Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells

Toxicology 256 (2009) 118–127

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Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells R. Jayaraj a,1 , Nimesh Gupta b , P.V. Lakshmana Rao a,∗ a b

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India Division of Virology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India

a r t i c l e

i n f o

Article history: Received 3 October 2008 Received in revised form 12 November 2008 Accepted 13 November 2008 Available online 25 November 2008 Keywords: Apoptosis Okadaic acid AIF pathway Caspase Mitochondrial membrane potential Protein phosphatase inhibitor

a b s t r a c t Okadaic acid (OA) is the major component of diarrhetic shell fish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. We investigated the signal transduction pathways involved in OA induced cell death in HeLa cells. OA induced cytotoxicity and apoptosis at IC50 of 100 nM. OA treatment resulted in time dependent increase in reactive oxygen species and depleted intracellular glutathione levels. Loss of mitochondrial membrane permeability led to translocation of bax, cytochrome-c and AIF from mitochondria to cytosol. The cells under fluorescence microscope showed typical apoptotic morphology with condensed chromatin, and nuclear fragmentation. We investigated the mitochondrial-mediated caspase cascade. The time dependent activation and cleavage of of bax, caspases-8, 10, 9, 3 and 7 was observed in Western blot analysis. In addition to caspase-dependent pathway AIF mediated caspase-independent pathway was involved in OA mediated cell death. OA also caused time dependent inhibition of protein phosphatase 2A activity and phosphorylation of p38 and p42/44 MAP kinases. Inhibitor studies with AcDEVO-CHO and Z-VAD-FMK could not prevent the phosphorylation of p38 and p42/44 MAP kinases. Our experiments with caspase inhibitors Ac-DEVD-CHO, Z-IETD-FMK and Z-VAD-FMK inhibited capsase-3, 8 cleavages but did not prevent OA-induced apoptosis and DNA fragmentation. Similarly, pretreatment with cyclosporin-A and N-acetylcysteine could not prevent the DNA fragmentation. In summary, the results of our study show that OA induces multiple signal transduction pathways acting either independently or simultaneously leading to apoptosis. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Okadaic acid (OA, C44 H44 O13 ) extracted from common black sponge Halachondria okaddai is a potent inhibitor of protein phosphatases, PP1 and PP2A (Cohen et al., 1990). OA is concentrated in shellfish and constitute the major toxins associated with diarrheic shellfish poisoning (DSP). The symptoms of DSP are usually selflimiting, subsiding in 1–3 days. However, these compounds still represent a risk to consumers in many parts of the world. This toxin is a potential threat to human health even at concentrations too low

Abbreviations: OA, okadaic acid; GSH, glutathione; PARP, poly(ADP-ribose) polymerase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; OPT, orthopththaldialdehyde; PBS, phosphate buffered saline; DCF-DA, 2,7-dichlorofluorescein diacetate; CHAPS, (3[(3-cholamidopropyl) dimethlylammonio]-1-propanesulfate); NAC, N-acetylcysteine; CsA, cyclosporin-A; MAPK, mitogen-activated protein kinase; LDH, lactate dehydrogenase. ∗ Corresponding author. Fax: +91 751 2341148. E-mail address: [email protected] (P.V.L. Rao). 1 Present address: Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Centre, Houston, TX 77030, USA. 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.11.013

to induce acute toxicity, since it has been reported as a potent tumor promoter and it could also be an initiator (Creppy et al., 2002). OA has been shown to possess tumor-promoting activity in several models of two-stage carcinogenesis (Fujiki and Suganuma, 1993). Failure to accurately undergo apoptosis can cause severe anomalies, ranging from autoimmune disease to cancer. Tumor promotion and apoptosis represent long term and acute effects of okadaic acid in cells and tissues. As there is a delicate balance in the cell between cell survival and death, any protein with a role in cell proliferation might also play a role in apoptosis if it is stimulated or inhibited (Santoro et al., 1998). There are conflicting reports on effect of okadaic acid on both cell proliferation and apoptosis in many cell types (Rossini, 2000). There are numerous cell death mechanisms that are tissuespecific and cell type-specific. These are regulated by large number of extra cellular and intracellular signals governed by the environment of the cell. Mitochondria play an important role in integration and transmission of death signals, activating caspases and other cell death execution events (Green, 2005). Two apoptotic pathways are relatively well understood at the molecular level: the intrinsic or mitochondrial pathway and extrinsic pathway. Activation of intrinsic pathway involves release of pro-apoptotic factors like

