Normal tissue toxicity markedly reduces the therapeutic index of genotoxic anticancer agents, including ionizing radiation. Countermeasures against tissue damage caused by radiation are limited by their potential to also protect malignant cells and tissues. Here, we tested a panel of signal transduction modifiers for selective radioprotection of normal but not tumor tissues. These included three inhibitors of GSK3 (LiCl, SB216763, and SB415286) and two inhibitors of NF-κB (ethyl pyruvate and RTA 408). Among these, the thiol-reactive triterpenoid RTA 408 emerged as a robust and effective protector of multiple organ systems (gastrointestinal, skin, and hemopoietic) against lethal doses of radiation. RTA 408 preserved survival and proliferation of intestinal crypt cells in lethally irradiated mice while reducing apoptosis incidence in crypts and villi. In contrast, RTA 408 uniformly inhibited growth of established CWR22Rv1, LNCaP/C4-2B, PC3, and DU145 xenografts either alone or combined with radiation. Antitumor effects in vivo were associated with reduced proliferation and intratumoral apoptosis and with inhibition of NF-κB–dependent transcription in PC3 cells. Selective protection of normal tissue compartments by RTA 408 critically depended on tissue context and could not be replicated in vitro. Collectively, these data highlight the potential of RTA 408 as a cytoprotective agent that may be safely used in chemoradiation approaches. Mol Cancer Ther; 13(12); 2968–77. ©2014 AACR.

Radiotherapy is the most common therapeutic modality across a wide range of malignant diseases, including prostate cancer. However, the delivery of curative radiation doses is hampered by acute or chronic “collateral damage” affecting normal tissues. When treating tumors in the abdominal cavity, toxicity to the intestine and the bladder are often dose limiting (1). Highly targeted methods to deliver radiation specifically to disease sites alleviate radiation toxicity, yet 40% to 50% of patients with locally advanced prostate cancers recur locally following treatment (2). Hence, protection of normal tissue will be a critical element of future dose-escalation trials in patients with locally advanced prostate cancer. Existing radiation protectors, including amifostine (3), are of limited utility in protecting the small and large intestines against radiation effects.

Inflammation is a key element of the radiation response of normal and tumor tissues and is commonly associated with increased activity of NF-κB (4, 5). Previously, we demonstrated that several inhibitors of canonical NF-κB activation improved survival of lethally irradiated zebrafish embryos and preserved gastrointestinal morphology and function (6). Inhibitors of glycogen synthase kinase (GSK)3 similarly protect normal tissues, including the gastrointestinal tract (7, 8). While the role of GSK3β in cell stress responses is complex (for review, see ref. 9), it has been implicated in modifying NF-κB–dependent transcription of genes encoding proinflammatory proteins (10, 11).

Here, we performed a side-by-side comparison of radioprotective properties of five compounds targeting either GSK3 and/or NF-κB with a focus on the gastrointestinal tract. We report that the triterpenoid RTA 408 provides robust radiation protection to the murine gastrointestinal system and markedly improves overall survival of lethally irradiated mice. Importantly, normal tissue protection by RTA 408 is contrasted by inhibition of human prostate cancer xenograft growth in mice.

Materials and cells

Compounds were obtained from the following sources: Ethyl pyruvate and lithium chloride (Sigma-Aldrich), SB216763 and SB415286 (Tocris Bioscience), amifostine (Medimmune), and 2-cyano-3,12- dioxooleana-1,9 (11)-dien-28-oic acid (CDDO) derivative RTA 408 (REATA Pharmaceuticals). Prostate cancer cells (PC3, LNCaP/C4-2B, DU145, and CWR22Rv1) were originally obtained from ATCC or from Dr. Thomas Pretlow (Case Western Reserve University, Cleveland, OH) and generously provided by Dr. Marja Nevalainen (Thomas Jefferson University, Philadelphia, PA), and immortalized NHPrE-1 and BHPrE-1 prostate epithelial cells were a gift from Dr. Simon Hayward (Vanderbilt University Medical Center, Nashville, TN). Normal primary prostate epithelial cells (PrEC) were from Lonza. The prostate cancer cell lines were authenticated on a regular basis by monitoring cell morphology, androgen responsiveness, and the expression of cell line–specific markers. Normal primary epidermal keratinocyte cultures were established using standard protocols. Cells were routinely tested for mycoplasma contamination using the MycoSensor PCR Assay Kit (Stratagene). Tumor cells were grown in RPMI1640 supplemented with 10% FBS (Corning Cellgro). Normal and immortalized prostate epithelial cells and primary keratinocytes were grown in specialty media (Lonza). For in vivo imaging, PC3 prostate carcinoma cells were stably transfected with reporter plasmids [pGL4.51(luc2/CMV/Neo) and pNL3.2.NF-κB-RE(NlucP/NF-κB-RE/Hygro), Promega] encoding firefly luciferase (FLuc) and NanoLuc luciferase, respectively, and luciferase reporter activity tested using reporter-specific in vitro assays (Promega).

Toxicity studies in mice

C57Bl/6 mice (6–8 weeks old) were from Charles River Laboratories. Mice were kept in pathogen-free conditions and handled in accordance with the requirements of the Guidelines for Animal Experiments and after approval of the experimental protocols by the Institutional Animal Care and Use Committee of Thomas Jefferson University (Philadelphia, PA). Ionizing radiation (IR) was administered at doses ranging from 5 to 30 Gy using a 250-kVp X-ray machine (PanTak) with 50-cm source-to-skin distance and a 2-mm copper filter. The dose rate was approximately 1.4 Gy/minute. Drugs were uniformly administered by intraperitoneal injection for up to 2 days before IR treatment, and on days 1, 2, and 3 after IR treatment as indicated. For comparison of RTA 408 and amifostine, mice received one dose (17.5 mg/kg) of RTA 408 24 hours before IR (whole body, 9 Gy), one dose 1 hour before IR, and 2 additional doses 24 and 48 hours after IR; amifostine was injected once (250 mg/kg) 15 minutes before IR. All injections were done intraperitoneally. Animals were euthanized at the end of the observation period, or when weight loss reached or exceeded 20% of the initial weight, or if they showed signs of severe morbidity (lethargy, hunched posture, and/or shivering or severe diarrhea). Kaplan–Meier survival curves were compared by using the log-rank (Mantel–Cox) test.

