Cyclic Pifithrin-α SensitizesWild Type p53 TumorCells to AntimicrotubuleAgent–Induced Apoptosis1

Valentina Zuco and Franco Zunino

Department of Experimental Oncology and Laboratories,Fondazione IRCCS Istituto Nazionale dei Tumori, 20133Milan, Italy

AbstractAs a consequence of multiple functions of p53, its activation in response to cytotoxic stress may have pro-apoptotic or protective effects depending on the nature of lesions. We have previously shown that mutationalinactivation of p53 results in sensitization to paclitaxel. In this study, we used cyclic pifithrin-α, a transcriptionalinhibitor of p53, to further investigate the relevance of p53 function in the response of tumor cells to microtubuleinhibitors. Using drug concentrations causing only antiproliferative effects, the combination of antimicrotubuleagents with subtoxic pifithrin-α doses resulted in increase of sensitivity of two wild type p53 cell lines, associatedwith a substantial M phase cell accumulation and marked sensitization to apoptosis. Pifithrin-α had no sensitizingeffect in p53 defective cells or a marginal effect in normal human fibroblasts. The apoptotic response to the com-bination was concomitant with p21 down-regulation, Polo-like kinase 1 up-regulation, p34cdc2 kinase dephosphory-lation, and cdc25C phosphatase phosphorylation, supporting mitotic arrest. Sensitization to paclitaxel-inducedapoptosis was also achieved by p53-siRNA transfection in wild type p53 H460 cells. Pifithrin-α did not enhancethe apoptotic response after p53 down-regulation. The results support a protective role of the transcriptionalactivity of p53 in response to mitotic spindle damage. The inhibition of transcriptional activity of p53 may havetherapeutic implications in the treatment of p53 wild type tumors with antimitotic agents.

Neoplasia (2008) 10, 587–596

Introductionp53 function is a transcription factor, and it induces the expression ofgenes involved in cell cycle control, DNA repair, and apoptosis [1–4]. The p53 tumor suppressor plays a central role in the response todiverse stress stimuli [1–5]. Cell fate after activation of p53 dependson the biologic context and on the nature of response to the cyto-toxic stress. P21WAF1, a p53 target protein, has been implicated as amajor determinant of cell fate under stress conditions (e.g., DNAdamage), because it participates in both G1 and G2 checkpoints.p53 can be activated also in response to mitotic spindle damage[6–11]. Checkpoint mechanisms play a critical role in controlling cellcycle progression and DNA repair under stress conditions, such asexposure to cytotoxic agents. Therefore, cell cycle checkpoint func-tion is considered to be an important determinant of cellular sen-sitivity to various antitumor agents.The mutation of p53 is a frequent event in most tumors (∼50%)

resulting in the inactivation of its transactivating function. p53 mu-tations may affect the apoptotic response to some p53-activatingagents such as DNA-damaging agents (e.g., platinum compounds

and alkylating agents) [12,13]. However, the influence of p53 statusis likely dependent on the nature of cytotoxic stress, because wildtype p53 also has a protective function through the activation ofp53-dependent checkpoints [14]. Indeed, we have found that ovariancarcinoma cells selected for resistance to cisplatin and characterized

Abbreviations: CPP32, caspase 3; DMSO, dimethyl sulfoxide; MPM-2, mitotic pro-tein monoclonal-2; PBS, phosphate-buffered saline; PFT, pifithrin-α; PI, propidiumiodide; Plk1, Polo-like kinase 1; SDS-PAGE, sodium dodecyl sulfate–polyacrylamidegel electrophoresis; TUNEL, terminal deoxynucleotidyl transferase–mediated nickend labelingAddress all correspondence to: Franco Zunino, Fondazione IRCCS Istituto Nazionaledei Tumori, via Venezian 1, 20133 Milan, Italy. E-mail: franco.zunino@istitutotumori.mi.it1This work was partially supported by the Associazione Italiana per la Ricerca sulCancro, Milan, by the Ministero della Salute, Rome, by the Fondazione Italo Monzino,Milan, Italy.Received 5 February 2008; Revised 19 March 2008; Accepted 20 March 2008

Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00DOI 10.1593/neo.08262

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Volume 10 Number 6 June 2008 pp. 587–596 587

by mutational inactivation of p53 are hypersensitive to paclitaxel[15]. In ovarian carcinoma cells with mutant p53, paclitaxel treat-ment caused apoptosis after progressing to a G1-like status andDNA endoreduplication [16].The present study is an additional attempt to analyze the impact of

p53 function on cellular response to antimicrotubule agents with theuse of cyclic pifithrin-α (PFT), a chemical inhibitor of p53 [17,18].The results indicate that treatment with the PFT analog in combina-tion with various antimicrotubule agents enforced cell accumulationin mitosis, resulting in a dramatic increase of apoptosis without evi-dence of progression to a G1-like status. The available results supportthat the transcriptional activity of p53 has a protective role in re-sponse to mitotic spindle damage.

