Sodium pyruvate protects against H2O2 mediated apoptosis in human neuroblastoma cell line-SK-N-MC

Journal of Chemical Neuroanatomy 26 (2003) 109 /118 www.elsevier.com/locate/jchemneu

Sodium pyruvate protects against H2O2 mediated apoptosis in human neuroblastoma cell line-SK-N-MC Jayashree C. Jagtap a, Anmol Chandele a, B.A. Chopde b, Padma Shastry a,* a

Department of Biotechnology, National Center for Cell Science, Government of India An Autonomous Institution, NCCS Complex, Pune University Campus, Ganeshkhind, Pune 411007, India b Department of Microbiology, Pune University, NCCS Complex, Ganeshkhind, Pune 411007, India Received 14 May 2002; received in revised form 12 February 2003; accepted 12 April 2003

Abstract Free radicals are involved in neuronal damage. The present study was aimed to investigate the protective effect of sodium pyruvate */a free radical scavenger against hydrogen peroxide (H2O2) induced apoptosis in human neuroblastoma cell line-SK-NMC. On exposure to H2O2 (0.025 mM) cells exhibited apoptosis within 24 h, demonstrating a high caspase 3 activity by 3 h followed by cleavage of PARP that was maximum at 24 h. A break down in the mitochondrial membrane potential was observed 3 h onwards. Sodium pyruvate protected cells significantly (P B/0.05) against apoptosis in a dose dependent manner as assessed for cell viability by dye exclusion method and apoptosis by TUNEL. Sodium pyruvate significantly inhibited caspase 3 activity, cleavage of PARP and breakdown of mitochondrial membrane potential. These data suggest that sodium pyruvate protects neuronal damage caused by H2O2. # 2003 Elsevier B.V. All rights reserved. Keywords: Neuroprotection; Caspase 3; PARP; Mitochondrial transmembrane potential

1. Introduction Nerve cell death is the central feature of the human neurodegenerative diseases. Apoptosis or programmed cell death is morphologically distinct and genetically controlled form of cell death. This includes shrinkage of the cell, nuclear condensation, DNA fragmentation and the formation of nuclear and cytoplasmic fragments known as apoptotic bodies, which are subsequently phagocytozed by cells like the macrophages (Raff, 1998). Recent evidences indicate that it is the process of apoptosis rather than necrosis which primarily contributes to nerve cell death in neurodegeneration (Tatton and Olanow, 1999). Programmed cell death or apoptosis can be triggered by a variety of stimuli (Wertz and Hanely, 1996; Jabs, 1999), which include reactive oxygen species (ROS) like superoxide anions and * Corresponding author. Tel.: /91-20-569-0931/41/51/61/22; fax: / 91-20-569-2259. E-mail addresses: [email protected], [email protected] (P. Shastry). 0891-0618/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0891-0618(03)00037-1

hydrogen peroxide (Zettl et al., 1997; Jabs, 1999). Cellular antioxidants act in concert to detoxify these species but when the balance is disrupted, a condition referred to as oxidative stress is induced. It has been demonstrated that oxidative stress plays an important role as a mediator of apoptosis (Hivert et al., 1998; Skaper et al., 1998). Hydrogen peroxide, a byproduct of oxidative stress has been implicated to trigger apoptosis in various cell types (Clement et al., 1998; Kitamura et al., 1999; Palomba et al., 1999) leading to major neurodegenerative diseases */Alzheimer’s disease (AD), Parkinson’s disease (PD) and amylotrophic lateral sclerosis (ALS). Recently it has been shown that the amyloid b peptide (Ab) produces hydrogen peroxide (H2O2) through metal ion reduction in AD (Huang et al., 1999a,b). Hydrogen peroxide is a useful agent for inducing oxidative stress in mammalian cells because it has a low reactivity and can easily penetrate cellular membranes causing further ROS production within the cells (Janseen et al., 1997; Li et al., 1997). Cells have numerous protective mechanisms that maintain the concentrations

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of ROS within a range compatible for survival. The brain can efficiently metabolize superoxides but may have difficulties in eliminating the hydrogen peroxide produced. It has been hypothesized that some trophic factors may upregulate neuronal antioxidants (Pan and Perez-Polo, 1993; Jackson et al., 1994; Mattson and Furukawa, 1996). Sodium pyruvate is a natural oxidant scavenger abundantly present in mammalian cells (Salahudeen et al., 1991; Shostak et al., 2000). We investigated the protective effect of sodium pyruvate on apoptosis induced by H2O2 using human neuroblastoma cells as a model (Shastry et al., 2001). In the present study, we also assessed the role of caspase 3 activity, expression of PARP and break down in mitochondrial membrane potential in H2O2 induced apoptosis.

