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Nitric Oxide Sensitizes Ovarian Tumor Cells to Fas-Induced Apoptosis
Gynecologic Oncology 73, 257–264 (1999) Article ID gyno.1999.5374, available online at http://www.idealibrary.com on
Nitric Oxide Sensitizes Ovarian Tumor Cells to Fas-Induced Apoptosis 1 Hermes J. Garba´n and Benjamin Bonavida 2 Department of Microbiology, Immunology, and Molecular Genetics, and Jonsson Comprehensive Cancer Center, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, California 90095-1747 Received August 17, 1998
receptor is expressed constitutively in the normal folliculi of the ovary, endometrial gland cells of the uterus, and columnar epithelium of the uterine cervix [1]. During reproductive life more than 99% of the ovarian follicles do not ovulate and are eliminated from the ovary by follicular atresia. Atresia is achieved by autodegeneration of thecal and granulosa cells that comprise the follicle, by the process of apoptosis. Moreover, the sphingomyelin-ceramide cycle can lead to cell suicide by Fas-mediated apoptosis, not only in the immune system, but also in thecal/interstitial cells of the ovarian follicles [2]. It has been shown that ovarian surface epithelial cells (OSE) are specially susceptible to Fas-mediated apoptosis among the normal mouse corpora lutea cultures containing luteal, stromal, endometrial cells, and fibroblasts and also OSE exposed to the Fas-agonist antibody JO2. In vivo, OSE undergo programmed cell death before ovulation and rapidly proliferate to repair the surface of the ovulatory follicle after ovulation [3]. It is noteworthy that most ovarian cancers are derived from the OSE. Thus, Fas/Apo-1 might not only transmit apoptotic signals but also play a more general role in growth control in both normal ovarian tissue and oncogenesis in the ovary. A recent report demonstrated that the majority of ovarian carcinoma cell lines screened for Fas expression were positive and their sensitivity to the anti-Fas agonist antibody correlated with the level of Fas expression [4]. Furthermore, the downregulation of Fas expression and subsequent resistance to antiFas were observed in the drug-resistant human ovarian carcinoma IGR-OVI/VCR, the human breast carcinoma cell line MCF7Adr, and the leukemic lymphoblast CEM/VLB cells, suggesting that the alteration of Fas expression following drugresistance is not restricted to one cell type [5]. The Fas receptor (CD95/APO-1) [6] has been recognized as a central receptor for apoptosis, particularly in mediating nonspecific T-cell cytotoxicity and activation-induced cell death in the peripheral immune system. Fas is a classical type I transmembrane receptor and belongs to the tumor necrosis factor (TNF) receptor superfamily [7].
Fas-mediated apoptosis represents one major mechanism by which tumor cells can be eliminated by activated cytotoxic immune lymphocytes. Previously, we have reported that interferon-g (IFN-g) sensitizes human ovarian carcinoma cell lines to Fasmediated apoptosis. Furthermore, IFN-g, together with many other proinflammatory cytokines (TNF-a, IL-1b, LPS, etc.), can stimulate the induction of inducible nitric oxide synthase (iNOS) and the generation of nitric oxide (NO). In this study, we examined whether nitric oxide is a mediator of IFN-g-induced sensitization of human ovarian carcinoma cell lines (A2780 and AD10) to Fas-mediated apoptosis and whether NO regulates the expression of the Fas receptor. Treatment of quiescent A2780 and AD10 ovarian carcinoma cells with IFN-g alone induced the expression of iNOS mRNA as examined by RT-PCR. There was accumulation of nitrite in the culture medium of IFN-g-treated cells, suggesting the generation of NO x. Like IFN-g, the use of exogenous sources of NO (S-nitroso-N-acetylpenicillamine (SNAP)) mimicked the sensitization of both cell lines to anti-Fas cytotoxic antibody (CH11) by IFN-g. Endogenously produced NO, by IFN-g pretreatment or exogenous nitrodonors, resulted in the upregulation of Fas receptor mRNA and protein expression. Blocking iNOS activity by N G-monomethyl-L-arginine (L-NMA) significantly reduced the sensitization, Fas mRNA, and protein expression observed with IFN-g pretreatment of the tumor cells. These findings demonstrate that sensitization of human ovarian carcinoma cell lines to Fas-mediated apoptosis by IFN-g can be due, in part, to the induction of iNOS and the subsequent upregulation of Fas gene expression by reactive nitrogen intermediates. Thus, the sensitivity of tumor cells to Fas-L-mediated cytotoxic immune lymphocytes can be regulated by the induction of NO or intermediates. © 1999 Academic Press Key Words: nitric oxide; nitric oxide synthase; Fas; apoptosis; ovarian carcinoma; tumor immunobiology.
INTRODUCTION Fas/Apo-1 (CD95 3) expression is not restricted to cells of the hematopoietic lineage. It has been observed that the Fas 1 This work was supported in part by the Boiron Research Foundation, the UCLA Gene Therapy Program, and the National Council of Science and Technology of Venezuela (CONICIT-Venezuela) (H.G.). 2 To whom reprint requests should be addressed. Fax: (310) 206-3865. E-mail: [email protected].
Abbreviations used: IFN-g, interferon gamma; iNOS, inducible nitric oxide synthase; NO, nitric oxide; IL-1b, interleukin 1-beta; LPS, lipopolysaccharide; SNAP, S-nitroso-N-acetylpenicillamine; L-NMA, NG-monomethyl-L-arginine; TNF, tumor necrosis factor; SNP, sodium nitroprusside; NOC18: DETA NONOeate.
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0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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The agonistic anti-Fas antibody CH11 exerts a cytolytic activity in sensitive cells expressing the Fas antigen, especially when combined with IFN-g. It was observed that treatment of the human colon carcinoma cell line HT-29 with IFN-g induced the expression of the Fas antigen on the cell surface and rendered the cells susceptible to the killing effect of the antiFas antibody [6]. Furthermore, we have demonstrated that IFN-g-treatment sensitizes several Fas-resistant tumor cell lines, including the ovarian carcinoma cell line AD10, to Fas-mediated cytotoxicity. In all cases, there was concurrent upregulation of Fas antigen expression with IFN-g-mediated sensitization to Fas killing [8]. Although IFN-g potentiates the cytolytic activity of TNF, the expression of TNF receptor type I mRNA was not affected by treatment with IFN-g [9], while that of the Fas mRNA in HT-29 cells was greatly stimulated by the same treatment [7]. These results suggest a specific regulatory mechanism of IFN-g on Fas gene expression, probably at the transcriptional level. However, the underlying mechanism by which IFN-g exerts this gene regulatory function remains unclear. It has been shown that IFN-g alone or in combination with TNF-a, interleukin 1b (IL-1b), and bacterial lipopolysaccharide (LPS) can induce the expression of nitric oxide synthase type II (iNOS) in a wide variety of tissue organs and in some tumor cell lines [10, 11]. The inducible type of nitric oxide synthase (iNOS) is considered to be a central molecule in the regulation of the immune response to tumors [12]. Further, iNOS is responsible for the production of large amounts of nitric oxide (NO), an unusual messenger. In an attempt to understand the mechanism by which IFN-g sensitizes ovarian tumor cells to Fas-mediated apoptosis, and based on the above findings, we hypothesized that IFN-gmediated induction of iNOS and NO is involved in the regulation of Fas expression and sensitization to Fas agonist CH11 apoptosis. The present study was undertaken to test this hypothesis using ovarian carcinoma cell lines. The following were examined: (a) the induction of iNOS and NO upon treatment with IFN-g, (b) the role of NOS blockers in the inhibition of the IFN-g-induced sensitization to Fas-mediated apoptosis, (c) the role of NO donors in mimicking IFN-gmediated effects, and (d) the role of NO in the regulation of Fas expression. MATERIALS AND METHODS Cell cultures and lines. The AD10 cell line is an Adriamycin-resistant, MDR phenotype-expressing subline derived from the ovarian carcinoma cell line A2780 and both were obtained from Dr. Ozols (Fox Chase Cancer Center, Philadelphia, PA). Cell cultures were maintained as monolayers on plastic dishes in DMEM (Life Technologies, Bethesda, MD), supplemented with 10% heat-inactivated FCS (Life Technologies, Bethesda, MD), 1% L-glutamine (Life Technologies), 1% pyruvate (Life Technologies), 1% nonessential amino acids
