Melatonin modulates apoptosis and TRPM2 channels in transfected cells activated by oxidative stress

Physiology & Behavior 107 (2012) 458–465

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Melatonin modulates apoptosis and TRPM2 channels in transfected cells activated by oxidative stress Ömer Çelik a, Mustafa Nazıroğlu a, b,⁎ a b

Department of Biophysics, Medical Faculty, Suleyman Demirel University, Isparta, Turkey Neuroscience Research Center, Suleyman Demirel University, Isparta, Turkey

H I G H L I G H T S ► TRPM2 currents, apoptosis and Ca2 + influx values in transfected cells were increased compared with controls. ► However, melatonin modulated the values. ► The current study is the first to compare treatment with melatonin to TRPM2 currents in the transfected cells.

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Article history: Received 13 April 2012 Received in revised form 5 August 2012 Accepted 26 September 2012 Keywords: Melatonin Oxidative stress Ca2 + signaling TRPM2 channel antagonist Apoptosis

a b s t r a c t Transient receptor potential melastatin-like 2 (TRPM2) is a non-selective Ca2+ permeable cation channel and is known to be activated by H2O2, one of the most important indicators of intracellular oxidative stress. A neurohormone melatonin may have a modulator role on TRPM2 channels activated by oxidative stress because it is a strong antioxidant. In this study we investigated the effects of melatonin on apoptosis, whole cell currents and Ca2+ influx arising from TRPM2 channels activated by H2O2. In whole-cell patch clamp experiments, TRPM2 channels in transfected Chinese hamster ovary (CHO) cells were activated by H2O2. However, the currents were inhibited either by intracellular or by extracellular melatonin. When intracellular melatonin was introduced by pipette, TRPM2 channel currents were not activated by H2O2 although H2O2-induced Ca 2+ gating and release were not blocked 2-aminoethyldiphenyl borate (2-APB). Cytosolic Ca 2+ release was measured by Fura-2 and was higher in H2O2 groups than in control. Melatonin also inhibited apoptosis in the transfected cells. In conclusion, we observed modulator roles of intracellular and extracellular melatonin on Ca 2+ influx and apoptosis through a TRPM2 channel in transfected CHO cells. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Calcium ion (Ca2+) homeostasis is one of the most important factors of cellular physiological function. It is involved in such diverse functions as cellular proliferation, apoptosis, physiological signal transduction and production of oxidative stress [1]. The cytosolic free Ca2+ [Ca2+]i concentration is controlled by a number of membrane-bound ion channels located both on the plasma and intracellular membranes [2,3]. In fact, Ca2+ is the most ubiquitous and studied of second messenger and has enormous

Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; ADPR, ADP-ribose; CHO, Chinese Hamster Ovary; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglion; GFP, green fluorescent protein; GSH, glutathione; GSH-Px, glutathione peroxidase; LP, lipid peroxidation; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; TRP, transient receptor potential; TRPM2, transient receptor potential melastatin-like 2. ⁎ Corresponding author at: Department of Biophysic, Medical Faculty, Süleyman Demirel University, Dekanlık Binasi, TR-32260 Isparta, Turkey. Tel.: +90 2462113708; fax: +90 246 2371165. E-mail addresses: [email protected] (Ö. Çelik), [email protected] (M. Nazıroğlu). 0031-9384/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.physbeh.2012.09.013

physiological and pathological implications in cellular function. Ca2+ signaling needs a complex system of refinement since Ca2+ is a simple cation with two positive charges that is able to translate a variety of extracellular signals in many different cell types to produce often very different responses [1]. Transient receptor potential (TRP) channels are a group of nonselective cation channels that have important functions in cellular systems [4]. One subgroup of the TRP channels is denoted as TRP melastatin 2 (TRPM2). The TRPM2 channel protein has two distinct domains with one functioning as an ion channel and the other as an ADP-ribose (ADPR)-specific pyrophosphatase [5]. The TRPM2 channel is also a redox-sensitive Ca2+-permeable cation channel, and Ca2+ influx through TRPM2 is induced by H2O2 [6]. When expressed in the cell lines such as transfected Chinese Hamster Ovary (CHO) cells [7–9], transfected human embryonic kidney293 (HEK) cell lines [6] and dorsal root ganglion (DRG) neurons [10–12], TRPM2 channels can also be gated by oxidative stress. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced and released by the pineal gland in association with the suprachiasmatic

