Rokitamycin Induces a Mitochondrial Defect and Caspase-Dependent Apoptosis in Human T-Cell Leukemia Jurkat Cells

Masayuki Fukui1, Yukitoshi Nagahara2, Yoshiaki Nishio2, Tsutomu Honjoh3, and Takahisa Shinomiya4,* 1Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS 66160, USA 2Department of Biotechnology, College of Science and Engineering, Tokyo Denki University, Saitama 350-0394, Japan 3Morinaga Institute of Biological Science, Inc., Kanagawa 236-0003, Japan 4Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China

Received September 26, 2008; Accepted March 9, 2009

Abstract. Macrolides are a well-known family of oral antibiotics whose antibacterial spectrum of activity covers most relevant bacterial species responsible for respiratory infectious disease. In recent years, it has been reported that macrolides have not only bactericidal activity but also direct immunomodulating activity in mammals. In this study, we observed new physiological activity of macrolides and examined whether various macrolides induce apoptosis in human leukemia cell lines. We investigated the effects of 13 different macrolides on the viability of Jurkat and HL-60 cells. Among all the macrolides used in this study, rokitamycin, a semisynthetic macrolide with a 16-member ring, effectively induced cell death. Rokitamycin induced DNA fragmentation and caspase activation, resembling the progression of apoptosis. Moreover, rokitamycin reduced the mitochondrial transmembrane potential and released cytochrome c from mitochondria to the cytosol, suggesting that mitochondrial perturbation is involved in rokitamycin- induced apoptosis. These results suggest that rokitamycin possesses not only bactericidal activity but also pro-apoptotic activity in human leukemia cells.

Keywords: macrolide, rokitamycin, apoptosis, mitochondria, caspase

Introduction azithromycin (AZM), for patients with DPB have also been reported (7, 8). Several actions of 14-member Macrolides are antibiotics with bactericidal effects. macrolide antibiotics provide support for these clinical In general, macrolides are much better known for their effects (2, 3, 6, 9, 10). A number of other studies have activity against Gram-positive bacteria and Mycoplasma suggested that macrolides have direct immuno- pneumoniae than for their activity against Gram- modulatory effects other than antibiotic effects (11). negative bacteria. Macrolides bind to bacterial 50S This immunomodulating action of macrolides may ribosome subunits, thus inhibiting bacterial expansion contribute to its clinical effectiveness in the treatment of (1). There is, however, an increasing body of evidence such inflammatory diseases as DPB (12, 13) and certain demonstrating that macrolides may have activities not dermatologic disorders (14). Recent studies suggest that related to their antibiotic properties. a direct immunomodulating role of macrolides can be Long-term therapy, with low doses (not sensitive to assumed as follows: neutrophil chemotaxis inhibition bacteria) of erythromycin (ERY), has been shown to (15 – 18), oxidant generation (15, 19, 20), phagocytosis be effective in treating patients with diffuse panbron- progression (21, 22), and apoptosis induction (23). chiolitis (DPB) (2 – 6). Similar clinical effects of other Recently, apoptosis induction in activated lympho- 14-member macrolides such as clarithromycin (CLR) cytes by macrolides has been considered to play an and roxithromycin (RXM) or a 15-member macrolide, important role in immunomodulation (24). Aoshiba et al. have proposed that cAMP is increased in ERY- *Corresponding author. [email protected] induced apoptosis (23). Jun et al. have reported that Published online in J-STAGE on April 29, 2009 (in advance) Fas ligand expression is enhanced in ciprofloxacin- and doi: 10.1254/jphs.08267FP RXM-induced apoptosis (25). However, details regard-

