cpth6, a thiazole derivative, induces histone ... · maria giulia rizzo2, and donatella del bufalo1...

Cancer Therapy: Preclinical

CPTH6, a Thiazole Derivative, Induces HistoneHypoacetylation and Apoptosis in Human Leukemia Cells

Daniela Trisciuoglio1, Ylenia Ragazzoni1, Andrea Pelosi2, Marianna Desideri1, Simone Carradori4,Chiara Gabellini1,3, Giovanna Maresca7, Riccardo Nescatelli5, Daniela Secci4, Adriana Bolasco4,Bruna Bizzarri4, Chiara Cavaliere5, Igea D'Agnano7, Patrizia Filetici6, Lucia Ricci-Vitiani8,Maria Giulia Rizzo2, and Donatella Del Bufalo1

AbstractPurpose: We previously identified novel thiazole derivatives able to reduce histone acetylation and

histone acetyltransferase (HAT) activity in yeast. Among these compounds, 3-methylcyclopentylidene-

[4-(40-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) has been selected and used throughout this study.

Experimental Design: The effect of CPTH6 on histone acetylation, cell viability and differentiation, cell-

cycle distribution, and apoptosis in a panel of acute myeloid leukemia and solid tumor cell lines has been

evaluated.

Results:Here, we showed thatCPTH6 leads to an inhibition ofGcn5 andpCAFHATactivity.Moreover, it

inhibits H3/H4 histones and a-tubulin acetylation of a panel of leukemia cell lines. Concentration- and

time-dependent inhibition of cell viability, paralleled by accumulation of cells in the G0/G1 phase and

depletion from the S/G2M phases, was observed. The role of mitochondrial pathway on CPTH6-induced

apoptosis was shown, being a decrease ofmitochondrialmembrane potential and the release of cytochrome

c, from mitochondria to cytosol, induced by CPTH6. Also the involvement of Bcl-2 and Bcl-xL on CPTH6-

induced apoptosis was found after overexpression of the two proteins in leukemia cells. Solid tumor cell

lines from several originswere shown tobe differently sensitive toCPTH6 treatment in terms of cell viability,

and a correlation between the inhibitory efficacy onH3/H4histones acetylation and cytotoxicity was found.

Differentiating effect on leukemia and neuroblastoma cell lines was also induced by CPTH6.

Conclusions: These results make CPTH6 a suitable tool for discovery of molecular targets of HAT

and, potentially, for the development of new anticancer therapies, which warrants further investigations.

Clin Cancer Res; 18(2); 475–86. �2011 AACR.

Introduction

Epigenetic changes, including histone modifications,often occur in cancer (1). Among the post-translationalmodifications of histones, one regards the acetylation ofspecific lysine e-amino groups in the N-terminal tail of the

core chromosomal histones H2A, H2B, H3, and H4. His-tone acetylation increases accessibility of several factors tothe chromatin at specific genes or over vast regions of thegenome (2, 3), and it is involved in the regulation of severalcell functions, such as gene transcription (4, 5), DNA repair(6), and replication (7). Histone acetyltransferases (HAT)are the enzymes responsible for histone acetylation, whichcan be reversed by the activity of histone deacetylases(HDAC), enzymes that hydrolyze the acetyl groups (8).The histone acetylation–deacetylation balance is accuratelymaintained through an equilibrium of HAT and HDACenzymatic activities in normal cells. On the contrary, irreg-ular pattern of histone acetylation is often found associatedwith cancer, and it has been hypothesized to modulatethe expression of oncogenes and tumor suppressor genes(9–12). Therefore, both protein acetylation and deacetyla-tion pathways represent attractive targets for cancer therapy.High hyperacetylation of histones in oral squamous andhepatocellular carcinoma patient samples has beenreported (13, 14). Translocation of HAT genes (15), muta-tion of the HAT p300 (16), overexpression of AIB-1, a HATcoactivator of a nuclear hormone receptor (17), represent

Authors' Affiliations: 1Experimental Chemotherapy Laboratory and2Molecular Oncogenesis Laboratory, Regina Elena National Cancer Insti-tute; Departments of 3Anatomy, Histology, Forensic Medicine, Orthope-dics, Section of Histology and Medical Embryology, 4Chemistry andPharmaceutical Technologies, 5Chemistry and 6Institute of Molecular Biol-ogy and Pathology, CNR, "Sapienza" University; 7Institute of Cell Biologyand Neurobiology-CNR Santa Lucia Foundation-IRCCS; and 8Departmentof Hematology, Oncology, and Molecular Medicine, Istituto Superiore diSanit�a, Rome, Italy

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Donatella Del Bufalo, Experimental Chemother-apy Laboratory, Regina Elena National Cancer Institute, Via delle Messid'Oro 156, 00158 Rome, Italy. Phone: 39-06 5266 2575; Fax: 39-06 52662592; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0579

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 475

on November 3, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 8, 2011; DOI: 10.1158/1078-0432.CCR-11-0579

http://clincancerres.aacrjournals.org/

examples of deregulated HATs in several tumor histotypes.These observations suggest that specific and relatively non-toxic inhibitors of acetyltransferases could be considered asnew generation of therapeutic agents for cancer. AlthoughHDAC inhibitors have been extensively studied and severalare currently in clinical trials (18), there is little informationavailable on inhibitors of HATs (HATi; ref. 19). Amongthem, several compounds have been described to elicitantiproliferative, proapoptotic, antitumoral, and antiangio-genic activity toward different tumor histotypes, and theymay represent new therapeutic agents for cancer treatmentand prevention (20–23). Recently, a set of newly synthe-sized molecules derived from thiazole has been selected asmodulators of the HATs in yeast-based drug screening (24).Among these compounds, the thiazole derivative CPTH6has been chosen to evaluate its possible antitumoral activityin a large panel of leukemic and solid tumor cell lines.