Materials and Methods

Cell Culture and DrugsThe human ovarian carcinoma cell line IGROV-1 and the cisplatin-

resistant subline IGROV-1/Pt1, selected by exposure to increasingdrug concentration [12], the human lung adenocarcinoma cell lineH460, the human prostate cell line PC3, and the human osteo-sarcoma cell line SAOS were grown in RPMI 1640 (Lonza, Vierviers,Belgium) containing 10% fetal bovine serum (Life Technologies,Inc., Gaithersburg, MD) in 5% CO2 at 37°C. Human normal em-bryonic lung fibroblasts were kindly provided by E. Prosperi (Univer-sity of Pavia, Italy) and grown in Eagle’s minimum essential medium(Lonza) supplemented with 10% fetal bovine serum (Life Tech-nologies). Paclitaxel (Indena, Milan, Italy), thiocolchicine, and PFT(Calbiochem EMD Chemicals, Inc., La Jolla, CA) were dissolved indimethyl sulfoxide (DMSO); vinorelbine (Navelbine, Pierre Fabre,Castres, France) was dissolved in H2O before use. In all experiments,cells were exposed to PFT 2 hours before treatment with antimicro-tubule agents. The highest final concentration of DMSO in culturemedium was 0.5%.

Antiproliferative ActivityCells were seeded in duplicate into six-well plates. After 24 hours,

cells were exposed to the cyclic form of PFT and to antimicrotubuleagents or to both simultaneously for 72 hours. After treatment, ad-herent cells were trypsinized and counted by a cell counter (CoulterElectronics, Luton, UK) [19]. Analysis of drug interaction was doneby a modified method of Drewinko et al. [20]: Drewinko index>1 indicates greater than additive effects (synergism; the higher thevalue, the greater the degree of synergy), Drewinko index =1 indi-cates additivity, and Drewinko index <1 indicates antagonism. Inall experiments, control cells were treated with the solvent at thesame concentration used in drug-exposed cells. When indicated,the method described by Chou and Talalay [21] was also applied.

Cell Cycle, Apoptosis, and CPP32 ActivityFor cell cycle analysis, cells were trypsinized, fixed in 70% etha-

nol, stained in phosphate-buffered saline (PBS) containing 10 μg/ml propidium iodide (PI; Sigma, St. Louis, MO) and RNase A(66 U/ml; Sigma) for 18 hours, analyzed by FACScan flow cytometer(Becton Dickinson, Mountain View, CA). For apoptosis detection,adherent and floating cells were harvested and analyzed for DNAfragmentation by terminal deoxynucleotidyl transferase–mediatednick end labeling (TUNEL) assay (Roche, Mannheim, Germany) ac-

cording to the manufacturer’s recommendation. Apoptosis was as-sessed by flow cytometer, and the results were analyzed using theCellQuest software (Becton Dickinson). For the analysis of CPP32activity (Oncogene Sciences, Uniondale, NY), adherent and floatingcells were harvested and processed as described by the manufacturer.After washing, cells were resuspended in wash buffer, the CPP32 ac-tivity was detected by flow cytometer (FACScan), and the resultswere analyzed using the CellQuest software.

Western Blot AnalysisAdherent and floating cells were lysed in hot sodium dodecyl sul-

fate (SDS) sample buffer, and whole-cell lysates were prepared forSDS–polyacrylamide gel electrophoresis (PAGE): 0.125 M Tris–HCl (pH 6.8), 5% SDS, 1 mM phenylmethylsulfonyl fluoride,10 μg/ml pepstatin, 12.5 μg/ml leupeptin, 100 Komberg inter-national units of aprotinin, 1 mM sodium orthovanadate. Sampleswere separated by SDS-PAGE and were transferred onto nitro-cellulose filters. Blots were preblocked for 2 hours at room tem-perature in PBS containing 5% (w/v) dried nonfat milk. Filterswere incubated overnight at 4°C with antibody anti–mitotic proteinmonoclonal 2 (MPM-2) and –Polo-like kinase 1 (Plk1; Upstate Bio-technology, Lake Placid, NY); anti–Raf-1 (E-10, sc-7267), –cyclinB1 (GNS1, sc-245), –cdc-25C (C-20, sc-327), –SNK/Plk2 (H-90,sc-25421), and –cyclin A (BF683, SC-239) (all from Santa Cruz Bio-technology, Santa Cruz, CA); anti–Bcl-2 and -p53 (Dako, Glostrup,Denmark); anti-p21 (Neomarker, Union City, CA); anti–PARP-1(Oncogene Sciences); anti–cytochrome C and -Rb (BD Pharmingen,Becton Dickinson); anti–cleaved caspase 3 and anti–phospho-AKT(Ser473; Cell Signaling Technology, Beverly, MA); anti-cdc2 (Tyr15-phospho–specific; New England BioLabs, Beverly, MA); anti-actinand –β-tubulin (Sigma); anti-HSP70 mitochondrial (Abcam, Ltd.,Cambridge, UK); and anti–PKBα/AKT (Transduction Laboratories,Lexington, NY).

