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[Cancer Research 65, 4799-4808, June 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Synthetic Triterpenoids Cooperate with Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand to Induce Apoptosis of Breast Cancer Cells

Marc L. Hyer1, Rhonda Croxton1, Maryla Krajewska1, Stanislaw Krajewski1, Christina L. Kress1, Meiling Lu2, Nanjoo Suh3, Michael B. Sporn4, Vincent L. Cryns2, Juan M. Zapata1 and John C. Reed1

1 The Burnham Institute, La Jolla, California; 2 Departments of Medicine and Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; 3 Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey; and 4 Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire

Requests for reprints: John C. Reed, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3100; Fax: 858-646-3194; E-mail: jreed{at}burnham.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIPL, and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL; TNF10, Apo-2L) is a member of the TNF family of cytokines. TRAIL induces rapid apoptosis of many types of cancer cells while sparing normal cells (1). To date, five members of the human TNF receptor (TNFR) superfamily have been identified that bind TRAIL. The death receptors DR4 (death receptor 4, TRAIL-R1, TNFR10A; ref. 2) and DR5 (TRAIL-R2, TNFR10B; ref. 3) contain conserved cytoplasmic death domains and are capable of binding TRAIL and initiating death signals. The decoy receptors DcR1 (TRAIL-R3, TNFR10C; ref. 4) and DcR2 (TRAIL-R4, TNFR10D; ref. 5) have close homology to the extracellular domains of DR4 and DR5, however, DcR1 lacks a transmembrane domain and a death domain, and DcR2 has a truncated, nonfunctional death domain. Hence, both DcR1 and DcR2 bind TRAIL but do not transmit death signals. Finally, TRAIL binds osteoprotegerin (TNFR11B), which is a soluble protein incapable of signaling (6).

Following TRAIL engagement with either DR4 or DR5, the ligated death receptors cluster and microaggregate within the cell membrane, thereby initiating formation of the death-inducing signaling complex (7). The functional death-inducing signaling complex is composed minimally of death receptors (DR4 and DR5), adapter protein FADD, and caspase 8 or 10 (reviewed in refs. 1, 8, 9). Active caspases 8 and 10 cleave and activate downstream effector caspases (3, 6, and 7), which ultimately cut vital cellular substrates resulting in apoptosis (reviewed in ref. 8).

FLICE-like inhibitory protein (FLIP) is an antiapoptotic protein that has been detected in two isoforms, FLIPL (55 kDa) and FLIPS (28 kDa; ref. 10). Similar to procaspases 8 and 10, the FLIP proteins contain a tandem pair of death effector domains, but they lack a catalytically active protease domain and thus can operate as trans-dominant inhibitors of caspases 8 and 10. During death-inducing signaling complex formation, FLIP is preferentially recruited to the death receptor complex where it binds FADD and thwarts activation of caspases 8 and 10.

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO; ref. 11) and its imidazole derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im; ref. 12) are synthetic triterpenoids synthesized from the naturally occurring triterpene oleanolic acid. Both CDDO and CDDO-Im have been shown to suppress cellular proliferation and induce apoptosis of leukemia (13, 14), multiple myeloma (15), breast cancer (16), squamous cell carcinoma (17), and osteosarcoma (18) cells in culture. Previously CDDO has been shown to sensitize prostate, ovarian, colon (19), and leukemia (20) cells to TRAIL-mediated apoptosis. In the current report, we show that CDDO and CDDO-Im sensitize breast cancer cell lines to TRAIL-mediated apoptosis while having no effect on normal human mammary epithelial cells (HMEC). Moreover, CDDO and CDDO-Im down-regulate the antiapoptotic protein FLIPL and up-regulate the death receptors DR4 and DR5 in breast cancer cells, rendering them sensitive to TRAIL. Finally, the combination of CDDO-Im and TRAIL is well tolerated in mice and reduces tumor burden in a mouse xenograft model of breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. T47D, PPC-1, OVCAR-3, PC-3M-LN4 (21), and LNCaP-LN3 (21) cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA). Mouse embryonic fibroblasts, MDA-MB-468, and MCF-7 cells were cultured in DMEM with high glucose (Irvine Scientific). RPMI 1640 and DMEM were supplemented with L-glutamine (1.8 and 3.6 mmol/L, respectively), penicillin G (10,000 units/mL), streptomycin sulfate (10,000 µg/mL), and 10% heat-inactivated fetal bovine serum (FBS; Tissue Culture Biologicals, Tulare, CA). HMECs (Cambrex, Walkersville, MD) were grown in MEGM according to the instructions of the manufacturer.

Production of recombinant soluble TRAIL. Competent BL-21 cells (Novagen, Madison, WI) were transformed with pET15b plasmid (Novagen) containing a partial TRAIL cDNA encoding amino acids 95 to 281 with an inframe Flag and His6 tag (22). After inducing TRAIL expression by adding 2 mmol/L isopropyl ß-D-1-thiogalactopyranoside to bacteria in log-phase growth, the recombinant His6-tagged protein was purified on Ni2+-NTA columns under native conditions using the QIAexpress system (Qiagen, Valencia, CA) as previously described (23, 24). Purified TRAIL was stored in aliquots at –80°C in 10% glycerol.

