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Plasma-activated medium-induced intracellular zinc liberation causes death of SH-SY5Y cells
Archives of Biochemistry and Biophysics 584 (2015) 51e60
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Plasma-activated medium-induced intracellular zinc liberation causes death of SH-SY5Y cells Hirokazu Hara*, Miko Taniguchi, Mari Kobayashi, Tetsuro Kamiya, Tetsuo Adachi Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2015 Received in revised form 21 August 2015 Accepted 23 August 2015 Available online 28 August 2015
Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons. Plasma-activated medium (PAM), which is prepared by the irradiation of cell-free medium with nonthermal atmospheric pressure plasma, induces cell death in various types of cancer cell. Since PAM contains reactive oxygen species (ROS), its anti-cancer effects are thought to be attributable to oxidative stress. Meanwhile, oxidative stress has been shown to induce the liberation of zinc (Zn2þ) from intracellular Zn2þ stores and to provoke Zn2þ-dependent cell death. In this study, we thus examined whether Zn2þ is involved in PAM-induced cell death using human neuroblastoma SH-SY5Y cells. Exposure to PAM triggered cell death in SH-SY5Y cells. The cell-permeable Zn2þ chelator N,N,N0 ,N0 -tetrakis(2pyridinylmethyl)-1,2-ethanediamine (TPEN) protected against PAM-induced cell death. Zn2þ imaging using the fluorescent Zn2þ probe FluoZin-3 revealed that PAM elicited a rise of intracellular free Zn2þ. In addition, PAM stimulated PARP-1 activation, mitochondrial ROS generation, and the depletion of intracellular NADþ and ATP. These findings suggest that PAM-induced PARP-1 activation causes energy supply exhaustion. Moreover, TPEN suppressed all of these events elicited by PAM. Taken together, we demonstrated here that Zn2þ released from intracellular Zn2þ stores serves as a key mediator of PAMinduced cell death in SH-SY5Y cells. © 2015 Elsevier Inc. All rights reserved.
Keywords: Zinc Non-thermal atmospheric pressure plasma PARP-1 Cell death Reactive oxygen species Zinc-binding protein
1. Introduction A variety of trials of the use of non-thermal atmospheric pressure plasma for medical treatment, referred to as plasma medicine, are currently in progress [1e4]. Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons [2]. Since high-dose plasma irradiation has been shown to induce severe cell injury in various types of cancer cell, its application is thought to be useful for cancer therapy. The indirect plasma irradiation method using plasma-activated medium (PAM), which is prepared by plasma irradiation of cell-free medium, is equally as effective to kill cancer cells as the direct plasma irradiation method [3,5,6]. PAM contains reactive oxygen species (ROS), reactive nitrogen oxide species (RNS), and their decomposition products [5,7]. Therefore, the anti-cancer effects of PAM have been thought to be attributable to oxidative stress. However, the mechanism underlying PAM-induced cell death is not fully understood.
* Corresponding author. E-mail address: [email protected] (H. Hara). http://dx.doi.org/10.1016/j.abb.2015.08.014 0003-9861/© 2015 Elsevier Inc. All rights reserved.
Zinc (Zn2þ) is the second most abundant trace metal in the human body. Since most intracellular Zn2þ is bound to Zn2þbinding proteins including metallothionein (MT) and zinc fingercontaining transcription factors, the concentration of intracellular free Zn2þ is very low [8,9]. This metal usually coordinates with cysteine thiols and imidazole nitrogen atoms of histidine in Zn2þbinding sites. Cysteine thiol is easily oxidized, and consequently Zn2þ-thiol clusters are thought to be critical targets for ROS and RNS [10,11]. In fact, oxidants have been shown to promote the release of Zn2þ from Zn2þ-saturated MT in cell-free systems [12]. Moreover, it has been demonstrated that oxidative stress induced by exposure to ROS and RNS causes intracellular Zn2þ liberation from Zn2þ pools such as MT in neurons or other cell types, leading to Zn2þ-dependent cell death [13e16]. Therefore, Zn2þ plays a critical role in oxidative stress-induced cytotoxicity, even though Zn2þ itself is a redox-inert metal. Intracellular free Zn2þ is very toxic and has been reported to inhibit the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the electron transport chain in mitochondria [17], stimulate the production of ROS [18e20], and activate p38 mitogen-activated protein kinase (MAPK) [21]. These findings suggest that the perturbation of
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intracellular Zn2þ homeostasis caused by oxidative stress affects several cellular processes. More recently, we have demonstrated that ROS included in PAM, especially hydrogen peroxide, are involved in PAM-induced cell death in human lung adenocarcinoma epithelial A549 cells [5]. Exposure to PAM causes overactivation of poly(ADP-ribose) polymerase-1 (PARP-1), which is an enzyme activated in response to DNA damage, ultimately leading to PARP-1-dependent cell death. Indeed, PARP-1 has so far been reported to be closely related to oxidative stress-induced cell death [22]. Additionally, it has been shown that PARP-1 inhibitors protect against Zn2þ-induced neuronal cell death [23,24]. Therefore, PARP-1 is thought to play a critical role in the neurotoxicity associated with Zn2þ. In this study, we found that human neuroblastoma SH-SY5Y cells were very sensitive to PAM. Hence, we aimed to determine whether Zn2þ modulates PAM-induced cytotoxicity in SH-SY5Y cells. 2. Materials and methods 2.1. Materials Zinc sulfate and N,N,N0 ,N0 -tetrakis(2-pyridinylmethyl)-1,2ethanediamine (TPEN), and pyrithione were purchased from Wako Pure Chemical (Osaka, Japan). FluoZin-3 AM was purchased from Life Technologies (Waltham, MA). 3,4-Dihydro-5[4-(1piperindinyl)butoxy]-1(2H)-isoquinoline (DPQ), anti-MT antibody (UC1MT), and anti-actin antibody were purchased from Merck Millipore (Billerica, MA). Anti-poly(ADP-ribose) mouse IgG monoclonal antibody (10H) was purchased from Immuno-Biological Laboratories (Fujioka, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and 2,20 -dithiodipyridine (DTDP), and rotenone were purchased from Sigma Aldrich (St. Louis, MO). 2.2. Preparation of plasma-activated medium (PAM) PAM was prepared as described in our previous report [5]. Briefly, we used an irradiation system that consists of a power controller/gas flow regulator, an argon (Ar) gas cylinder, and a plasma source head (PN-120 TPG; NU Global, Nagoya, Japan) in this study. DMEM without pyruvate (D5796; Sigma Aldrich) in a dish (35-mm diameter) was irradiated with non-thermal atmosphericpressure plasma for 3 min at a flow rate of 2 L/min. The distance between the plasma source and the surface of the medium was fixed at L ¼ 3 mm. PAMs were stored at 80 C until use. We have previously demonstrated that the storage of PAM at 80 C hardly affects cytotoxic effects of PAM [5].
Cell viability was measured using an MTT assay. The medium was replaced with fresh medium containing MTT (final concentration 0.5 mg/mL), and was then incubated for 2 h at 37 C. The optical density was measured at 570 nm with a microplate reader. The experiments for cell viability were carried out in quadruplicate. The results are expressed as percentages relative to untreated cells. 2.5. RT-PCR SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~7.0 105 cells/dish. The next day, cells were treated with or without CdCl2 (5 mM). Total RNA was extracted from the treated cells with TRIzol reagent (Invitrogen, Carlsbad, CA). Firststrand cDNA was synthesized from 1 mg of total RNA. Aliquots of the cDNA solution (1 mL) were amplified using the following specific primers: MT (forward primer: 50 -ATGGACCCCAACTGCTCCTG30 , and reverse primer: 50 -CAGCAGGTGCACTTGTCCGA-30 ) and GAPDH (forward primer: 50 -GAAGGTGAAGGTCGGAGTC-30 , and reverse primer: 50 -CAAAGTTGTCATGGATGACC-30 ). PCR was carried out as follows: initially 2 min at 94 C, followed by 24 (MT) or 18 (GAPDH) cycles of 40 s at 94 C, 40 s at 58 C, and 1 min at 72 C. Aliquots of the PCR mixtures were separated on 2% agarose gel and stained with ethidium bromide. 2.6. Western blotting SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~7.0 105 cells/dish. The next day, cells were treated with or without CdCl2 (5 mM). After the treatment, cells were washed twice with ice-cold PBS, collected using 150 mL of 1 sodium dodecyl sulfate (SDS) Laemmli sample buffer containing 25 mM dithiothreitol, and heated for 5 min in a boiling water bath. Iodoacetamide was added to the lysate at a final concentration of 25 mM, and then this lysate was incubated for 15 min at 50 C. Aliquots of the prepared samples were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) in a 15% polyacrylamide gel. After being transferred onto a polyvinylidene difluoride (PVDF) membrane, the blotted membrane was blocked using PBS containing 1% bovine serum albumin (BSA). The membrane was sequentially incubated with each primary antibody (1:3000), biotin-conjugated secondary antibody (1:3000), and ABC reagents (Vector Laboratories, Inc., Burlingame, CA, USA) (1:5000). Finally, proteins were detected using Super-signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA) or ImmunoStar LD (Wako Pure Chemical) and imaged using an LAS-3000 (FUJIFILM).