(Life Technologies), and 1% fungibact solution (Irvine Scientific, Irvine, CA). The cells were preincubated with the iNOS inhibitor, N G-monomethyl-L-arginine (L-NMA; final concentration 1 mM; Sigma Chemical Co., St. Louis, MO) or an equimolar concentration of its biologically inactive D-enantiomer, DNMA (Sigma Chemical Co.) for 18 h prior to IFN-g induction. RT-PCR. Total RNA was extracted and purified from approximately 5 3 10 5 cells for each different condition by a single-step guanidinium thiocyanate– chloroform method with STAT 60 reagent (Tel-Test “B,” Inc., Friendswood, TX). One microgram of total RNA was reverse transcribed to firststranded cDNA for 1 h at 42°C with SuperScript II reverse transcriptase [200 U] and random hexamer primers [20 mM] (Life Technologies). Amplification of 1/10 of these cDNA by PCR was performed using the following gene-specific primers: Fas receptor sense (59-ATG CTG GGC ATC TGG ACC CT39), Fas receptor antisense (59-GCC ATG TCC TTC ATC ACA CAA-39) [338 bp expected product]; iNOS sense (59CCG AGC CCG AAC ACA CAG AAC-39) and iNOS antisense (59-GGG TTG GGG GTG TGG TGA TGT-39) [462 bp, expected product]. Internal control for equal cDNA loading in each reaction was assessed using the following gene-specific glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) primers: G-3-PDH sense (59-GAA CAT CAT CCC TGC CTC TAC TG-39), G-3-PDH antisense (59-GTT GCT GTA GCC AAA TTC GTT G-39) [355 bp expected product]. PCR amplifications were carried out using the Hot Start/Ampliwax method as described by the supplier (Perkin–Elmer) with the following temperature cycling parameters: 94°C/45 s; 65°C/2 min for 26 cycles; and a final extension at 72°C/10 min. The amplified products were resolved by 1.5% agarose gel electrophoresis and their relative concentrations were assessed by densitometric analysis (BIOSOFT, Cambridge, UK) of the ethidiumbromide-stained image. FACS. Surface Fas antigen expression on tumor cells was determined by flow cytometry. Briefly, harvested cells were washed with cold buffer consisting of PBS without Ca 11 or Mg 11 with 2% heat-inactivated FCS and 0.1% sodium azide. Cells (2 3 10 5 per sample) were pretreated with human AB serum (Gemini Bioproducts, Calabasas, CA) for 1 h, washed twice, and resuspended in 50 ml of PBS. The cells were incubated with 10 mg/mL of anti-Fas monoclonal antibody FITC-conjugated (Pharmingen, San Diego, CA) or isotype control antibody for 1 h. The cells were then washed twice and fixed in 2% paraformaldehyde solution (Sigma Chemical Co.) and flow cytometry was conducted at the FACScan facility of the UCLA Department of Microbiology and Immunology. Cytotoxicity. Sensitization to Fas-mediated apoptosis was assessed using the agonist anti-Fas monoclonal antibody CH11 (IgM) [0.01, 0.1, and 1 mg/mL] (Kamiya Biomedical, Thousand Oaks, CA) in a 24-h incubation assay. The lactate dehydrogenase (LDH)-based CytoTox 96 Assay (Promega, Madison, WI) was used to determine cytotoxicity. Briefly, 1 3 10 4
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cells/sample, in quadruplicate, were distributed into a 96-well flat-bottom microtiter plate (Costar, Cambridge, MA). After the initial incubation for each different experimental condition, released LDH into the culture supernatants was measured with a 30-min coupled enzymatic assay which results in the conversion of a tetrazolium salt (INT) into a red formazan product that is read at 490 nm in an automated plate reader (Emax, Molecular Devices, Sunnyvale, CA). Percentage cytotoxicity was calculated using the spontaneous release-corrected OD as follows: % cytotoxicity 5 (OD of experimental well/OD of maximum release control well) 3 100. Nitrite/nitrate determination. Nitric oxide generation was monitored indirectly by levels of nitrite/nitrate (NO 22/NO 32) released into the culture medium as determined by the diazotization reaction of Griess with NaNO 2 as standard [13]. Briefly, 50-ml aliquots of cell culture supernatants from each sample were mixed with one volume of Griess reagent [1% sulfanilamide; 0.1% naphthylethylenediamine dihydrochloride; 2.5% H 3PO 4] and incubated at room temperature for 10 min. The absorbance at 550 nm was measured in an automated plate reader (Emax, Molecular Devices). Nitrite concentrations were calculated by comparison with OD 550 values of standard solutions of sodium nitrite prepared in culture medium. Protein expression. Cell extracts for iNOS and Fas receptor analysis were prepared by lysing 5 3 10 6 cells in 1 mL phosphate buffer solution [10 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 0.01% leupeptin]. Cell lysates were boiled (3 min) with 1 volume gel loading buffer [50 mM Tris/10% sodium dodecyl sulfate (SDS)/10% glycerol/ 10% 2-mecaptoethanol/2 mg/mL bromophenol blue] and centrifuged at 1 3 10 4 g for 10 min. Protein concentrations of the supernatants were determined according to Bradford [14], and total protein equivalents for each sample were separated on 12% SDS–polyacrylamide minigels (Bio-Rad, Richmond, CA) and transferred to nitrocellulose membranes (Amersham Corp., Arlington Heights, IL). Nonspecific immunoglobulin G (IgG) binding sites were blocked with 5% dried milk protein, and samples were then incubated with the antibody to iNOS [1:1000] (Transduction Laboratories, Lexington, KY) or CD95 [1:500] (Pharmingen). Relative concentrations were assessed by densitometric analysis (BIOSOFT) of the bands detected using a horseradish peroxidase-conjugated secondary antibody coupled to a ECL-chemiluminescent system (Amersham Corp.). Apoptosis (acridine orange/ethidium bromide staining). Characteristic apoptotic morphological changes were assessed by fluorescent microscopy using the acridine orange and ethidium bromide staining (AO/EB) method [15]. Briefly, adherent cells, under different experimental conditions, were cultured in 24-well plates and washed with PBS once prior to staining. Monolayers of adherent cells were covered with 100 ml of AO/EB solution (4 mg/mL of each). Immediately after
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adding the AO/EB solution, each sample was examined under an inverted/fluorescent microscope. Statistical analysis. The experimental values were expressed as the mean 6 standard error of the mean for the number of separate experiments indicated in each case. Oneway analysis of variance was used to compare variances within groups and among them. Bartlett’s tests were used to establish the homogeneity of variance on the basis of the differences among standard deviations. Whenever needed, post hoc unpaired multiple comparison test (Bonferroni’s test) and Student’s t test were used for comparison between two groups. Significant differences were considered for those probabilities , 5% (P , 0.05). RESULTS Induction of iNOS expression in IFN-g-treated ovarian carcinoma cells. We examined whether the ovarian carcinoma cell lines, A2780 and AD10, can be induced to generate iNOS upon treatment with IFN-g. Quiescent cultured cells were treated with increasing concentrations of human recombinant IFN-g for 18 h. Significant level of iNOS mRNA was observed by semiquantitative RT-PCR in 1 U/mL of IFN-g-treated AD10 cells (Fig. 1A). iNOS mRNA levels increased as a function of increasing concentrations of IFN-g. A plateau of iNOS mRNA