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[Cancer Research 65, 2890-2898, April 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

A Novel Ring-Substituted Diindolylmethane,1,1-Bis[3'-(5-Methoxyindolyl)]-1-(p-t-Butylphenyl) Methane, Inhibits Extracellular Signal-Regulated Kinase Activation and Induces Apoptosis in Acute Myelogenous Leukemia

Rooha Contractor1, Ismael J. Samudio1, Zeev Estrov2, David Harris2, James A. McCubrey4, Stephen H. Safe3,5, Michael Andreeff1 and Marina Konopleva1

Departments of 1 Blood and Marrow Transplantation and 2 Leukemia, University of Texas M.D. Anderson Cancer Center; 3 Institute for Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas; 4 Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and 5 Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas

Requests for reprints: Marina Konopleva, Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Unit 448, 1400 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-1628; Fax: 713-794-4747; E-mail: mkonople{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane derivative, 1,1-bis[3'-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in acute myelogenous leukemia (AML) cells. DIM #34 inhibited AML cell growth via the induction of apoptosis and abrogated clonogenic growth of primary AML samples. Exposure to DIM #34 induced loss of mitochondrial inner transmembrane potential, release of cytochrome c into the cytosol, and caspase activation. Bcl-2–overexpressing, Bax knockout, and caspase-9–deficient cells were partially resistant to cell death, suggesting the involvement of the intrinsic apoptotic pathway. Furthermore, DIM #34 transiently inhibited the phosphorylation and activity of the extracellular signal-regulated kinase and abrogated Bcl-2 phosphorylation. Because other methylene-substituted diindolylmethane analogues have been shown to transactivate the nuclear receptor peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), we studied the role of PPAR{gamma} in apoptosis induction. Cotreatment of cells with a selective PPAR{gamma} antagonist or with retinoid X receptor and retinoic acid receptor ligands partially modulated apoptosis when combined with DIM #34, suggesting PPAR{gamma} receptor-dependent and receptor-independent cell death. Together, these findings suggest that diindolylmethanes are a new class of compounds that selectively induce apoptosis in AML cells through the modulation of the extracellular signal-regulated kinase and PPAR{gamma} signaling pathways.

Key Words: Diindolylmethane • AML • apoptosis • PPAR{gamma} • ERK


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute myelogenous leukemia (AML) is usually treated with chemotherapeutic regimens that may include cytosine-arabinoside and anthracycline analogues (1). Although these standard treatments induce remissions in most patients, there is still the likelihood of relapse and the development of resistant disease. As a result, many novel agents do not improve survival of patients once relapse occurs (1–3), which enforces the need for more effective treatments for AML, particularly the ones that exploit apoptosis pathways.

Apoptosis resistance is a mechanism that can contribute to leukemogenesis and drug resistance (4). Apoptosis is regulated by two major pathways, both of which lead to the activation of proteases known as caspases (5). The extrinsic or death receptor-mediated pathway is regulated via the activation of caspase-8. The intrinsic or mitochondria-dependent apoptosis pathway is regulated by the Bcl-2 family of proteins. Proapoptotic Bcl-2 family members can promote the release of cytochrome c from mitochondria resulting in the activation of caspase-9. Both extrinsic and intrinsic pathways are frequently dysregulated in AML (6). Bcl-2 protein is reportedly overexpressed in AML and associated with drug resistance and/or poor response to chemotherapy (7, 8). Recent studies have shown that the post-translational phosphorylation of Bcl-2 at Ser70 is required for its antiapoptotic activity, and protein kinase C and mitogen-activated protein kinase (MAPK), specifically the extracellular signal-regulated kinases (ERK1/2), can phosphorylate Bcl-2 (9, 10).

Diindolylmethane, an anticancer agent found in cruciferous vegetables, is formed by acid-catalyzed condensation and dimerization of indole-3-carbinol (11). Diindolylmethane has been shown to cause apoptosis and/or cell cycle arrest in several human cancer cell lines, including breast (12), prostate (13), and colon (14). Recent studies have shown that several analogues of diindolylmethane [i.e., 1,1-bis(3-indolyl)-1-(p-substituted phenyl) methanes] inhibit the proliferation of MCF-7 cells, which is believed to be mediated through the activation of the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}; ref. 15). PPAR{gamma} is a ligand-activated transcription factor belonging to the steroid receptor superfamily. PPAR{gamma} becomes an active transcription factor on ligand binding and the formation of a heterodimeric complex with retinoid X receptors (RXR; ref. 16). PPAR{gamma} plays a major role in the regulation of several metabolic pathways, including adipogenesis, insulin resistance, and atherosclerosis. Agonists for this receptor cause proliferation inhibition, differentiation, and/or apoptosis in several cancer cells, including breast cancer cells (17), colon cancer cells (18) and leukemias (19). Taken together, these data suggest that analogues of diindolylmethane may be effective in the treatment of various types of cancer.