2. Materials and methods 2.1. Cell cultures and reagents Human neuroblastoma cell line-SK-N-MC was obtained from American Type Culture Collection (Rockville, USA). Sodium pyruvate was procured from Sigma Chemicals (USA). The cells were cultured in Eagle’s minimum essential medium with Earle’s salt MEM (E) supplemented with 10% fetal bovine serum (FBS, Trace, Australia), penicillin (200 units/ml), and streptomycin (0.2 mg/ml) and maintained at 37 8C in a humidified atmosphere with 5% CO2. Cells were seeded at a density of 0.5 /106 cells/ml in 6-well plates, 1 /104 cells/100 ml in 96-well plates or 1.5 /106 cells/5 ml in 25 cm2 flasks for a period of 24 h before treatment. 2.2. Induction of apoptosis Hydrogen peroxide (H2O2 50% w/v) was used to induce apoptosis in SK-N-MC cells. The cells were treated with serial concentrations of H2O2 (0.005/0.05 mM) prepared in MEM (E) containing 10% FBS for different time periods up to 24 h.

2.4. Determination of apoptosis by flow cytometry Untreated control and H2O2 treated cells were harvested at the respective time points and washed with PBS (1 /). Cell pellets were fixed with 70% chilled ethanol for 30 min at 4 8C. The ethanol was washed thoroughly and the cells were resuspended in 5 mg/ml RNase A at RT for 20 min followed by 450 ml of 50 mg/ ml propidium iodide and incubated in dark at RT for 2 h. The PI stained cells were acquired and analyzed by FACS vantage equipped with a 488 nm argon laser. 2.5. DNA fragmentation by TUNEL assay Terminal deoxynucleotidal transferase mediated dUTP-biotin nick end labeling (TUNEL) assay was performed with FragEL DNA fragmentation detection kit (Oncogene research). The untreated control and H2O2 treated cells were harvested at appropriate time intervals and fixed with 70% ethanol. The cells were washed and rehydrated with TBS (1 /). The cells were equilibrated for 30 min with 1 / equilibration buffer provided with the kit. The labeling reaction was carried out at 37 8C for 3 h. The cells were washed and analyzed on FACS vantage. The percentage of TUNEL positive cells was scored with respect to the untreated control cells. 2.6. Mitochondrial transmembrane potential The mitochondrial potential of untreated and treated cells was measured using Mitocapture Apoptosis Detection Kit (Oncogene Research) by flow cytometry. The cells were harvested and washed with 1/ PBS. The cell pellet was resuspended in MitocaptureTM dye dissolved in incubation buffer (1 ml/ml). After incubation with 5% CO2 at 37 8C for 20 min, the cells were centrifuged and resuspended in incubation buffer for flowcytometric analysis using FL-1 parameter for disruption of mitochondrial membrane potential. 2.7. Caspase 3 activity colorimetric assay

2.3. Determination of cell viability The loss of membrane integrity was determined by the inability of cells to exclude the vital dye-trypan blue. Cells (0.5 /106/ml) were plated in 6-well plate and treated with H2O2 at different concentrations for 24 h and the viability was assessed by trypan blue dye exclusion method. The cells were dislodged and viable cells were counted by diluting the cell suspension 1:2 with trypan blue (0.4%). The percent viability was calculated with respect to untreated control cells that were considered to be 100%.