2.3. Cell culture
2.7. Zn2þ imaging
Human neuroblastoma SH-SY5Y, lung adenocarcinoma epithelial A549, and breast cancer MCF-7 cells were cultured in the growth medium (DMEM supplemented with 10% fetal calf serum, 100 units/mL penicillin G, and 0.1 mg/mL streptomycin) in a humidified 5% CO2/95% air incubator at 37 C.
SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~6 105 cells/dish. The next day, cells were loaded with the fluorescent Zn2þ indicator FluoZin-3 AM (1 mM) and Hoechst 33342 (1 mg/mL; Dojindo, Kumamoto, Japan) in serum-free DMEM for 30 min. After washout of the fluorescent dye, serum-free DMEM (1 mL) was added to the dish. The cells loaded with FluoZin-3 were treated with DTDP (100 mM) or PAM (500 mL) in serum-free DMEM (1.5 mL). Time-lapse fluorescence imaging of live cells was performed at 30-s intervals for 20 min under a confocal laser fluorescence microscope (LSM700, Carl Zeiss) with Plan-Apochromat 10 /0.45 or 20 /0.75 objective. The fluorescence of FluoZin-3 was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The images were quantified using ZEN software (Carl Zeiss).
2.4. Cytotoxicity assay SH-SY5Y (1.5 104 cells/well), A549 (0.8 104 cells/well), and MCF-7 (1.5 104 cells/well) cells were seeded in a 96-well plate. The next day, cells were treated with serum-free DMEM (100 mL) containing various volumes of PAM for 1 h. The treatment with PAM was terminated by replacing PAM-containing DMEM with the growth medium and then cells were further cultured for 16e20 h.
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2.8. Detection of PARP-1 activity
3. Results
PARP-1 activity was evaluated by the immunostaining of poly(ADP-ribose), a product of this enzymatic reaction. SH-SY5Y cells (~2 105 cells) growing on a coverslip in a 4-well plate were treated with PAM (50 mL) in serum-free DMEM (500 mL) for 1 h. The cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 3% BSA in PBS, the cells were incubated for 1 h with anti-poly(ADPribose) mouse IgG monoclonal antibody (10H) (1:50). After the cells had been washed with PBS, they were incubated for 1 h with Alexa Fluor 488 goat anti-mouse IgG (1:400). Nuclei were stained with the dye Hoechst 33342 (1 mg/mL). Fluorescence was visualized using a confocal laser fluorescence microscope (LSM700).
3.1. PAM induces cell death in SH-SY5Y cells
2.9. Detection of mitochondrial ROS SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~6 105 cells/dish. Cells were treated with serum-free DMEM (1.5 mL) containing PAM (100 mL) for 1 h. After washout of the PAM, the cells were stained with MitoSOX (5 mM), a fluorescent probe for the detection of mitochondrial ROS, for 20 min. After washing twice with Hank's balanced salt solution, the fluorescence of MitoSOX was detected at an excitation wavelength of 488 nm and an emission wavelength of 580 nm using a confocal fluorescence microscope (LSM700). Quantification of the image was performed using NIH Image J Software. 2.10. Measurement of intracellular NADþ SH-SY5Y cells were seeded in a 24-well plate at a density of ~2 105 cells/well. The next day, cells were treated with serumfree DMEM (1 mL) containing PAM (50 mL) for 1 h. After the treatment with PAM, cells were cultured in the growth medium for another 1.5 h. Intracellular NADþ levels were measured using the EnzyChrom NADþ/NADH assay kit (BioAssay Systems, Hayward, CA). The treated cells were washed twice with cold PBS, and then lysed with the supplied NADþ extraction buffer. NADþ was extracted from the lysate according to the manufacturer's protocol. The protein contents in the lysate were determined using Bio-Rad protein assay reagent.
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Previous reports have demonstrated that PAM triggers cell injuries in various tumor cells such as A549 cells and glioblastoma [3,5]. First, to elucidate the effect of PAM on the viability of human neuroblastoma SH-SY5Y cells, we exposed cells to various doses of PAM for 1 h and cell viability was measured 20 h later using MTT assay. As shown in Fig. 1, PAM induced cell death in a dosedependent manner. Recently, we and others showed that cell death caused by exposure to PAM is due to ROS included in PAM [5,6]. Therefore, to determine the involvement of ROS in PAMinduced SH-SY5Y cell death, we investigated the effect of the antioxidant N-acetylcysteine (NAC) on this phenomenon. Consistent with previous reports, NAC protected cells against PAMinduced cell death (Fig. 1). This result indicates that ROS included in PAM might participate in the cytotoxicity elicited by exposure to PAM. 3.2. Involvement of intracellular Zn2þ in PAM-induced cell death in SH-SY5Y cells In general, most intracellular Zn2þ exists in a complex with Zn2þ-binding proteins. However, it has been reported that oxidative stress caused by ROS and RNS stimulates the release of Zn2þ from intracellular Zn2þ stores such as MT, leading to Zn2þ-dependent cell death [13,14,25]. Therefore, SH-SY5Y cells were exposed to PAM in the presence of the cell-permeable Zn2þ chelator TPEN in order to determine whether Zn2þ is involved in PAM-induced cell death. As shown in Fig. 2A, TPEN dose-dependently suppressed PAM-induced cell death. To test cytotoxic effects of PAM in other cell lines, human lung (A549) and breast (MCF-7) cancer cells were exposed to PAM in the same way. PAM elicited cell death and TPEM prevented PAM cytotoxicity in both cells (Fig. 2B). However, SHSY5Y cells were more sensitive to PAM compared to A549 and MCF-7 cells. Preincubation of TPEN with an equimolar concentration of Zn2þ (Zn-TPEN) abolished the protective effect of TPEN on PAM-induced
2.11. Measurement of intracellular ATP SH-SY5Y cells were seeded in a 24-well plate at a density of ~2 105 cells/well. The next day, cells were treated with serumfree DMEM (1 mL) containing PAM (50 mL) for 1 h and then cultured in the growth medium for another 3 h. Cells were washed twice with ice-cold PBS, harvested, and then sonicated in PBS. Immediately following sonication, ATP was extracted with perchloric acid (final concentration 0.5 M). The extracts were neutralized and then centrifuged. The supernatants were diluted with 10 mM TriseHCl pH 7.8 (1:100), and then aliquots (10 mL) of the diluted samples were subjected to ATP measurement. Intracellular ATP levels were measured using the ENLITEN rLuciferase/ Luciferin Reagent (Promega, Madison, WI). The luminescence was detected using a luminometer. The protein contents in the lysate after sonication were determined using Bio-Rad protein assay reagent. 2.12. Statistics Data were analyzed using ANOVA followed by post hoc Bonferroni tests. A P value less than 0.05 was considered significant.
Fig. 1. PAM induces cell death in SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells were exposed to various doses of PAM (0, 2.5, 5, 10, and 20 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with or without NAC (2 mM) for 30 min, and NAC was present during PAM exposure at a final concentration of 1 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. untreated cells); ##, P < 0.01.
