Anovelsmallmoleculewithpotentanticanceractivity ... · Zinc has regulatory and structural functions in a large num-ber of enzymes and transcription factors. The structural functions

A novel small molecule with potent anticancer activityinhibits cell growth by modulating intracellularlabile zinc homeostasis

Mario Huesca, Lisa S. Lock, Aye Aye Khine,Stéphane Viau, Robert Peralta, I. Howard Cukier,Hongnan Jin, Raed A. Al-Qawasmeh, Yoon Lee,Jim Wright, and Aiping Young

Lorus Therapeutics Inc., Toronto, Ontario, Canada

AbstractML-133 is a novel small molecule with potent antiprolifera-tive activity, as shown in cancer cell lines and in a humancolon tumor xenograft model. ML-133 reduces the con-centration of intracellular labile zinc in HT-29 colon cancercells, leading to induction of the Krüppel-like factor 4 tran-scription factor. Krüppel-like factor 4 displaces the positiveregulator SP1 from the cyclin D1 promoter, thereby nega-tively regulating the expression of cyclin D1 and promotingthe G1-S phase arrest of cell proliferation. The antiproli-ferative and antitumor activity of ML-133 described inthe present study suggests modulation of intracellular zinchomeostasis as a potential strategy for the treatment ofseveral cancer types, and ML-133 represents a promisingnew class of antitumor agents that deserves further devel-opment. [Mol Cancer Ther 2009;8(9):2586–96]

IntroductionZinc has regulatory and structural functions in a large num-ber of enzymes and transcription factors. The structuralfunctions involve a highly stable association of zinc tofolded protein domains, whereas a more dynamic, ex-changeable labile zinc pool is involved in the regulatoryfunctions (1). Intracellular zinc homeostasis is regulated bysensor proteins, such as the metal-responsive transcriptionfactor 1, which regulates the transcription of zinc-sensitivegenes, including membrane transporter proteins, involvedin the cellular and vesicular influx and efflux of zinc, andmetallothionein and thionein, which play an important role

in the storage and distribution of intracellular zinc (2).Although labile zinc has been shown to be involved in sev-eral cellular pathways related to the regulation of cell fate,these mechanisms are not well characterized (3). Reductionof intracellular labile zinc has been associated with theinduction of apoptosis, decreased cell proliferation, and al-tered cell cycle progression in a number of cancer cell types,including mammary adenocarcinoma (4), melanoma (5), co-lon adenocarcinoma (6), and lymphocytic leukemia (7–10).

Studies of zinc-responsive gene regulation induced by in-tracellular labile zinc depletion in colon carcinoma HT-29cells identified Krüppel-like factor 4 (KLF4, also known asGKLF) as one of the genes whose expression is most signif-icantly changed (up-regulated) among >10,000 target genestested (6). KLFs are members of the SP/XKLF family oftranscription factors defined by an amino acid binding do-main at the C termini that comprises three C2H2-type zincfingers with similarity to the developmental gene Krüppelof Drosophila melanogaster (11). KLFs play an important rolein mammalian morphogenesis by controlling the prolifera-tion and/or differentiation of distinct cell lineages (12). Theexpression and function of KLFs are relatively tissue re-stricted (11), with KLF4 mainly expressed in epithelial cellsof the gastrointestinal tract, lung, testis, and skin, with afunctional role in skin barrier and gastric epithelial homeo-stasis (13), and development (14).

KLF4 mRNA is significantly reduced in colorectal cancercompared with normal matched tissues (15), and inductionof KLF4 expression in a colorectal cancer cell line results indiminished tumorigenicity (16). Furthermore, overexpressionof KLF4 causes cell cycle arrest at G1-S transition in RKO hu-man colon carcinoma cells (7). In addition, KLF4 is down-regulated in adenomas from the APCmin+/- mouse model ofcolorectal cancer, and crossing APCmin+/- mice with KLF4+/-

heterozygotes resulted in significantly more adenomas thanin APCmin+/- mice alone (17). Taken together, these resultsindicate a role of KLF4 as tumor suppressor factor in coloncancer. A similar function for KLF4 has also been reportedin bladder cancer (18), gastric cancer (19), esophageal cancer(20), pancreatic cancer (21), and adult T-cell leukemia (22).

Here we present the characterization of the anticanceractivity of the compound ML-133, selected from a novel seriesof 2-indolyl imidazol [4,5-d] phenanthroline derivatives withmetal chelation activity that exhibits a potent and selectiveantitumor activity against multiple cancer cell types (23).ML-133 reduces the concentration of intracellular labile zincin HT-29 colon cancer cells, leading to the induction of KLF4expression.KLF4displaces thepositive regulator SP1 from thecyclin D1 promoter, thereby negatively regulating the expres-sion of cyclin D1 and promoting the arrest of cell proliferation.

Received 11/20/08; revised 6/29/09; accepted 6/29/09; publishedOnlineFirst 9/15/09.

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Current address for A. Khine: NOVX Systems Canada Inc., Markham L3R6G3, Canada; S. Viau: Trillium Therapeutics Inc., Toronto M9W 4Y9,Canada; and for R.A. Al-Qawasmeh: University of Jordan, Amman 11942,Jordan.

