A novel small molecule with potent anticancer activity inhibits cell growth by modulating intracellular labile zinc homeostasis

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Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104
                 Published Online First on September 15, 2009 as 10.1158/1535-7163.MCT-08-1104

2586

       A novel small molecule with potent anticancer activity
       inhibits cell growth by modulating intracellular
       labile zinc homeostasis

       Mario Huesca, Lisa S. Lock, Aye Aye Khine,                                 in the storage and distribution of intracellular zinc (2).
       Stéphane Viau, Robert Peralta, I. Howard Cukier,                           Although labile zinc has been shown to be involved in sev-
       Hongnan Jin, Raed A. Al-Qawasmeh, Yoon Lee,                                eral cellular pathways related to the regulation of cell fate,
       Jim Wright, and Aiping Young                                               these mechanisms are not well characterized (3). Reduction
                                                                                  of intracellular labile zinc has been associated with the
       Lorus Therapeutics Inc., Toronto, Ontario, Canada                          induction 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-
       Abstract                                                                   lon adenocarcinoma (6), and lymphocytic leukemia (7–10).
       ML-133 is a novel small molecule with potent antiprolifera-                   Studies of zinc-responsive gene regulation induced by in-
       tive activity, as shown in cancer cell lines and in a human                tracellular labile zinc depletion in colon carcinoma HT-29
       colon tumor xenograft model. ML-133 reduces the con-
                                                                                  cells identified Krüppel-like factor 4 (KLF4, also known as
       centration of intracellular labile zinc in HT-29 colon cancer
                                                                                  GKLF) as one of the genes whose expression is most signif-
       cells, leading to induction of the Krüppel-like factor 4 tran-
                                                                                  icantly changed (up-regulated) among >10,000 target genes
       scription factor. Krüppel-like factor 4 displaces the positive
                                                                                  tested (6). KLFs are members of the SP/XKLF family of
       regulator SP1 from the cyclin D1 promoter, thereby nega-
                                                                                  transcription factors defined by an amino acid binding do-
       tively regulating the expression of cyclin D1 and promoting
                                                                                  main at the C termini that comprises three C2H2-type zinc
       the G1-S phase arrest of cell proliferation. The antiproli-
                                                                                  fingers with similarity to the developmental gene Krüppel
       ferative and antitumor activity of ML-133 described in
                                                                                  of Drosophila melanogaster (11). KLFs play an important role
       the present study suggests modulation of intracellular zinc
                                                                                  in mammalian morphogenesis by controlling the prolifera-
       homeostasis as a potential strategy for the treatment of
                                                                                  tion and/or differentiation of distinct cell lineages (12). The
       several cancer types, and ML-133 represents a promising
                                                                                  expression and function of KLFs are relatively tissue re-
       new class of antitumor agents that deserves further devel-
                                                                                  stricted (11), with KLF4 mainly expressed in epithelial cells
       opment. [Mol Cancer Ther 2009;8(9):2586–96]
                                                                                  of the gastrointestinal tract, lung, testis, and skin, with a
                                                                                  functional role in skin barrier and gastric epithelial homeo-
       Introduction                                                               stasis (13), and development (14).
       Zinc has regulatory and structural functions in a large num-                  KLF4 mRNA is significantly reduced in colorectal cancer
       ber of enzymes and transcription factors. The structural                   compared with normal matched tissues (15), and induction
       functions involve a highly stable association of zinc to                   of KLF4 expression in a colorectal cancer cell line results in
       folded protein domains, whereas a more dynamic, ex-                        diminished tumorigenicity (16). Furthermore, overexpression
       changeable labile zinc pool is involved in the regulatory                  of KLF4 causes cell cycle arrest at G1-S transition in RKO hu-
       functions (1). Intracellular zinc homeostasis is regulated by              man colon carcinoma cells (7). In addition, KLF4 is down-
       sensor proteins, such as the metal-responsive transcription                regulated in adenomas from the APCmin+/- mouse model of
       factor 1, which regulates the transcription of zinc-sensitive              colorectal cancer, and crossing APCmin+/- mice with KLF4+/-
       genes, including membrane transporter proteins, involved                   heterozygotes resulted in significantly more adenomas than
       in the cellular and vesicular influx and efflux of zinc, and               in APCmin+/- mice alone (17). Taken together, these results
       metallothionein and thionein, which play an important role                 indicate a role of KLF4 as tumor suppressor factor in colon
                                                                                  cancer. A similar function for KLF4 has also been reported
                                                                                  in bladder cancer (18), gastric cancer (19), esophageal cancer
       Received 11/20/08; revised 6/29/09; accepted 6/29/09; published            (20), pancreatic cancer (21), and adult T-cell leukemia (22).
       OnlineFirst 9/15/09.                                                          Here we present the characterization of the anticancer
       Note: Supplementary material for this article is available at Molecular    activity of the compound ML-133, selected from a novel series
       Cancer Therapeutics Online (http://mct.aacrjournals.org/).
                                                                                  of 2-indolyl imidazol [4,5-d] phenanthroline derivatives with
       Current address for A. Khine: NOVX Systems Canada Inc., Markham L3R
       6G3, Canada; S. Viau: Trillium Therapeutics Inc., Toronto M9W 4Y9,         metal chelation activity that exhibits a potent and selective
       Canada; and for R.A. Al-Qawasmeh: University of Jordan, Amman 11942,       antitumor activity against multiple cancer cell types (23).
       Jordan.
                                                                                  ML-133 reduces the concentration of intracellular labile zinc
       Requests for reprints: Mario Huesca, Lorus Therapeutics Inc., 2 Meridian
       Road, Toronto, Ontario M9W 4Z7 Canada. Phone: 416-798-1200, ext.           in HT-29 colon cancer cells, leading to the induction of KLF4
       311; Fax: 416-798-2200. E-mail: mhuesca@lorusthera.com                     expression. KLF4 displaces the positive regulator SP1 from the
       Copyright © 2009 American Association for Cancer Research.                 cyclin D1 promoter, thereby negatively regulating the expres-
       doi:10.1158/1535-7163.MCT-08-1104                                          sion of cyclin D1 and promoting the arrest of cell proliferation.