Tubulin Polymerization AssayCells were seeded at a density of 1 × 106 cells in 10-cm Petri

dishes. The day after, cells were exposed to paclitaxel/PFT combinedtreatment and, 24 hours later, were processed for the tubulin poly-merization assay. Samples were prepared as described by Blagosklonnyet al. [22] and Cassinelli et al. [16] and separated by SDS-PAGE(10% resolving gel and 3% stacking gel), and tubulin distributionwas analyzed by immunoblot analysis using a mouse anti–β-tubulinantibody (Sigma).

Analysis of Cytochrome C Release andSubcellular FractionationH460 and IGROV-1 cells were exposed to paclitaxel (IC50 or

IC80) alone or combined with the cyclic form of PFT. Cells werewashed in PBS added with 0.1 mM ice-cold sodium orthovanadateand then incubated in permeabilization buffer (20 mM HEPES–KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mMEGTA, 250 mM sucrose, 0.5 mM phenylmethylsulfonyl fluoride,10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml trypsin in-hibitor) for 15 minutes in ice. Cells were homogenized using a glassDounce and pestle for approximately 50 strokes. After two centrifuga-tion cycles at 3000 rpm for 10 minutes, the supernatants were againcentrifuged at 14,000 rpm for 30 minutes. Cytosolic fraction wasanalyzed by immunoblot analysis.

588 Sensitization to Apoptosis by p53 Inhibition Zuco and Zunino Neoplasia Vol. 10, No. 6, 2008

Immunofluorescence AnalysisH460 and IGROV-1 cell lines were treated with either PFT or

paclitaxel alone or with their combination for 24 hours. After washingwith PBS, the cells were fixed in 2% paraformaldehyde for 30 min-utes and permeabilized with ice-cold methanol for 20 minutes at−20°C. After blocking with PBA (PBS containing 1% bovine serumalbumin), anti-p53 antibody (DO7; Dako) was incubated for 1 hourat room temperature followed by incubation with Alexa Fluor488 goat antimouse IgG secondary antibody (Molecular Probes,Eugene, OR) for 1 hour. Images were observed and analyzed by con-focal microscopy (Microradiance 2000; Bio-Rad Laboratories, Inc.,Hercules, CA) equipped with Kr/Ar (488 nm), HeNe (543 nm),and diode (638 nm) lasers. Images (525 × 525 pixels) were obtainedusing a ×60 oil immersion lens and were analyzed using Image-ProPlus software (Version 6.0; Media Cybernetics, Inc., Bethesda, MD).Reported images represent a single Z-section of the samples (23–51 stacks), with 0.48-μm step. The pinhole diameter was regulatedaccording to the value suggested by the acquisition software to obtainthe maximum resolution power.

Small Interfering RNA TransfectionSmall interfering RNAs were synthesized by Invitrogen Corp.

(Carlsbad, CA). The p53-siRNA consisted of a mixture of twosiRNA duplexes targeting for a different region of the p53 mRNA(Validated stealth RNAi DuoPak). A pool of two nontarget-ing siRNA duplexes was used as a negative control medium GCduplex (Invitrogen).Transfection of cells with siRNA duplexes was performed using

Lipofectamine 2000 Reagent (Invitrogen). H460 were transfectedwith control siRNA or p53-siRNA at a final concentration of100 nM for 24 hours. After silencing, cells were replated and treatedon the second day with PFT for 2 hours and, after that, with pacli-taxel, as indicated. Gene silencing effects were evaluated by Westernblot also on the day of the treatment.