Requests for reprints: Mario Huesca, Lorus Therapeutics Inc., 2 MeridianRoad, Toronto, Ontario M9W 4Z7 Canada. Phone: 416-798-1200, ext.311; Fax: 416-798-2200. E-mail: [email protected]

Copyright © 2009 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-08-1104

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Materials and MethodsChemical Synthesis

ML-133 was synthesized as described elsewhere (23).In vitro Cell Line Cancer Screen

To evaluate the potential antitumor activity of ML-133and to prioritize the selective activity on particular typesof tumor cell lines, the antiproliferative activity of ML-133was tested by the in vitro cell-line cancer screen at the Na-tional Cancer Institute (NCI; ref. 24). The detailed method isdescribed at the NCI Developmental Therapeutics Programwebsite.1

In vivo Hollow Fiber Assay

To assess the initial drug efficacy of ML-133 in vivo, theactivity of ML-133 was tested by NCI hollow fiber assay(25) on a panel of 12 tumor cell lines; (breast: MDA-MB-231, MDA-MB-435; glioma: U251, SF-295; ovarian: OV-CAR-3, OVCAR-5; colon: COLO-205, SW-620; melanoma:LOX-IMVI, UACC-62; and lung: NCI-H23, NCI-H522).The detailed method is described at the NCI DevelopmentalTherapeutics Program website.2

Cell Culture Maintenance

HT-29 colon carcinoma cell line was purchased fromATCC (Manassas, VA) and maintained in McCoy's 5Amodified 1× medium (Sigma, Oakville, Ontario, Canada),supplemented with 2 mmol/L L-glutamine (Gibco, GrandIsland, NY), 10% fetal bovine serum (Multicell, WisentInc., St-Bruno, Quebec, Canada), and antibiotic-antimycoticsolution (Multicell) at 37°C in a 5% CO2-humidifiedincubator.Cell Proliferation Inhibition Assay

Cells (2 × 103/well) in 100 μL of growth medium wereseeded in 96-well cell culture plates and incubated overnightat 37°C. The medium was removed and replaced with a totalvolume of 100 μL growth medium containing indicated con-centrations of ML-133 or metal supplements, or 0.1% DMSOvehicle control, as described in the respective experiments.After incubation of the cells at 37°C for 5 d, cell viabilitywas quantitated with the use of sodium 3’-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) colorimetric assay(Roche Applied Science, Penzberg, Germany). XTT labelingreagent (1 mg/mL) was mixed with electron-coupling reagent,following the manufacturer's instructions, and 50 μL of themixture was added directly to the cells. The plates were furtherincubated at 37°C for 4 h, and the absorbance of each wellwas measured at 490 nm with a multiwell spectrophotometer(Bio-Tek Instruments Inc., Winooski, VT). The data wereadjusted relative to the blank and expressed as a percentageof cell growth compared with the vehicle control.Int race l lu lar Z inc Measurement by Zinquin

Fluorescence Assay

HT-29 cells were harvested by trypsinization, and 8.0 ×106 cells in 1 mL volume of PBS were aliquoted into Eppen-dorf tubes. The cells were incubated at room temperature

for 30 min with indicated concentrations of ML-133 or0.1% DMSO vehicle control, followed by incubation withzinquin (Biotium Inc., Hayward, CA) at 30 μmol/L finalconcentration at room temperature for 30 min before themeasurement of zinquin-Zn2+ complex fluorescence. Thecell suspensions in triplicates were transferred to a 96-wellplate (Corning #3603), and the fluorescence count was mea-sured in a Fluoroskan Ascent luminescence spectrofluorom-eter (Thermo Electron Corporation, Vantaa, Finland) at355 nm excitation and 485 nm emission wavelengths.Cu/Zn Superoxide Dismutase Assay

HT-29 cells were seeded in 6-well dishes (2.5 × 105 cellsper well) and incubated overnight. The culture mediumwas removed and replaced with growth medium containingthe indicated concentrations of ML-133, the copper-specificchelator 2,3,2-tetramine, or 0.1% DMSO vehicle control.After 24 h, cells were lysed in 200 uL of cold lysis buffer(20 mmol/L HEPES, pH 7.2, containing 1 mmol/L EGTA,210 mmol/L mannitol, 70 mmol/L sucrose) and centrifugedat 1, 500 × g for 5 min at 4°C. The supernatant was thencentrifuged at 10,000 × g for 15 min at 4°C to separateCu/Zn superoxide dismutase (SOD; cytosolic SOD) fromMn SOD (mitochondrial SOD). Protein concentration wasdetermined by Bradford assay (Bio-Rad), and samples werediluted to 10 uL with sample buffer from the SOD assay kit(Cayman Chemical, Ann Arbor, MI). Assay was doneaccording to the manufacturer's instructions, and absor-bance was measured at 450 nm. Results are expressed aspercentage SOD activity relative to DMSO control.RNA Preparation and Real-time PCR