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cell death (Fig. 2C). TPEN is known to chelate other metals such as iron ion (Fe2þ). However, preincubation of TPEN with an equimolar concentration of Fe2þ (Fe-TPEN) protected against PAM cytotoxicity as well as TPEN (Fig. 2C). The affinities of TPEN for Zn2þ and Fe2þ are shown to be Ka ¼ 1015.58 M1 and 1014.61 M1, respectively [26]. These results indicated that cell death caused by PAM is attributable to intracellular Zn2þ. Zn2þ is an essential nutrient, whereas it has been reported that free Zn2þ is toxic to a variety of cells [17,27]. When SH-SY5Y cells were treated with ZnSO4 in the presence or absence of pyrithione, a specific Zn2þ ionophore, which rapidly allows Zn2þ to enter the cells, Zn2þ leaded to cell death in cells treated with ZnSO4 and pyrithione (Fig. 2D). Therefore, the accumulation of intracellular Zn2þ might be responsible for PAMinduced cell death of SH-SY5Y cells. On the other hand, oxidative stress has been shown to raise intracellular calcium (Ca2þ) via Ca2þ influx thought the activation of voltage-gated Ca2þ channels (VGCCs) [28] or Ca2þ release from intracellular pools [29]. To rule out the involvement of Ca2þ in PAM-induced cell death, SH-SY5Y cells were exposed to PAM in the presence or absence of verapamil, a VGCC inhibitor, or dantrolene, a ryanodine receptor inhibitor. As shown in Fig. 2E, these inhibitors failed to abolish PAM cytotoxicity. MT is a family of small cysteine-rich metal binding proteins and plays a major role in the regulation of intracellular Zn2þ homeostasis and heavy metal detoxification. Therefore, we hypothesized that newly synthesized apo-MT, which can sequester Zn2þ, improves Zn2þ perturbation elicited by PAM exposure and exerts a protective effect against PAM cytotoxicity. Cadmium (Cd2þ) is known to be a potent inducer of the MT gene. Indeed, treatment of SH-SY5Y cells with Cd2þ markedly increased MT mRNA and protein expression (Fig. 2F). As expected, PAM-induced cell death was suppressed in cells pretreated with Cd2þ (Fig. 2G). 3.3. PAM triggers intracellular Zn2þ liberation We next examined whether PAM causes the elevation of intracellular free Zn2þ ([Zn2þ]i) in SH-SY5Y cells. To monitor alterations of the liberated Zn2þ, we employed the fluorescent Zn2þ indicator FluoZin-3. SH-SY5Y cells loaded with FluoZin-3 were exposed to DTDP, a thiol oxidant, which is known to elicit Zn2þ liberation, or PAM. As expected, DTDP markedly increased the fluorescence intensity of FluoZin-3, indicating the rise of [Zn2þ]i (Fig. 3A). This elevation was rapidly abolished by the addition of TPEN. PAM also increased the fluorescence, but to a lesser extent than DTDP, and the addition of TPEN abolished PAM-induced augmentation of the fluorescence (Fig. 3A). These results indicate that PAM triggers Zn2þ
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liberation in SH-SY5Y cells. To determine where Zn2þ liberation occurs, we measured fluorescence intensities of FluoZin-3 (Zn2þ) and Hoechst 33324 (nucleus) within the cross-section of single cell after PAM exposure. As shown in Fig. 3B, the accumulation of free Zn2þ was observed in not only the cytoplasm but also the nucleus. Previously, it has been reported that ROS contribute to the liberation of Zn2þ from Zn2þ-binding proteins [10,11]. As described above, ROS included in PAM were closely related to PAM-induced cell death in SH-SY5Y cells. To elucidate whether PAM-containing ROS participate in the rise of [Zn2þ]i, cells loaded with FluoZin-3 were exposed to PAM in the presence or absence of NAC, followed by monitoring of the alteration in fluorescence intensity. As shown in Fig. 3C, NAC abolished PAM-induced elevation of [Zn2þ]i. 3.4. Involvement of PARP-1 in PAM-induced cell death There is evidence that various stresses such as oxidants and genotoxin cause the activation of PARP-1 and sequentially the accumulation of poly(ADP-ribose) in the nucleus [22]. Many studies have shown that overactivation of PARP-1 under severe stress conditions results in cell death [30e32]. To determine whether exposure to PAM stimulates PARP-1 activation in SH-SY5Y cells, we performed immunocytochemistry in order to detect the accumulation of poly(ADP-ribose) in the nucleus. As shown in Fig. 4A, PAM promoted the nuclear accumulation of poly(ADP-ribose), indicating that PAM stimulates PARP-1 activation. To clarify the relationship between Zn2þ and PARP-1 activation, we tested the effect of TPEN on PAM-induced PARP-1 activation. TPEN suppressed the nuclear accumulation of poly(ADP-ribose) (Fig. 4A). We next determined whether PAPR-1 activation is related to PAM-induced cell death. When SH-SY5Y cells were exposed to PAM in the presence of the PARP-1 inhibitor DPQ, this inhibitor prevented PAM-induced cell death (Fig. 4B). These results indicate that PARP-1 plays a critical role in such death. In addition, we also examined the effect of DPQ on PAM-induced Zn2þ liberation. As shown in Fig. 4C, however, DPQ failed to suppress the rise of [Zn2þ]i, suggesting that PAM-induced Zn2þ liberation occurrs upstream of PARP-1 activation. 3.5. PAM stimulates mitochondrial ROS generation It has been demonstrated that exposure to hydrogen peroxide triggers robust delayed generation of ROS in mitochondria and mitochondrial ROS cause PARP-1 activation [33]. To determine whether the exposure of SH-SY5Y cells to PAM elicits mitochondrial ROS generation, we assayed ROS production using a fluorescent probe for mitochondrial ROS, MitoSOX. As shown in Fig. 5A,
Fig. 2. Involvement of intracellular Zn2þ in PAM-induced cell death. A) Effect of TPEN on PAM cytotoxicity. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with different concentrations of TPEN (1.25, 2.5, 5, and 10 mM) for 20 min, and TPEN was present during PAM exposure at final concentrations of 0.63, 1.25, 2.5, and 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01; ##, P < 0.01 (vs. cells treated with PAM alone). B) Cytotoxic effects of PAM in A549 and MCF-7 cells. A549 and MCF-7 cells were exposed to PAM (15 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with TPEN (10 mM) for 20 min, and TPEN was present during PAM exposure at a final concentration of 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with PAM alone). C) Effect of Zn-TPEN on PAM cytotoxicity. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for a further 20 h. Zn-TPEN and Fe-TPEN were prepared by incubation of TPEN (10 mM) with equimolar concentrations of ZnSO4 and FeCl2, respectively. Cells were pretreated with or without TPEN, Zn-TPEN, or Fe-TPEN for 20 min, and they were present during PAM exposure at a final concentration of 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control without PAM); ##, P < 0.01 (vs. cells treated with PAM alone); yy, P < 0.01. D) Zn2þ toxicity toward SH-SY5Y cells. SHSY5Y cells were treated with ZnSO4 (20 mM) in the presence or absence of pyrithione (Pyri, 1 mM) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with Zn2þ alone). E) No involvement of Ca2þ in PAM-induced cell death. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with verapamil (Ver, 20 mM) or dantrolene (Dan, 20 mM) for 30 min, and these inhibitors were present during PAM exposure at a final concentration of 10 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. cells treated with PAM alone). F) Cd2þ induces MT expression in SH-SY5Y cells. SH-SY5Y cells were treated with or without CdCl2 (5 mM) for 1.5 h, followed by culture of the cells in the growth medium for 6 h. After the treatment, RT-PCR and Western blotting were performed. These results are representative of three independent experiments. G) Overexpression of MT protects against PAM-induced cell death. SH-SY5Y cells pretreated with or without CdCl2 (5 mM) for 1.5 h, followed by culture of the cells in the growth medium for 8 h. The Cd2þ-treated cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for a further 20 h. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control without Cd2þ); ##, P < 0.01.
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Fig. 3. A) PAM-induced Zn2þ liberation. SH-SY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) for 20 min. After washout, serum-free DMEM (1 mL) was added to the dish. The cells were exposed to DTDP (final conc. 100 mM) or PAM (500 mL). Fifteen minutes later, TPEN was added to the dish at a final concentration of 10 mM. The fluorescence was detected using a confocal microscope. This result is representative of three experiments. B) Distribution of intracellular Zn2þ after PAM exposure. Upper panel; SH-SY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) and Hoechst 33342 (1 mg/mL) for 20 min. After washout, serum-free DMEM (1 mL) was added to the dish, followed by the exposure to PAM (500 mL). Fifteen minutes later, the fluorescence was detected using a confocal microscope. Bar ¼ 10 mm. Lower panel; fluorescence profile of the cross-section indicated by red arrow. The solid and dashed lines correspond to FluoZin-3 (Zn2þ) and Hoechst 33342 (nucleus), respectively. C) Effect of NAC on PAM-induced Zn2þ liberation. SHSY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) and NAC (1 mM) for 30 min. After washout, serum-free DMEM (1 mL) was added to the dish. The cells were exposed to PAM (500 mL) in the presence or absence of NAC (1 mM) for 15 min. The fluorescence was detected using a confocal microscope. Values are means ± SD of 15 cells in a single experiment. This result is representative of three experiments. **, P < 0.01; ##, P < 0.01 (vs. cells treated with PAM alone). NS, not significant.
exposure to PAM increased the intensity of MitoSOX fluorescence. The ROS generation was suppressed in the presence of TPEN (Fig. 5A). In addition, we examined the effect of DPQ on PAMinduced ROS generation in mitochondria. Unexpectedly, DPQ also prevented mitochondrial ROS generation (Fig. 5A). Moreover, to verify the involvement of mitochondria in PAM-induced ROS generation, SH-SY5Y cells were exposed to PAM in the presence of a low concentration of rotenone, a mitochondrial complex I inhibitor. As shown in Fig. 5B, rotenone reduced mitochondrial ROS generation caused by PAM. 3.6. PAM causes depletion of intracellular NADþ and ATP It is well known that PARP-1 consumes NADþ to form poly(ADPribose). Therefore, marked PARP-1 activation induced by severe stress causes energy supply exhaustion, leading to cell death. We examined whether the exposure to PAM influences the contents of intracellular NADþ and ATP. As shown in Fig. 6A, PAM exposure caused declines in intracellular NADþ and ATP levels in SH-SY5Y cells. We next examined the contribution of Zn2þ to PAM-induced NADþ and ATP depletions. TPEN ameliorated the reductions of NADþ and ATP levels (Fig. 6A). It has been reported that supplementation of an NADþ precursor, nicotinamide, and a TCA cycle
substrate, pyruvate, prevents PARP-1-mediated cell death [23,24]. Therefore, we hypothesized that energy substrates such as nicotinamide and pyruvate confer protection against PAM-induced cell death. As expected, these agents reduced PAM-induced cytotoxicity (Fig. 6B). 4. Discussion PAM contains various kinds of ROS, such as hydrogen peroxide, and decomposition products of RNS, such as nitrite and nitrate. Recently, we have demonstrated that ROS included in PAM, especially hydrogen peroxide, are responsible for PAM-induced cell death in A549 cells [5]. In this study, we found that PAM induced a rise of [Zn2þ]i in SH-SY5Y cells and that liberated Zn2þ elicited PARP-1 activation, mitochondrial ROS generation, and intracellular NADþ and ATP loss, and ultimately led to Zn2þ-dependent cell death. There is evidence that oxidative stress triggers Zn2þ release from 2þ Zn -binding proteins including MT [10,11]. Although most intracellular Zn2þ is coordinated to cysteine thiols and imidazole nitrogen atoms of histidine in Zn2þ-binding sites, oxidative thiol modification allows the release of Zn2þ from these proteins. Previous reports have demonstrated that ROS/RNS cause intracellular
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Fig. 4. PAM-induced PARP-1 activation is Zn2þ-dependent. A) Effect of TPEN on PAM-induced PARP-1 activation. SH-SY5Y cells growing on a coverslip were exposed to PAM (50 mL/ 500 mL) in a 4-well plate for 1 h. Cells were pretreated with TPEN (10 mM) for 20 min, and TPEN was present during PAM exposure. After the exposure, the cells were fixed, and immunocytochemistry was performed using anti-poly(ADP-ribose) antibody. The nucleus was stained with Hoechst 33342. The fluorescence was visualized using a confocal microscope. Bar ¼ 20 mm. PAR; poly(ADP-ribose). B) Effect of PARP-1 inhibitor on PAM cytotoxicity. SH-SY5Y cells were exposed to various doses of PAM for 1 h, followed by culture of the cells in the growth medium for a further 20 h. Cells were pretreated with or without DPQ (10 mM) for 30 min, and DPQ was present during PAM exposure at a final concentration of 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. untreated cells); ##, P < 0.01. C) Effect of PARP-1 inhibitor on PAM-induced Zn2þ liberation. SH-SY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) and DPQ (5 mM) for 30 min. After washout, serum-free DMEM (1 mL) with or without DPQ was added to the dish. The cells were exposed to PAM (500 mL). Fifteen minutes later, TPEN was added to the dish at a final concentration of 10 mM. DPQ was present at a final concentration of 5 mM prior to and during PAM exposure. This result is representative of three experiments.