                                                                                                     Mol Cancer Ther 2009;8(9). September 2009

                   Downloaded from mct.aacrjournals.org on January 15, 2021. © 2009 American Association for Cancer
                                                              Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

                                                                                                    Molecular Cancer Therapeutics    2587

Materials and Methods                                                for 30 min with indicated concentrations of ML-133 or
   Chemical Synthesis                                                0.1% DMSO vehicle control, followed by incubation with
   ML-133 was synthesized as described elsewhere (23).               zinquin (Biotium Inc., Hayward, CA) at 30 μmol/L final
   In vitro Cell Line Cancer Screen                                  concentration at room temperature for 30 min before the
   To evaluate the potential antitumor activity of ML-133            measurement of zinquin-Zn2+ complex fluorescence. The
and to prioritize the selective activity on particular types         cell suspensions in triplicates were transferred to a 96-well
of tumor cell lines, the antiproliferative activity of ML-133        plate (Corning #3603), and the fluorescence count was mea-
was tested by the in vitro cell-line cancer screen at the Na-        sured in a Fluoroskan Ascent luminescence spectrofluorom-
tional Cancer Institute (NCI; ref. 24). The detailed method is       eter (Thermo Electron Corporation, Vantaa, Finland) at
described at the NCI Developmental Therapeutics Program              355 nm excitation and 485 nm emission wavelengths.
website.1                                                               Cu/Zn Superoxide Dismutase Assay
   In vivo Hollow Fiber Assay                                           HT-29 cells were seeded in 6-well dishes (2.5 × 105 cells
   To assess the initial drug efficacy of ML-133 in vivo, the        per well) and incubated overnight. The culture medium
activity of ML-133 was tested by NCI hollow fiber assay              was removed and replaced with growth medium containing
(25) on a panel of 12 tumor cell lines; (breast: MDA-MB-             the indicated concentrations of ML-133, the copper-specific
231, MDA-MB-435; glioma: U251, SF-295; ovarian: OV-                  chelator 2,3,2-tetramine, or 0.1% DMSO vehicle control.
CAR-3, OVCAR-5; colon: COLO-205, SW-620; melanoma:                   After 24 h, cells were lysed in 200 uL of cold lysis buffer
LOX-IMVI, UACC-62; and lung: NCI-H23, NCI-H522).                     (20 mmol/L HEPES, pH 7.2, containing 1 mmol/L EGTA,
The detailed method is described at the NCI Developmental            210 mmol/L mannitol, 70 mmol/L sucrose) and centrifuged
Therapeutics Program website.2                                       at 1, 500 × g for 5 min at 4°C. The supernatant was then
   Cell Culture Maintenance                                          centrifuged at 10,000 × g for 15 min at 4°C to separate
   HT-29 colon carcinoma cell line was purchased from                Cu/Zn superoxide dismutase (SOD; cytosolic SOD) from
ATCC (Manassas, VA) and maintained in McCoy's 5A                     Mn SOD (mitochondrial SOD). Protein concentration was
modified 1× medium (Sigma, Oakville, Ontario, Canada),               determined by Bradford assay (Bio-Rad), and samples were
supplemented with 2 mmol/L L-glutamine (Gibco, Grand                 diluted to 10 uL with sample buffer from the SOD assay kit
Island, NY), 10% fetal bovine serum (Multicell, Wisent               (Cayman Chemical, Ann Arbor, MI). Assay was done
Inc., St-Bruno, Quebec, Canada), and antibiotic-antimycotic          according to the manufacturer's instructions, and absor-
solution (Multicell) at 37°C in a 5% CO 2 -humidified                bance was measured at 450 nm. Results are expressed as
incubator.                                                           percentage SOD activity relative to DMSO control.
   Cell Proliferation Inhibition Assay                                  RNA Preparation and Real-time PCR
   Cells (2 × 103/well) in 100 μL of growth medium were                 Total RNA from HT-29 cells or HT-29 tumor xenografts
seeded in 96-well cell culture plates and incubated overnight        was extracted with the use of TRIzol (Invitrogen Life Tech-
at 37°C. The medium was removed and replaced with a total            nologies, Carlsbad, CA) following the manufacturer's
volume of 100 μL growth medium containing indicated con-             instructions. First-strand cDNA was synthesized from 200
centrations of ML-133 or metal supplements, or 0.1% DMSO             ng total RNA in a Biometra Tpersonal Thermal Cycler
vehicle control, as described in the respective experiments.         (Abgene, Epsom, United Kingdom), with the use of pd(N)
After incubation of the cells at 37°C for 5 d, cell viability        6 random hexamer (Amersham Biosciences, Piscataway, NJ)
was quantitated with the use of sodium 3’-[1-(phenyl-                and the SuperScript II Reverse Transcriptase kit (Invitrogen)
amino-carbonyl)-3,4-tetrazolium}-bis (4-methoxy-6-nitro)             according to the manufacturer's protocol. Real-time PCR
benzene sulfonic acid hydrate (XTT) colorimetric assay               was done with the ABI Prism 7000 Sequence Detection
(Roche Applied Science, Penzberg, Germany). XTT labeling             System (Applied Biosystems Inc., Foster City, CA) with
                                                                     the use of 5 μL of cDNA synthesized by the abovemen-
reagent (1 mg/mL) was mixed with electron-coupling reagent,
                                                                     tioned procedure and with respective human TaqMan Gene
following the manufacturer's instructions, and 50 μL of the
                                                                     Expression Assays (ActB; KLF4, Hs00358836_m1; cyclin D1,
mixture was added directly to the cells. The plates were further
                                                                     Hs00277039_m1; transferrin receptor C, Hs00174609; Sp1,
incubated at 37°C for 4 h, and the absorbance of each well
                                                                     Hs00412720_m1) by following the ABI TaqMan Universal
was measured at 490 nm with a multiwell spectrophotometer
                                                                     PCR Master Mix protocol. Alteration in the respective gene
(Bio-Tek Instruments Inc., Winooski, VT). The data were
                                                                     expression was normalized with β-actin gene expression
adjusted relative to the blank and expressed as a percentage
                                                                     in the same sample with the use of the comparative cycle
of cell growth compared with the vehicle control.
                                                                     threshold method. Fold changes in the respective genes were
   Intracellular Zinc Measurement by Zinquin
                                                                     expressed relative to the corresponding gene level of the
Fluorescence Assay
                                                                     indicated control, as described in respective experiments.
   HT-29 cells were harvested by trypsinization, and 8.0 ×
                                                                        Cell Cycle Analysis by Flow Cytometry
106 cells in 1 mL volume of PBS were aliquoted into Eppen-
                                                                        HT-29 cells (1 × 106) in 10 mL volume of growth medium
dorf tubes. The cells were incubated at room temperature
                                                                     were seeded in 100-mm dishes and incubated overnight at
                                                                     37°C. Cells were treated with the indicated concentrations
1
    http://dtp.nci.nih.gov                                           of ML-133 or 0.1% DMSO vehicle control, and after 24 h,
2
    http://dtp.nci.nih.gov                                           cells were detached with 0.05% Trypsin-EDTA (Multicell),