In the current study, we analyzed the cytotoxic effects of several novel ring-substituted diindolylmethane derivatives on leukemic cell lines and primary blasts from patients with AML. We observed that one such compound, 1,1-bis[3'-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), was a very potent apoptogenic agent in these cells. Our results suggest that DIM #34 triggers apoptosis in leukemic cells through the inhibition of MAPK signaling and activation of the intrinsic and possibly caspase-independent apoptotic pathways that may be partially mediated by the activation of PPAR{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies. DIM #34 and N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide (T007; ref. 20), a selective PPAR{gamma} antagonist, were synthesized as described previously (15). All-trans retinoic acid (ATRA) was purchased from Sigma Chemical Co. (St. Louis, MO). The RXR-specific ligands LG100268 and LG0100069 were kindly provided by Dr. Reid Bissonnette (Ligand Pharmaceuticals, San Diego, CA). The small molecule Bcl-2 inhibitor ethyl 2-amino-6-bromo-4-[1-cyano-2-ethoxy]-4H-chromene-3-carboxylate (HA14-1) was purchased from Maybridge (Cornwall, United Kingdom).

Annexin V-phycoerythrin was purchased from Caltag Laboratories (Burlingame, CA). Mouse IgG1 FITC and phycoerythrin were purchased from BD Biosciences (San Jose, CA).

Caspase-3, caspase-8, caspase-9 (anti-human and anti-mouse), Akt, phospho-Akt (Ser473), phospho-Bcl-2 (Ser70), and phospho-ERK1/2 antibodies were purchased from Cell Signaling Technologies, Inc. (Beverly, MA). Cytochrome c and Bax antibodies were purchased from BD Biosciences. Bcl-2 antibody was purchased from DAKO (Carpinteria, CA). PPAR{gamma} and ERK2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Chemicon International (Temecula, CA). Goat anti-mouse and anti-rabbit horseradish peroxidase–conjugated secondary antibodies were purchased from Bio-Rad (Hercules, CA).

Cell lines and primary acute myelogenous leukemia samples. U937, HL-60, Jurkat, and I2.1 cells (a Jurkat clone with a caspase-8 mutation; ref. 21) were purchased from the American Type Culture Collection (Rockville, MD). HL-60 cells stably transfected with Bcl-2 (HL-60/Bcl-2) or empty vector control (HL-60/neo) were kindly provided by Dr. K. Bhalla (Moffitt Cancer Center, University of South Florida, Tampa, FL; ref. 22). Caspase-9 knockout mouse embryonic fibroblast (MEF) cells (23) and caspase-3 knockout mice (24) were provided by Dr. R. Flavell (Yale University School of Medicine, New Haven, CT). HCT116 Bax +/– and –/– cells were provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD). Parental TF1 cells were obtained from American Type Culture Collection. TF1/{Delta}Raf-1 ER cells (DD, cells with estrogen-inducible mutated {Delta}Raf-1) were derived as described (25).

Bone marrow or peripheral blood samples were obtained for in vitro studies from patients with newly diagnosed or recurrent AML during routine diagnostics under informed consent in accordance with regulations and protocols approved by the Human Subjects Committee of the University of Texas M.D. Anderson Cancer Center (Houston, TX). Mononuclear cells were separated by Ficoll-Hypaque (Sigma Chemical) density-gradient centrifugation. The clinical features of the patients are listed in Table 1. Cells were either used for colony assays, as described below, or cultured in AIM-V medium (Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS), 1 mmol/L L-glutamine, and 50 µg/mL penicillin/streptomycin (Life Technologies).


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  		Table 1. Clinical data for patients

 
Cell culture. U937, HL-60, HL-60/neo, HL-60/Bcl-2, Jurkat, Jurkat I2.1, TF1, and TF1/{Delta}Raf-1 ER (DD) cells were cultured in RPMI 1640. Wild-type (WT) MEF (CD1 strain) and caspase-3 and caspase-9 knockout MEFs were cultured in DMEM. HCT116 Bax +/– and –/– cells were cultured in McCoy's 5A medium. All media were supplemented with 10% FBS (Gemini Bio-Products, Woodland, CA), 1 mmol/L L-glutamine, and 50 µg/mL penicillin/streptomycin. In addition, medium for MEFs was supplemented with nonessential amino acid solution (Life Technologies) for MEM, medium for TF1 with granulocyte-macrophage colony-stimulating factor (11.2 units/mL medium), and medium for TF1/{Delta}Raf-1 ER (DD) cells with 1 µmol/L ß-estradiol (Sigma Chemical).

Leukemic cell lines and mononuclear cells from AML patients were cultured at a density of 3.0 x 105 cells/mL in medium supplemented with 5% FBS and treated with either DIM #34 or vehicle (DMSO final concentration, 0.1%). HCT116 and MEFs were plated at a density of 1.0 x 105 cells/mL in medium supplemented with 5% FBS, allowed to attach for 24 hours, and then treated with either DIM #34 or DMSO. DIM #34 was dissolved in DMSO to yield a stock of 10 mmol/L, which was diluted into the culture medium to the indicated concentrations. In all experiments, cells were treated in log-phase growth.

Viability assay. This assay was done using the WST cell proliferation kit (Roche Diagnostics Co., Indianapolis, IN). Briefly, the WST reagent (a tetrazolium salt, which is cleaved to a colored formazan product by mitochondrial dehydrogenases in metabolically active cells) was added to the cells (treated for 72 hours with 0.1% DMSO or DIM #34) and the absorbance was measured in a plate reader.

