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Cisplatin Alters Nitric Oxide Synthase Levels in Human Ovarian Cancer Cells: Involvement in P53 Regulation and Cisplatin Resistance

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Cisplatin Alters Nitric Oxide Synthase Levels in Human Ovarian Cancer Cells: Involvement in P53 Regulation and Cisplatin Resistance British Journal of Cancer (2008) 98, 1803 – 1809 & 2008 Cancer Research UK All rights reserved 0007 – 0920/08 $30.00 www.bjcancer.com Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance 1,2,3 1,2,4 3,5 *,1,2 EL Leung , M Fraser , RR Fiscus and BK Tsang 1 Reproductive Biology Unit and Division of Gynecologic Oncology, Department of Obstetrics and Gynecology and Cellular and Molecular Medicine, 2 University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9; Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 3 4E9; Department of Physiology, Faculty of Medicine and Centre of Gerontology and Geriatrics, Chinese University of Hong Kong, Shatin, NT, Hong Kong The present study determines if (1) basal protein levels of nitric oxide (NO) synthases (eNOS, iNOS, and nNOS) are different in cisplatin-sensitive (OV2008) and counterpart cisplatin-resistant (C13*) human ovarian cancer cells, (2) cisplatin alters NOS levels, (3) NO donor causes apoptosis and p53 upregulation, (4) NO donor sensitises C13* cells to cisplatin via p53 upregulation (determined by p53 siRNA gene-knockdown), and (5) inhibition of endogenous NOS alters cisplatin-induced apoptosis. Basal iNOS levels were higher in OV2008 cells than in C13* cells. Cisplatin upregulated iNOS, but dramatically reduced eNOS and nNOS, in OV2008 cells only. Failure of cisplatin to upregulate iNOS and downregulate eNOS/nNOS in cisplatin-resistant C13* cells may be an aetiological factor in the development of resistance. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased p53 protein levels and induced apoptosis in both cell types, and enhanced cisplatin-induced apoptosis in C13* cells in a p53-dependent manner (i.e., enhancement blocked by p53 siRNA). Specific iNOS inhibitor 1400W partially blocked cisplatin-induced apoptosis in OV2008 cells. G In cisplatin-resistant C13* cells, blocking all NOSs with N -amino-L-arginine dramatically changed these cells from cisplatin-resistant to cisplatin-sensitive, greatly potentiating cisplatin-induced apoptosis. The data suggest important roles for the three NOSs in regulating chemoresistance to cisplatin in ovarian cancer cells. British Journal of Cancer (2008) 98, 1803–1809. doi:10.1038/sj.bjc.6604375 www.bjcancer.com Published online 27 May 2008 & Translational Therapeutics 2008 Cancer Research UK Keywords: nitric oxide; ovarian cancer; apoptosis; NOS; p53; cisplatin Cisplatin (CDDP) is a widely used first-line chemotherapeutic At high concentrations (typically 4100 mM), NO donors agent for ovarian cancer (Siddik, 2003), and the induction of generate toxic concentrations of NO (e.g., 4100 nM), inducing apoptosis is a key determinant of CDDP sensitivity. Indeed, we and apoptosis in many mammalian cells (Fiscus, 2002; Fiscus et al, others have demonstrated that failure of CDDP to induce apoptosis 2002), including cancer cells (Wink et al, 1998). However, NO at is a major component of CDDP resistance (Sasaki et al, 2000; Yuan lower, more physiological levels (e.g., 1–20 nM) often has the et al, 2003; Fraser et al, 2003a). CDDP induces DNA crosslinks, opposite effect, preventing (or attenuating) the onset of apoptosis upregulation of p53, and subsequent p53-dependent apoptosis in many mammalian cells, including lung fibroblasts (Wink et al, (Siddik, 2003). However, in CDDP-resistant ovarian cancer cells, 1993), human B lymphocytes (Mannick et al, 1994), and many failure to properly regulate p53 can contribute to the resistance normal and transformed neural cells (Fiscus, 2002; Fiscus et al, (Fraser et al, 2003a, b). Furthermore, basal activity of soluble 2002). In most cells, the anti-apoptotic effects of low-level NO are guanylyl cyclase (sGC), an intracellular receptor for nitric oxide attributed to the stimulation of sGC with subsequent elevation of (NO), suppresses apoptosis in ovarian cancer cells, partly via cyclic guanosine monophosphate (cGMP) levels and increased inhibition of p53 accumulation and activation (Fraser et al, 2006). activation of protein kinase G (PKG). The anti-apoptotic role of We hypothesised that basal sGC activity and regulation of p53 may cGMP/PKG signalling pathway has been further established using depend on endogenous NO formed by one of the three NO natriuretic peptides, atrial natriuretic peptide, and B-type/brain synthases (NOSs), eNOS (also called NOS3), iNOS (also called natriuretic peptide (BNP), which activate cGMP/PKG via an NOS2), or nNOS (also called NOS1) and that resistance to CDDP in alternate pathway (i.e., activation of particulate guanylyl cyclase). ovarian cancer