Total RNA from HT-29 cells or HT-29 tumor xenograftswas extracted with the use of TRIzol (Invitrogen Life Tech-nologies, Carlsbad, CA) following the manufacturer'sinstructions. First-strand cDNA was synthesized from 200ng total RNA in a Biometra Tpersonal Thermal Cycler(Abgene, Epsom, United Kingdom), with the use of pd(N)6 random hexamer (Amersham Biosciences, Piscataway, NJ)and the SuperScript II Reverse Transcriptase kit (Invitrogen)according to the manufacturer's protocol. Real-time PCRwas done with the ABI Prism 7000 Sequence DetectionSystem (Applied Biosystems Inc., Foster City, CA) withthe use of 5 μL of cDNA synthesized by the abovemen-tioned procedure and with respective human TaqMan GeneExpression Assays (ActB; KLF4, Hs00358836_m1; cyclin D1,Hs00277039_m1; transferrin receptor C, Hs00174609; Sp1,Hs00412720_m1) by following the ABI TaqMan UniversalPCR Master Mix protocol. Alteration in the respective geneexpression was normalized with β-actin gene expressionin the same sample with the use of the comparative cyclethreshold method. Fold changes in the respective genes wereexpressed relative to the corresponding gene level of theindicated control, as described in respective experiments.Cell Cycle Analysis by Flow Cytometry

HT-29 cells (1 × 106) in 10 mL volume of growth mediumwere seeded in 100-mm dishes and incubated overnight at37°C. Cells were treated with the indicated concentrationsof ML-133 or 0.1% DMSO vehicle control, and after 24 h,cells were detached with 0.05% Trypsin-EDTA (Multicell),

1 http://dtp.nci.nih.gov2 http://dtp.nci.nih.gov

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collected by centrifugation at 1,000 g for 4 min, washedonce with PBS, and fixed in 70% ethanol at -20°C for 4 h.The fixed cells were centrifuged at 800 g for 3 min, washedonce with cold PBS containing 2% fetal bovine serum, andtreated with 3 mg/mL ribonuclease (Sigma) and 50 μg/mLpropidium iodide (Sigma) for 30 min at 37°C. The fluores-cence counts of the stained cells were analyzed with theuse of a FACScan flow cytometer and the CellQuest pro-gram (BD Biosciences, San Jose, CA). Data were analyzedwith the use of Modfit software (Verity Software House,Topsham, ME).SDS-PAGE and Western Blot Analysis

Whole cell protein extract was prepared from HT-29 cells(5.5 × 105 cells in 35-mm culture dishes) in lysis buffer(50 mmol/L HEPES, pH 8.0, 0.5% Triton X-100, 150 mmol/LNaCl, 10% glycerol, 2 mmol/L EGTA, 1.5 mmol/L MgCl2). Extracted proteins (10 μg/lane) were resolved on12% SDS-PAGE and transferred to nitrocellulose mem-branes. The following antibodies were used: anti-cyclinD1 rabbit monoclonal antibody (Lab Vision, Fremont,CA; 1:1,000), anti-KLF4 rabbit polyclonal antibody (SantaCruz Biotechnology Inc., Santa Cruz, CA; 1:500), anti-Sp1rabbit polyclonal (Santa Cruz Biotechnology Inc.), andanti–glyceraldehyde-3-phosphate dehydrogenase mousemonoclonal antibody (Biodesign International, Saco, ME;1:10,000), followed by a 1:2,000 dilution of donkey anti-rabbit or 1:20,000 dilution of goat anti-mouse horseradishperoxidase–conjugated secondary antibodies (AmershamBiosciences, Arlington Heights, IL), respectively, and visu-alized with the use of the ECL Plus Western blottingdetection system (Amersham Biosciences).Chromatin Immunoprecipitation

Cell lysates from HT-29 cells grown in three 15-cm cultureplates were prepared at the end of the indicated experi-ments, and chromatin immunoprecipitation assays weredone with the use of anti-Sp1 or anti-KLF4 antibodies (SantaCruz Biotechnology Inc.) and a ChIP-IT kit (Active Motif,Carlsbad, CA) by following the manufacturer's instructions.The primers were synthesized at Invitrogen and encompassthe -231 to -92 region of the cyclin D1 promoter: 5′ primer(5′-CGGACTACAGGGGCAA-3′) and 3′ primer (5′-GCTCCAGGACTTTGCA-3′).Small Interfering RNA Transfection

Predesigned KLF4 siRNA (ID #115492) was from Ambion(Austin, TX), whereas the nonspecific double-stranded RNA[5′r(CUAGGGUAGACGAUGAGAG)d(TT)3′] and [3′d(TT)r(GAUCCCAUCUGCUACUCUC)5′] were synthesized atQiagen (Cambridge, MA) based on the sequence of an un-related gene. HT-29 cells were transfected with indicatedconcentrations of small interfering RNA with the use ofLipofectamine 2000 transfection reagent (Invitrogen) ac-cording to the manufacturer's instructions. At the end ofthe incubation period, the transfection medium was supple-mented with a complete growth medium, and the cells wereincubated at 37°C for 24 h before the indicated experiments.For the measurement of gene expression by real-time PCR,the cells (3 × 105 cells in 35-mm culture dishes) were trans-fected with 100 nmol/L small interfering RNA for 6 h, and