Growth inhibition of prostate cancer xenografts

Prostate carcinoma cells were inoculated by subcutaneous injection (5 × 106 per mouse) into the lower abdominal skin of male Foxn1nu (nude) mice (6–8 weeks old; Charles River Laboratories). Tumor progression was monitored by caliper measurements and by in vivo live imaging (see below). Xenografts were allowed to grow for 2 to 3 weeks before treatment. RTA 408 (17.5 mg/kg) or vehicle control (DMSO) were administered intraperitoneally three times per week until the end of the observation periods. To assess effects of the combination of RTA 408 and IR, radiation (5 Gy) was administered at different time points as indicated. Tumor volumes were calculated by multiplying the two longest planar axes measured by the depth of the tumor (as determined by caliper measurements). Mixed effects regression models were used to determine statistical significance of tumor growth data over time.

In situ imaging of xenografts and image analysis

For FLuc-based in vivo live imaging, mice were injected intraperitoneally with 200 μL d-luciferin in PBS (15 mg/mL) per 20 g of mouse body weight 15 minutes before imaging, and imaged using an IVIS In Vivo Imaging System (Caliper Life Sciences). For in vivo imaging of the NanoLuc luciferase under the control of the NF-κB response element, 100 μL of the NanoGlo substrate (10 μg; Promega) were injected via tail vein. Image analysis and quantitation was done using Living image 4.2 software (Caliper LifeSciences). Luciferase-positive areas on individual images were selected as regions of interest (ROI) with a 14% threshold. Planar spectral images were automatically analyzed by the software. Total counts for all pixels inside the ROI were recorded. At least 3 animals from each experimental group were used for each time point.

Histology, immunohistochemistry, and in situ apoptosis detection

For histologic, immunofluorescent, and direct fluorescent analyses, tissue samples (e.g., small intestines, tumors) were embedded in the optimal cutting temperature compound (Tissue-Tek), and cryosectioned (7 μm). Hematoxylin and eosin staining was done on ethanol/acetic acid–fixed slides. Apoptosis incidence was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the In Situ Cell Death Detection Kit (Roche Applied Science) and 4′,6-diamidino-2-phenylindole (DAPI) counterstain. Quantitative analysis was done using ImagePro software (Media Cybernetics) on at least 6 different and independent microscopic fields for each treatment condition. Indirect immunofluorescence was performed by incubation with primary antibodies for cleaved PARP (Asp214) Human Specific (Cell Signaling Technology), M30CytoDEATH (Roche Applied Science), or Ki-67 (Abcam) for 1 hour at room temperature or overnight at 4°C followed by secondary antibodies labeled with either Alexafluor 488 or Alexafluor 594 (BD Biosciences). Sections were counterstained with DAPI and slides were mounted using anti-fade Fluorosafe reagent (Calbiochem).

In vitro cell growth and viability assays

Cells were seeded in 96-well plates at 15,000 cells per well; after 24 hours, RTA 408 or vehicle were added in triplicate. After 72 hours, attached cells were fixed with 70% ethanol, stained with crystal violet solution (0.2% crystal violet in 2% ethanol), and quantitated by measuring absorbance (ODA595). Metabolic activity was determined after 72 hours by addition of WST-1 reagent (Roche; 10 μL per 100 μL supernatant) for at least 3 hours at 37°C, followed by measuring absorbance at ODA450 with a reference of ODA650 and using wells containing media without cells for background subtraction. Statistical differences between treatment groups were determined using one-way ANOVA with Tukey posttest correction (GraphPad Prism).

Immunoblot analyses

Prostate carcinoma cell lines were treated for 24 hours with RTA 408 or vehicle at the concentrations indicated. Immunoblots were reacted with (i) PARP-1 primary antibody (C2-10; Santa Cruz Biotechnology) and IRDye 800CW goat anti-mouse IgG1-specific secondary antibody; (ii) cleaved caspase-3 (Asp175; 5A1E) primary antibody (Cell Signaling Technology) and IRDye 800CW donkey anti-rabbit IgG (H + L) secondary antibody; (iii) cleaved caspase-8 (Asp384; 11G10) primary antibody (Cell Signaling Technology) and IRDye 800CW goat anti-mouse IgG1-specific secondary antibody, or (iv) M30CytoDEATH primary antibody (Roche Applied Science) and IRDye 800CW goat anti-mouse IgG2b-specific secondary antibody; all secondary antibodies were from LICOR. Filters were analyzed on a LICOR Odyssey imaging system.

Colony formation assays

Cells were seeded at clonogenic densities in T-25 flasks and treated with RTA 408 at various concentrations or DMSO, at 24 and 1 hour before radiation exposure. IR was administered at 0, 2, 4, 6, and 8 Gy. All treatments were performed in biologic triplicate. After IR, flasks were incubated for 2 weeks. Colonies were identified by crystal violet staining; those containing ≥50 cells were counted. The data were fit to a linear quadratic model for cell survival by using GraphPad Prism software and the equation Y = exp(−a × xb × x2) (12). Statistically significant differences between drug and control curves were determined by using two-way ANOVA.