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nucleus and peripheral tissues and is considered a potent antioxidant that detoxifies a variety of ROS in many pathophysiological states [13]. Chemically, melatonin and its metabolites can function as endogenous free radical scavengers and broad-spectrum antioxidants [14,15]. Oxidative stress-induced pathophysiologic conditions such as ischemia/reperfusion injury, neuronal excitotoxicity, and chronic inflammation can be a direct cause of cell death but melatonin has been shown to counteract such pathophysologic conditions [13,16,17]. Melatonin has been reported to modulate the L-type voltage gated channelopathies [18,19], as well as apoptosis [15,20]. However, the mechanisms by which melatonin are involved in Ca2+ influx is continuing to be explored. We hypothesized that, if melatonin is a potent scavenger, it might prevent or ameliorate the experimental oxidative stress-induced cellular oxidative injury through regulation of TRPM2 channels and Ca2+ influx, counteracting the impairment of endogenous antioxidant systems. The molecular mechanisms by which antioxidants lead to inhibition of TRPM2 Ca 2+ channels need to be elucidated in detail. Therefore, the present study was aimed at elucidating the role of melatonin in modulation of the effects of H2O2-induced oxidative stress on TRPM2 cation channels and to our knowledge, there has been no prior study on the interaction of melatonin and TRPM2 channels in cell lines. 2. Materials and methods 2.1. Cell culture and transfection CHO cells K1 were obtained from the Cell Culture Bank of Şap Institute (Ankara, Turkey) and cultured in Ham's F12 medium (1×) (Biochrome, Berlin, Germany), supplemented with 10% (v/v) fetal calf serum (Biochrome), and 4 mM L-glutamine (Biochrom, Berlin, Germany). Penicillin (50 U/ml)-streptomycin (50 mg/ml) combination in whole-cell experiments was added to the medium. Cells were seeded on glass cover slips (TPP Cell culture and Labour Technique, Trasadingen, Switzerland) at a density of b10 3 cells/mm2 and grown for 24 h. Subsequently, the pcDNA3-EGFP-TRPM2/TRPM2ΔC expression (5 μg) constructs were transiently transfected into the CHO cells, using the Trans-Fast transfection reagent (7.5 μl; Promega, Mannheim, Germany). As controls, cells were transfected with pcDNA3enhanced green fluorescent protein (GFP) vector alone. Electrophysiological studies were carried out 24–48 h after transfection in cells visibly positive for enhanced GFP. 2.2. Cell viability (MTT) assay Cell viability in transfected CHO cells was evaluated by MTT assay based on the ability of viable cells to convert a water-soluble, yellow tetrazolium salt into a water-insoluble, purple formazan product. The enzymatic reduction of the tetrazolium salt happens only in living, metabolically active cells but not in dead cells. Cells were seeded on plates at a density of 1 × 10 6 cells per well. They were incubated at 37 °C with different doses (0.001, 0.010, 0.10 and 1.0 mM) of H2O2 and harvested at different incubation times (30 min, 1 h, 2 h, 5 h, 10 h and 24 h). The medium was removed and MTT (15 μl) was added into each well and then incubated for 45 min at 37 °C in a shaking water bath. The supernatant was discarded and dimethyl sulfoxide (DMSO, 500 μl) was added to dissolve the formazan crystals. Treatments were carried out in duplicate. Absorbance values were measured in a spectrofluorometer cuvette at 490 nm and 650 nm, and expressed as a percentage of the control. 2.3. Electrophysiology Patch clamp techniques have been described in detail elsewhere [15,16]. The cells were studied with the patch-clamp technique in the whole-cell mode, using an EPC 10 USB equipped with a personal

