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Published OnlineFirst April 6, 2010; DOI: 10.1158/1535-7163.MCT-09-0971

Research Article Molecular
Cancer
Therapeutics

SIRT Inhibitors Induce Cell Death and p53 Acetylation throughTargeting Both SIRT1 and SIRT2

Barrie Peck1, Chun-Yuan Chen3, Ka-Kei Ho1, Paolo Di Fruscia2, Stephen S. Myatt1, R. Charles Coombes1,Matthew J. Fuchter2, Chwan-Deng Hsiao3, and Eric W.-F. Lam1

Abstract

Authors'CyclotronCampus aLondon, UAcademia S

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Corresponry, DepartmCollege LonW12 0NN,E-mail: eric

doi: 10.115

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SIRT proteins play an important role in the survival and drug resistance of tumor cells, especially duringchemotherapy. In this study, we investigated the potency, specificity, and cellular targets of three SIRTinhibitors, Sirtinol, Salermide, and EX527. Cell proliferative and cell cycle analyses showed that Sirtinoland Salermide, but not EX527, were effective in inducing cell death at concentrations of 50 μmol/L or overin MCF-7 cells. Instead, EX527 caused cell cycle arrest at G1 at comparable concentrations. In vitro SIRT assaysusing a p53 peptide substrate showed that all three compounds are potent SIRT1/2 inhibitors, with EX527having the highest inhibitory activity for SIRT1. Computational docking analysis showed that Sirtinol andSalermide have high degrees of selectivity for SIRT1/2, whereas EX527 has high specificity for SIRT1 but notSIRT2. Consistently, Sirtinol and Salermide, but not EX527, treatment resulted in the in vivo acetylation of theSIRT1/2 target p53 and SIRT2 target tubulin in MCF-7 cells, suggesting that EX527 is ineffective in inhibitingSIRT2 and that p53 mediates the cytotoxic function of Sirtinol and Salermide. Studies using breast carcinomacell lines and p53-deficient mouse fibroblasts confirmed that p53 is essential for the Sirtinol and Salermide-induced apoptosis. Further, we showed using small interfering RNA that silencing both SIRTs, but not SIRT1and SIRT2 individually, can induce cell death in MCF-7 cells. Together, our results identify the specificity andcellular targets of these novel inhibitors and suggest that SIRT inhibitors require combined targeting of bothSIRT1 and SIRT2 to induce p53 acetylation and cell death. Mol Cancer Ther; 9(4); 844–55. ©2010 AACR.

Introduction

The development and progression of cancer involvesboth epigenetic and genetic changes leading to the alter-ation of gene expression and thus cell phenotype. It iswell established that alteration in the epigenome of thecell through promoter hypermethylation and histonedeacetylation is a common facet of tumorigenesis. In-deed, previous studies have reported that the promotersof tumor suppressor genes, such as p16INK4A and p15INK4B

frequently show increases in DNA methylation and/orhistone deacetylation, thereby leading to gene silencing(1). However, emerging evidence also suggests that

Affiliations: 1Department of Surgery and Cancer, MRCBuilding, Imperial College London, Hammersmith Hospitalnd 2Department of Chemistry, Imperial College London,nited Kingdom; and 3Institute of Molecular Biology,inica, Taipei, Taiwan

plementary material for this article is available at Molecularrapeutics Online (http://mct.aacrjournals.org/).

ding Author: Eric W.-F. Lam, Cancer Research-UK Laborato-ent of Surgery and Cancer, MRC Cyclotron Building, Imperialdon, Hammersmith Hospital Campus, Du Cane Road, LondonUK. Phone: 44-20-8383-5829; Fax: [email protected]

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the role of acetylation as a protein posttranslational mod-ification, independent of histone modification, may alsoplay a critical role in cell fate and thus tumorigenesis.For example, the activities of the tumor suppressorsp53 and FOXO3a are both in part regulated by theiracetylation status. The degree of acetylation is largelymediated by a balance between histone acetyl transferaseand histone deacteylase (HDAC) activity, in a substrate-specific manner.Sirtuins (also called SIRT) are NAD-positive–depen-

dent class III HDACs that share extensive homologieswith the yeast HDAC Silent Information Regulator 2(Sir2; ref. 2). In yeast (3), nematode worms (4), and fruitflies (5), SIRT/Sir2 activity is crucial for life span extensionin response to metabolic and other environmental stresses(6–9). In mammals, seven Sir2 homologues (Sirtuin 1-7 orSIRT1-7) have been identified, which possess primarilyHDAC (SIRT1, SIRT2, SIRT3, SIRT5) or monoribosyltrans-ferase activity (SIRT4 and SIRT6; refs. 10, 11), which targethistone and various nonhistone proteins in distinct sub-cellular locations.The mammalian SIRT1 is the direct homologue of

the yeast Sir2 and has a wide range of substrates and cel-lular functions. SIRT1 can induce chromatin silencingthrough deacetylation of histones H1, H3, and H4 (12),and can modulate cell survival by regulating the tran-scriptional activities of p53 (13), NF-κB (14), FOXOproteins (15, 16), and p300 (17). In contrast to SIRT1,