Cell viability assay. Cells (5.0 x 103-2.0 x 104) were seeded in 96-well plates and the next day (50-75% cell confluency) treated with various concentrations of CDDO, CDDO-Im, and TRAIL. Cytotoxicity was determined using the CellTiter96 AQueous one solution cell proliferation assay (Promega, Madison, WI) according to the instructions of the manufacturer. Plating cells at various dilutions confirmed assays were done within the linear range of the assay. For some experiments, the TRAIL-neutralizing antibody (clone 2E5, Abcam Ltd., Cambridge, United Kingdom) was preincubated with recombinant TRAIL for 30 minutes before challenging cells. LNCaP LN3 cells were seeded on polylysine-coated plates to maximize cell adherence.

Apoptosis assay. Cells (5 x 105) were seeded in six-well plates and the next day treated with CDDO, CDDO-Im, TRAIL, DR4 monoclonal antibody (TRAIL-R1), DR5 (TRAIL-R2), or various combinations of these reagents. After 12 or 24 hours, both adherent and floating cells were collected and stained with Annexin V and propidium iodide using the Annexin V-FITC apoptosis detection kit (Biovision, Mountain View, CA) per instructions of the manufacturer. Ten thousand cells per treatment were analyzed using a flow cytometer (Becton Dickinson FACSort, Franklin Lakes, NJ). TRAIL-R1 and TRAIL-R2 antibodies were kindly provided by Human Genome Sciences (Rockville, MD; ref. 25).

Cell surface DR4/DR5 analysis. Cells (3.2 x 106 T47D and 2.0 x 106 MDA-MB-468) were seeded in 100-mm dishes and the next day treated with CDDO or CDDO-Im. After 18 hours, adherent cells were washed once with PBS (pH 7.4), detached using a trypsin-free chelating solution (26), and resuspended in ice-cold fluorescence-activated cell sorting (FACS) buffer (3% heat-inactivated FBS in PBS). Following centrifugation, cells were resuspended in FACS buffer yielding 220,000 cells/50 µL and incubated on ice for 15 minutes with 50 µg/mL human {gamma}-globulin (Cappel, Aurora, OH). Then cells were incubated in the dark on ice with saturating concentrations of phycoerythrin-labeled anti-DR4, anti-DR5, or immunoglobulin G1 isotype control antibodies (eBioscience) per instructions of the manufacturer. After 1 hour, cells were washed once with FACS buffer and analyzed by flow cytometry. A total of 10,000 events were analyzed for each treatment.

Reverse transcription-PCR. Reverse transcription PCR (RT-PCR) was done on total RNA prepared from T47D and MDA-MB-468 cells using the RNeasy Mini Kit (Qiagen). Primer sequences for each of the genes analyzed are as follows: DR4 forward primer: 5'-TGTTGTTGCATCGGCTCAGGTTGT-3', DR4 reverse primer: 5'-GAGGCGTTCCGTCCAGTTTTGTTG-3'; DR5 forward primer: 5'-GAGCGGCCCCACAACAAAAGAGGT-3', DR5 reverse primer: 5'-CAAGACTACGGCTGCAACTGTGAC-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, all purchased from Clontech, (Palo Alto, CA). The linear range for GAPDH was determined to be between 20 and 30 cycles when 100 ng of total RNA were provided as template.

RT-PCR was done using the SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA), using 100 ng total RNA as template in each reaction. The thermocycler was programmed as follows: reverse transcription reaction, 50°C for 30 minutes; post reverse transcription denaturation, 94°C for 2 minutes, 25 cycles of 94°C for 30 seconds, 54°C for 30 seconds, 72°C for 45 seconds; elongation step, 72°C for 10 minutes, then samples were held at 4°C. The primer pairs for all genes were specifically selected such that all reactions could be done simultaneously using a 54°C annealing temperature. RT-PCR products were analyzed using a 1.0% agarose gel and stained with ethidium bromide for visualization by UV transillumination. Gels were imaged using a ChemiImager 4000 (Alpha Innotech, San Leandro, CA) equipped with a multiImage light cabinet. Software from Alpha Innotech was used to quantify bands, normalizing data relative to GAPDH.

Immunoblotting. Breast cancer cells at 4 x 106/100-mm dish or HMEC at 1.5 x 106/100-mm dish were seeded and treated 1 day later with various agents (note: for DR4 and DR5 immunoblotting 3.2 x 106 T47D and 2.0 x 106 MDA-MB-468 cells were used, similar to FACS analysis). For caspase-independent experiments, cells were pretreated for 1 hour with 100 µmol/L z-VAD-fmk (MP Biomedicals, Irvine, CA). Cells were washed once with PBS, scraped into radioimmunoprecipitation assay buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Sigma, St. Louis, MO), incubated on ice for 30 minutes, passed eight times through a 21-gauge needle, further incubated on ice for 30 minutes, pelleted by centrifugation at 15,000 x g for 20 minutes, and the supernatant was stored at –80°C. The DC Protein Assay (Bio-Rad, Hercules, CA) was used to determine protein concentrations. Cell lysates (35-75 µg) were subjected to SDS-PAGE (8-12% gels) and blotted onto 0.45 µm nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Membranes were probed with the following antibodies: 1:1,000 (v/v) anti-FADD (Upstate, Lake Placid, NY), 1:1,000 (v/v) anti–polyADP ribosylpolymerase (PARP) clone C2-10 (BD Biosciences, San Jose, CA), 1:1,000 (v/v) anti–caspase-8 clone 5F7 (Upstate), 1:500 (v/v) anti–FLIP antibody Dave-2 (Alexis), 1:500 (v/v) anti–FLIP antibody NF6 (Alexis), 1:1,000 (v/v) anti–TRAIL-R2 (Axxora, San Diego, CA), 1:500 (v/v) anti–DR4/TRAIL-R1 (Upstate), 1:2,000 (v/v) monoclonal anti–{alpha}-tubulin clone DM1A (Sigma), and 1:1,000 (v/v) anti-BID (Cell Signaling, Beverly, MA). Secondary antibodies used were all horseradish peroxidase conjugated (Amersham, Piscataway, NJ) and used at 1:2,000 (v/v) dilution. Proteins were visualized using an enhanced chemiluminescence substrate (supersignal; Pierce, Rockford, IL).