The lysates of untreated control and H2O2 treated cells were prepared in cell lysis buffer (10 mM Tris /HCl, 10 mM NaH2PO4 pH 7.5, 130 mM NaCl, 1% Triton X100 and 10 mM NaPPi). Caspase 3 activity was determined by caspase 3 colorimetric assay kit (Oncogene Research) according to the manufacturer instructions. The cleavage of substrate DEVD-pNa by caspase 3 was measured as an increase in the absorbance at 405 nm. The results were expressed as fold increase in per unit caspase 3 activity compared with that of the positive control provided by the kit.

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2.8. SDS-PAGE and Western blotting for PARP cleavage Cells were lysed at 4 8C using cell lysis buffer (Tris pH 7.5, 1 M NaCl, 1% Triton X 100, 0.5 M EDTA, 0.1% sodium azide, 10 mM sodium fluoride with 1 / protease inhibitory cocktail containing 1.6 mg/ml Benzamidine HCl, 1 mg/ml phenanthroline, aprotinin, leupeptin, pepstatin and PMSF). The lysates were sonicated and centrifuged at 12 000 rpm for 10 min and the resultant supernatant was stored at /70 8C. The 20 mg of cell lysates was separated on 12.5% SDS-PAGE and electroblotted onto a nitrocellulose membrane. The blots were blocked with 3% BSA at RT for 1 h followed by incubation with an appropriate dilution of the antiPARP rabbit polyclonal antibody (Santa Cruz Biotechnology) at RT for 2 h. The blots were washed and incubated with HRP labeled secondary antibody at RT for 1 h. Enhanced chemiluminescence (ECL) plus kit (Amersham) detected the protein of interest. 2.9. Statistical analysis The degree of significance in the groups was evaluated with one way ANOVA. A P -value of B/0.05 was considered significant.

3. Results 3.1. Cell viability Initial experiments were carried out to study the dose dependent effect of H2O2 on the cell viability in SK-NMC cells using concentrations ranging from 0.005 to 0.05 mM. A significant decrease in cell viability was observed by trypan blue dye exclusion method when treated for 24 h with 0.025 mM H2O2. A concentration of 0.05 mM was toxic (/99% dead cells) and lower concentrations had no effect on cell survival (Fig. 1A). 3.2. Determination of apoptosis To confirm whether H2O2 induced cell death was due to apoptosis, cells were stained with propidium iodide and the extent of apoptosis was measured. The hypodiploid population was considered apoptotic and DNA content (DNA fragmentation) was quantified by flowcytometry. The results showed that no apoptosis was induced with lower concentrations (0.005 and 0.01 mM) H2O2. A significant increase in apoptotic population is demonstrated in cells treated with 0.025 mM (42.19/ 11.6, P B/0.005) (Fig. 1B). With 0.05 mM H2O2 cells showed very high level of apoptosis, confirming that the dose was toxic to cells. All further experiments were done using 0.025 mM concentration.

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The time period required for induction of apoptosis was studied by treating SK-N-MC cells for different time periods from 6 to 24 h with 0.025 mM H2O2 and cell viability was measured. Data analyzed from these experiments showed a significant decrease (P B/0.05) in viability with treatment as early as 9 h (Fig. 2). 3.3. Protective effect of sodium pyruvate against H2O2 induced apoptosis We assessed the effect of a free radical scavenger */ sodium pyruvate (used in range of concentrations, 0.125 /1.0 mM) on apoptosis induced by exposure of cells to H2O2 for 24 h. The cell viability was determined by dye exclusion method. Significant protection (P B/ 0.05) against cell death induced by H2O2 was seen with all doses of sodium pyruvate used in the experiments. The effect was dose dependent (Fig. 3A) and almost 100% protection was achieved with 1 mM treatment. The protective effect of sodium pyruvate was also assessed by flowcytometry. The cells were stained with propidium iodide and the hypodiploid cells were gated as the apoptotic population. It was observed that 0.5 and 1 mM sodium pyruvate protected the cells against H2O2 mediated apoptosis (P B/0.05). The percent apoptotic cells (mean9/S.E.M.) with 0.5 and 1 mM were 15.989/3.085 and 5.349/0.47, respectively, compared with 33.59/5.94 with H2O2 alone. Fig. 3B shows the panel of a representative data. The result for apoptosis was confirmed by TUNEL assay which is considered more sensitive as compared with propidium iodide staining. In this assay, DNA fragments generated by apoptosis are detected by the addition of fluorescein isothiocyanate (FITC) labeled deoxynucleotides by the TdT enzyme at the 3? OH end. The percent cells positive are determined as the measure of the extent of DNA fragmentation. As our time kinetic study showed that cell death was seen as early as 9 h following H2O2 treatment and as apoptotic cells are TUNEL positive before cell viability is lost, the experiments were done with treatment of cells for 9 h with H2O2 alone or in combination with sodium pyruvate. We observed 59.34% cells were FITC positive with H2O2 alone which decreased to 15.4% when cells were treated in combination with sodium pyruvate (1 mM), confirming the protective effect of sodium pyruvate (Fig. 4). 3.4. Detection of mitochondrial transmembrane potential The mitochondria are regarded as key player in the execution of the cell death machinery and form the central point during cell death. One of the parameters that are altered during apoptosis is the transmembrane potential of the mitochondria. We, therefore, performed experiments to investigate the mechanism of protection by sodium pyruvate. The effect of H2O2 alone and in