Inhibitors of canonical NF-κB activation and of GSK3 improve survival of lethally irradiated mice

We performed a side-by-side comparison of several NF-κB and GSK3 inhibitors on mice challenged with a lethal whole body radiation dose (8 Gy; Fig. 1). All of the compounds reportedly protect against or mitigate radiation injury in different experimental settings either in vitro or in vivo. They included two inhibitors of canonical NF-κB signaling (ethyl pyruvate; ref. 13) and RTA 408 (ref. 14; Fig. 1A), and three GSK3 inhibitors, including lithium chloride (LiCl; ref. 8), SB415286 (8), and SB216763 (ref. 8; Fig. 1B, C). Ethyl pyruvate interferes with NF-κBp65 signaling by covalently modifying a reactive cysteine residue (Cys36) of the NF-κBp65 subunit (15). RTA 408 is a variant of the triterpenoid CDDO that reversibly and covalently modifies reactive cysteine residues on multiple proteins, including several of potential relevance to radiation protection. Specifically, binding of CDDO to Cys179 in IKKβ leads to inhibition of canonical NF-κB signaling (16) and binding to KEAP1 leads to increased levels of the transcription factor Nrf2 and of multiple antioxidant and phase II defense enzymes (17). RTA 408 was included in the screen because we previously observed robust radioprotection of zebrafish embryos by another variant of CDDO (CDDO-TFEA; ref. 6). SB415286 and SB216763 are ATP-competitive GSK3 inhibitors (18), whereas LiCl increases inhibitory phosphorylation of GSK3 (19). To allow direct side-by-side comparison all drugs were given using a standardized regimen, i.e., for 1 day and 1 hour before radiation and daily for 3 days after. Drug dosages were guided by published results and administration was by intraperitoneal injection. We observed various levels of radiation protection with each of the compounds tested. The CDDO derivative RTA 408 provided robust and consistent levels of radiation protection [100% at 30 days post-IR (8 Gy)] either as a single compound (Fig. 1A) or in combination with the GSK3 inhibitor SB216763 (Fig. 1C). In agreement with an earlier report (13), ethyl pyruvate also markedly increased survival of lethally irradiated mice (Fig. 1A). Interestingly, the survival advantage provided by RTA 408 was compromised when combined with SB415286 but not when combined with SB216763 (Fig. 1C). Similarly, survival of lethally irradiated mice treated with a combination of RTA 408 and LiCl was slightly lower than survival of mice receiving RTA 408 alone (Fig. 1C). Finally, RTA 408 produced levels of radiation protection similar to amifostine, the only currently approved radiation protector (Fig. 1D). These results encouraged us to further investigate tissue protection provided by RTA 408 alone.

Figure 1.

Effects of signal transduction modifiers on survival of lethally irradiated mice. A, survival of mice pretreated (24 hours) with the NF-κB inhibitors ethyl pyruvate (EP; 70 mg/kg) or RTA 408 (17.5 mg/kg) and receiving 8 Gy single dose whole body radiation. Ethyl pyruvate was administered 15 minutes before IR and on days 1 to 3 after IR. RTA 408 was administered 1 day and 1 hour preirradiation and on days 1 to 3 after IR. B, survival of mice treated with the GSK3 inhibitors LiCl (40 mg/kg), SB415286 (1 mg/kg), and SB216673 (1 mg/kg) and receiving 8 Gy single dose whole body radiation; treatment schedule for these compounds was 2 and 1 day before IR and daily on days 1 to 3 after IR. C, effects of select combinations of NF-κB and GSK3 inhibitors on survival of irradiated mice (8 Gy); treatment schedules for individual compounds as described under A. D, survival of irradiated mice (whole body, 9 Gy) treated with RTA 408 (17.5 mg/kg) at 24 and 1 hour before IR and on days 1 to 2 after IR or with amifostine (250 mg/kg) administered 15 minutes before IR. Experimental groups in each panel consisted of 5 animals each. P value summaries refer to pairwise comparisons between IR and (IR + drug) treatment groups, generated by using the log-rank (Mantel–Cox) test. *, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ns, not significant.

Figure 1.

Effects of signal transduction modifiers on survival of lethally irradiated mice. A, survival of mice pretreated (24 hours) with the NF-κB inhibitors ethyl pyruvate (EP; 70 mg/kg) or RTA 408 (17.5 mg/kg) and receiving 8 Gy single dose whole body radiation. Ethyl pyruvate was administered 15 minutes before IR and on days 1 to 3 after IR. RTA 408 was administered 1 day and 1 hour preirradiation and on days 1 to 3 after IR. B, survival of mice treated with the GSK3 inhibitors LiCl (40 mg/kg), SB415286 (1 mg/kg), and SB216673 (1 mg/kg) and receiving 8 Gy single dose whole body radiation; treatment schedule for these compounds was 2 and 1 day before IR and daily on days 1 to 3 after IR. C, effects of select combinations of NF-κB and GSK3 inhibitors on survival of irradiated mice (8 Gy); treatment schedules for individual compounds as described under A. D, survival of irradiated mice (whole body, 9 Gy) treated with RTA 408 (17.5 mg/kg) at 24 and 1 hour before IR and on days 1 to 2 after IR or with amifostine (250 mg/kg) administered 15 minutes before IR. Experimental groups in each panel consisted of 5 animals each. P value summaries refer to pairwise comparisons between IR and (IR + drug) treatment groups, generated by using the log-rank (Mantel–Cox) test. *, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ns, not significant.