Flow cytometric analysis of apoptosis. Apoptosis was determined by the flow cytometric measurement of phosphatidylserine exposure using Annexin V-phycoerythrin (26). The mitochondrial inner transmembrane potential ({Delta}{psi}m) was determined by measuring CMXRos retention (red fluorescence) while simultaneously adjusting for mitochondrial mass (MitoTracker Green, green fluorescence; ref. 27). Briefly, cells were stained with 300 nmol/L CMXRos and 100 µmol/L MitoTracker Green (both from Molecular Probes, Eugene, OR) for 1 hour at 37°C. Caspase activation was measured by flow cytometry using a FITC-conjugated, cell-permeable peptide that irreversibly and selectively binds to activated caspases (Caspatag, Intergen, Purchase, NY).

Western blot analysis. Cells were lysed at a density of 1 x 106 per 50 µL in protein lysis buffer (0.25 mol/L Tris-HCl, 2% SDS, 4% ß-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue). For determination of phosphospecific proteins, cells were lysed in buffer containing 150 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 10 mmol/L NaF, 5 mmol/L sodium pyrophosphate, 10 mmol/L ß-glycerophosphate, 1% Triton X-100, 10 mmol/L iodacetamide, 1 mmol/L Na3VO4, 0.1% NaN3, and 3 mmol/L phenylmethylsulfonyl fluoride. For preparation of cytosolic extracts, cells were resuspended in ice-cold extraction buffer [25 mmol/L Tris-HCl, 5 mmol/L MgCl2 (pH 7.4)], permeabilized on ice for 5 minutes, and centrifuged at 10,000 x g for 10 minutes. The supernatant was the cytosolic fraction. All lysis buffers were supplemented with a protease inhibitor cocktail (Roche Diagnostics). Cell lysates were then loaded onto a 10% to 12% SDS-PAGE gel (Bio-Rad). After electrophoresis, proteins were transferred to Hybond-P membranes (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) followed by immunoblotting. Signals were detected using a PhosphorImager (Storm 860, version 4.0, Molecular Dynamics, Sunnyvale, CA).

In vitro extracellular signal-regulated kinase assay. The effect of DIM #34 on MAPK was determined using an in vitro MAPK assay kit from Upstate Biotechnology (Lake Placid, NY). This assay detects a substrate [myelin basic protein (MBP)] that is phosphorylated by the immunoprecipitated enzyme (MAPK). For each control or treated sample, ERK1/2 was immunoprecipitated from 15 x 106 cells using a specific anti-ERK1/2 antibody and anti-MAPK1/2 agarose beads. The ERK-containing agarose pellet was resuspended in assay buffer containing an inhibitor cocktail (protein kinase C inhibitor peptide, protein kinase A inhibitor peptide, and compound R24571) to block the effects of possible contaminating non-ERK kinases, dephosphorylated MBP (20 µg) as a substrate, and a MgCl2-ATP cocktail. Phosphorylation of MBP was determined using an anti-phospho-MBP antibody. The amount of total ERK immunoprecipitated from each sample was determined using an anti-ERK2 antibody.

Acute myelogenous leukemia blast colony and colony-forming unit granulocyte-macrophage assays. AML bone marrow cells were isolated by gradient centrifugation and plated in duplicate at a density of 1 x 105 to 2 x 105/mL in 1% methylcellulose in Iscove's modified Dulbecco's medium (Methocult, Stem Cell Technologies, Vancouver, British Columbia, Canada) containing 10% FBS and the following human recombinant growth factors: erythropoietin (3 units/mL), interleukin-6 (10 ng/mL), interleukin-3 (10 ng/mL), granulocyte-macrophage colony-stimulating factor (10 ng/mL), and stem cell factor (50 ng/mL). DIM #34 was added at the start of cultures at concentrations of 5 to 20 µmol/L.

In three experiments, mononuclear cells isolated from normal bone marrow (1 x 104/mL) were plated as described above. The colony-forming capacity of AML and normal samples was evaluated under a stereo or inverted microscope after 8 to 10 days of culture at 37°C in a 5% CO2 humidified environment. A colony was defined as a cluster of ≥40 cells: blasts [colony-forming unit (CFU)] or erythrocyte [blast-forming unit-erythroid (BFU-E)], granulocyte, monocyte [CFU granulocyte-macrophage (CFU-GM)], or the mixed population [CFU-granulocyte erythrocyte monocyte macrophage (GEMM)].

Statistics. Results are expressed as means ± SE of three separate replicate experiments, unless otherwise indicated. Levels of significance were evaluated by a two-tailed, paired, Student's t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening of diindolylmethanes. We compared the effects of several methylene-substituted diindolylmethane analogues containing various phenyl groups with the following p-substituents: X = CF3 (#1), Br (#2), F (#3), tBu (#4), OCH3 (#5), N(CH3)2 (#6), H (#7), OH (#8), C6H5 (#9), CN (#10), and CH3 (#11). The most active compounds as inhibitors of cell viability of U937 (Fig. 1A, inset i) and HL-60 cells (data not shown) were #4 and #9, which have been identified previously as PPAR{gamma} agonists in breast, pancreatic, and colon cancer cells (15, 28, 29). We further investigated two indole ring-substituted analogues of #4 and #9 containing a 5-methoxy substituent in the phenyl ring (#34 and #14, respectively). The results showed that the 5-methoxy derivatives were also active as inhibitors of cell viability (Fig. 1A, inset ii). Based on these preliminary data, we selected compounds #4, #9, #14, and #34, having an IC50 of 5 µmol/L at 72 hours, for further analysis. Further studies showed that compounds #4, #9, and #14 predominantly inhibited cell growth without apoptosis induction (data not shown). In contrast, DIM #34 induced apoptosis in leukemic cells; therefore, DIM #34 was selected as the lead compound for further studies. It should be noted that similar to compounds #4 and #9 both of the indole ring-substituted analogues (#34 and #14) also activate PPAR{gamma} in cancer cell lines (data not shown).