69 70 M Fukui et al ing the overall apoptotic induction mechanism by concentration of 0.5 mg/ml and further incubated for macrolides remain obscure. 1 h. The net absorbance at 570 nm, which was propor- In the present study, we attempted to observe the tional to the viable cell number, was then measured apoptotic ability of various macrolides using human using a microplate reader. The absorbance of culture leukemia cell lines. We investigated the effects of wells without macrolides was set as 100%. stimulation by 13 different macrolides on the viability of human leukemia cell lines. Among all the macrolides, Determination of mitochondrial transmembrane poten- semisynthetic macrolides, with its 16-member rokita- tial (ΔΨm) on intact cells mycin (RKM), was found to effectively induce cell The ΔΨm value was determined using 3,3'-dihexyloxa- death in a dose-dependent manner. Therefore, the carbocyanide iodide (DiOC6(3); Invitrogen, Carlsbad, present study was undertaken to evaluate the effects of CA, USA) as described (26). RKM and other macrolides on the apoptosis of leukemia and to determine what mechanism underlies this effect. DNA fragmentation Apoptosis was determined by assaying DNA frag- Materials and Methods mentation by agarose gel electrophoresis. Cells were rinsed once with PBS and dissolved in 50 mM Tris-HCl Macrolides buffer, pH 7.8, containing 10 mM EDTA, 0.5% sodium ERY, oleandomycin (OL), RXM, triacetyloleando- lauryl sarcocinate, and 0.5 mg/ml RNase A. After mycin (TAO), and AZM (Sigma, St. Louis, MO, USA); incubation at 50°C for 1 h, Proteinase K was added at a CLR (Taisho Pharmaceutical, Tokyo); josamycin (JM), concentration of 1 mg/ml and then the mixture was leucomycin (LM), spiramycin (SPM), midecamycin further incubated at 50°C for 30 min. Chromosomal (MDM), and tylosin (TYL) (Wako Pure Chemical DNA was analyzed by 2% agarose gel electrophoresis. Industries, Osaka); miocamycin (MOM; Meiji Seika Kaisha, Tokyo); and RKM (Asahi Kasei, Tokyo) were Preparation of mitochondria and cytosol dissolved in dimethyl sulfoxide (DMSO, Wako) and Mitochondria and cytosol fraction was collected as subsequently diluted in RPMI 1640 or DMEM medium. described before (27). Briefly, cells (1 × 107) were washed with PBS, resuspended in 5 volumes of cell-free Cells system buffer (CFS buffer) (220 mM mannitol, 68 mM The human myelogenous leukemia cell line HL-60 sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, was provided by the Human Science Resources Bank 2mM MgCl2, 5 mM sodium pyruvate, 0.1 mM phenyl- (Osaka). The human leukemia T-cell line Jurkat was a methylsulfonyl fluoride, and 10 mM HEPES-NaOH, gift from T. Miyashita (Kitasato Univ., Kanagawa). The pH 7.4) and swollen on ice for 20 min. Cells were dis- human leukemia B-cell line BALL-1 was supplied by rupted by 10 – 15 aspirations through a 22-gauge needle Cell Resource Center for Biomedical Research, Tohoku and centrifuged at 750 × g for 5 min at 4°C to remove Univ. (Sendai). Jurkat, HL-60, and BALL-1 were nuclei. The supernatant was centrifuged again (10,000 × cultured in RPMI 1640 medium supplemented with 10% g, 10 min, 4°C) to recover mitochondria. The 10,000 × g FCS and 75 mg/L kanamycin sulfate. Human osteo- supernatant was ultracentrifuged at 100,000 × g for sarcoma cell line Saos-2 cells were purchased from the 30 min at 4°C, and the remaining supernatant was American Type Culture Collection (Manassas, VA, used as the cytosol fraction. Both samples were used USA) and cultured in DMEM medium supplemented immediately or stored at −80°C until immunodetection with 10% FCS and 75 mg/L kanamycin sulfate. All cell of cytochrome c. lines were maintained at 37°C in a humidified chamber under an atmosphere of 95% air and 5% CO2. Western blots Whole cell lysate, mitochondria and cytosol fractions Assessment of macrolide cytotoxicity were mixed in the same volume of SDS sample buffer To assess the susceptibility to macrolides, the relative (4% SDS, 125 mM Tris, pH 6.8, 10% glycerol, 0.02 mg number of viable cells (percentage of viable cells) was /ml bromophenol blue, and 10% 2-mercaptoethanol) determined by colorimetric assay using 3-[4,5-dimethyl- and heated at 100°C for 5 min. Proteins were separated thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) by 4% – 20% gradient SDS-PAGE and electrically (Sigma) according to the manufacturer’s protocol. In transferred to a polyvinylidene difluoride membrane brief, cells were cultured at 2 × 104 cells/well in flat- (Millipore, Bedford, MA, USA). After blocking the bottomed, 96-well plates with macrolides. Twenty-four membrane using 5% skimmed milk, caspase-3, caspase- hours later, MTT was added to each well at the final 8, caspase-9, cytochrome c, Bax, phospholylated (phos- Rokitamycin-Induced Leukemia-Cell Apoptosis 71 pho)-SAPK/JNK, and native-SAPK/JNK were immuno- detected using anti-caspase-3 Ab (1:1000; Cell Signal- ing Technology, Beverly, MA, USA), anti-caspase-8 Ab (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-caspase-9 Ab (1:200, Santa Cruz), anti- cytochrome c Ab (1:1500; BD Bioscience, San Jose, CA, USA), anti-Bax Ab (1:1000, Santa Cruz), and anti- phospho-SAPK/JNK Ab and anti-native-SAPK/JNK Ab (each diluted 1:1000, Cell Signaling Technology). Thereafter, HRP-conjugated anti-mouse IgG or anti- rabbit IgG was applied as second Abs, and positive bands were detected by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK).