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computer with patch-master USB software (HEKA, Lamprecht, Germany). Pipettes were made of borosilicate glass (Sutter Instrument Borosilicate Glass with filament. O.D.: 1.5 mm, I.D. 0.86 mm and 10 cm in length, Novato, CA, USA). The standard extracellular bath solution contained (in mM): 145 NaCl, 1.0 MgCl2, 1.0 CaCl2, 5 KCl, 10 HEPES, 10 glucose, with the pH adjusted with KOH to 7.4. For Na+ free solutions, Na + was replaced by 150 mM NMDG (N-methyl-D-glucamine) and the pH was adjusted with HCl. The osmolarity of the solution was 300 mosmol/l. The pipette solution contained in mM: 145 L-glutamic acid, 8 NaCl, 8 EDTA, 1 MgCl2 and 10 HEPES (pH 7.2) (adjusted with CsOH). The calcium concentration was adjusted to 1 μM. Cells were held at a potential of −60 mV, and current–voltage (I–V) relations were obtained from voltage ramps from −90 to +60 mV applied over 400 ms. H2O2 and melatonin and all other chemicals were obtained from Sigma. Melatonin was dissolved in DMSO, further diluted with PBS (final DMSO concentration b 0.2%) and added to the medium for Ca 2+ signaling and apoptosis experiments or to extracellular or intracellular buffers for electrophysiological experiments at the concentration to be tested, as previously described [20]. After addition of melatonin to standard extracellular bath solution, the pH of the solution was adjusted with KOH to 7.4. The melatonin was added to the cell dishes extracellularly (in the bath). In previous studies, melatonin at concentrations of 0.3–3.0 mM induced protective effects against H2O2-induced oxidative stress in cell culture [21,22]. For the present study, cell cultures were incubated with two different doses (0.3 and 1.0 mM) of melatonin for 2 h before H2O2 addition because transfer of the antioxidants into the cell is slow [21,22]. In some experiments, melatonin (0.2 mM) was also included in the patch pipette. 2.4. 2-aminoethyldiphenyl borate (2-APB) treatments TRP channels can be indirectly blocked by certain chemicals. However, for most TRP channels including TRPM2, the range of suitable pharmacological modulators is limited. Recently, 2-aminoethyl diphenylborinate (2-APB) was described as a TRPM8 channel blocker in addition to its actions as an inositol 1,4,5-triphosphatase (InsP3) receptor antagonist [23]. Thus, oxidative stress-induced Ca2+ influx in the TRPM2transfected CHO cells might also be modulated by 2-APB. To test this idea, some cells in the Ca2+ signaling experiments were pre-incubated with 2-APB (0.05 mM) for 1 min [11]. Stock 2-APB was dissolved in DMSO and stored at − 33 °C. Before the patch-clamp and Ca 2+ signaling experiments, 2-APB in extracellular bath solutions was diluted to reach the required final concentrations. All experiments were carried out at room temperature (approx 22 °C). After addition of 2-APB to standard extracellular bath solution, the pH was adjusted to 7.4 with KOH. The 2-APB was added to the patch-chamber (in the bath). 2.5. Measurement of intracellular free calcium concentration ([Ca 2+]i) The CHO cells at a density of 1 × 10 5 cells per ml were loaded with 4 μM fura-2/AM in loading buffer and incubated for 45 min at 37 °C in the dark, then washed twice with phosphate buffer and incubated for an additional 30 min at 37 °C to complete probe de-esterification. The cells were then re-suspended in loading buffer at a density of 1 × 10 5 cells per ml according to a procedure published elsewhere [24,25]. The six experimental groups were exposed to H2O2 for stimulation of Ca 2+ release and entry. Fluorescence was recorded from 2 ml aliquots of magnetically stirred cellular suspension at 37 °C by using a spectrofluorometer (Carry Eclipsys, Varian Inc, Sydney, Australia) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca 2+]i were monitored by using the fura-2 340/380 nm fluorescence ratio and were calibrated according to the method of Grynkiewicz et al. [25]. Ca 2+ mobilization in the CHO cells was estimated using the integral of the rise in [Ca 2+]i for 150 s after addition of H2O2 [26].

