Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells

Cellular Signalling 16 (2004) 81 – 88 www.elsevier.com/locate/cellsig

Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells Dong-Seok Kim a, Sang-Eun Jeon b, Kyoung-Chan Park b,* a

b

Research Division for Human Life Sciences, Seoul National University, South Korea Department of Dermatology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, South Korea Received 2 May 2003; received in revised form 19 May 2003; accepted 20 May 2003

Abstract The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or ( )-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRPproduced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways. D 2003 Elsevier Inc. All rights reserved. Keywords: Indole-3-acetic acid; Horseradish peroxidase; Free radical; Apoptosis

1. Introduction Indole-3-acetic acid (IAA) is the principal form of the plant growth hormone, auxin, found in higher plants, and is a key regulator of plant cell division, elongation and differentiation [1]. Recently, it was reported that the combination of IAA and horseradish peroxidase (HRP) is cytotoxic to mammalian cells, and it was suggested that it could be used as a novel cancer therapy [2 –4]. Interestingly, neither IAA nor HRP alone show a cytotoxic effect at concentrations, which lead to cell death when they are

Abbreviations: DCFH-DA, 2,7-dichlorofluorescin diacetate; EGCG, ( )-epigallocatechin gallate; HRP, horseradish peroxidase; IAA, indole-3acetic acid; JNK, c-Jun N-terminal kinase; NAC, N-acetylcysteine; MAP, mitogen-activated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; ROS, reactive oxygen species. * Corresponding author. Tel.: +82-2-3668-7474; fax: +82-2-3675-1187. E-mail address: [email protected] (K.-C. Park). 0898-6568/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0898-6568(03)00091-3

in combination [2]. IAA only becomes cytotoxic after oxidative decarboxylation by HRP [5], which is a hemecontaining peroxidase and can oxidize a wide variety of substrates including IAA [6 –8]. The reaction between IAA and HRP proceeds through a complex mechanism that remains to be elucidated [5]. It has been reported that IAA activated by HRP produces free radicals, such as, indolyl, skatolyl, and peroxyl radicals [2,9]. In addition, it has been proposed that cellular lipid peroxyl radicals induce an apoptotic signaling cascade [10]. IAA also stimulates the production of reactive oxygen species (ROS) such as O2
and H2O2 [11,12] and ROS is known to induce lipid peroxidation, which causes structural changes in plasma membranes and initiates cellular damage and apoptosis [13,14]. Thus, the proposed IAA/HRP combination may enhance cellular oxidative stress, and lead to cell death [15]. Moreover, it has also been reported that the IAA/ HRP damages nucleic acids [16,17]. However, the molecular mechanisms responsible for its cytotoxicity are little known.

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The mitogen-activated protein (MAP) kinases are activated by extracellular stimuli [18]. In particular, p38 MAP kinase and c-Jun N-terminal kinase (JNK) can be stimulated by UV, free radicals, heat shock, osmotic shock, ischemia and many others [19 – 22]. Further studies have shown that MAP kinase activation is involved in the apoptotic effects of these stress agents. Moreover, it is believed that ROS is a key mediator in the activation of the p38 and JNK pathway [23 –25]. The apoptotic signaling pathways in apoptosis are potential targets for cancer therapy [26]. Cells undergoing apoptotic cell death show many characteristic biochemical and morphological features [27,28]. These features distinguish apoptosis from the changes observed in cells undergoing pathological, necrotic cell death, and which are caused by a family of cysteine-dependent aspartate-specific proteases, known as the caspases [29,30]. Two main apoptotic pathways have been identified in mammalian cells. Death receptors such as Fas (also known as CD95) and tumor necrosis factor receptor 1 (TNFR1) activate caspase-8, whereas caspase-9 is activated via the mitochondrial pathway. Activated caspase-8 or caspase-9 can cleave and activate executive caspases, like caspase-3 [31 – 34], the activated form of which mediates the cleavage of protein substrates, such as, poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, which in turn results in the morphological nuclear changes associated with apoptosis [35]. In the present study, we investigated the effects of the IAA/HRP on the death of G361 human melanoma cells, with a view toward its use as a possible therapeutic in cancer. We found that IAA/HRP treatment induces apoptosis in G361 cells. Furthermore, we examined the apoptotic mechanism induced by IAA/HRP, particularly with respect to the caspases and PARP.

