Cilostazol Enhances Casein Kinase 2 Phosphorylation and Suppresses Tumor Necrosis Factor- -Induced Increased Phosphatase and Tensin Homolog Deleted from Chromosome 10 Phosphorylation and Apoptotic Cell Death in SK-N-SH Cells

This study shows the signaling pathway by which cilostazol suppresses tumor necrosis factor(TNF)-induced the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) phosphorylation and apoptosis via casein kinase 2 (CK2) phosphorylation in the SK-N-SH cells (neuroblastoma cells). Cilostazol (10 M) fully restored cell proliferation with suppression of DNA fragmentation induced by TNFand emodin, a CK2 inhibitor, which were antagonized by iberiotoxin, a maxi-K channel blocker. Under application of TNFor emodin, increased PTEN phosphorylation and decreased phosphorylation of CK2/Akt/ cyclic AMP response element-binding protein (CREB), and CK2 activity were significantly reversed by cilostazol ( 1–100 M), all of which were antagonized by iberiotoxin. 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) and (3S)-( )-(5chloro-2-methoxyphenyl-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one (BMS 204352) maxi-K channel openers significantly elevated CK2 activities that were reversible by iberiotoxin.SK-N-SHcellstreatedwithantisenseCK2oligodeoxynucleotide showed a prominent DNA fragmentation with little responsiveness to TNFin the phosphorylation of PTEN, indicative of the essential role of p-CK2/CK2 in cell proliferation, and the decreased cell viability of these cells was not restored by cilostazol. It is suggested that the action of cilostazol promoting cell survival is ascribed to the maxi-K channel openingcoupled up-regulation of CK2 phosphorylation and downregulation of PTEN phosphorylation with resultant increased phosphorylation of Akt and CREB. Cilostazol is known to increase the intracellular level of cyclic AMP by inhibiting its hydrolysis by type III phosphodiesterase (Kimura et al., 1985). Recently, Kim et al. (2002) addressed the in vitro inhibition of lipopolysaccharide-induced apoptosis by cilostazol in human umbilical vein endothelial cells, in that they demonstrated a reversal by cilostazol of the lipopolysaccharide-induced decrease in Bcl-2 protein and increases in Bax protein production and cytochrome c release. The neuroprotective effect of cilostazol was documented against cerebral ischemic infarct in rats (Choi et al., 2002). The phosphatase and tensin homolog deleted from chromosome 10 (PTEN) has both protein phosphatase (phosphoserine/threonine and phospho-tyrosine) and phosphoinositide 3-phosphatase activities (Myers et al., 1997; Maehama and Dixon, 1998), and negatively regulates the phosphatidylinositol-3 kinase pathway by catalyzing degradation of the phosphatidylinositol (3,4,5)-triphosphate (Stambolic et al., 1998). Increasing numbers of reports have shown that a signaling cascade mediated by phosphatidyl inositol-3 kinase/Akt (PKB, serine/threonine kinase) up-regulates cyclic AMP response element-binding protein (CREB), and in turn Bcl-2 promoter activity in association with enhanced Bcl-2 protein expression, and promotes cell survival by growth factors (Dudek et al., 1997; Crowder and Freeman, 1998; Walton et al., 1999; Pugazhenthi et al., 2000). Most recently, Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.058768. ABBREVIATIONS: PTEN, phosphatase and tensin homolog deleted from chromosome 10; CREB, cyclic AMP response element-binding protein; CK2, casein kinase 2; p-CK2, phosphorylated casein kinase 2; p-PTEN, phosphorylated phosphatase and tensin homolog deleted from chromosome 10; AS, antisense; ODN, oligodeoxynucleotide; MEM, minimal essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; TNF, tumor necrosis factor; NS-1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2one; BMS 204352, (3S)-( )-(5-chloro-2-methoxyphenyl-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one. 0022-3565/04/3081-97–104$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 1 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 58768/1116388 JPET 308:97–104, 2004 Printed in U.S.A. 97 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from Hong et al. (2003) have shown that cilostazol increases the K current in SK-N-SH cells by activating the maxi-K channels, and increase in neuronal cell survival by cilostazol is linked to its ability to decrease PTEN phosphorylation and to increase Akt/CREB phosphorylation. Protein kinase CK2 (CK2, formerly known as casein kinase 2) has been extensively studied in recent years for its potential role in multiple functional activities, including regulation of cell growth and proliferation. It is a ubiquitous protein Ser/Thr kinase, localized in the cell cytoplasm and nucleus, existing as a heterotetramer