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RTA 408 protects mice against gastrointestinal syndrome and death after lethal doses of radiation

Next, we investigated the effect of RTA 408 on the small intestine in C57Bl/6 mice irradiated at a dose (9 Gy) that causes death from gastrointestinal syndrome within 10 to 15 days (20). We observed that RTA 408 preserved the integrity of the mucosal lining of the small intestine of lethally irradiated mice (Fig. 2A). Mice that did not succumb to gastrointestinal syndrome lived beyond 30 days after IR, consistent with radiation protection of multiple organs, including the hemopoietic system. Furthermore, as determined by Ki-67 staining RTA 408–treated mice revealed robust proliferation in the crypt area at 2 and 7 days after IR, whereas radiation alone (9 Gy) markedly reduced proliferation in this tissue compartment concurrent with extensive tissue destruction (Fig. 2A). RTA 408 also significantly reduced radiation-induced apoptosis in both villi and crypts as determined by TUNEL staining (Fig. 2B). This effect extended to the skin, in which RTA 408 similarly reduced the apoptosis incidence caused by radiation exposure (Supplementary Fig. S1).

Figure 2.

RTA 408 reduces radiation-associated damage to the mucosal lining of the small intestine after single dose (9 Gy) radiation exposure. RTA 408 (17.5 mg/kg) was administered on days 1 to 3 after IR. A, assessment of gastrointestinal morphology was performed on tissue sections sampled days 2 and 7 after radiation exposure. Parallel sections were subjected to staining with Ki-67–reactive antibody to ascertain the proliferative state of the gastrointestinal stem cell compartment located in the crypt areas; scale bars, 100 μm. B, effects of RTA 408 on radiation-induced apoptosis incidence in the gastrointestinal mucosa. RTA 408 (17.5 mg/kg) was administered 24 hours after IR (9 Gy) and small intestine tissues sampled at 48 hours. Apoptosis incidence was determined by TUNEL staining and cell nuclei were counterstained with DAPI; scale bars, 100 μm. Results shown in B represent mean ± SD of at least 6 fields per condition analyzed. The number of TUNEL-positive cells was significantly (Student t test; *, P < 0.05) reduced in irradiated animals receiving RTA 408 when compared with vehicle-treated, irradiated animals.

Figure 2.

RTA 408 reduces radiation-associated damage to the mucosal lining of the small intestine after single dose (9 Gy) radiation exposure. RTA 408 (17.5 mg/kg) was administered on days 1 to 3 after IR. A, assessment of gastrointestinal morphology was performed on tissue sections sampled days 2 and 7 after radiation exposure. Parallel sections were subjected to staining with Ki-67–reactive antibody to ascertain the proliferative state of the gastrointestinal stem cell compartment located in the crypt areas; scale bars, 100 μm. B, effects of RTA 408 on radiation-induced apoptosis incidence in the gastrointestinal mucosa. RTA 408 (17.5 mg/kg) was administered 24 hours after IR (9 Gy) and small intestine tissues sampled at 48 hours. Apoptosis incidence was determined by TUNEL staining and cell nuclei were counterstained with DAPI; scale bars, 100 μm. Results shown in B represent mean ± SD of at least 6 fields per condition analyzed. The number of TUNEL-positive cells was significantly (Student t test; *, P < 0.05) reduced in irradiated animals receiving RTA 408 when compared with vehicle-treated, irradiated animals.

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RTA 408 inhibits cell growth and survival of human prostate cancer in vivo

To address whether the cytoprotective effects of RTA 408 extended to tumor cells, we first tested the effects of RTA 408 on four different prostate cancer cell lines representing advanced, androgen-independent tumor stages (LNCaP/C4-2B, CWR22Rv1, DU145, and PC3). We observed robust tumor growth inhibition by RTA 408 of established xenografts (tumor size > 30 mm3 when treatment commenced) of all four cell lines tested even in the absence of radiation. Tumor growth, as determined by caliper measurements, is shown in Fig. 3A and representative in vivo tumor images at different days after treatment in Supplementary Fig. S2. In marked contrast to the protective effects observed in normal tissues, RTA 408 did not radioprotect PC3 xenotransplants. Rather, when used in combination with radiation, RTA 408 amplified the antitumor effect of radiation alone (P = 0.001; Fig. 3B). In vivo imaging revealed complete tumor growth inhibition in animals that received both radiation and RTA 408 at 17.5 mg/kg (Fig. 3B, see insert) but not in mice treated with 5 mg/kg RTA 408 (not shown). RTA 408 induced high levels of intratumoral apoptosis as determined by detection of fragmented DNA (TUNEL), cleaved PARP, and the caspase-3 cleavage product of cytokeratin18 (Fig. 4A). The antibodies used to detect cleaved PARP and cytokeratin18 do not crossreact with mouse tissues indicating that RTA 408 induced apoptosis of human tumor cells in situ. RTA 408–dependent inhibition of PC3 xenografts was associated with significantly reduced proliferation as determined by Ki-67 staining (Fig. 4B).

Figure 3.

RTA 408 inhibits growth and survival of human prostate cancer xenotransplants. A, effects of RTA 408 on growth of DU145, PC3, LNCaP/C4-2B, and CWR22Rv1 cells in vivo. RTA 408 was administered (3 times per week at 17.5 mg/kg) after tumors had reached volumes exceeding 25 to 30 mm3. Experimental groups consisted of 5 animals each. Results represent mean ± SD of these groups. P value summaries refer to tumor growth trajectories over time in RTA 408 and control groups. B, inhibition of PC3 xenograft growth and survival by combined radiation and RTA 408 treatment. Tumor-bearing mice were treated for 2 weeks with either RTA 408 or RTA 408 and IR. IR (5 Gy) was administered twice on days 1 and 8 and RTA 408 (17.5 mg/kg) was administered 1 day before and for 3 days after each IR in the combination group. RTA 408 administration (3 times weekly) was continued for an additional 4 weeks. Results represent mean ± SD of groups of 5 animals each. Tumor growth trajectories over time were compared between treatment and control groups, and among treatment groups. The insert shows representative images of tumors in situ at treatment start and 40 days after. Chemiluminescence was detected by IVIS bioimaging of PC3 cells constitutively expressing FLuc. *, P = 0.01 to 0.05; **, P = 0.001 to 0.01.