2. Materials and methods 2.1. Materials Indole-3-acetic acid, horseradish peroxidase, ascorbic acid, ( )-epigallocatechin gallate (EGCG), N-acetylcysteine (NAC) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). 2,7-Dichlorofluorescin diacetate (DCFH-DA) was from Calbiochem (San Diego, CA). Antibodies that recognize phospho-specific JNK1/2 (Thr183/Tyr185, G-7, sc-6254), total JNK2 (D-2, sc-7345), phospho-specific p38 (Try182, D-8, sc-7973), total p38 (A-12, sc-7972), phospho-specific c-Jun (sc-822), caspase-9 (sc-8355), caspase8 (sc-7890), caspase-3 (sc-7272), p53 (sc-126), Bax (B-9, sc-7480), Bcl-2 (C-2, sc-7382) and actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-PARP antibody from BD Pharmingen (San Diego, CA).

2.2. Cell cultures The lightly pigmented human melanoma cell line, G361 (ATCC, Rockville, MD), was grown in RPMI supplemented with 10% FBS and 1% penicillin –streptomycin (10,000 U/ml and 10,000 Ag/ml, respectively) in 5% CO2 at 37 jC. 2.3. MTT assay for cell viability Cells (4  104 cells/well) were seeded into 24-well plates. After serum starvation for 24 h, the cells were incubated with test substances for indicated times at 37 jC in 5% CO2, 100 Al/well of MTT solution (5 mg/ml) was added and the plates were incubated for another 4 h. Supernatants were removed and formazan crystals were solubilized in 1 ml of dimethylsulfoxide. Optical density was determined at 540 nm using an ELISA reader (TECAN, Salzburg, Austria). 2.4. Crystal violet assay Cell viability was also confirmed using a crystal violet assay [36]. After incubating cells with the test substances for 24 h, the culture medium was removed and replaced with 0.1% crystal violet in 10% ethanol for 5 min at room temperature. The cells were rinsed four times in distillated water, and adherent crystal violet was extracted with 95% ethanol. Absorbance was determined at 590 nm using an ELISA reader. 2.5. Free radical determination The formation of free radicals was determined using DCFH-DA, which is oxidized by free radicals to dichlorofluorescein (DCF) [37,38]. To activate DCFH-DA, 350 Al of a 1 mM stock of DCFH-DA in ethanol was mixed with 1.75 ml of 0.01 N NaOH and allowed to stand for 20 min before adding 17.9 ml of 25 mM sodium phosphate buffer (pH 7.2). The reaction mixture contained 150 Al of activated DCFH-DA solution, 20 Al of IAA, 10 Al of HRP and 20 Al of ascorbic acid or EGCG. Absorbance was determined at room temperature every 10 min at 490 nm, after subtracting the 590 nm background using an ELISA reader. 2.6. Flow cytometric analysis Twenty-four hours after irradiation, we collected the culture supernatant, which contained floating dying cells and apoptotic cells, and the harvested adherent cells by a brief trypsinization. The two fractions were combined and washed with PBS. Labeling of the cells with FITC-conjugated annexin V and propidium iodide (PI) was performed using a TACS Annexin V-FITC kit (Trevigen, Gaithersburg, MD), and flow cytometric analyses were performed on a

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Fig. 1. Cytotoxic effect of IAA/HRP in G361 human melanoma cells. (A) After serum starvation, cells were treated with varying concentrations (1 – 500 AM) of IAA in the absence (n) and in the presence (o) of HRP (1.2 Ag/ml). After 24 h, cell viability was measured by MTT assay. (B) Cells were treated with IAA (500 AM) and HRP (1.2 Ag/ml) with or without the indicated concentrations of ascorbic acid. After 24 h, an MTT assay was performed. (C) Cells were treated with IAA (500 AM) and HRP (1.2 Ag/ml) with or without ascorbic acid (500 AM). At the indicated time points after treatment, an MTT assay was performed. Control (n), IAA + HRP ( ), IAA + HRP + ascorbic acid (D). (D) Cells were treated with IAA (500 AM) and HRP (1.2 Ag/ml) with or without the indicated concentrations of EGCG. After 24 h, an MTT assay was performed. (E) Cells were treated with IAA (500 AM) and HRP (1.2 Ag/ml) with or without NAC (10 mM). After 24 h, an MTT assay was performed. Data represent the means F S.D. of triplicate assays expressed as percentages of the control. Each experiment was repeated at least twice independently and representative results are shown. **P < 0.01, *P < 0.05 compared to the untreated control.

