Novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid–nitric oxide (GIT-27NO) induces p53 mediated apoptosis in human A375 melanoma cells

Nitric Oxide 19 (2008) 177–183

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Novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid–nitric oxide (GIT-27NO) induces p53 mediated apoptosis in human A375 melanoma cells Sanja Mijatovic a,1, Danijela Maksimovic-Ivanic a,1, Marija Mojic a, Graziella Malaponte b, Massimo Libra b, Vera Cardile c, Djordje Miljkovic a, Ljubica Harhaji a, Darrin Dabideen d, Kai Fan Cheng d, Ylenia Bevelacqua b, Marco Donia b, Gianni Garotta e, Yousef Al-Abed b, Stanislava Stosic-Grujicic a, Ferdinando Nicoletti c,* a

Department of Immunology, Institute for Biological Research ‘‘Sinisa Stankovic”, Belgrade University, Belgrade, Serbia Department of Biomedical Sciences, University of Catania, Via Androne, 83, 95124, Catania, Italy c Department of Physiological Sciences, University of Catania, Catania, Italy d Laboratory of Medicinal Chemistry, North Shore Long Island Jewish Health System, NY, USA e Ganial Immunotherapeutics Inc., Wilmington, DE 19801, USA b

a r t i c l e

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Article history: Received 3 February 2008 Revised 6 April 2008 Available online 19 May 2008 Keywords: A375 melanoma cells Apoptosis VGX-1027 GIT-27NO Nitric oxide YY1 p53

a b s t r a c t In this study we evaluated the effects of the new NO donating compound (S,R)-3-phenyl-4,5-dihydro-5isoxazole acetic acid–nitric oxide (GIT-27NO) on the A375 human melanoma cell line. Treatment with the drug led to concentration-dependent reduction of mitochondrial respiration and number of viable cells in cultures. Decreased cell viability correlated with release and internalization of NO and was neutralized by the extracellular scavenger hemoglobin. GIT-27NO neither influenced cell division nor induced accidental or autophagic cell death. Early signs of apoptosis were observed upon coculture with the drug, and resulting in marked accumulation of hypodiploid cells, suggesting that the induction of apoptosis is one primary mode of action of the compound in A375 cells. GIT-27NO significantly inhibited the expression of the transcription repressor and apoptotic resistant factor YY1 and, in parallel, augmented the presence of total p53. The capacity of GIT-27NO to induce p53-mediated apoptosis along with inhibition of YY1 repressor in A375 melanoma cells indicates that GIT-27NO possesses an important anti-cancer pharmacological profile. The findings suggest the potential therapeutic use of GIT-27NO in the clinical setting. Ó 2008 Elsevier Inc. All rights reserved.

Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) represent a class of agents synthesized by covalent attachment of the NO moiety to the parental compound through an aromatic or aliphatic spacer [1]. Unlike conventional NSAIDs, they exhibit less adverse effects, such as gastrointestinal toxicity, while retaining beneficial activities of their parent compounds. It has also been shown that the so-modified NO-NSAID may acquire 30- to 5000-fold greater anti-cancer effects than the NO-deprived parental compound both in vitro and in vivo [2–7], and that the acquisition of these anti-cancer properties is not secondary to differences in chemical structure of the modified NSAIDs [8].

Abbreviations: VGX-1027, (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid; GIT-27NO, (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid–nitric oxide; NONSAIDs, nitric oxide-donating non-steroidal anti-inflammatory drugs; YY1, Yin Yang 1. * Corresponding author. Fax: +39 095 320267. E-mail address: [email protected] (F. Nicoletti). 1 These authors equally contributed to this manuscript. 1089-8603/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2008.04.004

