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Originally published In Press as doi:10.1074/jbc.M312381200 on February 26, 2004

J. Biol. Chem., Vol. 279, Issue 19, 20267-20276, May 7, 2004
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Down-regulation of Survivin in Nitric Oxide-induced Cell Growth Inhibition and Apoptosis of the Human Lung Carcinoma Cells*

Jui-I Chao{ddagger}, Pao-Chen Kuo, and Tzu-Sheng Hsu

From the Molecular Toxicology Laboratory, Institute of Pharmacology and Toxicology, College of Life Sciences, Tzu Chi University, Hualien 970, Taiwan

Received for publication, November 12, 2003 , and in revised form, February 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Survivin is expressed in most tumor cells and has been associated with both anti-apoptosis and mitotic progression. However, the mechanism of regulation of the survivin expression remains unclear. In this study we investigated the expression and regulation of survivin in the nitric oxide (NO)-exposed human lung carcinoma cells. The lung carcinoma cell lines CL3, H1299, and A549 but not normal lung fibroblast expressed high levels of survivin proteins. NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) decreased the survivin expression. SNAP (0.4 mM, 24h)and SNP (1 mM, 24 h) significantly induced cytotoxicity and apoptosis in lung carcinoma cells. Furthermore, SNAP inhibited the cell growth and increased the fractions of G2/M phase. The levels of cyclin B1 and phospho-cdc2-(Thr-161) proteins were inhibited in the NO-exposed cells. The cdc25 phosphatase inhibitors (Cpd 5 and NSC 663284) and the cdc2 kinase inhibitors (alsterpaullone and purvalanol A) enhanced SNP-induced cytotoxicity and the decrease in survivin expression. However, overexpression of survivin by a pOTB7-survivin vector reduced SNP-induced cell growth inhibition and cytotoxicity. In addition, SNP activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The specific p38 MAP kinase inhibitor, SB202190, significantly decreased the cytotoxicity and increased the survivin levels in NO donor-treated and inducible NOS-transfected cells. Conversely, anticancer agents including quercetin, arsenite, and cisplatin but not genistein increased the levels of survivin protein. Our results indicated for the first time that NO inhibited the expression of survivin, which was down-regulated by the p38 MAP kinase pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Survivin is expressed in the embryo stage and most human tumor cells, but it is undetectable in normal adult cells (1–3). The gene map position of survivin is located on the 17q25 of chromosome and is expressed as a 16.5-kDa protein (1, 4, 5). Survivin is a member of the inhibitors of apoptosis (IAP)1 family (6, 7) that contains a single zinc binding motif, called baculoviral IAP repeat (4, 8, 9). The homology of survivin to IAP has been demonstrated by the observation that exhibits anti-apoptotic activity and inhibits the activity of caspases (4, 5, 9, 10). In addition, survivin has been shown to maintain the integrity of microtubule (2, 11), which is expressed at the G2/M transition stage and concentrates on the mitotic spindle (1–3, 12). Moreover, the inhibition of survivin has been found to correlate with the cell cycle defects (2, 3, 13). The survivin activity resulted from the phosphorylation of Thr-34 by the mitotic kinase complex cdc2-cyclin B1 (3, 10). However, the precise mechanism of the regulation of survivin gene and protein expression of the cancer cells is still not clear.

Nitric oxide (NO) is an important signaling messenger that has been shown to play important roles in many physiological and pathological conditions (14–16). Endogenous NO is generated from L-arginine by three major types of NO synthase (NOS), i.e. endothelial NOS), neural NOS, and inducible NOS (iNOS) (17, 18). It has been shown that NO can induce apoptosis in a variety of tumor cells (19, 20). NO triggers apoptosis by mechanisms involving induction of stress proteins, mitochondrial disruption, release of cytochrome c, and caspase activation (21–25). In a recent study, NO has been shown to induce an increase in protein levels of p21 and a G2/M cell cycle checkpoint (26). The p38 mitogen-activated protein (MAP) kinase pathway can mediate the cell growth arrest and apoptosis in response to different stimuli (27–29). It has been found that the p38 MAP kinase mediates NO-induced apoptosis (29–31). The specific inhibitor of p38 MAP kinase, SB203580, can inhibit the iNOS mRNA expression and the NO production in macrophages (30). Furthermore, p38 MAP kinase regulates NO-induced apoptosis that associates with the p53 accumulation and the caspase-3 activation in chondrocytes (29).

In this study we investigated the expression and regulation of survivin in the NO-induced cell growth inhibition and apoptosis in the human lung cancer cells and its possible mechanisms. We further studied the role of p38 MAP kinase in the survivin expression on cell survival of the NO-exposed cells. Using NO donors or transfection of an iNOS expression vector, we found that the survivin expression in the lung carcinoma cells was down-regulated via the p38 MAP kinase pathway and may involve the inhibition of cdc2-cyclin B1 complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies—Cpd 5 and NSC 663284 compounds were kindly provided by Dr. C. Chen of National Dong-Hwa University. Arsenite, cisplatin, genistein, quercetin, S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO) were purchased from Sigma. Alsterpaullone, purvalanol A, and SB202190 were purchased from Calbiochem. Anti-phospho-p38 (9211S), anti-phospho-cdc2-(Thr-161) (9114S), and anti-XIAP (2042) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-Bcl-2 (100), anti-cdc2 (17), anti-ERK-2 (C-14), anti-p38 (C-20), and anti-survivin (FL-142) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-cyclin B1 (Ab-2) was purchased from Oncogene Sciences (Cambridge, MA).