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2588 Anticancer Activity of ML-133

       collected by centrifugation at 1,000 g for 4 min, washed        RNA was extracted at the end of 24-h incubation in a com-
       once with PBS, and fixed in 70% ethanol at -20°C for 4 h.       plete medium. For the cell proliferation experiment, ML-133
       The fixed cells were centrifuged at 800 g for 3 min, washed     or DMSO vehicle control was added to the cells (2 × 103
       once with cold PBS containing 2% fetal bovine serum, and        cells per well in 96-well plates) at the end of 24-h incubation
       treated with 3 mg/mL ribonuclease (Sigma) and 50 μg/mL          in a complete medium, following 6-h transfection with
       propidium iodide (Sigma) for 30 min at 37°C. The fluores-       100 nmol/L small interfering RNA. The experiment was
       cence counts of the stained cells were analyzed with the        stopped 3 d later.
       use of a FACScan flow cytometer and the CellQuest pro-             KLF4 Overexpression
       gram (BD Biosciences, San Jose, CA). Data were analyzed            HT-29 cells were transfected with either KLF4 expression
       with the use of Modfit software (Verity Software House,         plasmid (Origene, Rockville, MD) or empty vector (0.8 μg/
       Topsham, ME).                                                   1 × 104 cells) with the use of Lipofectamine (Gibco) according
          SDS-PAGE and Western Blot Analysis                           to the manufacturer's instructions. Twenty-four hours post
          Whole cell protein extract was prepared from HT-29 cells     transfection, cells were either lysed for Western blot, or trea-
       (5.5 × 10 5 cells in 35-mm culture dishes) in lysis buffer      ted with 1 μmol/L ML-133 or DMSO for 4 d, followed by cell
       (50 mmol/L HEPES, pH 8.0, 0.5% Triton X-100, 150 mmol/L         proliferation inhibition assay (measured by XTT assay).
       NaCl, 10% glycerol, 2 mmol/L EGTA, 1.5 mmol/L Mg                   In vivo Antitumor Activity in HT-29 Xenograft Mouse
       Cl2). Extracted proteins (10 μg/lane) were resolved on          Model
       12% SDS-PAGE and transferred to nitrocellulose mem-                CD-1 athymic nude mice (four per group) were injected
       branes. The following antibodies were used: anti-cyclin         s.c. with HT-29 cells (3 × 106 cells in 0.1 mL PBS). At 5 d after
       D1 rabbit monoclonal antibody (Lab Vision, Fremont,             tumor cell inoculation, the mice were treated i.p. or p.o. with
       CA; 1:1,000), anti-KLF4 rabbit polyclonal antibody (Santa       200 μL vehicle control or 100 mg/kg ML-133 for 5 days, fol-
       Cruz Biotechnology Inc., Santa Cruz, CA; 1:500), anti-Sp1       lowed by a 10-d interval, for two cycles (days 5 to 9 as the
       rabbit polyclonal (Santa Cruz Biotechnology Inc.), and          first cycle and days 20 to 24 as the second cycle). The tumor
       anti–glyceraldehyde-3-phosphate dehydrogenase mouse             size was measured with the use of calipers during the
       monoclonal antibody (Biodesign International, Saco, ME;         course of treatment. The mice were sacrificed at 34 d
       1:10,000), followed by a 1:2,000 dilution of donkey anti-       after tumor cell inoculation. The tumor tissues were excised,
       rabbit or 1:20,000 dilution of goat anti-mouse horseradish      frozen immediately, and stored at -80°C until RNA extrac-
       peroxidase–conjugated secondary antibodies (Amersham            tion. Gene expression studies in xenografted tumors were
       Biosciences, Arlington Heights, IL), respectively, and visu-    done after 5-d treatment with ML-133.
       alized with the use of the ECL Plus Western blotting               In vitro 4-(2-Pyridylazo)Resorcinol Zinc Binding Assay
       detection system (Amersham Biosciences).                           ZnCl2 or CuCl2 (final concentration of 10 μmol/L) was
          Chromatin Immunoprecipitation                                incubated with the indicated concentrations of ML-133,