One possible mechanism by which NO-NSAIDs develop anticancer activities is may depend on their capacity to induce apoptosis through generation of oxidative stress [9]. It is well documented that NO is involved in several pathways responsible for induction of apoptosis [10,11]. In addition, NO controls transcriptional events, serves as intracellular second messenger, and is capable of altering the activity of numerous enzymes relevant for cell proliferation, differentiation and death [12,13]. For instance, NO inhibited Fas-induced apoptosis in transformed hematopoietic derived cells and also interrupted programmed cell death through direct blockade of caspases. However, in other circumstances, NO sensitized ovarian and other solid tumor cells to Fas-mediated apoptosis through regulation of Fas gene expression and signaling toward apoptosis [12]. It is known that Fas expression is negatively regulated by Yin Yang 1 (YY1), a multifunctional transcription factor involved in the regulation of many mammalian genes, which acts as repressor of the Fas promoter [12]. It was also recently documented that drug-induced sensitization of tumor cells to tumor necrosis factor receptor apoptosis induced ligand (TRAIL) is mediated, in part, by

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inhibition of YY1 followed by an up-regulation of DR5 expression; the promoter of DR5 has in fact one putative binding site for the transcription repressor YY1 [14]. It is well documented that YY1 is crucial in embryogenesis, differentiation, replication, and cellular proliferation. Depending on the context, YY1 can initiate, activate, and repress transcription. Control of YY1 activity by transcription factors and cytoplasmatic proteins has been the focus of much attention [15]. The presence of zinc-finger domains renders YY1 sensitive to NO mediated S-nitrosylation of cysteine and thus interrupts its binding to DNA [16,17]. Hence, if YY1 overexpression plays a key role in cellular proliferation, development of apoptosis resistance and malignant transformation, targeting YY1 expression and activity could represent a novel avenue for more specific antitumor therapy [15]. Since NO compromises YY1 binding affinity, NO chimeric drugs represent ideal candidate for specific YY1-targeted therapy. In contrast to conventional NO-NSAIDs such as NO-aspirin which possesses incorporated spacer endowed with own antitumor capacity, the novel isoxazole derivative, GIT-27NO (Fig. 1), generated by modifying the parental antiinflammatory compound VGX-1027, is generated by direct linking of NO to the original compound [18–21]. We have very recently reported that this chemical modification results in marked NOdependent tumoricidal activity of the drug both upon in vitro and in vivo conditions [21]. Determined by cell specificity, NO released from the compound, in association with reactive oxygen species, selectively affected MAP kinases pathways and promoted different type of programmed cell death. In the present study we evaluated the anti-melanoma activity of GIT-27NO on the A375 cell line as well as the intracellular events targeted by the drug. Experimental procedures Reagents and cells Acridine orange was obtained from Labo-Moderna (Paris, France) and carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Molecular Probes (Eugene, USA). Other reagents were purchased from Sigma (St. Louis, USA) unless otherwise indicated. GIT-27NO and VGX-1027 were synthesized as described elsewhere [19,21] and were purchased from GaNiAl Immunotherapeutics (Locust Valley, NY, USA). GIT-27NO (M = 328 g/mol) and VGX-1027 (M = 205 g/mol) were stored at +4 °C as a 5 mg/ml stock solution in 2.5% DMSO–H2O, and were diluted in culture medium immediately before use. Control cell cultures were treated with an adequate volume of DMSO. A375 human melanoma cell line was obtained from American Type Culture Collection (Rockville, MD, USA) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 2.0 mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin, and 25 lg/ml fungizone (Sigma–Aldrich, Italy), and incubated at 37 °C and 5% CO2/95% air. Cells from confluent cultures were detached using 0.25% trypsin–1 mM EDTA and seeded in complete DMEM medium. Cells were seeded at 1  104 cells/well in 96-well plate for the viability tests, 2.5  105 cells/well in 6-well plate for Flow cytometry, or 3  104 cells/well in 4well chamber slide, cultivated overnight, and treated with GIT-27NO as indicated.

