Free Radical Biology & Medicine 48 (2010) 1090–1099
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Free Radical Biology & Medicine
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Original Contribution
Induction of caspase-independent apoptotic-like cell death of mouse mammarytumor TA3Ha cells in vitro and reduction of their lethality in vivo by the novelchemotherapeutic agent GIT-27NO
Sanja Mijatovic a, Danijela Maksimovic-Ivanic a, Gordana Timotijevic b, Djordje Miljkovic a, Marco Donia c,Massimo Libra c, Marinella Coco d, James McCubrey e, Yousef Al-Abed f, Aleksandra Korac g,Stanislava Stosic-Grujicic a, Ferdinando Nicoletti c,⁎a Department of Immunology, Institute for Biological Research “Sinisa Stankovic,” Belgrade University, 11000 Belgrade, Serbiab Institute of Molecular Genetics and Genetic Engineering, Belgrade University, 11000 Belgrade, Serbiac Department of Biomedical Sciences, University of Catania, 95125 Catania, Italyd Department of Physiological Sciences, University of Catania, 95125 Catania, Italye Department of Microbiology & Immunology, Brody School of Medicine at East Carolina University, Greenville, NC 27858, USAf Laboratory of Medicinal Chemistry, North Shore Long Island Jewish Health System, Manhasset, NY 11030, USAg Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia
The new chemical entity GIT-27NO was created by the covalent linkage of a NO moiety to the anti-inflammatory isoxazoline VGX-1027. The compound has been shown to possess powerful anticancer effectsboth in vitro and in vivo. However, its effects on nonsolid and metastatic forms of tumors have not yet beeninvestigated. We have studied the effects of GIT-27NO on the highly invasive mouse mammary TA3Ha cellline in vitro and in vivo. In contrast to the conventional exogenous NO donor sodium nitroprusside, GIT-27NO successfully enhanced intracellular NO concentration in TA3Ha cells. Intracellular accumulation of NOwas followed by marked decrease in TA3Ha cell viability accompanied by typical apoptotic features.Interestingly, inverted membrane phosphatidylserine residues, reduced volume of nucleus, condensedchromatin, and terminal fragmentation of DNA were associated with inhibited caspase-3 activity andtranscription of the genes encoding caspase-3, -8, and -9. In parallel, GIT-27NO rapidly but transientlyprevented the loss of p53 through phosphorylation on Ser 20 and provided the necessary signals for theexecution of downstream processes without p53 de novo synthesis. The caspase-independent apoptotic-likedeath process triggered by GIT-27NO could be mediated by markedly down-regulated expression of theantiapoptotic Bcl-2 molecule observed in TA3Ha cells exposed to GIT-27NO. In agreement with these in vitrodata, GIT-27NO efficiently suppressed the growth of the ascites form and associated lethality of tumorinduced by TA3Ha cells in mice.
Increasing evidence indicates that, in addition to their primarypharmacological anti-inflammatory activity, some nonsteroidal anti-inflammatory drugs (NSAIDs) may also be effective in cancerprevention, particularly in colon, rectal, and stomach cancer [1–3].Stimulation of apoptosis, cell growth suppression, and inhibition ofangiogenesis are induced by these compounds even in COX-negativecells, indicating that COX-independent mechanisms are involved intheir antitumor effects [4–7]. However, numerous side effects, mainlyin the esophagus, stomach, and small intestine, limit the application ofthese drugs [8]. An approach frequently used to overcome drug
toxicity is chemical modification of NSAIDs through covalentattachment of a nitric oxide (NO) moiety to the original structurevia addition of a molecular carrier [8]. It has been shown that thesedesigned hybrid nitrates possess hundreds to thousands fold higheranticancer potential in comparison to the parental drugs [8–11]. Thisdoes not simply result from NO release from the NO-conjugatedNSAID, but is also the consequence of the molecule carrier, which isusually a quinone molecule. However, in addition to beneficialantitumor effects, this type of molecule might be also genotoxic,which could compromise the safety and therapeutic application of thedrugs [10–12]. Therefore we have recently described a procedure tosynthesize an isoxazoline compound with such structural modifica-tion that avoids the involvement of a molecule “carrier” (Supplemen-tary Fig. S1) [13]. The new drug, GIT-27NO, is composed of theparental drug (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid
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(named VGX-1027) and a NO-donating group. The original com-pound, VGX-1027, exhibits powerful anti-inflammatory effects inseveral animal models of acute and chronic immunoinflammatorydisease [14,15]. The chemical intervention performed on VGX-1027resulted in significant changes in its primary mode of action but alsogenerated a completely new chemical entity. Whereas the parentalanti-inflammatory drug did not elicit any antitumor effects over abroad range of doses tested, the modified substance acquiredpowerful tumoricidal effects [13,16,17]. GIT-27NO acted with almostequal efficiency in several tumor cell lines from different species andcells representing different stages of differentiation. The compounddown-regulates tumor cell viability through induction ofprogrammed cell death types I and II accompanied by significantcellular uptake of NO, consequent generation of reactive oxygenspecies, high levels of tyrosine residue nitration, and cellular-specificactivation of MAP kinase family members [13,16]. The antitumorproperties of GIT-27NO are clearly dependent on the release of highlyreactive NO upon contact with cells or cellular products. Although theprecise trigger for NO release is still unclear, the stimulus might act inthe extracellular compartment, as the removal of NO by extracellularscavenger hemoglobin completely abolished the tumoricidal action ofGIT-27NO [13]. Anticancer properties of GIT-27NO were confirmed invivo in syngeneic and xenograft tumor models such as solidmelanoma and hormone-independent prostate cancer models[13,17].