          Cell lysates from HT-29 cells grown in three 15-cm culture   EDTA, EGTA, N,N,N'N'-tetrakis[2-pyridylmethyl]-ethylene-
       plates were prepared at the end of the indicated experi-        diamine, or 2,3,2-tetramine in a total volume of 100 μL for
       ments, and chromatin immunoprecipitation assays were            15 min in 0.2 mol/L Tris-HCl (pH 7.5), followed by the
       done with the use of anti-Sp1 or anti-KLF4 antibodies (Santa    addition of 4-(2-pyridylazo)resorcinol (Sigma; 100 μmol/L
       Cruz Biotechnology Inc.) and a ChIP-IT kit (Active Motif,       final concentration). The color development of 4-(2-
       Carlsbad, CA) by following the manufacturer's instructions.     pyridylazo)resorcinol–metal ion complex was measured at
       The primers were synthesized at Invitrogen and encompass        500 nm with the use of a multiwell spectrophotometer
       the -231 to -92 region of the cyclin D1 promoter: 5′ primer     (Bio-Tek Instruments Inc.; ref. 26).
       (5′-CGGACTACAGGGGCAA-3′) and 3′ primer (5′-                        Statistical Analysis
       GCTCCAGGACTTTGCA-3′).                                              All data in quantitative assays represent the mean ± SD
          Small Interfering RNA Transfection                           from triplicate samples. The data are representatives of at
          Predesigned KLF4 siRNA (ID #115492) was from Ambion          least three independent experiments, and were analyzed
       (Austin, TX), whereas the nonspecific double-stranded RNA       by two-tailed Student's t tests. Differences were considered
       [5′r(CUAGGGUAGACGAUGAGAG)d(TT)3′] and [3′d(TT)                  statistically significant at P < 0.05.
       r(GAUCCCAUCUGCUACUCUC)5′] were synthesized at
       Qiagen (Cambridge, MA) based on the sequence of an un-
       related gene. HT-29 cells were transfected with indicated       Results
       concentrations of small interfering RNA with the use of            Compound ML-133 Exhibits In vitro and In vivo Tumor
       Lipofectamine 2000 transfection reagent (Invitrogen) ac-        Growth Inhibition
       cording to the manufacturer's instructions. At the end of          1H-imidazol [4,5-f][1,10] phenanthroline, 2-(2-methyl-1H-
       the incubation period, the transfection medium was supple-      indol-3-YL) (ML-133; Fig. 1A) was selected from a library of
       mented with a complete growth medium, and the cells were        novel compounds based on its potent and selective antipro-
       incubated at 37°C for 24 h before the indicated experiments.    liferative effects against particular cancer types (23). ML-133
       For the measurement of gene expression by real-time PCR,        showed consistent growth inhibition of colon, leukemia,
       the cells (3 × 105 cells in 35-mm culture dishes) were trans-   non–small cell lung, renal, and prostate cancer cell lines in
       fected with 100 nmol/L small interfering RNA for 6 h, and       a screening test of 50 cell lines done at the NCI Developmental

                                                                                          Mol Cancer Ther 2009;8(9). September 2009

                 Downloaded from mct.aacrjournals.org on January 15, 2021. © 2009 American Association for Cancer
                                                            Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

                                                                                                                       Molecular Cancer Therapeutics           2589

Figure 1. Antiproliferative activity of ML-133. A, chemical structure of ML-133. B, NCI antitumor in vitro screening. The cell growth inhibition activity
of 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-133
showed consistent inhibition of non–small cell lung cancer, colon cancer, leukemia, and prostate cancer cell lines. Growth inhibition by 50% (GI50) is the
drug 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.