Figure 3.

RTA 408 inhibits growth and survival of human prostate cancer xenotransplants. A, effects of RTA 408 on growth of DU145, PC3, LNCaP/C4-2B, and CWR22Rv1 cells in vivo. RTA 408 was administered (3 times per week at 17.5 mg/kg) after tumors had reached volumes exceeding 25 to 30 mm3. Experimental groups consisted of 5 animals each. Results represent mean ± SD of these groups. P value summaries refer to tumor growth trajectories over time in RTA 408 and control groups. B, inhibition of PC3 xenograft growth and survival by combined radiation and RTA 408 treatment. Tumor-bearing mice were treated for 2 weeks with either RTA 408 or RTA 408 and IR. IR (5 Gy) was administered twice on days 1 and 8 and RTA 408 (17.5 mg/kg) was administered 1 day before and for 3 days after each IR in the combination group. RTA 408 administration (3 times weekly) was continued for an additional 4 weeks. Results represent mean ± SD of groups of 5 animals each. Tumor growth trajectories over time were compared between treatment and control groups, and among treatment groups. The insert shows representative images of tumors in situ at treatment start and 40 days after. Chemiluminescence was detected by IVIS bioimaging of PC3 cells constitutively expressing FLuc. *, P = 0.01 to 0.05; **, P = 0.001 to 0.01.

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Figure 4.

Effects of RTA 408 on apoptosis incidence and proliferation in PC3 prostate cancer xenografts treated with RTA 408. A, apoptosis incidence was determined by TUNEL and by immunohistochemical detection of cleaved PARP and cytokeratin 18 (M30) at different time intervals after treatment with RTA 408 commenced; scale bars, 100 μm. Quantitative analysis of the results obtained on day 15 of treatment was performed by averaging the number of positive cells in at least 6 different fields. Results are expressed as mean ± SD. Statistically significant (P < 0.05) differences were determined by Student t test. B, RTA 408–dependent inhibition of PC3 prostate cancer cell proliferation. Proliferating cells were detected by staining with Ki-67 antibody (red). Cell nuclei were counterstained with DAPI; scale bars, 100 μm. Tumors were harvested 15 days after treatment initiation.

Figure 4.

Effects of RTA 408 on apoptosis incidence and proliferation in PC3 prostate cancer xenografts treated with RTA 408. A, apoptosis incidence was determined by TUNEL and by immunohistochemical detection of cleaved PARP and cytokeratin 18 (M30) at different time intervals after treatment with RTA 408 commenced; scale bars, 100 μm. Quantitative analysis of the results obtained on day 15 of treatment was performed by averaging the number of positive cells in at least 6 different fields. Results are expressed as mean ± SD. Statistically significant (P < 0.05) differences were determined by Student t test. B, RTA 408–dependent inhibition of PC3 prostate cancer cell proliferation. Proliferating cells were detected by staining with Ki-67 antibody (red). Cell nuclei were counterstained with DAPI; scale bars, 100 μm. Tumors were harvested 15 days after treatment initiation.

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RTA 408 decreases growth and survival of human prostate cancer cells in vitro

Next, we examined dose-dependent effects of RTA 408 on prostate cancer cells in vitro. Within 24 hours of exposure, RTA 408 (1 μmol/L) induced varying degrees of apoptosis in all four prostate cancer cell lines as determined by detection of cleaved caspase-3, cytokeratin18, and PARP-1 in both attached and, more prominently, in cells detached from substrate (Supplementary Fig. S3). In contrast, caspase-8 cleavage was only marginally detected in DU145 cells at the higher doses of RTA 408 (0.5–1 μmol/L) tested but not the other three cell lines. As determined by clonogenic survival assays, RTA 408 radiosensitized all four prostate cancer cell lines under investigation with the strongest effects observed in DU145 and LNCaP/C4-2B cells (Supplementary Fig. S4). As assessed by crystal violet staining and by WST assay, RTA 408 reduced viability of all four prostate cancer cell lines in a dose-dependent fashion (Fig. 5A). The IC50 for inhibition of in vitro growth and survival of these cell lines ranged from 250 to 750 nmol/L. Interestingly, RTA 408 also inhibited in vitro growth and survival of PrEC, the immortalized NHPr-E1 and BHPrE-1 prostate cells and, primary human epidermal keratinocytes (NHEK-1, -2, and -3; Fig. 5B). The IC50 as determined by crystal violet staining for the normal or premalignant cells was in the range of 125 to 250 nmol/L. Compromised cell viability was associated with substrate detachment of normal prostate epithelial cells and keratinocytes, as well as control PC3 cells (Supplementary Fig. S5). Collectively, these results highlight a broad spectrum of inhibitory effects of RTA 408 on benign and malignant prostate cells and on normal prostate epithelial cells and keratinocytes in vitro. The effects on normal epithelial cells in vitro are in marked contrast to tissue protection of normal epithelial tissues in irradiated mice.

Figure 5.

In vitro effects of RTA 408 on prostate cancer cell (A) and normal cell (B) growth and survival. Loss of viability was determined by assessing the numbers of adherent cells using crystal violet stain or measuring metabolic activity (WST assay). Experiments were performed in triplicate and repeated two times. Results shown are mean ± SD of triplicates of a representative experiment. Results are presented as percentages of vehicle control. Analysis by using one-way ANOVA with Tukey posttest correction (GraphPad Prism) revealed significant differences (P < 0.05 to P < 0.001) between vehicle- and RTA 408–treated groups (typically at ≥0.5 μmol/L or 0.25 μmol/L in tumor or normal cell lines, respectively).

Figure 5.