The aim of this study was to determine whether GIT-27NO is alsocapable of exerting its anticancer effects in nonsolid tumors as well asin tumors with highmetastatic potential. For this purpose we used themodel of terminal peritoneal cancer induced by intraperitoneal (ip)injection of TA3Ha mouse mammary epithelial cells [18]. Our studyprovides clear evidence that TA3Ha cells are highly sensitive to GIT-27NO treatment, which resulted in caspase-independent apoptosisthrough immediate stabilization of p53 intracellular content anddecreased expression of Bcl-2. Moreover, GIT-27NO significantlyextended the survival rate of mice bearing ascites tumors inducedby TA3Ha cells.
Materials and methods
Animals
Female strain A inbredmice, 5–6weeks of age, were obtained fromCharles River (Lecco, Italy) and kept under standard (non-SPF)laboratory conditionswith free access to food andwater. The handlingof animals and the study protocol were in accordance withinternational guidelines and approved by the local InstitutionalAnimal Care and Use Committee.
Reagents and cells
Fetal calf serum (FCS), RPMI 1640, phosphate-buffered saline(PBS), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactic acid, phenazine methosul-fate, propidium iodide (PI), p-iodonitrotetrazolium, andβ-nicotinamideadenine dinucleotide were obtained from Sigma (St. Louis, MO, USA).Annexin V (AnnV)–FITC was from Biotium (Hayward, CA, USA).VGX-1027 and GIT-27NO were synthesized as described else-where [5,6] and were obtained from Ganial Immunotherapeutics(Wilmington, DE, USA). The TA3Ha mouse mammary tumor cell linewas kindly provided by Professor M.S. Pappalardo (Department ofPharmaceutical Science, School of Pharmacy, School of Medicine,University of Catania, Catania, Italy) and routinely maintained inRPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin, and 25 μg/ml fungizone (Sigma)or in Hepes-buffered RPMI 1640 medium supplemented with 10%FCS, 2 mM L-glutamine, 0.01% sodium pyruvate, 5×10−5 M
2-mercaptoethanol, and antibiotics (culture medium) at 37°C in ahumidified atmosphere with 5% CO2. After conventional trypsiniza-tion, adherent and suspended cells were collected and seeded at1×104/well in 96-well plates for viability tests and measurement ofnitrite accumulation, 2.5×105/well in 6-well plates for flowcytometry and real-time PCR, and 1×106/25-cm3
flask for Westernblot analysis and apoptosis assays. Control cell cultures contained anamount of DMSO equal to that of the solution with the highestconcentration of GIT-27NO used in each experiment.
MTT and lactate dehydrogenase (LDH) assays
In the MTT assay, cell respiration, and thus cell viability, wasassessed based on the mitochondrial-dependent reduction of MTT tothe colored formazan product [19]. TA3Ha cells were treated withvarious doses of GIT-27NO for 24 h, and the conversion of MTT toformazan was monitored at 570 nm. Cell viability was expressed as apercentage of the control value (untreated cells), which wasarbitrarily set to 100%. As a marker of necrotic cell death, wemeasured the release of the intracellular enzyme LDH, whichmediates the conversion of 2,4-dinitrophenylhydrazine into a visiblehydrazone precipitate in the presence of pyruvate [20]. TA3Ha cellswere treated with various doses of GIT-27NO. After a 24-h incubationperiod, the amount of LDH released into the cell supernatant wascalculated using the formula [(E − C)/(T − C)]×100, where E is theexperimental absorbance of treated or untreated culturesmeasured at492 nm, C is the absorbance of the medium without cells, and T is theabsorbance corresponding to the maximal (100%) LDH release ofTriton-lysed cells.