RNA was extracted at the end of 24-h incubation in a com-plete medium. For the cell proliferation experiment, ML-133or DMSO vehicle control was added to the cells (2 × 103

cells per well in 96-well plates) at the end of 24-h incubationin a complete medium, following 6-h transfection with100 nmol/L small interfering RNA. The experiment wasstopped 3 d later.KLF4 Overexpression

HT-29 cells were transfected with either KLF4 expressionplasmid (Origene, Rockville, MD) or empty vector (0.8 μg/1 × 104 cells) with the use of Lipofectamine (Gibco) accordingto the manufacturer's instructions. Twenty-four hours posttransfection, cells were either lysed for Western blot, or trea-ted with 1 μmol/L ML-133 or DMSO for 4 d, followed by cellproliferation inhibition assay (measured by XTT assay).In vivo Antitumor Activity in HT-29 Xenograft Mouse

Model

CD-1 athymic nude mice (four per group) were injecteds.c. with HT-29 cells (3 × 106 cells in 0.1 mL PBS). At 5 d aftertumor cell inoculation, the mice were treated i.p. or p.o. with200 μL vehicle control or 100 mg/kg ML-133 for 5 days, fol-lowed by a 10-d interval, for two cycles (days 5 to 9 as thefirst cycle and days 20 to 24 as the second cycle). The tumorsize was measured with the use of calipers during thecourse of treatment. The mice were sacrificed at 34 dafter tumor cell inoculation. The tumor tissues were excised,frozen immediately, and stored at -80°C until RNA extrac-tion. Gene expression studies in xenografted tumors weredone after 5-d treatment with ML-133.In vitro 4-(2-Pyridylazo)Resorcinol Zinc Binding Assay

ZnCl2 or CuCl2 (final concentration of 10 μmol/L) wasincubated with the indicated concentrations of ML-133,EDTA, EGTA, N,N,N'N'-tetrakis[2-pyridylmethyl]-ethylene-diamine, or 2,3,2-tetramine in a total volume of 100 μL for15 min in 0.2 mol/L Tris-HCl (pH 7.5), followed by theaddition of 4-(2-pyridylazo)resorcinol (Sigma; 100 μmol/Lfinal concentration). The color development of 4-(2-pyridylazo)resorcinol–metal ion complex was measured at500 nm with the use of a multiwell spectrophotometer(Bio-Tek Instruments Inc.; ref. 26).Statistical Analysis

All data in quantitative assays represent the mean ± SDfrom triplicate samples. The data are representatives of atleast three independent experiments, and were analyzedby two-tailed Student's t tests. Differences were consideredstatistically significant at P < 0.05.

ResultsCompound ML-133 Exhibits In vitro and In vivo Tumor

Growth Inhibition

1H-imidazol [4,5-f][1,10] phenanthroline, 2-(2-methyl-1H-indol-3-YL) (ML-133; Fig. 1A) was selected from a library ofnovel compounds based on its potent and selective antipro-liferative effects against particular cancer types (23). ML-133showed consistent growth inhibition of colon, leukemia,non–small cell lung, renal, and prostate cancer cell lines ina screening test of 50 cell lines done at the NCIDevelopmental

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Therapeutic Program, with average growth inhibitionby 50% values of 0.33, 0.75, 0.74, 0.93, and 0.13 μmol/L,respectively (Fig. 1B). The anticancer activity of ML-133was also shown by the NCI hollow fiber assay, a solid tumorefficacy model based on the cell growth of 12 human tumorcell lines encased in biocompatible hollow fibers implantedin mice (25). This method was statistically validated withthe use of a “training set” of standard anticancer compoundsto represent the score achieved by clinically used anticanceragents. ML-133 produced a total growth inhibition score of32 (compounds with a total score of ≥20 are considered

significantly active), also showing a positive cytocidal effect.Furthermore, ML-133 showed in vivo antitumor activity in ahuman colon carcinoma (HT-29) xenograft model (Fig. 1C)when it was given p.o. or i.p. into athymic nude mice, with71% (P = 0.0032) and 69% (P = 0.007) tumor growthinhibition, respectively.ML-133 Preferentially Chelates Labile Zinc In vivoAs with other 1,10-phenanthroline–containing com-

pounds (27), 2-indolyl imidazol [1,10] phenanthroline de-rivatives exhibit metal chelation properties. To assesswhether metal chelation plays a role in ML-133–mediated

Figure 1. Antiproliferative activity of ML-133. A, chemical structure of ML-133. B, NCI antitumor in vitro screening. The cell growth inhibition activityof ML-133 was tested against a panel of 50 cell lines derived from different cancer types as part of the in vitro cell line cancer screen at the NCI. ML-133showed consistent inhibition of non–small cell lung cancer, colon cancer, leukemia, and prostate cancer cell lines. Growth inhibition by 50% (GI50) is thedrug concentration resulting in 50% inhibition of net cell growth. C, in vivo efficacy in a xenograft model of colon carcinoma HT-29 in CD-1 nude mice. CD-1 athymic nude mice harboring HT-29 xenografted human colon tumors (HT-29) were treated with ML-133 p.o. and i.p. at a dose of 100 mg/kg. Data,mean tumor volume ± SE for each treatment group.