[24]. MTT and CV absorption were assessed by an automated microplate reader at 570 nm, while the pyruvate-mediated conversion of 2,4-dinitrophenylhydrazine into visible hydrazone precipitate in LDH assay was measured at 492 nm. The results of MTT and crystal violet assay were presented as % of control values obtained in untreated cell cultures. The % of LDH release was determined using the following formula: (E  C)/(TC)  100, where E is the experimental absorbance of cell cultures, C is the control absorbance of cell-free culture medium, and T is the absorbance corresponding to the maximal (100%) LDH release of Triton-lysed cells. Determination of cell proliferation The rate of cell proliferation was determined using flow cytometric analysis of cells labeled with CFSE [25]. Briefly, cells were detached, loaded with 1.5 lM CFSE for the 15 min at 37 °C, washed two times and then seeded in 6-well plates at 2  105 cells/well. Cells were treated with the drug for 24 and 48 h, trypsinized and washed two times. Finally, the cells were resuspended in PBS and analyzed by flow cytometry. Green fluorescence emission from cells illuminated with excitation light of 488 nm was measured with a FACSCalibur (BD, Heidelberg, Germany) and analyzed using CellQuest software. Determination of apoptotic and autophagic cell death Apoptotic cell death was assessed by flow cytometry analysis of either DNA fragmentation in cells stained with the DNA-binding dye propidium-iodide (PI) or position of phosphatidylserine with annexin V, and microscopic evaluation of propidium-iodide stained fixed cells [26]. For cell cycle analysis, after 24 h incubation in 6-well plate, the cells were detached with trypsin, washed and fixed in 70% ethanol at 4 °C for 30 min. After removing the ethanol by repeated washing in PBS, the cells were resuspended in PBS containing 1 mg/ml RNase and PI (40 lg/ml) and kept at 37 °C in the dark for 30 min. Red fluorescence was analyzed with FACSCalibur flow cytometer (BD, Heidelberg, Germany), using a peak fluorescence gate to exclude cell aggregates. Cell distribution among cell cycle phases was determined with Cell Quest Pro software (BD) and hypodiploid cells in sub-G0/G1 compartment were considered apoptotic. Early apoptosis was detected upon 8 h of incubation of cells with the drug with ApoAlertÒAnnexinV-FITC kit that was used according to the manufacturer’s (Clonotech Laboratories, Mountain View, CA) instruction. For morphological examination, at the end of incubation period, the cells were fixed with 4% paraformaldehyde 15 min at RT, subsequently washed with PBS and stained with 20 lg/ml PI for 2 min. Finally, the cells were washed, covered with fluorescent mounting media (Dako, Glostrup, Denmark) and analyzed under the fluorescent microscope. Determination of autophagy is based on the detection of acidic vesicular organelles, stained by the vital dye acridine orange. The intensity of red fluorescence is proportional to the acidity and volume of present organelles, while the cytoplasm and nucleolus emit bright green and dim red [27]. For the detection of autophagy, the cells were cultured 24 h in the presence of the drug, detached with trypsin at the end of cultivation and stained with 1 lg/ml acridine orange for 15 min at RT. At the end of incubation period the cells were washed and resuspended in PBS. Green (FL1) and red (FL3) fluorescence emission from 104 cells was measured with a FACSCalibur and analyzed using CellQuest software. Measurement of intracellular NO 4-Amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM diacetate, Molecular Probe, Leiden, The Netherlands) is used as intracellular NO indicator. Briefly, after 2 h of cultivation in the presence of GIT-27NO the cells were incubated with diluted DAF-FM diacetate (2 lM) 1 h at 37 °C, washed and additionaly incubated 15 min at 37 °C in phenol red- and serum-free RPMI for the completition of de-esterification of intracellular diacetates. Finally, the cells were washed and resuspended in PBS. Green (FL1) fluorescence emission from 104 cells was measured with a FACSCalibur and analyzed using CellQuest software.

Cell viability determination by the MTT, crystal violet and LDH release assays Cell-based ELISA Mitochondrial-dependent reduction of 3-4,5-dimethylthiazol-2-yl)-2,5-iphenyltetrazolium bromide (MTT) to formazan reflects mitochondrial activity of cultured cells [22], intensity of crystal violet (CV) staining is directly proportional to the number of adherent cells [23], while the release of cytosolic lactate dehydrogenase (LDH) indicates the loss of membrane integrity that occurs in necrotic cells

Fig. 1. GIT-27NO structure.