Therapeutic Program, with average growth inhibition                             significantly active), also showing a positive cytocidal effect.
by 50% values of 0.33, 0.75, 0.74, 0.93, and 0.13 μmol/L,                       Furthermore, ML-133 showed in vivo antitumor activity in a
respectively (Fig. 1B). The anticancer activity of ML-133                       human colon carcinoma (HT-29) xenograft model (Fig. 1C)
was also shown by the NCI hollow fiber assay, a solid tumor                     when it was given p.o. or i.p. into athymic nude mice, with
efficacy model based on the cell growth of 12 human tumor                       71% (P = 0.0032) and 69% (P = 0.007) tumor growth
cell lines encased in biocompatible hollow fibers implanted                     inhibition, respectively.
in mice (25). This method was statistically validated with                         ML-133 Preferentially Chelates Labile Zinc In vivo
the use of a “training set” of standard anticancer compounds                       As with other 1,10-phenanthroline–containing com-
to represent the score achieved by clinically used anticancer                   pounds (27), 2-indolyl imidazol [1,10] phenanthroline de-
agents. ML-133 produced a total growth inhibition score of                      rivatives exhibit metal chelation properties. To assess
32 (compounds with a total score of ≥20 are considered                          whether metal chelation plays a role in ML-133–mediated

Mol Cancer Ther 2009;8(9). September 2009

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                                                        Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

2590 Anticancer Activity of ML-133

       cell growth inhibition, HT-29 cells were incubated with ML-                    Cu/Zn SOD, which protects cells from the effects of
       133 in the presence or absence of metal ions (100 μmol/L).                     superoxide anions (29). Cu/Zn SOD activity was decreased
       ML-133–mediated cell growth inhibition was completely                          in a dose-response manner in HT-29 cells treated with the
       blocked by Zn 2+ or Cu 2+ supplementation, partially                           copper-specific chelator 2,3,2-tetramine. In contrast, HT-29
       blocked by Fe 2+, and was not affected by addition of                          cells treated with ML-133 showed no significant dose-
       Fe3+, Mg2+, or Ca2+ (Fig. 2A). However, the results ob-                        dependent changes in Cu/Zn SOD activity (Fig. 2C), indi-
       tained from adding supplemental metals to cells merely                         cating that chelation of intracellular copper by ML-133
       indicate whether a metal is capable of blocking the active                     does not occur significantly in vivo.
       site of ML-133 through chelation and do not represent                            Expression of the iron-sensitive transferrin receptor 1
       a physiologic cellular environment. To assess whether                          gene (30) was not significantly altered by ML-133 treatment,
       ML-133 affects the endogenous levels of metals in vivo,                        in contrast to the iron chelator desferoxamine, which
       metal-specific assays were undertaken.                                         up-regulated the expression of this gene after 16 hours
          Zinquin, a zinc-specific fluorophore, has been used to                      (Fig. 2D), indicating that chelation of intracellular iron by
       detect the intracellular changes in zinc available for cellular                ML-133 does not occur significantly in vivo.
       reactions (28). ML-133 decreased the fluorescence produced                       Overall, these results indicate that ML-133–mediated
       by zinquin-Zn2+ complex formation in a dose-dependent                          HT-29 cell growth inhibition is mainly associated with
       manner (Fig. 2B), indicating that ML-133 does reduce the                       the reduction of intracellular zinc levels.
       concentration of endogenous intracellular labile zinc in                         ML-133–Mediated Cell Cycle Arrest Involves Cyclin D1
       HT-29 cells.                                                                   Repression
          The activity of copper-dependent enzymes is commonly                          Cell cycle analysis by flow cytometry of HT-29 cells trea-
       used to assess the copper status of animal tissues and cells.                  ted with ML-133 showed a dose-dependent increase in the
       Copper functions as the active center of the cuproenzyme                       percentage of cells in the G 1 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/L
       ML-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 cell
       growth inhibition relative to cells treated with DMSO and metal supplements B, ML-133 dose-dependent decrease in endogenous intracellular labile
       zinc pool measured by zinquin assay in HT-29 treated cells C, comparison of intracellular copper levels in HT-29 cells treated with the copper chelator
       2,3,2-tetramine and ML-133 measured by Cu/Zn SOD activity. Results, percentage of SOD activity relative to DMSO-treated control. D, comparison of
       intracellular 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.