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SIRT Inhibitors Target SIRT1/2 to Activate p53


SIRT2 is predominantly a cytoplasmic protein and is ableto deacetylate several cytoplasmic substrates, includingα-tubulin (18, 19).Both SIRT1 and SIRT2 may have a role in the develop-

ment of cancer. For example, SIRT1 can activate stressdefense and DNA repair mechanisms, thus allowingthe preservation of the genomic integrity (20). Con-versely, it has also been shown that SIRT1 overex-pression can enhance tumor growth and promote cellsurvival in response to stress and drug resistance. Never-theless, SIRT1 is upregulated in a spectrum of cancers,including lymphomas, leukemia and soft tissue sarco-mas, prostate cancer, and lung and colon carcinomas(21–23). Overexpression of SIRT2 can significantly pro-long the mitotic (M) phase and delay mitotic exit. Inconsequence, it has been proposed that SIRT2 mightfunction as a mitotic checkpoint protein in G2-M toprevent the induction of chromosomal instability, par-ticularly in response to microtubule inhibitor–mediatedmitotic stress (24). Consistently, tumors with high levelsof SIRT2 are refractory to chemotherapy, especially mi-crotubule poisons (25).The dual role of SIRTs in modulating the acetylation of

tumor suppressor proteins and chromatin renders themattractive therapeutic targets for anticancer drug devel-opment (20). However, despite the development of sev-eral effective SIRT inhibitors, little is known about thespecific mechanisms of action and cellular targets of theseproteins. In the present study, we have used breast cancercells as a model system, and profiled the in vitro andin vivo activity of several recently developed potent SIRTinhibitors. Moreover, we have identified the tumor sup-pressor p53 as the critical target of the SIRT inhibitor–induced cell death in breast cancer cells.

Materials and Methods

Cells and cell cultureThe human breast carcinoma cell lines MCF-7 originat-

ed from the American Type Culture Collection and wereacquired from Cancer Research UK, in which they weretested and authenticated. These procedures includecross-species checks, DNA authentication, and quaran-tine. Cell lines used in the present study were in culturefor <6 mo and were maintained in DMEM/F12 media(Sigma) containing 10% FCS and 2 mmol/L glutamine(10% CO2; 37°C). All final concentrations of solventwere identical between and within experiments; SIRT in-hibitors were dissolved in stock solutions of 20 mmol/Lin DMSO, and for treatments, the compounds werediluted in DMSO to the appropriate concentrations andadded to cells [(0.5% (v/v) final DMSO concentration].All cells were <70% confluent and growing exponentiallybefore transfection and treatment. The number ofcells seeded was adjusted to attain this level of con-fluency depending on the culture volume and cell type,but was consistent within experiments. The population

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doubling time was 16 to 19 h with cell confluency main-tained at <80%.

Compound synthesisSIRT inhibitors EX527 (6-Chloro-2,3,4,9-tetrahydro-1H-

carbazole-1-carboxamide; ref. 26), Sirtinol (2-[(2-Hydro-xynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide; ref. 27), and Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide; ref. 28) were prepared according toreported procedures. Nicotinamide was acquired fromSigma UK.

Small interfering RNA transfectionMCF-7 cells were transfected with 100 nmol/L SIRT1

or SIRT2 small interfering RNA (siRNA; Dhamarcon)using oligofectamine (Invitrogen). Twenty hours post-transfection, transfected cells were examined by cell cycleanalysis.

Sulforhodamine B assayApproximately 3,000 cells were seeded in each well

of the 96-well plate. All treatment compounds wereadded 24 h following seeding; this is designated 0 h in thesulforhodamine B (SRB) assays. After culture, 100 μL oftrichloroacetic acid were added to each well and incu-bated for 1 h at 4°C. The plates were then washed withdeionized water thrice before incubation at room temper-ature for 1 h with 0.4% SRB in 1% acetic acid. The plateswere then washed with deionized water and air dried.Tris base (10 mmol/L) was then added to the wells to sol-ubilize the bound SRB dye, and the plates were then readat 492 nm using the Anthos 2001 plate read (JenconsScientific Ltd.).

Cell cycle analysisCell cycle analysis was done by propidium iodide

staining, as previously described (29). Both adherentand floating cells were harvested and stained with pro-pidium iodide (0.2 mg/mL) in the presence of DNase-free RNase for flow cytometry analysis. The cell cycleprofile was analyzed using the Cell Diva software(Becton Dickinson UK Ltd.).