Measurement of intracellular NO and nitrite accumulation
Cells were treated with various concentrations of the drug. Nitriteaccumulation, an indirect measure of NO release, was determined incell culture supernatants after 2, 4, or 24 h using the Griess reaction, asdescribed previously [21]. 4-Amino-5-methylamino-2′,7′-difluoro-fluorescein diacetate (DAF-FM diacetate; Molecular Probes, Leiden,The Netherlands) was used as an indicator of intracellular NO. Briefly,after 2, 4, and 24 h of cultivation in the presence of GIT-27NO orsodium nitroprusside (SNP), the cells were incubated with dilutedDAF-FM diacetate (2–5 μM) for 1 h at 37°C, washed, and thenincubated for 15min at 37°C in phenol red- and serum-free RPMI 1640for the completion of deesterification of intracellular diacetates.Finally, the cells were washed and PBS was added. Green (FL1)fluorescence emission from104 cellswasmeasuredwith a FACSCaliburand analyzed using CellQuest software or the plate reader Chameleon.
Electron microscopy
TA3Ha cells treated 18 h with GIT-27NO were fixed with 2.5%glutaraldehyde in PBS at room temperature for 30 min and thenrinsed three times with PBS. After fixation, the cells were incubated inosmium tetroxide (1%) for 30 min, dehydrated in a series of ethanolwashes, and then embedded in araldite. One-micrometer-thicksections were stained with toluidine blue, mounted in DPX, andobserved under a Leica light microscope. Thin sections were cut with adiamond knife on a Leica UC6, mounted on a copper grid, and viewedwith an electron microscope (Philips CM12, Eindhoven, TheNetherlands).
Detection of apoptosis by annexin V–FITC/PI double staining, cell-cycledistribution analyses, PI staining, and DNA fragmentation assay
Early apoptotic cell deathwas assessed by flow cytometric analysisof phosphatidylserine with AnnV. Late apoptotic cell death wasdetected in cells stained with the DNA-binding dye PI or by DNA
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fragmentation assays. Cells were incubated in the presence or absenceof the drug for the indicated times, trypsinized, and then stained withAnnV and PI, as previously described [16]. For detection of autophagy,
Fig. 1. GIT-27NO-induced, NO-mediated decrease in viability of TA3Ha cells. (A) Cells(1×104 cells/well) were treated with a range of concentrations of either VGX-1027 orGIT-27NO for 24 h, after which cell viability was determined by MTT assays. The dataare presented as means±SD from representative of three independent experiments.⁎pb0.05, compared to untreated cultures. (B) Intracellular NO was determined by flowcytometry of DAF-FM diacetate fluorescence after 2 and 4 h of incubation of cellswithout (control) or with GIT-27NO (75 μM). (C) Cells were incubated with variousconcentrations of GIT-27NO for 24 h, at which point nitrite accumulation wasdetermined in culture supernatants. Results were calculated as indicated underMaterials and methods and presented as means±SD from representative of threeindependent experiments (⁎pb0.05).
the cells were cultured under the indicated conditions and thenstained with 1 μg/ml acridine orange as described previously [13].Green (FL1, 510–530 nm) and red (FL3, N650 nm) fluorescenceemission from 104 cells was measured with a FACSCalibur andanalyzed using CellQuest software. For morphological characteriza-tion of apoptosis, PI staining of cells on chamber slides was performedexactly as previously described [13]. For detection of DNA fragmen-tation, cells were treated with GIT-27NO for 24 h, and nucleic acidfragments were detected as previously described [22] without thefinal step of treatment with RNase.
Intracellular detection of the active form of caspase-3 by flow cytometry
FITC-conjugated caspase-3 monoclonal antibody (BD Pharmingen,San Diego, CA, USA) was used to detect the active form of caspase-3.TA3Ha cells were treated with GIT-27NO or left untreated. After 13 hof incubation, intracellular staining was performed, following themanufacturer's instructions, and then analyzed by flow cytometry.