consisting of , , and subunits. CK2 catalyzes the phosphorylation of a large number of proteins, and its phosphorylation modulates their activities positively or negatively (Pinna, 1990; Allende and Allende, 1995). Nevertheless, little information is known regarding implication of CK2 activation and the signaling pathway including PTEN, Akt, and CREB phosphorylation in relation with cell survival. Therefore, it is inferred that, if activation/phosphorylation of protein kinase CK2 regulates increase in Akt/CREB phosphorylation via inactivation of PTEN, these may provide the favorable signals for cell survival. In this study, we assessed the protective effect of cilostazol as a maxi-K channel opener against TNFand emodin (CK2 inhibitor)-induced reduction in proliferation in the SK-N-SH cells (human neuroblastoma). Here, to identify whether cilostazol regulates the CK2 phosphorylation (p-CK2) and PTEN phosphorylation (pPTEN) as a maxi-K channel opener, we observed these parameters in the absence and presence of iberiotoxin, a maxi-K channel blocker. We further assessed the changes in p-CK2, p-PTEN, Akt phosphorylation (p-Akt), and CREB phosphorylation (p-CREB) levels in the wild-type and antisense (AS) CK2 oligodeoxynucleotide (ODN)-treated SKN-SH cells by Western blot analysis in the absence and presence of TNFand emodin, a CK2 inhibitor. Materials and Methods Neuronal Cell Cultures. SK-N-SH (KCLB 30011, human brain neuroblastoma) cells were cultured in Eagle’s minimal essential medium (MEM) with 2 mM L-glutamine and 1.0 mM sodium pyruvate supplemented with 10% heat-inactivated fetal bovine serum. Cells were grown to confluence at 37°C in 5% CO2. Sense and Antisense Oligodeoxynucleotides. The AS and sense are phosphorothioate analogs of ODN to the 5 end of the different subunits of CK2. These were synthesized commercially (Bioneer Corporation, Daejon, South Korea). AS phosphorothioate ODN had the following sequences: complimentary to CK2 -subunit mRNA (CK2 702–721, target sequence site 702–721): 5 -CCAAACTCCACATATCCAAA, complimentary to CK2 mRNA (CK2 144–163, 144–163 target sequence site): 5 -GTCCCGACATGTCAGACAGG, complimentary to CK2 mRNA (CK2 124–143, target sequence site 124–143): 5 -GAGCTGCTCATCTTCACGTC. Sense phosphorothioate ODN had the following sequences: complimentary to CK2 mRNA (target sequence site 144–163): 5 -CCTGTCTGACATGTCGGGAC. Oligodeoxynucleotide Treatment of SK-N-SH Cells. SKN-SH cells were rendered quiescent by a low concentration of fetal bovine serum (0.5%) for 15 h. ODN was added to the medium (final concentration, 100 g/ml) 2 h before treatment of drugs. Cell Proliferation Assay. For mitochondrial tetrazolium assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; MTT], cells were seeded 1 10 cells/well in 96-well tissue culture plates. The confluent cells were incubated in the MEM medium with 1% fetal bovine serum plus drugs and then exposed to TNFfor 24 h. Thereafter, 20 l/well of MTT solution (5 mg/ml phosphate-buffered saline) was added and incubated for 2 h. The medium was aspirated and replaced with 150 l/well of ethanol/dimethyl sulfoxide solution (1:1). The plates were shaken for 20 min and the optical density was measured at a wavelength 570 nm and a reference wavelength 630 nm using enzyme-linked immunosorbent assay (Bio-Tek Instruments, Winooski, VT). DNA Fragmentation Assay. After incubation of the cells in the absence and presence of the drugs for 3 h, cells ( 1–5 10) were exposed to TNF(50 ng/ml) for 24 h. At harvest, trypsinized cells were pelleted by centrifugation. Cells were lysed in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% sodium dodecyl sulfate, and 0.5 mg/ml proteinase K). Digestion was continued for 1 to 3 h at 55°C, followed by addition of RNase A to 0.1 mg/ml and running dye (10 mM EDTA, 0.25% bromphenol blue, 50% glycerol). Equivalent amounts of DNA ( 15–20 g) were loaded into wells of 1.6% agarose gel and electrophoresed in 0.5 TAE buffer (40 mM Tris-acetate, 1 mM EDTA) for 2 h at 6 V/cm. DNA was visualized by ethidium bromide staining. Gel pictures were taken by the UV transillumination with the Polaroid camera. Western Blot Analysis. The confluent cells received MEM medium with 1% fetal bovine serum plus cilostazol 3 h before stimulation with TNFand then were exposed to TNFfor 1 h. The cells were lysed in lysis buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 g/ml phenylmethylsulflonyl fluoride, 1 g/ml aprotinin, and 1% Triton X-100. After centrifugation at Fig. 1. A, effect of cilostazol on the TNFand emodin-induced decreased cell proliferation measured by MTT conversion in SK-N-SH cells. Values are means S.E.M. from three experiments performed in quadruplicate. Significant differences were shown between control and TNF/emodintreated groups by two-way repeated measures analysis of variance followed by Tukey’s multiple comparison tests as a post hoc comparison (P 0.001). B, amelioration of the TNFand emodin-induced DNA fragmentation by cilostazol ( 1–100 M) in the SK-N-SH cells. Representative agarose gel electrophoresis showing DNA laddering feature after exposure of the SK-N-SH cells to TNF(50 ng/ml) and emodin (10 M) in the absence and presence of iberiotoxin (1 M). M represents the 100-base pair DNA ladder markers. The results were confirmed with four different preparations. 