Materials and Methods

Cell culturesHuman acute myeloid leukemia (AML) and solid tumor

cell lines were obtained from different sources and culturedas reported in Supplementary Materials.

Generation of Bcl-2, Bcl-xL, and cFLIP stablyoverexpressing cells

The HA-cFLIP, bcl-xL, and bcl-2 cDNAs were clonedunder the cytomegalovirus promoter of a lentiviral vector,which carried the Enhanced Green Fluorescent Proteinreporter gene under the 3-phosphoglycerate kinase promot-er. Recombinant lentiviruses were produced as described

previously (25). Transduced cells were sorted for greenfluorescence (FACS Aria; Becton Dickinson)

Reagents preparation and treatmentCPTH6 (3-methylcyclopentylidene-[4-(40-chlorophe-

nyl)thiazol-2-yl]hydrazone; Supplementary Fig. S1A) wassynthesized as previously reported (24), dissolved indimethyl sulfoxide (DMSO; Sigma-Aldrich) at the con-centration of 100 mmol/L and diluted to the final con-centrations in complete medium. For all the experiments,cells were treated with 1% DMSO as control. After 24hours from seeding, exponentially growing AML andsolid tumor cells were treated with CPTH6 at concentra-tions ranging from 1 to 100 mmol/L for 24 to 120 hours.For some experiments, treatment with CPTH6 was fol-lowed or preceded by exposure to the HDAC inhibitortrichostatin A (TSA; Sigma-Aldrich), while pan-caspaseinhibitors z-VAD-fmk (100 mmol/L; Calbiochem) andqVD (40 mmol/L; Santa Cruz Biotechnology) were added1 hour before CPTH6 treatment.

Curcumin (Sigma-Aldrich), anacardic acid (Alexis Bio-chemical), andMB-3 (Alexis Biochemical)were dissolved inDMSO and stored as stock at �20�C. Vitamin D3 and All-Trans-Retinoic Acid (ATRA; Sigma-Aldrich) were dissolvedin ethanol and DMSO, respectively, and stored as stock at�80�C. Because of light sensitivity of ATRA, all incubationswere done under subdued lighting.

Measurement of HAT activityTo evaluate the effect of CPTH6on in vitroHAT enzymatic

activity, 1 mg of Gcn5, pCAF, p300, or CBP recombinantproteins (Enzo Life Science), 2 mg of H3 or H4 histones(New England Biolabs) and 20 mmol/L Acetyl-CoA (Sigma-Aldrich), containing 0.01 mCi/mL [3H]Acetyl-CoA (Perki-nElmer) were incubated in HAT assay buffer (50 mmol/LTris-HCl pH 8.0, 0.1 mmol/L EDTA, 1 mmol/L dithiothrei-tol and 10% glycerol) at 30�C for 1 hour in the presence of800 mmol/L CPTH6 or 2% DMSO, as control. The reactionmixture was then blotted in triplicate onto P-81 filter papers(Upstate) that were washed, dried, and placed in vialscontaining scintillation fluid. Radioactive counts wererecorded on Tri-Carb 2800TR Liquid Scintillation Analyzer(PerkinElmer). To characterize the inhibition kinetics ofCPTH6, HAT assays were done using 2 mg of histones withconcentrations of [3H]Acetyl-CoA from 20 to 80 mmol/L.

To evaluate HAT activity in CPTH6-treated cells, nuclearprotein extracts (50 mg) were prepared as previouslyreported (26) and subjected to colorimetric HAT ActivityAssay Kit (Enzo Life Science) following manufacturer’sinstructions.

Measurement of histone deacetylase activityThe effect of CPTH6 on in vitro HDAC activity and on

cellular HDAC activity was analyzed using Epiquik HDACActivity/Inhibition Assay Colorimetric Kit (Epigentek) fol-lowing manufacturer’s instructions. Nuclear extracts ofU-937 cells, obtained by using EpiQuik Nuclear ExtractionKit I (Epigentek), were used as source of HDAC activity.

Translational Relevance

Recent studies strongly support the histone acetyla-tion as a promising therapeutic target with a strongpreclinical rationale in hematologic disease and solidtumors. Although a growing body of evidence shows adirect relationship between histone acetyltransferases(HAT) activity anddevelopment or progressionof cancerdisease, only limited progress has beenmade in the fieldof HAT inhibitors. Our study identifies the thiazolederivative 3-methylcyclopentylidene-[4-(40-chlorophe-nyl)thiazol-2-yl]hydrazone (CPTH6) as a novel Gcn5and pCAF HAT inhibitor. CPTH6 treatment in humanacute myeloid leukemia (AML) and colon carcinomacells significantly decreased histones acetylation and cellviability and activated apoptosis. Differentiation of AMLandneuroblastoma cellswas also evidenced afterCPTH6treatment. Overall, these results are of both biologicaland clinical significance, and they identify a new inhib-itor of histone acetylation, targeting HAT activity andinducing cell death and differentiation in AML thatshould be further studied for leukemia therapy.

Trisciuoglio et al.