Western blot analysis
Cells (106) were seeded in flasks (25 cm3), incubated in 0.5% FCSRPMI 1640 overnight, and then treated with the drug for the indicatedtimes. Whole-cell lysates were prepared in a solution containing62.5 mM Tris–HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mMdithiothreitol, and 0.01% w/v bromophenol blue and were separatedby electrophoresis on 12% SDS–polyacrylamide gels. The sampleswere electrotransferred to polyvinylidene difluoride membranes at5 mA/cm2, using a semidry blotting system (Fastblot B43; Bio-Rad,Goettingen, Germany). The blots were blocked with 5% w/v nonfatdry milk in PBS with 0.1% Tween 20 and then probed with specificantibodies to p53, pSer20-p53, and β-actin (Santa Cruz Biotechnology,Santa Cruz, CA, USA); Bax and Bcl-2 (eBioscience, San Diego, CA, USA);or active caspase-3 (R&D Systems, Minneapolis, MN, USA), followedby incubation with secondary antibody (ECL donkey anti-rabbit HRPlinked; GE Healthcare, Buckinghamshire, UK). Bandswere detected bychemiluminescence (ECL; GE Healthcare).
RNA isolation and RT-PCR analysis
Total RNA from TA3Ha cells was isolated with an RNA isolator(Metabion, Martinsried, Germany) according to the manufacturer'sinstructions. RNA (1 μg) was reverse transcribed using Moloneymurine leukemia virus reverse transcriptase and random primers(both from Fermentas, Vilnius, Lithuania). PCR amplification of cDNA(1 μl per 20 μl of reaction) was carried out in a real-time PCR machine(Applied Biosystems, Foster City, CA, USA) using SYBR Green PCRmaster mix (Applied Biosystems) as follows: 10 min at 50°C for dUTPactivation and 10 min at 95°C for initial denaturation of cDNA,followed by 40 cycles, each consisting of 15 s of denaturation at 95°Cand 60 s for primer annealing and chain extension. Primer pairs werecaspase-3, 5′-TCTGACTGGAAAGCCGAAACT-3′ and5′-AGGGACTGGAT-GAACCACGAC-3′; caspase-8, 5′-TCAACTTCCTAGACTGCAACCG-3′ and5′-CTCAATTCCAACTCGCTCACTT-3′; caspase-9, 5′-TCCTGGTACATCGA-GACCTTG-3′ and 5′-AAGTCCCTTTCGCAGAAACAG-3′; Bcl-2, 5′-TCGCA-GAGATGTCCAGTCAG-3′ and 5′-CCTGAAGAGTTCCTCCACCA-3′; Bax,5′-TGAAGACAGGGGCCTTTTTG-3′ and 5′-AATTCGCCGGAGACACTCG-3′; and β-actin, 5′-GGACCTGACAGACTACC-3′ and 5′-GGCATA-GAGGTCTTTACGG-3′. The expression level of each genewas calculatedaccording to the formula 2−(Cti− Cta), where Cti is the cycle threshold ofthe gene of interest and Cta is the cycle threshold value of β-actin.
Tumor induction and treatment with GIT-27NO
Mouse ascites were induced in strain A mice by ip injection ofcultured tumorigenic TA3Ha cells. After conventional trypsinization,
Fig. 2. GIT-27NO favored the internalization of NO. Cells (1×104 cells/well) were treated with a range of concentrations of either SNP or GIT-27NO. (A, B) Nitrite accumulation at theindicated time points, (C) intracellular NO by DAF-FM diacetate fluorescence, and (D) cell viability by MTT assays after 24 h were determined. The data are presented as means±SDfrom representative of three independent experiments. ⁎pb0.05, compared to untreated cultures.
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adherent and suspended cells were collected, washed in serum-freeRPMI 1640 medium, and inoculated (1.4×103) in 0.5 ml of sterile PBSinto the peritoneal cavity of the mice. On day 7 postinoculation (p.i.),the mice were randomly divided into experimental and controlgroups (each n=16). The mice in the experimental group weretreated with GIT-27NO ip at a dose of 40 mg/kg for 21 consecutivedays, beginning 7 days after the tumor cell challenge. A negativecontrol group was treated with GIT-27NO vehicle under the sameconditions used for experimental mice. An additional group of micewas treated with cisplatin ip at the dose of 6 mg/kg once a week aspositive control treatment. Another group was left untreated.
The survival times and body weights for each group weremonitored until day 28 p.i. Body weight variation was determinedevery day for each animal in relation to its weight at day 0.
