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Induction of mitochondria-dependent apoptosis through the inhibition of mevalonate pathway in human breast cancer cells by YM529, a new third generation bisphosphonate
Cancer Letters 253 (2007) 89–96 www.elsevier.com/locate/canlet
Induction of mitochondria-dependent apoptosis through the inhibition of mevalonate pathway in human breast cancer cells by YM529, a new third generation bisphosphonate Hiroo Nakajima a, Junji Magae b,*, Mie Tsuruga b, Koichi Sakaguchi a, Ikuya Fujiwara a, Mitsuhiko Mizuta c, Kiyoshi Sawai a, Hisakazu Yamagishi d, Naruhiko Mizuta a a
Department of Endocrine and Breast Surgery, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan b Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan c Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan d Department of Digestive Surgery, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan Received 15 November 2006; accepted 15 January 2007
Abstract YM529, a new third generation bisphosphonate, induced apoptosis of a human breast cancer cell line, MX-1. Cytotoxic activity of YM529 was more potent than that of incadronate. YM529 activated caspase-9, but not caspase-8, and induced the release of cytochrome c into cytosol. YM529 increased Bax expression and decreased Bcl-2 expression, while it did not induce caspase-8-dependent Bid truncation. Farnesyl pyrophosphate prevented YM529-mediated cytotoxicity. These results suggest that YM529 is a potent therapeutic agent for human breast cancers, activating the mitochondria-dependent apoptotic pathway through the inhibition of protein farnesylation. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Apoptosis; Breast cancer; YM529; Bcl-2; Bax; Farnesylation
1. Introduction Apoptosis requires the activation of caspases, which are ubiquitously and constitutively expressed as inactive zymogens in the cytosol [1]. Caspases can be divided into initiator caspases (caspase-2, -8, -9 and -10) and effector caspases (caspase-3, -6 and -7). * Corresponding author. Tel.: +81 4 7144 9142; fax: +81 4 7144 9007. E-mail address: [email protected] (J. Magae).
The initiator caspases activate the downstream effector caspases, which subsequently degrade certain cellular proteins, resulting in cell death. The initiator caspases are activated through a process mediated by mitochondria or by death receptors on the cell surface. The mitochondria-mediated process begins with mitochondrial permeability transition, followed by the release of apoptogenic factors such as cytochrome c and apoptosis-inducing factor [1,2]. The initiator caspase-9 then becomes activated through the formation of the cytochrome c/Apaf-1/
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.01.008
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procaspase-9-containing apoptosome complex in cytosol, leading to the activation of downstream effector caspases [3]. Bcl-2 family proteins regulate the mitochondrial permeability transition by interacting with mitochondrial channels such as voltagedependent anion channel [4,5]. Pro-apoptotic members, such as Bax and Bid, induce the release of apoptotic factors, whereas anti-apoptotic members, such as Bcl-2 and Bcl-xl, prevent their release. In contrast, the death receptor-mediated process activates an apoptotic pathway that is dependent on the initiator caspase-8 [6]. Cross-linking of the extracellular domains of Fas by the Fas ligand results in Fas trimerization and intracellular recruitment of the adapter molecule Fas-associated death domain protein (FADD) to the receptor cluster. Recruitment is facilitated by homotypic interaction between death domains located in Fas and FADD. Receptor-bound FADD oligomerizes and utilizes a second domain, the death effector domain, to recruit procaspase-8. The Fas/FADD/ procaspase-8 complex is referred to as the deathinducing signaling-complex (DISC) [7,8]. DISC catalyzes the proteolytic conversion of procaspase-8 into active caspase-8, which activates the downstream effector caspases, triggering cell death. The death receptor-mediated process and mitochondrial pathways are connected by Bid, a proapoptotic Bcl-2 family member. Bid truncated by caspase-8 translocates from the cytosol to the mitochondria and induces cytochrome c release [9,10]. Whether apoptosis proceeds through the death receptor-mediated pathway or the mitochondrial pathway depends on the cell properties and the conditions of stimulation [11]. Bone metastasis commonly occurs in breast cancer patients; in fact, more than 80% of women with advanced breast cancer ultimately develop bone metastases, which account for significant morbidity and mortality [12]. Because patients with advanced breast cancer will eventually develop serious bone-related symptoms, various combination therapies such as analgesics, radiation therapy, and systemic treatments such as hormone therapy or chemotherapy are routinely given. However, even with these therapies, it is difficult to cure the cancer and alleviate the symptoms. To improve upon these conventional therapies, bisphosphonates, analogues of endogenous pyrophosphates in which the central atom of oxygen is replaced with a carbon atom, have been developed and used for patients with bone metastasis. In accordance with their structural innovation, bis-
phosphonates are classified as first, second, or third generation. Their main function is inhibition of osteoclast-mediated bone resorption [13,14] through the prevention of osteoclast maturation [15–17] and/ or induction of apoptosis in osteoclasts [18–20]. In addition to their direct effects on osteoclasts, bisphosphonates may directly induce apoptosis in metastasized breast cancer cells. In animal models of metastatic disease, bisphosphonates reduce the tumor burden in bone [21,22]. Bisphosphonates also inhibit the adhesion of breast cancer cells to bone in vitro [23] and directly induce apoptosis in human breast cancer cell lines in vitro [24]. The features of apoptosis, such as DNA fragmentation, decreased Bcl-2/ Bax radio, poly(ADP ribose) polymerase (PARP) cleavage [24], and caspase activation [22,25,26], have been identified in different in vitro systems. The simple bisphosphonates that closely resemble pyrophosphate such as clodronate, etidronate and tiludronate can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulates intracellularly, resulting in induction of apoptosis. By contrast, nitrogen-containing bisphosphonate such as pamidronate, alendronate, and risedronate, appear to act as analogues of isoprenoid diphosphate lipid, thereby inhibiting farnesyl pyrophosphate synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme prevents the biosynthesis of isoprenoid lipids that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signaling proteins [27]. Thus, cytotoxic activity and growth arrest by nitrogen-containing bisphosphonates is prevented by the addition of geranylgeraniol and farnesol [28,29], and inhibitors of mevalonate pathway induce apoptosis in the manner similar to bisphosphonates. In the present study, we investigated the signal transduction of apoptosis in breast cancer cells induced by YM529, a third-generation nitrogencontaining bisphosphonate. Our results suggest that YM529 induces cytochrome c release into the cytosol from mitochondria by modulating Bcl-2 family expression, leading to the activation of caspase-9 and subsequent effector-caspase dependent apoptosis, while the DISC-dependent apoptotic pathway is not affected. Because the apoptosis induced by YM529 was partially prevented by the addition of farnesyl pyrophosphate, inhibition of mevalonate pathway by YM529 is assumed to evoke apoptosis and subsequent activation of mitochondria-dependent apoptosis pathway.