In vitro effects of RTA 408 on prostate cancer cell (A) and normal cell (B) growth and survival. Loss of viability was determined by assessing the numbers of adherent cells using crystal violet stain or measuring metabolic activity (WST assay). Experiments were performed in triplicate and repeated two times. Results shown are mean ± SD of triplicates of a representative experiment. Results are presented as percentages of vehicle control. Analysis by using one-way ANOVA with Tukey posttest correction (GraphPad Prism) revealed significant differences (P < 0.05 to P < 0.001) between vehicle- and RTA 408–treated groups (typically at ≥0.5 μmol/L or 0.25 μmol/L in tumor or normal cell lines, respectively).

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Inhibition of NF-κB activity by RTA 408 in vivo

The selective antitumor activity of RTA 408 on prostate cancer cell lines in vivo raises the question which molecular target(s) are responsible for this effect. In prostate cancer, deregulated NF-κB signaling is associated with disease progression, contributes to expression of both prostate specific antigen (PSA; 21) and androgen receptor (22), and is prevalent in castrate-resistant and metastatic tumors (23–26). Conversely, disrupting NF-κB signaling by forced expression of a phosphorylation-deficient IκB radiosensitizes PC3 prostate cells in vitro (27). Other NF-κB inhibitors, including curcumin (28), parthenolide (29), and SN52 (30), similarly inhibit prostate cancer growth and survival. On the basis of this prior work, we used NF-κB-NLuc- and control CMV-Fluc -reporter constructs to measure NF-κB activity in transfected PC3 tumors in vivo, before and after treatment with RTA 408 and/or IR (Fig. 6A and B). As expected, radiation induced NF-κB activity in tumor tissue (Fig. 6, panel 4). In the posttreatment group, the ratio of NF-κB-NLuc- to CMV-Fluc activity in RTA 408–treated mice (Fig. 6, panel 6) was significantly lower compared with that observed in mice treated with vehicle alone (Fig. 6, panel 2). In addition, the ratio of NF-κB-NLuc- to CMV-Fluc activity in mice treated with RTA 408 and IR combined (Fig. 6, panel 8) was significantly lower compared with that observed in mice treated with IR alone (Fig. 6, panel 4). Hence, at tumor growth–inhibitory concentrations, RTA 408 effectively inhibited transcription of an NF-κB–responsive reporter construct in PC3 cells in vivo.

Figure 6.

In vivo imaging of NF-κB reporter activity in PC3 xenografts treated with either IR, or RTA 408, or a combination of IR and RTA 408. A, representative images of mice showing expression of FLuc under control of a CMV promoter and corresponding images showing activity of an NF-κB–responsive promoter driving NanoLuc (NLuc) expression are shown. Pretreatment images were acquired 2 days before treatment with either RTA 408 or IR (panels 1, 3, 5, and 7). Posttreatment images were acquired from the same mice either untreated or after short-term (1 hour) treatment with RTA 408 (17.5 mg/kg) or after IR (5 Gy) or RTA 408 (17.5 mg/mL; 2 days) and IR (5 Gy) as indicated (panels 2, 4, 6, 8). Images in the posttreatment group were acquired 1 hour after IR exposure. B, quantitative representation of NF-κB activity expressed as the ratio of NanoLuc to firefly luciferase (n = 2/group). Labeling of the x-axis refers to treatment groups as shown in A. Results shown are mean ± SD of duplicate mice in each group. This experiment was repeated with comparable results.

Figure 6.

In vivo imaging of NF-κB reporter activity in PC3 xenografts treated with either IR, or RTA 408, or a combination of IR and RTA 408. A, representative images of mice showing expression of FLuc under control of a CMV promoter and corresponding images showing activity of an NF-κB–responsive promoter driving NanoLuc (NLuc) expression are shown. Pretreatment images were acquired 2 days before treatment with either RTA 408 or IR (panels 1, 3, 5, and 7). Posttreatment images were acquired from the same mice either untreated or after short-term (1 hour) treatment with RTA 408 (17.5 mg/kg) or after IR (5 Gy) or RTA 408 (17.5 mg/mL; 2 days) and IR (5 Gy) as indicated (panels 2, 4, 6, 8). Images in the posttreatment group were acquired 1 hour after IR exposure. B, quantitative representation of NF-κB activity expressed as the ratio of NanoLuc to firefly luciferase (n = 2/group). Labeling of the x-axis refers to treatment groups as shown in A. Results shown are mean ± SD of duplicate mice in each group. This experiment was repeated with comparable results.

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These observations extend and confirm previous reports describing in vitro growth inhibition of human prostate cancer cells by CDDO variants. Specifically, Deeb and colleagues described proapoptotic effects of CDDO, CDDO-methyl(ME), and CDDO-imidazole(IM) in cultured human LNCaP, PC3, and DU145 and murine TRAMPC-1 prostate cancer cells in vitro (31, 32). Furthermore, Gao and colleagues described CDDO-dependent chemoprevention of prostate cancer development in transgenic TRAMP mice in which the SV40 T antigen is expressed by prostate epithelial cells (33). Tumor growth inhibition by CDDO derivatives extends to other tumor types ranging from leukemias (34–37) to solid malignancies (38–42). A common denominator of these tumor types is deregulated NF-κB activity, which is effectively inhibited by RTA 408 not only in vitro but also in vivo. It remains to be determined whether inhibition of survival pathways beyond NF-κB plays a role in prostate cancer inhibition by RTA 408. For example, the Akt/mTOR pathway is also reportedly inhibited by CDDO-ME in prostate cancer cells (43).