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FACSCalibur (Becton Dickinson, San Jose, CA). The fluorescences of the FITC-labeled and PI-labeled cells were measured. 2.7. Western blot analysis Cells were grown in 100 mm culture dishes, starved of serum for 24 h, treated with the test substances at the time points indicated, and lysed in cell lysis buffer [62.5 mM Tris – HCl (pH 6.8), 2% SDS, 5% h-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, protease inhibitors (Roche, Mannheim, Germany), 1 mM Na3VO4, 50 mM NaF and 10 mM EDTA]. Ten micrograms of protein per lane was separated by SDS-polyacrylamide gel electrophoresis and blotted onto PVDF-membranes, which were then saturated with 5% dried milk in Tris-buffered saline containing 0.4% Tween 20. Blots were incubated with the appropriate primary antibodies at a dilution of 1:1000, and then further incubated with horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence plus kit (Amersham International, Little Chalfont, UK). 2.8. Statistics Differences between results were assessed for significance using the Student’s t-test.

3. Results 3.1. IAA/HRP combination induces cell death in human melanoma cells, and this is restored by antioxidants We first examined the effects of IAA alone and of the IAA/HRP combination on the cell viability of G361 human melanoma cells. Cell viability was measured using the MTT assay 24 h after treatment. As shown in Fig. 1A, in the presence of 1.2 Ag/ml HRP, IAA caused cell death. About 50% of cells were died at 100 AM IAA, and almost all cells died after treatment with 500 AM IAA for 24 h. However, IAA alone was not cytotoxic at any concentrations tested up to 500 AM for 24 h. We confirmed these observations by crystal violet assay and obtained similar results (data not shown). Ascorbic acid, an antioxidant, almost completely abrogated the cytotoxic effect of IAA/HRP when it was added at concentrations higher than 500 AM (Fig. 1B). In contrast, IAA, HRP or ascorbic acid alone had no effect on cell viability. To confirm the cytoprotective effect of ascorbic acid, a time course analysis was performed. The viability of cells treated with IAA/HRP decreased in a time-dependent manner. From 3 h after IAA/HRP (500 AM/1.2 Ag/ml) treatment, cells showed significantly reduced viability compared with an untreated control; moreover, ascorbic acid abrogated this cytotoxic effect throughout the 24-h treatment

Fig. 2. Free radical production by IAA/HRP. The formation of free radicals was determined using DCFH-DA, which is oxidized by free radicals to DCF, as described in Section 2. (A) Increasing amounts of IAA were added in the presence of 1.2 Ag/ml of HRP. (B) Increasing doses of HRP were added in the presence of 500 AM of IAA. The values shown are means F S.D. of triplicate wells. (C) The effects of ascorbic acid (500 AM) or EGCG (50 AM) on the free radical production induced by IAA (500 AM) and HRP (1.2 Ag/ml). Absorbance was determined at the indicated time points. The data shown represent the means of triplicate wells.

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period (Fig. 1C). We also examined the effects of other antioxidants on IAA/HRP-induced cell death. As shown in Fig. 1D, EGCG restored the cytotoxicity of IAA/HRP when present at concentrations higher than 10 AM, as did NAC another antioxidant (Fig. 1E). 3.2. The IAA/HRP increases the formation of free radicals Indolyl, skatolyl and peroxyl radicals are produced when IAA is oxidatively activated by HRP [17]. Hence, we measured the production of free radicals at concentrations of IAA and HRP varying from 0 to 1000 AM and 0 to 9.6 Ag/ml, respectively (Fig. 2A,B). In the presence of 1.2 Ag/ ml of HRP, the production of free radicals was increased by IAA concentration-dependently (Fig. 2A), in the presence of 500 AM of IAA; HRP additions also increased the free radical formation in a concentration-dependent manner (Fig. 2B). Neither IAA nor HRP alone had an influence on free radical formation. We next examined the inhibitory effects of antioxidants on the formation of free radicals. As shown in Fig. 2C, at 500 AM IAA and 1.2 Ag/ml HRP both EGCG and ascorbic acid suppressed IAA/HRP-induced free radical production. 3.3. IAA/HRP activates the stress-regulated signaling pathways We next examined the changes of the intracellular JNK and p38 MAP kinase cascade by IAA/HRP treatment. IAA/ HRP (500 AM/1.2 Ag/ml) resulted in the activation of JNK, which remained substantially unchanged from 2 to 4 h (Fig. 3A). p38 MAP kinase was also activated by this combination at 4 h after treatment. However, virtually no activation of JNK or p38 MAP kinase occurred within 1 h of treatment (data not shown).