Results

Cell Growth InhibitionThe effects of the combination of the p53 inhibitor of cyclic PFT

and antimicrotubule agents were investigated in two human cancercell lines with wild type p53 (IGROV-1 and H460), in a cisplatin-resistant subline (IGROV-1/Pt) characterized by a mutant p53 andin two p53 defective cell lines (PC3 prostate carcinoma and SAOSosteosarcoma). Cells were pretreated for 2 hours with subtoxic PFTconcentrations (in the range of IC10–IC30) followed by a simulta-neous 72-hour exposure to the antimitotic agent (paclitaxel, vino-relbine, or thiocolchicine; Figure 1). The IGROV-1 cells were lesssensitive to the antimitotic agents than the H460 cell (IC50 valuesfor paclitaxel 0.12 ± 0.02 and 0.03 ± 0.004 μM, respectively). Incontrast, IGROV-1 cells, characterized by a higher level of p53 ex-pression, were more sensitive to the antiproliferative effects of PFT.The pattern of sensitivity of these cell lines was apparently consistentwith the level of p53 protein. Dose–response curves of various anti-microtubule agents in combination with subtoxic concentrations ofPFTrevealed a supraadditive effect in both IGROV-1 and H460 cells(Figure 1, A and B). The combination of PFTwas synergic with bothmicrotubule-stabilizing and -destabilizing drugs. The synergistic in-teraction was more marked in the H460 cell line. The analysis ofdrug interaction according to the method of Drewinko et al. [20]

and to that of Chou and Talalay [21] confirmed that the antimitoticagents/PFT combination was synergic. Figure 1C shows the analy-sis of interaction for paclitaxel and PFT, according to the methodof Chou and Talalay [21]. On the contrary, PFT did not affect thedose–response curve of paclitaxel in a cisplatin-resistant subline(IGROV-1/Pt) carrying the mutant p53, in prostate PC3 cells, andin osteosarcoma SAOS cells characterized by the loss of p53 expres-sion (Figure 1D), suggesting that the sensitization observed inIGROV-1 or in H460 cells was related to the inhibition of the func-tional p53 by PFT. To better support this interpretation, we studiedthe effect of paclitaxel in H460 cells after transfection with p53-siRNA (Figure 1E ). p53 silencing substantially reduced the ac-cumulation of the p53 protein caused by paclitaxel treatment andresulted in a sensitization to paclitaxel comparable to the effect ofPFT/paclitaxel combination in control cells. Under the same treat-ment conditions, PFT produced only a marginal sensitization topaclitaxel of “normal” human fibroblasts (Figure 1F ).

Cell Cycle AnalysisBecause induction of mitotic arrest is a hallmark of microtubule

inhibitors, we examined the cell cycle perturbation of H460 andIGROV-1 cells exposed to paclitaxel, vinorelbine, thiocolchicine(IC50), or to their combination with PFT. Whereas PFT itself didnot induce modifications of the cell cycle distribution of H460 cells,at the concentrations used, the antimicrotubule agents caused onlyinhibition of cell proliferation with a moderate increase of cellsin G2/M. In spite of this behavior, H460 cells exhibited a normalmitotic spindle checkpoint, because higher concentrations (IC80)caused mitotic arrest (not shown). A similar behavior has beendescribed in other cell lines [23,24]. Pretreatment with PFT and24 hours of simultaneous treatment with a low concentration ofthe antimicrotubule drug caused a cell accumulation in G2/M inH460 cells (Figure 2A). In IGROV-1 cells, paclitaxel alone causedan evident increase in the number of cells in the G2/M phase(Figure 2A). The accumulation of cells in the G2/M phase was en-hanced with the PFT combined treatment in a dose-dependent man-ner. In particular, treatment with paclitaxel alone resulted in 50% cellaccumulation in G2/M, and the combination with PFT (10 μM) en-hanced the number of G2/M phase cells to 80%. The combinationinduced a marked increase of sub-G1 fraction in both cell lines. Atdrug concentrations that produced a low (<20%) extent of mitosisin wild type p53 cells, the addition of PFT resulted in a marked in-crease of mitotic cells as determined by the analysis with PI staining(Figure 2B). Again, the effect of combined treatment on the cell cyclewas more evident in H460 cells that were also more responsive to thedrug combination.

Tubulin PolymerizationTo investigate the drug effects on the microtubule system, we

compared cells treated with paclitaxel (IC50) or with PFT combi-nation for 24 hours (Figure 2C ). In H460 and IGROV-1 cells, thepaclitaxel/PFT combination enhanced the tubulin polymerizationinduced by paclitaxel. This effect is consistent with the increase ofmitotic cells.