Statistical analysis
The results are presented as means±SD of triplicate observationsfrom one representative of at least three experiments with similarresults, unless indicated otherwise. The significance of the differencesbetween various treatments was evaluated by analysis of variance,followed by the Student–Newman–Keuls test. For the in vivo studythe log-rank test was applied. Values of pb0.05 were consideredstatistically significant.
Results
GIT-27NO suppresses TA3Ha cell growth in vitro; participation of the NOliberated from the drug
TA3Ha cells were treated with various concentrations of eitherGIT-27NO or its parental compound VGX-1027 for 24 h. The results ofthe MTT assay, presented in Fig. 1A, clearly indicate that GIT-27NO, incontrast to VGX-1027, dramatically decreased the number of viablecells in the cultures. The observed effects coincided with significantcellular uptake of drug-generated NO as early as 2 h after the start ofincubation (Fig. 1B). A dose-dependent increase in nitrite accumula-tion in the cell culture supernatants was detected after 24 h ofcultivation in the presence of the NO-modified compound (Fig. 1C).Thus, it was evident that decreased viability was related to drug-delivered NO.
GIT-27NO favors intracellular NO uptake
Next we compared the capacity of GIT-27NO and of theconventional NO donor SNP to deliver NO into the cells. Cells weretreated with either GIT-27NO or SNP and the accumulation of nitrites,intracellular uptake of NO, and cell viability were estimated at theindicated time points (Figs. 2A, 2B, 2C, and 2D). A significant amount
Fig. 3.GIT-27NO-induced apoptotic cell death in TA3Ha cells. (A) Cells were incubated with various concentrations of GIT-27NO for 24 h, at which point LDH release was determinedin the culture supernatants. Results were calculated as indicated under Materials and methods and presented as means±SD from representative of three independent experiments.Cells were incubated without (control) or with 75 μM GIT-27NO and then subjected to: (B) acridine orange staining (top) or AnnV/PI double staining (bottom), (C, D) cell cycleanalysis, (E) electron microscopy after staining with toluidine blue (left) or fluorescence microscopy after PI staining (right), or (F) DNA fragmentation assay. The assays wereperformed after 18 (B, C, D, E) or 24 h (A, F).
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of nitrites was detected even after 2 h of treatment with both GIT-27NO and SNP. After an additional 22 h, the level of accumulatednitrites produced from SNP-originated NO was seven times higher incomparison to GIT-27NO (Figs. 2A and 2B). Surprisingly, intracellularuptake was not correlated with the quantity of measured nitrites.Whereas intracellular NO was not detected in cells treated with SNP,even at the highest doses tested, a remarkable amount of internalizedNO was identified upon the treatment of Ta3Ha cells with GIT-27NO(Fig. 2C). Concordantly, MTT assay revealed that cell viabilityreciprocally correlated with detected intracellular NO (Fig. 2D).These results clearly indicate that the advantage of the molecularstructure of the novel drug relies on its capacity to successfully deliverNO into the cell.
GIT-27NO-induced cell death in TA3Ha cells is accompanied by stronginhibition of caspase expression and activity
The observed decrease in number of viable cells upon treatmentwith GIT-27NO was not accompanied by an intensified release of LDHenzyme, indicating that cell membrane permeability was notdisturbed during the first 24 h of incubation (Fig. 3A). This rules outnecrotic cell death as a primary mode of drug action in TA3H cells andanticipates that other types of cell death or inhibition of proliferationhave been activated in these cells upon their in vitro exposure to GIT-27NO. To determine the cause of the decreased viability of GIT-27NO-treated TA3Ha cells, staining with AnnV/PI, as well as acridine orange,was performed. Whereas flow cytometric analysis of acridine orange-
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stained cells treated with 75 μM GIT-27NO eliminated the possibilitythat autophagy contributed to drug-promoted reduction of TA3Ha cellviability (Fig. 3B, top), AnnV/PI staining revealed significant increasesin AnnV+/PI− cells and a subsequent elevation in the number ofdouble-positive cells, in comparison to untreated controls (Fig. 3B,bottom). These results suggest that GIT-27NO promotes apoptosiswith typical early apoptotic characteristics, including the abnormalpresence of inverted phosphatidylserine residues on the outer side ofthe cell membrane. Furthermore, analysis of cell cycle distribution inGIT-27NO-treated cells revealed intensive accumulation of hypodip-loid cells in the sub-G1 compartment (Fig. 3D) in comparison tocontrol cells (Fig. 3C). While untreated cells were round, with large,mostly euchromatic nuclei, the cells treated with GIT-27NO weremarkedly altered, with lobated or segmented nuclei typical ofapoptotic nuclear destruction (highly dense nucleoplasm with smallclumps of residual chromatin at the inner border of the nuclearmembrane) accompanied by formation of apoptotic bodies (Fig. 3E,left). In some of them nuclear destruction was associated withmassive cytoplasm vacuolization. Significant shrinkage of nucleus,high condensation of chromatin, and presence of apoptotic bodies in
Fig. 4. GIT-27NO reduced caspase activity and transcription. Cells were incubated without(A, left) flow cytometry or (right) Western blot analysis. Densitometric analysis of data from-8, and -9 was performed at the indicated time points. The data are presented as meansuntreated cultures.