expression was reached with 100 U/mL of IFN-g. In contrast to AD10, it was not possible to detect any iNOS expression upon IFN-g treatment of the parental A2780 cell line (data not shown). These results demonstrate the inducibility of functional iNOS in the AD10 cell line. The activity of iNOS and generated NO was monitored by the release of NO 22/NO 32 into the cell culture medium as determined by the Griess reaction. This activity was demonstrated to be specific for NOS by blocking the generation of NO 22/NO 32 using the NOS inhibitor L-NMA (1.0 mM) prior to the induction in AD10 by IFN-g (Fig. 1B). IFN-g-mediated sensitization to Fas-mediated apoptosis is blocked by NOS inhibitors. We first examined the sensitivity of the ovarian carcinoma cell line A2780 and the Adriamycinresistant subline AD10 to Fas-mediated apoptosis using the Fas agonistic antibody CH11. Quiescent cultured cells were pretreated with increasing concentrations of IFN-g (0, 10, 100, and 1000 U/mL) 18 h prior to performing the cytotoxicity assay with increasing concentrations of the anti-Fas agonist antibody CH11 (0, 0.01, and 0.1 mg/mL). The parental cell line A2780 (Fig. 2A) exhibited a lower capacity for being sensitized by IFN-g when compared with the Adriamycin-resistant AD10. IFN-g-pretreated AD10 cells were sensitized an average of 10-fold higher compared with the untreated control group (Fig. 2B). These results confirm previous observations seen in other ovarian carcinoma and tumor cell lines [8]. IFN-g alone or in combination with other proinflammatory cytokines like TNF-a, IL-1, and LPS has been shown to be
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NO donors sensitize ovarian carcinoma cells to Fas-mediated apoptosis. The role of the endogenously generated NO in the IFN-g-mediated sensitization to Fas-induced apoptosis was corroborated by the use of an exogenous source of NO, which mimics the production of NO by iNOS. Ovarian carcinoma cells were cultured in the presence or in the absence of three different NO donors, namely sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine, and DETA NONOeate (NOC18), in equimolar concentrations of 10 and 100 mM for 24 h before the cytotoxicity assay with the Fas-agonistic antibody. In all cases, NO donors sensitized ovarian cells to Fas-mediated cytotoxicity in the same way as IFN-g-mediated sensitization. The findings with SNAP are shown in Fig. 4. The sensitization was dependent on the concentration of the NOS inhibitor used. These results establish a correlation between the generation of NO by IFN-g and sensitization of ovarian carcinoma cell lines to Fas-mediated cytotoxicity. Sensitization of AD10 to CH11-mediated cytotoxicity was due to apoptosis. The morphological pattern was examined by staining the ovarian carcinoma cells with AO/EB. Pretreated cells with IFN-g for 18 h and then treatment with 0.1 mg/mL
FIG. 1. IFN-g induces iNOS expression in AD10 ovarian carcinoma cells. (A) AD10 cells (5 3 10 5/well) were cultured for 24 h in 6-well plates supplemented with 1% FCS prior to incubation with increasing concentrations of IFN-g (1–1000 U/mL) and FCS concentration restored to 10%. Induction of iNOS mRNA was detected by RT-PCR at 18 h of incubation. (B) Using the Griess reaction, production of NO was measured as accumulation of nitrites and nitrates in the culture medium of AD10 cells (2 3 10 5/well in 12-well plates) under the same conditions as above in the presence (ANOVA: P 5 0.096) or in the absence (ANOVA: P 5 0.0005) of the NOS inhibitor L-NMA (1.0 mM). **P , 0.005.
effective in the induction of the inducible form of nitric oxide synthase in several tumor cell lines [10, 11]. In order to examine the possible role of iNOS in the mechanism of sensitization to Fas-mediated cytotoxicity, ovarian carcinoma cells were incubated in the presence or in the absence of the competitive NOS inhibitor L-NMA (1 mM) 6 h prior to IFN-g treatment. NOS inhibition significantly decreased IFN-g-mediated sensitization to Fas-mediated apoptosis in AD10 cells (Fig. 3B), suggesting that iNOS induction by IFN-g is an important component in the process of sensitization. In contrast, A2780 cells did not respond to NOS blocking and preserved their sensitization to Fas-mediated apoptosis achieved with IFN-g treatment (Fig. 3A).
FIG. 2. IFN-g sensitizes ovarian carcinoma cell lines to Fas-mediated cytotoxicity. Cytotoxicity of the Fas-agonistic antibody CH11 was assessed by LDH release into the culture medium at 24 h of A2780 (A) and AD10 (B) cells (1 3 10 4 cells/well) cultured in 96-well plates supplemented with 10% FCS and incubated for 24 h, prior to the cytotoxicity assay, in the presence of IFN-g [0 U/mL (blank bars), 1 U/mL (solid bars), 10 U/mL (slashed bars), and 100 U/mL (dotted bars)]. **P , 0.005; ***P , 0.001.
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In order to confirm that the endogenous generation of NO by the induction of iNOS was responsible for the upregulation of Fas, AD10 cells were treated with 10 and 100 U/mL of IFN-g in the presence or absence of the NOS inhibitor L-NMA (1.0 mM). Fas mRNA expression was markedly reduced by NOS blocking (Fig. 6B). Further, the upregulation of Fas receptor was corroborated by Western blot (Fig. 6C) and by flow cytometry (Fig. 6D). IFN-g-mediated upregulation of Fas expression was blocked by NOS inhibitors and was restored by the NO donor (SNAP, 100 mM). Altogether, these results demonstrate a strong correlation between the generation of NO by AD10 ovarian carcinoma cells and upregulation of Fas receptor expression. DISCUSSION This study provides several lines of evidence that indicate that the sensitization to Fas-mediated apoptosis observed in the AD10 ovarian carcinoma cell line treated with IFN-g is due in
FIG. 3. NOS inhibitor blocks IFN-g-induced sensitization of AD10 cells to Fas-mediated cytotoxicity. Cytotoxicity of the Fas-agonistic antibody CH11 was assessed by LDH release into the culture medium at 24 h of A2780 (A) and AD10 (B) cells (1 3 10 4 cells/well) cultured in 96-well plates supplemented with 10% FCS and incubated for 24 h with IFN-g (10 U/mL, solid bars) in the presence (slashed bars) or absence of the NOS inhibitor L-NMA (1.0 mM). The NOS inhibitor effectively blocked the IFN-g-induced sensitization to Fas-mediated apoptosis (P , 0.001) in AD10 cells whereas in A2780, it was ineffective. **P , 0.005; ***P , 0.001.
of the Fas-agonistic antibody CH11 for 6 h resulted in a greater frequency of cells undergoing apoptosis (Fig. 5) compared with the untreated control group (Fig. 5). When the cells were incubated in the presence of the NOS inhibitor L-NMA (1 mM) 6 h prior to IFN-g pretreatment, the frequency of cells undergoing apoptosis (Fig. 5) was significantly reduced and was less than that of cells incubated in the absence of the NOS inhibitor. Furthermore, treatment of AD10 cells with SNAP prior to exposure to CH11 also increased the frequency of cells undergoing characteristic apoptosis (Fig. 5), whereas the addition of the NO donor alone did not increase the number of apoptotic cells (Fig. 5). NO upregulates Fas receptor expression. We examined the role of NO in the regulation of Fas receptor expression in AD10. First, AD10 cells were exposed to increasing concentrations of an NO donor (SNAP: 1, 10, and 100 mM) for 18 h and the relative Fas mRNA expression was determined by RT-PCR. Clearly, SNAP upregulated Fas receptor mRNA expression (Fig. 6A).
FIG. 4. NO donor sensitizes ovarian carcinoma cell lines to Fas-mediated cytotoxicity. Cultured A2780 (A) and AD10 (B) cells (1 3 10 4 cells/well) in 96-well plates supplemented with 10% FCS were incubated in the presence of the photoactivated SNAP (0, 10, and 100 mM) for 24 h and cytotoxicity of the Fas-agonist antibody (CH11) was assessed by LDH release into the culture medium. Significant sensitization was observed in AD10 and A2780 cells using 100 mM of SNAP. **P , 0.005; ***P , 0.001.