The mechanistic basis for the dual and opposite effects of RTA 408 on normal and a broad range of malignant tissues remains to be investigated further. Of particular relevance to cytoprotection, CDDO covalently attaches to KEAP1, disrupts KEAP1/Nrf2 interaction, and triggers Nrf2-dependent transcription of a host of genes encoding antioxidant enzymes (16). Nrf-2 activation by the CDDO derivatives CDDO-ethylamide (EA) and CDDO-ME has been proposed to improve survival of irradiated mice (44). We observed that topical application of RTA 408 markedly reduced radiation dermatitis in mice associated with significant increases in Nrf2 target genes and significant decreases in NF-κB target genes (45). Interestingly, radiation protection of normal prostate epithelial cells contrasted by growth inhibition of prostate cancer cells in vivo has been very recently described for dimethylamineparthenolide (DMAPT; ref. 46). DMAPT and its parent compound parthenolide alkylate reactive cysteines on multiple protein targets, including KEAP1, and inhibit canonical NF-κB signaling by interacting with IκB and the NFκBp65 subunit (47–49). In contrast to RTA 408, parthenolide or DMAPT reportedly did not inhibit cultured normal or immortalized prostate cells to the same extent as their malignant counterparts and this difference has been attributed to differential effects of DMAPT on KEAP1-dependent oxidation status in normal and malignant cells (46). This difference between RTA 408 and DMAPT suggests that tissue protection by RTA 408 as seen in vivo is not primarily due to cell-autonomous effects but likely depends on environmental factors provided by the tissue context in vivo. A precedent for “contextual” antitumor effects of CDDO-ME has been established previously (50). Specifically, CDDO-ME inhibited myeloid-derived suppressor cells in the tumor microenvironment associated with improved immune responses. Regardless of the relative contribution of cell-intrinsic or “environmental” antitumor mechanisms, the results obtained for DMAPT (46) and RTA 408 (this study) validate the concept of selective radiosensitization of tumor tissues by thiol-reactive compounds (5).

K. Ward has ownership interest (including patents) in Reata Pharmaceuticals. U. Rodeck received a commercial research grant from REATA Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K. Ward, A.P. Dicker, U. Rodeck

Development of methodology: V. Alexeev, A. Linnenbach

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): V. Alexeev, E. Lash, A. Bitterman, A. Linnenbach, U. Rodeck

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V. Alexeev, L. Corsini, A. Bitterman, A.P. Dicker, A. Linnenbach

Writing, review, and/or revision of the manuscript: V. Alexeev, K. Ward, A.P. Dicker, A. Linnenbach, U. Rodeck

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Lash, A. Aguillard, L. Corsini, A.P. Dicker, U. Rodeck

Study supervision: U. Rodeck

The authors thank Phyllis Wachsberger (Thomas Jefferson University, Philadelphia, PA) for advice on performing clonogenic assays, Simon Hayward (Vanderbilt University, Nashville, TN) for providing immortalized prostate cells, and Marja Nevalainen (Thomas Jefferson University, Philadelphia, PA) for providing prostate cancer cells.

This work was supported by DoD grant W81XWH-12-1-0477 and a pilot project under NIH grant U19A1091175. Additional support was provided by the Prostate Cancer Foundation and by REATA Pharmaceuticals.

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.