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Ca 2+ release is expressed in nanomolar quantities taking a sample every second as previously described [24]. 2.6. Apoptosis assay Apoptosis tests in CHO cells were assessed by use of a commercial kit (APOPercentage, Biocolor Ltd, County Antrim, United Kingdom). Fluorescence of the samples was measured at excitation and emission wavelengths of 530 nm and 580 nm, respectively (with a cut-off of 570 nm). 2.7. Statistical analyses All results were expressed as means ± standard deviation (SD). Significance in the groups was firstly checked by ANOVA- Kruskal

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Wallis test. Then, significant values in the groups were assessed with Mann-Whitney U test. Data were analyzed using the SPSS statistical program (version 9.05 software, SPSS Inc. Chicago, Illinois, USA). P-values of less than 0.05 were regarded as significant. 3. Results 3.1. Effects of H2O2 Effects of H2O2 on TRPM2 currents continuously recorded at −60 mV are shown in Figs. 1B and 2. TRPM2 currents were activated by extracellular H2O2 (10 mM). The currents were non-selective cation currents with a reversal potential close to 0 mV, as previously found to be typical of TRPM2 currents, and were readily blocked in the inside direction when the Na+ in the bath was replaced by the large impermanent cation

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Fig. 1. Effects of extracellular melatonin on H2O2-induced TRPM2 channel activation in transfected cells. The time when the bath solution was changed to a solution with NMDG+ as the main cation is shown. The holding potential was −60 mV. A. Original recordings from a control cell (transfected with the vector only). B. A cell expressing TRPM2 currents stimulated by H2O2 in the bath (chamber) with subsequent addition of 2-APB to the bath. C, D and E. The transfected cells in C and D groups were incubated with extracellular 300 μM (C) and 1 mM (D) melatonin, before H2O2 addition. In E, 200 μM melatonin was included in the intracellular pipette solution. B-1, C-1 and D-1. Current voltage relationships of NMDG and H2O2 through TRPM2 channel in presence of various extracellular cations (same experiments as in panels B, C and D). W.C. whole cell.

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NMDG+. No such currents were ever observed when cells were studied that were transfected with the vector only i.e. not with TRPM2; such negative control data were obtained on every experimental day of studying TRPM2 (Fig. 1A). Current densities were significantly (pb 0.001) higher in H2O2 group than in control. When H2O2 was added to the bath, there was a typical delay of 2 to 4 min before TRPM2 cation currents started to develop. Within 5 min, the currents reached a clear plateau in many experiments (Fig. 1B). 3.2. Both extracellular and intracellular melatonin inhibited H2O2-mediated activation of TRPM2 in the transfected CHO cells We next tested whether the antioxidants melatonin would prevent or attenuate the induction of TRPM2 currents by H2O2. Firstly, two doses (300 μM and 1 mM) of melatonin incubation were tested. The two doses of the antioxidant hormone lead to an inhibition of TRPM2 currents. The results of these experiments are presented in Fig. 1C and D. Secondly, we tested the effects of intracellular melatonin on H2O2-induced currents in the transfected cells. The channels were totally protected against H2O2-induced currents when melatonin was applied to the cytosolic side of the channels through the patch pipette (Fig. 1E). Current densities were significantly lower in extracellular (p b 0.05) and intracellular (p b 0.001) groups than in the H2O2 group. Current densities were also significantly (p b 0.001) lower in intracellular than in extracellular melatonin treated groups (Fig. 2). The data with melatonin would support the idea that H2O2 acts by initiating a metabolic cascade resulting in the production of cytosolic factors such as oxidative stress that are responsible for the activation of TRPM2 channel activity.