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cyt-c, Smac/Diablo, Endonuclease G and AIF from the mitochondrial intermembrane space, which amplifies the apoptotic cascade to the final destruction of the cell (Kinnally and Antonsson, 2007). The extrinsic and intrinsic pathways initially appeared to be independent. However, it is now clear that a cross talk exists between the two pathways, which are mediated by the BH3-only protein Bid (Li et al., 1998). Both extrinsic and the intrinsic routes to apoptosis ultimately lead to cell shrinkage, chromatin condensation, nuclear fragmentation, blebbing and phosphatidylserine exposure on the surface of the plasma membrane. Mitogen-activated protein kinases (MAPKs) are important intermediates in signalling pathways that transduce extra cellular stimulation into intracellular responses. MAPKs have been implicated in a number of physiological processes including cell growth, differentiation, and apoptosis (Yang et al., 2000). Based on the structural differences, the MAPK family has been classified into three major subfamilies; the extra-cellular signal-regulated (ERK), the Cjun N terminal kinase (JNK) and the p38 kinase. JNK and p38 are activated in response to chemical and environmental stress. The balance between ERK1/2 pathway and stress activated SAP/JNK and p38 MAPK pathways have been proposed to be a fundamental determinant of cell survival or apoptosis (Honkanen and Golden, 2002; Van Hoof and Goris, 2003). It has long been recognized that OA exerts its cellular effects by binding and inhibiting type 1 and 2A ser/thr protein phosphatases. OA is known to induce apoptosis in many cell types like intestinal cells, neuronal, leukemic cells, etc. (Leira et al., 2002; Lago et al., 2005). Much less is known on molecular mechanisms, and the components involved in the apoptotic responses induced by the toxin. The characterization of the phenomenon, in fact has been generally performed by morphological criteria, and limited information is available on biochemical parameters of OA-induced apoptosis (Rossini et al., 2001). The present study was carried out with following objectives (a) delineate the multiple signal transduction pathways involved in OA-induced apoptosis in HeLa cells; (b) effect of OA on protein phosphatase activity and phosphorylation of MAP kinases; (c) effect of caspase inhibitors on MAP kinases, mitochondrial dependent and mitochondrial independent caspase cascade. The results of our study conclusively show involvement of multiple signal transduction pathways in OA-induced apoptosis. 2. Materials and methods 2.1. Chemicals Okadaic acid, Proteinase K, RNAse, propidium iodide, CHAPS, DTT, PMSF, 4methyl umbellyferyl phosphate (4-MUP) were obtained from Sigma Chemical Co. Reduced glutathione (GSH) was from Acros (Belgium). O-Phthaldehyde (OPT) MTT, ethidium bromide, Hoechst 33342 were purchased from Fluka (USA). The antibodies were obtained from Sigma Chemical Co., USA, Calbiochem, Cell Signaling and Dako. JC-1 dye was obtained from Molecular Probes, USA. All other chemicals were obtained from Sigma Chemical Co. (St. Louis, USA) unless otherwise mentioned.

2.2. Cell cultures and chemical treatment HeLa cells were obtained from NCCS, Pune. The cells were grown in minimum essential medium (Eagle) with out tryptose phosphate broth and supplemented with 2 nM l-glutamine. Earle’s BSS adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate 90%, fetal calf serum 10%, and gentamycin (80 ␮g/ml). Cells were maintained at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 in an incubator. To study the effect of okadaic acid on signal transduction pathways, the HeLa cells were grown in either 6 well/24 well tissue culture plates or 25 mm2 tissue culture flasks (Grenier) and treated with okadaic acid dissolved in minimum amount of DMSO (0.1%) then diluted in PBS. The treatments were given in serum free medium for different time duration. After the treatment the cells were detached from the flasks using cell scraper and washed with PBS. Then the cells were processed for various biochemical end points following procedures described below. Each treatment consisted of four replicates and each experiment was repeated at least three times.

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2.3. Cell viability assays (CVDE, LDH leakage) For IC50 determination of okadaic acid, the cells were grown (1–2 × 106 cells/well) in 24 well tissue culture plates and were exposed to various concentrations of toxins for 24 h. The 50% inhibitory concentration (IC50 ) of OA was determined from the plots of viability of cells by crystal violet dye exclusion assay. For all subsequent time course studies IC50 of okadaic acid was used. LDH activity in the culture media was measured spectrophotometrically as an index of plasma membrane damage and loss of membrane integrity (Rao et al., 1998). Enzyme activity was expressed as the percentage of extra cellular LDH activity of the total LDH activity of the cells. For viability and LDH leakage treatment duration was 24 h and for all subsequent experiments the duration was up to 16 h. 2.4. Measurement of intracellular GSH Intracellular levels of reduced glutathione were determined using the method of Hisin and Hilf (1976). Cells after treatment were washed with PBS and scrapped into 6.5% trichloroacetic acid. Phosphate-EDTA buffer (4.5 ml) of pH 8.0 was added to 0.5 ml of 100,000 × g supernatant. The final assay mixture (2 ml) contained 100 ␮l of the diluted supernatant, 1.8 ml phosphate-EDTA buffer pH 8.0 and 100 ␮l o-phthalaldehyde solution containing 100 ␮g of OPT, after thorough mixing and incubation at room temperature for 15 min, fluorescence was read at Ex-350 nm and Em-420 nm in Shimadzu RF-5000 spectrofluorometer. The reduced form of GSH was used as a standard. Data were expressed as nanomole GSH per 106 cells. 2.5. Measurement of intracellular ROS The intracellular ROS was estimated by fluorescent probe, 2,7dichlorofluorescein diacetate (DCFH-DA). The dye diffuses through the cell membrane and hydrolysed by intracellular esterases to non-fluorescent dichlorofluorescin (DCFH), which is oxidised to fluorescent DCF in presence of ROS (LeBel et al., 1992). The OA is added at 100 nM concentrations, as co-treatment with DCFH-DA at a final concentration 5 ␮M and incubated at 37 ◦ C up to 3 h. The fluorescence intensity was measured at different time intervals with Ex-485 nm and Em-530 nm. The DCF fluorescence intensity is proportional to the amount ROS (peroxy radicals) formed intracellularly. 2.6. Determination of mitochondrial membrane potential The changes in mitochondrial membrane potential were monitored with dye 5,5 ,6,6 -tetra chloro 1,1 ,3,3 -tetraethyl benzamidazolocarbocyanin iodide (JC-1, Molecular Probes). JC-1 emits light at red and green wavelengths according to its concentration taken up into the mitochondria. At high concentration JC-1 aggregated form emits a red light, and at low concentration the monomer form emits green light. The mitochondria imaging of cells with or without OA treatment is carried out as described previously with minor modifications (Yermolaieva et al., 2004). Briefly, the cells were plated on glass cover slips, the day before the experiments. After the cells grown to 80% confluence, the OA (100 nM) treatment is given in plain medium (without FCS). After 4 h of OA treatment, the culture medium was replaced with plain medium containing JC-1 (10 ␮g/ml) dye, and the cells were incubated at 37 ◦ C in a 5% CO2 incubator for 15 min. The cells were washed once with phosphate buffered saline and observed immediately in a fluorescence microscope (Carl Zeiss, Axiomot 2) using blue filter. For membrane potential ( m ) measurements, the cells were seeded to a 24 well plate. The following day, the growth medium was removed and the cells were incubated in plain medium (without FCS) with or without OA for different time points. At the end of incubation, the medium was removed and 500 ␮l of plain medium containing JC-1 dye (10 ␮g/ml) was added to each well. The cells were incubated at 37 ◦ C in 5% CO2 incubator for 15 min, and then the staining solution was removed. The cells were washed once with phosphate buffered saline and resuspended in 1.5 ml of phosphate buffered saline. The fluorescence was measured in a Shimadzu RF-5000 spectrofluorometer. The measurement for red fluorescence was taken at Ex-550 nm and Em-590 nm and for green fluorescence was measured at Ex-490 nm and Em-535 nm. The mitochondrial membrane potential is expressed as JC-1 fluorescence units in terms of red fluorescence to green fluorescence ratio. 2.7. Morphological determination of apoptosis and DNA fragmentation analysis Following various treatments, apoptotic nuclei were quantified using fluorescence staining (Rao et al., 2002). Briefly, at designated time points, media were removed and HeLa cells were washed once with PBS. The cells were then incubated with 10 ␮g/ml Hoechst 33342 (used to identify nuclear fragmentation) and 5 ␮g/ml propidum iodide (to identify non-viable cells) in PBS for 30 min at 37 ◦ C. Cells were then visualized with fluorescence microscope (Carl Zeiss, Axiomot 2) and fragmented nuclei in viable cells were counted. For each treatment group, 800–1000 nuclei were counted. Data were expressed as percentage of viable cells with apoptotic nuclei. DNA fragmentation was qualitatively analyzed by gel electrophoresis (Gong et al., 1994). Briefly, 1–2 × 106 cells were pelleted from the medium, washed once with Hank’s balanced salt solution (HBSS), re-suspended in 1 ml of HBSS, diluted with