Tumor xenograft experiments. MDA-MB-468 cells (4.8 x 106) resuspended in 100 µL of serum-free DMEM were s.c. injected into the flanks of 4-week-old female Balb/c nu/nu mice using a 23-gauge needle. When tumor volumes reached 25 mm3 (about 14 days), animals were treated daily for 14 days with i.p. injections of CDDO-Im [in 10% Cremephor-El (Sigma), 80% PBS, and 10% DMSO], then 6 hours later (where indicated) given i.p. injections of 5 mg/kg/d of TRAIL (in PBS). Tumor volume was measured every other day using vernier calipers and tumor volume calculated using the following formula: (long axis x short axis2)/2.

Tissue and blood analysis. Mice were anaesthetized (n = 5/group) using Avertin and blood collected via cardiac puncture. Serum chemistry and blood cell analysis were done by the animal care program diagnostic laboratory at the University of California, San Diego. Anaesthetized mice were then transcardinally perfused with ice-cold PBS (pH 7.4) for 2 minutes, followed by cold zinc-containing buffered formalin (Z-fix, Anatech, Inc., Battle Creek, MI) for 5 to 10 minutes. After perfusion, tissues were immediately removed, postfixed in Z-fix, and embedded in paraffin. Dewaxed tissue sections (4.0 µm) were stained using H&E. For proliferating cell nuclear antigen (PCNA) staining, tumor sections were stained with a mouse monoclonal anti-PCNA antibody (DakoCytomation, Inc., Carpinteria, CA) and SG-Vector chromagen (Vector Lab, Inc., Burlingame, CA) yielding a black color. Nuclear red (DakoCytomation) was used as a counterstain. The detection of nuclei with fragmented DNA by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) was accomplished using the ApopTag Peroxidase In situ Apoptosis Detection Kit (Chemicon, Temecula, CA) according to the instructions of the manufacturer. Methyl green was used as a counterstain for TUNEL staining. The percentage of PCNA and TUNEL staining in tissues was quantified using image analysis software (Image Pro Plus).

Statistical analysis. Unless otherwise noted, data were analyzed using a nonparametric one-way ANOVA with a Bonferroni posttest. The confidence interval was set at 95% unless otherwise indicated.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CDDO and CDDO-Im sensitize breast cancer cells to TRAIL-induced apoptosis. We examined breast, prostate, and ovarian cell lines for TRAIL sensitivity using an extracellular domain of recombinant soluble TRAIL (amino acids 95-281), which had been tagged with both FLAG and His6 (22). In brief, cells were treated for 24 hours with TRAIL and then assessed for cell viability using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Consistent with previous reports, PPC-1 (27), OVCAR-3 (19), and PC-3M LN4 cells were found to be TRAIL sensitive whereas LNCaP LN3, T47D (27), MCF-7 (27), MDA-MB-468 (27), and HMEC (27) cells were TRAIL resistant (Fig. 1A). To confirm the specificity of these results, we preincubated recombinant TRAIL with a TRAIL neutralizing antibody, which completely abrogated TRAIL-induced killing (data not shown). Thus, whereas some prostate and ovarian cancer lines are intrinsically sensitive to TRAIL, three of three breast cancer lines were determined to be TRAIL resistant.



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  		Figure 1. Triterpenoids sensitize tumor cells to TRAIL. A, cell lines were cultured with various concentrations of His6-FLAG–tagged recombinant TRAIL. After 24 hours, the MTS assay was used to determine cell viability. Points, mean percentage relative to control-treated cells (n = 3); bars, SE. B, MDA-MB-468, T47D, and HMEC cells were treated simultaneously with various concentrations of either CDDO or CDDO-Im, in combination with either 200 ng/mL TRAIL ({circ}) or vehicle ({bullet}), and then analyzed 24 hours later for cell viability by MTS assay. Points, mean percent of control cells (n = 3); bars, SE. C, MDA-MB-468 cells were sequentially treated, first with various concentrations of CDDO/CDDO-Im for 24 hours, then cultured without ({bullet}) or with ({circ}) 100 ng/mL TRAIL for 24 hours. Percentage of viable cells was determined by MTS assay. Points, mean (n = 3); bars, SE.