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FIG. 5. NO sensitizes AD10 cells to Fas-mediated apoptosis. Acridine orange/ethidium bromide staining revealed that IFN-g-pretreated (100 U/mL) AD10 cells (1 3 10 5 cells/well) cultured in 12-well plate for 18 h increased the amount of cells undergoing characteristic apoptosis (chromatin condensation and margination, membrane blebbing, etc.). These findings demonstrate sensitization to Fas-mediated apoptosis (c) when compared with background killing by CH11 alone (0.1 mg/mL) (b) or with the control-untreated group (a). Sensitization was blocked by incubating IFN-g-treated AD10 cells in the presence of the NOS inhibitor L-NMA (1.0 mM) (d). Treatment of AD10 cells with the NO donor SNAP (100 mM) for 18 h also sensitized these cells to be killed by apoptosis by CH11 (f), whereas the addition of SNAP in the absence of CH11 did not affect the cultured cells (e).
part to the generation of nitric oxide, or its reaction products, by the induction of iNOS in these cells. NOS inhibitors blocked this sensitization. The use of nitrodonors mimicked the IFN-g-mediated sensitization to the Fas-agonist antibody, CH11. There was a concurrent upregulation of Fas receptor, either upon induction of iNOS or upon treatment of the ovarian cell lines with nitrodonors. The observed upregulation of Fas receptor was abrogated by the use of NOS inhibitors suggesting a strong correlation that might account for the sensitization to the Fas-agonist antibody CH11. Nitric oxide is a potent and pleiotropic free radical molecule that has been involved in a wide variety of physiological and pathophysiological functions [16]. NO is generated in low levels by two constitutive nitric oxide synthases (eNOS and nNOS) and in much greater levels by the inducible NO synthase (iNOS) [17]. It has been shown that NO might participate in the apoptosis process by either inhibiting or promoting some apoptotic events [18 –21]. There has been a long debate about the specific role that nitric oxide might play in apoptosis. Recently, several studies have referred that nitric oxide is a novel and potent inhibitor of apoptosis. For instance, endogenous NO synthesis or exposure to low levels of NO donors was first shown to inhibit apoptosis in human B lymphocytes [21], and similar findings have been reported in splenocytes [18], eosinophils [19, 22], and endothelial cells [23]. NOS inhibitors have also been directed toward the specific disruption of the Fas-induced apoptotic mechanism. Basal NOS activity in human leukocytes was
shown to inhibits Fas-induced apoptosis via a cGMP-independent mechanism and further inhibition of caspase activation [24, 25]. Recent studies, examining the involvement of CD95/ CD95L system and signal transduction pathway in NO-induced apoptosis in human neoplastic lymphoid cells, demonstrated that NO triggers the death receptor system by regulating the expression of ligands involved in apoptosis like CD95L and TRAIL/APO-2L with unaltered expression of CD95 receptor after NO treatment [26]. In contrast, our results herein with ovarian carcinoma cells clearly demonstrate that NO, far from protecting tumor cells from Fas-induced apoptosis, synergized with the Fas agonist antibody CH11 in the induction of apoptosis. Differences among the effects of NO in the various systems used above are noteworthy. NO appears to inhibit Fas-induced apoptosis in transformed cells derived from the hematopoietic lineage, whereas it mediates sensitization to Fas apoptosis as observed here in ovarian tumor cells. This dichotomy represents a crucial point of divergence between the two cell types and needs to be considered when making strategies toward the use of NO as an anti-tumor agent. It has been shown that some tumor cells can be prompted to generate endogenously NO by the induction of iNOS upon treatment with proinflammatory cytokines alone or in combination [11, 27, 28]. Herein, we demonstrate the inducibility of the ovarian carcinoma cell line AD10 to generate NO by iNOS and the susceptibility of this cell line to be sensitized to Fas-mediated apoptosis. Furthermore, we have found that the
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FIG. 6. Upregulation of Fas receptor expression on AD10 cells by NO. (A) Upregulation of Fas receptor mRNA was detected by RT-PCR in AD10 cells (5 3 10 5/well) cultured in a 12-well plate supplemented with 10% FCS in the presence of increasing concentrations of SNAP (lane 1, control; lane 2, 1 mM; lane 3, 10 mM; lane 4, 100 mM). Relative expression of Fas receptor mRNA amplicons was assessed by densitometric analysis of the ethidiumbromide-stained DNA band resolved in agarose gel electrophoresis (A, bottom). (B) IFN-g-mediated upregulation of Fas mRNA expression (lane 1, control; lane 2, 10 U/mL; lane 3, 100 U/mL) was reduced by incubation off cells with the NOS inhibitor L-NMA (1.0 mM) for 24 h (lane 4, IFN-g 10 U/mL 1 L-NMA; lane 5, IFN-g 100 U/mL 1 L-NMA). Relative densitometric values of a representative experiment are shown in the lower panel. (C) Upregulation of Fas receptor protein level (lane 1, control; lane 2, IFN-g 100 U/mL) was detected by Western immunoblot in IFN-g-induced AD10 cells (1 3 10 7/plate) cultured in a 100-mm plate supplemented with 10% FCS. IFN-g-mediated upregulation of Fas protein level was markedly reduced in the presence of the NOS inhibitor (lane 3, IFN-g 100 U/mL 1 L-NMA 1.0 mM) and restored by the addition of SNAP (lane 4, SNAP 100 mM). (D) Increased expression of Fas surface molecule was detected by flow cytometry on A2780 and AD10 (2 3 10 5 cells/well) cultured in 12-well plates supplemented with 10% FCS in the presence of IFN-g (10 U/mL) for 18 h (solid bars) when compared with untreated control cells (blank bars). This increased expression was partially blocked by the NOS inhibitor (slashed bars, IFN-g 10 U/mL 1 L-NMA 1.0 mM) and restored by incubation with the NO donor (dotted bars, SNAP 100 mM).
parental cell line A2780, which is not able to express iNOS upon IFN-g stimulation, was not sensitized at the same level when compared with the Adriamycin-resistant cell line AD10, which is induced by IFN-g alone to express iNOS. However, when nitrodonors were used as exogenous sources of NO, a similar sensitization to Fas-mediated apoptosis was observed in both cell lines as well in two other human prostate cancer cell lines (data not shown). This differential response to endoge-
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nous versus exogenous sources of NO suggests the susceptibility of tumor cells to be sensitized by NO despite their capacity to be induced to express iNOS. Previous studies demonstrated the influence of IFN-g in the regulation of Fas receptor expression on the tumor cell surface [8]. Furthermore, studies searching for the role of NO in vascular smooth muscle cell apoptosis demonstrated that NO induces upregulation of Fas antigen expression via a cGMPindependent mechanism [29]. Likewise, it has been shown that NO primes pancreatic b cells for Fas-induced apoptosis by increasing the surface CD95 receptor expression [30]. However, it is not clear how NO can interact with the transcriptional machinery in order to regulate Fas gene expression. In this study, evidence is provided that suggests the presence of a correlation between IFN-g-mediated sensitization of ovarian carcinoma cell lines to Fas-induced apoptosis and the generation of NO. The sensitization achieved by IFN-g treatment was blocked by the NOS inhibitor (L-NMA, 1 mM) and was restored by the NO donor (SNAP, 100 mM). Furthermore, the modulation of Fas expression correlated with either the endogenous production of NO by iNOS or the exogenous presence of the NO donor. Altogether, these results suggest an important role of NO in the regulation of Fas gene expression. NO has been increasingly implicated in the control of the activity of the transcription machinery. Reactive nitrogen intermediates can serve as intracellular second messengers to induce IL-8 gene expression [30 –32]. AP-1 (a common regulatory element found along the Fas promoter) can be attenuated by its proteolytic decomposition, which might be mediated by S-nitrosylation of AP-1 [33, 34]. Thus, the regulation of the Fas gene expression by NO observed in the ovarian carcinoma cell lines may be explained by alteration of the composition of the transcriptional regulatory units by reactive nitrogen intermediates. In summary, the present study shows that the AD10 ovarian carcinoma cell line, when stimulated with IFN-g, can express iNOS and produce NO. The generation of NO correlates with the sensitization of AD10 cells to Fas-induced apoptosis and can be blocked by the NOS inhibitor, thus implicating the role of NO in the IFN-g-mediated sensitization to Fas-induced killing. Moreover, the use of NO donor bypassed the inability of the parental cell line A2780 to express iNOS and sensitized those cells to the Fas agonist antibody. Sensitization was concomitantly observed with upregulation of Fas gene expression. In contrast to the role of NO in protecting against apoptosis in cells of the hematopoietic lineage, our findings demonstrate that NO plays a role in the sensitization of tumor cells to Fas-mediated apoptosis. Such sensitization is due to the regulation of Fas gene expression and/or signaling toward apoptosis. Our data raise the possibility that NO generation (NO-based therapies) can be used to control tumor cell death by apoptotic-mediated mechanisms.
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We thank Dr. Jon Fukuto for his thorough discussions of this study and his fine NO chemistry comments. We are grateful to the members of the UCLA Women’s Reproductive Cancer Program, The Multidisciplinary Ovarian Cancer Research Group, for their advice. The authors also thank Ms. Samantha Nguyen for preparing the manuscript.