Determination of isolate mitochondrial membrane potential and cytochrome c release Determination of isolated mitochondria was described before (27, 28). Briefly, to determine ΔΨm, isolated mitochondria were washed and resuspended in CFS buffer at a concentration of approximately 25 mg mitochondria protein in 100 ml CFS buffer. Protein concentration was determined by the BCA protein assay kit (Pierce, Rockford, IL, USA). After an incubation with or without macrolides for 30 min at 37°C, the mitochondrial suspensions were further incubated for 15 min with DiOC6 (3). Mitochondrial ΔΨm was examined by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA, USA). To immunodetect cytochrome c, isolated mitochondria were washed and resuspended in CFS buffer at a concentration of approximately 100 mg Fig. 1. The cytotoxic effects of 13 macrolide antibiotics on human mitochondrial protein in 100 ml CFS buffer. After leukemia cell lines. Human T lymphoma cell line (Jurkat) (a) and incubating with or without macrolides for 60 min at human acute promyelocytic cell line (HL-60) (b) were treated with μ μ 37°C, reaction mixtures were centrifuged (10,000 × g, macrolides at 10 M (open bars) or 40 M (closed bars) for 24 h, ° respectively. The relative number of viable cells (percentage of viable 30 min, 4 C). The mitochondria pellet and supernatant cells) was determined by a MTT colorimetric assay, as described were separated and immediately used or stored at −80°C in Materials and Methods. Vertical lines indicate the S.D. (n = 3). c: until the immunodetection of cytochrome c. Human B lymphoma cell line (BALL-1), T lymphoma cell line (Jurkat), and myelogenous leukemia cell line (HL-60) were each treated with the indicated doses of RKM for 24 h. The relative Results number of viable cells (percentage of viable cells) was determined by a MTT colorimetric assay, as described in Materials and Methods. Effects of macrolides on cell viability Vertical lines indicate the S.D. (n = 3). We investigated the effects of macrolides on Jurkat human T leukemia cell line and HL-60 human acute promyelocytic cell lines. Cells were exposed to 13 RKM-induced DNA fragmentation and caspase activa- macrolides, respectively, for 24 h. One of the 16-ring tion member macrolides, RKM, exhibited strong cytocidal We investigated whether the observed leukemia cell activity in Jurkat, but the others did not (Fig. 1a). RKM death was from apoptosis or necrosis. The presence of also reduced cell viability in HL-60 (Fig. 1b). Further- DNA degradation of nucleosomal units and sub-G1 more, the dose-dependent effect of RKM was deter- DNA content cells in 40 μM RKM–treated Jurkat cells mined. Various leukemia cell lines (human B leukemia indicates that the form of cell death was apoptosis cell line BALL-1, human T leukemia cell line Jurkat, (Fig. 2: a and b). The mass of sub-G1 DNA content human acute promyelocytic cell line HL-60) showed cells was not significantly altered by MOM (another 16- dose-dependent decreases in cell viability, with a similar member macrolide)-treatment (Fig. 2b). We then tested trends (Fig. 1c). whether RKM can induce apoptotic-specific proteases, 72 M Fukui et al