 
Previously, it was shown that CDDO and TRAIL cooperate to induce apoptosis of ovarian, prostate, and colon cancer cell lines (19). Because breast cancer cell lines were found to be TRAIL resistant, we sought to determine whether synthetic triterpenoids, CDDO and CDDO-Im, would also cooperate with TRAIL to induce apoptosis of MDA-MB-468 and T47D breast cancer cells. Comparisons were made with normal mammary epithelial cells (HMEC). TRAIL-resistant cells were treated simultaneously with subtoxic doses of either CDDO or CDDO-Im, in combination with low-dose TRAIL (≤250 ng/mL), and cell viability was determined after 24 hours. Both CDDO and CDDO-Im converted the TRAIL-resistant breast cancer cells to TRAIL sensitive (Fig. 1B). In contrast, CDDO and CDDO-Im did not sensitize normal HMECs to TRAIL-induced apoptosis (Fig. 1B). Furthermore, by using a sequential treatment protocol, where cells were first treated with CDDO or CDDO-Im for 24 hours followed by TRAIL treatment, we were able to reduce the triterpenoid dose needed to sensitize breast cancer cell lines to the low nanomolar range (Fig. 1C).

MTS assays determine the relative number of viable cells, which can be affected by both cell death and cell proliferation. To directly evaluate the effects of triterpenoids on cell death, breast cancer cells were cultured with CDDO or CDDO-Im in combination with TRAIL and then subjected to Annexin V/propidium iodide staining. Using the accepted criterion that apoptotic cells are Annexin V–positive/propidium iodide–negative, we found that the combination of TRAIL and either CDDO or CDDO-Im induced both MDA-MB-468 and T47D breast cancer cells to undergo apoptosis at frequencies that were more than additive, compared with cells treated with TRAIL or triterpenoids individually (Fig. 2). Apoptosis induced by the combination of TRAIL and triterpenoids was completely blocked when cells were preincubated for 30 minutes with 50 µmol/L z-VAD-fmk (data not shown), a broad-spectrum caspase inhibitor, thus confirming a caspase-dependent mechanism. Accumulation of Annexin V–positive/propidium iodide–positive cells in cultures treated with triterpenoids and TRAIL could be due to secondary necrosis of cells that originally succumbed to apoptosis.



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  		Figure 2. Combined treatment with triterpenoids and TRAIL induces apoptosis of breast cancer cells. MDA-MB-468 and T47D cells were treated with either CDDO (0.5 µmol/L for MDA-MB-468 and 0.85 µmol/L for T47D cells) or CDDO-Im (0.5 µmol/L), in combination with 100 ng/mL TRAIL, and assayed for apoptosis after 12 hours by Annexin V/propidium iodide staining. Annexin V (x-axis) versus propidium iodide (y-axis) staining. Regions corresponding to viable (bottom left), apoptotic (bottom right), and necrotic (top right) cells are shown. Representative of three independent experiments.

 
CDDO and CDDO-Im sensitize breast cancer cells to agonistic anti-DR4 and anti-DR5 monoclonal antibodies. TRAIL can stimulate apoptosis through either of the two death receptors, DR4 (2) or DR5 (3), also known as TRAIL-R1 and TRAIL-R2. We explored the effects of CDDO and CDDO-Im on apoptosis induction of breast cancer cell lines using agonistic monoclonal antibodies that bind selectively to either DR4 or DR5. Like TRAIL, neither anti-DR4 nor anti-DR5 induced significant amounts of apoptosis in cultures of MDA-MB-468 (Fig. 3) or T47D (data not shown) breast cancer cells. In contrast, addition of CDDO or CDDO-Im to cultures sensitized breast cancer cells in a concentration-dependent manner to apoptosis induction by both anti-DR4 and anti-DR5, with DR4 more potent than DR5 (Fig. 3). The combination of anti-DR4 and anti-DR5 was not superior to anti-DR4 alone or to TRAIL (Fig. 3).



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  		Figure 3. Triterpenoids sensitize breast cancer cells to anti-DR4 and anti-DR5 monoclonal antibodies. MDA-MB-468 cells were treated with various concentrations of either CDDO (A) or CDDO-Im (B), in combination with TRAIL (100 ng/mL), anti-DR4 antibodies (5.0 µg/mL), anti-DR5 antibodies (5.0 µg/mL), both anti-DR4 and anti-DR5, or vehicle control. Cells were stained for Annexin V/propidium iodide after 24 hours and the percentage of Annexin V–positive cells was determined. Representative of three independent experiments.

 
TRAIL-mediated apoptosis is blocked upstream of caspase-8 in MDA-MB-468 and T47D cells. To pinpoint the defect in TRAIL-mediated apoptosis in breast cancer cells, we analyzed (a) caspase-8 cleavage, a proximal event in the TRAIL-induced apoptotic pathway; (b) expression of FADD (28), an adapter protein bridging caspase-8 to DR4/DR5; and (c) BID (29) cleavage, a caspase-8 substrate, following TRAIL treatment of MDA-MB-468 and T47D cells. TRAIL treatment alone failed to induce detectable caspase-8 and BID processing or to alter FADD levels, as did treatment with either CDDO or CDDO-Im (Fig. 4). However, treatment with TRAIL in combination with either CDDO or CDDO-Im promoted robust caspase-8 and BID processing. TRAIL and CDDO or CDDO-Im, in combination but not individually, also induced proteolytic processing of PARP, converting the 116-kDa protein to the 85-kDa form indicative of caspase-mediated cleavage (Fig. 4). These data indicate that the block in the TRAIL-mediated apoptotic pathway occurs upstream or at the level of caspase-8 activation.