Cell Culture—The A549 cell line was derived from human lung carcinoma of a 58-year-old Caucasian male (32). The H1299 cell line was p53-null and was derived from a non-small cell lung adenocarcinoma tumor (33, 34). The CL3 cell line, from a non-small-cell lung adenocarcinoma tumor of a 60-year-old male patient in Taiwan, was established by Dr. P. C. Yang of the National Taiwan University Hospital (35). The lung adenocarcinoma cells were kindly provided by Dr. J. L. Yang of the National Tsing-Hua University. The TSGH8301 cell line was derived from a bladder cancer of a male patient in Taiwan, which was purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). The PC12 cell line, derived from rat adrenal pheochromocytoma cells (36), was a gift of Dr. D. I. Yang of Tzu Chi University. These cell lines were cultured in RPMI 1640 medium (Invitrogen) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, L-glutamine (0.03%, w/v), sodium bicarbonate (2.2%, w/v), and 10% fetal bovine serum. The cells were maintained at 37 °C and 5% CO2 in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).

Cytotoxicity Assay—The cells were plated in 96-well plates at a density of 1 x 104 cells/well for 16–20 h. Then the cells were treated with 0–0.8 mM SNAP or 0–2 mM SNP for 24 h in serum-free RPMI 1640 medium. After drug treatment, the cells were washed twice with phosphate-buffered saline (PBS) and were cultured in complete RPMI 1640 medium (containing 10% serum) for 2 days. Subsequently, the medium was replaced, and the cells were treated with 500 µg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) in complete RPMI 1640 medium for 4 h. The surviving cells converted MTT to formazan that generates a blue-purple color when dissolved in dimethyl sulfoxide (Me2SO) (37). The intensity was measured at 545 nm using a plate reader for enzyme-linked immunosorbent assays. The absorbance of each treatment was from the average of six repeats (wells). The relative percentage of survival was calculated by dividing the absorbance of treatment by that of the control in each experiment.

Apoptosis Assay—The adherent cells were cultured on coverslips. After exposure to SNAP (0.4 mM, 24 h), the cells were washed twice with isotonic PBS (pH 7.4). Annexin V-fluorescein isothiocyanate (FITC) binding assay kit (Strong Biotech, Taipei, Taiwan) was used for the examination of apoptosis. The cells were stained with annexin V-FITC in binding buffer containing propidium iodide for 30 min in the dark. Then the cells were washed three times with isotonic PBS (pH 7.4) and analyzed using fluorescence microscope.

During the apoptotic process, the enrichment of mono- and oligonucleosomes in cytoplasm of the apoptotic cell is due to DNA degradation before plasma membrane breakdown (38). The cell death detection ELISAplus kit (Roche Applied Science) was based on the quantitative sandwich enzyme-immunoassay using antibodies directed against DNA and histones. Exponentially growing cells were plated at a density of 1 x 105 cells/p60 dish for 16–20 h. Then the cells were treated with or without 20 µM cPTIO for 1 h before exposure to 0–2 mM SNP for 24 h in serum-free RPMI 1640 medium. After treatment, the cells were washed twice with PBS and cultured in complete RPMI 1640 medium for 2 days. The cells were then analyzed for apoptosis, determined by the amount of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. The intensity was measured at 405 nm using a plate reader for enzyme-linked immunosorbent assays.

NO Production Assay—NO production was detected by measuring the stable NO metabolite, nitrite, in cultured medium using a spectrophotometric method based on the Griess reaction (39). After exposure to SNAP or SNP, the 100-µl culture supernatant was mixed with 100 µlof Griess reagent (0.1% naphthylethylenediamine dihydrochloride, 1% sulfanilamide, and 1.25% H3PO4). After incubation for 5–10 min at room temperature, nitrite concentrations were measured at 550 nm using a plate reader for enzyme-linked immunosorbent assays.

Cell Cycle Analysis—Asynchronized-CL3 and -H1299 cells were plated at a density of 1.5 x 106 cells per 60-mm Petri dish in complete RPMI 1640 medium for 16–20 h. Then the cells were treated with 0–0.8 mM SNAP for 24 h in serum-free medium. To examine the synchronized cells, CL3 cells were grown to 80–90% confluence and re-cultured in serum-free RPMI 1640 medium for 48 h. The cells were trypsinized and plated at a density of 1.5 x 106 cells per 60-mm Petri dish in complete RPMI 1640 medium containing 1 µg/ml aphidicolin. After 24 h of aphidicolin treatment, the cells were washed twice with PBS and replaced with fresh RPMI medium. After incubation for 4 h, the cells were exposed to 0.4 mM SNAP for 2 h in serum-free medium. At the end of treatment, the cells were collected and fixed with ice-cold 70% ethanol overnight at 4 °C. After centrifugation, the cell pellets were treated with 4 µg/ml propidium iodide solution containing 100 µg/ml RNase and 1% Triton X-100 for 30 min and placed at 4 °C before flow cytometry analysis. Samples were analyzed in a FACScalibur system (BD Biosciences) using CellQuest software.