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2. Materials and methods 2.1. Reagents Incadronate and YM529 were obtained from Yamanouchi Pharmaceutical Company (Tokyo, Japan). Geranylgeranyl pyrophosphate and farnesyl pyrophosphate were purchased from Sigma Chem. (St. Louis, MO, USA).
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lowed by the addition of 1.5 lg/ml of propidium iodide (PI, Nakarai Tesque, Kyoto, Japan). After excluding the debris by brief spinning (1000 rpm, 1 min), the morphology of the stained cells was examined using an MX50 fluorescence microscope (Olympus, Tokyo, Japan) with an ultraviolet filter. Cells with a blue condensed or fragmented nucleus were scored as apoptotic cells, and cells stained with PI were scored as dead cells.
2.2. Cell culture A human breast cancer cell line, MX-1, was kindly provided by Kyowa Hakko Inc. (Tokyo Japan). MDAMB-231 and ZR75-1 were purchased from American Type Culture Collection. It was maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 1% penicillinstreptomycin and 10% fetal bovine serum (BioWhittaker, MD, USA) at 37 C under a humidified atmosphere containing 5% carbon dioxide. 2.3. MTT dye reduction assay Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) dye reduction assay measuring mitochondrial respiratory function [30,31]. Breast cancer cells that were exponentially grown (5 · 104 cells/well) were plated in 96-well flatbottom plates (Nunc. Roskilde, Denmark) and allowed to adhere for 24 h. After incubation for 48 h with the drug, cells were incubated with MTT dye (1 mg/ml) for 2 h and solubilized with 20 ll of 10% SDS. The optical densities of the samples were measured at 550 nm. 2.4. DNA fragmentation assay DNA fragmentation was detected using TACSTM Apoptotic DNA Laddering Kits (Trevigen, MD, USA), according to the manufacturer’s instructions. MX-1 cells (1 · 106cells) were lysed. Their DNA was extracted and then separated by 2% agarose gel electrophoresis, followed by ethidium bromide staining to visualize the laddered DNA. 2.5. Determination of mitochondria membrane potential MX-1 cells (1 · 106 cells/ml) were incubated with 3,3 dihexyloxacarbocyanide iodide (DiOC6(3), Molecular Probes, Inc., Eugene, OR, USA) at 40 nM for 20 min at 37 C, and the green fluorescent intensity was analyzed using FACScan flow cytometry (Becton Dickinson, Mountain View, CA). 0
2.7. Western blotting Western blotting was performed as described previously [32]. Cell pellets were lysed in 2 · SDS sample buffer containing 100 mM dithiothreitol and then boiled for 5 min. A uniform amount of each sample (10 lg of protein) was resolved by SDS–PAGE (12% acrylamide). Proteins in each gel were transferred to Immobilon-P membrane (Milipore, Bedford, MA). After the blots were blocked for 1 h in 5% non-fat dry milk in 20 mM Tris, 0.8% NaCl, 0.1% Tween 20, pH 7.5, the blots were incubated with the primary antibody for 1 h and then with the appropriate horseradish peroxidase-conjugated secondary antibody (MBL, Nagoya, Japan). Signals were visualized by fluorescence emission using commercial detection kits (Roche Diagnosis, Mannheim, Germany), according to the manufacturer’s instructions. Antibodies for Bcl2, Bax, and PARP were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies for Bid, caspase3, caspase-8, and caspase-9 were purchased from MBL (Nagoya, Japan). Antibodies for cytochrome c were purchased from BD Pharmingen, Becton Dickinson (San Jose, CA, USA). 2.8. Cytochrome c detection assay Cells were washed twice with ice-cold PBS (pH 7.4) and suspended in 100 ll of ice-cold mitochondrial buffer (20 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and a cocktail of protease inhibitors) containing 250 mM sucrose [33]. The nuclei and unbroken cells were separated from the cytosol fraction by centrifugation at 10,000g for 10 min. The resultant cytosol fractions from 10 mg protein-equivalent cells were resolved on SDS–PAGE (12% acrylamide) and visualized by Western blotting. 2.9. Statistics
2.6. Assay for apoptotic morphology MX-1 cells (2 · 105/well) were collected in a 1.5 ml Eppendorf tube and stained with 3 lg/ml of Hoechst 33342 (Sigma, St. Lewis, MO, USA) for 15 min, fol-
Difference between given two experimental groups were statistically evaluated using double-sided t-test. Difference giving p values less than 0.05, are considered statistically significant.