For measuring p53 expression, a modified method for cell-based ELISA by Versteeg et al. [28] was employed. At the end of incubation, the cells were fixed in 4% paraformaldehyde, endogenous peroxydase was quenched with 1% H2O2 in PBS containing 0.1% Triton X-100 (PBST), and unspecific binding of antibodies blocked with PBST solution containing 10% FCS. Primary rabbit polyclonal antibody specific for total p53 at a concentration 1:250 (Santa Cruz Biotechnology, Santa Cruz, CA) was applied in PBST supplemented with 2% bovine serum albumin (PBSTB), followed by secondary peroxidase-conjugated donkey anti-rabbit IgG (1:2500 in PBSTB; Amersham Biosciences, Buckinghamshire, UK). Both incubations were at 37 °C for 1 h. The absorbance at 450 nm was measured in an automated microplate reader 15 min after incubation with peroxydase substrate TMB and subsequently to addition of 0.1 M HCl. The obtained absorbances were corrected for the cell number determined by CV staining, as described in the original protocol. The results are presented as relative expression in comparison with the control value, which was arbitrarily set to 1.

S. Mijatovic et al. / Nitric Oxide 19 (2008) 177–183 Immunocytochemical analysis of p53 At the end of cultivation period the cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS and endogenous peroxydase was quenched with 3% H2O2–10% methanol in PBS. After the blocking of unspecific binding of antibodies with PBST solution containing 5% FCS, the cells were incubated with primary polyclonal anti-p53 antibody (1:250) over night at 4 °C. Detection was performed with rabbit extravidin peroxidase staining kit (Sigma) according to the manufacturer’s instructions and diaminobenzidine (R&D Systems, Minneapolis, MN) as a substrate. The cells were counterstained with Mayer’s hematoxylin and slides were mounted with glycergel mounting medium (Dako, Glostrup, Denmark). Western blotting The expression of YY1 was determined by Western blot analysis. A375 cells were cultured in 75-cm2 flasks and treated 1–2 days before reached the confluence. Drug solutions were prepared in DMSO and stored at +4 °C (stock solution) and diluted in culture medium immediately before use. The subconfluent cells were treated for 72 h with 37.5, 75, and 150 lM of GIT-27NO. Control cultures were treated with DMSO alone. Briefly, the untreated or treated and harvested cells were washed twice with ice-cold PBS, collected with lysing buffer containing 50 mM Tris–HCl, pH 7.5 plus 20 mM EDTA and 0.5% SDS, and, after 30 min on ice, homogenized and centrifuged at 13,000g for 15 min. Thirty micrograms of total protein, present in the supernatant, was loaded into a lane and separated on a 4–12% Novex Bis–Tris gel by electrophoresis (NuPAGE, Invitrogen, Italy). The separated proteins were then transferred to nitrocellulose membrane (Invitrogen, Italy) in a wet system. The transfer of proteins was verified by staining the nitrocellulose membrane with Ponceau S and staining the Novex Bis–Tris gel 7 with Brillant blue R. Membranes were blocked in Tris-buffered saline containing 0.01% Tween-20 (TBST) and 5% non-fat dry milk at 4 °C overnight. Respectively, a primary rabbit polyclonal anti-YY1 (1:100 dilution in 5% milk, Santa Cruz Biotechnology, Italy), and rabbit polyclonal a-tubulin antibody (1:1000 dilution in 5% milk, Sigma–Aldrich) were incubated overnight at 4 °C. Antibodies were detected with horseradish peroxidase-conjugated secondary antibody using the enhanced chemiluminescence detection Supersignal West Pico Chemiluminescent Substrate (Pierce, France). The bands were measured densitometrically and their relative density was calculated based on density of a-tubulin band in each sample. The values were expressed as arbitrary densitometric units corresponding to signal intensity. Statistical analysis The significance of the differences between various treatments was analyzed by ANOVA followed by Student–Newman–Keuls test for multiple comparisons. A p value less than 0.05 was considered significant.