98 Kim et al. at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from 12,000 rpm, 50 g of total protein was loaded into 8 or 10% SDSpolyacrylamide gel electrophoresis gel, and transferred to nitrocellulose membrane (Amersham Biosciences Inc., Piscataway, NJ). The blocked membranes were then incubated with the indicated antibody, and the immunoreactive bands were visualized using chemiluminescent reagent as recommended by the Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical, Rockford, IL). The signals of the bands were quantified using the GS-710 calibrated imaging densitometer (Bio-Rad, Hercules, CA). The results were expressed as a relative density. Polyclonal antibodies against CREB, p-CREB, and CK2 were from the Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and polyclonal antibodies against PTEN, p-PTEN (Ser380/Thr382/383), Akt, and p-Akt (Ser473) were from the Cell Signaling Technology (Beverly, MA). Polyclonal antibodies against p-CK2 were from the Calbiochem (San Diego, CA). CK2 and PTEN mRNA Expression. The expression of CK2 and PTEN mRNA was determined by reverse transcription-polymerase chain reaction. Polymerase chain reaction primers for amplification of CK2 were designed based on the sequences obtained (sense, 5 CCT CGA GAA TAC TGG GAT TAC GAG-3 ; antisense, 5 -AGC CAT CCA CTA ACC ACT TAC AGG-3 ) (Wirkner et al., 1998). Reverse transcription-polymerase chain reaction was carried out in a total volume of 50 l containing reverse transcription reaction, 10 Taq polymerase buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0 at 25°C, 1.0% Triton X-100, 25 mM MgCl2, 100 pmol of primers, and 1 U of Taq polymerase; Promega, Madison, WI). The conditions were 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s), and extension at 72°C (90 s), followed by a 10-min extension reaction at 72°C. The primer for sense PTEN was 5 -TCT ACT CCT CCA ACT CAG GAC-3 , and for antisense PTEN was 5 -CAT TAT CCG CAC GCT CTA TAC-3 (Konu et al., 2001). The conditions were 35 cycles of denaturation at 94°C (1 min), annealing at 60°C (1 min), and extension at 72°C (1 min), followed by a 10-min extension reaction at 72°C. Endogenous CK2 Activity in Cell Lysate. CK2 activity was assayed by using casein kinase 2 assay kit (Upstate Biotechnology, Lake Placid, NY). The 2.5 to 5 g of proteins from the cleared lysate was used for the assay of CK2 activity, measured by addition of the specific peptide R3D3SD3 (1 mM) in the presence of 20 mM MOPS, pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovaradate, 1 mM dithiothreitol, and 10 l of [ -P]ATP (100 Ci) in a total volume of 50 l, and the radioactivity was measured by scintillation counting. CK2 activity value represents pmol phosphate incorporated into CK2 substrate peptide per min per nanogram of CK2 according to the manual of the product company. Drugs. Cilostazol (OPC-13013), (6-[4-(1-cyclohexyl-1H-tetrazol-5yl) butoxy]-3,4-dihydro-2-(1H)-quinolinone), generously donated from Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan), and dissolved in dimethyl sulfoxide as a 10 mM stock solution. TNF(Upstate Biotechnology) was dissolved in the phosphate-buffered saline as a 10 g/ml stock solution. Others were NS-1619 (Sigma/ RBI, Natick, MA) and iberiotoxin (Upstate Biotechnology). MTT and Fig. 2. Effect of cilostazol on the p-CK2/CK2, p-PTEN/PTEN, p-Akt/Akt, and p-CREB/CREB levels in the presence of TNFin SK-N-SH cells. A, concentration dependently decreased p-CK2 levels in response to TNF( 10–100 ng/ml) in the SK-N-SH cells. B and C, representative Western blot assay and densitometric analysis. Cilostazol ( 1–100 M) significantly increased the p-CK2, p-Akt, and p-CREB in the SK-N-SH cells, whereas the p-PTEN level was significantly suppressed by cilostazol. Application of iberiotoxin ( 0.3–3 M) reversed the whole cilostazol effects. CK2, PTEN, Akt, and CREB expressions were little changed. Values are means S.E.M. from four experiments. ##, P 0.01 versus none; , P 0.05; , P 0.01; , P 0.001 versus TNFalone; †, P 0.05; ††, P 0.01 versus TNFplus 10 M cilostazol. Cilostazol Suppresses TNF-Induced Neuronal Cell Death 99 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from