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  		Figure 1. DIM #34 inhibits cell growth, induces apoptosis, decreases {Delta}{psi}m, and activates caspases. A, effects of p-substituted diindolylmethane analogues on the viability of leukemic (U937) cells. Cells cultured at a density of 3.0 x 105/mL in medium supplemented with 5% FBS were treated with 5 and 10 µmol/L diindolylmethanes. DMSO (0.1%) was used as a control. Cell viability of U937 cells was assessed by the WST assay at 72 hours following treatment with (i) 1,1-bis(3'-indolyl)-1-(p-X-phenyl) methanes or (ii) 1,1-bis[3'-(5-methoxyindolyl)]-1-(p-X-phenyl) methanes. Mean of three replicate experiments. B, U937 cells were treated with 2.5, 5, 7.5, and 10 µmol/L DIM #34 and viable cell number was assessed by trypan blue exclusion at 24, 48, and 72 hours following treatment with DIM #34. Points, mean of three replicate experiments; bars, SE. C, apoptosis in U937 cells was determined using Annexin V staining. Reduction in {Delta}{psi}m was determined by decrease of CMXRos staining. Activation of caspases was measured using the Caspatag peptide. Columns, mean of three replicate experiments; bars, SE. D, cleavage of caspase-3, caspase-8, and caspase-9; release of cytochrome c (Cyt c); and cleavage of Bax after DIM #34 treatment were assessed by Western blot. GAPDH was used as a loading control.

 
DIM #34 inhibits the growth of U937 and HL-60 leukemic cells. U937 (Fig. 1B) and HL-60 (data not shown) cells were exposed to 2.5, 5, 7.5, and 10 µmol/L DIM #34 or vehicle (DMSO) for 72 hours and the growth curves were constructed at 24, 48, and 72 hours. Concentrations of 2.5 and 5 µmol/L had no effect on cell growth. However, 7.5 µmol/L resulted in a steady time-dependent decline in cell number. A similar pattern of threshold growth-inhibitory effect was seen with diindolylmethane derivatives in other cell types (15, 28, 29). This loss of viability was more pronounced in cells treated with 10 µmol/L DIM #34, with the majority of the cells dying at 24 hours. Taking into consideration the possibility of overestimating growth-inhibitory effects of the compounds by the WST assay, which measures activity of the mitochondrial respiratory chain, we chose to use growth-inhibitory concentrations of 7.5 and 10 µmol/L DIM #34 for further mechanistic studies.

DIM #34 induces apoptosis, decreases {Delta}{psi}m, and activates caspases in AML cells. To study the mechanisms of growth inhibition by DIM #34, we did time course studies of apoptosis induction and cell cycle distribution and examined the sequence of molecular changes in U937 cells. DIM #34 (7.5 µmol/L) caused apoptosis in a time-dependent fashion, as evidenced by Annexin V positivity, with essentially 90% of cells undergoing apoptosis at 48 hours (Fig. 1C). DIM #34 had no effect on cell cycle distribution (as determined by propidium iodide staining; data not shown). DIM #34 also induced a time-dependent decrease in {Delta}{psi}m and caspase activation at 8, 12, 24, and 48 hours (Fig. 1C). Western blot analysis showed that DIM #34 induced the cleavage and activation of caspase-9 at 12 hours followed by cleavage and activation of caspase-3 and caspase-8 at 24 hours (Fig. 1D). These data suggest the initial activation of the intrinsic apoptotic pathway (caspase-9) followed by the subsequent cleavage of caspase-8. To further characterize effects of DIM #34 on mitochondrial events related to apoptosis, we evaluated cytochrome c release induced by DIM #34. DIM #34 induced release of cytochrome c into the cytosol in a time-dependent manner. This release was observed as early as 8 hours and paralleled caspase-9 activation. In addition, cleavage of Bax to the active p18 fragment was observed 24 hours after treatment with 7.5 µmol/L DIM #34 (Fig. 1D).

Role of caspases in DIM #34–induced apoptosis. To dissect the role of caspases in DIM #34–induced apoptosis, we used genetically modified caspase-deficient cells. We examined caspase-8 mutant Jurkat cells (I2.1; ref. 21), which do not express the caspase-8 protein (Fig. 2A). Cells treated with 7.5 µmol/L DIM #34 seemed to be slightly protected from apoptosis at 48 hours; however, cells exposed to 10 µmol/L DIM #34 were not protected compared with WT Jurkat cells. Western blot analysis showed that caspase-9 and caspase-3 were cleaved in both WT and caspase-8 mutant Jurkat cells (Fig. 2B). These results support the notion that DIM #34 does not require caspase-8 activation for execution of apoptosis. In contrast, caspase-3 and caspase-9 knockout MEF cells were resistant to 7.5 µmol/L DIM #34 but only partially protected against 10 µmol/L of the drug (Fig. 2C). This would suggest that caspase-independent apoptosis could be triggered by acute exposure to DIM #34.