cells exposed to GIT-27NO were further confirmed by PI staining(Fig. 3E, right). Finally, DNA ladder analysis confirmed the presence ofextensive nucleosomal DNA fragmentation (Fig. 3F). However, flowcytometry (Fig. 4A, left) and Western blot analyses (Fig. 4A, right)revealed that the level of the active form of caspase-3 wassignificantly lower in GIT-27NO-treated cells than in controls.Specifically, basal activity of caspase-3 was almost totally abrogatedafter only 2 h of treatment with GIT-27NO, and this persisted for anadditional 11 h (Fig. 4A, right). The observed down-regulation ofcaspase-3 activity was accompanied by reduced transcription ofcaspase-3, -8, and -9 (Fig. 4B). These results clearly indicate thatapoptosis-like cell death triggered by GIT-27NO in TA3Ha cells wasnot mediated by increased caspase activity.
GIT-27NO-induced apoptosis coincides with rapid mobilization of p53and diminished expression of Bcl-2
Having demonstrated that NO-mediated cytotoxicity of GIT-27NOwas caspase-independent, we determined whether there was arelationship between the death processes triggered by the compound
(control) or with 75 μM GIT-27NO, and intracellular, active caspase-3 was detected byrepresentative experiments is presented in arbitrary units. (B) RT-PCR for caspase-3,
±SD from representative of three independent experiments. ⁎pb0.05, compared to
Fig. 5. GIT-27NO temporarily prevents loss of p53 and abolishes Bcl-2 expression. Cells were incubated without (0) or with 75 μM GIT-27NO, and Western blot analyses of (A, left)p53 and Ser20p-p53, (B, left) Bax, and (C, left) Bcl-2 protein expression are presented. Densitometric analysis of data from representative experiments is presented as fold increaserelative to control (A) or arbitrary units (B and C). RT-PCR for Bax (B, right) and Bcl-2 (C, right) was performed at the indicated time points. The data are presented as means±SDfrom representative of three independent experiments. ⁎pb0.05, compared to untreated cultures.
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and the levels of available p53 and pro- and antiapoptotic members ofthe Bcl-2 family, which are crucial for sensitivity and propagation ofcaspase-independent forms of cell death [23,24]. First we performedWestern blot analysis to determine the levels of total p53 as well asSer20p-p53 proteins. The results revealed a marked transient increasein phosphorylation of p53 at Ser 20 (Fig. 5A). Because a high rate ofphosphorylation of p53 on Ser 20 prevents p53 degradation andenlarges the available p53 pool, the data indicate that the destructivesignal induced by GIT-27NO included a temporary recruitment of the
cellular p53 pool. At the last time point tested (13 h of GIT-27NOtreatment), the level of the phosphorylated form of p53 decreased. Inaddition, a decrease in the level of total p53 proteinwas also observed,which may be at least due to ubiquitin-dependent p53 proteolysis.The transcription of two major members of the Bcl-2 family ofmolecules, proapoptotic Bax and cell-protective Bcl-2, is regulated byp53. To delineate the sensitivity of cells to NO-triggered caspase-independent cell death, the levels of mRNA encoding these genes aswell as of their protein products were determined. The obtained
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results reveal a slight inhibition of Bax protein that was notsynchronized with appropriate changes in its gene expression(Fig. 5B). In addition, the cell-protective molecule Bcl-2 was radicallyrepressed at both protein and transcriptional levels (Fig. 5C). Takentogether, the results presented here strongly support the hypothesisthat GIT-27NO activates NO-mediated caspase-independent apopto-sis via rapid but temporary engagement of available p53, diminishedBcl-2 activity, and subsequent increase in the Bax/Bcl-2 ratio.