Cell Growth Assay—CL3 and H1299 cells were separately plated onto a p60 Petri dish at a density of 1 x 106 cells in complete RPMI 1640 medium for 18 h. Then the cells were treated with 0–0.8 mM SNAP for 24 h. At the end of the treatment, the cells were trypsinized, and the total cell number was counted.

Mitotic Index Analysis—CL3 cells were plated at a density of 1 x 105 cells/p60 Petri dish for 16–20 h. Subsequently, the cells were treated with 0–0.8 mM SNAP for 24 h. After drug treatment, the cells were trypsinized and treated with hypotonic 0.05% KCl solution for 5–10 min. After centrifugation, the supernatant was removed, and the cell pellet was fixed with methanol/acetic acid solution (3:1, v/v). The cell suspension was dropped onto a clean slide and stained with a 10% Giemsa solution (Sigma). A total of 200 cells were examined for the calculation of mitotic index in each treatment.

Indirect Immunofluorescence Analysis—The cells were cultured on coverslip, which was kept in a 60-mm Petri dish for 16–20 h before treatment. After exposure to SNAP (0.4 mM, 24 h), the cells were washed twice with isotonic PBS (pH 7.4) and fixed in ice-cold 4% paraformaldehyde solution in PBS for overnight at 4 °C. The coverslips were washed three times with PBS, and nonspecific binding sites were blocked in PBS containing 10% normal goat serum, 0.25% Triton X-100 for 1 h. The cells were incubated with rabbit anti-survivin antibody (1:250) in PBS containing 0.25% Triton X-100 and 10% normal goat serum overnight at 4 °C and washed three times with 0.25% Triton X-100 in PBS. Then the cells were incubated with goat anti-rabbit Cy5TM (1:500) (Amersham Biosciences) in PBS containing 0.25% Triton X-100 and 10% normal goat serum for 3 h at 37 °C and washed 3 times with 0.25% Triton X-100 in PBS. The nuclei were stained with 2.5 µg/ml Hoechst 33258 (Sigma) for 10 min. Finally, the samples were stored in the dark at 4 °C until examined under a fluorescence microscope. The images were digitally processed for presentation by Adobe Photoshop software (Version 6.0), Adobe Systems (San Jose, CA).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis—Cells were plated at a density of 1–2 x 106 cells per 60-mm Petri dish in culture medium. After treatment the cells were washed twice with PBS. Total cellular RNA was purified using RNA extraction kit (Geneaid, Taoyuan, Taiwan) according to the manufacturer's protocol. RNA concentrations were determined by spectrophotometry. cDNAs were synthesized by Moloney murine leukemia virus reverse transcriptase with oligo(dT)15 primer (Promega, Madison, WI). Each reverse transcript was amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The primer pairs used for amplification of survivin (40) were 5'-GGCATGGGTGCCCCGACGTTG-3' (sense) and 5'-CAGAGGCCTCAATCCATGGCA-3' (antisense) and for GAPDH were 5'-CGGAGTCAACGGATTTGGTCGTAT-3' (sense) and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (antisense). RT-PCR was performed by a DNA thermal cycler, 5331/Mastercycler gradient (Eppendorf, Hamburg, Germany). The initial denaturation was performed at 95 °C for 5 min followed by 35 cycles at 95 °C for 1 min, 55 °C for 1 min, and finally 72 °C for 1 min. The PCR products were visualized on 2% agarose gels with ethidium bromide staining under UV transillumination, and photographs were taken (Ezcatcher EZC-2002, Medclub Scientific Co., Taipei, Taiwan). The amplification products of survivin and GAPDH were 439 and 306 bp, respectively.

Western Blot Analysis—Western analyses of Bcl-2, cdc2, phospho-cdc2, cyclin B1, ERK-2, p38, phospho-p38, survivin, and XIAP were performed using specific antibodies. Cells were lysed in the ice-cold cell extraction buffer (pH 7.6) containing 0.5 mM dithiothreitol, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2, 75 mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, and 0.1% Triton X-100. The protease inhibitors including 1 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 100 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride were added to the cell suspension. The cell extracts were gently rotated at 4 °C for 30 min. After centrifugation, the pellets were discarded, and supernatant protein concentrations were determined by the BCA protein assay kit (Pierce). Equal amounts of proteins (20–60 µg/well) were subjected to electrophoresis by 10–12% sodium dodecyl sulfate-polyacrylamide gels. After electrophoretic transfer of proteins onto polyvinylidene difluoride membranes, they were sequentially hybridized with primary antibody and followed with a horseradish peroxidase-conjugated second antibody (Bio-Rad). Finally, the protein bands were visualized using the enhanced chemiluminescence detection system (PerkinElmer Life Sciences). To verify equal protein loading and transfer, ERK-2 and p38 were used as the protein loading control.