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  		Figure 2. Effects of DIM #34 in caspase-deficient cells. Apoptosis was determined using Annexin V staining. Columns, mean of three replicate experiments; bars, SE. A, WT and caspase-8 mutant Jurkat cells were treated with 7.5 and 10 µmol/L DIM #34 for 48 hours. DMSO (0.1%) was used as a control. B, cleavage of caspase-3, caspase-8 (C8), and caspase-9 was monitored by Western blot analysis. GAPDH was used as a loading control. C, MEFs were plated at a density of 1.0 x 105/mL in medium supplemented with 5% FBS, allowed to attach for 24 hours, and then treated with 7.5 or 10 µmol/L DIM #34 for 48 hours. DMSO (0.1%) was used as a control. Blot shows levels of caspase-3 (C3) and caspase-9 (C9) WT and knockout (KO) MEFs.

 
Role of Bcl-2 in DIM #34–induced apoptosis. Bcl-2 is an antiapoptotic protein that protects from apoptosis initiated by the intrinsic pathway. We studied the effects of DIM #34 on HL-60/Bcl-2 cells that overexpressed Bcl-2 protein at twice the level seen in HL-60/neo cells (determined by Western blot analysis). HL-60/Bcl-2 cells exposed to 10 µmol/L DIM #34 were completely resistant to apoptosis for up to 72 hours compared with HL-60/neo controls, which underwent apoptosis, with essentially all of the cells becoming apoptotic after 72 hours (Fig. 3A). We then tested whether the inhibition of Bcl-2 function by the small molecule Bcl-2 inhibitor HA14-1 (30) could sensitize HL-60/Bcl-2 cells to DIM #34–induced apoptosis. Whereas HL-60/Bcl-2 cells did not undergo apoptosis in response to 10 µmol/L HA14-1 alone, when combined with DIM #34 (1:1 ratio of 10 µmol/L) apoptosis induction was observed in 62% of treated cells (Fig. 3B).



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  		Figure 3. DIM #34 induces apoptosis in HL-60/neo but not HL-60/Bcl-2 cells. A, HL-60/neo and HL-60/Bcl-2 cells were treated with 10 µmol/L DIM #34 for 24, 48, and 72 hours. Western blot shows levels of Bcl-2 protein in HL-60/neo and HL-60/Bcl-2 cells. Apoptosis was determined using Annexin V staining. Columns, mean of three replicate experiments; bars, SE. B, HL-60/neo and HL-60/Bcl-2 cells were exposed to DIM #34, HA14-1, or their combination at a 1:1 ratio and induction of apoptosis was assessed by Annexin V staining at 48 hours. C, HCT116 Bax +/– and –/– cells were plated at a density of 1.0 x 105/mL in medium supplemented with 5% FBS, allowed to attach for 24 hours, and then treated with 10 µmol/L DIM #34 for up to 72 hours. DMSO (0.1%) was used as a control. Western blot shows levels of Bax in +/– and –/– HCT116 cells. *, P < 0.05.

 
Because Bax is one of the critical mediators of mitochondrial damage, we compared the effects of DIM #34 on Bax knockout HCT116 colon cancer cell line. Bax –/– cells exhibited only partial resistance to DIM #34–induced apoptosis compared with Bax +/– cells (P < 0.05 at 48 and 72 hours; Fig. 3C). This would suggest that Bax was not required as a primary signaling intermediate in DIM #34–induced cell death.

DIM #34 inhibits Bcl-2 and extracellular signal-regular kinase phosphorylation and activation. Post-translational modifications of Bcl-2 significantly affect its prosurvival functions. Because Bcl-2 overexpression protected against DIM #34–mediated cell death, we proposed that the mechanism of activation of the mitochondrial apoptotic pathway by DIM #34 may involve inhibition of Bcl-2 function that is abrogated in cells with forced overexpression of Bcl-2. We therefore studied effects of DIM #34 on Bcl-2 phosphorylation.

Treatment of U937 cells with 7.5 µmol/L DIM #34 did not change total Bcl-2 protein levels for up to 48 hours but inhibited the activation of Bcl-2 at 4 hours as evidenced by inhibition of Bcl-2 phosphorylation (Fig. 4A). Because ERK acts as a Bcl-2 kinase, we further tested the effects of DIM #34 on the activity of ERK1/2. Treatment of U937 cells with 7.5 µmol/L DIM #34 inhibited the activation of ERK1/2 starting at 4 hours of treatment as shown by the inhibition of ERK1/2 phosphorylation. However, DIM #34 had no effect on ERK1/2 total protein levels (Fig. 4A) and no changes in Akt activity were seen (data not shown).