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10 ml of ice-cold 70% ethanol and stored at −20 ◦ C for 24 h. The cells were then pelleted by centrifugation (800 × g for 10 min) and ethanol was removed completely. The pellet was resuspended and the cells were lysed in 50 ␮l of phosphate-citrate buffer (192 parts of 0.2 M Na2 HPO4 and 8 parts of 0.1 M citric acid, pH 7.8). After incubation at room temperature for 30 min, the cell lysate was centrifuged (1000 × g for 5 min) and the supernatant was concentrated using a Speed Vac concentrator. A 5 ␮l 0.25% NP-40 in distilled water was added to each sample followed by 3 ␮l of RNase A (1 mg/ml in water) and the suspension was incubated at 37 ◦ C for 30 min followed by 5 ␮l of proteinase K (1 mg/ml) and incubated for further 30 min at 37 ◦ C. DNA extracted from control and treated cells were electrophoresed on a 1.6% agarose gel impregnated with ethidium bromide. 1 kb DNA ladder (Promega, USA) served as molecular size standard.

PBST buffer for 1 h at room temperature followed by anticytochrome-c antibody (Oncogene, mouse antibody, 1:1000) or anti AIF antibody (Sigma, rabbit antibody, 1:1000) at 4 ◦ C. The membranes were developed by chemiluminescent method as described above.

2.8. Estimation of caspase-3 activity

For inhibition studies, the cells were pre incubated with caspase-3 specific inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 ␮M, capsase-8 inhibitor Z-IETD-FMK at 40 ␮M, and broad-spectrum caspase inhibitor Z-VAD-FMK, 40 ␮M concentration 2 h prior to OA treatment. The incubation of immunosupressive compound cyclosporin-A (CsA) at 1 ␮g/ml and antioxidant N-acetyl cysteine (NAC) at 10 ␮g/ml concentration was done 1 h prior to OA treatment. The cells were then incubated for 8 h. After the incubation time, the cells were processed for quantitative estimation of caspase-3 activity and ROS generation.

The activities of the caspases were measured in a fluorimetric assay modified from Nicholson et al. (1995). After different treatments, cells were harvested with phosphate-buffered saline and centrifuged at 1500 RPM for 5 min. Cell pellets were resuspended in 2 ml buffer containing 59 mM Tris–HCl, 1 mM EDTA, 10 mM EGTA, and lysed with 10 ␮M digitonin. After incubation at 37 ◦ C for 10 min, the lysates were centrifuged at 1000 × g for 5 min and the supernatants were stored at −70 ◦ C till assayed. Protein concentration was estimated (Lowry et al., 1951) and lysates (100 ␮g protein) were assayed in caspase assay buffer [312.5 mM HEPES (pH 7.5), 31.25% sucrose and 0.3125% CHAPS] with or without protease inhibitors. The reaction was started with addition of 50 ␮M caspase-3 substrate, acetyl-Asp-Glu-Val-Asp amino-methylcoumarin (Ac-DEVD-AMC), and the reaction was followed for 60 min. Fluorescence was measured at excitation 360 nm and emission 460 nm in spectrofluorometer (Shimadzu RF 5000). Fluorescence intensity was calibrated with standard concentrations of AMC. Protease activity was calculated from the slope of the curve and expressed as pmol/min/mg protein.