Transfection—The pOTB7-survivin expression vector was from IMAGE clone (ID 2961114) and was purchased from American Type Culture Collection (ATCC, MGC-8592). The piNOSL8-CMV expression vector (41) and the pcDNA3.1 control vector were kindly provided by Dr. D. I. Yang (Tzu Chi University). These plasmids were employed for transient transfections in CL3 cells. The vectors of pOTB7-survivin and piNOSL8-CMV expressed human survivin and mouse iNOS, respectively. CL3 cells (1 x 106 cells/p60 dish) were transfected with 3 µg of expression vector or control vector by using LipofectAMINETM 2000 (Invitrogen) in 1.5 ml of serum-free DMEM medium for 5 h at 37 °C in a CO2 incubator according to the manufacturer's recommendations. Then 1.5 ml of DMEM medium with 20% fetal bovine serum was added without removing the transfection mixture and incubation proceeded for an additional 24 h. After transient transfection, the cells were subjected to cell growth, Western blot analysis, or MTT assay as described above.

Quantitative Analysis—The percentage of cell cycle phases was analysis by ModFit LT software (Version 2.0, BD Biosciences). Each experiment was repeated at least 3 times. For comparing the level of Western blot protein between samples, a densitometer (Molecular Dynamics, Personal Densitometer ST) was used for estimating the intensity of each band on the X-film. Each experiment was repeated at least 3 times. A gel digitizing software, Un-Scan-It gel (Version 5.1, Silk Scientific, Inc.), was used to quantify the intensity of each band on the gel from the RT-PCR products of samples. Each experiment was repeated 2–5 times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
NO Mediates Cytotoxicity, Apoptosis, and Decreased Survivin Expression in the Lung Cancer Cells—As shown in Fig. 1A, SNAP and SNP significantly generated NO in both CL3 and H1299 human lung cancer cells. Concentrations of SNAP (0.4 and 0.8 mM) and SNP (1 and 2 mM) increased the NO production by 7–16-fold (Fig. 1A). To determine the involvement of NO in the regulation of cell survival, the cells were exposed to increased concentrations of SNAP (0–0.8 mM) or SNP (0–2 mM), and the percentage of cell survival was measured by MTT assay. As shown in Fig. 1B, SNAP induced cell death in a concentration-dependent manner, and ~40, 55, and 65% of cells survived after exposure to 0.4 mM SNAP for 24 h in CL3, H1299, and A549 cells, respectively. Similarly, SNP concentration dependently induced cytotoxicity in these cell lines (Fig. 1B). We further assessed apoptosis from the cells that had been exposed to NO donors. SNAP-treated H1299 cells were heavily stained with annexin V-FITC (early apoptotic cells), which displayed a green color (Fig. 1C, lower right picture) and induced chromatin condensation and nuclear fragmentation (Fig. 2B), indicating these that cells were in the apoptotic stage. The late apoptotic or necrotic cells were stained with annexin V-FITC and propidium iodide, which displayed a yellow color (Fig. 1C, lower right picture). In contrast, the untreated cells did not stain with fluorochrome (Fig. 1C, upper right picture). In addition, apoptosis was measured using the cell death detection ELISAplus kit. As shown in Fig. 1D, the extent of apoptosis was concentration-dependently increased after exposure to 1–2 mM SNP for 24 h in CL3 cells. Pretreatment with an NO scavenger, cPTIO (42, 43), significantly reduced SNP-induced apoptosis (Fig. 1D). In the absence of SNP, the concentration of cPTIO used was without effect on apoptosis in CL3 cells (results not shown).



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  		FIG. 1.
Effects of NO donors on the cytotoxicity and apoptosis in the lung carcinoma cells. A, the cells were treated with 0–0.8 mM SNAP or 0–2 mM SNP for 24 h. The concentration of NO was measured by detecting the stable NO metabolite, nitrite, using a spectrophotometric method. The data were the mean ± S.E. from four experiments. B, cell survival was measured by MTT assay. Results were obtained from 12–20 experiments, and the bar represents the mean ± S.E. C, the H1299 cells were treated with 0.4 mM SNAP for 24 h. Annexin V-FITC displayed green fluorescence. The nuclei were stained with propidium iodide, which displayed red fluorescence. D, the levels of apoptosis were assayed by the cell death detection ELISAplus kit. Results were obtained from three experiments, and the bar represents S.E. p < 0.05 (*), p < 0.01 (**) and p < 0.01 (##), indicate significance between untreated and NO-donor treated samples. #, p < 0.05 between SNP alone and pretreatment with cPTIO.