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Toxic effects of H2O2 were observable at a 1 mM dose for 5 h after incubation. Hence, the transfected cells in Ca 2+ signaling (Fura-2) analysis were incubated with 1 mM H2O2 for 5 h. 3.5. Effects of extracellular melatonin and 2-APB on H2O2-induced Ca 2+ signaling in the transfected CHO cells Effects of melatonin on [Ca 2+]i release in the CHO cells are shown in Figs. 4 and 5. Intracellular [Ca 2+]i release levels in the CHO cells were significantly (p b 0.001) higher in the H2O2 group than in control. The [Ca 2+]i release in the cells was significantly (p b 0.001) lower with the four concentrations (300 μM and 1–3 mM) of H2O2 + melatonin groups than in the H2O2 alone group. The [Ca 2+]i release in the cells was significantly (p b 0.05) lower in the 1 mM melatonin group than in 300 μM melatonin group. The [Ca 2+]i concentration in the cells was also significantly (p b 0.001) lower in the 2 mM melatonin group than in 1 mM melatonin group and it was lower (p b 0.001) in the 3 mM melatonin group than in the 2 mM melatonin group. Hence the results indicated that melatonin has dose dependent protective effects against H2O2-induced [Ca 2+]i concentration through regulation of TRPM2 cation channels. 3.6. Effects of different doses of melatonin on apoptosis Effects of different concentrations of melatonin on apoptosis in the transfected CHO cells are shown in Fig. 6. The extent of apoptosis in the CHO cells was significantly (p b 0.001) higher in the H2O2 group than in the control. The apoptosis significantly (p b 0.001) reduced by each of four concentrations of melatonin. Hence the results indicated that melatonin has dose dependent protective effects against H2O2-induced apoptosis.

3.3. Effects of TRPM2 antagonist in the CHO cells

4. Discussion

We tested whether the extracellular 2-APB would prevent or attenuate the TRPM2 currents induced by oxidative stress. The 2-APB did not inhibit H2O2-induced TRPM2 currents and current densities were significantly (p b 0.001) higher in 2-APB groups than in the control group, and there was no significant statistical difference between H2O2 + 2-APB and H2O2 groups. The results of the transfected cell experiments are presented in Figs. 1B, C, D and 2. Hence unlike melatonin, 2-APB did not induce blocker effects on H2O2-induced TRPM2 currents, in contrast to melatonin.

Changes in redox status are involved in normal cellular function, but can also induce necrotic/apoptotic processes in radiation injury, reperfusion injury during ischemia, and neurodegenerative diseases. Regulation of [Ca 2 +]i concentration is also critical in both normal and pathological cellular responses [1–4]. In the current study, TRPM2 channels were constitutively activated by H2O2. Different strategies have been used to inhibit H2O2-induced and TRPM2 gated channels, but these have not included melatonin administrations. In this study we have shown for the first time that melatonin inhibited the H2O2-induced TRPM2 channel gated Ca 2 + influx and apoptosis in the transfected cell line system. In the present study, TRPM2 channels were activated by H2O2. Oxidative stress causes the generation of ROS including H2O2 as by-products of the consumption of molecular oxygen in the redox system [1–4]. Physiologically, these ROS are mostly trapped within

3.4. Cell viability results on toxic doses of H2O2 Effects of H2O2 on the cell viability of the transfected CHO cells are shown in Fig. 3. Cells were incubated with increasing concentrations of H2O2 (1 μM-1 mM) for various periods of time (30 min–24 h).

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Fig. 2. Effects of melatonin (MEL) and 2-APB on H2O2-induced currents in TRPM2 transfected CHO cells. For each of the four applications, the initial current density as well as maximal current density after administration of H2O2 was administrated was divided by the cell capacitance, a measure of cell size. The numbers in parentheses indicate the number of observations per group. Significant (apb 0.001 versus control. bpb 0.05 and cpb 0.001 versus H2O2) stimulation and inhibition of currents are indicated with letters. (mean±SD).

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Fig. 3. Effect of H2O2 on cell viability in the TRPM2-transfected CHO cells. Cells were incubated with increasing concentrations of H2O2 (1, 10, 100 μM and 1 mM) for various periods of time (30 min, 1, 2, 5, 10 and 24 h). Toxic effect of H2O2 first appeared at the 1 mM dose and 5 h of incubation.

mitochondria and rapidly scavenged by endogenous or cell membrane antioxidants such as melatonin [13,14]. Yet under metabolic stress, ROS can be overproduced and cause damage to mitochondria [16,20]. Consequently, ROS may diffuse through the cytoplasm and cause further deleterious effects on other cellular processes. Hence, TRPM2 channels in the current study were likely gated by H2O2 due to damage structure to intracellular component(s) and subsequent activation of the N domain of TRPM2 channels. Based on various experimental findings, it has been deduced that poly(ADP-ribose) polymerase (PARP) activation is one of the key components for activation of TRPM2 channels. Activation of PARP is now viewed as an important effector of oxidative stress [27]. Under physiological conditions of oxidative stress, excessive DNA single-strand breakage is triggered by ROS leading to over-activation of PARP [28]. Activated PARP initiates an energy-consuming cycle resulting in rapid depletion of the intracellular pools of NAD+ and ATP hampering