Modulation of Biochemical Markers of Mitotic ArrestTo gain further insight into the mechanism of cell arrest in the

M phase, we performed additional investigation with PFT/paclitaxelcombination. In cells exposed for 24 hours to the paclitaxel/PFT

Neoplasia Vol. 10, No. 6, 2008 Sensitization to Apoptosis by p53 Inhibition Zuco and Zunino 589

Figure 1. Antiproliferative effects of paclitaxel (PTX), vinorelbine (VIN), or thiocolchicine (TIO) in H460 (A); IGROV-1 (B); IGROV/Pt1, PC3,SAOS (D) and human normal fibroblasts (F) in the absence (▪) or presence of 20 μM (•), 10 μM (▾), and 2 μM (○) cyclic PFT. Cells werepretreated with the indicated concentrations of PFT for 2 hours before the combined treatment for 72 hours. Pifithrin-α was used at twosubtoxic concentrations (i.e., 2 μM PFT was equivalent to IC15 in IGROV-1 cells; 10 μM PFT was equivalent to IC10 in H460 and SAOSand to IC20 in IGROV-1, IGROV-1/Pt, human fibroblasts and PC3; 20 μM PFT was equivalent to IC20 in H460 and SAOS and to IC30 inIGROV-1/Pt and PC3). (E) Effects of paclitaxel (▪) and its combination with PFT 10 μM (▾) in H460-negative control cells and effectsof paclitaxel in p53-siRNA–transfected cells (□). The p53 expression analysis is also shown. The negative control cells are referred tocells transfected with RNAi-negative control duplex containing 48% GC. Cell transfection was performed as indicated in the Materialsand Methods section. (Inset C) combination index calculated according to the Chou and Talalay analysis [21] for paclitaxel/PFT interaction.

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combination, the mitotic arrest was confirmed by the increase ofmitosis-specific phosphorylated epitopes recognized by MPM-2 anti-body (Figure 2D). This enhancement was less evident in IGROV-1cells, because paclitaxel alone produced an up-regulation of phospho-proteins in keeping with the accumulation of cells in the M phase.

A hyperphosphorylation of Raf-1 and Bcl-2 is a biochemical hall-mark of the cellular response to microtubule inhibitors, because theseevents are associated with the M phase [22,25]. In H460 cells, thePFT/paclitaxel combination treatment for 24 hours induced a parallelmobility shift of Bcl-2 and Raf-1 in SDS-PAGE, thereby supporting a

Figure 2. Cell cycle perturbation and analysis of mitotic arrest and of mitotic biochemical markers in cells pretreated for 2 hours with PFTfollowed by simultaneous 24-hour exposure to antimicrotubule agents (IC50). (A) Cell cycle distribution of H460 and IGROV-1. The rep-resentative of at least three independent experiments is shown. (B) Mitotic index in PI-stained H460 and in IGROV-1 cells determined byfluorescence microscope analysis. The results are expressed as a percentage of the total cell population and are the mean ± SD of threeindependent experiments. (C) Analysis of the tubulin polymerization in H460 and in IGROV-1 cells. Cell fractionation was performed asindicated in the Materials and Methods section. Soluble cytosolic (S) or polymerized (P) tubulin was detected in cell fractions by Westernblot analysis after 24 hours of exposure. (D) Activation of mitosis-related factors in H460 and IGROV-1 cells. Western blot analysis of celllysates was performed at the end of treatment (24 hours). β-Tubulin is shown as a control for protein loading.

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drug-induced phosphorylation of the two proteins (Figure 2D). In ad-dition, the combined treatment induced dephosphorylation of p34cdc2

kinase (at Tyr15), phosphorylation of cdc25C phosphatase, andup-regulation of cyclin B1. In IGROV-1 cells, no change in the phos-phorylation of Raf-1 and Bcl-2 was detected between the treatmentwith paclitaxel alone or with the combination. However, after thecombination treatment, cells showed a reduction of the phosphoryla-tion of p34cdc2 kinase and an increase of phosphorylation of cdc25C.Pifithrin-α itself caused a down-regulation of the phosphorylation ofp34cdc2, most evident at 10 μM. Moreover, paclitaxel combined withthe higher dose of PFT induced an increase of the cyclin B1 proteinlevels. This event supported that PFT induced in paclitaxel-treatedcells a mitotic arrest without progression to the G1-like phase.The activation of Plk1 (a member of the Polo-like kinase family)

is implicated in various processes that contribute to the activation ofcyclin B/p34cdc2 complex [26]. After 24 hours of exposure, in H460cells paclitaxel alone did not modify the Plk1 expression, whereas thePFT/paclitaxel combination caused a substantial up-regulation ofPlk1. In IGROV-1 cells, paclitaxel alone caused a moderate increaseof Plk1 protein, in keeping with the appearance of G2/M peak,MPM-2 reactivity, and phosphorylation of Raf-1 and Bcl-2. The PFTcombination produced only a moderate increase of Plk1 expression.Plk2, another member of the Polo-like kinase family activated

during the DNA damage checkpoint in G2 phases, has been reportedto be a p53 target, and the p53-dependent activation of Snk/Plk2prevents mitotic catastrophe after spindle damage [8]. However, nomodulation of Plk2 could be detected in H460 and in IGROV-1cells in our experimental conditions (data not shown).