GIT-27NO inhibits TA3Ha-induced peritoneal cancer development
To evaluate the potential therapeutic properties of the drug in vivo,we used a model of intraperitoneally implanted TA3Ha-inducedtumors in mice. Tumors induced in this model grew rapidly in themidst of ascites, and the animals began to die 15–17 days after tumorimplantation. Daily treatment with GIT-27NO, at a dose of 40 mg/kg,began 7 days after tumor induction and continued for an additional21 days. At day 28 after TA3Ha-cell implantation, the mortality rate ofthe untreated group reached 81.25% (Figs. 6A and 6B). Vehicletreatment was associated with 75% lethality, whereas the mortality ofGIT-27NO- and cisplatin-treated, tumor-bearing animals reachedrespectively 38 (pb0.05 vs vehicle ) and 25% (pb0.01 vs vehicle)throughout the observation period (Figs. 6A and 6B). The body weightof tumor-bearing mice increased by 12–15% from the beginning of thetreatment until the end of the study. The vehicle-treated mice and theuntreated mice showed a slightly higher body weight increase thanthemice treatedwith GIT-27NO and cisplatin, probably because of thesevere neoplastic ascites induced by the massive presence of cancercells in the peritoneal cavity (data not shown). These data clearlyindicate that treatment with GIT-27NO inhibited cancer progression.Taken together, this evidence indicates that treatment of peritoneal
Fig. 6. GIT-27NO reduced lethality in mice with peritoneal cancer. TA3Ha cells(1.4×103) were injected intraperitoneally into strain A mice. GIT-27NO wasadministered intraperitoneally at a dose of 40 mg/kg for 21 consecutive days,beginning on day 7 after cell implantation. (A) Mortality rate 28 days after tumorchallenge and (B) the kinetics of a representative experiment. ⁎pb0.05 and ⁎⁎pb0.01 vsvehicle group.
cancer induced by TA3Ha cell injection was highly efficient, inconcordance with the observed efficacy of the compound in vitro.
Discussion
We have demonstrated that the ascitic tumor induced by theTA3Ha cell line is sensitive to GIT-27NO treatment and we providenovel information regarding the mode of action of GIT-27NO in thissetting. High expression of hyaluron receptors and mucins such ascarbohydrate glycoprotein–epiglycanan on the membranes of TA3Hacells confers on them an extremely invasive phenotype capable ofescaping specific immune responses [18]. Despite these complexbiochemical characteristics of TA3Ha cells, GIT-27NO efficientlyreleased and delivered NO inside the cells.
It is likely that increased intracellular NO concentrations inducedby GIT-27NO in TA3Ha cells are closely related to the precisemechanism of NO release by the compound as well as to its in vitroand in vivo antitumor efficacy [13]. Under similar in vitro conditions,the exogenous donor SNP released significantly larger amounts of NOin comparison to GIT-27NO. However, the SNP-released NO did notenter the TA3Ha intracellular compartment and, as a consequence,cell viability was not changed. In parallel, the same tumor cell lineexhibited dose-dependent sensitivity to GIT-27NO treatment in vitroto an extent similar to that observed in other rodent and human celllines [13,16,17]. Most importantly, the observed decrease in TA3Hacell growthwhen exposed to GIT-27NO in vitro was further confirmedin vivo. Data obtained in tumor-bearing mice revealed an impressivereduction of lethality even when the mice were first treated with GIT-27NO 7 days after tumor challenge, under a therapeutic regime.
Analyses of cell cycle distribution and AnnV/PI double staining, aswell as morphological characterization of cells by PI staining andelectron microscopy, revealed that GIT-27NO triggered apoptotic celldeath. The presence of phosphatidyl serine on the outer surface of thecell membrane was associated with a reduction in nuclear volume,condensation of chromatin, and nuclear and DNA fragmentation as aterminal event in the drug-initiated cascade. In addition, intracellularstaining and Western blot analysis ruled out the involvement ofcaspase-3 in this process. Toxic amounts of NO released from twodifferent exogenous donors, such as SNAP and SIN-1, induce apoptosisin PC12 and HeLa cells, which could not be prevented by caspaseinhibitors [23]. In agreement with these data our findings clearlyindicate that GIT-27NO uses the same caspase-independent pathwayto accomplish its tumoricidal action.
The most frequent death processes in a caspase-inhibitedenvironment are necrotic and autophagic cell death. In this setting,the absence of massive LDH release as well as the rare presence ofnecrotic-like cells revealed by morphological evaluation of GIT-27NO-treated TA3Ha cells and unchanged levels of autophagosomes indicatethat neither necrotic nor autophagic cell death could primarily explainthe reduced viability of TA3Ha cells exposed to the compound. Theabove suggests that DNA fragmentation, as the end step in the deathprocess triggered by GIT-27NO in the absence of caspase-3 activity,might be executed through caspase-independent endonucleases suchas AIF, Topo II, EndoG, and DNase I [25].