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mitochondrial respiration, eventually leading to cellular energy crisis and cell death [29]. ADP-ribose (ADPR) is synthesized from NAD + through mitochondrial PARP activation [4,5]. TRPM2 contains a characteristic structural feature known as a Nudix domain in its C-terminal cytosolic tail [3,6–8]. A nudix domain is a consensus region that is known to be present in a class of pyrophosphatases that degrade nucleoside diphosphates [3]. Wehage et al. [6] and Hara et al. [30] reported that H2O2 evokes Ca 2+ influx by increasing ADPR levels and by subsequent binding of NAD+ directly to the Nudix motif in the cytosolic C-terminus of TRPM2. TRPM2 is also known to respond to intracellular ADPR, a metabolite of NAD+, via direct binding to the Nudix domain [3,6–12]. Since H2O2 is a strong oxidant, it is possible that intracellular H2O2 can oxidize NADH to NAD+ in H2O2-treated cells. In one study, when PARP enzyme activity was inhibited in rat primary striatal cultures, the TRPM2 channels were also inhibited [31]. However, melatonin attenuates apoptotic cell death through a PARP-mediated mechanism [21,22].

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Fig. 5. Effects of melatonin (MEL) on the rate of rise of [Ca2+]i concentrations in the transfected CHO cells. Stimulation was performed by hydrogen peroxide (H2O2, 100 μM). The cells were incubated with different doses (300 μM, 1, 2 and 3 mM) of melatonin. ap b 0.001 versus control. bp b 0.001 versus H2O2 group. cp b 0.05 versus 300 μM melatonin group. d p b 0.001 versus 300 μM and 1 mM melatonin groups. ep b 0.001 versus 300 μM, 1 and 2 mM melatonin groups.

Melatonin, a naturally mitochondria-targeted protective molecule with minimal cytotoxicity has not been shown to possess pro-oxidant effects in the presence of transition metals. While highly lipophilic antioxidants such as vitamin E and β-carotene are primarily retained in plasma membranes, melatonin seemingly targets the mitochondria. Melatonin possesses redox properties due to the presence of an electron-rich aromatic ring which allows the indole to function as an electron donor. Thus, through efficient removal of ROS which are mainly formed in the mitochondria, melatonin effectively reverses mitochondrial oxidative stress [13–20].

Melatonin inhibited H2O2-induced TRPM2 gating and apoptosis in the transfected cells. The H2O2 gates the channel dependently of ADPR and the activation of TRPM2 by H2O2 is probably linked to the activity of PARP-1, an enzyme transferring multiple ADPR groups to proteins [5]. Evidence for this intracellular pathway resulting in TRPM2 activation has been confirmed by the use of inhibitors of PARP-1, which were able to interfere with the H2O2 induced TRPM2 activation [31]. Similarly, combined treatment of melatonin with the anticancer drug, puromycin, reduced the expression of anti-apoptotic proteins, such as bcl-2 and bcl-x(L), and also induced caspase-3 activation and PARP

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Fig. 6. Effects of different doses of melatonin on apoptosis values in the transfected CHO cells (n = 8, mean ± SD).