Apoptotis Induction and Caspase ActivationWe tested the hypothesis that the sensitization effect of PFT was

related to an enhanced cell susceptibility to antimicrotubule agent–induced apoptosis. The CPP32 activity assay and the TUNEL assayfor the analysis of apoptotic response were performed in cells exposedto low concentrations of the antimicrotubule agents (paclitaxel,vinorelbine, or thiocolchicine), which caused only antiproliferativeeffects, or to their combination with subtoxic PFT concentrationsfor 72 hours. In both H460 and IGROV-1 cells, the FACScan analy-sis of CPP32-positive cells supported the increase in the activationof CPP32 after 72 hours of combined treatments (Figure 3A). In-deed, PFT induced a remarkable increase of apoptosis in paclitaxel-,vinorelbine-, or thiocolchicine-treated cells (Figure 3B). A sensitiza-tion to paclitaxel-induced apoptosis was not observed in “normal”human fibroblasts (Figure 3B), thus supporting a low susceptibilityto apoptosis of normal cells.Because proapoptotic stimuli induced by mitotic spindle damage

involved mitochondrial pathway, we performed Western blot analy-sis of the release of cytochrome C in H460 and IGROV-1 cells at24 hours after treatment (Figure 3C ). Compared with the effect ofpaclitaxel alone, an increased release of cytochrome C at 24 hourswas found in the combined treatment in a PFT dose-dependentmanner. According to the release of cytochrome C after 24 hoursof combined treatment, the apoptosis activation was further sup-ported by Western blot analysis of caspase cleavage (Figure 3D).Barely detectable caspase activation was induced by the two drugsgiven alone. In contrast, in cells treated with a drug combination,a marked cleavage of caspase 3 was associated with the cleavage ofthe caspase substrate PARP-1, thus confirming an enhancement ofthe caspase-dependent apoptotic pathways. The synergistic activa-

tion of caspase, already observed at 24 hours after treatment, wasmore marked after 48 hours. The release of cytochrome C andcaspase 3 activation were concomitant with the appearance of mitoticcells. Because under the same conditions approximately 80% of thetreated cells are arrested in mitosis (Figure 2, A and B), this findingsupports the interpretation that cells treated with the combinationunderwent apoptosis during mitosis.In contrast to control H460 cells, the p53-siRNA–transfected cells

was able to undergo apoptosis after 24 hours of exposure to a lowdose of paclitaxel (0.03 μM), thus supporting the proposed protec-tive role of p53 (Figure 3E ). As expected, as a consequence of sup-pression of the p53 expression, this effect was not enhanced by theaddition of PFT under these conditions.

Expression of p53 and p21WAF1/Cip1

The expression level of p53 and p21WAF1/Cip1 was investigated byWestern blot analysis 24 hours after drug treatment. As shown inFigure 4A, paclitaxel determined a marked increase of p53 andp21 expressions in both IGROV-1 and H460 cells. Pifithrin-α hadno effect on paclitaxel-induced p53 activation but had reduced theup-regulation of p21 induced by paclitaxel, likely because of theinhibition of p53 transcriptional activity. At the used subtoxic con-centrations, PFT itself was unable to modify the basal expression ofp21. As expected, a marginal or no induction of p21 was detected inH460 cells after p53 down-regulation with p53-siRNA (Figure 4B).Analysis by confocal microscopy revealed an increased amount ofp53 in the nuclei of cells exposed to a low paclitaxel concentration(IC50; not shown). In contrast, the combination of PFT with pacli-taxel resulted in a widespread distribution of p53 in the cytoplasm.

Modulation of AKTBecause AKT is involved in the cellular response to taxanes [16,27],

we examined the state of activation of this kinase in H460 andIGROV-1 cells treated with paclitaxel alone or with PFT combinationfor 24 hours (Figure 5). In both cell lines, AKT phosphorylation atSer473 was marginally modulated by paclitaxel or PFT alone. On thecontrary, the combination of paclitaxel with PFT resulted in a sub-stantial decrease of AKT phosphorylation in H460 or IGROV-1 cells,associated with a down-regulation of AKT protein itself.