Clin Cancer Res; 18(2) January 15, 2012 Clinical Cancer Research476




Assessment of cell viabilityExponentially growing AML and solid tumor cells were

seeded in duplicate in 6-well (2 � 105 cells per well) orin sextuplicate in 96-well (3 � 103 cells per well) cultureplates (Nunc), respectively, and after 24 hours CPTH6was added to the medium at concentrations rangingfrom 5 to 100 mmol/L for 24 to 120 hours. Cell viabilityof AML and solid tumor cells was evaluated by trypanblue exclusion and by MTT (mitochondrial respirationanalysis; Sigma-Aldrich) assays, respectively. AML cellsviability was calculated for each concentration of CPTH6used as "number of CPTH6-treated cells/number of con-trol cells" �100. MTT was added to each well at the finalconcentration of 0.5 mg/mL and after 4 hours of incu-bation at 37�C, the formazan salt was dissolved with 200mL isopropylic alcohol. The absorbance of each well wasmeasured with ELISA reader (DASIT) at 570 nm wave-length and the viability was calculated for each concen-tration of CPTH6 used as "OD of CPTH6-treated cells/OD of control cells" �100. The concentration of CPTH6that causes 50% of cell viability inhibition (IC50) wasalso calculated.

Cytofluorimetric analyses of cell-cycle, apoptosis,mitochondrial membrane potential and reactiveoxygen species productionTo analyze cell-cycle phases distribution, cells were col-

lected by centrifugation, fixed in cold 70% ethanol, andstained in a PBS solution containing propidium iodide (PI;62.5 mg/mL; Sigma-Aldrich), and RNase A (1.125 mg/mL;Sigma-Aldrich). Samples were acquired with a FACScaninstrument (Becton Dickinson) and the percentage of cellsin the different phases of cell-cycle and in sub-G1 compart-ment was calculated using ModFit LT software (BectonDickinson).Apoptosis was assayed by using Terminal deoxynucleo-

tidyl transferase–mediated fluorescein-dUTP nick-endlabeling (TUNEL)-based commercial Kit (Roche Diagnos-tic) or by staining cells with the Annexin V–fluoresceinisothiocyanate apoptosis kit (eBioscience), according to themanufacturer’s instructions. Mitochondrial membranepotential (MMP) was assessed using Mito-ID Detection Kit(Enzo Life Science), according to manufacturer’s instruc-tions. Reactive oxygen species (ROS) production was mea-sured using dihydroethidium staining (DHE; MolecularProbes). For detection of apoptosis, MMP and ROS, at theend of incubations with the respective reagents, about20,000 eventswere acquired and gatedusing forward scatterand side scatter to exclude cell debris, and the percentage ofcells relatives to the different analyses was calculated usingCellQuest software (Becton Dickinson).

Western blot analysisCells were lysed and total extracts were fractionated by

SDS-PAGE, transferred to a nitrocellulose filter, and sub-jected to immunoblot assay, as previously described (27).Cytochrome c release into the cytosol was detected aspreviously described (27). Immunodetection was done

using the antibodies reported in Supplementary Materials.Horseradish peroxidase–conjugated secondary antibodiesbinding was visualized by enhanced chemiluminescenceaccording to manufacturer’s specification and recorded onautoradiography film (Amersham Biosciences).

Assessment of cell differentiationAML cell differentiation was analysed by morphologic

and functional analyses [CD11b, CD14, alpha-naphthylacetate esterase (aNAE)] and neuroblastoma cell differen-tiation was evaluated by immunofluorescence analysis(NF-200), as reported in Supplementary Materials.

In vivo toxicity and pharmacokinetic analysisAssessment of in vivo tolerability and pharmacokinetic

parameters of CPTH6 was done as reported in Supplemen-tary Materials.

StatisticsExperiments were replicated 3 times and the data were

expressed as means � SD, unless otherwise indicated.Differences between groups were analyzed with a 2-sidedpaired or unpaired t test, and they were considered to bestatistically significant for P < 0.05.

Results

CPTH6 is a specific Gcn5/pCAF inhibitor and reduceshistones and a-tubulin acetylation

To identify the specific target(s) of CPTH6, a thiazolederivative (Supplementary Fig. S1A) previously shown toinhibit histone acetylation in yeast (24), we analyzed theeffect of the compound on in vitro HAT activity of humanrecombinant p300, CBP, pCAF, and Gcn5 enzymes. Asshown in Fig. 1A, CPTH6 exerted a significant inhibitoryeffect on HAT activity of both pCAF and Gcn5, whereas itdid not affect p300 and CBP HAT activity. As expected,anacardic acid, a well known pan-HAT inhibitor, almostcompletely inhibited HAT activity of all enzymes analyzed.The inhibitory effect of CPTH6 on Gcn5 activity is quitesimilar to the one observed forMB-3, a butyrolactonewhichhas been described as a specific Gcn5 HAT inhibitor (28).Using histone H4 as substrate, CPTH6 showed a significantinhibitory effect on the activity of pCAF and Gcn5 (Sup-plementary Fig. S1B), whose preference for histone H3 hasbeen previously shown (28). We also found that theincrease of acetyl-CoA concentration reverted the inhibitoryeffect of CPTH6 on Gcn5 HAT activity (Supplementary Fig.S1C). Moreover, HDAC activity was not affected by CPTH6exposure (Supplementary Fig. S1D and E).

By usingU-937 cell line, we found that 50mmol/LCPTH6decreasesHAT activity by about 40%and75%after 6 and18hours of treatment, respectively (Fig. 1B) and reduces acet-ylation levels of bothH3andH4histones (Fig. 1C),withoutaffecting their total expression (Fig. 1F). We also evaluatedthe effect of lower concentrations of CPTH6: reduced acet-ylation of histone H3 was observed after 72 and 120 hoursof exposure to 10/20 and 5 mmol/L CPTH6, respectively,

CPTH6 Induces Apoptosis in Leukemia Cell Lines

www.aacrjournals.org Clin Cancer Res; 18(2) January 15, 2012 477




whereas no effect on H3 acetylation was observed aftertreatment with 1 mmol/L CPTH6 (Fig. 1D and E).