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  		Figure 4. Triterpenoids collaborate with TRAIL to promote proteolytic processing of caspase-8, BID, and PARP. The effects of CDDO (0.7 µmol/L) and CDDO-Im (0.7 µmol/L), with and without TRAIL (200 ng/mL), on FADD, caspase-8, BID, and PARP were evaluated by immunoblotting in T47D and MDA-MB-468 cells. Both adherent and floating cells were collected for analysis after 18 hours of treatment. Cell lysates were prepared and normalized for total protein content. For PARP, black arrow indicates the 116 kDa full-length PARP and white arrow indicates the 85 kDa cleaved fragment.

 
CDDO and CDDO-Im down-regulate FLIPL. Previously we showed that CDDO down-regulates the antiapoptotic protein FLIP in epithelial cancer cells (19) and leukemias (13, 20). We therefore examined FLIP expression in breast cancer cells following CDDO and CDDO-Im treatment. Both CDDO and CDDO-Im induced dose-dependent reductions in the levels of FLIPL in MDA-MB-468 and T47D cells (Fig. 5A). However, FLIPL down-regulation by CDDO was consistently more robust compared with CDDO-Im, suggesting differences in the interactions of these compounds with cellular targets relevant to FLIPL regulation. Down-regulation of FLIPL by CDDO and CDDO-Im was caspase independent because z-VAD-fmk failed to prevent triterpenoid-induced reductions in FLIPL (Fig. 5B). Note that the FLIPS expression in these breast cancer cells was very low (data not shown) and triterpenoids did not alter the expression.



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  		Figure 5. CDDO and CDDO-Im down-regulate FLIPL, A, cells were treated for 18 hours with various concentrations of CDDO or CDDO-Im. Lysates were prepared, normalized for total protein content, and analyzed by SDS-PAGE/immunoblotting using anti-FLIP and anti-tubulin antibodies. B, cells were pretreated for 1 hour with 100 µmol/L z-VAD-fmk before CDDO and CDDO-Im treatment for 18 hours (CDDO concentrations used: 0.4 µmol/L for MDA-MB-468 and 1.0 µmol/L for T47D; CDDO-Im concentrations used: 0.75 µmol/L for MDA-MB and 1.0 µmol/L for T47D cells). Cell lysates were prepared and analyzed by immunoblotting as above.

 
Comparison of the concentrations of CDDO or CDDO-Im required to down-regulate FLIP (Fig. 5) with the doses that sensitized breast cancer cells to TRAIL-induced apoptosis (Fig. 1B) suggests that FLIP may not be the only target of these triterpenoids, which is relevant to apoptosis sensitization. For example, in contrast to MDA-MB-468 cells, the concentrations of triterpenoids capable of reducing FLIP levels in T47D cells were less than the doses needed to sensitize to TRAIL-induced apoptosis.

We therefore explored whether triterpenoids can modulate TRAIL sensitivity in cells lacking FLIP. Using flip+/– and flip–/– mouse embryonic fibroblasts (30), we explored the requirement for FLIP for CDDO-Im to sensitize to TRAIL. Absence of FLIP rendered mouse embryonic fibroblasts sensitive to TRAIL-induced apoptosis, whereas FLIP-expressing mouse embryonic fibroblasts were intrinsically resistant to TRAIL (Fig. 6A), consistent with FLIP playing an important role in controlling TRAIL resistance. However, when added to cultures of TRAIL-treated mouse embryonic fibroblasts, CDDO-Im promoted TRAIL-induced apoptosis in both flip+/– and flip–/– cells (Fig. 6B). These data therefore suggest that triterpenoids can sensitize cells to TRAIL through FLIP-independent mechanisms.



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  		Figure 6. Studies with flip–/– cells. A, mouse embryonic fibroblasts collected from flip+/– ({circ}) and flip–/– ({bullet}) embryos were treated for 24 hours with TRAIL, and then assayed for cell viability using the MTS assay. B, flip+/– and flip–/– mouse embryonic fibroblasts were cultured with various concentrations of CDDO-Im in the presence of TRAIL (250 ng/mL) for 24 hours and then assayed for cell viability using the MTS assay. Points, mean percentage relative to control-treated cells (n = 3); bars, SE. Representative of three independent experiments.

 
CDDO and CDDO-Im up-regulate surface DR4 and DR5. To search for other mechanisms by which triterpenoids might alter sensitivity of breast cancer cells to TRAIL, we evaluated the effects of CDDO and CDDO-Im on expression of proteins involved in TRAIL-mediated apoptosis, including DR4, DR5, FADD, DAP3, BID, and caspases 8 and 10. CDDO and CDDO-Im did not alter expression of FADD, DAP3, BID, or caspases in the absence of TRAIL (Fig. 4 and data not shown). In contrast, CDDO-Im, and to a lesser extent CDDO, induces increases in one or both TRAIL death receptors (DR4 and DR5).

Figure 7 shows results from experiments where we examined the effects of triterpenoids on cell surface expression of DR4 and DR5 in MDA-MB-468 and T47D, using flow cytometry. CDDO-Im increased cell surface DR4 and DR5 expression in a concentration-dependent manner on both MDA-MB-468 and T47D cells (Fig. 7). At equimolar concentrations, CDDO was less potent than CDDO-Im at altering cell surface DR4/DR5 levels, substantially increasing DR5 on T47D but not MDA-MB-468, and causing only a slight increase in DR4 expression on either MDA-MB-468 or T47D cells (Fig. 7).