Western blottingBoth adherent and floating cells were harvested for

Western blot analysis and 20 μg of protein were loadedof each sample. Cells were lysed and SDS-PAGE gel elec-trophoresis was done as previously described (30). Theantibodies against β-tubulin (H-235), p21 (SC-6248),and SIRT2 (H-95) were purchased from Santa CruzBiotechnology (Autogen Bioclear). Antibodies againstacetyl-Lysine (#9441), acetyl-p53 (lys382; #2525), andp53 (1C12) were purchased from Cell Signaling Technol-ogy. The antibody against SIRT1 (E104) was purchasedfrom Abcam. Primary antibodies were detected usinghorseradish peroxidase linked to anti-mouse and anti-rabbit conjugates as appropriate (DAKO), and visualized

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using the enhanced chemiluminescence detection system(Amersham Biosciences).

In vitro SIRT activity assayThe Biomol hSIRT1/hSIRT2 activity assay (AK-555/

AK-556) was used to deduce the activity potential ofSIRT inhibitors. The assay was done essentially asdescribed (31) and according to the manufacturer'sinstructions (Biomol International). All compoundswere preincubated with the hSIRT1/hSIRT2 beforecommencing the reaction through the addition of the“Fluor de Lys” deacetylase substrate. Deacetylation ofK382-p53/K320-p53 was used as a marker of HDACactivity. All compounds were prepared fresh in DMSOto 10 mmol/L, 24 h before the assay was preformed.Fluorescence was read (excitatory, 360; emission, 460)using the Fluoroskan Ascent FL fluorometer (ThermoFisher) using the Ascent software. The dose concentra-tions were done in triplicate. The amounts of acetylationwere derived from the levels of fluorescence (displayedin arbitrary fluorescence units). In addition, the percen-tages of inhibition were calculated from the arbitraryfluorescence unit of the treated assays and nontreatedcontrols.

Three-dimension modelingTo prepare the docking-related files including protein

and inhibitors, the three dimensional structure ofHuman SIRT1 was modeled by the Protein Homology/analogy Recognition Engine (Phyre; ref. 32). The modeledstructure of SIRT1 is based on the structure of yeastSir2 (PDB entry is 2HJH) already containing nicotin-amide and Zn, with the original human SIRT1 aminoacid sequence from residues 212 to 529. The three-dimensional structure of SIRT2 used for docking wastaken from the human Sirtuin C-pocket (PDB entry is1J8F). The three inhibitor structures, Sirtinol, Salermide,and EX527, were generated by the Dundee PRODR G2Server (33). The docking studies were done using thesite feature docking algorithm from the LibDock (34)software of the Discovery Studio package (version 2.1).The force field was applied to the SIRT1 and SIRT2using the in-built CHARMm force field calculation. Thebinding region for the docking of SIRT2 is the same apreviously described (28), which was a 10-Å spherearound the Gln167. The docking region used for SIRT1was designed in a 10-Åf sphere around the residueof Gln345, which is genetically conserved to the Gln167of SIRT2. All the docking studies were carried out bydefault setting of the LibDock software and then thegenerated results of these docking studies were subjectedto the “Score Ligand Poses” built-in function of theDiscovery Studio to evaluate and rank the docking re-sults. The best pose of each docking study was chosenbased on the LigScore2 (35). Furthermore, the bestpose of each docking study was energy minimized withthe protein structure by the “Ligand Minimization”function of the Discovery Studio. All the structural pre-

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senting figures were created with the PyMOL program(DeLano Scientific).

Results

Sirtinol and salermide induce cell death whereasEX527 triggers G1 arrestTo gain insights into the anticancer potential of the

three novel synthetic SIRT inhibitors EX527 (26), Sirtinol(27), and Salermide (Supplementary Fig. S1; ref. 28), wefirst investigated the effects of these SIRT inhibitorson the proliferation of the breast carcinoma cell lineMCF-7. The natural SIRT inhibitor nicotinamide wasalso included as a reference compound. SRB resultsshowed that concentrations of 25 μmol/L and over ofeither Sirtinol or Salermide could significantly inhibitMCF-7 cell proliferation (≥80% of inhibition), whereasonly ≥100 μmol/L of EX527 could effectively repressMCF-7 cell proliferation (Fig. 1A). Even at 100 μmol/L,nicotinamide failed to elicit any substantial inhibitoryeffect on MCF-7 cells (Fig. 1A). To confirm these pro-liferative results, we next examined the effects ofthese SIRT inhibitors on cell cycle progression andtreated cycling MCF-7 cells with 50 μmol/L of eachof the four SIRT inhibitors for 0, 24, and 48 hours.Flow analysis results showed that both Sirtinol andSalermide effectively induced cell death, whereas EX527treatment caused an increase in G1 cell population,with no detectable cell death (Fig. 1B). These cell pro-liferation data were supported by morphologic exam-ination using light microscope showing that onlySirtinol and Salermide could induce extensive cell deathat 50 μmol/L (Fig. 1C).

