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), 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 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 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 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 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 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 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 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
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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. Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-08-1104 Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2009/09/16/1535-7163.MCT-08-1104.DC1 E-mail alerts Sign up to receive free email-alerts related to this article or journal. Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at pubs@aacr.org. Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2009/09/08/1535-7163.MCT-08-1104. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site. Downloaded from mct.aacrjournals.org on January 15, 2021. © 2009 American Association for Cancer Research.
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