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  		Figure 7. CDDO and CDDO-Im increase cell surface expression of DR4 and DR5. Cell surface DR4 and DR5 expression was measured by flow cytometry on MDA-MB-468 and T47D cells following CDDO or CDDO-Im treatment for 18 hours using anti-DR4 and anti-DR5 antibodies conjugated to phycoerythrin (shaded no line, DMSO vehicle treatment with isotype matched control antibody; thin line, DMSO vehicle with either DR4 or DR5 staining; thick line, triterpenoid with either DR4 and DR5 staining). Bar graphs indicate fold increase in mean fluorescence intensity of DR4 and DR5 surface expression relative to vehicle control. Triterpenoid concentrations used range from 0.25 to 1.0 µmol/L (except for CDDO-treated T47D cells where 0.5-2.5 µmol/L was used). Representative of two independent experiments.

 
Next, we analyzed the effects of CDDO and CDDO-Im on DR4 and DR5 protein expression by immunoblotting. CDDO-Im increased cellular levels of DR5 protein in a dose-dependent manner (Fig. 8A), consistent with the observed increase in surface DR5 detected by flow cytometry. Note that the DR5 antibody used in these experiments recognized two DR5 splice variants, with approximate molecular weights of 46-kDa [DR5S(Short)] and 52-kDa [DR5 L(Long)], similar to previous reports (3, 1). The increase in DR5S was more striking than DR5L. Densitometry analysis indicated that CDDO-Im increased DR5S protein levels by 2.0- to 3.5-fold relative to untreated cells (Fig. 8A). In contrast to DR5, CDDO-Im did not detectably alter DR4 levels as assessed by immunoblotting (Fig. 8A), despite increasing DR4 levels on the cell surface (Fig. 7). However, because the extent of DR4 up-regulation on the cell surface was more modest than DR5 (Fig. 7), the failure to detect an increase in total cellular DR4 protein levels may be due to limitations in the sensitivity of immunoblotting methods compared with flow cytometry. We cannot exclude the possibility, however, that CDDO-Im induces redistribution of DR4, causing more receptors to traffic to the cells surface. Increases in DR4 and DR5 proteins were not evident by immunoblot analysis of cell lysates derived from MDA-MB-468 and T47D breast cancer cells treated with CDDO (data not shown). Thus, whereas a modest increase in surface DR4 and DR5 was detected by flow cytometry analysis of CDDO-treated breast cancer cells, we were unable to confirm an increase in total cellular DR4 and DR5 by immunoblotting, suggesting either a limitation in the sensitivity of the immunoblotting method employed or possibly receptor redistribution.



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  		Figure 8. Analysis of effects of triterpenoids on DR4 and DR5 expression. A, immunoblotting was used to quantify total DR4 and DR5 protein levels in whole cell lysates following 18 hours of CDDO-Im treatment, using cell lysates normalized for total protein content. Graph (bottom) indicates relative DR5S expression (normalized to both loading and vehicle controls), as measured by scanning densitometry. B, semiquantitative RT-PCR was used to quantify the DR5 mRNA levels following 16 hours of CDDO-Im treatment (normalized to both loading and vehicle controls). Graph (bottom) represents densitometry quantification of the data.

 
To determine whether CDDO-Im regulates DR5 gene expression at the mRNA level, we did semiquantitative RT-PCR to detect DR5 mRNA in MDA-MB-468 and T47D cells following 16 hours of CDDO-Im treatment. Figure 8B shows that CDDO-Im increased DR5 mRNA levels in both of these breast cancer cell lines, with MDA-MB-468 more affected than T47D. Image analysis of the ethidium-stained gels suggested that CDDO-Im induced a modest 25% to 75% increase in DR5 mRNA levels. These data suggest that CDDO-Im either stabilizes DR5 mRNA or increases DR5 mRNA transcription.

In addition to analyzing cell surface DR4 and DR5 death receptors, we also examined the cell surface TRAIL decoy receptors, DcR1 and DcR2, on T47D and MDA-MB-468 cells following CDDO and CDDO-Im treatments (data not shown). CDDO did not alter the number of decoy receptors in MDA-MB-468 and T47D cells. In contrast, CDDO-Im induced changes in surface decoy receptor expression in both breast cancer cell lines. For T47D cells, CDDO-Im increased surface DcR1 by 1.3-fold but decreased surface DcR2 by 0.7-fold, suggesting no net gain of surface decoy receptors. For MDA-MB-468 cells, CDDO-Im increased DcR1 by 1.4-fold and DcR2 by 1.2-fold on the cell surface (data not shown). Because CDDO-Im induced a 1.3-fold increase in DR5 and 1.7-fold in DR4, the ratio of death receptors (DR4 and DR5) to decoy receptors (DcR1 and DcR2) presumably remains in favor of death receptors in MDA-MB-468 cells. We speculate, therefore, that CDDO-Im influences death receptor up-regulation to a greater extent than decoy receptor up-regulation, creating a more TRAIL-sensitive environment.