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We further investigated whether antioxidants could inhibit IAA/HRP-induced JNK and p38 activation, in an attempt to explain the effect of antioxidants upon the apoptosis-inducing effect of IAA/HRP. As shown in Fig. 3B, ascorbic acid or EGCG inhibited IAA/HRP-stimulated JNK and p38 phosphorylation and suppressed c-Jun phosphorylation resulted from JNK activation. Based on these results, we suggest that the activation of JNK and p38 MAP kinase play an important role in IAA/HRP-induced cell death, and that ascorbic acid or EGCG may exert an effect by inhibiting JNK and p38 activation. 3.4. IAA/HRP activates caspases, cleaves PARP and induces apoptosis The MTT assay is a convenient screening assay for the measurement of cell death, but it does not discriminate between apoptosis and necrosis. One of the early events of the apoptotic process involves the translocation of phosphatidylserine. Annexin V can bind to phosphatidylserine on the surface of cells undergoing apoptosis. Thus, to confirm whether IAA/HRP-induced melanoma cell death is due to apoptosis, flow cytometry was used to count apoptotic and viable cells. Annexin V binding and propidium iodide (PI) uptake revealed various cellular states. At 24 h after IAA/HRP treatment, the cells were categorized into four populations; vital cells (annexin V /PI ), early apoptotic cells (annexin V +/PI ), late apoptotic cells (annexin V+/PI+), and necrotic cells (annexin V /PI+). IAA/HRP treatment (b) induced apoptosis significantly compared to the untreated control (a) and almost all cells were judged to be in early or late apoptosis (Fig. 4A). However, ascorbic acid (c) or EGCG treated cells (d) were resistant to IAA/HRP and remained in a vital state (annexin V /PI ) (Fig. 4A).

Fig. 3. Effects of IAA/HRP on the JNK and the p38 pathways. (A) After serum starvation, G361 cells were stimulated with IAA (500 AM) and HRP (1.2 Ag/ ml). Samples were collected at the time points indicated after IAA/HRP treatment. Cell lysates were then subjected to Western blot analysis with antibodies against phospho-specific JNK or p38 MAP kinase. Equal protein loadings were confirmed by reaction with phosphorylation-independent JNK2 or p38 MAP kinase antibodies, respectively. (B) The cells were stimulated with IAA (500 AM) and HRP (1.2 Ag/ml) in the absence and in the presence of ascorbic acid (500 AM) or EGCG (50 AM). Samples were collected for Western blot analysis 4 h after IAA/HRP treatment. Cell lysates were then Western blotted with antibodies against phospho-specific JNK, c-Jun and p38. Equal protein loadings were confirmed by reaction with actin antibody.