Fig. 3. Caspase-3–dependent apoptosis induced by RKM in Jurkat Fig. 2. DNA fragmentation induced by RKM in Jurkat cells. a: × 5 × 5 cells. a: Jurkat cells (2 10 /ml) were preincubated with or without Jurkat cells (2 10 /ml) were incubated with the indicated concentra- 10 μM pan-caspase inhibitor Z-VAD-fmk or 20 μM caspase-3 tions of RKM for 20 h. The cells were then lysed, and DNA was specific inhibitor Ac-DEVD-fmk for 60 min and then incubated with prepared. DNA fragmentation was analyzed by agarose gel electro- 40 μM RKM for 6 h. Cells were lysed, and caspase-3, caspase-8, and phoresis. Data are representative of three independent experiments. × 5 caspase-9 were detected by Western blotting. Data are representative b: Jurkat cells (2 10 /ml) were incubated with the indicated concen- of 3 independent experiments. b: Jurkat cells (2 × 105/ml) were trations of MOM or RKM for 20 h. Cells were treated with 0.1% μ μ preincubated with or without 10 M pan-caspase inhibitor Z-VAD- TritonX-100 and thereafter stained with 5 g/ml PI. DNA content fmk for 60 min and then incubated with 40 μM RKM for 6 h. was examined by flow cytometry. Data are representative of 3 Thereafter, cells were lysed and DNA was prepared. DNA frag- independent experiments. mentation was analyzed by agarose gel electrophoresis. Data are representative of 3 independent experiments. caspases activation. In RKM-treated Jurkat cells, a specific inhibitor, Ac-DEVD-fmk, suggesting that large fragment of active caspase-3 (17, 19-kDa) was mitochondria perturbation is the major pathway for detected (Fig. 3a). Moreover, RKM cleaved one of the RKM-induced apoptosis (Fig. 3a). caspase-3–cleaving upstream caspases, caspase-9 (17- kDa fragment) (Fig. 3a). The pan-caspase inhibitor Z- RKM-induced apoptosis in intact cells via a mito- VAD-fmk inhibited caspase-3 and -9 activation and chondrial pathway DNA degradation in RKM-treated Jurkat cells, suggest- Because RKM induced caspase-9 activation, we ing that RKM-induced DNA fragmentation is caspase- examined the release of cytochrome c from mito- dependent (Fig. 3: a and b). Another caspase-3–cleav- chondria, which is the major event of mitochondria- able upstream caspase, caspase-8, was also activated by mediated apoptosis. Similar to the results of cytotoxicity RKM treatment (Fig. 3a). However, RKM-induced and DNA fragmentation assay (Figs. 1 and 2), treatment caspase-8 activation was inhibited by a caspase-3 with 40 μM RKM induced cytochrome c release from Rokitamycin-Induced Leukemia-Cell Apoptosis 73

Fig. 5. p53-Independent apoptosis was induced by RKM in Saos-2 cells. Saos-2 cells (2 × 104) were incubated with MOM or RKM at the indicated concentrations for 6 h. The relative number of viable cells (percentage of viable cells) was determined by an MTT assay. Vertical lines indicate the S.D. (n = 3).

independent. The reduction in the ΔΨm value, which is another major event in mitochondrial damage, was observed with DiOC6 (3) staining. The level of ΔΨm was dose- dependently reduced by RKM-treatment (Fig. 4b).