Zn2þ liberation in neurons or other cell types, leading to cytotoxicity [13e15]. We have more recently demonstrated that ROS are important mediators in PAM-induced cell death [5]. These findings prompted us to determine the contribution of intracellular free Zn2þ to PAM-induced cell death. Herein, we showed that Zn2þ chelation using TPEN protected against PAM cytotoxicity (Fig. 2A). In addition, overexpression of MT induced by Cd2þ conferred protection against PAM-induced cell death (Fig. 2G). Most, if not all, of
newly synthesized thionein (the apo-form of MT) has the ability to sequester Zn2þ. On the other hand, however, since MT is also known to be an antioxidant, it could also function as a radical scavenger. These results suggest that Zn2þ liberated by PAM exposure is an important mediator of PAM-induced cell death. Actually, Zn2þ imaging using FluoZin-3 revealed that PAM elevated [Zn2þ]i in SH-SY5Y cells. The rise in [Zn2þ]i caused by PAM exposure was observed in cells bathed in serum-free DMEM (Fig. 3A).
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Fig. 5. PAM stimulates mitochondrial ROS generation. A) Effect of TPEN and DPQ on PAM-induced ROS generation. SH-SY5Y cells (35-mm dish) were incubated with TPEN (10 mM) or DPQ (10 mM) for 30 min in serum-free DMEM (1 mL) and subsequently PAM (100 mL) was added to the dish. One hour later, the cells were stained with MitoSOX (5 mM) for 20 min, and then the fluorescence was observed using a confocal microscope. TPEN or DPQ was present during PAM exposure. Values are means ± SD of 25 cells in a single experiment. This result is representative of three experiments. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with PAM alone). Bar ¼ 100 mm. B) Effect of rotenone on PAMinduced ROS generation. SH-SY5Y cells (35-mm dish) were incubated with TPEN (10 mM) or rotenone (Rote; 9 nM) for 30 min in serum-free DMEM (1 mL) and subsequently PAM (100 mL) was added to the dish. One hour later, the cells were stained with MitoSOX (5 mM) for 20 min, and then the fluorescence was observed using a confocal microscope. Rotenone was present during PAM exposure. Values are means ± SD of 25 cells in a single experiment. This result is representative of three experiments. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with PAM alone). Bar ¼ 100 mm.
Since DMEM does not contain Zn2þ, we could rule out the possibility that Zn2þ sources are present in the extracellular space. Moreover, we found that the accumulation of [Zn2þ]i was observed in the nucleus as well as in the cytoplasm of SH-SY5Y cells exposed to PAM. Berendji et al. [34] also demonstrated that the treatment of mouse fibroblast L929 cells with RNS triggers Zn2þ release all over the cell. These findings suggested the possibility that PAM causes oxidative modification of cysteine residues in not only cytosolic proteins such as MT but also nuclear proteins such as zinc fingercontaining transcription factors. Consistent with previous studies using other oxidants [13,25], the rise of [Zn2þ]i also occurred immediately after PAM exposure (within 15 min) under our experimental conditions. Therefore, we consider that liberated Zn2þ acts as an important initial signal in the PAM-induced cell death process. Intracellular Zn2þ has been shown to stimulate various signaling pathways such as the MAPK (p38 and ERK) pathway in various cell types [21,27,35,36]. However, under our experimental conditions, p38 and ERK pathway inhibitors had no effect on PAM-induced cytotoxicity (data not shown). Therefore, the MAPK pathway is unlikely to be involved in PAM-induced cell death in SH-SY5Y cells. It has been demonstrated that oxidative stress-induced DNA damage activates PARP-1, leading to PARP-1-dependent cell death
[22]. Therefore, the activation of PARP-1 is thought to be closely related to the cell death process. When cortical neurons are exposed to excessive Zn2þ, PARP-1-dependent neurotoxicity has been shown to occur [23,24]. In this study, we found that the exposure of SH-SY5Y cells to PAM elicited PARP-1 activation. Additionally, PARP-1 inhibitors prevented PAM-induced cell death. Our present results suggest that PARP-1 plays a key role in PAM cytotoxicity. The activation of PARP-1 was inhibited in the presence of TPEN, whereas PARP-1 inhibitor DPQ did not affect the PAMinduced rise of [Zn2þ]i. Therefore, Zn2þ is likely to function as an upstream regulator for PARP-1 activation. Unfortunately, however, the details of how Zn2þ activates PARP-1 are currently unknown. On the other hand, it has been reported that excessive Zn2þ influx increases ROS production in neurons [18e20]. We found that PAM stimulated ROS generation in mitochondria and TPEN suppressed the generation of mitochondrial ROS (Fig. 5). Therefore, it is likely that the liberated Zn2þ modulates PARP-1 activation via ROS signaling. Unexpectedly, however, PARP-1 inhibitors as well as TPEN decreased PAM-induced ROS generation in SH-SY5Y cells. PARP-1 activation has also been shown to induce ROS production, leading to cell injury [37,38]. Thus, we cannot rule out the possibility that mechanisms other than ROS are involved in PARP-1 activation following Zn2þ liberation.
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energy supply exhaustion caused by Zn2þ liberation and subsequent PARP-1 activation. Meanwhile, Zn2þ has been shown to inhibit GAPDH, which is a key enzyme in glycolysis [17,24]. Therefore, not only PARP-1 activation but also GAPDH inhibition might be involved in PAM-induced cytotoxicity. In conclusion, we demonstrated here that Zn2þ released from intracellular sources serves as a key mediator of PAM-induced cell death in SH-SY5Y cells. Our findings suggest that the conversion of PAM stimulus to a Zn2þ signal is a critical step in the PAM-induced cell death process. Therefore, it is likely that intracellular Zn2þ contents affect the efficacy of plasma irradiation against cancer therapy. At present, however, how liberated Zn2þ activates its downstream signaling is not fully understood. Thus, further investigations are needed to understand Zn2þ signaling in the PAMinduced cell death process. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (to H.H.; No. 26460630) and in part by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (to T.A.; No. 25108511). References
Fig. 6. PAM elicits depletion of intracellular NADþ and ATP. A) Effect of PAM on intracellular NADþ and ATP levels. SH-SY5Y cells (24-well plate) were exposed to PAM (50 mL/500 mL) for 1 h in serum-free DMEM. One and a half (NADþ) and three (ATP) hours after the exposure, intracellular NADþ and ATP contents were measured. Cells were pretreated with TPEN (10 mM) for 20 min, and TPEN was present during PAM exposure. **, P < 0.01 (vs. control); ##, P < 0.01. B) Effect of nicotinamide and pyruvate on PAM-induced cell death. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h in serum-free DMEM, followed by culture of the cells in the growth medium for a further 20 h. Cells were pretreated with vehicle (open columns), nicotinamide (3 mM; hatched columns), or pyruvate (3 mM; gray columns) for 30 min, and they were present during PAM exposure at a final concentration of 1.5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. #, P < 0.05; ##, P < 0.01 (vs. cells treated with PAM alone).