                                                                                                            Mol Cancer Ther 2009;8(9). September 2009

                   Downloaded from mct.aacrjournals.org on January 15, 2021. © 2009 American Association for Cancer
                                                              Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

                                                                                                           Molecular Cancer Therapeutics     2591

                                                                              ML-133–Mediated Up-Regulation of the KLF4
                                                                           Transcription Factor
                                                                              Studies of zinc-regulated gene expression in HT-29 colon
                                                                           carcinoma cells indicate that KLF4 shows the most
                                                                           pronounced change in expression among other zinc
                                                                           finger–containing transcription factors under reduced zinc
                                                                           level conditions (6). Because KLF4 is known to inhibit cell
                                                                           proliferation by blocking G1-S progression of the cell cycle
                                                                           through transcriptional repression of cyclin D1 (7, 31), we
                                                                           addressed the question of whether KLF4 is involved in the
                                                                           cell growth inhibition mechanism of ML-133. Increased
                                                                           expression of the KLF4 gene was detected in HT-29 cells
                                                                           after 4-hour treatment with ML-133, with a peak at 16 hours
                                                                           (Fig. 4A), and this effect was partially reversed upon zinc
                                                                           supplementation (Fig. 4B), which supports the role of
                                                                           ML-133–mediated zinc level reduction in the induction of
                                                                           KLF4 gene expression. KLF4 protein was also increased
                                                                           after treatment of HT-29 cells with ML-133 for 16 hours
                                                                           (Fig. 4C). Moreover, ML-133 induced KLF4 gene expression
                                                                           in 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 the
                                                                           activator SP1 for binding to transcriptional control se-
                                                                           quences in the cyclin D1 promoter (31). Therefore, we exam-
                                                                           ined whether this mechanism was involved in the
                                                                           repression of cyclin D1 by ML-133 in HT-29 colon cancer
                                                                           cells with the use of the chromatin immunoprecipitation as-
                                                                           say. ML-133 treatment produced increased binding of KLF4
                                                                           to the cyclin D1 promoter and displacement of SP1, as
                                                                           shown by decreased SP1 binding (Fig. 4D), indicating that
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-     induction of KLF4 by ML-133 represses Sp1-dependent
centrations of ML-133; a dose-dependent G1-S phase cell cycle arrest       constitutive cyclin D1 transcription in vivo. ML-133 had no
was observed as measured by flow cytometry (*, P < 0.05 at 1 and           significant effect on the mRNA or protein expression of
5 μmol/L ML-133 compared with control). B, decreased protein expres-
sion of cyclin D1 detected by Western blotting at the indicated time       SP1 (Supplementary Fig. S1A and B). These results provide
points post treatment with 1 μmol/L ML-133. Densitometric analysis         a molecular link between reduction of intracellular zinc
was done to compare cyclin D1 protein levels to glyceraldehyde-3-
phosphate dehydrogenase protein levels to normalize the results. These
                                                                           levels, cyclin D1 down-regulation, and cell growth inhibi-
results are representative of three replicate experiments. C, decreased    tion produced by ML-133.
gene expression of cyclin D1 determined by real-time PCR after treatment      KLF4 Up-Regulation Contributes to ML-133–Mediated
with 1 μmol/L ML-133 for 16 h and 25 μmol/L ZnCl 2 . (*, P < 0.05
versus ML-133).                                                            Growth Inhibition
                                                                              In an effort to evaluate the biological significance of KLF4
                                                                           up-regulation by ML-133, we transiently transfected HT-29
                                                                           cells with a KLF4 expression vector (Fig. 5A) and examined
60.8% ± 3.4% and 66.2% ± 4.0% at 1 and 5 μmol/L ML-133                     the impact of KLF4 overexpression on ML-133–mediated cell
concentrations, respectively, compared with 45.1% ± 2.4% in                growth inhibition (Fig. 5B). KLF4-expressing cells were
cells treated with the vehicle control (0.1% DMSO; Fig. 3A),               growth inhibited relative to vector-transfected cells in the
indicating that ML-133 can arrest HT-29 cells in the G1-S                  absence of ML-133 (P < 0.05). Importantly, the effect of ML-
phase of the cell cycle. Because cyclin D1 is a key regulator              133 on growth inhibition was enhanced as a result of KLF4
of the G1-S phase progression, we examined its expression in               overexpression (P = 0.01). As a flip side to the overexpression
HT-29 cells treated with ML-133. Cyclin D1 protein expres-                 of KLF4, we examined the effect of decreased KLF4 expres-
sion was decreased in a time-dependent manner by treat-                    sion with the use of KLF4-targeted small interfering RNA
ment of HT-29 cells with 1 μmol/L ML-133 (Fig. 3B).                        (Fig. 5C). In the absence of ML-133, cell growth was not sig-
Moreover, cyclin D1 gene expression was reduced by ML-                     nificantly altered by KLF4 knockdown, probably due to the
133 treatment, and importantly, this effect was partially re-              relatively low basal level of KLF4 in the cells. Importantly,
versed by supplementation with 25 μmol/L Zn2+ (Fig. 3C),                   ML-133–mediated cell growth inhibition was significantly
indicating that the ML-133–mediated reduction in cyclin                    muted as a result of KLF4 knockdown (P = 0.01; Fig. 5D).
D1 gene expression is mediated by the reduction of labile                  Together, these results support the role of KLF4 in mediating
intracellular zinc.                                                        the effect of ML-133 on the growth inhibition of cancer cells.

Mol Cancer Ther 2009;8(9). September 2009

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2592 Anticancer Activity of ML-133

       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-dependent
       increase 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 the
       presence of 25 μmol/L ZnCl2. (*, P < 0.05 versus ML-133). C, increased protein expression of KLF4 was detected by Western blotting after treatment
       with the indicated concentrations of ML-133 for16 h compared with 0.01% DMSO vehicle control. D, transcription factor binding activity determined by
       chromatin immunoprecipitation assay showed increased KLF4 binding and decreased Sp1 binding to the cyclin D1 promoter (-231 to -92) upon ML-133
       treatment.