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3. Results 3.1. Induction of apoptosis by YM529 in human breast cancer cells MX-1 is a poorly differentiated duct cell carcinoma isolated from a human breast cancer in 1974. MX-1 is a typical estrogen receptor (ER)-negative breast cancer, and has been used for screening for anti-breast cancer agents in National Cancer Institute in USA [34,35] Because no effective therapy regimen has been established for ER-negative breast cancers, we have evaluated therapeutic efficacy of anti-cancer agents on MX-1 cells [36]. MX-1 cells were incubated for 48 h in the presence of bisphosphonates, and the cell viability was determined by MTT dye reduction assay. Incadronate, a second-generation bisphosphonate used clinically for breast cancer patients, only slightly decreased cell viability even at a 100 lM concentration. However, YM529, the new third-generation bisphosphonate, exhibited significant cytotoxicity in a dose-dependent manner; YM529 decreased the cell viability to less than 50% at 100 lM (Fig. 1a). We also evaluated cytotoxic effect of YM529 on other human breast cancer cell lines. MDA-MB-231 expresses no ER, whereas ZR75-1 is a typical ER-positive cell line. YM529 had significant cytotoxic effect on these cell lines, though they were less
sensitive than MX-1 (Fig. 1b). The cytotoxic effect of YM529 is at least partially due to the induction of apoptosis, because the typical DNA ladder formation that is characteristic of apoptosis was detected in the DNA extracted from MX-1 cells treated with YM529 (Fig. 1c). Apoptotic character of YM529-mediated cytotoxicity was further confirmed with reduction of mitochondrial membrane potential detected by fluorescent dye DiO6(3), and morphological studies. Consistent with induction of DNA fragmentation, YM529 significantly reduced fluorescence intensity above 50 lM in a dose dependent manner, suggesting that YM529treated cells decreased mitochondrial membrane potential (Fig. 1d). Fluorescent microscopic analysis of MX-1 cells stained with Hoechst 33342 and PI demonstrated condensation and segmentation of the nuclei in MX-1 cells treated with YM529 at 50 and 100 lM concentrations (Fig. 2), supporting the results of the DNA fragmentation assay. Apoptotic nuclei were observed even in living cells, as well as in dead cells, as judged by PI exclusion. Similar apoptotic morphology was observed with MDA-MB231 and ZR75-1 treated with 100 lM YM529 (data not shown). These findings suggest that YM529 kills human breast cancer cells through apoptosis but not through necrosis.
Fig. 1. YM529 induces apoptosis of human breast cancer cell lines. (a and b) MX-1 was treated with YM529 (closed circles) or incadronate (open circles) for 48 h (a). MDA-MB-231 (open circles), ZR75-1 (open squares) or MX-1 (open triangles) was treated with YM529 for 48 h (b). Cell viability was quantified by MTT dye reduction assay. Results are shown as the means ± SE of 4 independent experiments. (c) Cells were treated with YM529 for 48 h, and DNA fragmentation was analyzed by 2% agarose gel electrophoresis. (d) Cells were treated with YM529 for 48 h, and mitochondrial membrane potential was determined as described in Section 2 (closed area). Control fluorescence profile of cells treated with vehicle is shown in each panel (open area).
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Fig. 2. Morphology of MX-1 cells treated with YM529. MX-1 cells were treated with YM529, stained with Hoechst 33342 and PI, and observed under a fluorescence microscope (a). The percentage of cells with apoptotic nuclei (left panel) and the percentage of cells stained with PI (right panel) were determined (b). Results are shown as the means ± SE of 4 independent experiments. Bars, 10 lM. * Indicates a statistically significant difference when compared to the control culture (p < 0.05, double-sided t-test).
3.2. YM529 induces apoptosis of human breast cancer cells through mitochondria-dependent apoptosis pathway To characterize the apoptosis induced by YM529, cellular protein was analyzed with Western blotting. PARP is a representative substrate for effector caspases, and its cleavage is a marker of caspase-dependent apoptosis [37]. Treatment with YM529 at concentrations greater than 25 lM cleaved the 116-kDa intact PARP into the 85-kDa product, a fragment generated by activated caspase-3. Consistent with the PARP cleavage, 32-kDa pro-caspase-3 was cleaved to generate 17-kDa active caspase-3. Along with the activation of caspase-3, caspase-9, an initiator caspase downstream of the mitochondrial pathway, was cleaved from a 45-kDa pro-enzyme to an active fragment of 37 kDa. In contrast, the 55-kDa procaspase-8, an initiator caspase downstream of the DISCdependent apoptotic pathway, was not cleaved into an active fragment. This result suggests that YM529 induces apoptosis of human breast cancer cells through the mitochondria-dependent pathway but not through the DISCdependent pathway. Mitochondria permeability transition results in the release of cytochrome c from the mitochondrial internal space into cytoplasm [1]. Treatment with YM529 at concentrations greater than 25 lM induced the cytoplasmic release of cytochrome c, confirming the activation of the mitochondria-dependent pathway. Mitochondrial permeability transition is regulated by the Bcl-2 family members [4]. The Western blotting results demonstrated that YM529 at concentrations greater than 25 lM increased the expression of a proapoptotic protein, Bax, and decreased the expression of an anti-apoptotic protein, Bcl-2, in a dose-dependent manner. Another pro-apoptotic protein, Bid, which is cleaved by caspase-8 in a DISC-dependent manner, was not truncated by treatment with YM529. This result is consistent with the observation that YM529 did not induce activation of the DISC-dependent apoptotic pathway (see Fig. 3).