16. 17. 18.
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Nitric Oxide Sensitizes Ovarian Tumor Cells to Fas-Induced Apoptosis 1 Hermes J. Garba´n and Benjamin Bonavida 2 Department of Microbiology, Immunology, and Molecular Genetics, and Jonsson Comprehensive Cancer Center, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, California 90095-1747 Received August 17, 1998
receptor is expressed constitutively in the normal folliculi of the ovary, endometrial gland cells of the uterus, and columnar epithelium of the uterine cervix [1]. During reproductive life more than 99% of the ovarian follicles do not ovulate and are eliminated from the ovary by follicular atresia. Atresia is achieved by autodegeneration of thecal and granulosa cells that comprise the follicle, by the process of apoptosis. Moreover, the sphingomyelin-ceramide cycle can lead to cell suicide by Fas-mediated apoptosis, not only in the immune system, but also in thecal/interstitial cells of the ovarian follicles [2]. It has been shown that ovarian surface epithelial cells (OSE) are specially susceptible to Fas-mediated apoptosis among the normal mouse corpora lutea cultures containing luteal, stromal, endometrial cells, and fibroblasts and also OSE exposed to the Fas-agonist antibody JO2. In vivo, OSE undergo programmed cell death before ovulation and rapidly proliferate to repair the surface of the ovulatory follicle after ovulation [3]. It is noteworthy that most ovarian cancers are derived from the OSE. Thus, Fas/Apo-1 might not only transmit apoptotic signals but also play a more general role in growth control in both normal ovarian tissue and oncogenesis in the ovary. A recent report demonstrated that the majority of ovarian carcinoma cell lines screened for Fas expression were positive and their sensitivity to the anti-Fas agonist antibody correlated with the level of Fas expression [4]. Furthermore, the downregulation of Fas expression and subsequent resistance to antiFas were observed in the drug-resistant human ovarian carcinoma IGR-OVI/VCR, the human breast carcinoma cell line MCF7Adr, and the leukemic lymphoblast CEM/VLB cells, suggesting that the alteration of Fas expression following drugresistance is not restricted to one cell type [5]. The Fas receptor (CD95/APO-1) [6] has been recognized as a central receptor for apoptosis, particularly in mediating nonspecific T-cell cytotoxicity and activation-induced cell death in the peripheral immune system. Fas is a classical type I transmembrane receptor and belongs to the tumor necrosis factor (TNF) receptor superfamily [7].
Fas-mediated apoptosis represents one major mechanism by which tumor cells can be eliminated by activated cytotoxic immune lymphocytes. Previously, we have reported that interferon-g (IFN-g) sensitizes human ovarian carcinoma cell lines to Fasmediated apoptosis. Furthermore, IFN-g, together with many other proinflammatory cytokines (TNF-a, IL-1b, LPS, etc.), can stimulate the induction of inducible nitric oxide synthase (iNOS) and the generation of nitric oxide (NO). In this study, we examined whether nitric oxide is a mediator of IFN-g-induced sensitization of human ovarian carcinoma cell lines (A2780 and AD10) to Fas-mediated apoptosis and whether NO regulates the expression of the Fas receptor. Treatment of quiescent A2780 and AD10 ovarian carcinoma cells with IFN-g alone induced the expression of iNOS mRNA as examined by RT-PCR. There was accumulation of nitrite in the culture medium of IFN-g-treated cells, suggesting the generation of NO x. Like IFN-g, the use of exogenous sources of NO (S-nitroso-N-acetylpenicillamine (SNAP)) mimicked the sensitization of both cell lines to anti-Fas cytotoxic antibody (CH11) by IFN-g. Endogenously produced NO, by IFN-g pretreatment or exogenous nitrodonors, resulted in the upregulation of Fas receptor mRNA and protein expression. Blocking iNOS activity by N G-monomethyl-L-arginine (L-NMA) significantly reduced the sensitization, Fas mRNA, and protein expression observed with IFN-g pretreatment of the tumor cells. These findings demonstrate that sensitization of human ovarian carcinoma cell lines to Fas-mediated apoptosis by IFN-g can be due, in part, to the induction of iNOS and the subsequent upregulation of Fas gene expression by reactive nitrogen intermediates. Thus, the sensitivity of tumor cells to Fas-L-mediated cytotoxic immune lymphocytes can be regulated by the induction of NO or intermediates. © 1999 Academic Press Key Words: nitric oxide; nitric oxide synthase; Fas; apoptosis; ovarian carcinoma; tumor immunobiology.
INTRODUCTION Fas/Apo-1 (CD95 3) expression is not restricted to cells of the hematopoietic lineage. It has been observed that the Fas 1 This work was supported in part by the Boiron Research Foundation, the UCLA Gene Therapy Program, and the National Council of Science and Technology of Venezuela (CONICIT-Venezuela) (H.G.). 2 To whom reprint requests should be addressed. Fax: (310) 206-3865. E-mail: [email protected].
Abbreviations used: IFN-g, interferon gamma; iNOS, inducible nitric oxide synthase; NO, nitric oxide; IL-1b, interleukin 1-beta; LPS, lipopolysaccharide; SNAP, S-nitroso-N-acetylpenicillamine; L-NMA, NG-monomethyl-L-arginine; TNF, tumor necrosis factor; SNP, sodium nitroprusside; NOC18: DETA NONOeate.
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The agonistic anti-Fas antibody CH11 exerts a cytolytic activity in sensitive cells expressing the Fas antigen, especially when combined with IFN-g. It was observed that treatment of the human colon carcinoma cell line HT-29 with IFN-g induced the expression of the Fas antigen on the cell surface and rendered the cells susceptible to the killing effect of the antiFas antibody [6]. Furthermore, we have demonstrated that IFN-g-treatment sensitizes several Fas-resistant tumor cell lines, including the ovarian carcinoma cell line AD10, to Fas-mediated cytotoxicity. In all cases, there was concurrent upregulation of Fas antigen expression with IFN-g-mediated sensitization to Fas killing [8]. Although IFN-g potentiates the cytolytic activity of TNF, the expression of TNF receptor type I mRNA was not affected by treatment with IFN-g [9], while that of the Fas mRNA in HT-29 cells was greatly stimulated by the same treatment [7]. These results suggest a specific regulatory mechanism of IFN-g on Fas gene expression, probably at the transcriptional level. However, the underlying mechanism by which IFN-g exerts this gene regulatory function remains unclear. It has been shown that IFN-g alone or in combination with TNF-a, interleukin 1b (IL-1b), and bacterial lipopolysaccharide (LPS) can induce the expression of nitric oxide synthase type II (iNOS) in a wide variety of tissue organs and in some tumor cell lines [10, 11]. The inducible type of nitric oxide synthase (iNOS) is considered to be a central molecule in the regulation of the immune response to tumors [12]. Further, iNOS is responsible for the production of large amounts of nitric oxide (NO), an unusual messenger. In an attempt to understand the mechanism by which IFN-g sensitizes ovarian tumor cells to Fas-mediated apoptosis, and based on the above findings, we hypothesized that IFN-gmediated induction of iNOS and NO is involved in the regulation of Fas expression and sensitization to Fas agonist CH11 apoptosis. The present study was undertaken to test this hypothesis using ovarian carcinoma cell lines. The following were examined: (a) the induction of iNOS and NO upon treatment with IFN-g, (b) the role of NOS blockers in the inhibition of the IFN-g-induced sensitization to Fas-mediated apoptosis, (c) the role of NO donors in mimicking IFN-gmediated effects, and (d) the role of NO in the regulation of Fas expression. MATERIALS AND METHODS Cell cultures and lines. The AD10 cell line is an Adriamycin-resistant, MDR phenotype-expressing subline derived from the ovarian carcinoma cell line A2780 and both were obtained from Dr. Ozols (Fox Chase Cancer Center, Philadelphia, PA). Cell cultures were maintained as monolayers on plastic dishes in DMEM (Life Technologies, Bethesda, MD), supplemented with 10% heat-inactivated FCS (Life Technologies, Bethesda, MD), 1% L-glutamine (Life Technologies), 1% pyruvate (Life Technologies), 1% nonessential amino acids