2.12. Analysis of protein phosphatase 2A activity The protein phosphatase 2A activity in OA treated cells was estimated using Serine/threonine Phosphatase assay system (Promega Corporation, USA), following manufacturer’s protocol. 2.13. Inhibitor studies

2.14. Statistical analysis All data are expressed as mean ± S.E. from four replicates per treatment. Data were analyzed by one-way ANOVA followed by Dunnet’s test for comparison between control and treatment groups. For the experiments on effect of inhibitors on ROS and capsase-3 activity, data were analyzed by one-way ANOVA followed by Student Newman Keul’s multiple comparison test. The level of significance was set at p ≤ 0.05. All the results shown in this article were obtained from at least three independent experiments with a similar pattern.

2.9. Preparation of cell lysates for immunoblotting For studies on caspases and other cellular proteins, control and OA treated cells grown in 25 cm2 tissue culture flasks were lysed in lysis buffer (10 mM HEPES pH 7.4, 42 mM KCl, 5 mM MgCl2 , 0.1 mM EDTA, 0.1 mM EGTA, 5 mM DTT, 2 mM PMSF, 1× complete protease inhibitor cocktail) containing 0.5% CHAPS. Cellular debris was spun down at 1400 × g for 20 min, and the supernatants were used as whole cell protein extracts. For PARP cleavage, cell extracts were prepared following the procedure of Shah et al. (1995). Briefly, control and okadaic acid treated cells grown in 25 cm2 tissue culture flasks were washed once with PBS, suspended at ∼5 × 106 cells/ml in sample buffer (6 M urea, 62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 5% ␤mercaptoethanol (freshly added), 2% SDS, 0.00125% bromophenol blue), sonicated for 15 s, and incubated at 65 ◦ C for 15 min. 2.10. Western blotting analysis Protein concentration of cell extracts was determined by Bio-Rad DC protein assay. Fifty micrograms protein from each sample was separated on SDS–PAGE and electrophoretically transferred on to a nitrocellulose membrane filter, using an electro-blotting apparatus. Membranes were incubated in blocking solution containing 5% non-fat dry milk in PBST buffer (PBS buffer containing 0.1% Tween-20) for 1 h at room temperature, followed by incubation for over night at 4 ◦ C in platform shaker with various primary antibodies at specified dilutions: monoclonal anti-PARP antibody at 1:2000 (clone C-2-10, Sigma) directed against uncleaved (116 kDa) and the cleaved form of PARP (85 kDa), anti-caspase-3, rabbit polyclonal antiserum (dilution 1:2000)) recognizing whole caspase-3 (34 kDa) as well as large (20 kDa) and small sub units (18 kDa); anticaspase-10, purified IgG fraction of polyclonal rabbit antiserum (1:2000) which recognizes uncleaved (58 kDa) and cleaved forms (23–17 kDa); anticaspase-9, rabbit polyclonal antibody (1:2000); anticaspase7, rabbit polyclonal antibody (1:1000); anticaspase-8, rabbit polyclonal antibody (1:2000); anti Bax mouse antibody (1:1000); and anti 42/44 (1:1000) or antiphospho p42/44 MAP kinase (1:1000) rabbit polyclonal antibody and anti p38 (1:1000) or anti-phospho p38 MAP kinase (1:1000) rabbit polyclonal antibody. Monoclonal anti-␤-actin (Clone AC-74) which detects ␤-actin (42 kDa) was used as protein loading control. All antibodies are diluted in PBST with 5% milk powder. The membranes were washed four times in PBST for 15 min, followed by incubation for 2 h in horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibody used at 1:50,000 dilution. The membranes were washed again and developed using an enhanced chemiluminescent detection system according to manufacturer’s protocol and the image was taken on X-ray films. 2.11. Analysis of cytochrome-c and AIF translocation For the analysis of cytochrome-c and AIF, mitochondrial and cytosolic fractions were prepared according to ‘cytochrome-c release apoptosis assay kit’ (Oncogene, USA) following manufacturer’s protocol. These mitochondrial and cytosolic fractions were used for Western blot studies. The samples were subjected to SDS-PAGE in a 12% gel and transferred electrophoretically to nitrocellulose membrane. The membranes were blocked using a blocking solution containing 5% non-fat dry milk powder in

3. Results 3.1. OA induced cytotoxicity In order to determine the IC50 of OA, the cells were treated with different concentrations of OA (10 nM–200 nM) in a 24 well tissue culture plate for 12 h and viability was determined by crystal violet dye exclusion assay. OA induced a dose dependent decrease in viability with increasing concentration of OA in HeLa cells (Fig. 1A). The IC50 was determined as 100 nM and used in all further experiments. OA induced cytotoxicity was evaluated in a time course experiment by treating the cells with 100 nM of toxin and viability was determined by intracellular LDH leakage assay (Fig. 1B). The viability profile by intracellular LDH leakage shows a significant increase in LDH activity at 4 h and reached the maximum after 16 h. 3.2. Effects on ROS and GSH levels The HeLa cells treated with OA showed a significant increase in ROS levels as early as 15 min post-treatment (Fig. 2A). DCF-DA dye used in the experiment detects peroxy radicals and not other oxygen species like super oxide anion (O2 •− ) and nitric oxide. ROS levels were similar at 15, 30 and 60 min. Though there was decrease at 120 min, the levels were still significantly higher than control. Further treatment up to 4 h did not show any difference from 120 min (data not shown). Cellular GSH levels were measured in the cells after OA treatment and the results are summarized in Fig. 2B. There was significant GSH depletion starting at 2 h with more than 50% depletion at 16 h compared to control. 3.3. Effects on mitochondrial membrane potential (

m)