Fig. 3. Protein expression in MX-1 cells treated with YM529. MX-1 cells were incubated with YM529 for 48 h, and protein expression was analyzed by Western blotting.
3.3. Inhibition of mevalonate pathway evokes YM529mediated apoptosis in human breast cancer cells It is known that nitrogen-containing bisphosphonates including YM529 inhibit mevalonate pathway as analogues of isoprenoid diphosphate lipids [27,38], and thus, growth arrest and apoptosis induced by nitrogen-containing bisphosphonates are overridden by the addition of isoprenoids including geranygeraniol and farnesiol [28,29]. To confirm involvement of mevalonate pathway in YM529-mediated apoptosis in MX-1 cells, cells were treated with YM529 in the presence of geranylgeranyl pyrophosphate or farnesyl pyrophosphate, and assessed cytotoxic activity with MTT assay (Fig. 4). While geranylgeranyl pyrophosphate only negligibly affected cytotoxic effect of YM529 on MX-1 cell, addition of farnesyl pyrophophate significantly recovered cell viability in the
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Fig. 4. Farnesyl pyrophosphate overrides cytotoxic effect of YM529 on MX-1 cells. Cells were treated with YM529 (100 lM) for 48 h in the absence or the presence of lipid pyrophosphates (50 lM). Cell viability was quantified by MTT dye reduction assay. Results are shown as the means ± SE of 4 independent experiments. In the presence of YM529, differences between control and farnesyl pyrophosphate-treated group, and between geranylgeranyl pyrophosphate-treated group and farnesyl pyrophosphate-treated group are statistically significant (*, p < 0.05).
presence of YM529. The result suggests that inhibition of mevalonate pathway by YM529 evokes activation of apoptotic pathway in the human breast cancer cell line.
4. Discussion The aim of the present study is evaluation of YM529, a newly developed third generation bisphosphosphonate as a therapeutic agent for malignant human breast cancer. We found that YM529 was a more potent apoptotic inducer than incadronate, a second-generation bisphosphonate used clinically for breast cancer patients. We also provided evidence that YM529 caused apoptosis in a human breast cancer cell line through the mitochondria-dependent apoptotic pathway, independent of the DISC-dependent pathway. Our results demonstrate that direct apoptosis induced in cancer cells, in addition to the inhibition of osteoclast-mediated bone resorption [39,40], is an advantageous aspect of YM529 in breast cancer therapy. Inhibition of the mevalonate pathway [26,28,29,38], downregulation of Bcl-2 expression [24], or caspases-3 activation
[22,25,26] has been proposed as a mechanism for apoptosis induced by bisphosphonates. In agreement with these reports, we observed activation of caspase-3, reduction of mitochondrial membrane potential, enhancement of Bax expression, reduction of Bcl-2 expression, and prevention of apoptosis by the addition of farnesyl pyrophosphate in the cells treated with YM529. These results suggest that apoptosis of breast cancer cells induced by YM529 is mediated by mitochondria-dependent pathway through the modulation of Bcl-2 family member expression, and that all these events are evoked by the inhibition of mevalonate pathway. It is known that nitrogen-containing bisphosphonates inhibits isoprenoid synthesis [27]. It is assumed that they act as analogues of isoprenoid diphosphate lipids, and inhibits farnesyl pyrophophate synthase, resulting in the inhibition of posttranslational farnesylation of small G proteins including Ras, Rac and Rho family proteins [41]. Inhibitors of mevalonate pathway such as mevastatin and lovastatin, induce apoptosis in the manner similar to nitrogen-containing bisphosphonates; they decrease Bcl-2 expression and increase Bax expression [28,29,42]. Therefore, it is likely that the primary target of YM529 is inhibition of mevalonate pathway, which results in modulation of Bcl-2 family expression, and subsequently induces mitochondria-dependent apoptosis. Cellular protein expression is regulated by transcription activation and/or degradation. Signal transduction pathway initiated with Ras activation modulates activity of transcription factors including AP-1. MX-1 has high AP-1 activity, and inhibition of the AP-1 transcription activity selectively induces apoptosis of MX-1 cells [36]. The Bcl-2 promoter region contains the consensus motif of AP-1, and Bcl-2 expression and AP-1 binding activity were correlated in proliferating glandular cells of the human uterine endothelium [43]. Bisphosphonates inhibit the transcription of vascular endothelial growth factor through the inhibition of AP-1 in human endothelial cells exposed to advanced glycation end product [44]. Thus, it is possible that YM529 suppresses Bcl-2 transcription in MX-1 cells through the inhibition of AP-1. Additional studies of YM529-mediated modulation of transcription and protein stability for the Bcl-2 family proteins might elucidate how small G-proteins regulate mitochondria-dependent apoptotic pathway, and such information may provide important insights into the therapeutic strategy against highly malignant
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tumors including estrogen receptor and Her2-negative breast cancer cells like MX-1.