PARP-1 activation consumes intracellular NADþ to form poly(ADP-ribose), leading to energy supply exhaustion. PAM decreased the intracellular levels of NADþ and ATP in SH-SY5Y cells. This result is consistent with the findings of our previous study using A549 cells. The addition of energy substrates such as nicotinamide and pyruvate rescued PAM-induced cell death (Fig. 6B). On the other hand, pyruvate itself is known to have a radical scavenging effect [39]. This might be the reason why pyruvate was more protective against PAM cytotoxicity than nicotinamide. TPEN ameliorated PAM-induced depletion of intracellular NADþ and ATP. These findings suggest that PAM-induced cell death is due to
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Plasma-activated medium-induced intracellular zinc liberation causes death of SH-SY5Y cells Hirokazu Hara*, Miko Taniguchi, Mari Kobayashi, Tetsuro Kamiya, Tetsuo Adachi Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2015 Received in revised form 21 August 2015 Accepted 23 August 2015 Available online 28 August 2015
Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons. Plasma-activated medium (PAM), which is prepared by the irradiation of cell-free medium with nonthermal atmospheric pressure plasma, induces cell death in various types of cancer cell. Since PAM contains reactive oxygen species (ROS), its anti-cancer effects are thought to be attributable to oxidative stress. Meanwhile, oxidative stress has been shown to induce the liberation of zinc (Zn2þ) from intracellular Zn2þ stores and to provoke Zn2þ-dependent cell death. In this study, we thus examined whether Zn2þ is involved in PAM-induced cell death using human neuroblastoma SH-SY5Y cells. Exposure to PAM triggered cell death in SH-SY5Y cells. The cell-permeable Zn2þ chelator N,N,N0 ,N0 -tetrakis(2pyridinylmethyl)-1,2-ethanediamine (TPEN) protected against PAM-induced cell death. Zn2þ imaging using the fluorescent Zn2þ probe FluoZin-3 revealed that PAM elicited a rise of intracellular free Zn2þ. In addition, PAM stimulated PARP-1 activation, mitochondrial ROS generation, and the depletion of intracellular NADþ and ATP. These findings suggest that PAM-induced PARP-1 activation causes energy supply exhaustion. Moreover, TPEN suppressed all of these events elicited by PAM. Taken together, we demonstrated here that Zn2þ released from intracellular Zn2þ stores serves as a key mediator of PAMinduced cell death in SH-SY5Y cells. © 2015 Elsevier Inc. All rights reserved.
Keywords: Zinc Non-thermal atmospheric pressure plasma PARP-1 Cell death Reactive oxygen species Zinc-binding protein
1. Introduction A variety of trials of the use of non-thermal atmospheric pressure plasma for medical treatment, referred to as plasma medicine, are currently in progress [1e4]. Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons [2]. Since high-dose plasma irradiation has been shown to induce severe cell injury in various types of cancer cell, its application is thought to be useful for cancer therapy. The indirect plasma irradiation method using plasma-activated medium (PAM), which is prepared by plasma irradiation of cell-free medium, is equally as effective to kill cancer cells as the direct plasma irradiation method [3,5,6]. PAM contains reactive oxygen species (ROS), reactive nitrogen oxide species (RNS), and their decomposition products [5,7]. Therefore, the anti-cancer effects of PAM have been thought to be attributable to oxidative stress. However, the mechanism underlying PAM-induced cell death is not fully understood.
* Corresponding author. E-mail address: [email protected] (H. Hara). http://dx.doi.org/10.1016/j.abb.2015.08.014 0003-9861/© 2015 Elsevier Inc. All rights reserved.
Zinc (Zn2þ) is the second most abundant trace metal in the human body. Since most intracellular Zn2þ is bound to Zn2þbinding proteins including metallothionein (MT) and zinc fingercontaining transcription factors, the concentration of intracellular free Zn2þ is very low [8,9]. This metal usually coordinates with cysteine thiols and imidazole nitrogen atoms of histidine in Zn2þbinding sites. Cysteine thiol is easily oxidized, and consequently Zn2þ-thiol clusters are thought to be critical targets for ROS and RNS [10,11]. In fact, oxidants have been shown to promote the release of Zn2þ from Zn2þ-saturated MT in cell-free systems [12]. Moreover, it has been demonstrated that oxidative stress induced by exposure to ROS and RNS causes intracellular Zn2þ liberation from Zn2þ pools such as MT in neurons or other cell types, leading to Zn2þ-dependent cell death [13e16]. Therefore, Zn2þ plays a critical role in oxidative stress-induced cytotoxicity, even though Zn2þ itself is a redox-inert metal. Intracellular free Zn2þ is very toxic and has been reported to inhibit the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the electron transport chain in mitochondria [17], stimulate the production of ROS [18e20], and activate p38 mitogen-activated protein kinase (MAPK) [21]. These findings suggest that the perturbation of
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intracellular Zn2þ homeostasis caused by oxidative stress affects several cellular processes. More recently, we have demonstrated that ROS included in PAM, especially hydrogen peroxide, are involved in PAM-induced cell death in human lung adenocarcinoma epithelial A549 cells [5]. Exposure to PAM causes overactivation of poly(ADP-ribose) polymerase-1 (PARP-1), which is an enzyme activated in response to DNA damage, ultimately leading to PARP-1-dependent cell death. Indeed, PARP-1 has so far been reported to be closely related to oxidative stress-induced cell death [22]. Additionally, it has been shown that PARP-1 inhibitors protect against Zn2þ-induced neuronal cell death [23,24]. Therefore, PARP-1 is thought to play a critical role in the neurotoxicity associated with Zn2þ. In this study, we found that human neuroblastoma SH-SY5Y cells were very sensitive to PAM. Hence, we aimed to determine whether Zn2þ modulates PAM-induced cytotoxicity in SH-SY5Y cells. 2. Materials and methods 2.1. Materials Zinc sulfate and N,N,N0 ,N0 -tetrakis(2-pyridinylmethyl)-1,2ethanediamine (TPEN), and pyrithione were purchased from Wako Pure Chemical (Osaka, Japan). FluoZin-3 AM was purchased from Life Technologies (Waltham, MA). 3,4-Dihydro-5[4-(1piperindinyl)butoxy]-1(2H)-isoquinoline (DPQ), anti-MT antibody (UC1MT), and anti-actin antibody were purchased from Merck Millipore (Billerica, MA). Anti-poly(ADP-ribose) mouse IgG monoclonal antibody (10H) was purchased from Immuno-Biological Laboratories (Fujioka, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and 2,20 -dithiodipyridine (DTDP), and rotenone were purchased from Sigma Aldrich (St. Louis, MO). 2.2. Preparation of plasma-activated medium (PAM) PAM was prepared as described in our previous report [5]. Briefly, we used an irradiation system that consists of a power controller/gas flow regulator, an argon (Ar) gas cylinder, and a plasma source head (PN-120 TPG; NU Global, Nagoya, Japan) in this study. DMEM without pyruvate (D5796; Sigma Aldrich) in a dish (35-mm diameter) was irradiated with non-thermal atmosphericpressure plasma for 3 min at a flow rate of 2 L/min. The distance between the plasma source and the surface of the medium was fixed at L ¼ 3 mm. PAMs were stored at 80 C until use. We have previously demonstrated that the storage of PAM at 80 C hardly affects cytotoxic effects of PAM [5].
Cell viability was measured using an MTT assay. The medium was replaced with fresh medium containing MTT (final concentration 0.5 mg/mL), and was then incubated for 2 h at 37 C. The optical density was measured at 570 nm with a microplate reader. The experiments for cell viability were carried out in quadruplicate. The results are expressed as percentages relative to untreated cells. 2.5. RT-PCR SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~7.0 105 cells/dish. The next day, cells were treated with or without CdCl2 (5 mM). Total RNA was extracted from the treated cells with TRIzol reagent (Invitrogen, Carlsbad, CA). Firststrand cDNA was synthesized from 1 mg of total RNA. Aliquots of the cDNA solution (1 mL) were amplified using the following specific primers: MT (forward primer: 50 -ATGGACCCCAACTGCTCCTG30 , and reverse primer: 50 -CAGCAGGTGCACTTGTCCGA-30 ) and GAPDH (forward primer: 50 -GAAGGTGAAGGTCGGAGTC-30 , and reverse primer: 50 -CAAAGTTGTCATGGATGACC-30 ). PCR was carried out as follows: initially 2 min at 94 C, followed by 24 (MT) or 18 (GAPDH) cycles of 40 s at 94 C, 40 s at 58 C, and 1 min at 72 C. Aliquots of the PCR mixtures were separated on 2% agarose gel and stained with ethidium bromide. 2.6. Western blotting SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~7.0 105 cells/dish. The next day, cells were treated with or without CdCl2 (5 mM). After the treatment, cells were washed twice with ice-cold PBS, collected using 150 mL of 1 sodium dodecyl sulfate (SDS) Laemmli sample buffer containing 25 mM dithiothreitol, and heated for 5 min in a boiling water bath. Iodoacetamide was added to the lysate at a final concentration of 25 mM, and then this lysate was incubated for 15 min at 50 C. Aliquots of the prepared samples were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) in a 15% polyacrylamide gel. After being transferred onto a polyvinylidene difluoride (PVDF) membrane, the blotted membrane was blocked using PBS containing 1% bovine serum albumin (BSA). The membrane was sequentially incubated with each primary antibody (1:3000), biotin-conjugated secondary antibody (1:3000), and ABC reagents (Vector Laboratories, Inc., Burlingame, CA, USA) (1:5000). Finally, proteins were detected using Super-signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA) or ImmunoStar LD (Wako Pure Chemical) and imaged using an LAS-3000 (FUJIFILM).
2.3. Cell culture
2.7. Zn2þ imaging
Human neuroblastoma SH-SY5Y, lung adenocarcinoma epithelial A549, and breast cancer MCF-7 cells were cultured in the growth medium (DMEM supplemented with 10% fetal calf serum, 100 units/mL penicillin G, and 0.1 mg/mL streptomycin) in a humidified 5% CO2/95% air incubator at 37 C.
SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~6 105 cells/dish. The next day, cells were loaded with the fluorescent Zn2þ indicator FluoZin-3 AM (1 mM) and Hoechst 33342 (1 mg/mL; Dojindo, Kumamoto, Japan) in serum-free DMEM for 30 min. After washout of the fluorescent dye, serum-free DMEM (1 mL) was added to the dish. The cells loaded with FluoZin-3 were treated with DTDP (100 mM) or PAM (500 mL) in serum-free DMEM (1.5 mL). Time-lapse fluorescence imaging of live cells was performed at 30-s intervals for 20 min under a confocal laser fluorescence microscope (LSM700, Carl Zeiss) with Plan-Apochromat 10 /0.45 or 20 /0.75 objective. The fluorescence of FluoZin-3 was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The images were quantified using ZEN software (Carl Zeiss).
2.4. Cytotoxicity assay SH-SY5Y (1.5 104 cells/well), A549 (0.8 104 cells/well), and MCF-7 (1.5 104 cells/well) cells were seeded in a 96-well plate. The next day, cells were treated with serum-free DMEM (100 mL) containing various volumes of PAM for 1 h. The treatment with PAM was terminated by replacing PAM-containing DMEM with the growth medium and then cells were further cultured for 16e20 h.
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2.8. Detection of PARP-1 activity
3. Results
PARP-1 activity was evaluated by the immunostaining of poly(ADP-ribose), a product of this enzymatic reaction. SH-SY5Y cells (~2 105 cells) growing on a coverslip in a 4-well plate were treated with PAM (50 mL) in serum-free DMEM (500 mL) for 1 h. The cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 3% BSA in PBS, the cells were incubated for 1 h with anti-poly(ADPribose) mouse IgG monoclonal antibody (10H) (1:50). After the cells had been washed with PBS, they were incubated for 1 h with Alexa Fluor 488 goat anti-mouse IgG (1:400). Nuclei were stained with the dye Hoechst 33342 (1 mg/mL). Fluorescence was visualized using a confocal laser fluorescence microscope (LSM700).
3.1. PAM induces cell death in SH-SY5Y cells
2.9. Detection of mitochondrial ROS SH-SY5Y cells were seeded in a dish (35-mm diameter) at a density of ~6 105 cells/dish. Cells were treated with serum-free DMEM (1.5 mL) containing PAM (100 mL) for 1 h. After washout of the PAM, the cells were stained with MitoSOX (5 mM), a fluorescent probe for the detection of mitochondrial ROS, for 20 min. After washing twice with Hank's balanced salt solution, the fluorescence of MitoSOX was detected at an excitation wavelength of 488 nm and an emission wavelength of 580 nm using a confocal fluorescence microscope (LSM700). Quantification of the image was performed using NIH Image J Software. 2.10. Measurement of intracellular NADþ SH-SY5Y cells were seeded in a 24-well plate at a density of ~2 105 cells/well. The next day, cells were treated with serumfree DMEM (1 mL) containing PAM (50 mL) for 1 h. After the treatment with PAM, cells were cultured in the growth medium for another 1.5 h. Intracellular NADþ levels were measured using the EnzyChrom NADþ/NADH assay kit (BioAssay Systems, Hayward, CA). The treated cells were washed twice with cold PBS, and then lysed with the supplied NADþ extraction buffer. NADþ was extracted from the lysate according to the manufacturer's protocol. The protein contents in the lysate were determined using Bio-Rad protein assay reagent.
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Previous reports have demonstrated that PAM triggers cell injuries in various tumor cells such as A549 cells and glioblastoma [3,5]. First, to elucidate the effect of PAM on the viability of human neuroblastoma SH-SY5Y cells, we exposed cells to various doses of PAM for 1 h and cell viability was measured 20 h later using MTT assay. As shown in Fig. 1, PAM induced cell death in a dosedependent manner. Recently, we and others showed that cell death caused by exposure to PAM is due to ROS included in PAM [5,6]. Therefore, to determine the involvement of ROS in PAMinduced SH-SY5Y cell death, we investigated the effect of the antioxidant N-acetylcysteine (NAC) on this phenomenon. Consistent with previous reports, NAC protected cells against PAMinduced cell death (Fig. 1). This result indicates that ROS included in PAM might participate in the cytotoxicity elicited by exposure to PAM. 3.2. Involvement of intracellular Zn2þ in PAM-induced cell death in SH-SY5Y cells In general, most intracellular Zn2þ exists in a complex with Zn2þ-binding proteins. However, it has been reported that oxidative stress caused by ROS and RNS stimulates the release of Zn2þ from intracellular Zn2þ stores such as MT, leading to Zn2þ-dependent cell death [13,14,25]. Therefore, SH-SY5Y cells were exposed to PAM in the presence of the cell-permeable Zn2þ chelator TPEN in order to determine whether Zn2þ is involved in PAM-induced cell death. As shown in Fig. 2A, TPEN dose-dependently suppressed PAM-induced cell death. To test cytotoxic effects of PAM in other cell lines, human lung (A549) and breast (MCF-7) cancer cells were exposed to PAM in the same way. PAM elicited cell death and TPEM prevented PAM cytotoxicity in both cells (Fig. 2B). However, SHSY5Y cells were more sensitive to PAM compared to A549 and MCF-7 cells. Preincubation of TPEN with an equimolar concentration of Zn2þ (Zn-TPEN) abolished the protective effect of TPEN on PAM-induced
2.11. Measurement of intracellular ATP SH-SY5Y cells were seeded in a 24-well plate at a density of ~2 105 cells/well. The next day, cells were treated with serumfree DMEM (1 mL) containing PAM (50 mL) for 1 h and then cultured in the growth medium for another 3 h. Cells were washed twice with ice-cold PBS, harvested, and then sonicated in PBS. Immediately following sonication, ATP was extracted with perchloric acid (final concentration 0.5 M). The extracts were neutralized and then centrifuged. The supernatants were diluted with 10 mM TriseHCl pH 7.8 (1:100), and then aliquots (10 mL) of the diluted samples were subjected to ATP measurement. Intracellular ATP levels were measured using the ENLITEN rLuciferase/ Luciferin Reagent (Promega, Madison, WI). The luminescence was detected using a luminometer. The protein contents in the lysate after sonication were determined using Bio-Rad protein assay reagent. 2.12. Statistics Data were analyzed using ANOVA followed by post hoc Bonferroni tests. A P value less than 0.05 was considered significant.
Fig. 1. PAM induces cell death in SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells were exposed to various doses of PAM (0, 2.5, 5, 10, and 20 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with or without NAC (2 mM) for 30 min, and NAC was present during PAM exposure at a final concentration of 1 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. untreated cells); ##, P < 0.01.