CDDO-Im cooperates with TRAIL to inhibit MDA-MB-468 xenograft tumor growth in vivo. To determine whether CDDO-Im in combination with TRAIL could inhibit tumor growth in vivo, MDA-MB-468 tumor–bearing mice were treated for 14 days with CDDO-Im (5 mg/kg/d), TRAIL (5 mg/kg/d), the combination of CDDO-Im and TRAIL, or vehicle control. The combination of CDDO-Im and TRAIL significantly inhibited tumor growth compared with either agent alone or vehicle control (Fig. 9). Following treatment termination, tumor growth was monitored for an additional 14 days. Tumors in the CDDO-Im and TRAIL treatment group resumed growth (data not shown), thus indicating the persistent need for CDDO-Im and TRAIL to maintain suppression of tumor growth in this xenograft model.



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  		Figure 9. CDDO-Im in combination with TRAIL suppresses MDA-MB-468 tumor growth in nude mice. Animals bearing pre-established tumors (n = 11 per group) were dosed daily for 14 days with i.p. injections of CDDO-Im (5 mg/kg/d) in the morning and TRAIL (5 mg/kg/d) in the afternoon. During the 14-day treatment, tumor volumes were estimated using measurements taken from external calipers (mm3). Points, mean; bars, SE. Statistical significance was assessed by ANOVA (P ≤ 0.01), and significant differences are indicated with asterisks, comparing the combination CDDO-Im and TRAIL group with the other three groups.

 
Histologic analysis of the tumors from mice sacrificed at day 15, following 14 days of treatment, indicated substantially fewer viable tumor cells in the animals treated with the combination of CDDO-Im and TRAIL compared with other groups. Areas of confluent necrosis, however, precluded accurate quantification of the percentage of TUNEL-positive cells. Proliferation marker analysis suggested fewer PCNA-immunopositive cells in tumors subjected to combination CDDO-Im/TRAIL treatment, implying either preferential killing of proliferating tumor cells or an antiproliferative effect (not shown).

In vivo toxicity associated with CDDO-Im and TRAIL combination therapy was analyzed using several different toxicology variables including animal weight, behavior, blood chemistry, and tissue analysis. Animal weight changes were recorded over 14 days of combination CDDO-Im and TRAIL treatment and during a 14-day follow-up, and compared with weight changes associated with single agent and vehicle control treatments. The combination of CDDO-Im and TRAIL treatment resulted in significant weight loss by day 3; however, by day 12, body weight had returned and surpassed baseline weight (Fig. 10A). Also, no significant weight changes were noted during the 14-day follow-up (data not shown). Single agent treatment with CDDO-Im, TRAIL, or vehicle did not induce significant weight loss over the 28-day experiment.



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  		Figure 10. Toxicology analysis of combination treatment with CDDO-Im and TRAIL. In vivo toxicity associated with CDDO-Im and TRAIL treatment was assessed in tumor-bearing mice treated with combination CDDO-Im (100 mg/kg/d) and TRAIL (5 mg/kg/d), single agent or vehicle control. A, animal weights were recorded during the 14 days of treatment. Points, mean (n = 11); bars, SE. In the combination CDDO-Im and TRAIL treatment group, statistically significant differences (*, P ≤ 0.05) were found on days 5 and 7, when compared with baseline weight (significance determined by a nonparametric Mann-Whitney test). B, animals were treated for 14 consecutive days as above (n = 5/group) and on day 15 the following blood chemistry variables were analyzed: sodium, potassium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, blood urea nitrogen, creatinine, and glucose. Columns, mean; bars, SE. No statistical differences were found.

 
Animal appearance and behavior (ruffled fur/lethargy) were observed over the 28-day experiment and no differences were noted comparing the control and treatment groups.

H&E staining was also used to ascertain toxicity to mouse tissues [brain, liver, spleen, lung, and kidney (n = 5/group)] following 14 consecutive days of CDDO-Im and TRAIL combination treatment, making comparisons with either agent alone or vehicle control treatment. No histologic differences were detected in brain, liver, and lung tissues among treatment groups. In contrast, mild renal changes were observed in three of five animals treated with TRAIL and in five of five animals treated with combination CDDO-Im and TRAIL (data not shown), and absent from vehicle- or CDDO-Im treated mice. These kidney changes were characterized by cytoplasmic eosinophilia or vacuolization, pyknotic/karyorrhetic nuclei, eosinophilic casts in tubules, and occasional tubular degeneration. In the CDDO-Im and TRAIL combination treatment group, spleens exhibited mild lymphoid cell hyperplasia with an increased proportion of white pulp lymphocytes in five of five animals (data not shown). The spleens from vehicle and single-drug groups maintained normal morphology.

To further ascertain in vivo toxicity, we analyzed serum chemistries, including electrolytes (sodium, potassium), glucose, liver transaminases (alanine aminotransferase, aspartate aminotransferase), alkaline phosphatase, and markers of renal function (blood urea nitrogen, creatinine), in tumor-bearing mice on day 15 following 14 consecutive days of treatment. No significant differences were noted when comparing the combination treatment group (CDDO-Im + TRAIL) with single agent treatment or vehicle control (Fig. 10B), suggesting that the combination of CDDO-Im and TRAIL is well tolerated in vivo.