          To validate the proposed molecular mechanism for ML-                     Discussion
       133–mediated cell growth inhibition in vivo, the levels of                  The present study shows the anticancer properties of the
       gene expression of KLF4 and cyclin D1 were determined                       novel small molecule ML-133. We have shown that chelation
       by real-time PCR in HT-29 tumors. These studies showed a                    of labile intracellular zinc is a key factor in the molecular
       consistent increase in KLF4 gene expression and                             events that lead to cell growth inhibition. The concentration
       decreased cyclin D1 expression in tumors grown in ML-                       of zinc in cells is controlled through a complex zinc homeo-
       133–treated CD-1 nude mice compared with tumors from                        static system. Total cellular zinc consists of a large pool of
       mice treated with the vehicle control (Fig. 5E). Taken together,            tightly bound zinc, and a small but measurable pool of
       these results indicate that ML-133 treatment reduces                        “free” zinc ions involved in regulatory functions. At least
       cyclin D1 expression through zinc-dependent up-regulation                   three factors control “free” zinc and the amplitudes of its
       of the transcription repressor KLF4, ultimately leading to cell             fluctuations: total zinc, zinc buffering, and redox buffering
       cycle arrest.                                                               capacity (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                                               6.5
       HCT-116                          Colon                                         0.20                                               5.9
       H-460                            Lung                                          2.90                                               3.4
       DU-145                           Prostate                                      0.35                                               9.2
       PC-3                             Prostate                                      0.30                                               2.3
       MDA-MB-231                       Breast                                        0.50                                               1.5
       MDA-MB-435                       Breast                                        0.35                                               9.1
       MOLT-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.

                                                                                                         Mol Cancer Ther 2009;8(9). September 2009

                   Downloaded from mct.aacrjournals.org on January 15, 2021. © 2009 American Association for Cancer
                                                              Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

                                                                                                                      Molecular Cancer Therapeutics          2593

the metallothionein-to-thionein ratio (33). In cells, metal-                   ment, after inducing the synthesis of metallothionein-Zn2+
lothionein is a dynamic protein with species constantly                        in TE671 cells with zinc, the addition of 25 μmol/L of the
changing due to Zn(II) transfer to apo-metalloproteins and                     zinc chelator TPEN for 30 minutes markedly reduced the
re-equilibration when thionein expression is induced.                          zinc content of the metallothionein pool without clearly
  We propose that ML-133 chelates zinc from the labile pool                    affecting the high–molecular weight zinc pool, which in-
of zinc, mainly from MTF1 and metallothionein. In agree-                       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 XTT assay. The results
are 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 interfering
RNA (siRNA) or nonspecific control siRNA treated with 1 μmol/L ML-133 or DMSO, determined by real-time PCR analysis. D, ML-133–mediated inhibition
of cell proliferation is impaired in HT-29 cells transfected with KLF4-targeted siRNA but not with nonspecific control siRNA (*, P = 0.01). E, KLF4 and
cyclin 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 four
control mice injected with vehicle control.

Mol Cancer Ther 2009;8(9). September 2009

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                                                       Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

2594 Anticancer Activity of ML-133

       suggests that metallothionein-Zn2+ is particularly labile         as KLF4 has been shown to repress transcription of other
       compared with the inertness of the high– molecular weight         genes through competition with Sp1, including histidine de-
       zinc pool (34). ML-133 is able to chelate Zn2+ in vitro (with a   carboxylase (43), Cyp1A1 (44), and ornithine decarboxylase
       similar affinity to EGTA), as shown by its ability to impair      (45). In addition to direct competition with Sp1 for binding
       the formation of a colored 4-(2-pyridylazo)resorcinol–Zn2+        to promoters, Ai et al. (43) have proposed that KLF4 could
       complex (Supplementary Fig. S2A). However, ML-133 che-            mediate transcriptional repression through several addi-
       lates zinc with a much lower affinity than TPEN, indicating       tional mechanisms. First, physical interaction between Sp1
       that ML-133 is unlikely to access the pool of tightly bound       and KLF4 has been shown (44), and this interaction might
       zinc. A zinc-chelating drug, such as ML-133, may interfere        disrupt the recruitment of the transcriptional coactivator
       with cellular zinc buffering, leading to perturbation of zinc     complex, resulting in transcriptional inhibition. Second,
       homeostasis.                                                      KLF4 might also interact directly with coactivator com-
          Exogenously added copper blocks ML-133–mediated cell           plexes, leading to failure of the recruitment of the complexes
       growth inhibition (Fig. 2A), and ML-133 is able to chelate        to the Sp1 binding site or to the inhibition of the activity of
       copper in vitro (with a similar affinity to EGTA), as shown       the coactivator complexes. However, all these possibilities
       by its ability to impair the formation of a colored 4-(2-pyri-    are not mutually exclusive (43).
       dylazo)resorcinol –Cu2+ complex (Supplementary Fig. S2B).            KLF4 is also known to act as a transcriptional repressor
       However, we do not believe that ML-133 is a chelator of           of other cell cycle promoters as well as a transcriptional
       copper in vivo. The results obtained from adding supple-          activator of several genes encoding inhibitors of the cell
       mental metals to cells merely indicate whether a metal is         cycle (46). Further studies are required to identify other
       capable of blocking the active site of ML-133 through che-        ML-133–responsive targets of KLF4 and their role in
       lation and do not represent a physiologic cellular environ-       ML-133–mediated cell growth inhibition. Overall, these
       ment. An excess of copper likely blocks the growth                results suggest that KLF4 is a molecular link between in-
       inhibitory activity of ML-133 by preventing ML-133 from           tracellular zinc depletion by ML-133, cyclin D1 down-
       accessing the labile zinc within the cell. In agreement,          regulation, G1-S phase arrest, and cell growth inhibition.
       ML-133 had no effect on copper status in vivo, as assessed           Further studies are also required to determine the mech-
       by Cu/Zn SOD activity (Fig. 2C). The activity of copper-          anism by which the expression of KLF4 is regulated in
       dependent enzymes, such as Cu/Zn SOD, is commonly                 response to zinc depletion. An obvious candidate for reg-
       used to assess the copper status of animal tissues and cells.     ulation of KLF4 expression is the zinc-sensitive transcrip-
       Copper is an essential, but potentially reactive and toxic        tion factor MTF1. Indeed, in HT-29 cells transfected with
       ion, and the free ionic copper concentration is extremely         MTF1 small interfering RNA, the ML-133–mediated induc-
       low in cells, estimated at
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