(Life Technologies), and 1% fungibact solution (Irvine Scientific, Irvine, CA). The cells were preincubated with the iNOS inhibitor, N G-monomethyl-L-arginine (L-NMA; final concentration 1 mM; Sigma Chemical Co., St. Louis, MO) or an equimolar concentration of its biologically inactive D-enantiomer, DNMA (Sigma Chemical Co.) for 18 h prior to IFN-g induction. RT-PCR. Total RNA was extracted and purified from approximately 5 3 10 5 cells for each different condition by a single-step guanidinium thiocyanate– chloroform method with STAT 60 reagent (Tel-Test “B,” Inc., Friendswood, TX). One microgram of total RNA was reverse transcribed to firststranded cDNA for 1 h at 42°C with SuperScript II reverse transcriptase [200 U] and random hexamer primers [20 mM] (Life Technologies). Amplification of 1/10 of these cDNA by PCR was performed using the following gene-specific primers: Fas receptor sense (59-ATG CTG GGC ATC TGG ACC CT39), Fas receptor antisense (59-GCC ATG TCC TTC ATC ACA CAA-39) [338 bp expected product]; iNOS sense (59CCG AGC CCG AAC ACA CAG AAC-39) and iNOS antisense (59-GGG TTG GGG GTG TGG TGA TGT-39) [462 bp, expected product]. Internal control for equal cDNA loading in each reaction was assessed using the following gene-specific glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) primers: G-3-PDH sense (59-GAA CAT CAT CCC TGC CTC TAC TG-39), G-3-PDH antisense (59-GTT GCT GTA GCC AAA TTC GTT G-39) [355 bp expected product]. PCR amplifications were carried out using the Hot Start/Ampliwax method as described by the supplier (Perkin–Elmer) with the following temperature cycling parameters: 94°C/45 s; 65°C/2 min for 26 cycles; and a final extension at 72°C/10 min. The amplified products were resolved by 1.5% agarose gel electrophoresis and their relative concentrations were assessed by densitometric analysis (BIOSOFT, Cambridge, UK) of the ethidiumbromide-stained image. FACS. Surface Fas antigen expression on tumor cells was determined by flow cytometry. Briefly, harvested cells were washed with cold buffer consisting of PBS without Ca 11 or Mg 11 with 2% heat-inactivated FCS and 0.1% sodium azide. Cells (2 3 10 5 per sample) were pretreated with human AB serum (Gemini Bioproducts, Calabasas, CA) for 1 h, washed twice, and resuspended in 50 ml of PBS. The cells were incubated with 10 mg/mL of anti-Fas monoclonal antibody FITC-conjugated (Pharmingen, San Diego, CA) or isotype control antibody for 1 h. The cells were then washed twice and fixed in 2% paraformaldehyde solution (Sigma Chemical Co.) and flow cytometry was conducted at the FACScan facility of the UCLA Department of Microbiology and Immunology. Cytotoxicity. Sensitization to Fas-mediated apoptosis was assessed using the agonist anti-Fas monoclonal antibody CH11 (IgM) [0.01, 0.1, and 1 mg/mL] (Kamiya Biomedical, Thousand Oaks, CA) in a 24-h incubation assay. The lactate dehydrogenase (LDH)-based CytoTox 96 Assay (Promega, Madison, WI) was used to determine cytotoxicity. Briefly, 1 3 10 4
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cells/sample, in quadruplicate, were distributed into a 96-well flat-bottom microtiter plate (Costar, Cambridge, MA). After the initial incubation for each different experimental condition, released LDH into the culture supernatants was measured with a 30-min coupled enzymatic assay which results in the conversion of a tetrazolium salt (INT) into a red formazan product that is read at 490 nm in an automated plate reader (Emax, Molecular Devices, Sunnyvale, CA). Percentage cytotoxicity was calculated using the spontaneous release-corrected OD as follows: % cytotoxicity 5 (OD of experimental well/OD of maximum release control well) 3 100. Nitrite/nitrate determination. Nitric oxide generation was monitored indirectly by levels of nitrite/nitrate (NO 22/NO 32) released into the culture medium as determined by the diazotization reaction of Griess with NaNO 2 as standard [13]. Briefly, 50-ml aliquots of cell culture supernatants from each sample were mixed with one volume of Griess reagent [1% sulfanilamide; 0.1% naphthylethylenediamine dihydrochloride; 2.5% H 3PO 4] and incubated at room temperature for 10 min. The absorbance at 550 nm was measured in an automated plate reader (Emax, Molecular Devices). Nitrite concentrations were calculated by comparison with OD 550 values of standard solutions of sodium nitrite prepared in culture medium. Protein expression. Cell extracts for iNOS and Fas receptor analysis were prepared by lysing 5 3 10 6 cells in 1 mL phosphate buffer solution [10 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 0.01% leupeptin]. Cell lysates were boiled (3 min) with 1 volume gel loading buffer [50 mM Tris/10% sodium dodecyl sulfate (SDS)/10% glycerol/ 10% 2-mecaptoethanol/2 mg/mL bromophenol blue] and centrifuged at 1 3 10 4 g for 10 min. Protein concentrations of the supernatants were determined according to Bradford [14], and total protein equivalents for each sample were separated on 12% SDS–polyacrylamide minigels (Bio-Rad, Richmond, CA) and transferred to nitrocellulose membranes (Amersham Corp., Arlington Heights, IL). Nonspecific immunoglobulin G (IgG) binding sites were blocked with 5% dried milk protein, and samples were then incubated with the antibody to iNOS [1:1000] (Transduction Laboratories, Lexington, KY) or CD95 [1:500] (Pharmingen). Relative concentrations were assessed by densitometric analysis (BIOSOFT) of the bands detected using a horseradish peroxidase-conjugated secondary antibody coupled to a ECL-chemiluminescent system (Amersham Corp.). Apoptosis (acridine orange/ethidium bromide staining). Characteristic apoptotic morphological changes were assessed by fluorescent microscopy using the acridine orange and ethidium bromide staining (AO/EB) method [15]. Briefly, adherent cells, under different experimental conditions, were cultured in 24-well plates and washed with PBS once prior to staining. Monolayers of adherent cells were covered with 100 ml of AO/EB solution (4 mg/mL of each). Immediately after
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adding the AO/EB solution, each sample was examined under an inverted/fluorescent microscope. Statistical analysis. The experimental values were expressed as the mean 6 standard error of the mean for the number of separate experiments indicated in each case. Oneway analysis of variance was used to compare variances within groups and among them. Bartlett’s tests were used to establish the homogeneity of variance on the basis of the differences among standard deviations. Whenever needed, post hoc unpaired multiple comparison test (Bonferroni’s test) and Student’s t test were used for comparison between two groups. Significant differences were considered for those probabilities , 5% (P , 0.05). RESULTS Induction of iNOS expression in IFN-g-treated ovarian carcinoma cells. We examined whether the ovarian carcinoma cell lines, A2780 and AD10, can be induced to generate iNOS upon treatment with IFN-g. Quiescent cultured cells were treated with increasing concentrations of human recombinant IFN-g for 18 h. Significant level of iNOS mRNA was observed by semiquantitative RT-PCR in 1 U/mL of IFN-g-treated AD10 cells (Fig. 1A). iNOS mRNA levels increased as a function of increasing concentrations of IFN-g. A plateau of iNOS mRNA expression was reached with 100 U/mL of IFN-g. In contrast to AD10, it was not possible to detect any iNOS expression upon IFN-g treatment of the parental A2780 cell line (data not shown). These results demonstrate the inducibility of functional iNOS in the AD10 cell line. The activity of iNOS and generated NO was monitored by the release of NO 22/NO 32 into the cell culture medium as determined by the Griess reaction. This activity was demonstrated to be specific for NOS by blocking the generation of NO 22/NO 32 using the NOS inhibitor L-NMA (1.0 mM) prior to the induction in AD10 by IFN-g (Fig. 1B). IFN-g-mediated sensitization to Fas-mediated apoptosis is blocked by NOS inhibitors. We first examined the sensitivity of the ovarian carcinoma cell line A2780 and the Adriamycinresistant subline AD10 to Fas-mediated apoptosis using the Fas agonistic antibody CH11. Quiescent cultured cells were pretreated with increasing concentrations of IFN-g (0, 10, 100, and 1000 U/mL) 18 h prior to performing the cytotoxicity assay with increasing concentrations of the anti-Fas agonist antibody CH11 (0, 0.01, and 0.1 mg/mL). The parental cell line A2780 (Fig. 2A) exhibited a lower capacity for being sensitized by IFN-g when compared with the Adriamycin-resistant AD10. IFN-g-pretreated AD10 cells were sensitized an average of 10-fold higher compared with the untreated control group (Fig. 2B). These results confirm previous observations seen in other ovarian carcinoma and tumor cell lines [8]. IFN-g alone or in combination with other proinflammatory cytokines like TNF-a, IL-1, and LPS has been shown to be
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NO donors sensitize ovarian carcinoma cells to Fas-mediated apoptosis. The role of the endogenously generated NO in the IFN-g-mediated sensitization to Fas-induced apoptosis was corroborated by the use of an exogenous source of NO, which mimics the production of NO by iNOS. Ovarian carcinoma cells were cultured in the presence or in the absence of three different NO donors, namely sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine, and DETA NONOeate (NOC18), in equimolar concentrations of 10 and 100 mM for 24 h before the cytotoxicity assay with the Fas-agonistic antibody. In all cases, NO donors sensitized ovarian cells to Fas-mediated cytotoxicity in the same way as IFN-g-mediated sensitization. The findings with SNAP are shown in Fig. 4. The sensitization was dependent on the concentration of the NOS inhibitor used. These results establish a correlation between the generation of NO by IFN-g and sensitization of ovarian carcinoma cell lines to Fas-mediated cytotoxicity. Sensitization of AD10 to CH11-mediated cytotoxicity was due to apoptosis. The morphological pattern was examined by staining the ovarian carcinoma cells with AO/EB. Pretreated cells with IFN-g for 18 h and then treatment with 0.1 mg/mL
FIG. 1. IFN-g induces iNOS expression in AD10 ovarian carcinoma cells. (A) AD10 cells (5 3 10 5/well) were cultured for 24 h in 6-well plates supplemented with 1% FCS prior to incubation with increasing concentrations of IFN-g (1–1000 U/mL) and FCS concentration restored to 10%. Induction of iNOS mRNA was detected by RT-PCR at 18 h of incubation. (B) Using the Griess reaction, production of NO was measured as accumulation of nitrites and nitrates in the culture medium of AD10 cells (2 3 10 5/well in 12-well plates) under the same conditions as above in the presence (ANOVA: P 5 0.096) or in the absence (ANOVA: P 5 0.0005) of the NOS inhibitor L-NMA (1.0 mM). **P , 0.005.