The safety of TRAIL is well documented by preclinical animal studies (reviewed in refs. 1, 31). However, much less is known about the in vivo safety of CDDO-Im. To determine whether higher CDDO-Im doses (>5 mg/kg/d) are safe in vivo, tumor-bearing mice were treated for 14 consecutive days with i.p. injections of 7.5 mg/kg CDDO-Im, 10.0 mg/kg CDDO-Im, or vehicle; then on day 15, blood and tissue samples were collected for analysis. CDDO-Im delivered at 10.0 mg/kg/d for 14 days was well tolerated in mice; only mild anemia (RBC count 12.5% below normal and hematocrit 15.5% below normal) was observed, without changes in WBC or platelet counts, serum chemistries, or tissue histology (data not shown). CDDO-Im caused a slight decrease in animal weight, peaking at day 5, but weight returned and surpassed baseline by the end of the treatment (data not shown). No changes in gross appearance or behavior (ruffled fur or lethargy) were noted over the 14 days of dosing. When taken together with the aforementioned experiments using CDDO-Im at 5 mg/kg/d, these data suggest that CDDO-Im, at doses of 5 to 10 mg/kg daily, is well tolerated in mice. In tumor-bearing mice, no inhibition of MDA-MB-468 xenograft growth was seen in animals treated with CDDO-Im (7.5-10 mg/kg/d) compared with vehicle control–treated mice (data not shown), thus emphasizing the importance of including TRAIL in combination with CDDO-Im to achieve tumor growth suppression in this breast cancer model.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented herein indicate that CDDO and CDDO-Im sensitize breast cancer cells to TRAIL-mediated apoptosis in vitro. Sensitization of tumor cells to TRAIL by CDDO and CDDO-Im was associated with up-regulation of cell surface death receptors DR4 and DR5 and with down-regulation of the antiapoptotic protein FLIPL. CDDO/CDDO-Im–mediated reductions in FLIPL occurred via a caspase-independent mechanism, as shown by the failure of z-VAD-fmk to abrogate this effect.

CDDO/CDDO-Im induced down-regulation of FLIPL and up-regulation of cell surface DR4 and DR5 in breast cancer cells. We speculate that both of these mechanisms influence TRAIL sensitivity in breast cancer cells. However, further studies are necessary to establish the functional significance of these triterpenoid-induced alterations in expression of FLIP and TRAIL receptors. Studies with flip–/– mouse embryonic fibroblast cells show that CDDO-Im sensitizes breast cancer cells to TRAIL even in the absence of FLIP, indicating that FLIP-independent mechanisms contribute to the TRAIL-sensitizing activity of this compound.

The mechanism by which CDDO and CDDO-Im alter cell surface DR4 and DR5 expression is currently unknown; it might involve disruption of intracellular redox balance leading to subsequent c-jun-NH2-kinase (JNK) activation. In this regard, recently, Yue et. al. (32) found that depletion of antioxidant glutathione (GSH) contributed to JNK activation and subsequent DR5 up-regulation. Moreover, it has been shown that CDDO, CDDO-Me, and CDDO-Im increase intracellular reactive oxygen species and decrease intracellular GSH levels, leading to JNK activation (14). Taken together, these findings raise the possibility that CDDO and its derivatives may up-regulate DR5 (perhaps also DR4) through effects on intracellular redox balance. Interestingly, the stability of the FLIPL protein is also known to be regulated by oxidative stress (33). Thus, triterpenoid-induced oxidative stress could conceivably provide a unifying mechanism to explain both up-regulation of TRAIL receptors and down-regulation of FLIP.

This report provides the first preclinical data concerning the in vivo efficacy and safety of the combination of a synthetic triterpenoid and TRAIL. Our data showed apparent synergy of CDDO-Im and TRAIL with respect to suppression of tumor growth in mice. At doses resulting in significant suppression of tumor xenograft growth, the combination of CDDO-Im and TRAIL was well tolerated in mice, causing little apparent toxicity to normal tissues. CDDO-Im used alone (at >5 mg/kg/d) caused mild anemia and weight loss, but overall was well tolerated, consistent with a previous report (12). TRAIL and the combination of TRAIL and CDDO-Im may provoke mild histologic changes in kidney, warranting further investigation of kidney toxicity following TRAIL or combination TRAIL and CDDO-Im treatment. Combination treatment with TRAIL and CDDO-Im also caused transient weight loss, which spontaneously reversed even with continued treatment. Altogether, these preclinical data suggest that CDDO-Im, alone and in combination with TRAIL, is safe in mice, thus setting the stage for more rigorous analysis of the combination of CDDO-Im and TRAIL in primates as an antecedent to human clinical trials.


   Acknowledgments
 
Grant support: Department of Defense Prostate Cancer Postdoctoral Traineeship Award (M.L. Hyer), NIH Specialized Programs of Research Excellence in Breast Cancer (V.L. Cryns and M. Lu), NIH award (CA-78814; M.B. Sporn), the National Foundation for Cancer Research (M.B. Sporn), and the California Breast Cancer Research Program (8WB-0079; J.C. Reed). M.B. Sporn is an Oscar M. Cohn Professor.

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.

We thank Mike Thomas, Steven Banares, Gennadi Glinsky, and Kelly Mckaig for advice and technical assistance; Robin Humphreys and colleagues at Human Genome Sciences for kindly providing the DR4 and DR5 agonistic antibodies; Wen-Chen Yeh and Tak Mak for providing the flip+/– and flip–/– mouse embryonic fibroblasts; Tadashi Honda for synthesizing the CDDO and CDDO-Im; and Judie Valois and Melanie Hanaii for manuscript preparation.

Received 9/13/04. Revised 2/11/05. Accepted 3/24/05.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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