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Induction of mitochondria-dependent apoptosis through the inhibition of mevalonate pathway in human breast cancer cells by YM529, a new third generation bisphosphonate Hiroo Nakajima a, Junji Magae b,*, Mie Tsuruga b, Koichi Sakaguchi a, Ikuya Fujiwara a, Mitsuhiko Mizuta c, Kiyoshi Sawai a, Hisakazu Yamagishi d, Naruhiko Mizuta a a
Department of Endocrine and Breast Surgery, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan b Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan c Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan d Department of Digestive Surgery, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan Received 15 November 2006; accepted 15 January 2007
Abstract YM529, a new third generation bisphosphonate, induced apoptosis of a human breast cancer cell line, MX-1. Cytotoxic activity of YM529 was more potent than that of incadronate. YM529 activated caspase-9, but not caspase-8, and induced the release of cytochrome c into cytosol. YM529 increased Bax expression and decreased Bcl-2 expression, while it did not induce caspase-8-dependent Bid truncation. Farnesyl pyrophosphate prevented YM529-mediated cytotoxicity. These results suggest that YM529 is a potent therapeutic agent for human breast cancers, activating the mitochondria-dependent apoptotic pathway through the inhibition of protein farnesylation. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Apoptosis; Breast cancer; YM529; Bcl-2; Bax; Farnesylation
1. Introduction Apoptosis requires the activation of caspases, which are ubiquitously and constitutively expressed as inactive zymogens in the cytosol [1]. Caspases can be divided into initiator caspases (caspase-2, -8, -9 and -10) and effector caspases (caspase-3, -6 and -7). * Corresponding author. Tel.: +81 4 7144 9142; fax: +81 4 7144 9007. E-mail address: [email protected] (J. Magae).
The initiator caspases activate the downstream effector caspases, which subsequently degrade certain cellular proteins, resulting in cell death. The initiator caspases are activated through a process mediated by mitochondria or by death receptors on the cell surface. The mitochondria-mediated process begins with mitochondrial permeability transition, followed by the release of apoptogenic factors such as cytochrome c and apoptosis-inducing factor [1,2]. The initiator caspase-9 then becomes activated through the formation of the cytochrome c/Apaf-1/
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.01.008
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procaspase-9-containing apoptosome complex in cytosol, leading to the activation of downstream effector caspases [3]. Bcl-2 family proteins regulate the mitochondrial permeability transition by interacting with mitochondrial channels such as voltagedependent anion channel [4,5]. Pro-apoptotic members, such as Bax and Bid, induce the release of apoptotic factors, whereas anti-apoptotic members, such as Bcl-2 and Bcl-xl, prevent their release. In contrast, the death receptor-mediated process activates an apoptotic pathway that is dependent on the initiator caspase-8 [6]. Cross-linking of the extracellular domains of Fas by the Fas ligand results in Fas trimerization and intracellular recruitment of the adapter molecule Fas-associated death domain protein (FADD) to the receptor cluster. Recruitment is facilitated by homotypic interaction between death domains located in Fas and FADD. Receptor-bound FADD oligomerizes and utilizes a second domain, the death effector domain, to recruit procaspase-8. The Fas/FADD/ procaspase-8 complex is referred to as the deathinducing signaling-complex (DISC) [7,8]. DISC catalyzes the proteolytic conversion of procaspase-8 into active caspase-8, which activates the downstream effector caspases, triggering cell death. The death receptor-mediated process and mitochondrial pathways are connected by Bid, a proapoptotic Bcl-2 family member. Bid truncated by caspase-8 translocates from the cytosol to the mitochondria and induces cytochrome c release [9,10]. Whether apoptosis proceeds through the death receptor-mediated pathway or the mitochondrial pathway depends on the cell properties and the conditions of stimulation [11]. Bone metastasis commonly occurs in breast cancer patients; in fact, more than 80% of women with advanced breast cancer ultimately develop bone metastases, which account for significant morbidity and mortality [12]. Because patients with advanced breast cancer will eventually develop serious bone-related symptoms, various combination therapies such as analgesics, radiation therapy, and systemic treatments such as hormone therapy or chemotherapy are routinely given. However, even with these therapies, it is difficult to cure the cancer and alleviate the symptoms. To improve upon these conventional therapies, bisphosphonates, analogues of endogenous pyrophosphates in which the central atom of oxygen is replaced with a carbon atom, have been developed and used for patients with bone metastasis. In accordance with their structural innovation, bis-
phosphonates are classified as first, second, or third generation. Their main function is inhibition of osteoclast-mediated bone resorption [13,14] through the prevention of osteoclast maturation [15–17] and/ or induction of apoptosis in osteoclasts [18–20]. In addition to their direct effects on osteoclasts, bisphosphonates may directly induce apoptosis in metastasized breast cancer cells. In animal models of metastatic disease, bisphosphonates reduce the tumor burden in bone [21,22]. Bisphosphonates also inhibit the adhesion of breast cancer cells to bone in vitro [23] and directly induce apoptosis in human breast cancer cell lines in vitro [24]. The features of apoptosis, such as DNA fragmentation, decreased Bcl-2/ Bax radio, poly(ADP ribose) polymerase (PARP) cleavage [24], and caspase activation [22,25,26], have been identified in different in vitro systems. The simple bisphosphonates that closely resemble pyrophosphate such as clodronate, etidronate and tiludronate can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulates intracellularly, resulting in induction of apoptosis. By contrast, nitrogen-containing bisphosphonate such as pamidronate, alendronate, and risedronate, appear to act as analogues of isoprenoid diphosphate lipid, thereby inhibiting farnesyl pyrophosphate synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme prevents the biosynthesis of isoprenoid lipids that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signaling proteins [27]. Thus, cytotoxic activity and growth arrest by nitrogen-containing bisphosphonates is prevented by the addition of geranylgeraniol and farnesol [28,29], and inhibitors of mevalonate pathway induce apoptosis in the manner similar to bisphosphonates. In the present study, we investigated the signal transduction of apoptosis in breast cancer cells induced by YM529, a third-generation nitrogencontaining bisphosphonate. Our results suggest that YM529 induces cytochrome c release into the cytosol from mitochondria by modulating Bcl-2 family expression, leading to the activation of caspase-9 and subsequent effector-caspase dependent apoptosis, while the DISC-dependent apoptotic pathway is not affected. Because the apoptosis induced by YM529 was partially prevented by the addition of farnesyl pyrophosphate, inhibition of mevalonate pathway by YM529 is assumed to evoke apoptosis and subsequent activation of mitochondria-dependent apoptosis pathway.