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Fig. 1. (A) The effect of different concentrations of H2O2 (0.005 /0.05 mM) on SK-N-MC cells treated for 24 h was determined by trypan blue dye exclusion method. Viability in controls was considered 100%. A significant reduction (P B/0.001) in cell viability is seen with 0.025 mM H2O2. The data is represented as mean9/S.D. of three independent experiments. (B) Induction of apoptosis by H2O2 by flow cytometry. SK-N-MC cells were treated with different concentrations of H2O2 (0.005 /0.05 mM) for 24 h. The panel shows (a) control cells, (b) /(f) cells treated with H2O2 at concentrations 0.005, 0.001, 0.02, 0.025 and 0.05 mM, respectively. X -axis is FL-2 region, which shows percent apoptotic cells on the markers representing subdiploid DNA content. The panel is representative of three similar experiments.

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Fig. 2. SK-N-MC cells were treated for 6, 9, 12, 24 h with 0.025 mM H2O2 and cell viability was determined at these time points by dye exclusion method. A significant decrease in percent cell viability is seen with 9 h treatment onwards (P B/0.05). Data is mean9/S.E.M. from three separate experiments.

combination with sodium pyruvate was studied using MitocaptureTM dye. The status of the membrane potential was determined by studying the aggregation of the MitocaptureTM dye in the mitochondria when reduced by mitochondrial enzymes. SK-N-MC cells treated with H2O2 alone and in combination with sodium pyruvate were harvested at different time periods */1, 3, 5, 6, 9 and 12 h post treatment (data not shown). A significant difference (P B/0.01) in the mean fluorescence intensity (MFI) between cells treated with H2O2 alone and in combination with sodium pyruvate was seen at 5 h (Fig. 5). This data suggested that sodium pyruvate protected the cells against H2O2 induced apoptosis by preventing the break down in transmembrane potential of the mitochondria.

3.6. Western blotting for PARP cleavage Poly (ADP-ribose) polymerase plays a pivotal role in maintaining genomic integrity. During apoptosis, many proteins undergo degradation by caspases. Among them the first protein described to be proteolyzed was PARP, which is converted from the 116-kD to fragments of 89 and 24-kDa (Schwartzman and Cidlowski, 1993). The cleavage of PARP on treatment with H2O2 alone and in combination with sodium pyruvate was studied by Western blotting. The cells treated with H2O2 alone showed a distinct band of cleaved PARP of 89 kD. The intensity of the cleaved fragment was higher with longer periods of treatment. Cells treated with sodium pyruvate in combination with H2O2 did not show the presence of the cleaved fragment (Fig. 7). The 89 kD was totally absent even at 24 h period demonstrating total protection by sodium pyruvate.

3.5. Detection of caspase 3 activity We next examined the status of caspase 3, a key protease in the execution of the apoptotic machinery. Caspase 3 cleaves a number of proteins that are essential for cell survival. The cleavage of DEVD-pNa and the release of chromophore group was assessed colorimetrically and units of active caspase 3 was calculated. The H2O2 treated SK-N-MC cells showed higher caspase 3 activity (39.49 units) as compared with the control cells (6.07 units). Treatment with sodium pyruvate induced protection in H2O2 treated cells and showed a decrease the caspase 3 activity to 8.73 units (Fig. 6). This data suggests the involvement of caspase 3 dependent pathway in H2O2 induced apoptosis in SK-N-MC cells.