The involvement of mitochondria in initiation of apoptosis in HeLa cells was evaluated recording changes in its membrane potential with JC-1 dye. The JC-1 red/green ratio signals recorded in the cells, with or without OA treatment at different time points is shown in Fig. 3A. In control cells, higher red/green ratio signal was observed, representing polarized mitochondria. A significant down fall in the ratio was observed from 30 min onwards, reaching

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Fig. 1. (A) Effect of okadaic acid (OA) on viability of HeLa cells. For determination of IC50 , the cells grown in 24 well plates were treated with varying okadaic acid concentration for 24 h. The IC50 was calculated as 100 nM. The values are mean ± S.E. of four replicates. * Significantly different from respective controls at p ≤ 0.05 by Dunnet’s test. For all subsequent experiments IC50 of 100 nM was used. (B) Time course effect 100 nM OA on HeLa cells by lactate dehydrogenase (LDH) leakage assay. The values are mean ± S.E. of four replicates. * Significantly different from respective controls at p ≤ 0.05 by Dunnet’s test. Results are representative of three separate experiments.

a sharp decrease at 2 h, which was continued at 4 and 8 h. The ratio of JC-1 emission intensity at 590 nm over that at 535 nm is commonly used to infer mitochondrial membrane potential ( m ) in a semi-quantitative and ratio metric manner (Smiley et al., 1991). The time dependent decrease in JC-1 red/green ratio after OA treatment indicates an alteration in mitochondrial membrane potential. The microscopic analysis of cells stained with JC-1 after OA treatment shows a distinct change in the fluorescence signal intensity from red to green (Fig. 3B) in treated cells. 3.4. Effect of OA on apoptosis and DNA fragmentation OA (100 nM) treated HeLa cells, stained with Hoechst 33342 and propidium iodide exhibited morphological changes typical of apoptosis including cell shrinkage, plasma membrane blebbing, chromatin condensation and nuclear fragmentation as compared to control cells with prominent rounded nuclei and defined plasma membrane contours. A distinguishing feature of apoptosis is the condensation and fragmentation of nuclear chromatin, which is monitored by fluorescence microscope. The OA treated cells showed the morphological signs of apoptosis after staining with Hoechst 33342 dye (Fig. 4A, i and ii). Quantitative estimation of

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Fig. 2. Effect of 100 nM OA on reactive oxygen species generation (A), and intracellular glutathione levels (B) in HeLa cells. The values are mean ± S.E. of four replicates. * Significantly different from respective controls at p ≤ 0.05 by Dunnet’s test. Results are representative of three separate experiments.

apoptotic cells induced by OA was evaluated in a time course experiment for 16 h. Data were expressed as percentage of viable cells with apoptotic nuclei. At 4 h post-treatment, significant number of apoptotic cells was observed with a four-fold increase in apoptotic cells reaching maximum by 16 h (Fig. 4B). The qualitative DNA fragmentation was carried out by agarose gel electrophoresis. The treated cells showed typical internucleosomal DNA fragmentation or ‘ladder’ formation at all time points tested (Fig. 4C). 3.5. Analysis of Cyt-c initiated caspase pathway Release of mitochondrial proteins like Bax, cyt-c and AIF was analyzed by immunoblotting. There was a time dependent increase in levels of Bax in OA treated cells at 8, 12, 16 h (Fig. 5A). To explore the events in the cyt-c initiated proteolytic cascade, we analyzed cyt-c release in cells separately in mitochondrial and cytosolic fractions. There was a time dependent decrease in mitochondrial fraction and corresponding increase in cytosolic fraction from 4 h (Fig. 5B). To explore the range of caspases activated upstream and down stream of cyt-c, the Western blot analysis of caspases-8, -10, 9, -3 and -7 were carried out after OA treatment in cells. Fig. 6 shows that detectable activation of most caspases in OA treated cells. Caspase-8 cleavage could be seen from 4 h onwards and procaspase-10 (58 kDa) and activated cleaved products of 23–17 kDa were observed from 2 h post-treatment. The uncleaved caspase-9 (47 kDa) was observed in control and cleaved product of 35 kDa was

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Fig. 3. Time course profile of 100 nM OA on mitochondrial membrane potential in HeLa cells. (A) The mitochondrial membrane potential is expressed as JC-1 fluorescence units in terms of red fluorescence to green fluorescence ratio. The values are mean ± S.E. of four replicates. * Significantly different from respective controls at p ≤ 0.05 by Dunnet’s test. (B) Fluorescence photomicrograph of change in MMP after OA treatment in HeLa cells. (i) Control cells (ii) OA treated cells (magnification ×100). Results are representative of three separate experiments.