Besides, CPTH6 downregulation of H3 acetylation wassimilar to that induced by MB-3 (about 40%), whereascurcumin, a pan-HAT inhibitor, induced higher reductionof H3 acetylation (about 70%; Supplementary Fig. S2A).

The ability of CPTH6 to decrease acetylation of histoneH3 specifically at lysine 18 in a dose-dependent fashionwasalso observed (Fig. 1F), whereas CPTH6 (50 and 100 mmol/L for 24 hours) did not affect H3 histone methylation atlysines 9 and 4 (Fig. 1G).

Tested ona-tubulin, a non-histone substrate, CPTH6wasalso able to decrease the acetylation level of the protein,without affecting the expression of total a-tubulin (Fig. 1I).

As reported in Fig. 1H, histone H3 hyperacetylationinduced by TSA, a well-known inhibitor of HDACs, wasreversed by CPTH6 treatment in a time-dependent mannersuch that after a 5-hour treatment, the level of acetylatedhistone H3 was similar to that of control cells. By contrast,pretreatment of cells withCPTH6was not able to counteractTSA-induced H3 histone and a-tubulin hyperacetylation(data not shown).

Figure 1. CPTH6 is aGcn5/pCAF inhibitor and reduces histones anda-tubulin acetylation in a time- and concentration-dependentmanner inU-937 cell line. A,HAT activity evaluated by radioactive assay of recombinant human p300, CBP, pCAF, andGcn5 using H3 histone as substrate and 20 mmol/L [3H]acetyl-CoAin presence of 2% DMSO (control), 800 mmol/L CPTH6, 20 mmol/L anacardic acid (AA) or 100 mmol/L MB-3. The values are expressed as percentageof enzymatic activity in CPTH6-treated versus control samples (100%). B, HAT activity evaluated by colorimetric assay using as enzyme donor thenuclear extract of U-937 control cells or treated with 50 mmol/L CPTH6 for the indicated hours. The values are expressed as percentage of HAT activity inCPTH6-treated versus control cells (100%). A and B, P values were calculated between control and treated samples (�, P < 0.05). C–G and I, Westernblot analysis of acetylated histoneH3, on Lys 9/14 (Ac-H3) or Lys 18 (Ac-H3 Lys18), acetylated histoneH4 on Lys 5,8,12,16 (Ac-H4),methylated histoneH3onLys 9 (Met-H3 Lys9) or Lys 4 (Met-H3 Lys4) and acetylated or total a-tubulin in protein extracts obtained after treatment with CPTH6 at the indicatedconcentrations and exposure times. H, Western blot analysis of acetylated histone H3 from cells pretreated with 250 nmol/L TSA for 24 hours and thenexposed to 100 mmol/L CPTH6 for the indicated times. C–H, HSP 72/73 and (I) a-tubulin expression was used as loading and transferring control. Westernblots representative of 2 independent experiments with similar results are shown.

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Different basal levels of H3 histone acetylation in AMLlines, and the ability of CPTH6 to decrease H3 and H4histones and a-tubulin acetylation were also shown in HL-60, KG1, andHEL97.1.7 leukemia cells (Supplementary Fig.S2B–G).

CPTH6differently affects in vitro cell viability of humancancer cell linesHaving corroborated the inhibitory effect of CPTH6 on

histone acetylation, we evaluated the in vitro viability of apanel 35 established human cancer cell lines of differenthistotypes exposed to increasing concentrations of CPTH6up to 100 mmol/L for times ranging from 24 to 72 hours(Fig. 2). As reported in Fig. 2A, 100 mmol/L CPTH6 for72 hours induced cell viability inhibition higher than 25%in 31 of 35 (about 82%) cell lines. An almost homogeneousresponse to CPTH6 in terms of cell viability inhibition wasobserved in glioblastoma (from 21%–47%), melanoma(from 21%–43%), neuroblastoma (from 21%–39%) andbreast (from 21%–49%), colon (from 67%–83%), andpancreas (from 41%–49%) carcinomas. On the contrary,a more heterogeneous response was evidenced in AML(from 51%–89%), lung (from 31%–69%), prostate (from0%–64%), and ovary (from 28%–79%) carcinoma celllines. As reported in Fig. 2B, a dose- and time-dependentreduction of cell viability by CPTH6 treatment was moreevident in some cell lines, such as U-937, HL-60, A2780,and HCT116 than in others (LAN-5 and U-87).As depicted in Fig. 3A, we observed a decrease of histone

H3 acetylation level upon 24 hours of treatment withCPTH6 in several "more sensitive" cells, in terms of reduc-

tion of cell viability (HL-60, HCT116, HT-29, H1299, andA2780) but not in other "less sensitive" (M14, MCF7, andU-87) ones. In agreement with these results, whereas longertreatments (72 hours) did not result in significant changesinH3 acetylation level in "less sensitive" cells (M14,MCF7),reduced acetylation levels of histone H3 in the "moresensitive" HCT116 line was already evident after 24 hoursCPTH6 treatment (Fig. 3B).

CPTH6 induces cell-cycle perturbation and apoptosisin AML cells

We next evaluated the cell-cycle profile after CPTH6treatment ofU-937andHL-60 leukemia cell lines. As shownin Fig. 4A and B, CPTH6 had a strong effect on cell-cycleprogression, being an accumulation in the G0/G1 phasewith depletion of cells from S and G2/M phases alreadyevident after 24 hours of treatment in both cell lines.

Consistent with the observed G0/G1 accumulation,CPTH6 modulated the expression of key proteins thatregulate cell-cycle progression (Fig. 4C): increased level ofcyclin-dependent kinase inhibitor p27 and transcriptionfactor E2F4, together with hypophosphorylation and re-duced expression of the tumor suppressor retinoblastomaproteins Rb and Rb2, respectively, were observed afterCPTH6 treatment.