 



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  		FIG. 2.
Effects of NO donors on the levels of survivin protein and gene expression. A, the total protein extracts in a variety of cell lines were prepared for Western blot analysis using anti-survivin and ani-ERK-2 antibodies. B, H1299 cells were treated with 0.4 mM SNAP for 24 h. The cells were incubated with rabbit anti-survivin antibody and then incubated with goat anti-rabbit Cy5. The survivin protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. C, CL3 and H1299 cells were treated with 0–2 mM SNP for 24 h, and whole cell extracts were prepared and subjected to immunoblot analysis. The relative protein levels under each treatment were the average of 3–9 independent experiments. D, CL3 cells were treated with 0.4–0.8 mM SNAP or 2 mM SNP for 24 h. The survivin and GAPDH levels were analyzed by the RT-PCR method. E, CL3 cells were treated with 1–2 mM SNP or 0.4–0.8 mM SNAP for 24 h. The total protein extracts were prepared for immunoblot analysis using anti-XIAP, Bcl-2, and ani-ERK-2 antibodies.

 
To examine the expression of survivin in tumor cells, total cellular proteins were extracted and subjected to immunoblot analysis. As shown in Fig. 2A, the tumor cell lines CL3, H1299, A549, and PC12 but not TSGH8301 expressed high levels of survivin protein. The survivin protein was undetectable in the human normal lung fibroblast that was used as a negative control (Fig. 2A). To further study the effect of NO donors on the survivin protein expression, cells were treated with SNAP or SNP and subjected to immunofluorescence staining and immunoblot analysis. As shown in Fig. 2B, the intensity of red fluorescence (Cy5) exhibited by survivin was significantly reduced when exposed to 0.4 mM SNAP for 24 h in H1299 cells. Furthermore, SNP reduced the level of survivin protein in both CL3 and H1299 cells in a concentration-dependent manner (Fig. 2C). ERK-2 protein was used as an internal control in this study. Additionally, the RT-PCR was used to examine the survivin gene expression. As shown in Fig. 2D, both SNAP and SNP decreased the level of survivin gene expression in CL3 cells. However, the protein levels of XIAP and Bcl-2 were not significantly decreased in NO donor-treated CL3 cells (Fig. 2E).

SNAP Induces Cell Growth Inhibition and Increases the G2/M Fractions in CL3 and H1299 Cells—To examine the effect of NO donors on tumor cell growth, CL3 and H1299 cells were plated at a density of 1 x 106 cells per p60 dish and treated with 0–0.8 mM SNAP for 24 h. Cell numbers were counted using a hemocytometer. As shown in Fig. 3A, SNAP inhibited cell growth in a concentration-dependent manner in both CL3 and H1299 cell lines. To further determine the possible involvement of NO in the regulation of cell cycle, the effect of SNAP on CL3 and H1299 cells was analyzed by flow cytometry. SNAP decreased the G1 fraction and increased the G2/M fraction in CL3 and H1299 cells (Fig. 3B). To further determine the level of the mitotic progression in SNAP-treated cells, the mitotic index was examined. SNAP concentration dependently increased the mitotic index of CL3 cells (Fig. 3C). About 15% of 0.8 mM SNAP-treated CL3 cells were in the mitotic phase, whereas less than 5% of the untreated cells were in the mitotic phase (Fig. 3C). We further examined the effect of SNAP on the aphidicolin-synchronized CL3 cells. The fractions of G2/M phase were significantly increased at 9 h after terminating the SNAP treatment. Also, SNAP inhibited the cell growth in the synchronized CL3 cells (results not shown).



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  		FIG. 3.
Effects of SNAP on cell growth and cell cycle progression in the lung carcinoma cells. A, CL3 and H1299 cells were treated with 0–0.8 mM SNAP for 24 h. The data represented the average values from three experiments. B, cells were treated with 0–0.8 mM SNAP for 24 h. Cells were trypsinized and subjected to flow cytometry analyses. Results were obtained from 5–6 experiments, and the bar represents S.E. C, CL3 cells were treated with 0–0.8 mM SNAP for 24 h. After treatment, the cells were trypsinized and then subjected to mitotic index analyses. Results were obtained from 6–9 experiments, and the bar represents S.E. p < 0.05 (*) and p < 0.01 (**) indicate significance between untreated and SNAP treated samples.

 
Overexpression of Survivin by a pOTB7-survivin Vector Reduces NO-induced Growth Inhibition and Cytotoxicity in CL3 Cells—An expression vector, pOTB7-survivin, and a control vector, pcDNA3.1, were employed for transient transfection in CL3 cells to examine the effect of survivin on NO-induced cell growth inhibition and cell death. As shown in Fig. 4A, the inhibition of cell growth caused by SNP was significantly reversed by the overexpression of survivin. To further investigate the role of survivin in NO-induced cytotoxicity, the pOTB7-survivin-transfected cells were treated with or without SNP (1 mM, 24 h), and the percentage of cell survival was estimated by MTT assay. The pOTB7-survivin vector-transfected cells were more resistant to SNP-mediated cytotoxicity than those control vector-transfected cells (Fig. 4B).