SIRT inhibitors display potent in vitro SIRT1/2inhibitory activitiesTo unravel the molecular basis for the difference in cy-

totoxicity between these SIRT inhibitors, we examinedthe in vitro inhibitory potential of these SIRT inhibitorson SIRT1 and SIRT2 using an in vitroHDAC activity fluo-rescent assay. The % values of SIRT activity at differentdoses (0–100 μmol/L) of inhibitors were first determinedagainst human recombinant SIRT1 and SIRT2 using asynthetic p53-derived peptide as a substrate, and fromthese data, the IC50 values for the SIRT inhibitors wereestablished (Fig. 2). The in vitro assay revealed thatSirtinol displayed a higher degree of inhibitory acti-vity toward SIRT1 than SIRT2, with IC50 of 37.6 and103.4 μmol/L, respectively. Salermide also exhibitedcomparable SIRT1 and SIRT2 inhibitory activity (IC50 =76.2 and 45.0 μmol/L, respectively). EX527 showed thehigher SIRT1 inhibitory activity (IC50 = 0.38 μmol/L)and SIRT2 inhibitory activity (IC50 = 32.6 μmol/L) com-pared with Sirtinol and Salermide, whereas nicotinamidehad relatively low potency against SIRT1 with an IC50 of85.1 but potently inhibited SIRT2 (IC50 = 1.16 μmol/L).These in vitro SIRT1/2 inhibition results are generallyin consensus with the previously published data on the

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efficacy of some of these SIRT inhibitors on SIRT1 andSIRT2 (26, 27, 36). However, these in vitro data couldnot completely explain for the failure of EX527 to inducecell death, as EX527 has higher SIRT2 inhibitory activitythan Sirtinol and higher SIRT1 inhibitory activity com-pared with Sirtinol and Salermide yet does not inducecell death.



Modeling of SIRT1/2 docking predicts sirtinol andsalermide binding efficiently to SIRT1/2, and ofEX527 to SIRT1 aloneNext, we analyzed the inhibitory functions of these

SIRT inhibitors using computational docking studies.Preliminary, computational docking studies were carriedout on Sirtinol, Salermide, and EX527 using both Gold

Figure 1. Effects of SIRT inhibitorson MCF-7 cell proliferation andsurvival. A, MCF-7 cells weretreated with 0 to 100 μmol/L ofnictotinamide, EX527, Sirtinol, orSalermide for 0, 24, 48, and 72 h.Cell proliferation was determinedby SRB assay. Points, mean ofthree independent experiments;bars, SD. B, MCF-7 cells wereeither nontreated or after treatmentwith 50 μmol/L of nicotinamide,EX527, Salermide, or Sirtinol for0, 24, and 48 h, and flowcytometric analysis of DNA contentwas done after propidium iodidestaining. Percentage of cells ineach phase of the cell cycle(sub-G 1, G1, S, and G2-M) isindicated. Representative datafrom three independentexperiments are shown.C, representative phase-contrastmicroscopy images of MCF-7cells nontreated or after treatmentwith 50 μmol/L of nicotinamide,EX527, Salermide, or Sirtinol(magnification, ×100).




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and LibDock in an effort to rationalize the observedSIRT1/SIRT2 selectivity differences. For these studies,the crystal structure of hSIRT2 (PBD code: 1J8F) was usedalongwith a hSIRT1model, and the inhibitors docked intothe C-pocket (Fig. 3). The hSIRT1 model was obtainedfrom the Phyre Web site (www.sbg.bio.ic.ac.uk/phyre/;structure 2HJH) and based on the homologous yeast pro-tein Sir2 (Fig. 3A). In terms of SIRT1 binding, both Sirtinoland Salermide show significant hydrogen bonding inter-actions with Gln 345 and His 363 (Fig. 3B). EX527 also dis-plays hydrogen bonding to Gln 345, with additional π-πinteractions with His 363 and Phe 273. In terms of SIRT2binding, Sirtinol and Salermide show comparable hydro-gen bonding interactionswithGln 167 (Fig. 4). Sirtinol alsodisplays π-π interactions withHis 187 and Phe 119, where-as Salermide has a hydrogen bond with His 187 and nointeraction with Phe 119. For EX527, we did not find anysignificant interactions with SIRT2, the closest interactionidentified being a Van derWaals force between EX527 andAla 85. Based on these results, it is plausible that the resi-dues Phe 273, Phe 297, Gln 345, and His 363 of SIRT1 andresidues Phe 96, Phe 119, Gln 167, and His 187 of SIRT2may play critical roles in inhibitor binding. Our dockingstudies suggest that both Sirtinol and Salermide can formkey interactions with these residues in the requisite bind-ing pocket, whereas EX-527 only forms strong interactionswith SIRT1. In conclusion, the computational modelingand docking studies suggest that that both Sirtinol and