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cell growth inhibition, HT-29 cells were incubated with ML-133 in the presence or absence of metal ions (100 μmol/L).ML-133–mediated cell growth inhibition was completelyblocked by Zn2+ or Cu2+ supplementation, partiallyblocked by Fe2+, and was not affected by addition ofFe3+, Mg2+, or Ca2+ (Fig. 2A). However, the results ob-tained from adding supplemental metals to cells merelyindicate whether a metal is capable of blocking the activesite of ML-133 through chelation and do not representa physiologic cellular environment. To assess whetherML-133 affects the endogenous levels of metals in vivo,metal-specific assays were undertaken.

Zinquin, a zinc-specific fluorophore, has been used todetect the intracellular changes in zinc available for cellularreactions (28). ML-133 decreased the fluorescence producedby zinquin-Zn2+ complex formation in a dose-dependentmanner (Fig. 2B), indicating that ML-133 does reduce theconcentration of endogenous intracellular labile zinc inHT-29 cells.

The activity of copper-dependent enzymes is commonlyused to assess the copper status of animal tissues and cells.Copper functions as the active center of the cuproenzyme

Cu/Zn SOD, which protects cells from the effects ofsuperoxide anions (29). Cu/Zn SOD activity was decreasedin a dose-response manner in HT-29 cells treated with thecopper-specific chelator 2,3,2-tetramine. In contrast, HT-29cells treated with ML-133 showed no significant dose-dependent changes in Cu/Zn SOD activity (Fig. 2C), indi-cating that chelation of intracellular copper by ML-133does not occur significantly in vivo.

Expression of the iron-sensitive transferrin receptor 1gene (30) was not significantly altered by ML-133 treatment,in contrast to the iron chelator desferoxamine, whichup-regulated the expression of this gene after 16 hours(Fig. 2D), indicating that chelation of intracellular iron byML-133 does not occur significantly in vivo.

Overall, these results indicate that ML-133–mediatedHT-29 cell growth inhibition is mainly associated withthe reduction of intracellular zinc levels.ML-133–Mediated Cell Cycle Arrest Involves Cyclin D1

Repression

Cell cycle analysis by flow cytometry of HT-29 cells trea-ted with ML-133 showed a dose-dependent increase in thepercentage of cells in the G1 phase of their cell cycle,

Figure 2. Reduction of intracellular zinc levels is associated with ML-133–mediated cell proliferation inhibition of HT-29 carcinoma cells. A, ZnCl2(Zn2+), CuCl2 (Cu2+), FeCl2 (Fe2+), FeCl3 (Fe3+), MgCl2 (Mg2+), or CaCl2 (Ca2+) were added (100 μmol/L) to cells simultaneously with 2.5 μmol/LML-133 or DMSO, and reversal of ML-133–induced cell growth inhibition was measured after 5 d by XTT assay. Results are presented as percentage cellgrowth inhibition relative to cells treated with DMSO and metal supplements B, ML-133 dose-dependent decrease in endogenous intracellular labilezinc pool measured by zinquin assay in HT-29 treated cells C, comparison of intracellular copper levels in HT-29 cells treated with the copper chelator2,3,2-tetramine and ML-133 measured by Cu/Zn SOD activity. Results, percentage of SOD activity relative to DMSO-treated control. D, comparison ofintracellular iron levels in HT-29 cells treated with the iron chelator desferoxamine (DFO) and ML-133, determined by the expression level of the iron-sensitive gene transferrin receptor 1. Fold change in gene expression is relative to 0.1% DMSO vehicle control at the same time point.



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60.8% ± 3.4% and 66.2% ± 4.0% at 1 and 5 μmol/L ML-133concentrations, respectively, compared with 45.1% ± 2.4% incells treated with the vehicle control (0.1% DMSO; Fig. 3A),indicating that ML-133 can arrest HT-29 cells in the G1-Sphase of the cell cycle. Because cyclin D1 is a key regulatorof the G1-S phase progression, we examined its expression inHT-29 cells treated with ML-133. Cyclin D1 protein expres-sion was decreased in a time-dependent manner by treat-ment of HT-29 cells with 1 μmol/L ML-133 (Fig. 3B).Moreover, cyclin D1 gene expression was reduced by ML-133 treatment, and importantly, this effect was partially re-versed by supplementation with 25 μmol/L Zn2+ (Fig. 3C),indicating that the ML-133–mediated reduction in cyclinD1 gene expression is mediated by the reduction of labileintracellular zinc.

ML-133–Mediated Up-Regulation of the KLF4

Transcription Factor

Studies of zinc-regulated gene expression in HT-29 coloncarcinoma cells indicate that KLF4 shows the mostpronounced change in expression among other zincfinger–containing transcription factors under reduced zinclevel conditions (6). Because KLF4 is known to inhibit cellproliferation by blocking G1-S progression of the cell cyclethrough transcriptional repression of cyclin D1 (7, 31), weaddressed the question of whether KLF4 is involved in thecell growth inhibition mechanism of ML-133. Increasedexpression of the KLF4 gene was detected in HT-29 cellsafter 4-hour treatment with ML-133, with a peak at 16 hours(Fig. 4A), and this effect was partially reversed upon zincsupplementation (Fig. 4B), which supports the role ofML-133–mediated zinc level reduction in the induction ofKLF4 gene expression. KLF4 protein was also increasedafter treatment of HT-29 cells with ML-133 for 16 hours(Fig. 4C). Moreover, ML-133 induced KLF4 gene expressionin various cancer cell types, including colon, lung, prostate,breast, leukemia, and melanoma (Table 1).