effective in the induction of the inducible form of nitric oxide synthase in several tumor cell lines [10, 11]. In order to examine the possible role of iNOS in the mechanism of sensitization to Fas-mediated cytotoxicity, ovarian carcinoma cells were incubated in the presence or in the absence of the competitive NOS inhibitor L-NMA (1 mM) 6 h prior to IFN-g treatment. NOS inhibition significantly decreased IFN-g-mediated sensitization to Fas-mediated apoptosis in AD10 cells (Fig. 3B), suggesting that iNOS induction by IFN-g is an important component in the process of sensitization. In contrast, A2780 cells did not respond to NOS blocking and preserved their sensitization to Fas-mediated apoptosis achieved with IFN-g treatment (Fig. 3A).
FIG. 2. IFN-g sensitizes ovarian carcinoma cell lines to Fas-mediated cytotoxicity. Cytotoxicity of the Fas-agonistic antibody CH11 was assessed by LDH release into the culture medium at 24 h of A2780 (A) and AD10 (B) cells (1 3 10 4 cells/well) cultured in 96-well plates supplemented with 10% FCS and incubated for 24 h, prior to the cytotoxicity assay, in the presence of IFN-g [0 U/mL (blank bars), 1 U/mL (solid bars), 10 U/mL (slashed bars), and 100 U/mL (dotted bars)]. **P , 0.005; ***P , 0.001.
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In order to confirm that the endogenous generation of NO by the induction of iNOS was responsible for the upregulation of Fas, AD10 cells were treated with 10 and 100 U/mL of IFN-g in the presence or absence of the NOS inhibitor L-NMA (1.0 mM). Fas mRNA expression was markedly reduced by NOS blocking (Fig. 6B). Further, the upregulation of Fas receptor was corroborated by Western blot (Fig. 6C) and by flow cytometry (Fig. 6D). IFN-g-mediated upregulation of Fas expression was blocked by NOS inhibitors and was restored by the NO donor (SNAP, 100 mM). Altogether, these results demonstrate a strong correlation between the generation of NO by AD10 ovarian carcinoma cells and upregulation of Fas receptor expression. DISCUSSION This study provides several lines of evidence that indicate that the sensitization to Fas-mediated apoptosis observed in the AD10 ovarian carcinoma cell line treated with IFN-g is due in
FIG. 3. NOS inhibitor blocks IFN-g-induced sensitization of AD10 cells to Fas-mediated cytotoxicity. Cytotoxicity of the Fas-agonistic antibody CH11 was assessed by LDH release into the culture medium at 24 h of A2780 (A) and AD10 (B) cells (1 3 10 4 cells/well) cultured in 96-well plates supplemented with 10% FCS and incubated for 24 h with IFN-g (10 U/mL, solid bars) in the presence (slashed bars) or absence of the NOS inhibitor L-NMA (1.0 mM). The NOS inhibitor effectively blocked the IFN-g-induced sensitization to Fas-mediated apoptosis (P , 0.001) in AD10 cells whereas in A2780, it was ineffective. **P , 0.005; ***P , 0.001.
of the Fas-agonistic antibody CH11 for 6 h resulted in a greater frequency of cells undergoing apoptosis (Fig. 5) compared with the untreated control group (Fig. 5). When the cells were incubated in the presence of the NOS inhibitor L-NMA (1 mM) 6 h prior to IFN-g pretreatment, the frequency of cells undergoing apoptosis (Fig. 5) was significantly reduced and was less than that of cells incubated in the absence of the NOS inhibitor. Furthermore, treatment of AD10 cells with SNAP prior to exposure to CH11 also increased the frequency of cells undergoing characteristic apoptosis (Fig. 5), whereas the addition of the NO donor alone did not increase the number of apoptotic cells (Fig. 5). NO upregulates Fas receptor expression. We examined the role of NO in the regulation of Fas receptor expression in AD10. First, AD10 cells were exposed to increasing concentrations of an NO donor (SNAP: 1, 10, and 100 mM) for 18 h and the relative Fas mRNA expression was determined by RT-PCR. Clearly, SNAP upregulated Fas receptor mRNA expression (Fig. 6A).
FIG. 4. NO donor sensitizes ovarian carcinoma cell lines to Fas-mediated cytotoxicity. Cultured A2780 (A) and AD10 (B) cells (1 3 10 4 cells/well) in 96-well plates supplemented with 10% FCS were incubated in the presence of the photoactivated SNAP (0, 10, and 100 mM) for 24 h and cytotoxicity of the Fas-agonist antibody (CH11) was assessed by LDH release into the culture medium. Significant sensitization was observed in AD10 and A2780 cells using 100 mM of SNAP. **P , 0.005; ***P , 0.001.