observed at 8, 12 and 16 h post-treatment. The caspase-3 cleavage in OA treated samples was observed at 8, 12 and 16 h post-treatment. The analysis of caspase-7 showed the activated cleaved products (20–23 kDa) at 8, 12 and 16 h time points. The ␤-actin expression was used as protein loading control. To directly assess the involvement of caspase-3 in downstream events of apoptosis its activity was determined in OA treated cells using fluorogenic caspase-3 substrate Ac-DEVD-AMC. OA treated cells showed no significant difference at 2 h but a 2-fold increase was observed at 4 h and reached a plateu by 8 h and no further increase was observed at 12 and 16 h (Fig. 7A). The activation of caspases was confirmed with Western blot analysis. Activation of caspase-3 leads to cleavage of number of proteins including PARP. Cleavage of PARP is an important indicator of apoptosis. The results on Western blotting analysis of OA treated cells showed uncleaved 116 kDa in control, 4, 8 and 12 h. The cleaved fragment was observed at 8, 12 and 16 h post-treatment (Fig. 7B). There was a correlation in caspase-3 cleavage time point, PARP cleavage and DNA fragmentation. 3.6. Analysis of AIF mediated caspase-independent pathway The translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus via cytosol is one of the mitochondrialmediated pathways, which is independent of caspase activation. We have analyzed the expression levels of AIF in both mitochondria and cytosol fraction of OA treated cells (Fig. 5B). There was a time dependent decrease in AIF levels of mitochondrial fraction with corresponding increase in cytosol. The base levels observed

in control cells may be due to release of AIF from small fraction of spontaneously dying cells. The additional bands probably represent the phosphorylated product of AIF. 3.7. Inhibition of protein phoshatase and activation of MAP kinases As OA is a potent inhibitor of PP1 and PP2A, we investigated the inhibition of protein phosphatase 2A and phosphorylation status of MAP kinases with specific antibodies against normal and phosphorylated p38 and p42/44 (ERK1/2) MAP kinases. A significant inhibition in the activity of PP2A was observed from 4 h onwards in OA treated cells. A 50% inhibition was observed in 8 h and reached the maximum inhibition by 12 h. Not much change was observed at 16 h time point (Fig. 8A). The p38 MAP kinase levels did not show any change in any of the time points compared to control but the phospho p38 MAP kinase levels showed a time dependent increase in the expression level (Fig. 8B). The analysis of p42/44 MAP kinase (ERK1/2) showed a time dependent decrease in expression levels. There was no distinct profile of phospho p42/44 MAP kinase except at 16 h time point. 3.8. Effect of capsase inhibitors on MAP kinases In order to understand the link between capsases and MAP kinases we pretreated the HeLa cells for 2 h with capsase inhibitors AC-DEVD-CHO (100 ␮M) and Z-VAD-FMK (40 ␮M) and then treated with OA for 8 h. The expression profiles of normal and phosphorylated MAP kinases are shown in Fig. 9. The pretreatment of

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Fig. 4. (A) Morphological features of OA (100 nM) induced apoptosis in HeLa cells. Fluorescence micrograph of the cells stained with Hoechst 33342 showing the nuclear fragmentation. (i) Control cells, (ii) OA treated cells after 12 h (magnification ×100); (B) time course effect of OA (100 nM) on percent apoptotic cells. The values are mean ± S.E. of four replicates. * Significantly different from respective control at p ≤ 0.05 by Dunnet’s test; (C) DNA agarose gel electrophoresis of OA (100 nM) treated cells. Lane M: 1 1 kb ladder; Lane 2 control; Lanes 3–6 OA treated cells at 4, 8, 12, 16 h, respectively. Results are representative of three separate experiments.

inhibitors Ac-DEVD-CHO and Z-VAD-FMK altered the phosphorylation pattern of p38 MAP kinase and P42/44 MAP kinase (Fig. 9). Z-VAD-FMK decreased the phosphorylation levels of p38 MAP kinase. But in the case of p42/44 MAP kinase Ac-DEVD-CHO partially inhibited the phosphorylation compared to Z-VAD-FMK.

Fig. 5. Time course effect of OA on release of mitochondrial proteins. (A) Blot showing the activation of Bax (21 kDa). (B) Expression levels of cytochrome-c and and apoptosis inducing factor (AIF) in mitochondrial and cytosolic fractions. ␤-Actin is shown as protein loading control. Results are representative of three separate experiments.

3.9. Effect of inhibitors on OA induced cell death To confirm the involvement of caspase-dependent and caspaseindependent pathway in OA-induced apoptosis, we pretreated the cells for 2 h with caspase-3 specific inhibitor Ac-DEVDCHO (100 ␮M), caspase-8 specific inhibitor Z-IETD-FMK (40 ␮M) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 ␮M) before OA treatment. In OA treated cells capsase-3 inhibitor could completely block the cleavage of caspase-3 (Fig. 10A). Caspase-8 inhibitor Z-IETD-FMK blocked the cleavage completely but caspase-3 inhibitor could not prevent caspase-8 cleavage (Fig. 10B). Both the inhibitors had no inhibitory effect on translocation of AIF into cytosol and DNA fragmentation. In addition to caspase inhibitors (IETD-FMK and Z-VAD-FMK), cyclosporine-A and NAC were tested for their effect on OA-induced apoptosis. Capsase-8 inhibitors, CsA and NAC could partially prevent OA induced ROS generation (Fig. 10C). The ROS generation was significantly higher in OA treated cells and reduced in inhibitor treated cells but is still higher than control cells. The effect of these compounds on caspase-3 activity showed that capsase-8 inhibitors could reduce the activity compared to OA but still significantly higher than control. The DNA fragmentation analysis of cells with and without caspase inhibitors showed that none of the inhibitors (Z-IETD-FMK, Z-VAD-FMK, CsA and NAC) could prevent OA-induced DNA fragmentation completely (Fig. 10D) our results with various inhibitors showed that OA-induced apoptosis mediated by caspasedependent and independent pathways.

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Fig. 6. Effect of OA on caspases in HeLa cells. Blots showing the procaspase and cleaved products of caspase-8, caspase-10, caspase-9, caspase-3. Caspase-7 shows only the cleaved product. ␤-Actin is shown as protein loading control. Results are representative of three separate experiments.