KLF4 has been shown to repress the constitutive expres-sion of the cyclin D1 gene through competition with theactivator SP1 for binding to transcriptional control se-quences in the cyclin D1 promoter (31). Therefore, we exam-ined whether this mechanism was involved in therepression of cyclin D1 by ML-133 in HT-29 colon cancercells with the use of the chromatin immunoprecipitation as-say. ML-133 treatment produced increased binding of KLF4to the cyclin D1 promoter and displacement of SP1, asshown by decreased SP1 binding (Fig. 4D), indicating thatinduction of KLF4 by ML-133 represses Sp1-dependentconstitutive cyclin D1 transcription in vivo. ML-133 had nosignificant effect on the mRNA or protein expression ofSP1 (Supplementary Fig. S1A and B). These results providea molecular link between reduction of intracellular zinclevels, cyclin D1 down-regulation, and cell growth inhibi-tion produced by ML-133.KLF4 Up-Regulation Contributes to ML-133–Mediated

Growth Inhibition

In an effort to evaluate the biological significance of KLF4up-regulation by ML-133, we transiently transfected HT-29cells with a KLF4 expression vector (Fig. 5A) and examinedthe impact of KLF4 overexpression on ML-133–mediated cellgrowth inhibition (Fig. 5B). KLF4-expressing cells weregrowth inhibited relative to vector-transfected cells in theabsence of ML-133 (P < 0.05). Importantly, the effect of ML-133 on growth inhibition was enhanced as a result of KLF4overexpression (P = 0.01). As a flip side to the overexpressionof KLF4, we examined the effect of decreased KLF4 expres-sion with the use of KLF4-targeted small interfering RNA(Fig. 5C). In the absence of ML-133, cell growth was not sig-nificantly altered by KLF4 knockdown, probably due to therelatively low basal level of KLF4 in the cells. Importantly,ML-133–mediated cell growth inhibition was significantlymuted as a result of KLF4 knockdown (P = 0.01; Fig. 5D).Together, these results support the role of KLF4 in mediatingthe effect of ML-133 on the growth inhibition of cancer cells.

Figure 3. ML-133–mediated cell cycle arrest involves cyclin D1 re-pression. A, HT-29 cells were treated for 24 h with the indicated con-centrations of ML-133; a dose-dependent G1-S phase cell cycle arrestwas observed as measured by flow cytometry (*, P < 0.05 at 1 and5 μmol/L ML-133 compared with control). B, decreased protein expres-sion of cyclin D1 detected by Western blotting at the indicated timepoints post treatment with 1 μmol/L ML-133. Densitometric analysiswas done to compare cyclin D1 protein levels to glyceraldehyde-3-phosphate dehydrogenase protein levels to normalize the results. Theseresults are representative of three replicate experiments. C, decreasedgene expression of cyclin D1 determined by real-time PCR after treatmentwith 1 μmol/L ML-133 for 16 h and 25 μmol/L ZnCl2. (*, P < 0.05versus ML-133).



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To validate the proposed molecular mechanism for ML-133–mediated cell growth inhibition in vivo, the levels ofgene expression of KLF4 and cyclin D1 were determinedby real-time PCR in HT-29 tumors. These studies showed aconsistent increase in KLF4 gene expression anddecreased cyclin D1 expression in tumors grown in ML-133–treated CD-1 nude mice compared with tumors frommice treated with the vehicle control (Fig. 5E). Taken together,these results indicate that ML-133 treatment reducescyclin D1 expression through zinc-dependent up-regulationof the transcription repressor KLF4, ultimately leading to cellcycle arrest.

DiscussionThe present study shows the anticancer properties of thenovel small molecule ML-133. We have shown that chelationof labile intracellular zinc is a key factor in the molecularevents that lead to cell growth inhibition. The concentrationof zinc in cells is controlled through a complex zinc homeo-static system. Total cellular zinc consists of a large pool oftightly bound zinc, and a small but measurable pool of“free” zinc ions involved in regulatory functions. At leastthree factors control “free” zinc and the amplitudes of itsfluctuations: total zinc, zinc buffering, and redox bufferingcapacity (32). Zinc buffering is determined by changes in

Table 1. Effects of ML-133 on growth of cancer cell lines and KLF4 gene expression

Cell line Type of cancer Growth inhibition IC50 (μmol/L) KLF4 induction (fold increase)

HT-29 Colon 0.71 6.5HCT-116 Colon 0.20 5.9H-460 Lung 2.90 3.4DU-145 Prostate 0.35 9.2PC-3 Prostate 0.30 2.3MDA-MB-231 Breast 0.50 1.5MDA-MB-435 Breast 0.35 9.1MOLT-4 Leukemia 0.42 >10*CCRF-CEM Leukemia 0.15 >10*SK-MEL-2 Melanoma 0.20 2.4

NOTE: Growth inhibition (IC50 values in μmol/L) and KLF4 induction after 16-h treatment (gene expression measured by real-time PCR relative to DMSO-treated control) in various cancer cell lines treated with ML-133.*KLF4 induction cannot be accurately determined due to the low basal levels of KLF4 in these cell lines.