The sub-G1 population (about 40% and 20% in U-937andHL-60, respectively, after 72hours of treatment)observ-ed in Fig. 4A and B is indicative of apoptosis. As shownin Fig. 4D, a time-dependent increase in the percentage ofapoptotic cells was induced by 50 and 100 mmol/L CPTH6in U-937 cells. The induction of apoptosis was confirmed

Figure 2. CPTH6 differently affectscell viability of human cancer celllines. Viability of (A) the indicated celllines treated with 100 mmol/L CPTH6for 72 hours and (B) U-937 andHL-60(leukemia), HCT116 (coloncarcinoma), A2780 (ovarycarcinoma), LAN-5 (neuroblastoma),and U-87 (glioblastoma) cell linestreated with concentrations ofCPTH6 ranging from5 to 100mmol/L,for 24 to 72 hours. A and B, theresults are reported as "viability ofCPTH6-treated cells/viability ofcontrol cells" � 100 and representthe average � SD of 3 independentexperiments.






by TUNEL assay (Fig. 4E) being the percentage of TUNEL-positive cells about 15% and 40% after treatment with 20and 100 mmol/L CPTH6, respectively.

Also CPTH2, a compound closely related to CPTH6 (24),reduced H3 and H4 histones acetylation, induced accumu-lation of cells in the G0/G1 cell-cycle phase, and activatedthe apoptotic program in U-937 cells (Supplementary Fig.S2H and I).

As reported in Fig. 5A and B, exposure to CPTH6 at dosesranging from 5 to 100 mmol/L reduced the expression ofprocaspases 8, 9, and 3 and PARP and induced their cleav-age. Moreover, CPTH6 treatment did not modulate procas-pase 1 expression and did not induce its cleavage. No effecton PARP protein was observed after treatment with 1 mmol/LCPTH6 (Fig. 5B). PARP andprocaspase 9 cleavagewas alsoobserved after exposure ofHL-60 line to 50 and 100 mmol/LCPTH6 for 48 hours (Fig. 5C).

Next, cells were pretreated for 1 hour with the pan-caspase inhibitors qVD and z-VAD-fmk and then exposedto 100 mmol/L CPTH6 for 72 hours, and the percentage ofapoptotic cells was evaluated. Both qVD and z-VAD-fmkdid not significantly reduce CPTH6-induced cell death,although as expected, both compounds inhibited stauros-porine-induced apoptosis by about 60% and 50%, respec-tively (data not shown).

To determine the role of the mitochondrial pathway, wechecked the release of cytochrome c from mitochondria tocytoplasm, ROS production, and the expression of someBcl-2 family proteins in U-937 cells. Cytochrome c releasewas evident after exposure to CPTH6 (Fig. 5E), and it wasassociated with a depolarization of the mitochondrialmembrane (Fig. 5F). By contrast, no modulation of Bcl-2, Bcl-xL, and Mcl-1 proteins (Fig. 5A) and less than 4% ofROS generation were observed after exposure to CPTH6(100 mmol/L for 72 hours; Fig. 5D).

The role of extrinsic and intrinsic apoptotic pathways onCPTH6-induced apoptosis was analysed by stably overex-pressing cFLIP or Bcl-2/Bcl-xL, respectively, in U-937 cells(Fig. 5G, Supplementary Fig. S3A). Stably cFLIP-overexpres-sing cells treated with 100 mmol/L CPTH6 for 72 hoursshowed a similar percentage of apoptosis (about 35%)when compared with mock-transfected cells (about40%), although as expected, cFLIP overexpression pre-vented TNFa-induced cell death (Supplementary Fig. S3Cand D). Induction of apoptosis by CPTH6 after forcedexpression of both Bcl-2 and Bcl-xL proteins was reducedby about 50% when compared with mock-transfected cells(Fig. 5G).

As reported in Supplementary Fig. S3E and F CPTH6exposure also induces the processing of PARP and thepan-caspase inhibitor z-VAD-fmk blocked PARP cleavagein HT-29 colon carcinoma. Moreover, overexpression ofBcl-2 partly attenuated the cleavage of PARP induced byCPTH6 treatment.On the contrary, exposure to 100 mmol/LCPTH6 for 72hours didnot result in significant reduction incell viability (lower than 25%), in cell-cycle perturbation,apoptotic activation and did not reduce H3 histone acety-lation in normal primary cells of different origins, such asmouse fibroblast NIH/3T3 and human endothelial HU-VE-C. (Supplementary Fig. S3A and B).

CPTH6 induces AML and neuroblastoma celldifferentiation

On the basis of the relevance of histone acetylationchanges on regulation of cell differentiation (29), we inves-tigatedwhetherCPTH6was able to induce differentiation inAML and neuroblastoma cells. After exposure to 20 and 50mmol/L CPTH6, U-937 cells displayed a more mature phe-notype (about 50%) relative to control cells (about 2%),based on size reduction, decreased nucleo/cytoplasmicratio, and condensed chromatin, all parameters indicativeof monocytic terminal differentiation, and similar resultswere also observed for HL-60 cells (Fig. 6A). Accordingto these results, a significantly increased esterase activity(Fig. 6B) and an increased expression of monocytic-specificmembrane antigen CD14 was observed in cells exposedto CPTH6 in a concentration-dependent manner whencompared with control cells, whereas the compound didnot modulate the specific differentiation marker of granu-locytic differentiation CD11b (Fig. 6C).