Salermide have high degrees of selectivity for SIRT1/2,whereas EX527 has a high specificity for SIRT1 butnot SIRT2.The present computational study is in general con-

sistent with the in vitro HDAC assay in that bothSirtinol and Salermide target SIRT1 and SIRT2, where-as EX527 has a high specificity for SIRT1. However,although EX527 showed strong in vitro inhibitory acti-vity toward SIRT2, the docking assay suggested littleaffinity between EX527 and SIRT2. Taken together,these findings suggest that Sirtinol and Salermide havea high affinity for SIRT1 and SIRT2, whereas EX527 isselective for SIRT1 but not SIRT2. These findings alsosuggested that both SIRT1 and SIRT2 have to be inhibitedto induce cell death in vivo as they target SIRT1 andSIRT2, whereas EX527 induced cell cycle arrest at G1

because it targets only SIRT1.

Sirtinol and salermide but not EX527 can inducep53 acetylation in vivoTo investigate further the distinct effects of the SIRT

inhibitors on cell fate, we performed Western blot analy-sis on MCF-7 cells treated with 50 μmol/L for 0, 24, and48 hours, to study their effects on the acetylation status ofthe SIRT1/2 target p53 (Fig. 4A). Interestingly, Sirtinoland Salermide but not EX527 could induce p53 acetyla-tion at lysine 382, which has been shown to correlate withp53 activation. Consistently, the p53 acetylation was also

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Figure 2. In vitro SIRT1 and SIRT2 inhibitionassays. Recombinant human SIRT1 and SIRT2were treated with various concentrations ofSirtinol, Salermide, EX 527, and nicotinamideas indicated, and their relative inhibitory po-tential (against DMSO as controls) was ana-lyzed and displayed as % of SIRT activity.Points, mean three independent experiments;bars, SD.


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accompanied by an induction of p53 stability. Thecontrol nicotinamide was ineffective in inducing p53acetylation at the concentrations studied. To examinethe effects of the compounds on SIRT2 activity, we in-vestigated the expression levels of acetylated tubulin,which is a specific SIRT2 target (18). The results showedthat only Sirtinol and Salermide but not EX527 andnicotinamide could induce tubulin acetylation. We nexttreated MCF-7 cells for 24 hours with a range of con-centrations of SIRT inhibitors and harvested the cells



for Western blot analysis (Fig. 4B). Similar to earlier data,the results showed that at concentrations of ≥50 μmol/L,Sirtinol and Salermide, but not EX527 and nicotinamide,are able to induce p53 acetylation and that only Sirtinoland Salermide but not EX527 and nicotinamide can in-duce tubulin acetylation. Notably, there was also a gen-eral decrease in protein levels in MCF-7 cells aftertreatment with 100 μmol/L Salermide and was likely tobe a result of protein degradation due to cell death afterdrug treatment. The failure of EX527 to induce p53 and

Figure 3. The three-dimensional structures ofSIRT1 and SIRT2. A, the crystal structure ofhSIRT2 (PBD code: 1J8F) was used along witha hSIRT1 model, and the inhibitors docked intothe C-pocket. The hSIRT1 model was obtainedfrom the Phyre Web site (structure 2HJH) andbased on the homologous yeast protein Sir2.B, structures of the complexes of inhibitorswith SIRT1 and SIRT2 modeled by docking theinhibitors onto the C-pocket of SIRT1 andSIRT2 structure after minimization.




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tubulin acetylation was not because of its lack of SIRT-in-hibitory activity as it could effectively cause global pro-tein acetylation as revealed by the acetyl-Lysine antibody.Together, these results suggest that although both Sirtinoland Salermide target SIRT1 and SIRT2, EX527 only inhi-bits SIRT1 and not SIRT2 effectively and that p53 acety-lation requires inhibition of both SIRT1 and SIRT2.