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2. Materials and methods 2.1. Reagents Incadronate and YM529 were obtained from Yamanouchi Pharmaceutical Company (Tokyo, Japan). Geranylgeranyl pyrophosphate and farnesyl pyrophosphate were purchased from Sigma Chem. (St. Louis, MO, USA).
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lowed by the addition of 1.5 lg/ml of propidium iodide (PI, Nakarai Tesque, Kyoto, Japan). After excluding the debris by brief spinning (1000 rpm, 1 min), the morphology of the stained cells was examined using an MX50 fluorescence microscope (Olympus, Tokyo, Japan) with an ultraviolet filter. Cells with a blue condensed or fragmented nucleus were scored as apoptotic cells, and cells stained with PI were scored as dead cells.
2.2. Cell culture A human breast cancer cell line, MX-1, was kindly provided by Kyowa Hakko Inc. (Tokyo Japan). MDAMB-231 and ZR75-1 were purchased from American Type Culture Collection. It was maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 1% penicillinstreptomycin and 10% fetal bovine serum (BioWhittaker, MD, USA) at 37 C under a humidified atmosphere containing 5% carbon dioxide. 2.3. MTT dye reduction assay Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) dye reduction assay measuring mitochondrial respiratory function [30,31]. Breast cancer cells that were exponentially grown (5 · 104 cells/well) were plated in 96-well flatbottom plates (Nunc. Roskilde, Denmark) and allowed to adhere for 24 h. After incubation for 48 h with the drug, cells were incubated with MTT dye (1 mg/ml) for 2 h and solubilized with 20 ll of 10% SDS. The optical densities of the samples were measured at 550 nm. 2.4. DNA fragmentation assay DNA fragmentation was detected using TACSTM Apoptotic DNA Laddering Kits (Trevigen, MD, USA), according to the manufacturer’s instructions. MX-1 cells (1 · 106cells) were lysed. Their DNA was extracted and then separated by 2% agarose gel electrophoresis, followed by ethidium bromide staining to visualize the laddered DNA. 2.5. Determination of mitochondria membrane potential MX-1 cells (1 · 106 cells/ml) were incubated with 3,3 dihexyloxacarbocyanide iodide (DiOC6(3), Molecular Probes, Inc., Eugene, OR, USA) at 40 nM for 20 min at 37 C, and the green fluorescent intensity was analyzed using FACScan flow cytometry (Becton Dickinson, Mountain View, CA). 0
2.7. Western blotting Western blotting was performed as described previously [32]. Cell pellets were lysed in 2 · SDS sample buffer containing 100 mM dithiothreitol and then boiled for 5 min. A uniform amount of each sample (10 lg of protein) was resolved by SDS–PAGE (12% acrylamide). Proteins in each gel were transferred to Immobilon-P membrane (Milipore, Bedford, MA). After the blots were blocked for 1 h in 5% non-fat dry milk in 20 mM Tris, 0.8% NaCl, 0.1% Tween 20, pH 7.5, the blots were incubated with the primary antibody for 1 h and then with the appropriate horseradish peroxidase-conjugated secondary antibody (MBL, Nagoya, Japan). Signals were visualized by fluorescence emission using commercial detection kits (Roche Diagnosis, Mannheim, Germany), according to the manufacturer’s instructions. Antibodies for Bcl2, Bax, and PARP were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies for Bid, caspase3, caspase-8, and caspase-9 were purchased from MBL (Nagoya, Japan). Antibodies for cytochrome c were purchased from BD Pharmingen, Becton Dickinson (San Jose, CA, USA). 2.8. Cytochrome c detection assay Cells were washed twice with ice-cold PBS (pH 7.4) and suspended in 100 ll of ice-cold mitochondrial buffer (20 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and a cocktail of protease inhibitors) containing 250 mM sucrose [33]. The nuclei and unbroken cells were separated from the cytosol fraction by centrifugation at 10,000g for 10 min. The resultant cytosol fractions from 10 mg protein-equivalent cells were resolved on SDS–PAGE (12% acrylamide) and visualized by Western blotting. 2.9. Statistics
2.6. Assay for apoptotic morphology MX-1 cells (2 · 105/well) were collected in a 1.5 ml Eppendorf tube and stained with 3 lg/ml of Hoechst 33342 (Sigma, St. Lewis, MO, USA) for 15 min, fol-
Difference between given two experimental groups were statistically evaluated using double-sided t-test. Difference giving p values less than 0.05, are considered statistically significant.