Also, SH-SY5Y and LAN-5 neuroblastoma cells treatedwith 50 mmol/L CPTH6 for 5 days showed the outgrowth ofneuritic processes, as evidenced by the expression ofNF200,

Figure 3. CPTH6 reduces histone acetylation in a cell type–dependentmanner. Western blot analysis of acetylated histone H3 (Ac-H3) in totalcell lysates from (A) HL-60 (leukemia), H1299 (lung carcinoma), HT-29and HCT116 (colon carcinoma), A2780 (ovary carcinoma), M14(melanoma), U-87 (glioblastoma) and MCF7 (breast carcinoma) cellstreatedwith100mmol/LCPTH6 for 24hours and from (B)M14,MCF7, andHCT116 cells treatedwith 100mmol/LCPTH6 for times ranging from24 to72 hours. HSP 72/73 expression was used as loading and transferringcontrol. Western blots representative of 2 independent experiments withsimilar results are shown.

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the characteristic marker of neuronal differentiation. Theformation of neurites induced by CPTH6 had significantnumber of branch points, and they occurred in themajorityof the cells showing multiple long and well-defined den-dritic processes (Fig. 6D). The length of the primary neuritewas similar to that obtained by treating the cells with ATRA(Fig. 6E). Also the percentage of cells with neurites out-growth increases in SH-SY5Y cells after exposure to CPTH6,ranging from 7.4% in control cells to 18.7% and 32% aftertreatment with 20 and 50 mmol/L CPTH6, respectively. A

slight decrease of H3 acetylation was also observed afterexposure of SH-SY5Y to CPTH6 (data not shown). More-over, 50 mmol/L CPTH6 for 5 days induced a reduction ofcell proliferation of about 30% and 50% in SH-S5Y5 andLAN5 cells, respectively.

In vivo toxicology and pharmacokinetic parameters ofCPTH6

When intraperitoneally injected inmice over 30days, 100and 300 mg/kg of CPTH6 did not produce any adverse

Figure 4. CPTH6 induces cell-cycleperturbation and apoptosis in AMLcells. Flow cytometric analysis ofcell-cycle distribution evaluated byPI staining in U-937 (A) and HL-60 (B)cells control or treated with 100mmol/L CPTH6 for the indicatedtimes. Thepercentageof cells in sub-G1 and in the different cell-cyclephases is shown. Each panel isrepresentative of 3 independentexperiments with comparableresults. C, Western blot analysis ofp27, E2F4, total Rb, total Rb2 proteinexpression in total cell lysates fromU-937 cell line treated with 50 or 100mmol/L CPTH6 for the indicatedtimes. HSP 72/73 expression wasused as loading and transferringcontrol.Western blots representativeof 2 independent experiments withsimilar results are shown. D, flowcytometric analysis of thepercentage of AnnexinV–positive/PI-negative cells andAnnexin V–positive/PI-positive cellsafter treatment with concentrationsof CPTH6 ranging from 10 to 100mmol/L for 24 to 72 hours. The resultsrepresent the average � SD of 3independent experiments. P valuewas calculated between control andtreated cells (�, P < 0.05). E, TUNELassay evaluated by flowcytofluorimetric analysis of U-937cells treated with 20 and 100 mmol/LCPTH6 for 72 hours. The percentageof TUNEL-positive cells is reported.A representative experiment of 3independent experiments withsimilar results is shown.






health effects as monitored by diet consumption, bodyweight loss, and postural and behavioral changes.

The in vivo biopharmaceutical profile of CPTH6 wasstudied by measuring pharmacokinetic parameters in plas-ma samples, obtained at fixed times after intraperitonealand intravenous delivery of 300 mg/kg CPTH6 (Supple-mentary Table SI, Supplementary Fig. S4). Results showedthat CPTH6 was absorbed very fast, and maximum plasmaconcentrations (Cmax 15.08 � 3.01 ng/mL) were observed45 minutes after CPTH6 injection. The compound admin-istration resulted in an AUCip of 13.07 � 1.96 ng/mL � h,compared with an AUCiv of 17.18 � 2.57 ng/mL � h.

Therefore, the absolute bioavailability (AUCip:AUCiv ratioof 0.76) of CPTH6 is very high. The eliminationhalf-lifewas1.30 � 0.4 hours.

Discussion

In this article, we showed that CPTH6 significantlyreduces H3 and H4 histones and a-tubulin acetylation, cellviability and HAT activity, and activates the apoptotic andthe differentiating programs of several cancer cell lines. Theanalysis of basal levels of H3histone acetylation in AML celllines indicated that this parameter did not correlate with the

Figure 5. CPTH6 induces caspases cleavage and cytochrome c release in U-937 cell line. A, Western blot analyses of procaspase 8, 9, 1, and 3 and PARPprotein cleavage, Bcl-xL, Bcl-2, andMcl-1 protein expression inU-937cells treatedwith 100mmol/LCPTH6 for the indicated times. B,Western blot analysis ofPARP protein cleavage in U-937 treated with concentrations of CPTH6 ranging from 1 to 10 mmol/L CPTH6 for 120 hours. C, Western blot analysis ofPARP and procaspase 9 protein cleavage in HL-60 cells treated with 50 and 100 mmol/L CPTH6 for 48 hours. D, flow cytometric analysis of ROS production,evaluated byDHE staining, inU-937 control cells and treatedwith 100mmol/LCPTH6 for 72 hours. Cells exposed to 3.5mmol/L H2O2 for 15minuteswas usedas a positive control. E, Western blot analyses of cytochrome c (cyto c) in mitochondrial (mito) or cytosolic (cyto) extracts from U-937 cells treated with100 mmol/L CPTH6 for 48 hours. A–C, E, HSP 72/73 or HSP 60 expression was used as loading and transferring control. Western blots representativeof 3 independent experiments with similar results are shown. F, flow cytometric analysis of mitochondrial membrane potential evaluated by Mito-IDstaining (grey line) in U-937 cells control or treated with 50 mmol/L CPTH6 for 24 hours. Black areas represent unstained cells. G, cytofluorimetric analysis ofthe percentage of apoptotic cells (sub-G1 peak) in U-937 control (U-937/control) and U-937 stably overexpressing cFLIP (U-937/cFLIP), Bcl-2 (U-937/bcl-2),andBcl-xL (U-937/bcl-xL) cells after treatmentwith 100mmol/LCPTH6 for 72 hours.P valuewas calculatedbetween control and treated cells (�,P < 0.05). Theresults represent the average � SD of 3 independent experiments.