The cytotoxic function of sirtinol and salermide isdependent on the presence of functional p53To examine the role of p53 in mediating the cytotoxic

and cytostatic effects of the SIRT inhibitors, we treatedbreast carcinoma cell lines (37), MCF-7 (wild-type p53),MDA-MB-231(mutant p53), BT474 (mutant p53), andZR-75-1 (very low p53 expression) with 50 μmol/L ofSIRT inhibitors for 0, 24, and 48 hours. Flow analysis re-sults showed that both Sirtinol and Salermide effectivelyinduced cell death in MCF-7 cells, but the ability of Sir-tinol and Salermide to induce cell death is compromisedin other breast cancer cell lines without functional p53(Fig. 5A). Notably, EX527 treatment caused a substantialincrease in G1 cell population with little cell death in allbreast carcinoma cell lines, with the exception of BT-474.To further confirm these results, we also studied the ef-fects of the SIRT inhibitors on the cell cycle status ofmouse embryo fibroblasts (MEF) derived from wild-type



and p53-deficient mice (Fig. 5B). Consistent with thebreast carcinoma cell line data, the flow cytometric re-sults indicated that although EX527 treatment caused asubstantial increase in G1 population with little cell deathin both the wild-type and p53−/− MEFs, Sirtinol and Sale-rmide only induced cell death efficiently in wild-type butnot p53−/− MEFs. We next examined the expression ofacetylated lysine, acetylated p53, and acetylated tubulinin the wild-type and p53−/− MEFs following SIRT inhibi-tor treatment. The Western blot results showed that Sir-tinol and Salermide, but not EX527, are able to inducep53 acetylation in the wild-type MEFs, and that Sirtinoland Salermide but not EX527 can induce tubulin acetyla-tion in both the wild-type and p53−/− MEFs. Westernblotting using the acetyl-Lysine antibody again revealedthat EX527 is more effective than Sirtinol and Salermidein inducing the acetylation of some proteins and some ofthese EX527 targets might account for its ability to in-duce G1 arrest. Cleaved caspase-3 was used as a markerof apoptosis, and the Western blot results showed thatthe loss of p53 decreases the cleavage and activation ofcaspase-3, reiterating our findings from the cell cycle anal-ysis. Together, these results suggest that functional p53is required for the cytotoxic function of Sirtinol andSalermide but is dispensable for the cytostatic effectsof EX527.

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Figure 4. Effects of SIRT inhibitorson p53, tubulin, and global lysineacetylation. A, MCF-7 cells weretreated with 50 μmol/L of SIRTinhibitors for 0, 24, and 48 h ofnicotinamide, Salermidem, Sirtinol,or EX527. The expression ofSIRT1, SIRT2, acetylated lysine,acetylated p53, p53, acetylatedtubulin, and tubulin wasanalyzed by Western blotting.B, MCF-7 cells were treatedwith increasing concentrations(conc; 0–100 μmol/L) andWestern blot analysis was done todetermine the protein expressionlevels of SIRT1, SIRT2, acetylatedp53, p53, and tubulin.

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Figure 5. Effects of SIRT inhibitors on cell cycle status and p53, tubulin, and global lysine acetylation. A, MCF-7, MDA-MB-231, BT474, and ZR-75-1breast carcinoma cells as well as the wild-type (WT) and p53−/− MEFs were treated with 50 μmol/L of SIRT inhibitors for 0, 24, and 48 h of Sirtinol,EX527, and Salermide. Flow cytometric analysis of DNA content was done after propidium iodide staining. Percentage of cells in each phase of thecell cycle (sub-G1, G1, S, and G2-M) is indicated. Representative data from three independent experiments are shown. B, the expression of cleaved (active)caspase-3, acetylated lysine, acetylated p53, p53, acetylated tubulin, and tubulin was analyzed by Western blotting in the SIRT inhibitor–treatedwild-type and p53−/− MEFs.

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Silencing of both SIRT1 and SIRT2 is required forinducing MCF-7 cell deathOur earlier results suggested that induction of cell

death by SIRT inhibitors in vivo requires the inactivationof both SIRT1 and SIRT2. To examine this conjecture,MCF-7 cells were transfected with either control siRNAor siRNA against SIRT1, SIRT2 or SIRT1, and SIRT2.The transfected cells were then incubation with or with-out 10 nmol/L of the chemotherapeutic drug paclitaxelfor 24 hour before the cells were subjected to cell cycleand Western blot analyses. The cell cycle analysisshowed that the siRNA-mediated knockdown of endog-enous SIRT1 or SIRT2 individually had no discernable ef-fect on the cell cycle status of MCF-7 cells, but thesilencing of endogenous SIRT1 and SIRT2 simultaneouslyinduced a noticeable G2-M phase cell cycle arrest as wellas cell death (Fig. 6A). Interestingly, treatment of MCF-7with 10 nmol/L paclitaxel also elicited a cell deathand cell cycle arrest profile similar to that inducedby SIRT1/2 knockdown. In addition, we discoveredthat silencing of SIRT2 alone actually abolished thepaclitaxel-induced G2-M cell cycle arrest and cell death.The Western blot analysis confirmed successful SIRT1and/or SIRT2 knockdown and showed that the SIRT1/2



knockdown–induced G2-M arrest and cell death wasassociated with an increase in p53 acetylation (Fig. 6B).Intriguingly, paclitaxel treatment also resulted in asimilar increase in p53 acetylation, and this p53 hyper-acetylation was again accompanied by cells undergoingG2-M arrest and cell death. In addition, we discoveredthat silencing of SIRT2 alone by specific siRNA sup-pressed paclitaxel-induced cell cycle arrest and celldeath as well as p53 acetylation. Together these resultssuggest that paclitaxel mediates the G2-M arrest–associated cell death through inducing the acetylationand activation of p53.