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  		Figure 4. DIM #34 inhibits Bcl-2 and ERK phosphorylation in U937 cells. A, U937 cells were treated with 7.5 µmol/L DIM #34 for the indicated times, and total and phosphospecific levels of Bcl-2 and ERK1/2 were monitored. B, U937 cells were treated in vivo with 7.5 µmol/L DIM #34 for the indicated times, and cell lysates were used in the in vitro kinase assay. MAPK activity was measured by the ability to phosphorylate the specific substrate (MBP) in the in vitro kinase assay. MBP phosphorylation was detected using a specific anti-phospho-MBP antibody. Total ERK immunoblot was used as a loading control. C, TF1 and TF1/{Delta}Raf-1 ER (DD) cells were treated with 7.5 µmol/L DIM #34 for 24 hours, and apoptosis was determined using Annexin V staining. Inset, ERK is constitutively phosphorylated in Raf-transformed cells. Columns, mean of three replicate experiments; bars, SE. *, P < 0.05.

 
We then investigated MAPK activity to further characterize functional effects of DIM #34 on ERK. DIM #34 abrogated ERK kinase activity as evidenced by the decrease in MBP phosphorylation as early as 4 hours (Fig. 4B). Equal amounts of immunoprecipitated ERK protein were confirmed by blotting the membrane with anti-ERK2 antibody. These data suggested that early inhibition of ERK signaling by DIM #34 resulted in activation of the mitochondrial apoptosis pathway and cell death.

To further examine the involvement of the MAPK/ERK kinase (MEK) pathway, we used genetically modified TF1 cells. These TF1/{Delta}Raf-1 ER (DD) cells are transfected with a retrovirus containing a mutated conditionally active human Raf-1 kinase (25). In this system, mutated Raf is fused to the hormone-binding domain of the human estrogen receptor. Treatment of these cells with estradiol activates the mutant Raf protein resulting in enhanced kinase activity, thus mimicking activation of the MEK pathway (ref. 25; Fig. 4C, inset). We have shown recently that TF1/{Delta}Raf-1 ER (DD) cells are exquisitely sensitive to the MEK inhibitor CI1040, suggesting dependence of these cells on activated Raf/MEK/ERK signaling pathway.6 Parental TF1 or modified TF1/{Delta}Raf-1 ER (DD) cells were treated with 7.5 µmol/L DIM #34 for 24 hours and apoptosis was determined by Annexin V flow cytometry. As shown in Fig. 4C, TF1/{Delta}Raf-1 ER (DD) cells were far more sensitive to the effects of DIM #34 at 24 hours. Whereas only 50% of the TF1 cells were Annexin V positive, 87% Annexin V positivity was noted at 24 hours in TF1/{Delta}Raf-1 ER (DD) cells (P < 0.001). As a control, when cells were treated with 10 and 25 µmol/L LY294002, a specific phosphatidylinositol 3-kinase inhibitor (31), there was no apoptosis in either TF1 or TF1/{Delta}Raf-1 ER (DD) cells for up to 72 hours, confirming the specificity of the observed effects.

DIM #34 induces apoptosis and selectively inhibits the colony formation of primary acute myelogenous leukemia cells. We next tested the effects of DIM #34 on clonogenic AML cell growth in the CFU-blast assay. The formation of surviving colonies of AML progenitors (Table 1, samples 1-5) was significantly reduced to 87 ± 2.27% at 5 µmol/L, 60 ± 8.6% at 10 µmol/L, and 11 ± 9.5% at 15 µmol/L; no colonies were recovered at 20 µmol/L (P < 0.05; Fig. 5A). In contrast, there was no inhibition of colony formation of normal bone marrow (n = 3) treated with up to 15 µmol/L DIM #34 (P > 0.05). Even at a concentration of 20 µmol/L, colony formation was only minimally reduced: CFU-GM, 81 ± 14% (Fig. 5B); BFU-E, 51 ± 9% (Fig. 5C); and CFU-GEMM, 65 ± 11% (P > 0.05; Fig. 5D). The difference in the inhibition of AML and normal granulocyte-macrophage progenitor cells was highly significant (P < 0.001), suggesting the preferential killing of leukemic progenitor cells.



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  		Figure 5. DIM #34 inhibits clonogenic progenitor growth. Points, mean of the percentage of colonies compared with the number in DMSO-treated control cells; bars, SE. Inhibition of colonies (CFU) in (A) AML bone marrow samples (n = 5), (B) CFU-GM, (C) BFU-E, and (D) CFU-GEMM in normal bone marrow samples (n = 3) in the presence of increasing concentrations of DIM #34 (5, 10, 15, and 20 µmol/L).

 
To directly determine the effects of DIM #34 on AML progenitors, we used a two-color CD34/Annexin V flow cytometry assay and found that DIM #34 directly induced apoptosis in AML progenitor cells (Table 1, samples 1-12; data not shown). At 7.5 and 10 µmol/L, apoptosis was induced in 11 of 12 AML samples (DMSO, 27 ± 5%; 7.5 µmol/L DIM #34, 48 ± 5%; 10 µmol/L DIM #34, 66 ± 6% CD34-positive/Annexin V–positive cells).

Relationship between peroxisome proliferator-activated receptor {gamma} and effects of DIM #34. Because of the reported transactivation of the nuclear receptor PPAR{gamma} by p-substituted analogues of diindolylmethane (15), we studied the role of PPAR{gamma} in DIM #34–induced apoptosis. We used the selective PPAR{gamma} antagonist T007 (20) to determine if apoptosis induced by DIM #34 resulted from effects on the nuclear receptor. Pretreatment of U937 cells with 2 µmol/L T007 followed by 6.5 µmol/L DIM #34 significantly diminished apoptosis (control, 4.2 ± 0.33; T007, 4.14 ± 0.27; DIM #34, 26.33 ± 1; DIM #34 + T007, 14.98 ± 0.89; P < 0.001; Fig. 6A). However, apoptosis was not inhibited at higher concentrations of DIM #34 (10 µmol/L), suggesting the contribution of PPAR{gamma}-dependent and PPAR{gamma}-independent mechanisms.