Results GIT-27NO, but not VGX-1027, inhibits A375 melanoma cell viability: Role of NO To examine the sensitivity of human melanoma A375 cells to GIT-27NO and its parental compound VGX-1027, MTT and CV tests were performed after 24 h of coculture of the cells in the presence of either compound. As seen on Fig. 2a and b, treatment of the cells with VGX-1027 did not affect the number of viable A375 cells. On the other hand, GIT-27NO strongly inhibited their viability in a concentration-dependent manner (Fig. 2a and b). Inhibition of cell growth was followed by elevated nitrite accumulation measured by the Griess reaction (not shown). Significant amount of intracellular NO, as a consequence of internalization of liberated NO by the cells, was detected after 2 h of culture in the presence of GIT-27NO (Fig. 2c). In order to confirm the crucial role of NO generated from the test compound in this setting, the cells were treated with the extracellular scavenger hemoglobin in the presence or absence of GIT-27NO and cell viability was determined by MTT after 24 h. Removal of NO by the scavenger resulted in recovering of cell viability even in the presence of higly toxic concentration of GIT-NO (150 lM) (Fig. 2d). This effect was further visualized by light microscopy (Fig. 2e). Thus, GIT-27NO exhibits strong NO-dependent tumoricidal activity against A375 cells.

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GIT-27NO induces type 1 programmed cell death In order to investigate the possible antiproliferative effect of GIT-27NO, the cells were stained with CFSE dye and further exposed to the 75 lM of GIT-27NO. After 48 h of incubation, the percentage of divided and nondivided cells, analyzed by Flow cytometry, did not differ from untreated controls. This indicates the irrelevance of drug to influence cell proliferation (Fig. 3a). To further explore the cause of decreased viability, we performed the LDH release assay for the evaluation of necrosis and acridine orange staining for detection of autophagic cell death, known as programmed cell death type II. Treatment of A375 melanoma with GIT-27NO neither affects cell membrane permeability nor promotes the formation of autophagosomes (Fig. 3b and c, respectively). On the other hand, time dependent accumulation of the cells in the subG compartment was observed upon GIT-27NO treatment (Fig. 3d). Concordantly, typical morphological appearance of apoptosis was visualized on PI stained cells (Fig. 3e). Phosphatidylserine translocation detected after 8 h of incubation with GIT27NO preceded previously mentioned apoptotic events (Fig. 3f), thus confirming the capacity of GIT-27NO to activate apoptotic pathways in A375 cells. GIT-27NO attenuates the expression of the transcription repressor YY1 We next analyzed the effect of GIT-27NO on YY1 protein expression. Treatment with GIT-27NO reduces significantly the expression of YY1 in melanoma cell line, while YY1 over-expression was observed in control cells (Fig. 4). The reduction of YY1 expression in treated cells was concentration-dependent. These findings provide evidence that one of the proposed antitumor mechanisms of GIT-27NO may be related to inhibition of the anti-apoptotic transcription repressor YY1. GIT-27NO increases p53 expression Having shown that GIT-27NO inhibits the expression of the transcription factor YY1 in A375 cells and knowing that YY1 acts as a negative regulator of p53 at multiple levels [29] we next studied the effects of GIT-27NO on p53 expression. After 24 h of culture with the drug, the levels of total p53 protein expression detected by cell-based ELISA were significantly higher in GIT-27NO-treated cells as compared to control cells (Fig. 5a). Significant expression of the tumor suppressor protein p53 was further confirmed by immunocytochemical analysis (Fig. 5b). Discussion This study provides clear-cut in vitro evidence for the anti-melanoma properties of the recently generated new chemical entity (NCE) GIT-27NO. In agreement with previous data [21], this study confirms that covalent addition of the NO moiety to the parental compound allows the generation of an NCE endowed with anticancer effects that are not observed with VGX-1027. The lack of antitumor effects of VGX-1027 is additionally proven by the fact that blocking NO activity by using the extracellular scavenger hemoglobin reverted the antitumor effect of GIT-27NO. This finding suggests the essential role of NO in mediating the antitumor effects of GIT-27NO, and also suggests that the remaining part of the drug may be devoided of anti-tumor effects. Intracellular staining with the specific dye DAF-FM diacetate showed that liberated NO rapidly entered into the cells. The absence of NO carrier that structurally differentiates GIT-27NO from other NO-NSAIDs offers theoretical advantages to the former over the latter and indicates that GIT-27NO may represent the prototype of a completely new class

