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WMC-79, a Potent Agent Against Colon Cancers, Induces Apoptosis Through a P53-Dependent Pathway

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WMC-79, a Potent Agent Against Colon Cancers, Induces Apoptosis Through a P53-Dependent Pathway 1617 WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway Teresa Kosakowska-Cholody,1 Introduction 1 2 W. Marek Cholody, Anne Monks, The bisimidazoacridones are bifunctional antitumor Barbara A. Woynarowska,3 agents with strong selectivity against colon cancers (1, 2). and Christopher J. Michejda1 Recent studies of the effect of bisimidazoacridones on sensitive colon tumors cells revealed that these com- 1 Molecular Aspects of Drug Design, Structural Biophysics pounds act as cytostatic agents that completely arrest cell Laboratory, Center for Cancer Research; 2Screening Technologies Branch, Laboratory of Functional Genomics, Science Applications growth at G1 and G2-M check points but do not trigger International Corporation, National Cancer Institute at Frederick, cell death even at high concentrations (10 Amol/L; ref. 3). Frederick, Maryland; and 3Department of Radiation Oncology, The chemical structure of bisimidazoacridones is symmet- University of Texas Health Science Center, San Antonio, Texas rical in that it consists of two imidazoacridone moieties held together by linkers of various lengths and rigidities. Abstract We recently reported on the synthesis of unsymmetrical variants of the original bisimidazoacridones (4). WMC-79 WMC-79 is a synthetic agent with potent activity (Fig. 1), a compound consisting of an imidazoacridone against colon and hematopoietic tumors. In vitro, the moiety linked to a 3-nitronaphthalimide moiety via agent is most potent against colon cancer cells that 1,4-bispropenopiperazine linker, was found to be a potent carry the wild-type p53 tumor suppressor gene (HCT- but selective cytotoxic agent in a variety of tumor cell f 116 and RKO cells: GI50 <1 nmol/L, LC50 40 nmol/L). lines (4). However, it was more toxic against tumor cell Growth arrest of HCT-116 and RKO cells occurs at the lines that carry the wild-type p53 tumor suppressor gene. G1 and G2-M check points at sublethal concentrations The p53 protein is a tightly regulated transcription factor (10 nmol/L) but the entire cell population was killed at that is elevated in response to DNA damage and has a 100 nmol/L. WMC-79 is localized to the nucleus where critical function in maintaining the integrity of the genome. it binds to DNA. We hypothesized that WMC-79 binding p53-driven cell cycle arrest prevents cells with altered DNA to DNA is recognized as an unrepairable damage in the from proliferating and p53-controlled apoptosis selectively tumor cells, which results in p53 activation. This eliminates severely damaged cells (5–8). Whether the cell triggers transcriptional up-regulation of p53-dependent enters growth arrest or undergoes apoptosis depends on genes involved in replication, cell cycle progression, the final integration of incoming signals with antagonistic growth arrest, and apoptosis as evidenced by DNA effects on cell growth. Many factors affect the cellular microarrays. The change in the transcriptional profile of response to activated p53. These include cell type, HCT-116 cells is followed by a change in the levels of oncogenic status of the cell, survival stimuli, intensity of cell cycle regulatory proteins and apoptosis. The stress signals, level of p53 expression, and interaction of recruitment of the p53-dependent apoptosis pathway p53 with specific proteins (9). was suggested by the up-regulation of p53, p21, Bax, We hypothesized that WMC-79 binding to DNA is DR-4, DR-5, and p53 phosphorylated on Ser15; down- recognized as a damage that is not readily repaired in the regulation of Bcl-2; and activation of caspase-8, -9, -7, tumor cells and which results in the activation of p53. and -3 in cells treated with 100 nmol/L WMC-79. The aim of this study was to investigate the molecular Apoptosis was also evident from the flow cytometric mechanism by which WMC-79 induces growth arrest and studies of drug-treated HCT-116 cells as well as from apoptosis in the sensitive HCT-116 and RKO colon cancer the appearance of nuclear fragmentation. However, cell lines and to determine the role of p53 in this whereas this pathway is important in wild-type p53 mechanism. colon tumors, other pathways are also in operation because colon cancer cell lines in which the p53 gene is mutated are also affected by higher concentrations Materials and Methods of WMC-79. [Mol Cancer Ther 2005;4(10):1617–27] Chemicals All mammalian cell culture reagents and trypan blue were purchased from Life Technologies, Invitrogen Cor- poration (Grand Island, NY). Other reagents were from Received 5/24/05; revised 7/11/05; accepted 7/25/05. Sigma-Aldrich (St. Louis, MO). Requests for reprints: Christopher J. Michejda, Molecular Aspects of Drug Cell Culture Design, Structural Biophysics Laboratory, Center for Cancer Research, +/+ National Cancer Institute at Frederick, Frederick, MD 21702. The human colon cancer cell lines HCT-116 p53 , HCT- Phone: 301-846-1216; Fax: 301-846-6231. E-mail: [email protected] 116 p53À/À, HCT-116 p21+/+, and HCT-116 p21À/À were Copyright C 2005 American Association for Cancer Research. a generous gift from Dr. Bert Vogelstein (Johns Hopkins doi:10.1158/1535-7163.MCT-05-0170 University, Baltimore, MD) and were maintained in Mol Cancer Ther 2005;4(10). October 2005 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. 