Figure 4. ML-133–mediated up-regulation of the KLF4 transcription factor.A, treatment of HT-29 cells with 1 μmol/L ML-133 produced a time-dependentincrease in the gene expression of KLF4. B, KLF4 gene expression induced by treatment with 1 μmol/L ML-133 for 16 h was partially reversed in thepresence of 25 μmol/L ZnCl2. (*, P < 0.05 versus ML-133). C, increased protein expression of KLF4 was detected by Western blotting after treatmentwith the indicated concentrations of ML-133 for16 h compared with 0.01% DMSO vehicle control. D, transcription factor binding activity determined bychromatin immunoprecipitation assay showed increased KLF4 binding and decreased Sp1 binding to the cyclin D1 promoter (-231 to -92) upon ML-133treatment.



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the metallothionein-to-thionein ratio (33). In cells, metal-lothionein is a dynamic protein with species constantlychanging due to Zn(II) transfer to apo-metalloproteins andre-equilibration when thionein expression is induced.

We propose that ML-133 chelates zinc from the labile poolof zinc, mainly from MTF1 and metallothionein. In agree-

ment, after inducing the synthesis of metallothionein-Zn2+

in TE671 cells with zinc, the addition of 25 μmol/L of thezinc chelator TPEN for 30 minutes markedly reduced thezinc content of the metallothionein pool without clearlyaffecting the high–molecular weight zinc pool, which in-cludes the majority of zinc-containing metalloproteins. This

Figure 5. KLF4 up-regulation contributes to ML-133–mediated growth inhibition. A, Western blot of KLF4 expression in vector-transfected and KLF4-transfected HT-29 cells. B, effect of KLF4 overexpression on ML-133–mediated cell growth inhibition of HT-29 cells as detected by XTTassay. The resultsare from three separate experiments (*, P < 0.05, statistically significant versus DMSO-treated vector-transfected cells; **, P = 0.01, statistically sig-nificant versus ML-133-treated vector-transfected cells). C, KLF4 gene expression after transfection of HT-29 cells with KLF4-targeted small interferingRNA (siRNA) or nonspecific control siRNA treated with 1 μmol/L ML-133 or DMSO, determined by real-time PCR analysis. D,ML-133–mediated inhibitionof cell proliferation is impaired in HT-29 cells transfected with KLF4-targeted siRNA but not with nonspecific control siRNA (*, P = 0.01). E, KLF4 andcyclin D1 gene expression determined by real-time PCR analysis in individual HT-29 tumors grown in athymic CD-1 nude mice treated with ML-133 for 5 d(i.p. administration of 100 mg/kg ML-133). Gene expression is represented as fold change relative to the average expression in tumors obtained from fourcontrol mice injected with vehicle control.



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suggests that metallothionein-Zn2+ is particularly labilecompared with the inertness of the high– molecular weightzinc pool (34). ML-133 is able to chelate Zn2+ in vitro (with asimilar affinity to EGTA), as shown by its ability to impairthe formation of a colored 4-(2-pyridylazo)resorcinol–Zn2+

complex (Supplementary Fig. S2A). However, ML-133 che-lates zinc with a much lower affinity than TPEN, indicatingthat ML-133 is unlikely to access the pool of tightly boundzinc. A zinc-chelating drug, such as ML-133, may interferewith cellular zinc buffering, leading to perturbation of zinchomeostasis.

Exogenously added copper blocks ML-133–mediated cellgrowth inhibition (Fig. 2A), and ML-133 is able to chelatecopper in vitro (with a similar affinity to EGTA), as shownby its ability to impair the formation of a colored 4-(2-pyri-dylazo)resorcinol –Cu2+ complex (Supplementary Fig. S2B).However, we do not believe that ML-133 is a chelator ofcopper in vivo. The results obtained from adding supple-mental metals to cells merely indicate whether a metal iscapable of blocking the active site of ML-133 through che-lation and do not represent a physiologic cellular environ-ment. An excess of copper likely blocks the growthinhibitory activity of ML-133 by preventing ML-133 fromaccessing the labile zinc within the cell. In agreement,ML-133 had no effect on copper status in vivo, as assessedby Cu/Zn SOD activity (Fig. 2C). The activity of copper-dependent enzymes, such as Cu/Zn SOD, is commonlyused to assess the copper status of animal tissues and cells.Copper is an essential, but potentially reactive and toxicion, and the free ionic copper concentration is extremelylow in cells, estimated at <1 ion per cell (35). Chaperoneproteins safeguard copper ions and make them availablefor incorporation into specific cuproenzymes (36). In con-trast to ML-133, the copper-specific chelator 2,3,2-tetramineimpaired Cu/Zn SOD activity in a dose-dependent man-ner, correlating with its higher affinity for copper in vitro(Supplementary Fig. S2B). ML-133 does not have a highenough affinity for copper to access this tightly boundmetal in vivo.