Fig. 8. (A) Time course effect of OA on protein phosphatase 2A activity in HeLa cells. The values are mean ± S.E. of four replicates. * Significantly different from respective control at p ≤ 0.05 by Dunnet’s test. (B) Effect of OA on phosphorylation status of MAP kinases. Antibodies specific for normal and phosphorylated proteins were used. Blot shows the intracellular levels of normal p38 and phosphorylated p38 MAP kinase and normal p42/44 and phosphorylated 42/44 MAP kinases. Results are representative of three separate experiments.

toxicity through general overphosphorylation of cellular proteins. Although it has been implicated that protein phosphorylation and caspases may play a role in okadaic acid induced apoptosis, the exact mechanism of OA-induced apoptosis remains unclear. In this study we investigated the signal transduction pathways involved in OA induced cell death in human cervical cancer (HeLa) cells.

Fig. 7. (A) Effect of OA on caspase-3 activity levels in in HeLa cells. The values are mean ± S.E. of four replicates. * Significantly different from respective control at p ≤ 0.05 by Dunnet’s test. (B) Time course profile of caspase-3 induced PARP cleavage. ␤-Actin is shown as protein loading control. Results are representative of three separate experiments.

4. Discussion Environmental toxins made by marine and freshwater microorganisms represent a significant health problem. Okadaic acid produced by marine dinoflagellate, is an enterortoxin involved in diarrhetic shellfish poisoning in humans. These toxins are inhibitors of protein phosphatases 1 and 2A, and exert their

Fig. 9. Effect of caspase-3 inhibitor Ac-DEVD-CHO and broad spectrum caspase inhibitor Z-VAD-FMK on phosphorylation of p38 MAP kinase and p42/44 MAP kinase. The cells were pretreated for 2 h with inhibitors before OA (100 ␮M) treatment. The OA treatment was done for 8 h.

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Fig. 10. (A) Effect of pre-treatment of caspase-3 inhibitor Ac-DEVD-CHO on caspase-3 activation. The immunoblot shows complete blockage of caspase-3 cleavage. Time point is 8 h post-treatment. (B) Effect of pre-treatment of caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK on cytosolic AIF levels and caspase-8 cleavage in HeLa cells. Time point is 8 h post-treatment. Results are representative of three separate experiments. (C) Effect of inhibitors on ROS generation and caspase-3 activity in OA treated cells. The cells were pre-treated for 2 h with Z-IETD-FMK—40 ␮M, Z-VAD-FMK—40 ␮M, CsA—1 ␮g/ml and NAC—10 ␮g/ml. After OA treatment caspase activity was measured at maximum activation time point of 8 h. The values are mean ± S.E. of four replicates. Means followed by different alphabet(s) are significantly different at p ≤ 0.05 by Student-Newman-Keuls multiple comparison test. (D) The DNA fragmentation profile with the same inhibitors in OA treated HeLa cells. Results show that none of the inhibitors could prevent DNA fragmentation. Lane 1—1 kb ladder, Lane 2—Control, Lane 3—OA, Lane 4—Z-IETD-FMK + OA, Lane 5—Z-VAD-FMK + OA, Lane 6—CsA + OA, Lane 7—NAC + OA. Time point is 8 h post-treatment. Results are representative of three separate experiments.

OA at 100 nM concentration showed time dependent cytotoxicity measured in terms of viability by CVDE and LDH leakage. OA induced rapid increase in ROS as early as 15 min. The ROS generation was more pronounced and persisted till 4 h (data not shown). OA treatment also caused a time dependent depletion of GSH levels. Our results show that GSH depletion persisted till 16 h in contrast to ROS levels. ROS burst occurs in the early stages after stimulation and is capable of functioning as an initial mediator in apoptotic pathway. Though ROS may be one of the triggers for GSH depletion, there are other mechanisms for GSH depletion. Apotosing cells get rid of GSH prior to apoptosis by promoting its efflux via specific carriers (Ghibelli et al., 1998). Inhibition of protein phosphatases 1 and 2A by OA leads to disruption of the cytoskeleton and cell death in several systems (Leira et al., 2002; Creppy et al., 2002). OA inhibits PP2A nearly 100-fold more than PP1. PP2A acts in the apoptotic signal transduction pathway not only upstream but also downstream of the effector caspase. In the present study, OA treatment caused a time dependent inhibition of PP2A activity in HeLa cells. This was supported by time dependent phosphorylation of p38 MAP kinases. Phosphorylation of MAP kinases plays an important role in the process of apoptosis (Cross et al., 2000). MAPKs also play a role in activating caspase cascade. Recent study (Kim and Chung, 2008) has shown that p38 MAPK signalling is linked to activation of caspase-8. Caspase-3 may also act as an upstream initiator of apoptosis. So we studied the effect of caspase inhibitors on phosphorylation status of MAP kinases. But our results showed that that these inhibitors could not completely prevent phosphorylation of both p38 and p42/44 (ERK1/2) MAP kinases. A complex, interactive network of signalling pathways regulates cell proliferation and survival. In the present study we have not evaluated the effect of OA on Ras-Raf-MEK-ERK pathway. It is well established that heat shock protein 90 (Hsp90) is responsible