RKM-induced apoptosis in p53-null Saos-2 cells Oncogene p53 is involved in mitochondria-mediated apoptosis by recruiting proapoptotic Bcl-2 family protein Bax to mitochondria (29). To determine whether Fig. 4. Cytochrome c was released into cytosol by incubating Jurkat 7 p53 activation is required for RKM-induced apoptosis, cells with RKM. a: Jurkat cells (2 × 10 ) were incubated with the indicated doses of RKM for 6 h. The cytosolic fraction was prepared we incubated Saos-2 (a p53-deficient human osteo- as described in Materials and Methods. Cytochrome c was detected sarcoma cell line) cells with RKM at the indicated by Western blotting. PC indicates the positive control for concentrations (Fig. 5). Exposure to RKM induced a cytochrome c detection (purified cytochrome c lane). b: Jurkat cells 7 dose-dependent cell death in Saos-2 cells. This finding (2 × 10 ) were preincubated with or without 10 μM Z-VAD-FMK for 60 min and then incubated with 40 μM RKM for 6 h. The cytosolic indicates that p53 activation is not required for RKM- fraction was prepared as described in Materials and Methods. induced apoptosis. Cytochrome c was detected by Western blotting. PC indicates the positive control for cytochrome c detection (purified cytochrome c 6 JNK/SAPK phosphorylation is not associated with lane). c: Jurkat cells (2 × 10 ) were incubated with MOM or RKM at 40 μM for 60 min. DiOC6 (3) (50 nM) was added 45 min before RKM-induced apoptosis ending the culture, and ΔΨm was analyzed by flow cytometry. Data Proapoptotic MAPK, JNK/SAPK, and p38 induce are representative of 3 independent experiments. mitochondria-mediated apoptosis (30 – 32). We further examined the possibility of JNK/SAPK activation by RKM. JNK/SAPK was phosphorylated within 2 h after mitochondria to the cytosol, but treatment with 20 μM exposure to RKM, and the phosphorylation status RKM did not (Fig. 4a). To determine whether RKM- reached a maximum 4 – 6 h later (Fig. 6a). To determine induced mitochondrial cytochrome c release or RKM- whether JNK/SAPK phosphorylation is required for induced caspase activation is the prior action, we used this apoptosis, we treated Jurkat cells with RKM after Z-VAD-fmk. After a 1-h preincubation with 10 μM pre-treatment with JNK inhibitor. Phosphorylation of Z-VAD-fmk, RKM-induced DNA fragmentation was JNK/SAPK was significantly blocked by JNK inhibitor completely blocked (Fig. 3b). However, Z-VAD-fmk (Fig. 6b), but DNA fragmentation was not (Fig. 6c). did not inhibit the RKM-induced mitochondrial Although RKM-induced JNK phosphorylation was not cytochrome c release (Fig. 4b). These results indicate inhibited by pre-treatment with Z-VAD-fmk, RKM- that RKM-induced mitochondrial cytochrome c release induced apoptosis was significantly inhibited. These precedes caspase activation, thus demonstrating that results indicate that RKM-induced JNK/SAPK phos- RKM-induced mitochondrial perturbation is caspase- phorylation is independent of RKM-induced apoptosis. 74 M Fukui et al