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  		Figure 6. PPAR{gamma}-mediated effects of DIM #34. A, U937 cells were pretreated with 2 µmol/L T007, a PPAR{gamma} antagonist, for 1 hour followed by treatment with 6.5 µmol/L DIM #34 for 24 hours. Apoptosis was determined using Annexin V staining. *, P < 0.001, compared with control; #, P < 0.001, compared with each other. Columns, mean of three replicate experiments; bars, SE. B, U937 cells were exposed for 24 hours to 1 µmol/L LG100268, LG0100069, or ATRA alone; 7.5 µmol/L DIM #34 alone; or combinations of LG100268, LG0100069, and ATRA and/or DIM #34. Apoptosis was determined using Annexin V staining. *, P < 0.05, compared with DIM #34. Columns, mean of three replicate experiments; bars, SE.

 
PPAR{gamma} heterodimerizes with nuclear RXR receptors, and the combination of ligands for both receptors enhances effects on downstream target genes (16, 32). Thus, we tested the combined effects of DIM #34 and retinoids/rexinoids with different specificities on the induction of apoptosis in U937 cells. We used ATRA, a retinoic acid receptor (RAR) ligand, and the RXR ligands LG100268 and LG0100069. U937 cells were exposed for 24 hours to 7.5 µmol/L DIM #34 alone; 1 µmol/L LG100268, LG0100069, or ATRA alone; or combinations of LG100268, LG0100069, and ATRA and/or DIM #34 for 24 hours (Fig. 6B). No apoptosis was induced by ATRA, LG100268, or LG0100069 alone. Apoptosis, however, was enhanced in cells treated with the combination of any of the three agents and 7.5 µmol/L DIM #34 (P < 0.05).

These data suggest that the effects of DIM #34 on leukemic cells are at least partially mediated by the PPAR{gamma} receptor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Diindolylmethane, a metabolite of indole-3-carbinol, is a phytochemical that contributes to the anticancer activity of cruciferous vegetables (15). Our study showed that DIM #34 inhibited cell growth of leukemic cells by inducing apoptosis. DIM #34 profoundly inhibited clonogenic cell growth of primary AML samples but spared normal hematopoietic progenitors. Previous studies with C-substituted diindolylmethanes in rodents showed mammary tumor growth inhibition at a dose of 1 mg/kg, and no toxicity was found at doses as high as 300 mg/kg (15).7 These data suggest that diindolylmethanes are relatively nontoxic compounds that cause preferential killing of cancer cells while sparing normal cells.

Our data suggest that DIM #34–induced apoptosis is mediated by multiple mechanisms. DIM #34 induced a decrease in the {Delta}{psi}m followed by the early cleavage of caspase-9, cytochrome c release, and later cleavage of both caspase-3 and caspase-8. This would suggest the activation of the intrinsic mitochondrial pathway of apoptosis. Caspase-8–deficient I2.1 cells were marginally protected from cytotoxicity, suggesting that caspase-8 activation occurs secondary to caspase-9 cleavage and serves as an amplification loop rather than an initiating event (33). However, caspase-3– and caspase-9–deficient MEFs were only partially resistant to cell death, suggesting that caspase-independent pathways contributed to DIM #34–induced apoptosis. These may include the release of a recently identified mitochondrial protein apoptosis-inducing factor (34), which can directly induce large-scale DNA degradation. Smac/Diablo and Omi/HtrA2, which are released from mitochondria in response to apoptotic stimuli, can inhibit members of the inhibitor of apoptosis protein (IAP) family by direct binding, thus promoting the activation of caspases and cell death (35). It is conceivable that DIM #34 affects mitochondrial integrity resulting in the release of mitochondrial proteins (i.e., apoptosis-inducing factor, endoG, Smac, and/or Omi) that may be involved in caspase-independent cell death, which may explain the prevention afforded by Bcl-2.