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3. Results 3.1. Induction of apoptosis by YM529 in human breast cancer cells MX-1 is a poorly differentiated duct cell carcinoma isolated from a human breast cancer in 1974. MX-1 is a typical estrogen receptor (ER)-negative breast cancer, and has been used for screening for anti-breast cancer agents in National Cancer Institute in USA [34,35] Because no effective therapy regimen has been established for ER-negative breast cancers, we have evaluated therapeutic efficacy of anti-cancer agents on MX-1 cells [36]. MX-1 cells were incubated for 48 h in the presence of bisphosphonates, and the cell viability was determined by MTT dye reduction assay. Incadronate, a second-generation bisphosphonate used clinically for breast cancer patients, only slightly decreased cell viability even at a 100 lM concentration. However, YM529, the new third-generation bisphosphonate, exhibited significant cytotoxicity in a dose-dependent manner; YM529 decreased the cell viability to less than 50% at 100 lM (Fig. 1a). We also evaluated cytotoxic effect of YM529 on other human breast cancer cell lines. MDA-MB-231 expresses no ER, whereas ZR75-1 is a typical ER-positive cell line. YM529 had significant cytotoxic effect on these cell lines, though they were less
sensitive than MX-1 (Fig. 1b). The cytotoxic effect of YM529 is at least partially due to the induction of apoptosis, because the typical DNA ladder formation that is characteristic of apoptosis was detected in the DNA extracted from MX-1 cells treated with YM529 (Fig. 1c). Apoptotic character of YM529-mediated cytotoxicity was further confirmed with reduction of mitochondrial membrane potential detected by fluorescent dye DiO6(3), and morphological studies. Consistent with induction of DNA fragmentation, YM529 significantly reduced fluorescence intensity above 50 lM in a dose dependent manner, suggesting that YM529treated cells decreased mitochondrial membrane potential (Fig. 1d). Fluorescent microscopic analysis of MX-1 cells stained with Hoechst 33342 and PI demonstrated condensation and segmentation of the nuclei in MX-1 cells treated with YM529 at 50 and 100 lM concentrations (Fig. 2), supporting the results of the DNA fragmentation assay. Apoptotic nuclei were observed even in living cells, as well as in dead cells, as judged by PI exclusion. Similar apoptotic morphology was observed with MDA-MB231 and ZR75-1 treated with 100 lM YM529 (data not shown). These findings suggest that YM529 kills human breast cancer cells through apoptosis but not through necrosis.
Fig. 1. YM529 induces apoptosis of human breast cancer cell lines. (a and b) MX-1 was treated with YM529 (closed circles) or incadronate (open circles) for 48 h (a). MDA-MB-231 (open circles), ZR75-1 (open squares) or MX-1 (open triangles) was treated with YM529 for 48 h (b). Cell viability was quantified by MTT dye reduction assay. Results are shown as the means ± SE of 4 independent experiments. (c) Cells were treated with YM529 for 48 h, and DNA fragmentation was analyzed by 2% agarose gel electrophoresis. (d) Cells were treated with YM529 for 48 h, and mitochondrial membrane potential was determined as described in Section 2 (closed area). Control fluorescence profile of cells treated with vehicle is shown in each panel (open area).
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Fig. 2. Morphology of MX-1 cells treated with YM529. MX-1 cells were treated with YM529, stained with Hoechst 33342 and PI, and observed under a fluorescence microscope (a). The percentage of cells with apoptotic nuclei (left panel) and the percentage of cells stained with PI (right panel) were determined (b). Results are shown as the means ± SE of 4 independent experiments. Bars, 10 lM. * Indicates a statistically significant difference when compared to the control culture (p < 0.05, double-sided t-test).
3.2. YM529 induces apoptosis of human breast cancer cells through mitochondria-dependent apoptosis pathway To characterize the apoptosis induced by YM529, cellular protein was analyzed with Western blotting. PARP is a representative substrate for effector caspases, and its cleavage is a marker of caspase-dependent apoptosis [37]. Treatment with YM529 at concentrations greater than 25 lM cleaved the 116-kDa intact PARP into the 85-kDa product, a fragment generated by activated caspase-3. Consistent with the PARP cleavage, 32-kDa pro-caspase-3 was cleaved to generate 17-kDa active caspase-3. Along with the activation of caspase-3, caspase-9, an initiator caspase downstream of the mitochondrial pathway, was cleaved from a 45-kDa pro-enzyme to an active fragment of 37 kDa. In contrast, the 55-kDa procaspase-8, an initiator caspase downstream of the DISCdependent apoptotic pathway, was not cleaved into an active fragment. This result suggests that YM529 induces apoptosis of human breast cancer cells through the mitochondria-dependent pathway but not through the DISCdependent pathway. Mitochondria permeability transition results in the release of cytochrome c from the mitochondrial internal space into cytoplasm [1]. Treatment with YM529 at concentrations greater than 25 lM induced the cytoplasmic release of cytochrome c, confirming the activation of the mitochondria-dependent pathway. Mitochondrial permeability transition is regulated by the Bcl-2 family members [4]. The Western blotting results demonstrated that YM529 at concentrations greater than 25 lM increased the expression of a proapoptotic protein, Bax, and decreased the expression of an anti-apoptotic protein, Bcl-2, in a dose-dependent manner. Another pro-apoptotic protein, Bid, which is cleaved by caspase-8 in a DISC-dependent manner, was not truncated by treatment with YM529. This result is consistent with the observation that YM529 did not induce activation of the DISC-dependent apoptotic pathway (see Fig. 3).