DiscussionIn this study, we have found that the inhibition of p53 transcrip-

tional function by a specific chemical inhibitor, cyclic PFT, resultedin a marked sensitization to antimitotic agent–induced apoptosis ofhuman tumor cells, carrying functional wild type p53. The sensiti-zation in the combined treatment was produced by all the testedantimicrotubule agents, including both microtubule-stabilizing drugs(taxanes) and compounds that inhibit tubulin assembly (e.g., vincaalkaloids), and occurred at concentrations that caused only anti-proliferative effects. Thus, subtoxic concentrations of the p53 inhib-itor converted the growth-inhibitory effect of the antimicrotubuleagent in a cytotoxic outcome as evidenced by a dramatic enhance-ment of the apoptotic response. Sensitization to paclitaxel was alsofound in wild type p53 cells after suppression of the p53 expressionby siRNA. No or only a marginal sensitization by PFTwas detectedin cells characterized by defective p53 or after down-regulation ofp53 by siRNA, supporting that sensitization to drug-induced apop-tosis by PFTwas closely related to the modulation of p53 functions,

592 Sensitization to Apoptosis by p53 Inhibition Zuco and Zunino Neoplasia Vol. 10, No. 6, 2008

Figure 3. Induction of apoptosis and analysis of apoptosis-related factors in H460 and in IGROV-1 cells exposed to the IC50 paclitaxel(PTX), vinorelbine (VIN), thiocolchicine (TIO), cyclic PFT, or their combination. Cells were pretreated with PFT for 2 hours and then ex-posed to the antimicrotubule agent. (A) Caspase 3 (CPP32) activity after 72 hours of treatment determined with the colorimetric assaykit (Calbiochem) according to the manufacturer’s protocol and analyzed by FACScan. The results, expressed as percentage of CPP32activity-positive cells, are the mean ± SD of two independent experiments. (B) Apoptosis determined by TUNEL assay and FACScananalysis after 72 hours of treatment. The percentages of TUNEL-positive cells are indicated in each panel. Representative of at leastthree independent experiments is shown. (C) Release of cytochrome C into the cytosol. The cytosolic extracts were prepared after24 hours of treatment with paclitaxel alone or combined with PFT. An antibody against the mitochondrial marker HSP70-mitochondrialwas used as protein control to ensure the subcellular fractionation process. As a control for HSP70-mitochondrial antibody, a mitochon-drial extract containing HSP70 (ctr+) is shown. (D) Cleavage of CPP32 and PARP-1. Western blot analysis with specific antibodies wasperformed after 24 hours of treatment. β-Tubulin is shown as a control of protein loading. The arrows indicate the cleavage products.(E) Caspase 3 activation and PARP fragmentation in response to paclitaxel (24 hours of exposure) after p53 down-regulation in p53-siRNA–transfected H460 cells.

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which could play a protective role. Our results are consistent with theobservation that activation of p53 by MDM2 antagonists providesprotection from cytotoxicity of paclitaxel [28].The two wild type p53 cell lines were characterized by a somewhat

different response to antimicrotubule agents in terms of perturbationof cell cycle progression. Indeed, the treatment with low concentra-tions of antimitotic agents causing only antiproliferative effects re-sulted in a G2/M phase accumulation that was moderate in H460cells but substantial in IGROV-1 cells. Under these conditions, noappreciable induction of apoptosis could be detected in both celllines, as indicated by the lack of release of cytochrome C and ofthe caspase activation. In contrast, the combination of the micro-tubule inhibitor with PFT enhanced the accumulation of cells inthe M phase, with a marked increase of apoptosis in both cell lines.

Low paclitaxel concentrations may cause inhibition of cell pro-liferation without mitotic arrest, because under these conditions,the drug may induce both p53 and p21 causing G1 and G2 arrests[23,24]. A similar behavior was observed in H460 cells, exhibitinga partial delay in G1 and a low accumulation in late G2, and inIGROV-1 cells, exhibiting a more evident G2/M arrest. Pifithrin-αabolished in both cell lines the paclitaxel-induced up-regulation ofp21 after the activation of p53. This event reflected the inhibitionof the transcriptional activity of p53, because p21 is a gene targetof p53. The implication of p21 in response to paclitaxel is consistentwith a parallel sensitization produced by p53 down-regulation inp53-siRNA–transfected cells. Down-regulation of p21 is expectedto allow the activation of the cyclin B/p34cdc2 complex [29–31],which is inactive in the G2 phase. Activation of the complex, indi-cated by the reduced phosphorylation of p34cdc2 at Tyr15, shouldallow a G2→M transition. However, a number of factors are knownto be involved in regulating mitotic entry. In particular, the activationof Plk1 is expected to promote the activation of cyclin B/p34cdc2