Fig. 7. RKM reduced ΔΨm directly in isolated Jurkat mitochondria. a: Mitochondria from Jurkat cells (25 mg/100 ml CFS buffer) were prepared as described in Materials and Methods. Mitochondria were incubated with no or 2.5 mM atractyloside (ATR: positive control) or 10 μM RKM for 30 min at 37°C. Mixtures were incubated with 50 nM DiOC6 (3) for 15 min, and ΔΨm was then analyzed by flow cytometry. b: Mitochondria were prepared as described in (a) and incubated with no or 2.5 mM atractyloside (ATR: positive control) or 10 or 20 μM RKM for 60 min. Mitochondria and mitochondrial supernatant were prepared, and cytochrome c was detected by Fig. 6. JNK-independent apoptosis was induced by RKM. a: Jurkat × 6 μ Western blotting and quantified by using Image J software (National cells (1.5 10 ) were incubated with 40 M RKM for the indicated Institutes of Health, Bethesda, MA, USA). Data are representative of times. Cells were lysed, and phospho-JNK/SAPK and native- 2 independent experiments. JNK/SAPK were detected by Western blotting. b: Jurkat cells (2 × 106) were preincubated with 10 μM Z-VAD-fmk or 100 nM JNK Inhibitor II for 60 min and then incubated with 40 μM RKM for 6 h. Cells were lysed, and phospho-JNK/SAPK and native-JNK/SAPK ΔΨ were detected by Western blotting. c: Jurkat cells (1 × 106) were RKM, m was reduced. Moreover, mitochondrial preincubated with 10 μM Z-VAD-fmk or 100 nM JNK Inhibitor II cytochrome c was examined in isolated mitochondria. for 60 min and then incubated with 40 μM RKM for 6 h. Cells were Isolated Jurkat mitochondria were incubated with RKM, treated with 0.1% TritonX-100 and thereafter stained with 5 μg/ml and cytochrome c in isolated mitochondria and mito- PI. DNA content was examined by flow cytometry. Data are representative of 3 independent experiments. chondrial supernatant was then detected by Western blotting (Fig. 7b). RKM was found to induce a release of cytochrome c from the mitochondria into the mitochondrial supernatant in a dose-dependent manner, RKM directly affects mitochondria and induces ΔΨm which was similar to the result in intact cells (Fig. 4a). reduction and cytochrome c release Cytochrome c release was induced by treatment with a We considered whether RKM directly affects mito- lower dose of RKM in isolated mitochondria than in chondria, similar to several other reagents with direct intact cells. These findings indicated that RKM directly effects (27, 33, 34). Mitochondria isolated from Jurkat affects mitochondria and induces ΔΨm reduction and cells were incubated with RKM and then stained with cytochrome c release. DiOC6 (3) to assess ΔΨm (Fig. 7a). After incubation with Rokitamycin-Induced Leukemia-Cell Apoptosis 75