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Fig. 2. GIT-27NO-induced NO-mediated decrease of viability of the A375 melanoma cell line. Cells (1  104 cells/well) were treated with different concentrations of either VGX-1027 or GIT-27NO for 24 h and thereafter cell viability was determined by the MTT (a) and CV (b) tests. The data are presented as mean ± SD from representative of three independent experiments (*p < 0.05, refers to untreated cells). Intracellular NO was determined by flow cytometry of DAF-FM diacetate fluorescence after 2 h of incubation of cells without or with GIT-27NO (75 lM) (c). Cells were incubated with or without hemoglobin (12.5 lM) and /or GIT-27NO (75 lM) and after the 24 h of incubation MTT assay (d) or light microscopy analyses (mag.  400) were performed (e).

of NO-releasing agents. For example, it was shown that the spacer that permitted NO binding to aspirin is endowed with expected tumoricidal properties, [20,26,30]. In addition, the entire toxicity of NO-aspirin is more complex than expected because the three components-NO, spacer and the parental compound contributed to it. NO delivered by GIT-27NO provoked the appearance of early apoptosis characterized by the inverted position of phosphatidyl serine and stable permeability of cell membrane. Marked timedependent hypodiploid cells accumulation in sub-G0/G1 compartment and deficiency of cell death triggered by autophagy or necrosis demonstrated that programmed cell death type I, better known as typical apoptosis, is probably the cause of diminished cell viability promoted by the drug. Apoptosis is described as one of the basic mechanism of action of NO-NSAIDs on different cell lines. NO-aspirin triggered apoptosis in colon cancer cells, and NO-sulindac induced apoptosis of bladder and prostate cancer cells [8,9,31]. Moreover, GIT-27NO promoted apoptosis in L929 fibrosarcoma cells since in B16 melanoma and C6 astrocytoma preferentially in-

duced the autophagic cell death [21]. Multifaceted role of NO in apoptosis induction as well as in modulation is well documented [10,11,13]. Depending on the cell type, source and amounts released, NO can act as both pro- and antiapoptotic mediator. Whilst NO often promotes apoptosis at high concentration, it may led to apoptosis resistance when present at low concentrations [13]. The combined action of NO and other oxygen radicals has been shown to cause DNA damage with up-regulation of p53 [32]. The role of NO in transcriptional regulation of many genes included in induction and apoptosis propagation has been the focus of much attention [32–37]. As a highly reactive and instable molecule, NO can influence the activity of different enzymes and transcription factors responsible for proliferation, differentiation and cell death through nitration and nitrosylation processes [13,32]. It has been shown that nitrosylation of the cysteine thiols groups and subsequent S-nitrosothiol formation in zinc-finger domains of YY1 repressor significantly inhibited its binding affinity to DNA sequence in promoters of genes which are under its control [12,16,17]. Diversity of effects of these interactions was underlined

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Fig. 3. GIT-27NO-induced apoptotic cell death of A375 melanoma cells. Cells were stained with CFSE (1.5 lM) and treated with GIT-27NO (75 lM). Percentage of divided cells was determined by flow cytometry after 48 h (a). Cells were incubated with different concentrations of GIT-27NO for 24 h, and LDH release assay was performed (b). After 24 h of culture in the presence of GIT-27NO (75 lM), cells were stained with either acridine orange (c) or with PI (d and e), and analyzed by flow cytometry for the presence of orange–red acidic autophagic vesicles (c), or by flow cytometry (d) and fluorescent microscope (e). Cells were incubated 8 h without or with GIT-27NO (75 lM), stained with annexin V and PI and analyzed by flow cytometry (f).