complex, because it is implicated in the phosphorylation and activa-tion of cdc25C, a phosphatase involved in the dephosphorylation ofp34cdc2 at Tyr15 [26,32]. Moreover, Plk1 regulates the degradationof Wee1 (implicated in the G2 checkpoint) after phosphorylation[26]. Indeed, under our conditions, down-regulation of p21 in cellstreated with PFT/paclitaxel combination was associated with theactivation of p34cdc2/cyclin B complex, an increase of cdc25C phos-phorylation, and up-regulation of Plk1, thus supporting a suppres-sion of G2 checkpoint and a facilitation of G2/M transition [26].A protective role of p21 against cytotoxicity of paclitaxel has beendescribed [31,33,34], but the cellular and molecular bases of thisprotection are not clearly defined. Besides its involvement in multiplecell cycle checkpoints, p21 could confer resistance to antimicrotubuleagents by a decrease of mitotic arrest and exit from abnormal mitosis[33,35,36]. In addition, p21 is implicated in the AKT-dependentsurvival pathway, because cytoplasmic accumulation of p21 inpaclitaxel-treated cells could be regulated by an AKT-dependent

Figure 4. Expression of p53 and p21waf1 in H460, IGROV-1 cells (A) and H460 cells transfected with RNAi-negative control duplex or withp53-siRNA (B) treated for 24 hours with paclitaxel (IC50, PTX) or cyclic PFT alone or their combination. In the combined treatment, PFTwere added 2 hours before treatment with PTX. (A) Whole-cell extracts were prepared and analyzed by Western blot analysis. β-Tubulinis shown as a control of protein loading. For p21WAF1, low and high film exposures are shown.

Figure 5. Down-regulation of AKT and inhibition of AKT phosphory-lation (Ser473) detected by Western blot analysis in H460 andIGROV-1 cells exposed to paclitaxel (IC50) and cyclic PFT in singleor combined treatment. Cells were pretreated for 2 hours withPFT followed by simultaneous 24-hour exposure to the antimicro-tubule agent. Filters were stripped and reprobed with the anti-body recognizing AKT protein or β-tubulin to control specific oroverall protein loading.

594 Sensitization to Apoptosis by p53 Inhibition Zuco and Zunino Neoplasia Vol. 10, No. 6, 2008

phosphorylation [37]. In our study, the down-regulation of p21 in-duced by PFT in paclitaxel-treated cells (Figure 4) was associatedwith the release of cytochrome C and the activation of caspase 3 after24 hours of combined treatment (Figure 3, C and D). Becausep21 has been implicated in the inhibition of the caspase activationprocess [38], the observed down-regulation may also have a pro-apoptotic effect.The observed sensitization of mitotic cells by PFT could reflect

at least in part a prevalence of apoptotic signals likely activated ina p53-independent manner. In particular, although a direct involve-ment of p53 in the modulation of the AKT-dependent survival path-way remains to be documented in our cell systems, the availableresults showed an inhibition of both up-regulation and phosphoryla-tion of AKT induced by paclitaxel. Because a protective role of AKTagainst taxane-induced apoptosis has been described in other cellsystems [27], it is conceivable that inactivation of AKT-mediated sur-vival pathway may favor a cell death process.In conclusion, the present observations of the sensitization to anti-

microtubule agents by a transcriptional inhibitor of p53 are consistentwith multiple and sometimes opposite functions of p53, depending onthe nature of cytotoxic stress. The available evidence supports that, inresponse to mitotic spindle damage, the transcription-mediated func-tions of p53 have a protective action. However, after inhibition of thetranscriptional activity, p53 could retain proapoptotic functions thatmight be prevalent over antiapoptotic signals. The sensitization oftumor cells with wild type p53 by selective inhibition of p53 functiondescribed in this study may have obvious implications in the antitumortherapy with antimitotic agents. Indeed, it supports the potential effi-cacy of regimens containing low (and less toxic) doses of antimitoticagents by combination with a nontoxic modulating agent. The use ofrational combinations could improve the efficacy of protracted treat-ment regimens with low doses of taxanes that are known to have anti-angiogenic and antitumor effects [39]. The use of p53 inhibitors hasbeen proposed as a chemoprotective strategy to reduce the side effectscaused by chemotherapy or radiotherapy or by other stress conditionsimplicating the activation of p53 [17]. The present study supports thepotential use of PFT as a chemosensitizing agent in combination withmicrotubule inhibitors. In contrast to the protective effect of PFTagainst toxicity of DNA-damaging agents, the combination with anti-microtubule drugs could enhance toxicity against normal tissue. Thepotential therapeutic advantages of the combination of p53 inhibitorswith antimicrotubule agents remain to be documented in in vivomodels, for improvement of the “therapeutic index.” However, itshould be emphasized that tumor cells seem to be more susceptibleto apoptotic death than nonmalignant cells, as supported by a lackof sensitization to paclitaxel-induced apoptosis in human “normal”fibroblasts (Figure 3B).

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