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FIG. 5. NO sensitizes AD10 cells to Fas-mediated apoptosis. Acridine orange/ethidium bromide staining revealed that IFN-g-pretreated (100 U/mL) AD10 cells (1 3 10 5 cells/well) cultured in 12-well plate for 18 h increased the amount of cells undergoing characteristic apoptosis (chromatin condensation and margination, membrane blebbing, etc.). These findings demonstrate sensitization to Fas-mediated apoptosis (c) when compared with background killing by CH11 alone (0.1 mg/mL) (b) or with the control-untreated group (a). Sensitization was blocked by incubating IFN-g-treated AD10 cells in the presence of the NOS inhibitor L-NMA (1.0 mM) (d). Treatment of AD10 cells with the NO donor SNAP (100 mM) for 18 h also sensitized these cells to be killed by apoptosis by CH11 (f), whereas the addition of SNAP in the absence of CH11 did not affect the cultured cells (e).
part to the generation of nitric oxide, or its reaction products, by the induction of iNOS in these cells. NOS inhibitors blocked this sensitization. The use of nitrodonors mimicked the IFN-g-mediated sensitization to the Fas-agonist antibody, CH11. There was a concurrent upregulation of Fas receptor, either upon induction of iNOS or upon treatment of the ovarian cell lines with nitrodonors. The observed upregulation of Fas receptor was abrogated by the use of NOS inhibitors suggesting a strong correlation that might account for the sensitization to the Fas-agonist antibody CH11. Nitric oxide is a potent and pleiotropic free radical molecule that has been involved in a wide variety of physiological and pathophysiological functions [16]. NO is generated in low levels by two constitutive nitric oxide synthases (eNOS and nNOS) and in much greater levels by the inducible NO synthase (iNOS) [17]. It has been shown that NO might participate in the apoptosis process by either inhibiting or promoting some apoptotic events [18 –21]. There has been a long debate about the specific role that nitric oxide might play in apoptosis. Recently, several studies have referred that nitric oxide is a novel and potent inhibitor of apoptosis. For instance, endogenous NO synthesis or exposure to low levels of NO donors was first shown to inhibit apoptosis in human B lymphocytes [21], and similar findings have been reported in splenocytes [18], eosinophils [19, 22], and endothelial cells [23]. NOS inhibitors have also been directed toward the specific disruption of the Fas-induced apoptotic mechanism. Basal NOS activity in human leukocytes was
shown to inhibits Fas-induced apoptosis via a cGMP-independent mechanism and further inhibition of caspase activation [24, 25]. Recent studies, examining the involvement of CD95/ CD95L system and signal transduction pathway in NO-induced apoptosis in human neoplastic lymphoid cells, demonstrated that NO triggers the death receptor system by regulating the expression of ligands involved in apoptosis like CD95L and TRAIL/APO-2L with unaltered expression of CD95 receptor after NO treatment [26]. In contrast, our results herein with ovarian carcinoma cells clearly demonstrate that NO, far from protecting tumor cells from Fas-induced apoptosis, synergized with the Fas agonist antibody CH11 in the induction of apoptosis. Differences among the effects of NO in the various systems used above are noteworthy. NO appears to inhibit Fas-induced apoptosis in transformed cells derived from the hematopoietic lineage, whereas it mediates sensitization to Fas apoptosis as observed here in ovarian tumor cells. This dichotomy represents a crucial point of divergence between the two cell types and needs to be considered when making strategies toward the use of NO as an anti-tumor agent. It has been shown that some tumor cells can be prompted to generate endogenously NO by the induction of iNOS upon treatment with proinflammatory cytokines alone or in combination [11, 27, 28]. Herein, we demonstrate the inducibility of the ovarian carcinoma cell line AD10 to generate NO by iNOS and the susceptibility of this cell line to be sensitized to Fas-mediated apoptosis. Furthermore, we have found that the
NITRIC OXIDE-MEDIATED SENSITIZATION TO FAS-INDUCED APOPTOSIS
FIG. 6. Upregulation of Fas receptor expression on AD10 cells by NO. (A) Upregulation of Fas receptor mRNA was detected by RT-PCR in AD10 cells (5 3 10 5/well) cultured in a 12-well plate supplemented with 10% FCS in the presence of increasing concentrations of SNAP (lane 1, control; lane 2, 1 mM; lane 3, 10 mM; lane 4, 100 mM). Relative expression of Fas receptor mRNA amplicons was assessed by densitometric analysis of the ethidiumbromide-stained DNA band resolved in agarose gel electrophoresis (A, bottom). (B) IFN-g-mediated upregulation of Fas mRNA expression (lane 1, control; lane 2, 10 U/mL; lane 3, 100 U/mL) was reduced by incubation off cells with the NOS inhibitor L-NMA (1.0 mM) for 24 h (lane 4, IFN-g 10 U/mL 1 L-NMA; lane 5, IFN-g 100 U/mL 1 L-NMA). Relative densitometric values of a representative experiment are shown in the lower panel. (C) Upregulation of Fas receptor protein level (lane 1, control; lane 2, IFN-g 100 U/mL) was detected by Western immunoblot in IFN-g-induced AD10 cells (1 3 10 7/plate) cultured in a 100-mm plate supplemented with 10% FCS. IFN-g-mediated upregulation of Fas protein level was markedly reduced in the presence of the NOS inhibitor (lane 3, IFN-g 100 U/mL 1 L-NMA 1.0 mM) and restored by the addition of SNAP (lane 4, SNAP 100 mM). (D) Increased expression of Fas surface molecule was detected by flow cytometry on A2780 and AD10 (2 3 10 5 cells/well) cultured in 12-well plates supplemented with 10% FCS in the presence of IFN-g (10 U/mL) for 18 h (solid bars) when compared with untreated control cells (blank bars). This increased expression was partially blocked by the NOS inhibitor (slashed bars, IFN-g 10 U/mL 1 L-NMA 1.0 mM) and restored by incubation with the NO donor (dotted bars, SNAP 100 mM).
parental cell line A2780, which is not able to express iNOS upon IFN-g stimulation, was not sensitized at the same level when compared with the Adriamycin-resistant cell line AD10, which is induced by IFN-g alone to express iNOS. However, when nitrodonors were used as exogenous sources of NO, a similar sensitization to Fas-mediated apoptosis was observed in both cell lines as well in two other human prostate cancer cell lines (data not shown). This differential response to endoge-
263
nous versus exogenous sources of NO suggests the susceptibility of tumor cells to be sensitized by NO despite their capacity to be induced to express iNOS. Previous studies demonstrated the influence of IFN-g in the regulation of Fas receptor expression on the tumor cell surface [8]. Furthermore, studies searching for the role of NO in vascular smooth muscle cell apoptosis demonstrated that NO induces upregulation of Fas antigen expression via a cGMPindependent mechanism [29]. Likewise, it has been shown that NO primes pancreatic b cells for Fas-induced apoptosis by increasing the surface CD95 receptor expression [30]. However, it is not clear how NO can interact with the transcriptional machinery in order to regulate Fas gene expression. In this study, evidence is provided that suggests the presence of a correlation between IFN-g-mediated sensitization of ovarian carcinoma cell lines to Fas-induced apoptosis and the generation of NO. The sensitization achieved by IFN-g treatment was blocked by the NOS inhibitor (L-NMA, 1 mM) and was restored by the NO donor (SNAP, 100 mM). Furthermore, the modulation of Fas expression correlated with either the endogenous production of NO by iNOS or the exogenous presence of the NO donor. Altogether, these results suggest an important role of NO in the regulation of Fas gene expression. NO has been increasingly implicated in the control of the activity of the transcription machinery. Reactive nitrogen intermediates can serve as intracellular second messengers to induce IL-8 gene expression [30 –32]. AP-1 (a common regulatory element found along the Fas promoter) can be attenuated by its proteolytic decomposition, which might be mediated by S-nitrosylation of AP-1 [33, 34]. Thus, the regulation of the Fas gene expression by NO observed in the ovarian carcinoma cell lines may be explained by alteration of the composition of the transcriptional regulatory units by reactive nitrogen intermediates. In summary, the present study shows that the AD10 ovarian carcinoma cell line, when stimulated with IFN-g, can express iNOS and produce NO. The generation of NO correlates with the sensitization of AD10 cells to Fas-induced apoptosis and can be blocked by the NOS inhibitor, thus implicating the role of NO in the IFN-g-mediated sensitization to Fas-induced killing. Moreover, the use of NO donor bypassed the inability of the parental cell line A2780 to express iNOS and sensitized those cells to the Fas agonist antibody. Sensitization was concomitantly observed with upregulation of Fas gene expression. In contrast to the role of NO in protecting against apoptosis in cells of the hematopoietic lineage, our findings demonstrate that NO plays a role in the sensitization of tumor cells to Fas-mediated apoptosis. Such sensitization is due to the regulation of Fas gene expression and/or signaling toward apoptosis. Our data raise the possibility that NO generation (NO-based therapies) can be used to control tumor cell death by apoptotic-mediated mechanisms.
´ N AND BONAVIDA GARBA
264 ACKNOWLEDGMENTS
We thank Dr. Jon Fukuto for his thorough discussions of this study and his fine NO chemistry comments. We are grateful to the members of the UCLA Women’s Reproductive Cancer Program, The Multidisciplinary Ovarian Cancer Research Group, for their advice. The authors also thank Ms. Samantha Nguyen for preparing the manuscript.
16. 17. 18.
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