Trisciuoglio et al.





sensitivity to CPTH6 in terms of cell viability. In fact, HL-60and KG1 cells that showed the highest and the lowest levelof H3 acetylation, respectively, possess a superimposableIC50 (about 50 mmol/L). By contrast, solid tumor cells weredifferently sensitive toCPTH6 in terms of cell viability. Eventhough colon carcinoma lines were more sensitive toCPTH6 than the majority of solid tumor cell lines, andsome lines belonging the same histotype, such as pancreascarcinoma, showed a similar response to CPTH6, weexclude that the effect of CPTH6 could be related to thetumor histotype. In fact, lung, prostate, and ovary carcino-ma lines showed a heterogeneous response to CPTH6 and

both cells with low and high sensitivity were found withinthe same tumor histotype.

CPTH6effect on cell viability seems tobe correlated to theextent to which the histone hypoacetylation occurs. In fact,reduction of cell viability in response to CPTH6 treatmentwas only observed in leukemia and solid tumor cell linesthat showed a decrease of their acetylation level of H3 andH4 histones after CPTH6 treatment.

Also H3 histone acetylation at lysine 18 (H3K18Ac) wasreduced by CPTH6 treatment in a concentration-dependentfashion. Given that changes in histones acetylation at sev-eral lysines can be predictive of cancer patients clinical

Figure6. CPTH6 inducesdifferentiation inAMLandneuroblastomacells. A, analysis ofmonocyte-like featuresevaluatedbyMay–Grunwald/Giemsastaining inHL-60 and U-937 cells treated with 20 and 50 mmol/L CPTH6 for 72 hours. Representative fields with the classical features of blastic phenotype such as theirregularly shaped nucleus (##) and monocytic phenotype, such as cells with horseshoe nuclear shape (�), rounded nucleus, and small cytoplasm (��) areshown. B, percentage of U-937 cells positive to the monocytic specific marker aNAE after treatment with 20 and 50 mmol/L CPTH6 for 72 hours.Data are expressed as means� SEM from 3 independent experiments in duplicate. P value was calculated between control and treated cells (�, P < 0.05). C,flow cytometric analysis of granulocytic CD11b and monocytic CD14 markers expression in U-937 cells untreated (green) and treated with 20 (fuchsia)or 50 mmol/L (blue) CPTH6, 2 mmol/L ATRA (black) or 50 nmol/L vitamin D3 (Vit D3, orange) for 72 hours. Dark blue areas represent negative control(no addition of primary antibody to the cells). The histograms are representative of 3 different experiments with similar results. D, fluorescent imagesof SH-SY5Y and LAN-5 cells control or treated with 50 mmol/L CPTH6 or 10 mmol/L ATRA for 5 days. Cells were immunostained with anti-NF200monoclonal antibody (red) and with the DNA dye DAPI (blue). Images are representative of 3 different experiments with similar results. E, neurite outgrowthof SH-SY5Y and LAN-5 cells after 5 days of treatment with CPTH6. Length of primary neurites was quantified in treated cells as fold increase versuscontrol. ATRA-treated cells were used as positive control of differentiation. Data are the means � SD of 5 randomly chosen fields in each treated or controlsamples. The experiments were repeated 3 times with similar results.






outcome (30, 31) and high global levels of H3K18Ac areassociated with an increased risk of tumor recurrence(32, 33), these data suggest the potential use of CPTH6 forcancer therapy.

High hyperacetylation of histones has been reported inseveral cancer patient samples (13, 14). We showed hypoa-cetylating activity of the compound also on histones hyper-acetylated by trichostatin, an HDAC inhibitor. On thecontrary, the fact that pretreatment of CPTH6 did notinfluence TSA-induced H3 histone hyperacetylation couldbe related to the "de novo" acetylation that occurs in ourexperimental model after removal of CPTH6 (data notshown).

Within cells, many proteins are acetylated in addition tohistones (34). In this context, CPTH6 was also able todecrease the acetylation level of a-tubulin, indicating thatthe potential activity of HAT inhibition may also havenonhistone molecular targets as reported for other HATinhibitors (35). Moreover, CPTH6 exerted a significantinhibitory effect on the HAT activity of both pCAF andGcn5.

The increase of the concentration of acetyl group donoracetyl-CoA reverted the effect of CPTH6 on HAT activity,suggesting that CPTH6 may elicit its inhibitory activitypreventing the binding of acetyl-CoA. Nevertheless, CPTH6did not affect HAT activity of p300 and CBP or the activityof enzymes involved in posttranslational modification ofhistones, such as HDAC. Consistent with these results,CPTH6-induced HAT activity inhibition and H3 histonehypoacetylation were similar to those induced by MB-3, aspecific Gcn5 HAT inhibitor (28).