Discussion

In this study, we have for the first time compared thepotencies, mechanisms of action, and cellular targets ofthe three recently developed SIRT inhibitors, Sirtinol,Salermide, and EX527. From cell proliferation and cellcycle assays, it was found that although Sirtinol andSalermide can effectively induce cell death, EX527 doesnot trigger cell death but a cell cycle arrest. The in vitroSIRT inhibitory assay shows that all three compoundspotently inhibited SIRT1, with EX527 at least two orders

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Figure 6. Effects of SIRT1/2silencing and paclitaxel on cellcycle progression and p53acetylation. MCF-7 cells wereeither untransfected (mock),transfected with nonspecific (NS)siRNA (100 nmol/L) or siRNA smartpool against SIRT1 (100 nmol/L),SIRT2 (100 nmol/L), or SIRT1 plusSIRT2 (100 nmol/L) for 24 h andthen incubated for another 24 hin the absence or presence of10 nmol/L paclitaxel. A, flowcytometric analysis of DNAcontent was done after propidiumiodide staining. Percentage ofcells in each phase of the cell cycle(sub-G1, G1, S, and G2-M) isindicated. Representative datafrom three independentexperiments are shown. B, theexpression levels of SIRT1, SIRT2,acetylated p53, p53, and tubulinwere determined by Westernblotting.

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of magnitude more potent than Sirtinol and Salermide.This finding is supported by computational dockingstudies showing that all three inhibitors bind SIRT1 withhigh affinity. However, despite the in vitro SIRT inhi-bitory assay showing high degrees of inhibitory activityagainst SIRT2 for all three compounds, only Sirtinoland Salermide exhibit high affinity for SIRT2 in the struc-tural studies. The structural-docking study results aresupported by the in vivo finding that EX527 failed tocause the reacetylation of tubulin, a critical cellular targetof SIRT2 (38). This finding is also consistent with ourin vivo results indicating that inactivation of both SIRT1and SIRT2 is required for the induction of cell deathand p53 acetylation and that EX527 neither triggers celldeath nor p53 acetylation in MCF-7 cells. The structuralin vitro activity discrepancy could be due to the factthat in the in vitro SIRT2 inhibitory assay, the recom-binant human SIRT2 is the single predominant Sirtuinspecies existing at high concentrations and the specificitymight be lost as a result. Alternatively, EX527 couldhave an altered selectivity toward the recombinant SIRT2protein under in vitro experimental conditions. Moreover,the in vitro assays used a synthetic p53 peptide insteadof a wild-type protein. These observations showedthat the in vitro SIRT assays are not always consistentwith findings in vivo, which may be based on cellcycle analysis, analysis of global acetylation profiles,SIRT isoform–specific target acetylation, and dockingstudies. Nevertheless, this also highlights the fact thatthe in vitro SIRT assay data have to be interpreted withcaution and should not be use alone in studying SIRTor inhibitor activity. It is important to note, however,that although the structural and computational dockingstudies provide a qualitative appreciation of possiblesources of inhibitor selectivity, further detailed biologicalstudy is required (for example, site-selective mutations)to verify these results in vivo. For example, althoughour results provide qualitative comparisons with a recentreport by Lara et al. (28), the precise binding modes ofthe inhibitors differ. Moreover, computational studiesby Huhtiniemi et al. (39) suggest that the conformationalfreedom of the “flexible loop” region of the C-pocketmay play a crucial role in substrate binding, some-thing that is not taken into account using static dockingtechniques.It is notable that in this study, we examined the cel-

lular potency and targets of the SIRT inhibitors usingthe MCF-7 breast carcinoma cell line because they havesubstantial levels of SIRT1 and SIRT2 as well as a func-tional p53. In mammalian cells, the transcription factorp53 functions as a tumor suppressor and is a target ofacetylation as well as SIRT1 and SIRT2. The acetylationof functional p53 leads to its activation and is usuallytriggered in response to oncogenic as well as envi-ronmental stress signals, including oxidative stress andchemotherapeutic drug treatments (40). Once activated,p53 triggers the apoptotic program to induce cell death.In cell-based in vivo studies, both Sirtinol and Salermide