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  		FIG. 4.
Effects of SNP on cell growth and cytotoxicity in the pOTB7-survivin vector-transfected cells. A, CL3 cells were transfected with a survivin expression vector or control vector. After transfection, the cells were plated at a density of 5 x 105 cells/p100 Petri dish for 18 h. Then the cells were exposed to 1 mM SNP for 24 h in serum-free medium, washed twice with PBS, and incubated for various times (0–4 days) before they were counted by a hemocytometer. B, cell survival was measured by MTT assay. Results were obtained from 3–6 experiments, and the bar represents S.E. *, p < 0.05 indicates significance between the control vector and the pOTB7-survivin vector-transfected cells from SNP treatment.

 
Inhibition of cdc2-Cyclin B1 Enhances NO Donor-induced Cell Death with Decreased Survivin Expression—Cells were treated with SNAP or SNP and subjected to immunoblot analysis and RT-PCR to study the involvement of cdc2-cyclin B1 in the NO donor-induced inhibition of survivin. Fig. 5A shows that 0.4 mM SNAP simultaneously inhibited the levels of cyclin B1 and survivin in CL3 and H1299 cells. However, the level of cdc2 protein was not significantly altered. We further examined the level of phospho-cdc2-(Thr-161) protein when CL3 cells were treated with SNP. SNP (1 mM, 24 h) significantly inhibited the protein level of phospho-cdc2-(Thr-161) (Fig. 5B). Also, SNAP (0.4–0.8 mM, 24 h) inhibited the protein level of phospho-cdc2-(Thr-161) in these cells (data not shown). The inhibitors of cdc25 phosphatase and cdc2 kinase were used to further examine the kinase activity of cdc2 involved in SNP-induced cell death. As shown in Fig. 5C, the cdc25 phosphatase inhibitors (1 µM both Cpd 5 and NSC 663284) and the cdc2 kinase inhibitors (0.3 µM alsterpaullone and 1 µM purvalanol A) enhanced SNP-induced cytotoxicity. In addition, 1 µM NSC 663284 or purvalanol A potentiated the SNP-induced decrease in survivin gene expression (Fig. 5D). In the absence of SNP, the concentrations of Cpd 5 (1 µM), NSC 663284 (1 µM), alsterpaullone (0.3 µM), and purvalanol A (1 µM) used in this study were without effect on the cytotoxicity in the CL3 cells (results not shown). However, higher concentrations of Cpd 5 and NSC 663284 (at 5 µM for 24 h) inhibited the survival of ~50% of the CL3 cells. Also, higher concentrations of alsterpaullone (1 µM) and purvalanol A (3 µM) inhibited the survival of ~27 and 61% of the CL3 cells, respectively (results not shown).



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  		FIG. 5.
Effects of inhibitors of cdc25 and cdc2 on NO-induced inhibition of the survivin expression and cytotoxicity. A, the cells were exposed to 0.4 mM SNAP for 24 h. The relative protein levels under each treatment were the average of 4–6 independent experiments. B, the CL3 cells were treated with or without 1 mM SNP for 24 h. The levels of phospho-cdc2-(Thr-161) and ERK-2 proteins were analyzed by Western blot. C, the CL3 cells were treated with 1 mM SNP or 1 mM SNP plus inhibitors (1 µM Cpd 5, 1 µM NSC 663284, 0.3 µM alsterpaullone, or 1 µM purvalanol A) for 24 h. The cell survival was measured by MTT assay. *, p < 0.05 indicates significance between SNP alone and SNP plus inhibitor. D, the CL3 cells were treated with 1 mM SNP in combination with 1 µM NSC 663284 or purvalanol A for 24 h. The survivin and GAPDH levels were analyzed by RT-PCR method.

 
p38 MAP Kinase Is Involved in the Regulation of Survivin Expression—We have investigated the possible role of p38 MAP kinase in NO-mediated decrease of survivin levels. CL3 cells were treated with SNP (0–2 mM) for 24 h, and p38 MAP kinase was assayed by the immunoblot using the phosphospecific antibody. As shown in Fig. 6A, SNP at 1 and 2 mM significantly increased the level of phospho-p38 protein in a concentration-dependent manner. To examine whether p38 MAP kinase activation is involved in regulating the expression of survivin induced by NO, the p38 specific inhibitor, SB202190, was used in this study. The inhibition of survivin expression by 2 mM SNP was reversed by pretreatment with SB202190 (20 µM, 2 h) (Fig. 6B). SB202190 also reversed the level of survivin protein in SNAP-treated CL3 cells (data not shown). In addition, 20 µM SB202190 blocked 0.4 mM SNAP-induced reduction in survivin gene expression in CL3 cells (Fig. 6C). SB202190 alone did not affect the survival of these cells; however, it significantly reduced the 0.8 mM SNAP-induced cell death (Fig. 6D).