Fig. 4. GIT-27NO down-regulates YY1 expression in A375 melanoma cells. Cells were treated 1–2 days before reached of the confluence with 10, 25, and 50 lg/ml of GIT-27NO for 72 h and, thereafter, total YY1 was determined by Western Blotting.

by numerous examples of genes regulations by different transcription factors such as YY1. YY1 plays a key role in embryonal development and differentiation as well as in basic cellular processes such as replication, proliferation, senescence, and response to genotoxic stress [15]. As regard to tumor biology, YY1 is thought to possess protumorigenic activity by down-regulating the process of cell death induced by some genotoxic stimuli. The regulatory role of YY1 in activation, progression and maintaining of malignancy has been demonstrated in different tumor models both in vitro and in vivo [15]. It has been therefore suggested that YY1 may represent a novel target of antitumor therapy. We presently demonstrated that along with down-regulating of viability and induction of programmed cell death type I in A375

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Fig. 5. GIT-27NO strongly enhanced p53 expression. Cells were treated with different concentrations of GIT-27NO for 24 h and thereafter total p53 was determined by cellbased ELISA (a) or by immunocytochemistry (b).

melanoma cells, GIT-27NO simultaneously silenced the expression of YY1. This could be related to the strong activation of p53 and observed G2/M arrest induced by the compound in A375 cells. Several data have also confirmed the relationship in tumorigenesis between YY1 and tumor suppressor gene p53. Furthermore, genetic ablation of YY1 in B lymphoma DT40 resulted in significantly increased expression of p53 and reduction of endogenous p53 ubiquitination. In agreement with this, increased existence of YY1 led to augment Hdm2 mediated ubiquitination of p53 both in vitro and in vivo [29,35]. We hypothesize that the antitumor effects of GIT-27NO secondary to NO release could cause weakened activity of preexisted YY1 and consequently up-regulation of p53 gene and other genes which are repressed by YY1. Furthermore, p53 is proposed to inhibit YY1 expression [36] and prohibitinrepressor of YY1 promoter binds to p53 binding site suggesting possible role of p53 in prolonged repression of YY1 [37]. The above leads us to hypothesize that GIT-27NO-mediated down-regulation of YY1 expression contributed to the observed up-regulation of p53 detected upon GIT-27NO exposure of A375 cells. Besides it’s own tumoricidal potential, the observed capacity of GIT-27NO to down-regulate YY1 could serve as a mechanism responsible for tumor sensitization to drug-mediated apoptosis and therefore indicates its application in combination with conventional chemotherapeutics. Acknowledgment This work was partly supported by the Serbian Ministry of Science (Grant No. 143029). References [1] J.E. Keeble, P.K. Moore, Pharmacology and potential therapeutic applications of nitric oxide-releasing non-steroidal anti-inflammatory and related nitric oxide-donating drugs, Brit. J. Pharmacol. 137 (2002) 295–310. [2] B. Rigas, K. Kashfi, Nitric-oxide-donating NSAIDs as agents for cancer prevention, Trends Mol. Med. 10 (2004) 324–330. [3] K. Kashfi, Y. Ryan, L.L. Qiao, J.L. Williams, J. Chen, P. Del Soldado, F. Traganos, B. Rigas, Nitric oxide-donating nonsteroidal anti-inflammatory drugs inhibit the growth of various cultured human cancer cells: evidence of a tissue typeindependent effect, J. Pharmacol. Exp. Ther. 303 (2002) 1273–1282. [4] N. Ouyang, J.L. Williams, G.J. Tsioulias, J. Gao, M.J. Iatropoulos, L. Kopelovich, K. Kashfi, B. Rigas, Nitric oxide-donating aspirin prevents pancreatic cancer in a hamster tumor model, Cancer Res. 66 (2006) 4503–4511. [5] N. Nath, G. Labaze, B. Rigas, K. Kashfi, NO-donating aspirin inhibits the growth of leukemic Jurkat cells and modulates beta-catenin expression, Biochem. Biophys. Res. Commun. 326 (2005) 93–99.

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