4. Discussion In the present study we examined the potential of sodium pyruvate in protection against hydrogen peroxide induced apoptosis in human neuroblastoma cell lineSK-N-MC. The study also focused on determining the typical changes in the apoptotic cascade like the caspase 3 activity, cleavage of PARP and alterations in mitochondrial membrane potential during apoptosis induced by H2O2 in these cells. Hydrogen peroxide has been widely used as an inducer for apoptosis and necrosis in different cell types (Hampton and Orrenius, 1997). Different concentrations of H2O2 have been used in

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Fig. 3. (A) Inhibition of cell damage induced by H2O2 by sodium pyruvate (Pyru¥Na). Cells were treated individually (H2O2) or in combination with different concentrations (0.125 /1 mM) of Pyru¥Na for 24 h. Cell viability was determined by dye exclusion method. Significant protection against cell death is seen with Pyru¥Na (P B/0.05) in dose dependent manner. The data is mean9/S.E.M. from three independent experiments. (B) Dose dependent protective effect of sodium pyruvate studied by propidium iodide staining. (a) Control SK-N-MC cells. (b) Cells treated with H2O2 (0.025 mM) individually or in combination with. (c) /(f) increasing concentrations of sodium pyruvate (0.125, 0.25. 0.5 and 1 mM). The panel is a representative of three similar experiments.

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Fig. 4. A representative panel demonstrating inhibition of apoptosis by sodium pyruvate by TUNEL assay in SK-N-MC cells. (a) Control (b) Cells treated with 0.025 mM H2O2. (c) Cells treated with 0.025 mM H2O2 and 1 mM sodium pyruvate. Apoptosis induced was determined 9 h post treatment by the extent of percentage FITC positivity.

different cell types depending on the sensitivity of the cells (Jiang et al., 1996; Uberti et al., 1999). In retinal neuronal culture, 100 mM H2O2 was shown to induce apoptosis as assessed by TUNEL (Xin et al., 2001). In our experimental set up, we found 0.05 mM H2O2 toxic to SK-N-MC cells with almost 98% non-viable cells on 24 h exposure to H2O2, therefore, all experiments for apoptosis were conducted using 0.025 mM. The protective effect of sodium pyruvate per se is not new. The concentration of pyruvate required for protection varies, depending on the cell type, the apoptotic agent and the apoptotic pathways operative in the system (Gupta et al., 1998). In rat model of transient forebrain ischemia, pyruvate was used for protection against zinc neurotoxicity. The effect was persistent and had no side effects (Lee et al., 2001). In cultured

mesothelial cells, 2 mM sodium pyruvate prevented the negative effect of H2O2 (Shostak et al., 2000). In our experiments with SK-N-MC cells, we observed a dose dependent effect of sodium pyruvate on apoptosis induced by H2O2 and total protection was achieved with 1 mM sodium pyruvate as assessed by cell viability, estimation of apoptotic population by hypodiploidy region and TUNEL assay. Caspases are family of cysteine proteases that play an important role during apoptosis. Cleavage of PARP is one of the earliest detectable proteolytic events that occur following high molecular fragmentation of chromatin DNA. (Lazebnik et al., 1994; D’Amours et al., 1998). Though PARP can be cleaved by almost all caspases, caspase 3 has been identified as the caspase responsible for much of the activity (Yu et al., 2001).

Fig. 5. Effect of H2O2 treatment on mitochondrial transmembrane potential in SK-N-MC cells, expressed as MFI. (a) Control (b) Cells treated with 0.025 mM H2O2 alone (c) Cells treated with 0.025 mM H2O2 and 1 mM sodium pyruvate (Pyru¥Na). Mitochondrial transmembrane rupture was detected by the increase in MFI at FL-1. H2O2 treated cells showed an increase in MFI as compared with the untreated controls (considered as 100%) and decreased when treated in combination with sodium pyruvate (P B/0.01) The data is mean9/S.D. calculated from three experiments.