One type of mitochondrial damage results from the disruption of heterodimers formed by proapoptotic and antiapoptotic proteins (e.g., Bcl-2/Bax or Bcl-xL/Bad), which leads to the permeabilization of the outer membrane. As such, Bax-induced cell death is not necessarily dependent on caspase activity (36). Bax knockout cells were not completely resistant to cell death induced by DIM #34, suggesting that this protein was not intimately associated with triggering the intrinsic apoptotic pathway. Although Bax was not up-regulated, it was cleaved and thus activated. Drug or growth factor withdrawal-induced cleavage of Bax to p18 Bax potently accelerates the apoptotic process in AML cells (37) and other cancer cell types (38, 39). Furthermore, forced overexpression of Bcl-2-mediated resistance to low doses of DIM #34 and inhibition of Bcl-2 function by HA14-1 sensitized HL-60/Bcl-2 cells to DIM #34–induced apoptosis. These findings suggest a model whereby DIM #34 interferes with Bcl-2/Bax interactions, likely freeing Bax and causing mitochondrial membrane permeabilization. In addition to BH3 mimetics, like HA14-1, which directly interfere with Bcl-2/Bax heterodimerization (40), conformational change of Bcl-2 mady also result in the inability of the protein to inhibit Bax. For example, we have recently shown inhibition of ERK enzymatic activity and Bcl-2 phosphorylation by the triterpenoid CDDO-Me (41). Strikingly, DIM #34 abrogated ERK kinase activity as early as 4 hours before {Delta}{psi}m loss and Annexin V positivity. Furthermore, TF1/{Delta}Raf-1 ER (DD) cells with constitutively activated Raf/MEK/ERK signaling were more sensitive to apoptosis induction by DIM #34. Of note, whereas TF1/{Delta}Raf-1 ER cells displayed Raf-driven constitutive ERK phosphorylation, no differences in Bcl-2 and PPAR{gamma} expression levels were detected in transformed cells compared with parental cells (data not shown). These results imply inhibition of ERK signaling by DIM #34, but further studies are needed to precisely map this action in the MAPK signaling pathway.

Antiapoptotic downstream targets of MAPK include p90RSK, cyclic AMP–responsive element binding protein, phospho-Bad, phospho-Bcl-2, X-linked IAP, and survivin. However, the observed activity in caspase-deficient cells and dependence of apoptosis on Bcl-2 suggests a specific target of DIM #34 upstream of mitochondria, thereby making IAP an unlikely choice. Our data show that DIM #34 caused Bcl-2 dephosphorylation following decrease in ERK activity. Of note, we have shown previously that Bcl-2 overexpression prevents apoptosis induction by the specific MEK inhibitor CI1040, whereas inhibition of Bcl-2 expression or function induces strikingly synergistic apoptosis in AML (42). Further studies will be required to determine the effects of DIM #34 on Bad phosphorylation. Bad is phosphorylated on its Ser112 residue by p90RSK, a downstream ERK target, indicating a link between Bad phosphorylation and MAPK signaling (43). We have shown recently that in the majority (41 of 42) of primary AMLs Bad is phosphorylated on both Ser112 and Ser136 sites, a process that converts its proapoptotic function into antiapoptotic (31, 44). Thus, inactivation of Bcl-2 and abrogation of Bad phosphorylation may likely explain apoptosis induction by DIM #34.

Potential cross-talk between MAPK signaling and nuclear receptors has been reported previously, showing that MAPK phosphorylates and inhibits activation of both PPAR{gamma} (45) and RXR (46). We therefore examined involvement of PPAR{gamma} in mediating the induction of apoptosis by DIM #34. Abrogation of PPAR{gamma} signaling by a specific PPAR{gamma} antagonist T007 significantly diminished DIM #34–induced apoptosis, suggesting a potential role for PPAR{gamma} in the induction of cell death. This notion was supported by increased cytotoxic effects when cells were cotreated with DIM #34 in combination with compounds that activate the heterodimeric PPAR{gamma} partner (RXR). It is conceivable that inhibition of ERK activity by DIM #34 could abrogate inhibitory phosphorylation of PPAR{gamma} and RXR nuclear receptors and thereby enhance the activity of PPAR{gamma}/RXR or RAR/RXR heterodimers. This hypothesis requires further investigation. Although the phosphorylation status of PPAR{gamma} in leukemias is unknown, our group reported that ERK1/2 is expressed and activated in the majority of primary AML cases (47), a finding that may result in the inhibition of nuclear receptor signaling. Of interest, recent studies in our laboratory revealed striking synergism between the specific MEK inhibitor CI1040 and RAR/RXR ligands (48).

In conclusion, our data provide the first evidence that a novel ring-substituted diindolylmethane, DIM #34, inhibits colony formation and induces apoptosis in AML cells while sparing normal hematopoietic progenitors. We identified the mechanisms of apoptosis induction as being related to mitochondrial depolarization and activation of the intrinsic apoptotic pathway, which seem to consist of caspase-dependent and caspase-independent mediators. We propose that inhibition of ERK phosphorylation and PPAR{gamma} is associated with the ability of DIM #34 to induce apoptosis. Further studies are required to dissect the interactions between PPAR{gamma} and MAPK signaling. Because both PPAR{gamma} and ERK1/2 are expressed in primary AML, these pathways represent attractive targets amenable to therapeutic intervention by small molecule inhibitors like DIM #34. Taken together, these results suggest that DIM #34 alone or in combination with chemotherapy or retinoids holds promise as a novel therapy for leukemias.


   Acknowledgments
 
Grant support: NIH grants CA55164, CA49639, CA16654, and CA100632 and Stringer Professorship for Cancer Treatment and Research (M. Andreeff); NIH grant ES09106 (S. Safe), and Leukemia and Lymphoma Society grant CF02007 (M. Konopleva).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors would like to thank Wendy Schober and Dr. Randall Evans for assistance with the flow cytometry measurements, Teresa McQueen for help in handling clinical samples, Dr. Numsen Hail, Jr., for help with preparation of the manuscript, and Rosemarie Lauzon and Tena Horton for their excellent administrative support.


   Footnotes
 
6 In preparation. Back

7 Unpublished data. Back

Received 10/21/04. Revised 1/18/05. Accepted 1/20/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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