1618 WMC-79 Induces Apoptosis in Colon Cancer Cells 35-mm glass-bottomed microwell dishes (MatTek Corpo- ration, Ashland, MA). The following day, cells were washed and fresh medium containing 100 nmol/L WMC- 79 was added. Cells were examined at different time points under a Zeiss 410 laser scanning confocal microscope. Areas were imaged using appropriate laser lines for WMC- 79 excitation (488 nm). Figure 1. Chemical structure of WMC-79. Western Blot Analysis Immunoblot analysis of cell protein lysates was done McCoy’s 5A media. Human colon carcinoma HCT-116 according to the protocol of the manufacturer (Santa Cruz +/+ +/+ +/À (p53 ), RKO (p53 ), HCT-15 (p53 ), COLO-205 Biotechnology, Inc., Santa Cruz, CA). Briefly, cells were À/À À/À (p53 ), and HT-29 (p53 ) cells were purchased from lysed on ice for 30 to 60 minutes in radioimmunopreci- the American Type Culture Collection (Rockville, MD). pitation assay buffer (1Â PBS, 1% igepal, 0.5% sodium HCT-116, RKO, and HT-29 cells were grown in DMEM; deoxycholate, 0.1% SDS) with freshly added inhibitors COLO-205 and HCT-15 cells were grown in RPMI 1640. All (10 Ag/mL phenylmethylsulfonyl fluoride, 50 Ag/mL media were supplemented with 10% heat-inactivated fetal aprotinin, and 1 mmol/L sodium orthovanadate). Cell bovine serum, 2 mmol/L L-glutamine, 100 units/mL lysate was passed through a 21-gauge needle followed by penicillin, and 100 Ag/mL streptomycin. The cells were centrifugation at 10,000 Â g for 10 minutes at 4jC. Protein grown at 37jC in a humidified atmosphere consisting of concentration was determined using Bio-Rad protein assay 5% CO2 and 95% air. (Bio-Rad Laboratories, Hercules, CA). Samples were Drugs and Drug Preparation Procedure mixed with 2Â Laemmli buffer, denaturated at 100jC for Stock solution of WMC-79 synthesized in our laboratory 3 minutes, and proteins were separated by electrophoresis (4) was freshly prepared by dissolving the free base form (NuPAGE 4-12% Bis-Tris Gel, Invitrogen, Life Technologies, of the compound in 2 equivalents of methanesulfonic acid Carlsbad, CA). Separated proteins were transferred to (as 10 mmol/L water solution) and then diluted with water polyvinylidene difluoride membrane (Millipore, Bedford, to a final concentration of 500 Amol/L. This solution was MA) and subjected to immunoblotting with various primary used to prepare 2 Amol/L working solution and its 10-fold antibodies. Positive antibody reactions were visualized with serial dilutions in appropriate complete tissue culture a horseradish peroxidase–conujugated secondary antibody media. and an enhanced chemiluminescence detection system CellViability (Amersham Pharmacia Biotech, Little Chalfont, United 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Kingdom) according to the protocol of the manufacturer. Bromide Assay. Cellular growth in the presence or The membrane was then deprobed and reprobed with an absence of experimental agents was determined using anti-actin antibody to confirm that all samples contained the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium similar amounts of proteins. TBS-0.05% Tween 20 was used bromide (MTT)–based CellTiter96 Non-Radioactive Cell as a wash buffer; 5% nonfat dry milk (Bio-Rad Laboratories) Proliferation Assay (Promega, Madison, WI) according to was dissolved in TBS-0.05% Tween 20 and was used as a the instruction of the manufacturer with small modifica- blocking solution. The following antibodies were used in tions as previously described (4, 10). Trypan blue exclusion this study: mouse anti-Bax (Ab-3), mouse anti-Bcl-2 (Ab-1), assay was used to determine the number of live/dead cells mouse anti–cyclin D1 (Ab-3), mouse anti-E2F1 (Ab-1), in HCT-116 cultures exposed to WMC-79. mouse anti-Mdm2 (Ab-2), mouse anti-p21 (Ab-1), mouse Fluorescence-Activated Cell Sorting. Tumor cells in anti-p53 (Ab-6), mouse anti-pRb (Ab-5), rabbit anti-caspase- exponential phase of growth were seeded at a density of 7 (AB-1), rabbit anti-DR4 (AB-1), rabbit anti-DR5 (AB-2), 0.5 Â 106 to 1 Â 106 cells in 25 or 75 cm2 T flasks, allowed to rabbit anti-phospho-p53(Ser15) (Ab-3; Oncogene Research attach for 24 hours, and then exposed to 10 or 100 nmol/L Products, Boston, MA), goat anti-actin (C-11), goat anti- WMC-79. At appropriate intervals, drug-treated and caspase-8 (C-20), rabbit anti-caspase-9 (PharMingen, San control cells (attached and floating) were collected and Diego, CA), mouse anti–cyclin A, mouse anti–cyclin B1, washed twice in ice-cold PBS containing 1% fetal bovine rabbit anti–cyclin E (Biosource International, Camarillo, serum.
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