but not EX527 can efficiently induce p53 acetylation andthis was associated with the induction of cell death inMCF-7 cells. Moreover, further studies using breast carci-noma cell lines with different p53 status and wild-typeand p53-deficient MEFs confirmed that Sirtinol andSalermide are ineffective in inducing apoptosis in theabsence of functional p53 expression. These findingssuggest that the tumor suppressor p53 is integral to thecytotoxic activity of the SIRT inhibitors Sirtinol andSalermide, and the failure of EX527 to trigger cell deathis because of its inability to acetylate and activate the en-dogenous p53. A cooperative role for SIRT1 and SIRT2to regulate stress-induced cell death pathways in a p53-independent manner has also been described (25). It isalso likely that SIRT inhibitors, such as Sirtinol andSalermide, also target SIRTs other than SIRT1 and SIRT2.Nevertheless, SIRT1 and SIRT2 will probably have amore prominent role in controlling cell growth and sur-vival as they exist in the same intracellular compartments(i.e., nucleus and cytoplasm) as most of the cell cycleand death regulators (41). The reason why Sirtinol andSalerimide are ineffective in causing global lysine acety-lation is unclear. However, because these global lysineacetylation patterns are associated with treatment withthe very potent SIRT1 inhibitor EX527, it could be thatthese acetylated patterns might represent proteins prefer-entially targeted by SIRT1 and/or other HDACs.Data from SIRT inhibitor experiments also suggest that

both SIRT1 and SIRT2 have to be inactivated to inducecell death in vivo, at least in SIRT1- and SIRT2-positivecells. This is confirmed by the siRNA-silencing experi-ments showing that not one but both SIRT1 and SIRT2have to be deleted to cause cell death. This observationis consistent with a recent study showing that despitethe interaction of SIRT1 with p53, deletion of SIRT1 inMEFs fails to induce p53 acetylation and activate the ap-optotic function of p53 (42). Notably, cell cycle profiles ofMCF-7 cells are different in response to treatment withEX-527 compared with SIRT-1 siRNA, and this mightsuggest that additional mechanisms of induction of cellcycle arrest by EX-527, which could be mediated by inhi-biting other HDACs. Intriguingly, treatment withthe chemotherapeutic drug paclitaxel also induces a G2-M arrest–associated cell death indistinguishable fromthat triggered by silencing of SIRT1 and SIRT2. The factthat this was also accompanied by p53 acetylation sug-gest that SIRT inhibitors, such as Sirtinol and Salermide,and paclitaxel function through common pathways andmediate their cytotoxic effects through targeting p53 andacetylation. Unexpectedly, silencing of SIRT2 is sufficientto repress the cytotoxic effects of paclitaxel and abolishp53 hyperacetylation. Similarly, SIRT1 knockdown couldalso prevent paclitaxel from causing the reacetylation oftubulin. The molecular mechanisms responsible are un-known and might involve compensatory mechanisms be-tween different SIRT proteins. It is also likely that SIRT2silencing will cause cell cycle arrest and chromatin con-densation as revealed by sirt2-deficient mouse studies




Peck et al.

854


(24). As a consequence, SIRT2 silencing alone may lessenthe potency of chemotherapeutic drugs, such as pac-litaxel, which preferentially target proliferating cells.Nevertheless, this observation further highlights the im-portance of p53 acetylation in the induction of cell deathand that SIRT inhibitors and paclitaxel might have syner-gistic or additive effects at suboptimal concentrations. Insummary, we have used a combination of in vitro SIRTinhibition assay, structural studies, and in vivo cell-basedsystems to delineate the substrate specificity and efficacyof three novel SIRT inhibitors, Sirtinol, Salermide, andEX527. Together, the results also provide evidence toshow that the cytotoxic effect of SIRT inhibitors ismediated predominantly through the inactivation ofboth SIRT1 and SIRT2 and the subsequent acetylationof the tumor suppressor p53. These data also suggestthat a redundancy of functions may exist between SIRT1



and SIRT2, and that SIRT1 and SIRT2 cooperate to de-acetylate the tumor suppressor protein p53 to attenuatecell death.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Biotechnology and Biological Sciences Research Council (B. Peck andE.W-F. Lam), Cancer Research UK (K.-K. Ho, P. Di Fruscia, S.S. Myatt,R.C. Coombes, M.J. Fuchter, and E.W-F. Lam).

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.

Received 10/19/2009; revised 12/21/2009; accepted 01/29/2010;published OnlineFirst 04/06/2010.

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2010;9:844-855. Published OnlineFirst April 6, 2010.Mol Cancer Ther Barrie Peck, Chun-Yuan Chen, Ka-Kei Ho, et al. Targeting Both SIRT1 and SIRT2SIRT Inhibitors Induce Cell Death and p53 Acetylation through

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