-7 and MDA-MB-231 cells. The cells were treated with 50 mM bile acids for 24 h and total cell lysates were prepared and immunoblotted. Western blots were detected with antibodies against: (A), p21 WAF1/CIP1; (B), p53; and (C), pRb, and ECL detection.

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undergo apoptosis, the speci®c degradation of several proteins, such as lamin B and PARP, followed by internucleosomal DNA degradation has been reported [24,25]. PARP, a nuclear protein implicated in DNA repair, is a 116 kDa polypeptide that converts nicotinamide adenine dinucleotide to nicotinamide and protein-linked ADP-ribose polymers [26,27], and one of the earliest proteins targeted for a speci®c cleavage to the signature 85 kDa fragment during apoptosis [28]. In synthetic retinoid CD437-induced apoptosis, PARP cleavage was shown in human prostate carcinoma cells [29]. A collapse of chromatin and the nuclear structure in apoptosis is also consistent with the degradation of lamins, which are a part of the nuclear envelope [30]. Glucocorticoids stimulate the rapid degradation of lamin B before DNA fragmentation in thymocytes undergoing apoptosis [24]. In tumor necrosis factor-induced apoptosis, the speci®c degradation of lamin B has also been observed in 3T3-like ®broblasts [31]. Additionally, ¯ow cytometry analysis has been used to detect and quantify cells undergoing apoptosis [32]. The decreased cell numbers in the treatment groups compared with the control group could be due to apoptotic cell death as well as cell growth inhibition. The observation of DNA ladder formation, lamin B and PARP cleavages and the augmentation of apoptotic sub-G1 population indicate that apoptosis is a major underlying process of the cytotoxic effects of novel bile acid derivatives. The Bcl-2 oncoprotein and other related proteins may play an important role in determining whether cells undergo apoptosis. Increased expression of Bax can induce apoptosis by suppressing the activity of Bcl-2 [33]. It was also reported that the ratio of Bcl-2 to Bax, rather than Bcl-2 alone, is important for the survival of drug-induced apoptosis [34]. Our data showed that Bcl-2 expression was not signi®cantly changed in MCF-7 cells treated with novel bile acids, whereas there was slight increase in Bax expression (Fig. 4). However, the expression level of Bax was slightly increased and that of Bcl-2 was signi®cantly decreased in MDA-MB-231 cells (Fig. 4). Taken together, the increased ratio of Bax to Bcl-2 might contribute to the initiation of apoptosis in novel bile acid-treated MCF-7 and MDA-MB-231 cells. The cytotoxic effect of novel bile acids could also be mediated by changes in cell cycle regulation. In mammalian cells, D-type cyclins are synthesized

during the G1 phase and rate-limiting factors for S phase entry [35±37]. The major catalytic partners of D-type cyclins are Cdk4 and Cdk6 [38]. In addition, Cdk inhibitors regulate cell cycle progression by association with cyclin/Cdk complexes [39,40], which may inhibit cyclin-containing complexes that decrease the cyclin-dependent activity in damaged cells destined to apoptosis [41]. Based on these reports, we next investigated the effects of these bile acids on the expression of cell cycle regulatory proteins. The bile acids selectively decreased the intracellular protein levels of D-type cyclins, but there was no change in the levels of Cdks (Fig. 6). Moreover, it was found that the bile acids selectively induced the expression of the Cdk inhibitor p21 WAF1/ CIP1 (Fig. 7A), but did not affect on the protein level of p53 (Fig. 7B). In addition to direct transcriptional induction by p53, recent studies have demonstrated that p21 WAF1/CIP1 was also induced independently from p53 by a variety of signals [17,18,42]. Another important downstream target of the G1 phase cyclin/ Cdk complex is the pRb, which is controlled by Cdkmediated phosphorylation [17,38]. pRb is also known to regulate the transcription of a variety of genes encoding growth-regulatory factors [43,44]. As shown in Fig. 7C, the levels of expression and phosphorylation form of pRb were decreased in both novel bile acid derivative-treated MCF-7 and MDA-MB231 cells. Although the detailed molecular mechanism of the apoptosis-inducing activity of novel bile acids studied is not the focus of this study, we can speculate as to the possible mechanisms based on the data of previous studies. Four bile acids, such as cholic acid (CA), CDCA, deoxycholic acid (DCA), and UDCA, exhibited distinct biological effects on the induction of apoptosis and inhibition of proliferation in several human cancer cells [1]. DCA induces apoptosis via a protein kinase C-dependent signaling pathway. However, neither CA nor UDCA could induce apoptosis. CDCA, DCA and UDCA inhibited cell growth at .50 mM. Toxic bile salts, such as glycochenodeoxycholate, induced rodent hepatocyte apoptosis via the direct activation of Fas [45]. Therefore, it is possible that the novel bile acids may induce apoptosis via a protein kinase C-dependent signaling pathway or directly bind to Fas receptors and induce apoptosis. Another possible mechanism is that the novel bile acid

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derivatives may affect Fas-associated death domain or ceramide on the cell membrane because the chemical structures of novel bile acid derivatives resemble glucocorticoids [46]. Moreover, it has been recently reported that CDCA exerted its action by binding to an orphan nuclear receptor, farnesoid X receptor (FXR), as a natural ligand [47±49]. Similarly, the novel CDCA derivatives may bind FXR, a new orphan nuclear receptor, or even possibly a ligand for a coactivator/corepressor type of molecule. Therefore, further experiments are required to clarify the structure/function of the novel UDCA and CDCA derivatives. In summary, we have demonstrated that novel UDCA and CDCA derivatives are capable of inhibiting cell proliferation and inducing apoptosis in MCF7 and MDA-MB-231 human breast carcinoma cells with different tumor suppressor p53 status. Novel bile acid derivative-induced growth inhibition and apoptosis were associated with the up-regulation of Bax and p21 WAF1/CIP1 in both cell lines; the effects were mediated via a p53-inpendent pathway. Additional studies are required to elucidate the molecular mechanism of the apoptosis-inducing activity of novel bile acids.

[2]

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Acknowledgements The authors would like to thank Dr Chia-Cheng Chang for critical reading of the manuscript. This study was supported by a grant (#HMP-98-D-40036) of the Good Health R&D Project, Ministry of Health & Welfare, South Korea. This work was partially supported by Pusan National University Research Grants in 1998 and 1999. H. Suh thanks the Korea Science and Engineering Foundation (960501-08-01-03) for the partial ®nancial support. K.W. Kim thanks the National Research Laboratory fund, the Ministry of Science and Technology, South Korea.

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