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It is well known that a variety of stress stimuli lead to caspase activation. Active effector caspases, such as caspase-3, mediate the cleavage of an overlapping set of protein substrates, resulting in the morphological features of apoptosis and the demise of the cell. Therefore, it was of interest to determine whether IAA/HRP could induce the activation of caspases and whether antioxidants could inhibit this process. As shown in Fig. 4B, we examined the proteolytic processing of caspase-9, -8 and -3, and PARP in response to IAA/HRP by Western blotting. Caspases become active when they are cleaved into processed fragments. Thus, we used antibodies directed against domains of the precursor forms of caspases, which did not recognize the processed forms. The precursor forms of caspase-9 and caspase-8 decreased considerably after 24 h of IAA/HRP treatment. Furthermore, almost all precursor forms of caspase-3 disappeared after treatment with IAA/HRP (500 AM/ 1.2 Ag/ml), indicating that the precursor form of caspase-3 was cleaved into active caspase-3. In contrast, ascorbic acid or EGCG treatment inhibited the cleavage of caspases induced by IAA/HRP. Caspase-3 is believed to be the most efficient PARP-cleaving caspase. Therefore, the proteolytic cleavage of PARP following IAA/HRP treatment was also confirmed by Western blotting. Accordingly, we found that the 116-kDa full-length PARP was converted to the apoptotic 85-kDa fragment (Fig. 4B). However, in accordance with the caspase-3 data, the 116-kDa form of PARP was not cleaved, when ascorbic acid or EGCG was treated. These findings suggest that caspases were activated by IAA/HRP and that the activated caspase cascade cleaved PARP in the IAA/HRP-treated cells, but not in cells treated with IAA/ HRP and ascorbic acid or EGCG. The tumor suppressor gene p53 and the proapoptotic protein Bax are known to be involved in apoptosis. However, our results show that IAA/HRP does not induce significant changes in p53 or Bax levels (Fig. 4B). In contrast, the antiapoptotic protein Bcl-2 was slightly reduced by IAA/HRP treatment and this was restored by antioxidants.

4. Discussion

Fig. 4. IAA/HRP-induced apoptosis is blocked by ascorbic acid or EGCG. After serum starvation, G361 cells were treated with IAA (500 AM) and HRP (1.2 Ag/ml) in the absence and in the presence of ascorbic acid (500 AM) or EGCG (50 AM). (A) After 24 h, the apoptotic cells were counted by flow cytometry, as described in Section 2; (a) control, (b) IAA/HRP, (c) IAA/HRP + ascorbic acid, (d) IAA/HRP + EGCG. (B) Samples were collected for Western blot analysis 24 h after IAA/HRP treatment. Cell lysates were then subjected to Western blot analysis with the indicated antibodies. Equal protein loadings were confirmed by reaction with actin antibody.

Although IAA/HRP-induced cell death and its potential applications in cancer therapy are recognized [4,5,39], the mechanism of this IAA/HRP-induced cytotoxic effect are substantially unknown. Therefore, we investigated the signal transduction pathways related to this apoptosis in order to elucidate the death mechanism. We chose G361 human melanoma cells to investigate the possible therapeutic use of IAA/HRP in human melanoma. In this study, the MTT assay showed that the viability of melanoma cells decreased with increasing IAA in the presence of HRP. In addition, flow cytometric analysis showed that the majority of IAA/HRPtreated cells suffered apoptotic cell death. In agreement with our results, it was also reported that the photoproducts of

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IAA induce apoptosis in human HL-60 and murine tumor cells [40]. Since JNK and p38 MAP kinase are important mediators of stress signals that ultimately lead to apoptosis [19,20,41], we first investigated the effects of IAA/HRP on stressregulated kinase pathways. Our results demonstrate that IAA/HRP results in the activation of JNK and p38. Interestingly, the activation of JNK and p38 MAP kinase is slow and delayed (ca. 2 and 4 h, respectively). To investigate the role of JNK and p38 on apoptosis in human melanoma, we made use of SP600125, a recently reported inhibitor of JNK [42,43], and of a p38 inhibitor, SB203580. The inhibition of p38 MAP kinase by SB203580 resulted in a slight protection against apoptosis, and SP600125 did not restore IAA/ HRP-induced apoptosis in G361 cells (data not shown). These results suggest that the activation of the stressregulated kinase pathway is not required for IAA/HRPinduced apoptosis. It was also reported that oxidative stress may be responsible for JNK and p38 activation [24,25,44]. Thus, we tried to suppress oxidative stress using ascorbic acid or EGCG and found these antioxidants inhibited JNK and p38 activation by IAA/HRP. These results indicate that free radicals are involved in the IAA/HRP-induced activation of JNK and p38. The role of the caspase pathway in apoptosis has been well established [29,30]. In mammalian cells, two main pathways are known to induce apoptosis. In the first pathway, death factors such as Fas ligand or tumor necrosis factor-a bind to cell surface death receptors (Fas or TNFR1) that transmit a death signal to cells, which results in the activation of caspase-8 [45]. In the second pathway, a variety of stimuli trigger the release of cytochrome c by mitochondria, binds to Apaf-1 and activates caspase-9 [46]. Our results indicate that IAA/HRP induced apoptotic cell death, and that IAA/HRP activates caspase-8, showing that this death-receptor pathway is involved in IAA-HRP-induced apoptosis. However, it is unlikely that IAA derivatives activate Fas or TNFR1 directly, though vanadate, an environmental toxicant, is reported to induce Fas aggregation and to activate caspase-8 [47]. Moreover, UVB also directly induces Fas activation, and the apoptosis of human keratinocytes [48], and JNK activation can induce Fas ligand expression in various cell types [49 –51]. In addition, oxidative stress induced Fas and Fas ligand expression in murine intestinal epithelial cells [52]. Therefore, we examined whether IAA/HRP increases Fas or Fas ligand production, but Western blot analysis showed no evidence of Fas or Fas ligand induction after IAA/HRP treatment (data not shown). Thus, the mechanism of the IAA/HRP-induced activation of caspase-8 requires further elucidation. In this study, we found that caspase-9 is activated by IAA/HRP treatment, indicating that IAA/HRP induces apoptosis via the mitochondrial pathway. However, activated caspase-8 can induce Bid cleavage, and C-terminal fragment of Bid binds to mitochondria and transduces apoptotic signals, suggesting that caspase-8 is also involved in the