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cleavage compared to puromycin treatment alone [21]. Cucina et al. [22] showed that melatonin induced caspase-independent apoptosis in MCF-7 breast carcinoma cells, which was accompanied by PARP cleavage. The membrane-permeable, noncompetitive inositol 1,4,5trisphosphate (IP3)-receptor inhibitor 2-APB has been widely used to probe for IP3-receptor involvement in Ca 2+ signaling pathways. 2-APB was found to inhibit capacitative calcium entry and TRPM2 currents at concentrations greater than 50 μM [23]. However, a number of recent studies in different cell types revealed other sites of action of 2-APB and that 2-APB increased cytosolic [Ca 2+]i concentrations by activation of nonselective cation channels [32]. It was also observed that 2-APB only partially inhibited store operated Ca 2+ entry (SOCE) and currents in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1 and these data revealed novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits [33]. Effects of 2-APB on TRPM2 channels in different cell system are also conflicting. For example, we observed inhibitory affects of 2-APB in rat DRG [10–14] but not in primary rat megakaryocytes [34]. In the current study, we observed negative results of 2-APB in the transfected cell line system. We did not observe any protective effects of 2-APB with/without melatonin on H2O2 currents in the CHO cells. Similarly, Tang et al. [35] reported that dantrolene, an antagonist of ryanodine (Ry) receptors in endoplasmic reticulum, almost abolished internal Ca2+ release, while 2-APB failed to block this Ca2+ release, indicating that released Ca2+ from mitochondria, which was induced by extracellular Na+ influx, further triggered much more Ca2+ release from endoplasmic reticulum. Apoptosis (or programmed cell death) is characterized by morphological changes such as DNA fragmentation and disintegration of the cell into apoptotic bodies that can be removed by phagocytosis [16,21]. Two major pathways have been described regulating apoptosis: the extrinsic pathway, in which cell plasma membrane receptors act as the starting point of the apoptotic process, and the intrinsic pathway, in which mitochondria play a central role. We observed that melatonin is able to reverse the cytosolic free Ca 2+ concentration, as well as the subsequent apoptosis inhibition, evoked by H2O2. In this regard, both in vitro and in vivo experiments have shown that melatonin can influence mitochondrial homeostasis. Melatonin stabilizes mitochondrial inner membrane [36] thereby improving electron transport chain activity. In fact, melatonin increases the activities of the brain and liver mitochondrial respiratory complexes I and IV in a time-dependent manner [37]. Recent finding indicates that melatonin is able to prevent mitochondrial cardiolipin oxidation/depletion, which controls several processes involved in mitochondrial bioenergetics, in mitochondrial steps of cell death, as well as in mitochondrial membrane stability and dynamics [38]. Similarly, Espino et al. [16] reported recently that melatonin is able to delay calcium overload-induced leukocyte apoptosis in advanced age likely due to its antioxidant properties. In conclusion, melatonin inhibited H2O2-induced TRPM2 channel gating. Hence, TRPM2 may be dependent on antioxidant redox cycles involving H2O2. Melatonin induced modulator effect on oxidative stress, the antioxidant redox system and Ca 2+ influx in the transfected CHO cells. The beneficial effects of melatonin on Ca2+ influx and apoptosis appeared to result from a decrease in TRPM2 cation channel activity. Oxidative injury through activation of TRPM2 cation channels have been causally linked to a variety of cellular diseases. We suggest that administration of melatonin, and thereby a reduction in TRPM2 cation channel activity, may provide a potential therapeutic approach for the prevention of cellular damage induced by products of oxidative stress. Acknowledgment MN formulated the present hypothesis and was responsible for writing the report. ÖÇ and BÇ were responsible for analysis of the data.

There is no conflict of interest in the current study. The authors wish to thanks Prof. Dr. James W. Putney (NIH, NC, USA) and Dr. Peter J Butterworth (King's College, London, UK) for polishing English of the manuscript. The study was supported by the Scientific Research Project Unit of Suleyman Demirel University (BAP-1882-D-09). Abstract of the study submitted in “Ion channel signaling Mechanisms” congress (October 31–November 4th 2011) in the city of Marrakesh, Morocco. References [1] Peng TI, Jou MJ. Oxidative stress caused by mitochondrial calcium overload. Ann N Y Acad Sci 2010;1201:183-8. [2] Putney JW, Smyth JT, Trebak M, Lemonnier L, Vazquez G, Gary S, et al. Activation and regulation of TRPC cation channels. Cell Membr Free Radic Res 2008;1:51-5. [3] Nazıroğlu M. TRPM2 cation channels, oxidative stress and neurological diseases: where are we now? Neurochem Res 2011;36:355-66. [4] Nazıroğlu M. 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