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cell death (Fig. 2C). TPEN is known to chelate other metals such as iron ion (Fe2þ). However, preincubation of TPEN with an equimolar concentration of Fe2þ (Fe-TPEN) protected against PAM cytotoxicity as well as TPEN (Fig. 2C). The affinities of TPEN for Zn2þ and Fe2þ are shown to be Ka ¼ 1015.58 M1 and 1014.61 M1, respectively [26]. These results indicated that cell death caused by PAM is attributable to intracellular Zn2þ. Zn2þ is an essential nutrient, whereas it has been reported that free Zn2þ is toxic to a variety of cells [17,27]. When SH-SY5Y cells were treated with ZnSO4 in the presence or absence of pyrithione, a specific Zn2þ ionophore, which rapidly allows Zn2þ to enter the cells, Zn2þ leaded to cell death in cells treated with ZnSO4 and pyrithione (Fig. 2D). Therefore, the accumulation of intracellular Zn2þ might be responsible for PAMinduced cell death of SH-SY5Y cells. On the other hand, oxidative stress has been shown to raise intracellular calcium (Ca2þ) via Ca2þ influx thought the activation of voltage-gated Ca2þ channels (VGCCs) [28] or Ca2þ release from intracellular pools [29]. To rule out the involvement of Ca2þ in PAM-induced cell death, SH-SY5Y cells were exposed to PAM in the presence or absence of verapamil, a VGCC inhibitor, or dantrolene, a ryanodine receptor inhibitor. As shown in Fig. 2E, these inhibitors failed to abolish PAM cytotoxicity. MT is a family of small cysteine-rich metal binding proteins and plays a major role in the regulation of intracellular Zn2þ homeostasis and heavy metal detoxification. Therefore, we hypothesized that newly synthesized apo-MT, which can sequester Zn2þ, improves Zn2þ perturbation elicited by PAM exposure and exerts a protective effect against PAM cytotoxicity. Cadmium (Cd2þ) is known to be a potent inducer of the MT gene. Indeed, treatment of SH-SY5Y cells with Cd2þ markedly increased MT mRNA and protein expression (Fig. 2F). As expected, PAM-induced cell death was suppressed in cells pretreated with Cd2þ (Fig. 2G). 3.3. PAM triggers intracellular Zn2þ liberation We next examined whether PAM causes the elevation of intracellular free Zn2þ ([Zn2þ]i) in SH-SY5Y cells. To monitor alterations of the liberated Zn2þ, we employed the fluorescent Zn2þ indicator FluoZin-3. SH-SY5Y cells loaded with FluoZin-3 were exposed to DTDP, a thiol oxidant, which is known to elicit Zn2þ liberation, or PAM. As expected, DTDP markedly increased the fluorescence intensity of FluoZin-3, indicating the rise of [Zn2þ]i (Fig. 3A). This elevation was rapidly abolished by the addition of TPEN. PAM also increased the fluorescence, but to a lesser extent than DTDP, and the addition of TPEN abolished PAM-induced augmentation of the fluorescence (Fig. 3A). These results indicate that PAM triggers Zn2þ
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liberation in SH-SY5Y cells. To determine where Zn2þ liberation occurs, we measured fluorescence intensities of FluoZin-3 (Zn2þ) and Hoechst 33324 (nucleus) within the cross-section of single cell after PAM exposure. As shown in Fig. 3B, the accumulation of free Zn2þ was observed in not only the cytoplasm but also the nucleus. Previously, it has been reported that ROS contribute to the liberation of Zn2þ from Zn2þ-binding proteins [10,11]. As described above, ROS included in PAM were closely related to PAM-induced cell death in SH-SY5Y cells. To elucidate whether PAM-containing ROS participate in the rise of [Zn2þ]i, cells loaded with FluoZin-3 were exposed to PAM in the presence or absence of NAC, followed by monitoring of the alteration in fluorescence intensity. As shown in Fig. 3C, NAC abolished PAM-induced elevation of [Zn2þ]i. 3.4. Involvement of PARP-1 in PAM-induced cell death There is evidence that various stresses such as oxidants and genotoxin cause the activation of PARP-1 and sequentially the accumulation of poly(ADP-ribose) in the nucleus [22]. Many studies have shown that overactivation of PARP-1 under severe stress conditions results in cell death [30e32]. To determine whether exposure to PAM stimulates PARP-1 activation in SH-SY5Y cells, we performed immunocytochemistry in order to detect the accumulation of poly(ADP-ribose) in the nucleus. As shown in Fig. 4A, PAM promoted the nuclear accumulation of poly(ADP-ribose), indicating that PAM stimulates PARP-1 activation. To clarify the relationship between Zn2þ and PARP-1 activation, we tested the effect of TPEN on PAM-induced PARP-1 activation. TPEN suppressed the nuclear accumulation of poly(ADP-ribose) (Fig. 4A). We next determined whether PAPR-1 activation is related to PAM-induced cell death. When SH-SY5Y cells were exposed to PAM in the presence of the PARP-1 inhibitor DPQ, this inhibitor prevented PAM-induced cell death (Fig. 4B). These results indicate that PARP-1 plays a critical role in such death. In addition, we also examined the effect of DPQ on PAM-induced Zn2þ liberation. As shown in Fig. 4C, however, DPQ failed to suppress the rise of [Zn2þ]i, suggesting that PAM-induced Zn2þ liberation occurrs upstream of PARP-1 activation. 3.5. PAM stimulates mitochondrial ROS generation It has been demonstrated that exposure to hydrogen peroxide triggers robust delayed generation of ROS in mitochondria and mitochondrial ROS cause PARP-1 activation [33]. To determine whether the exposure of SH-SY5Y cells to PAM elicits mitochondrial ROS generation, we assayed ROS production using a fluorescent probe for mitochondrial ROS, MitoSOX. As shown in Fig. 5A,
Fig. 2. Involvement of intracellular Zn2þ in PAM-induced cell death. A) Effect of TPEN on PAM cytotoxicity. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with different concentrations of TPEN (1.25, 2.5, 5, and 10 mM) for 20 min, and TPEN was present during PAM exposure at final concentrations of 0.63, 1.25, 2.5, and 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01; ##, P < 0.01 (vs. cells treated with PAM alone). B) Cytotoxic effects of PAM in A549 and MCF-7 cells. A549 and MCF-7 cells were exposed to PAM (15 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with TPEN (10 mM) for 20 min, and TPEN was present during PAM exposure at a final concentration of 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with PAM alone). C) Effect of Zn-TPEN on PAM cytotoxicity. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for a further 20 h. Zn-TPEN and Fe-TPEN were prepared by incubation of TPEN (10 mM) with equimolar concentrations of ZnSO4 and FeCl2, respectively. Cells were pretreated with or without TPEN, Zn-TPEN, or Fe-TPEN for 20 min, and they were present during PAM exposure at a final concentration of 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control without PAM); ##, P < 0.01 (vs. cells treated with PAM alone); yy, P < 0.01. D) Zn2þ toxicity toward SH-SY5Y cells. SHSY5Y cells were treated with ZnSO4 (20 mM) in the presence or absence of pyrithione (Pyri, 1 mM) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with Zn2þ alone). E) No involvement of Ca2þ in PAM-induced cell death. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for another 20 h. Cells were pretreated with verapamil (Ver, 20 mM) or dantrolene (Dan, 20 mM) for 30 min, and these inhibitors were present during PAM exposure at a final concentration of 10 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. cells treated with PAM alone). F) Cd2þ induces MT expression in SH-SY5Y cells. SH-SY5Y cells were treated with or without CdCl2 (5 mM) for 1.5 h, followed by culture of the cells in the growth medium for 6 h. After the treatment, RT-PCR and Western blotting were performed. These results are representative of three independent experiments. G) Overexpression of MT protects against PAM-induced cell death. SH-SY5Y cells pretreated with or without CdCl2 (5 mM) for 1.5 h, followed by culture of the cells in the growth medium for 8 h. The Cd2þ-treated cells were exposed to PAM (10 mL/100 mL) for 1 h, followed by culture of the cells in the growth medium for a further 20 h. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. control without Cd2þ); ##, P < 0.01.
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Fig. 3. A) PAM-induced Zn2þ liberation. SH-SY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) for 20 min. After washout, serum-free DMEM (1 mL) was added to the dish. The cells were exposed to DTDP (final conc. 100 mM) or PAM (500 mL). Fifteen minutes later, TPEN was added to the dish at a final concentration of 10 mM. The fluorescence was detected using a confocal microscope. This result is representative of three experiments. B) Distribution of intracellular Zn2þ after PAM exposure. Upper panel; SH-SY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) and Hoechst 33342 (1 mg/mL) for 20 min. After washout, serum-free DMEM (1 mL) was added to the dish, followed by the exposure to PAM (500 mL). Fifteen minutes later, the fluorescence was detected using a confocal microscope. Bar ¼ 10 mm. Lower panel; fluorescence profile of the cross-section indicated by red arrow. The solid and dashed lines correspond to FluoZin-3 (Zn2þ) and Hoechst 33342 (nucleus), respectively. C) Effect of NAC on PAM-induced Zn2þ liberation. SHSY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) and NAC (1 mM) for 30 min. After washout, serum-free DMEM (1 mL) was added to the dish. The cells were exposed to PAM (500 mL) in the presence or absence of NAC (1 mM) for 15 min. The fluorescence was detected using a confocal microscope. Values are means ± SD of 15 cells in a single experiment. This result is representative of three experiments. **, P < 0.01; ##, P < 0.01 (vs. cells treated with PAM alone). NS, not significant.
exposure to PAM increased the intensity of MitoSOX fluorescence. The ROS generation was suppressed in the presence of TPEN (Fig. 5A). In addition, we examined the effect of DPQ on PAMinduced ROS generation in mitochondria. Unexpectedly, DPQ also prevented mitochondrial ROS generation (Fig. 5A). Moreover, to verify the involvement of mitochondria in PAM-induced ROS generation, SH-SY5Y cells were exposed to PAM in the presence of a low concentration of rotenone, a mitochondrial complex I inhibitor. As shown in Fig. 5B, rotenone reduced mitochondrial ROS generation caused by PAM. 3.6. PAM causes depletion of intracellular NADþ and ATP It is well known that PARP-1 consumes NADþ to form poly(ADPribose). Therefore, marked PARP-1 activation induced by severe stress causes energy supply exhaustion, leading to cell death. We examined whether the exposure to PAM influences the contents of intracellular NADþ and ATP. As shown in Fig. 6A, PAM exposure caused declines in intracellular NADþ and ATP levels in SH-SY5Y cells. We next examined the contribution of Zn2þ to PAM-induced NADþ and ATP depletions. TPEN ameliorated the reductions of NADþ and ATP levels (Fig. 6A). It has been reported that supplementation of an NADþ precursor, nicotinamide, and a TCA cycle
substrate, pyruvate, prevents PARP-1-mediated cell death [23,24]. Therefore, we hypothesized that energy substrates such as nicotinamide and pyruvate confer protection against PAM-induced cell death. As expected, these agents reduced PAM-induced cytotoxicity (Fig. 6B). 4. Discussion PAM contains various kinds of ROS, such as hydrogen peroxide, and decomposition products of RNS, such as nitrite and nitrate. Recently, we have demonstrated that ROS included in PAM, especially hydrogen peroxide, are responsible for PAM-induced cell death in A549 cells [5]. In this study, we found that PAM induced a rise of [Zn2þ]i in SH-SY5Y cells and that liberated Zn2þ elicited PARP-1 activation, mitochondrial ROS generation, and intracellular NADþ and ATP loss, and ultimately led to Zn2þ-dependent cell death. There is evidence that oxidative stress triggers Zn2þ release from 2þ Zn -binding proteins including MT [10,11]. Although most intracellular Zn2þ is coordinated to cysteine thiols and imidazole nitrogen atoms of histidine in Zn2þ-binding sites, oxidative thiol modification allows the release of Zn2þ from these proteins. Previous reports have demonstrated that ROS/RNS cause intracellular
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Fig. 4. PAM-induced PARP-1 activation is Zn2þ-dependent. A) Effect of TPEN on PAM-induced PARP-1 activation. SH-SY5Y cells growing on a coverslip were exposed to PAM (50 mL/ 500 mL) in a 4-well plate for 1 h. Cells were pretreated with TPEN (10 mM) for 20 min, and TPEN was present during PAM exposure. After the exposure, the cells were fixed, and immunocytochemistry was performed using anti-poly(ADP-ribose) antibody. The nucleus was stained with Hoechst 33342. The fluorescence was visualized using a confocal microscope. Bar ¼ 20 mm. PAR; poly(ADP-ribose). B) Effect of PARP-1 inhibitor on PAM cytotoxicity. SH-SY5Y cells were exposed to various doses of PAM for 1 h, followed by culture of the cells in the growth medium for a further 20 h. Cells were pretreated with or without DPQ (10 mM) for 30 min, and DPQ was present during PAM exposure at a final concentration of 5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. **, P < 0.01 (vs. untreated cells); ##, P < 0.01. C) Effect of PARP-1 inhibitor on PAM-induced Zn2þ liberation. SH-SY5Y cells (35-mm dish) were incubated with FluoZin-3 AM (1 mM) and DPQ (5 mM) for 30 min. After washout, serum-free DMEM (1 mL) with or without DPQ was added to the dish. The cells were exposed to PAM (500 mL). Fifteen minutes later, TPEN was added to the dish at a final concentration of 10 mM. DPQ was present at a final concentration of 5 mM prior to and during PAM exposure. This result is representative of three experiments.