for chaperoning proteins involved in cell signalling, proliferation and survival. Raf-1 is part of a conserved signal transduction pathway involved in transmitting signals from cytosolic and transmembrane tyrosine kinases to MAP Kinases. Disruption of Hsp90 function inhibits Raf-1 signalling, in part by preventing newly synthesised Raf protein from reaching plasma membrane and possibly by inhibiting assembly of raf-1/MEK1 signalling unit (Goetz et al., 2003). Tucker et al. (2008) study showed that Hsp90 is involved in ERK activation in macrophages treated with OA. We examined the involvement of the mitochondrial pathway by monitoring the mitochondrial membrane potential, cytochrome-c release and Bax translocation. ROS are reported to directly activate mitochondrial permeability transition resulting in  m loss and the release of proapoptotic molecules cyt-c, AIF and EndoG (Daugas et al., 2000). A significant time dependent decrease in MMP was observed 30 min post-treatment. The decrease in MMP correlated well with ROS generation and cyt-c level as seen by Western blotting. There was decrease in cyt-c levels in mitochondrial fraction and corresponding increase in cytosol. Our results show that induction of ROS formation by OA causes onset of mitochondrial permeability transition. There are number of reports on effect of ROS and oxidative stress on mitochondrial membrane potential (Kim et al., 2004). Apart from reactive oxygen species, nitric oxide and [Ca2+ ]i are known to activate cell death signalling cascades in different cell types. Pro-apoptotic pathways of nitric oxide (NO) are compatible with established signalling circuits involved in mitochondria dependent cell death, with particular role on the involvement of tumor suppressor p53 as a target during cell death execution. The anti-apoptotic role of NO covers essential mechanisms such as protection via cGMP, cell death protective protein expression or radical–radical interactions (Br¨vne, 2003). Similarly [Ca2+ ]i plays a key role in both apoptotic and necrotic cell death. Release of intra-

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cellular Ca2+ stores and/or influx of extracellular Ca2+ can modulate cell death in many cell types (Waring, 2005). However, Ca2+ independent apoptosis could be found in some cell types (Chou et al., 2007). In order to explore the range of caspases activated upstream and down stream of cyt-c, we did the Western blot analysis of caspases8, -10, -9, -3 and -7. The time of activation of caspase 9, 3 and 7 correlates well with cyt-c release. Capsase-3 activation resulted in the PARP cleavage and DNA fragmentation in OA treated cells. Most pathways upstream of MMP are caspase-independent, and both caspase-dependent and caspase-independent paths become possible after mitochondrial damage. Recently, mitochondria mediated, caspase-independent apoptotic pathways were reported. Release of apoptosis-inducing factor (AIF), endonuclease G (EndoG) and HtrA2/Omi from mitochondria can trigger cell death in the absence of caspase activation (Lorenzo and Susin, 2004; Modjtahedi et al., 2006; Criollo et al., 2007). AIF is a flavoprotein that, in healthy cells is confined to mitochondria where it exerts a vital function in bioenergetic and redox metabolism. After mitochondrial membrane permeabilization AIF is released from mitochondria and translocates to cytosol first and then to the nucleus, where it triggers large scale (∼50 kb) DNA fragmentation. There are no earlier reports on caspase-independent and caspase-dependent pathways in OA-induced apoptosis simultaneously. So we tested certain inhibitors to confirm our findings. The pre-treatment with inhibitor DEVD-CHO could block completely block the caspase-3 cleavage. We also investigated whether caspase 8 activation occurred upstream or downstream of mitochondria. Cells were pre-treated caspase 8 specific inhibitor Z-IETD-FMK and caspase-3 inhibitor DEVD-CHO. Our results showed that cleavage of caspase-8 was inhibited by Z-IETD-FMK only and not by caspase 3 inhibitor. This shows that caspase-8 was cleaved upstream of mitochondria. Boudreau et al. (2007) showed that OA-induced apoptosis and DNA fragmentation in T-leukemic cells was prevented by pretreatment with caspase-8 and casapse-9 inhibitors. Complete blockage of OA induced procaspase-3 cleavage was observed in Z-VAD-FMK, CsA and NAC pretreated cells, but still DNA fragmentation was evident in all groups. Siu et al. (2008) have shown that, besides CsA’s well known inhibitory action of mitochondrial permeability transition due to binding to cyclophilin D, low concentrations of CsA can inhibit Bax activation and translocation to mitochondria. CsA, NAC and Z-VAD-FMK did prevent caspase-dependent features of apoptosis. But they did not prevent translocation of AIF to the nucleus and DNA fragmentation as determined by gel electrophoresis. The results indicate that mitochondria-nuclear translocation of AIF and subsequent DNA fragmentation is caspase independent. Our earlier studies with caspase inhibitors Ac-DEVD-CHO and Z-VAD-FMK could completely block ricin induced apoptosis and DNA fragmentation in HeLa cells (Rao et al., 2005). In conclusion, our experimental results clearly show at least three distinct signal transduction pathways are involved in OAinduced apoptosis in HeLa cells: (1) OA treatment cause generation of ROS leading to mitochondrial membrane permeability, release of bax, cty-c and activation of caspase cascade, DNA fragmentation and apoptosis. (2) OA treatment causes change of mitochondrial membrane permeability leading to release of AIF which enters nucleus and cleaves DNA resulting in DNA fragmentation and apoptosis. AIF mediated apoptosis pathway was not inhibited by caspase inhibitors. (3) The third pathway involves inhibition of protein phosphatase 2A by OA and subsequent phosphorylation of MAP kinases p38 and p42/44 leading to activation of caspase cascade and apoptosis. Our results show that despite their well-differentiated mechanisms, caspase-dependent and -independent pathways may operate together (or in parallel) in the HeLa cell line we have studied. This apparent redundancy ensures the cell suicide once

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