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Fig. 6. Inhibition of caspase 3 activity in H2O2 treated cells by sodium pyruvate. The cells were treated for 6 h with 0.025 mM H2O2 alone and in combination with 1 mM sodium pyruvate (Pyru¥Na). Cell lysates were prepared and caspase 3 was estimated by colorimetric assay. The high activity of caspase 3 observed in H2O2 treated cells was inhibited by sodium pyruvate. The data is representative of two similar experiments.

The sequential events in the apoptotic pathway suggest a defined cascade of proteolytic events. Secondly, ROI mediated DNA damage has been shown to cause the activation of poly-ADP-ribose transferase, step important in apoptosis (Schwartzman and Cidlowski, 1993). We, therefore, examined the effect of sodium pyruvate on caspase 3 activity and cleavage of PARP in cells exposed to H2O2. Caspase 3 activity showed 65% increase on 6 h exposure to H2O2 beginning at 3 h. Addition of sodium pyruvate along with H2O2 decreased the caspase 3 activity by 51%. The data on the kinetics of the enzyme activity are in line with an earlier report on Jurkat T lymphocytes treated with 50 mM H2O2 (Gobeil et al., 2001). The cleavage of PARP was analyzed in SK-N-MC cells treated with H2O2. We observed the appearance of the 89 kDa cleaved fragment as early as 3 h and the increase was most significant at 24 h. The data with PARP cleavage and caspase 3 activity suggests that H2O2 induced apoptosis is mediated by caspase 3 which then cleaves PARP. Further experiments confirmed that the protective effect of sodium pyruvate were by inhibition of caspase 3 activity resulting in no cleavage of the 116 kDa PARP. Our data with H2O2 corroborate the finding, demon-

strating increased caspase 3 activity and cleavage of PARP in a similar time dependent manner in HL-60 cells treated with comparable concentration of H2O2 (DiPietrantonio et al., 1999). There is increasing evidence for role of mitochondria in control of apoptosis induced by different stimuli. It is now well established that in cells undergoing apoptosis, events related to formation of apoptosome involving Apaf 1 and caspase 9 are initiated by release of cytochrome c , which is the result of increased permeability in mitochondrial membrane (Hu et al., 1999). Recent reports have also suggested that mitochondria could be a primary target of H2O2 induced apoptosis and that loss of membrane potential is an early event in the process (Takeyama et al., 2002). It was, therefore, of interest to examine the changes in the membrane potential in our model system with cells triggered to undergo apoptosis with H2O2 treatment. Interestingly, a significant loss in the membrane potential was seen by 5 h on exposure to H2O2. Further more, sodium pyruvate inhibited the loss in membrane potential in SK-N-MC cell undergoing apoptosis. This finding suggests that in SK-N-MC cells, mitochondria may be the target for the action of H2O2, which then initiates a cascade of events

Fig. 7. Inhibition of PARP cleavage by sodium pyruvate. SK-N-MC cells were treated with 0.025 mM H2O2 alone and in combination with 1 mM sodium pyruvate. The cells were harvested at 3, 6 and 24 h and PARP cleavage was determined by immunoblot analysis. Lane: 1 untreated controls; Lanes: 3, 5, 7, H2O2 alone, for 3, 6 and 24 h, respectively; and Lanes: 2, 4, 6 treated with combination of H2O2 and sodium pyruvate (1 mM).

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leading to DNA fragmentation and apoptosis. These data assume importance in the context that mitochondrial derived pathological free radicals have been implicated in numerous diseases and the aging process itself and that the human CNS is relatively deficient in oxidative defenses rendering it more susceptible to the ROS induced damage (Marklund et al., 1982; Martilla et al., 1988). In conclusion, sodium pyruvate may be useful as an efficient scavenger of hydrogen peroxide in future studies to determine the mechanism(s) of action of ROS and in development of treatment strategies for degenerative diseases.

Acknowledgements This work was carried out at NCCS supported by Department of Biotechnology, India. We thank Dr G.C. Mishra, Director, NCCS, Pune, for providing us with the necessary facilities and late Mr Atul Suple for his help in flow cytometric analysis.

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