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mitochondrial damage [53]. These results suggest that IAA/ HRP activates both the mitochondrial and death –receptor pathways, and that these two pathways work together to induce apoptosis. These two pathways converge at the activation of executive caspases such as caspase-3 [30,33], the activation of which mediates the cleavage of protein substrates, such as PARP and the nuclear lamins [35]. In addition, the present study demonstrates caspase-3 activation and PARP cleavage after IAA/HRP treatment. In summary, this study demonstrates that the combination of IAA and HRP induces apoptosis in human melanoma cells, and that free radicals are involved in IAA/HRPinduced apoptosis via both death receptor-mediated and mitochondrial apoptotic pathways.

Acknowledgements We would like to thank Dr. Dr. Hans Rainer Kuesell in New York for the motivation to conduct this research.

References [1] Goldsmith MH. Proc Natl Acad Sci U S A 1993;90:11442 – 5. [2] Folkes LK, Candeias LP, Wardman P. Int J Radiat Oncol Biol Phys 1998;42:917 – 20. [3] Greco O, Dachs GU. J Cell Physiol 2001;187:22 – 36. [4] Wardman P. Curr Pharm Des 2002;8:1363 – 74. [5] Folkes LK, Wardman P. Biochem Pharmacol 2001;61:129 – 36. [6] Gazaryan IG, Lagrimini LM, Ashby GA, Thorneley RN. Biochem J 1996;313(Pt. 3):841 – 7. [7] Candeias LP, Folkes LK, Porssa M, Parrick J, Wardman P. Biochemistry 1996;35:102 – 8. [8] Metodiewa D, de Melo MP, Escobar JA, Cilento G, Dunford HB. Arch Biochem Biophys 1992;296:27 – 33. [9] Candeias LP, Folkes LK, Porssa M, Parrick J, Wardman P. Free Radic Res 1995;23:403 – 18. [10] Kotamraju S, Hogg N, Joseph J, Keefer LK, Kalyanaraman B. J Biol Chem 2001;276:17316 – 23. [11] Pires de Melo M, Curi TC, Miyasaka CK, Palanch AC, Curi R. Gen Pharmacol 1998;31:573 – 8. [12] Kawano T, Kawano N, Hosoya H, Lapeyrie F. Biochem Biophys Res Commun 2001;288:546 – 51. [13] Bongarzone ER, Pasquini JM, Soto EF. J Neurosci Res 1995;41: 213 – 21. [14] de Kok TM, ten Vaarwerk F, Zwingman I, van Maanen JM, Kleinjans JC. Carcinogenesis 1994;15:1399 – 404. [15] Candeias LP, Folkes LK, Wardman P. Biochem Soc Trans 1995;23: 262S. [16] de Mello MP, de Toledo SM, Aoyama H, Sarkar HK, Cilento G, Duran N. Photochem Photobiol 1982;36:21 – 4. [17] Folkes LK, Dennis MF, Stratford MR, Candeias LP, Wardman P. Biochem Pharmacol 1999;57:375 – 82. [18] Chang L, Karin M. Nature 2001;410:37 – 40. [19] Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, et al. Curr Biol 1996;6:606 – 13. [20] Harper SJ, LoGrasso P. Cell Signal 2001;13:299 – 310. [21] Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Science 1995;270:1326 – 31. [22] Butterfield L, Storey B, Maas L, Heasley LE. J Biol Chem 1997;272: 10110 – 6.