                                                                                                                        Molecular Cancer Therapeutics            2595

   Modulation of intracellular zinc homeostasis by zinc-                         17. Ghaleb AM, McConnell BB, Nandan MO, Katz JP, Kaestner KH, Yang
                                                                                 VW. Haploinsufficiency of Kruppel-like factor 4 promotes adenomatous
specific chelators represents a potential new strategy for                       polyposis coli dependent intestinal tumorigenesis. Cancer Res 2007;67:
the treatment of certain types of cancer. We have shown                          7147–54.
that ML-133 is a chelator of zinc and that it potently inhi-                     18. Ohnishi S, Ohnami S, Laub F, et al. Downregulation and growth inhib-
bits multiple cell types with growth inhibition by 50%                           itory effect of epithelial-type Kruppel-like transcription factor KLF4, but
                                                                                 not KLF5, in bladder cancer. Biochem Biophys Res Commun 2003;308:
values in the nanomolar range as determined by the in vitro                      251–6.
cancer cell line screen of the NCI. Moreover, ML-133                             19. Wei D, Kanai M, Huang S, Xie K. Emerging role of KLF4 in human gas-
efficiently impairs tumor growth in a HT-29 colon tumor                          trointestinal cancer. Carcinogenesis 2006;27:23–31.
xenograph mouse model. In conclusion, we have identified                         20. Wang N, Liu ZH, Ding F, Wang XQ, Zhou CN, Wu M. Down-regulation
                                                                                 of gut-enriched Kruppel-like factor expression in esophageal cancer. World
a new zinc chelator, ML-133, as a potential anticancer thera-                    J Gastroenterol 2002;8:966–70.
peutic drug.                                                                     21. Wei D, Kanai M, Jia Z, Le X, Xie K. Kruppel-like factor 4 induces
                                                                                 p27Kip1 expression in and suppresses the growth and metastasis of
                                                                                 human pancreatic cancer cells. Cancer Res 2008;68:4631–9.
Disclosure of Potential Conflicts of Interest                                    22. Yasunaga J, Taniguchi Y, Nosaka K, et al. Identification of aberrantly
All authors are current or former employees of Lorus Therapeutics, Inc.          methylated genes in association with adult T-cell leukemia. Cancer Res
No other potential conflicts of interest were disclosed.                         2004;64:6002–9.
                                                                                 23. Huesca M, Young AH, Lee Y, et al., inventors. 2-Indolyl imidazo [4, 5-D]
                                                                                 phenanthroline derivatives and their use in the treatment of cancer. U S A
Acknowledgments                                                                  patent WO06126177. 2006.
We thank Tracy Wong, Michelle Liu, Stefanie Lau, Jason DeMelo, and               24. Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-flux anti-
Cindy Liu for their excellent technical assistance.                              cancer drug screen using a diverse panel of cultured human tumor cell
                                                                                 lines. J Natl Cancer Inst 1991;83:757–66.
                                                                                 25. Hollingshead MG, Alley MC, Camalier RF, et al. In vivo cultivation of
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                                                                                                            Mol Cancer Ther 2009;8(9). September 2009

                   Downloaded from mct.aacrjournals.org on January 15, 2021. © 2009 American Association for Cancer
                                                              Research.
Published OnlineFirst September 15, 2009; DOI: 10.1158/1535-7163.MCT-08-1104

A novel small molecule with potent anticancer activity
inhibits cell growth by modulating intracellular labile zinc
homeostasis
Mario Huesca, Lisa S. Lock, Aye Aye Khine, et al.

Mol Cancer Ther Published OnlineFirst September 15, 2009.

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