cells may involve altered expression of the NOSs. Atrial natriuretic peptide and BNP are exceptionally effective inhibitors of stress-induced apoptosis in PC-12 phaeochromo- *Correspondence: Dr BK Tsang, Chronic Disease Program, Ottawa cytoma and in NG108-15 neuroblastoma–glioma hybrid cells, Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, including suppressing the toxic/pro-apoptotic effects of high-level Canada K1Y 4E9; NO (Fiscus et al, 2001; Cheng Chew et al, 2003). Even basal E-mail: [email protected] levels of cGMP, generated primarily by basal sGC activity, and 4 Current address: Ontario Cancer Institute, Princess Margaret Hospital, subsequent partial activation of PKG are sufficient (in fact, Toronto, Ontario, Canada M5G 2M9. necessary) to prevent spontaneous apoptosis in certain 5 Current address: Nevada Cancer Institute, One Breakthrough Way, Las cells, including NG108-15 cells (Fiscus, 2002; Fiscus et al, 2002), Vegas, NV, 89135, USA. immortalised uterine epithelial cells (Chan and Fiscus, 2003), and Received 3 January 2008; revised 7 April 2007; accepted 8 April 2008; human ovarian cancer cells (Fraser et al, 2006). Because sGC is published online 27 May 2008 activated by NO at very low concentrations (i.e., 50% of maximal NOSs regulate chemoresistance/p53 in ovarian cancer EL Leung et al 1804 sGC activity stimulated by only 4 nM NO; Bellamy et al, 2002), low Protein extraction and western blotting endogenous level of NO, generated by one of the three NOSs, may be responsible for stimulating basal sGC activity and inhibiting Cells were pelleted and lysed in ice-cold lysis buffer (pH 7.4) apoptosis in these cells. containing 50 mM Hepes, 150 mM NaCl, 1.5 mM MgCl2,1mM EGTA, 100 mM NaF, 10 mM NaPPi, 10% glycerol, and 1% Triton X-100. Previously, melanoma cells (Tang and Grimm, 2004), transformed À1 b-cell line RINm5F (Tejedo et al, 2001), and transformed human Protease inhibitors PMSF (1 mM), aprotinin (125 000 IU ml ), and leukocytes (Mannick et al, 1997) were reported to generate Na3VO4 (1 mM) were added to the lysis buffer freshly. Cell lysates 1 endogenous NO capable of suppressing apoptosis. Endogenous NO were sonicated briefly for three cycles (5 s per cycle, 0 C). is also believed to be responsible for aggressive tumour progression, Sonicates were incubated on ice for 1 h and pelleted by metastasis, and poor survival in cancer patients with tumours centrifugation (15 000 g; 20 min). Protein concentration was exhibiting increased iNOS expression (Thomsen and Miles, 1998). determined using Bio-Rad DC Protein Assay Kits. Equal amounts However, other studies focusing on ovarian cancer have shown that of proteins (50–100 mg) were loaded and resolved by 15% overexpression of iNOS correlates with increased apoptosis (Son and SDS-PAGE and electrotransferred (110 V, 1 h) onto nitrocellulose Hall, 2000; Rieder et al,2001).Thus,endogenousNOmaycontribute membranes (Bio-Rad) as previously described (Fraser et al, 2006). to either anti-apoptotic or pro-apoptotic effects in cancer cells, Membranes were blocked (at room temperature, 1 h) with 5% most likely depending on cell type, local NO levels, source of NO Blotto (Tris-HCl (10 mM; pH 8.0), NaCl (150 mM), Tween 20 production, and other conditions (e.g., concurrent overproduction of (0.05%, v/v; TBS-Tween 20) containing skim milk (5%; w/v)), then superoxide anion, which can combine with NO to form the toxic pro- incubated overnight with primary antibodies iNOS, eNOS, nNOS, oxidant peroxynitrite) (Fiscus, 2002; Fiscus et al, 2002). and p53 (1 : 1000) or GAPDH (1 : 200 000)), and subsequently with The present study determines whether eNOS, iNOS, and nNOS the appropriate horseradish peroxidase-conjugated secondary Translational Therapeutics are involved in regulating p53 accumulation and CDDP resistance antibody (1 : 2000 in 5% Blotto; at room temperature for 1 h). in ovarian cancer cells. CDDP-sensitive OV2008 cells and its The membranes were washed with TBS-Tween 20 thrice (15 min resistant variant C13* cells were used. Effects of NO donor and per wash), peroxidase activity was visualised with ECL reagent NOS inhibitors on CDDP-induced p53 regulation and apoptosis (Amersham Biosciences), and exposed to Hyperfilm ECL (Amer- were also studied. Overall, the present study shows that all three sham Biosciences, Buckinghamshire, UK). Signal intensity was isoforms of NOS are expressed in both cell lines, but protein determined densitometrically by Scion Image Software (version levels are differentially regulated by CDDP in CDDP-sensitive and 4.02; Scion Corporation, Frederick, MD, USA). CDDP-resistant cells. Furthermore, the data suggest that iNOS contributes to CDDP-induced apoptosis in CDDP-sensitive cells, RNA interference whereas eNOS/nNOS contributes (in a p53-independent manner) to chemoresistance by suppressing CDDP-induced apoptosis in After plating cells
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