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  		FIG. 6.
Effects of SB202190 on the survivin expression and cytotoxicity in the NO-exposed CL3 cells. A, cells were treated with 0–2 mM SNP for 24 h. The protein levels of phospho-p38 and p38 were analyzed by Western blot using anti-phospho-p38 and anti-p38. B, cells were pretreated with 20 µM SB202190 for 2 h before exposure to 2 mM SNP for 24 h. The protein level of survivin was analyzed by Western blot. The relative protein levels under each treatment were the average of 3–6 independent experiments. C, cells were pretreated with 20 µM SB202190 for 2 h before exposure to 0.4 mM SNAP for 24 h. The level of survivin gene expression was analysis by RT-PCR. D, CL3 cells were pretreated with 20 µM SB202190 for 2 h before exposure to 0.8 mM SNAP for 24 h. The cell survival was measured by MTT assay. Results were obtained from 6–10 experiments, and the bar represents S.E. *, p < 0.05 indicates significance between SNAP alone and pretreatment with SB202190.

 
The iNOS Expression Induces the Cytotoxicity and Inhibits the Level of Survivin in CL3 Cells—To further examine the effect of iNOS expression on the protein level of survivin expression, an expression plasmid, piNOSL8-CMV, and a control vector, pcDNA3.1, were employed for this study. As shown in Fig. 7A, the piNOSL8-CMV-transfected CL3 cells exhibited lower levels of survivin protein. The reduction of survivin protein expression caused by iNOS transfection was significantly blocked by 20 µM SB202190 (Fig. 7A). To further investigate the role of p38 pathway in iNOS-induced cytotoxicity, CL3 cells were pretreated with SB202190 (20 µM, 2 h) before transfection with the piNOSL8-CMV plasmid, and the percentage of cell survival was estimated by MTT assay. The control vector or SB202190 alone did not affect the survival of these cells; however, SB202190 significantly reduced the cell death of the piNOSL8-CMV-transfected cells (Fig. 7B).



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  		FIG. 7.
Effects of iNOS expression vector on the cytotoxicity and the survivin protein expression in CL3 cells. A, CL3 cells were transfected with an iNOS expression vector or a control vector. The level of survivin was analyzed by Western blot using a specific antibody. The relative protein levels under each treatment were the average of three independent experiments. The concentration of SB202190 was 20 µM. B, cell survival was measured by MTT assay. Results were obtained from three experiments, and the bar represents S.E. *, p < 0.05 indicates significance between pcDNA3.1 and iNOS vector-transfected cells. #, p < 0.05 indicates significance between iNOS vector alone and pretreatment with SB202190.

 
Distinct Expression of Survivin Induced by Anticancer Agents—The CL3 cells were treated with genistein (30 µM), quercetin (10 µM), sodium arsenite (5 µM), and cisplatin (3 µM) for 24 h, and the whole cell extracts were analyzed for survivin expression using immunoblot assay. The concentrations of anticancer agents used inhibited the cell survival to a similar degree (Fig. 8). The survivin protein expression was significantly reduced when exposed to 1 mM SNP for 24 h. In contrast, quercetin, arsenite, and cisplatin increased the protein level of survivin in CL3 cells. However, the level of survivin protein was not altered by genistein (Fig. 8).



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  		FIG. 8.
Effects of several anticancer agents on the protein levels of survivin. The cells were treated with SNP (1 mM), genistein (30 µM), quercetin (10 µM), arsenite (5 µM), or cisplatin (3 µM) for 24 h. The level of survivin was analyzed by Western blot using specific antibody. The relative protein levels under each blot were the average of four independent experiments. The percentage of cell survival was measured by MTT assay.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Inhibitors of the survivin pathway have been intensely investigated for cancer therapy in recent years. The inhibition of survivin would prevent the anti-apoptosis and mitotic progression in tumor cells, providing important strategy in cancer therapy (3). NO has been shown to trigger apoptosis in a variety of tumor cells (19, 20). In the present study we found that exogenously supplied NO donors or endogenous NO generation by iNOS expression inhibited the survivin expression and induced cell death in the human lung carcinoma cells. Moreover, SNP induced phosphorylation of p38 MAP kinase in CL3 cells. The specific p38 inhibitor, SB202190, was effective in protecting CL3 cells from NO-mediated cell death and blocked the NO inhibition of survivin expression. The inhibition of survivin expression disrupted anti-apoptosis function and increased caspase-3 activity (2). p38 MAP kinase has been associated with the induction of apoptosis in numerous cell types and in response to many different cellular stresses (44–46). It has been shown that p38 MAP kinase can serve as a mediator of NO-induced, caspase-3-associated apoptosis (29–31). Furthermore, another p38 MAP kinase inhibitor, SB203580, has been observed to inhibit the iNOS mRNA expression and the NO production in macrophages (30). We found that iNOS induction inhibited the survivin protein expression, which was reversed by p38 MAP kinase inhibitor. Thus, our study suggests that NO inhibits survivin expression possibly via the p38 MAP kinase-dependent pathway and induces apoptosis in lung carcinoma cells.