Focusing our attention on the 2 representative leukemiacell lines U-937 and HL-60, we found that decreased cellviability observed after CPTH6 treatment was associatedat earlier time points to an increase of percentage ofcells in the G0/G1 compartment associated with a depletionin the S and G2/M phases. An accumulation of both cyclin-dependent kinase inhibitor p27Kip1 and transcription factorE2F4. Rb hypophosporylation and Rb2 reduced expression,cell-cycle proteins having important roles in blocking cellcycle in the G1 phase, were also observed.

CPTH6 also induced apoptosis in AML cells and reducedprocaspase 3, 9, and 8 expression and concomitantlyincreased their cleavage, indicative of enzyme activation.In line with this possibility, the PARP cleavage product(a downstream target of caspase 3) was increased aftertreatment with CPTH6, suggesting a role for caspase acti-vation in the cytotoxic effect of the compound. However,addition of the irreversible, cell-permeable pan-caspaseinhibitors, z-VAD-fmk and qVD, at concentrations thatprevent caspase activation, did not significantly reverse celldeath after exposure to CPTH6. The lack of an effect of thecaspase inhibitors was not due to an inappropriate use ofthe 2 inhibitors because they were able to reduce apoptosisinduced by staurosporine. Together, these data suggest thatcell death induced by CPTH6 may proceed independentlyfrom caspases activation. Our results are in agreement withprevious reports showing that caspase-independent path-

ways may play essential roles in apoptosis induced byhistone acetylation–targeted drugs (36, 37). It is possiblethat proapoptotic mitochondrial factors such as calpain,apoptosis-inducing factor, endonuclease G or Omi/HtrA2,which have been reported to participate in caspase-inde-pendent apoptosis (38, 39), may play a role in CPTH6-induced apoptosis.

The inability of caspase 8 inhibitor to affect CPTH6-induced cell death (data not shown), and the observationthat cFLIP overexpression did not prevent CPTH6-inducedapoptosis, rule out a role of caspase 8 in CPTH6-inducedapoptosis. This evidence is in line with data showing anon-apoptotic role of caspase 8, but further a role of thisenzyme in the regulation of U-937 differentiation (40),suggesting a model in which CPTH6 induces apoptosis anddifferentiation through the involvement of different path-ways. Activation of procaspase 8 we observed after CPTH6treatment could be considered as a possible downstream"bystander" event and support the evidence that in ourmodel, the intrinsic pathway is more important in protect-ing cells from death than the extrinsic pathway.

The role of mitochondrial pathway and Bcl-2/Bcl-xLproteins on CPTH6-induced apoptosis was also shown,being a decrease of MMP and the release of cytochrome cinduced by CPTH6 treatment, and being CPTH6-inducedapoptosis partially reduced after forced expression of Bcl-2and Bcl-xL proteins.

Processing of PARP and the ability of Bcl-2 to partlyattenuate the cleavage of PARP have also been shown inthe HT-29 colon carcinoma cell line.

The ability of CPTH6 to inducemonocytic differentiationin AML cells has also been shown bymorphologic changes,such as size reduction, decreased nuclear/cytoplasmic ratioand chromatin condensation, and by the increased expres-sion of the monocytic specific marker CD14 induced byCPTH6.

CPTH6 was also able to activate the differentiation pro-gram in neuroblastoma, which represents an almost uniquetumor cell type, given its capacity to differentiate upontreatment with different agents, upregulating a well-estab-lished set of differentiationmarker genes,making it possibleto monitor the differentiation process (41). Indeed, follow-ing CPTH6 treatment, we found amarked expression of theNF200, which represents one of the standard neuronalmarkers. These results are in agreement with papers report-ing the ability of several HATi/HDACi to modulate normaland tumor cell differentiation. Examples are representedby quinoline derivatives that induce granulocytic differ-entiation in leukemia cells (35), curcumin, which activelysuppresses differentiation in astrocytes while promotingdifferentiation into the neurons (42), and regulators ofnucleosomal histones acetylation state that induce neuro-blastoma cells differentiation (43–45).

In conclusion, the identification of new compoundspossessing HAT inhibitory activity can represent a tool forstudying the effects of HATs on target molecules. Moreover,(i) the fact that also CPTH2, a closely related compoundbelonging to the same class of CPTH6, inhibits H3

Trisciuoglio et al.





acetylation and induces cell-cycle perturbation and apopto-sis inU-937 cells; (ii) the lack or reduced effect of CPTH6onviability and its inability to induce cell-cycle perturbationand apoptosis in primary normal cells; (iii) the hypoace-tylating and proapoptotic effect of CPTH6 also at lowdoses;and (iv) the in vivo data about tolerability and pharmaco-kinetics, can provide information necessary to design moreefficient drugs potentially useful for cancer therapy and, inparticular, for leukemia and colon carcinoma, which in ourstudy represent the most responsive to CPTH6.

Disclosure of Potential Conflicts of Interest

Y. Ragazzoni, C. Gabellini, and A. Pelosi are PhD students of "Sapienza"University. Y. Ragazzoni and A. Pelosi are recipients of a fellowship from theItalian Foundation for Cancer Research.

Acknowledgments

The authors thank Dr. A. Petricca for secretarial assistance and Dr. E.Tabolacci for providing some reagents.

Grant Support

The work was supported by IFO 10/30/c/50 and IFO 11/30/r/25 ItalianAssociation for Cancer Research (D. Del Bufalo) and New Idea Award 2010IFO Project3 (D. Del Bufalo).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMarch9, 2011; revised September 29, 2011; acceptedOctober 30,2011; published OnlineFirst November 8, 2011.

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2012;18:475-486. Published OnlineFirst November 8, 2011.Clin Cancer Res Daniela Trisciuoglio, Ylenia Ragazzoni, Andrea Pelosi, et al. Apoptosis in Human Leukemia CellsCPTH6, a Thiazole Derivative, Induces Histone Hypoacetylation and

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