88

D.-S. Kim et al. / Cellular Signalling 16 (2004) 81–88

[23] Du J, Suzuki H, Nagase F, Akhand AA, Ma XY, Yokoyama T, et al. Free Radic Biol Med 2001;31:469 – 78. [24] Lee MW, Park SC, Yang YG, Yim SO, Chae HS, Bach JH, et al. FEBS Lett 2002;512:313 – 8. [25] Haddad JJ, Land SC. Br J Pharmacol 2002;135:520 – 36. [26] Huang P, Oliff A. Trends Cell Biol 2001;11:343 – 8. [27] Kerr JF, Wyllie AH, Currie AR. Br J Cancer 1972;26:239 – 57. [28] Wyllie AH, Morris RG, Smith AL, Dunlop D. J Pathol 1984;142: 67 – 77. [29] Hengartner MO. Nature 2000;407:770 – 6. [30] Nunez G, Benedict MA, Hu Y, Inohara N. Oncogene 1998;17: 3237 – 45. [31] Mehmet H. Nature 2000;403:29 – 30. [32] Ashkenazi A, Dixit VM. Science 1998;281:1305 – 8. [33] Stennicke HR, Jurgensmeier JM, Shin H, Deveraux Q, Wolf BB, Yang X, et al. J Biol Chem 1998;273:27084 – 90. [34] Green DR, Reed JC. Science 1998;281:1309 – 12. [35] Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, et al. Nature 1995;376:37 – 43. [36] Dooley TP, Gadwood RC, Kilgore K, Thomasco LM. Skin Pharmacol 1994;7:188 – 200. [37] Valkonen M, Kuusi T. J Lipid Res 1997;38:823 – 33. [38] McCune LM, Johns T. J Ethnopharmacol 2002;82:197 – 205. [39] Greco O, Dachs GU, Tozer GM, Kanthou C. J Cell Biochem 2002; 87:221 – 32. [40] Edwards AM, Barredo F, Silva E, De Ioannes AE, Becker MI. Photochem Photobiol 1999;70:645 – 9.

[41] Peus D, Vasa RA, Beyerle A, Meves A, Krautmacher C, Pittelkow MR. J Invest Dermatol 1999;112:751 – 6. [42] Bennett BL, Sasaki DT, Murray BW, O’Leary EC, Sakata ST, Xu W, et al. Proc Natl Acad Sci U S A 2001;98:13681 – 6. [43] Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, et al. J Clin. Invest 2001;108:73 – 81. [44] Katiyar SK, Afaq F, Azizuddin K, Mukhtar H. Toxicol Appl Pharmacol 2001;176:110 – 7. [45] Srinivasan A, Li F, Wong A, Kodandapani L, Smidt Jr R, Krebs JF, et al. J Biol Chem 1998;273:4523 – 9. [46] Soengas MS, Alarcon RM, Yoshida H, Giaccia AJ, Hakem R, Mak TW, et al. Science 1999;284:156 – 9. [47] Luo J, Sun Y, Lin H, Qian Y, Li Z, Leonard SS, et al. J Biol Chem 2003;278:4542 – 51. [48] Aragane Y, Kulms D, Metze D, Wilkes G, Poppelmann B, Luger TA, et al. J Cell Biol 1998;140:171 – 82. [49] Le-Niculescu H, Bonfoco E, Kasuya Y, Claret FX, Green DR, Karin M. Mol Cell Biol 1999;19:751 – 63. [50] Faris M, Kokot N, Latinis K, Kasibhatla S, Green DR, Koretzky GA, et al. J Immunol 1998;160:134 – 44. [51] Kolbus A, Herr I, Schreiber M, Debatin KM, Wagner EF, Angel P. Mol Cell Biol 2000;20:575 – 82. [52] Denning TL, Takaishi H, Crowe SE, Boldogh I, Jevnikar A, Ernst PB. Free Radic Biol Med 2002;33:1641 – 50. [53] Li H, Zhu H, Xu CJ, Yuan J. Cell 1998;94:491 – 501.