The inhibition of survivin has been related to cell cycle defects (2, 13). We found that SNAP induced the cell growth inhibition and increased the G2/M fractions in CL3 and H1299 cells. The activation of cdc2-cyclin B1 complex is required for the mitotic entry, and its inactivation occurs at late anaphase of the mitotic phase. However, SNAP decreased the level of cyclin B1 but did not significantly alter the cdc2 protein level in lung carcinoma cells. The activation of cdc2 is through phosphorylation of Thr-161 by cdc2-activating kinase (CAK) and dephosphorylation of Thr-14 and Tyr-15 by cdc25C phosphatase (47). Cpd 5 and NSC 663284 have been shown to inhibit the cdc25 phosphatases including cdc25C phosphatase that results in the inhibition of cdc2 kinase activity (48, 49). In addition, alsterpaullone and purvalanol A have been used as cdc2 kinase inhibitors (50). In this study, both cdc25 inhibitors (Cpd 5 and NSC 663284) and cdc2 kinase inhibitors (alsterpaullone and purvalanol A) enhanced SNP-induced cell death and the decrease in survivin levels. Our results suggest that NO decreases the level of cyclin B1 and inhibits cdc2 kinase activity, causing the inhibition of survivin activity with enhanced NO-induced cell death. Indeed, the survivin activity has recently been identified to result from the phosphorylation of Thr-34 by the mitotic kinase complex cdc2-cyclin B1 (10). Flavopiridol, a cyclin-dependent kinase inhibitor, suppresses the survivin phosphorylation on Thr-34 and enhances tumor cell apoptosis induced by anticancer agents, e.g. adriamycin and UVB irradiation (3). Furthermore, it has been reported that survivin is required in chromosome segregation and cytokinesis (13, 51, 52). In addition, survivin has been characterized as a chromosomal passenger protein that interacts with Aurora-B and INCENP (13, 53). The chromosomal passenger complex has been found to play a crucial role in the execution of cytokinesis (13, 53). Also, survivin-null cells display abnormal mitotic spindles and failure of cytokinesis (13). We suggest that the inhibition of the survivin expression by NO may be involved in the inhibition of cytokinesis.

The anticancer drugs or chemicals may induce chemo-resistance during cancer therapy in patients. It has been proposed that survivin may serve as a radio- and chemo-resistance factor (54). For example, treatments with adriamycin and taxol increased survivin expression in MCF-7 cells (3). In this study, several anticancer agents induced high levels of survivin protein expression. We found that quercetin, arsenite, and cisplatin but not genistein increased the levels of survivin protein in CL3 cells. It has been proposed that the balance between apoptosis (such as p53 pathway) and survival (such as AKT pathway) participates in the pathogenesis of a variety of cancers (55, 56). The H1299 cell is a p53-null cell line. In this study, we found that NO donors could decrease the survivin expression and induced apoptosis in H1299 cells. Furthermore, SNAP inhibited the cell growth and increased the fractions of G2/M phase in H1299 cells. Therefore, we suggest that NO mediates the inhibition of survivin expression through a p53-independent pathway. However, the mechanisms for the increase in survivin level caused by quercetin, arsenite, and cisplatin need further investigation.

In summary, we propose that the expression of survivin appears to be critical for anti-apoptosis and cell cycle progression, and the survivin gene and protein expression in lung carcinoma cells after NO exposure is down-regulated by p38 MAP kinase. Moreover, NO may inhibit the cdc2-cyclin B1 complex and may decrease the activation of survivin. Inhibitors of cdc25/cdc2 and p38 MAP kinase indicate the opposite role of cdc2-cyclin B1 and p38 MAP kinase on the regulation of survivin in lung cancer cells (Fig. 9). Understanding the mechanisms by which survivin, p38 MAP kinase, and cdc2-cyclin B1 pathways modulate NO-induced cell growth inhibition and apoptosis in cancer cells may contribute to the development of novel therapeutic strategies in such disease states.



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  		FIG. 9.
The model of NO-mediated inhibition of survivin expression.

 

   FOOTNOTES
 
* This work was supported by National Science Council, Taiwan, Grant NSC 91-2320-B-320-020, and Tzu Chi University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Molecular Toxicology Laboratory, Institute of Pharmacology and Toxicology, College of Life Sciences, Tzu Chi University, 701, Section 3, Chung-Yang Rd., Hualien 970, Taiwan. Fax: 886-3-8561465; E-mail: chaoji{at}mail.tcu.edu.tw.

1 The abbreviations used are: IAP, inhibitors of apoptosis; NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; MAP, mitogen-activated protein; SNAP, S-nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction; ERK, extracellular signal-regulated kinase; CMV, cytomegalovirus. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. L. Yang for advice and Dr. P. C. Yang for permission to use CL3 cell line. We also are indebted to Dr. C. Chen and Dr. D. I. Yang for providing the chemical inhibitors (Cpd 5 and NSC 663284) and the plasmids (piNOSL8-CMV and pcDNA3.1), respectively, and Dr. T. C. Tsou for providing TSGH8301 cell line and LipofectAMINETM 2000. In addition, we thank Drs. Ted H. Chiu and Tony J. F. Lee for reading the manuscript.



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