Zn2þ liberation in neurons or other cell types, leading to cytotoxicity [13e15]. We have more recently demonstrated that ROS are important mediators in PAM-induced cell death [5]. These findings prompted us to determine the contribution of intracellular free Zn2þ to PAM-induced cell death. Herein, we showed that Zn2þ chelation using TPEN protected against PAM cytotoxicity (Fig. 2A). In addition, overexpression of MT induced by Cd2þ conferred protection against PAM-induced cell death (Fig. 2G). Most, if not all, of
newly synthesized thionein (the apo-form of MT) has the ability to sequester Zn2þ. On the other hand, however, since MT is also known to be an antioxidant, it could also function as a radical scavenger. These results suggest that Zn2þ liberated by PAM exposure is an important mediator of PAM-induced cell death. Actually, Zn2þ imaging using FluoZin-3 revealed that PAM elevated [Zn2þ]i in SH-SY5Y cells. The rise in [Zn2þ]i caused by PAM exposure was observed in cells bathed in serum-free DMEM (Fig. 3A).
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Fig. 5. PAM stimulates mitochondrial ROS generation. A) Effect of TPEN and DPQ on PAM-induced ROS generation. SH-SY5Y cells (35-mm dish) were incubated with TPEN (10 mM) or DPQ (10 mM) for 30 min in serum-free DMEM (1 mL) and subsequently PAM (100 mL) was added to the dish. One hour later, the cells were stained with MitoSOX (5 mM) for 20 min, and then the fluorescence was observed using a confocal microscope. TPEN or DPQ was present during PAM exposure. Values are means ± SD of 25 cells in a single experiment. This result is representative of three experiments. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with PAM alone). Bar ¼ 100 mm. B) Effect of rotenone on PAMinduced ROS generation. SH-SY5Y cells (35-mm dish) were incubated with TPEN (10 mM) or rotenone (Rote; 9 nM) for 30 min in serum-free DMEM (1 mL) and subsequently PAM (100 mL) was added to the dish. One hour later, the cells were stained with MitoSOX (5 mM) for 20 min, and then the fluorescence was observed using a confocal microscope. Rotenone was present during PAM exposure. Values are means ± SD of 25 cells in a single experiment. This result is representative of three experiments. **, P < 0.01 (vs. control); ##, P < 0.01 (vs. cells treated with PAM alone). Bar ¼ 100 mm.
Since DMEM does not contain Zn2þ, we could rule out the possibility that Zn2þ sources are present in the extracellular space. Moreover, we found that the accumulation of [Zn2þ]i was observed in the nucleus as well as in the cytoplasm of SH-SY5Y cells exposed to PAM. Berendji et al. [34] also demonstrated that the treatment of mouse fibroblast L929 cells with RNS triggers Zn2þ release all over the cell. These findings suggested the possibility that PAM causes oxidative modification of cysteine residues in not only cytosolic proteins such as MT but also nuclear proteins such as zinc fingercontaining transcription factors. Consistent with previous studies using other oxidants [13,25], the rise of [Zn2þ]i also occurred immediately after PAM exposure (within 15 min) under our experimental conditions. Therefore, we consider that liberated Zn2þ acts as an important initial signal in the PAM-induced cell death process. Intracellular Zn2þ has been shown to stimulate various signaling pathways such as the MAPK (p38 and ERK) pathway in various cell types [21,27,35,36]. However, under our experimental conditions, p38 and ERK pathway inhibitors had no effect on PAM-induced cytotoxicity (data not shown). Therefore, the MAPK pathway is unlikely to be involved in PAM-induced cell death in SH-SY5Y cells. It has been demonstrated that oxidative stress-induced DNA damage activates PARP-1, leading to PARP-1-dependent cell death
[22]. Therefore, the activation of PARP-1 is thought to be closely related to the cell death process. When cortical neurons are exposed to excessive Zn2þ, PARP-1-dependent neurotoxicity has been shown to occur [23,24]. In this study, we found that the exposure of SH-SY5Y cells to PAM elicited PARP-1 activation. Additionally, PARP-1 inhibitors prevented PAM-induced cell death. Our present results suggest that PARP-1 plays a key role in PAM cytotoxicity. The activation of PARP-1 was inhibited in the presence of TPEN, whereas PARP-1 inhibitor DPQ did not affect the PAMinduced rise of [Zn2þ]i. Therefore, Zn2þ is likely to function as an upstream regulator for PARP-1 activation. Unfortunately, however, the details of how Zn2þ activates PARP-1 are currently unknown. On the other hand, it has been reported that excessive Zn2þ influx increases ROS production in neurons [18e20]. We found that PAM stimulated ROS generation in mitochondria and TPEN suppressed the generation of mitochondrial ROS (Fig. 5). Therefore, it is likely that the liberated Zn2þ modulates PARP-1 activation via ROS signaling. Unexpectedly, however, PARP-1 inhibitors as well as TPEN decreased PAM-induced ROS generation in SH-SY5Y cells. PARP-1 activation has also been shown to induce ROS production, leading to cell injury [37,38]. Thus, we cannot rule out the possibility that mechanisms other than ROS are involved in PARP-1 activation following Zn2þ liberation.
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energy supply exhaustion caused by Zn2þ liberation and subsequent PARP-1 activation. Meanwhile, Zn2þ has been shown to inhibit GAPDH, which is a key enzyme in glycolysis [17,24]. Therefore, not only PARP-1 activation but also GAPDH inhibition might be involved in PAM-induced cytotoxicity. In conclusion, we demonstrated here that Zn2þ released from intracellular sources serves as a key mediator of PAM-induced cell death in SH-SY5Y cells. Our findings suggest that the conversion of PAM stimulus to a Zn2þ signal is a critical step in the PAM-induced cell death process. Therefore, it is likely that intracellular Zn2þ contents affect the efficacy of plasma irradiation against cancer therapy. At present, however, how liberated Zn2þ activates its downstream signaling is not fully understood. Thus, further investigations are needed to understand Zn2þ signaling in the PAMinduced cell death process. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (to H.H.; No. 26460630) and in part by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (to T.A.; No. 25108511). References
Fig. 6. PAM elicits depletion of intracellular NADþ and ATP. A) Effect of PAM on intracellular NADþ and ATP levels. SH-SY5Y cells (24-well plate) were exposed to PAM (50 mL/500 mL) for 1 h in serum-free DMEM. One and a half (NADþ) and three (ATP) hours after the exposure, intracellular NADþ and ATP contents were measured. Cells were pretreated with TPEN (10 mM) for 20 min, and TPEN was present during PAM exposure. **, P < 0.01 (vs. control); ##, P < 0.01. B) Effect of nicotinamide and pyruvate on PAM-induced cell death. SH-SY5Y cells were exposed to PAM (10 mL/100 mL) for 1 h in serum-free DMEM, followed by culture of the cells in the growth medium for a further 20 h. Cells were pretreated with vehicle (open columns), nicotinamide (3 mM; hatched columns), or pyruvate (3 mM; gray columns) for 30 min, and they were present during PAM exposure at a final concentration of 1.5 mM. Cell viability was measured with an MTT assay. Values are means ± SD from four separate cultures. #, P < 0.05; ##, P < 0.01 (vs. cells treated with PAM alone).
PARP-1 activation consumes intracellular NADþ to form poly(ADP-ribose), leading to energy supply exhaustion. PAM decreased the intracellular levels of NADþ and ATP in SH-SY5Y cells. This result is consistent with the findings of our previous study using A549 cells. The addition of energy substrates such as nicotinamide and pyruvate rescued PAM-induced cell death (Fig. 6B). On the other hand, pyruvate itself is known to have a radical scavenging effect [39]. This might be the reason why pyruvate was more protective against PAM cytotoxicity than nicotinamide. TPEN ameliorated PAM-induced depletion of intracellular NADþ and ATP. These findings suggest that PAM-induced cell death is due to
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