It is well documented that NO down-regulates the activity ofcaspase-3-like enzymes through S-nitrosylation of their catalytic unitsand also via cGMP-dependent mechanisms that function eitherdownstream or upstream of the level of caspase-3-like proteases[26,27]. In addition, the expression of caspase genes also could beaffected by NO. An extremely large superfamily of nucleic acid bindingproteins responsible for regulation of gene expression possessesdiscrete elements in their structure, such as zinc-finger domains withhighly conserved pairs of cysteine and histidine residues, which makethese transcriptional factors sensitive to NO. Interaction with NOmodulates the primary biological functions of these transcriptionfactors in embryogenesis, cell differentiation, proliferation, and
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apoptosis [28,29]. In this context, NO-sensitive sites in the promotersof caspase genes are probably responsible for GIT-27NO-promotedsuppression of caspase-3, -8, and -9 genes.
Tumor suppressor protein p53 acts as one of the crucialmediators of the death process even if caspases are not involvedin propagation of apoptotic signals [30,31]. The results presented inour study illustrate well how a rapid cellular response to drugtreatment can be mediated by the time-limited action of availablep53. In our study the rapid phosphorylation on Ser 20 impedes itsdegradation and, during a limited window, can provide thenecessary quantity of this molecule for the execution of downstreamprocesses without de novo synthesis. Intracellular Mdm-2 binds top53, exports it out of the nucleus, and targets it for ubiquitin-dependent proteolysis [32,33]. However, a series of genetic andbiochemical experiments has implicated the phosphorylation of Ser20 as the critical posttranslational modification that leads todissociation of p53 from Mdm-2 in vitro, as well as its stabilizationin response to different proapoptotic stimuli in vivo [32–34]. It waspreviously reported that NO promotes nuclear retention of p53 inneuroblastoma cells, through inhibition of Mdm- mediated export[35]. In a similar manner, GIT-27NO-treated TA3Ha cells seem to becapable of providing a sufficient pool of p53 molecules to completethe apoptotic program within the first few hours without additionalsynthesis of this molecule. Consequent reduction of total p53expression observed after this period of time is probably linked toaccelerated proteolysis, which could occur by the decreased rate ofphosphorylation on Ser 20. Because ubiquitin-dependent proteolysisis not a unique mode of p53 degradation, our results cannot excludethe contributions of alternative, ubiquitin-independent pathways ofp53 proteolysis [36]. The decreased expression of the Bcl-2 genemediated by GIT-27NO is probably a consequence of temporaryabrogated p53 degradation as p53 can repress expression of the Bcl-2 gene [37]. However, a direct influence of GIT-27NO on thepreexisting Bcl-2 protein is also possible. The capacity of NO totrigger the carbonylation of Bcl-2 was described in insulin-secretingRINm5F as well as Jurkat cells under the influence of exogenous orendogenous NO [38,39]. The decreased amount of Bcl-2 present inTA3Ha cells exposed to GIT-27NO could importantly contribute tocaspase-independent apoptosis triggered by GIT-27NO [24]. Despitethe fact that proapoptotic members of the Bcl-2 family such as Baxwere described as enhancers of caspase-independent apoptosistriggered by NO [40], its expression was not considerably affectedby GIT-27NO in TA3Ha cells. The net effect of changes in pro- andantiapoptotic signals leading to a higher Bax/Bcl2 ratio indicates apharmacologically induced prevalence of the proapoptotic pathways.The high basal expression of Bax observed in this cell line could beone of the parameters that determine their sensitivity to atypicalapoptosis induced by GIT-27NO.
In summary, we conclude that GIT-27NO powerfully inhibits the invitro growth and reduces the in vivo lethality of the specific andhighly malignant TA3Ha cell line, which serves as a preclinical modelof highly invasive metastatic cancer. These results warrant additionalstudies for the use of GIT-27NO in human cancer.
Acknowledgments
This work was supported by the SerbianMinistry of Science (Grant143029). We thank Dr. Gianni Garotta (Ganial Immunotherapeutics,Inc., Wilmington, DE, USA) for providing GIT-27NO and Igor Golic(Faculty of Biology, University of Belgrade, Belgrade, Serbia) forperforming electron microscopy.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.freeradbiomed.2010.01.026.
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