Discussion (Fig. 7). Moreover, overexpression of Bcl-2 inhibits apoptotic cell death induced by RKM (data not shown). In this study, the 16-member macrolide RKM was These results suggest that RKM-induced mitochondria found to induce apoptosis in leukemia cells. Caspase-3 perturbation is essential for RKM-induced apoptosis. and caspase-9 were activated in Jurkat exposed to RKM The macrolide derivatives antimycin, oligomycin, and in vitro. In addition, the pan-caspase inhibitor Z-VAD- rapamycin influence mammalian mitochondrial activity fmk completely blocked this caspase activation and by binding to cytochrome b (40), F0F1-ATPase (41), and subsequent RKM-induced nuclear DNA fragmentation mammalian TOR (42, 43), respectively. Since macrolide (Fig. 3). Thus, caspase activation appears to be essential derivatives retain structural similarity, RKM (and other for RKM-induced apoptosis. macrolides) may influence mitochondria by binding to Lowe et al. have previously reported that ionizing mitochondrial proteins directly. So why does RKM radiation, 5-fluorouracil, etoposide, and adriamycin induce mammalian cell death specifically among the induce p53-dependent apoptosis in mouse embryonic other macrolides? Ishiguro et al. revealed that the 16- fibroblasts or thymocytes and that p53 tumor suppressor membered macrolide RKM penetrated into human is required for efficient execution of the death program polymorphonuclear leukocytes in a greater amount than (35, 36). We therefore tested whether p53 tumor the other macrolides that are 14- or 15-membered (44). suppressor is required for RKM-induced apoptosis. Cell Taking this into consideration, RKM’s higher potency death was induced in saos-2 cells (p53-null) by exposure to induce mammalian mitochondria perturbation, to RKM (Fig. 5). Moreover, in Jurkat cells, Bax (a pro- compared to the other less effective macrolides, may apoptotic Bcl-2 family protein affected by p53) expres- result from its greater ability to penetrate into the intra- sion was not altered after exposure to RKM (data not cellular space. Further analysis is necessary to elucidate shown). These findings indicate that p53 tumor suppres- the targeting molecule of RKM in mammalian cells and sor is not required for RKM-induced apoptosis. the mechanism underlying mitochondrial defects. Xia et al. demonstrated that activation of JNK/SAPK The RKM concentrations that we used in these studies and p38 are critical for the induction of apoptosis after were higher than the serum drug concentration, which withdrawal of nerve growth factor from rat PC-12 are achieved in serum clinically after oral ingestion. In pheochromocytoma cells (37). Recent studies have staphylococci, the MIC50s of macrolides used in this revealed that JNK/SAPK and p38 MAP kinase are acti- study are about 0.06 – 1 μg/ml, which are less than the vated by cytotoxic stresses such as UV radiation, X-ray, respective concentrations that we used in this investiga- heat shock, and osmotic shock, as well as by proinflam- tion (40 μM RKM is about 32 μg/ml). However, tissue matory cytokines such as tumor necrosis factor and /cellular drug concentrations might be much higher than interleukin-1 (38, 39). However, we demonstrated the serum drug concentrations. It has been reported that here that the JNK/SAPK-induced apoptosis pathway is ERY concentration has been concentrated up to 50-fold independent of the RKM-induced apoptosis pathway. in neutrophils (45). As previously mentioned, since Activation of JNK/SAPK was significantly blocked by RKM tended to penetrate into human polymorpho- the JNK inhibitor, but DNA fragmentation was not nuclear leukocytes more effectively than 14-membered (Fig. 6). Although RKM-induced activation of JNK ERY (44), RKM might affect not only bacteria but /SAPK was not inhibited by pre-treatment with Z-VAD- also to mammalian cells in vivo. A previous study fmk, RKM-induced apoptosis was significantly inhibited. reported that RKM induced human polymorphonuclear These results indicate that RKM-induced activation of chemotaxis at doses over 20 μg/ml (about 25 μM) (46). JNK/SAPK is independent of RKM-induced apoptosis. Similarly, we determined that RKM was toxic to leuko- Phosphorylation of p38 MAP kinase was not detected cytes at doses similar to those toxic to leukemia cells. after exposure to RKM (data not shown). The IC50 dose in leukocytes was 35 μM (data not shown) In this study, cytochrome c was released from the and that in Jurkat cells was 25 μM. However, the human mitochondrial intermembrane into the cytosol, and ΔΨm normal fibroblast cell line MRC-5 was resistant to RKM was reduced in RKM-treated Jurkat cells (Fig. 4). (IC50 = 70 μM) (data not shown). Further in vivo studies Cytochrome c release was not inhibited by pre-incubat- estimating the clinical relevance of RKM are warranted. ing the cells with Z-VAD-fmk. This evidence suggests In summary, RKM induces apoptosis more effectively that releasing cytochrome c from mitochondria and than the other 12 macrolides used in this study. RKM reducing ΔΨm are upstream events before caspase acti- directly induces mitochondrial defects and cytochrome c vation. Furthermore, studies using a cell-free system release. The released cytochrome c then binds to Apaf-1 revealed that RKM directly triggers the reduction of ΔΨm and activates further caspases, including caspase-9 and and the release of cytochrome c from mitochondria caspase-3. Activated caspases then execute apoptotic 76 M Fukui et al cell death. Tumor suppressor p53 and the MAPK family leukocyte function. Curr Pharm Des. 2004;10:3067–3080. are independent of this mechanism. This above- 12 Schultz MJ. Macrolide activities beyond their antimicrobial mentioned mitochondrial perturbation mechanism by effects: macrolides in diffuse panbronchiolitis and cystic RKM seems to occur without cell specificity in leukemia fibrosis. J Antimicrob Chemother. 2004;54:21–28. 13 Ichikawa Y, Ninomiya H, Koga H, Tanaka M, Kinoshita M, cells (Fig. 1c). 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