In this report, we provide evidence that KLF4 is criticalin mediating the effect of ML-133 on growth inhibition ofcancer cells. KLF4 is a stress-associated and differentia-tion-associated inhibitor of proliferation (37) with tumor-suppressive functions (18, 38, 39). Indeed, loss of KLF4expression is a frequent occurrence in various human can-cers (14, 15, 18, 40–42). We show here that KLF4 is signifi-cantly up-regulated in response to ML-133 in HT-29 cells(Fig. 4A), in multiple cancer cell types (Table 1), as well asin HT-29 xenograft tumor samples (Fig. 5E). Additionally,the growth inhibitory effect of ML-133 is enhanced whenKLF4 is overexpressed (Fig. 5B) and suppressed whenKLF4 is knocked down in HT-29 cells (Fig. 5D).

In response to ML-133, we have shown that KLF4 func-tions as a negative regulator of cyclin D1 through competi-tion with Sp1 at Sp1-binding motifs on the cyclin D1promoter (Fig. 4D). Competition between KLF4 and Sp1at the cyclin D1 promoter has already been established(31), and indeed, this seems to be a common mechanism

as KLF4 has been shown to repress transcription of othergenes through competition with Sp1, including histidine de-carboxylase (43), Cyp1A1 (44), and ornithine decarboxylase(45). In addition to direct competition with Sp1 for bindingto promoters, Ai et al. (43) have proposed that KLF4 couldmediate transcriptional repression through several addi-tional mechanisms. First, physical interaction between Sp1and KLF4 has been shown (44), and this interaction mightdisrupt the recruitment of the transcriptional coactivatorcomplex, resulting in transcriptional inhibition. Second,KLF4 might also interact directly with coactivator com-plexes, leading to failure of the recruitment of the complexesto the Sp1 binding site or to the inhibition of the activity ofthe coactivator complexes. However, all these possibilitiesare not mutually exclusive (43).

KLF4 is also known to act as a transcriptional repressorof other cell cycle promoters as well as a transcriptionalactivator of several genes encoding inhibitors of the cellcycle (46). Further studies are required to identify otherML-133–responsive targets of KLF4 and their role inML-133–mediated cell growth inhibition. Overall, theseresults suggest that KLF4 is a molecular link between in-tracellular zinc depletion by ML-133, cyclin D1 down-regulation, G1-S phase arrest, and cell growth inhibition.

Further studies are also required to determine the mech-anism by which the expression of KLF4 is regulated inresponse to zinc depletion. An obvious candidate for reg-ulation of KLF4 expression is the zinc-sensitive transcrip-tion factor MTF1. Indeed, in HT-29 cells transfected withMTF1 small interfering RNA, the ML-133–mediated induc-tion of KLF4 expression is partially blocked (data notshown), indicating that MTF1 is involved in the regulationof KLF4 expression. MTF1 is a cellular zinc sensor that co-ordinates the expression of genes involved in zinc homeo-stasis, including metallothionein. MTF1 DNA-bindingactivity, nuclear translocation, and occupancy of metalresponse elements in the promoter regions of genes are re-sponsive to intracellular zinc concentration (47). However,it is still unclear how genes are regulated in conditions oflow zinc availability, as MTF1 is transcriptionally active inconditions of high zinc availability. Importantly, MTF1 in-teracts or cooperates with a diverse set of factors, includ-ing NF-κB, hypoxia-inducible factor 1α, USF, SP1, heatshock transcription factor 1, and ribosomal protein S1, inaddition to forming a coactivator complex with p300/CBP and SP1 in a zinc-dependent manner (48). These inter-actions may be altered by ML-133–mediated changes in la-bile zinc concentrations affecting the transcription of targetgenes, including KLF4. Moreover, heat shock transcriptionfactor 1, a known transcriptional regulator of KLF4 in re-sponse to heat stress (49), can be negatively regulated byforming a complex with MTF1 (50). Interestingly, we haveshown with the use of a second-generation derivative ofML-133 that heat shock transcription factor 1 had the high-est activation level among 50 transcription factors tested inHT-29 colon cancer cells (data not shown), suggesting thatheat shock transcription factor 1 may also be involved inthe regulation of KLF4.



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Modulation of intracellular zinc homeostasis by zinc-specific chelators represents a potential new strategy forthe treatment of certain types of cancer. We have shownthat ML-133 is a chelator of zinc and that it potently inhi-bits multiple cell types with growth inhibition by 50%values in the nanomolar range as determined by the in vitrocancer cell line screen of the NCI. Moreover, ML-133efficiently impairs tumor growth in a HT-29 colon tumorxenograph mouse model. In conclusion, we have identifieda new zinc chelator, ML-133, as a potential anticancer thera-peutic drug.

Disclosure of Potential Conflicts of Interest

All authors are current or former employees of Lorus Therapeutics, Inc.No other potential conflicts of interest were disclosed.

Acknowledgments

We thank Tracy Wong, Michelle Liu, Stefanie Lau, Jason DeMelo, andCindy Liu for their excellent technical assistance.

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2009;8:2586-2596. Published OnlineFirst September 15, 2009.Mol Cancer Ther Mario Huesca, Lisa S. Lock, Aye Aye Khine, et al. homeostasisinhibits cell growth by modulating intracellular labile zinc A novel small molecule with potent anticancer activity

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