Fig. 3. Protein expression in MX-1 cells treated with YM529. MX-1 cells were incubated with YM529 for 48 h, and protein expression was analyzed by Western blotting.
3.3. Inhibition of mevalonate pathway evokes YM529mediated apoptosis in human breast cancer cells It is known that nitrogen-containing bisphosphonates including YM529 inhibit mevalonate pathway as analogues of isoprenoid diphosphate lipids [27,38], and thus, growth arrest and apoptosis induced by nitrogen-containing bisphosphonates are overridden by the addition of isoprenoids including geranygeraniol and farnesiol [28,29]. To confirm involvement of mevalonate pathway in YM529-mediated apoptosis in MX-1 cells, cells were treated with YM529 in the presence of geranylgeranyl pyrophosphate or farnesyl pyrophosphate, and assessed cytotoxic activity with MTT assay (Fig. 4). While geranylgeranyl pyrophosphate only negligibly affected cytotoxic effect of YM529 on MX-1 cell, addition of farnesyl pyrophophate significantly recovered cell viability in the
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Fig. 4. Farnesyl pyrophosphate overrides cytotoxic effect of YM529 on MX-1 cells. Cells were treated with YM529 (100 lM) for 48 h in the absence or the presence of lipid pyrophosphates (50 lM). Cell viability was quantified by MTT dye reduction assay. Results are shown as the means ± SE of 4 independent experiments. In the presence of YM529, differences between control and farnesyl pyrophosphate-treated group, and between geranylgeranyl pyrophosphate-treated group and farnesyl pyrophosphate-treated group are statistically significant (*, p < 0.05).
presence of YM529. The result suggests that inhibition of mevalonate pathway by YM529 evokes activation of apoptotic pathway in the human breast cancer cell line.
4. Discussion The aim of the present study is evaluation of YM529, a newly developed third generation bisphosphosphonate as a therapeutic agent for malignant human breast cancer. We found that YM529 was a more potent apoptotic inducer than incadronate, a second-generation bisphosphonate used clinically for breast cancer patients. We also provided evidence that YM529 caused apoptosis in a human breast cancer cell line through the mitochondria-dependent apoptotic pathway, independent of the DISC-dependent pathway. Our results demonstrate that direct apoptosis induced in cancer cells, in addition to the inhibition of osteoclast-mediated bone resorption [39,40], is an advantageous aspect of YM529 in breast cancer therapy. Inhibition of the mevalonate pathway [26,28,29,38], downregulation of Bcl-2 expression [24], or caspases-3 activation
[22,25,26] has been proposed as a mechanism for apoptosis induced by bisphosphonates. In agreement with these reports, we observed activation of caspase-3, reduction of mitochondrial membrane potential, enhancement of Bax expression, reduction of Bcl-2 expression, and prevention of apoptosis by the addition of farnesyl pyrophosphate in the cells treated with YM529. These results suggest that apoptosis of breast cancer cells induced by YM529 is mediated by mitochondria-dependent pathway through the modulation of Bcl-2 family member expression, and that all these events are evoked by the inhibition of mevalonate pathway. It is known that nitrogen-containing bisphosphonates inhibits isoprenoid synthesis [27]. It is assumed that they act as analogues of isoprenoid diphosphate lipids, and inhibits farnesyl pyrophophate synthase, resulting in the inhibition of posttranslational farnesylation of small G proteins including Ras, Rac and Rho family proteins [41]. Inhibitors of mevalonate pathway such as mevastatin and lovastatin, induce apoptosis in the manner similar to nitrogen-containing bisphosphonates; they decrease Bcl-2 expression and increase Bax expression [28,29,42]. Therefore, it is likely that the primary target of YM529 is inhibition of mevalonate pathway, which results in modulation of Bcl-2 family expression, and subsequently induces mitochondria-dependent apoptosis. Cellular protein expression is regulated by transcription activation and/or degradation. Signal transduction pathway initiated with Ras activation modulates activity of transcription factors including AP-1. MX-1 has high AP-1 activity, and inhibition of the AP-1 transcription activity selectively induces apoptosis of MX-1 cells [36]. The Bcl-2 promoter region contains the consensus motif of AP-1, and Bcl-2 expression and AP-1 binding activity were correlated in proliferating glandular cells of the human uterine endothelium [43]. Bisphosphonates inhibit the transcription of vascular endothelial growth factor through the inhibition of AP-1 in human endothelial cells exposed to advanced glycation end product [44]. Thus, it is possible that YM529 suppresses Bcl-2 transcription in MX-1 cells through the inhibition of AP-1. Additional studies of YM529-mediated modulation of transcription and protein stability for the Bcl-2 family proteins might elucidate how small G-proteins regulate mitochondria-dependent apoptotic pathway, and such information may provide important insights into the therapeutic strategy against highly malignant
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