1.
Kountouras
J
,
Zavos
C
. 
Recent advances in the management of radiation colitis
.
World J Gastroenterol
2008
;
14
:
7289
301
.
2.
Souhami
L
,
Bae
K
,
Pilepich
M
,
Sandler
H
. 
Impact of the duration of adjuvant hormonal therapy in patients with locally advanced prostate cancer treated with radiotherapy: a secondary analysis of RTOG 85–31
.
J Clin Oncol
2009
;
27
:
2137
43
.
3.
Simone
NL
,
Menard
C
,
Soule
BP
,
Albert
PS
,
Guion
P
,
Smith
S
, et al
Intrarectal amifostine during external beam radiation therapy for prostate cancer produces significant improvements in Quality of Life measured by EPIC score
.
Int J Radiat Oncol Biol Phys
2008
;
70
:
90
5
.
4.
Spitz
DRD
,
Hauer-Jensen
M
. 
Ionizing radiation-induced responses: where free radical chemistry meets redox biology and medicine
.
Antioxid Redox Signal
2014
;
20
:
1407
9
.
5.
Deorukhkar
A
,
Krishnan
S
. 
Targeting inflammatory pathways for tumor radiosensitization
.
Biochem Pharmacol
2010
;
80
:
1904
14
.
6.
Daroczi
B
,
Kari
G
,
Ren
Q
,
Dicker
AP
,
Rodeck
U
. 
Nuclear factor kappaB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos
.
Mol Cancer Ther
2009
;
8
:
2625
34
.
7.
Thotala
DK
,
Hallahan
DE
,
Yazlovitskaya
EM
. 
Inhibition of glycogen synthase kinase 3 beta attenuates neurocognitive dysfunction resulting from cranial irradiation
.
Cancer Res
2008
;
68
:
5859
68
.
8.
Thotala
DK
,
Geng
L
,
Dickey
AK
,
Hallahan
DE
,
Yazlovitskaya
EM
. 
A new class of molecular targeted radioprotectors: GSK-3beta inhibitors
.
Int J Radiat Oncol Biol Phys
2010
;
76
:
557
65
.
9.
Jacobs
KM
,
Bhave
SR
,
Ferraro
DJ
,
Jaboin
JJ
,
Hallahan
DE
,
Thotala
D
. 
GSK-3beta: A Bifunctional Role in Cell Death Pathways
.
Int J Cell Biol
2012
;
2012
:
930710
.
10.
Hoeflich
KP
,
Luo
J
,
Rubie
EA
,
Tsao
MS
,
Jin
O
,
Woodgett
JR
. 
Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation
.
Nature
2000
;
406
:
86
90
.
11.
Cremer
TJ
,
Shah
P
,
Cormet-Boyaka
E
,
Valvano
MA
,
Butchar
JP
,
Tridandapani
S
. 
Akt-mediated proinflammatory response of mononuclear phagocytes infected with Burkholderia cenocepacia occurs by a novel GSK3beta-dependent, IkappaB kinase-independent mechanism
.
J Immunol
2011
;
187
:
635
43
.
12.
Hall
EJ
,
Giaccia
AJ
. 
Cell survival curves. Radiobiology for the radiologist
.
Philadelphia, PA
:
Lippincott Williams & Wilkins
; 
2006
.
p.
30
46
.
13.
Epperly
M
,
Jin
S
,
Nie
S
,
Cao
S
,
Zhang
X
,
Franicola
D
, et al
Ethyl pyruvate, a potentially effective mitigator of damage after total-body irradiation
.
Radiat Res
2007
;
168
:
552
9
.
14.
Daroczi
B
,
Kari
G
,
McAleer
MF
,
Wolf
JC
,
Rodeck
U
,
Dicker
AP
. 
In vivo radioprotection by the fullerene nanoparticle DF-1 as assessed in a zebrafish model
.
Clin Cancer Res
2006
;
12
:
7086
91
.
15.
Han
Y
,
Englert
JA
,
Yang
R
,
Delude
RL
,
Fink
MP
. 
Ethyl pyruvate inhibits nuclear factor-kappaB-dependent signaling by directly targeting p65
.
J Pharmacol Exp Ther
2005
;
312
:
1097
105
.
16.
Ahmad
R
,
Raina
D
,
Meyer
C
,
Kharbanda
S
,
Kufe
D
. 
Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179
.
J Biol Chem
2006
;
281
:
35764
9
.
17.
Liby
K
,
Hock
T
,
Yore
MM
,
Suh
N
,
Place
AE
,
Risingsong
R
, et al
The synthetic triterpenoids, CDDO and CDDO-imidazolide, are potent inducers of heme oxygenase-1 and Nrf2/ARE signaling
.
Cancer Res
2005
;
65
:
4789
98
.
18.
Coghlan
MP
,
Culbert
AA
,
Cross
DA
,
Corcoran
SL
,
Yates
JW
,
Pearce
NJ
, et al
Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription
.
Chem Biol
2000
;
7
:
793
803
.
19.
Zhang
F
,
Phiel
CJ
,
Spece
L
,
Gurvich
N
,
Klein
PS
. 
Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3
.
J Biol Chem
2003
;
278
:
33067
77
.
20.
Saha
S
,
Bhanja
P
,
Liu
L
,
Alfieri
AA
,
Yu
D
,
Kandimalla
ER
, et al
TLR9 agonist protects mice from radiation-induced gastrointestinal syndrome
.
PLoS ONE
2012
;
7
:
e29357
.
21.
Chen
CD
,
Sawyers
CL
. 
NF-kappa B activates prostate-specific antigen expression and is upregulated in androgen-independent prostate cancer
.
Mol Cell Biol
2002
;
22
:
2862
70
.
22.
Zhang
L
,
Altuwaijri
S
,
Deng
F
,
Chen
L
,
Lal
P
,
Bhanot
UK
, et al
NF-kappaB regulates androgen receptor expression and prostate cancer growth
.
Am J Pathol
2009
;
175
:
489
99
.
23.
McCall
P
,
Bennett
L
,
Ahmad
I
,
Mackenzie
LM
,
Forbes
IW
,
Leung
HY
, et al
NFkappaB signalling is upregulated in a subset of castrate-resistant prostate cancer patients and correlates with disease progression
.
Br J Cancer
2012
;
107
:
1554
63
.
24.
Siddiqui
IA
,
Shukla
Y
,
Adhami
VM
,
Sarfaraz
S
,
Asim
M
,
Hafeez
BB
, et al
Suppression of NFkappaB and its regulated gene products by oral administration of green tea polyphenols in an autochthonous mouse prostate cancer model
.
Pharm Res
2008
;
25
:
2135
42
.
25.
Andela
VB
,
Gordon
AH
,
Zotalis
G
,
Rosier
RN
,
Goater
JJ
,
Lewis
GD
, et al
NFkappaB: a pivotal transcription factor in prostate cancer metastasis to bone
.
Clin Orthop Relat Res
2003
:
S75
85
.
26.
Jin
R
,
Sterling
JA
,
Edwards
JR
,
DeGraff
DJ
,
Lee
C
,
Park
SI
, et al
Activation of NF-kappa B signaling promotes growth of prostate cancer cells in bone
.
PLoS ONE
2013
;
8
:
e60983
.
27.
Pajonk
F
,
Pajonk
K
,
McBride
WH
. 
Inhibition of NF-kappaB, clonogenicity, and radiosensitivity of human cancer cells
.
J Natl Cancer Inst
1999
;
91
:
1956
60
.
28.
Chendil
D
,
Ranga
RS
,
Meigooni
D
,
Sathishkumar
S
,
Ahmed
MM
. 
Curcumin confers radiosensitizing effect in prostate cancer cell line PC-3
.
Oncogene
2004
;
23
:
1599
607
.
29.
Sun
Y
,
St Clair
DK
,
Fang
F
,
Warren
GW
,
Rangnekar
VM
,
Crooks
PA
, et al
The radiosensitization effect of parthenolide in prostate cancer cells is mediated by nuclear factor-kappaB inhibition and enhanced by the presence of PTEN
.
Mol Cancer Ther
2007
;
6
:
2477
86
.
30.
Xu
Y
,
Fang
F
,
St Clair
DK
,
Sompol
P
,
Josson
S
,
St Clair
WH
. 
SN52, a novel nuclear factor-kappaB inhibitor, blocks nuclear import of RelB:p52 dimer and sensitizes prostate cancer cells to ionizing radiation
.
Mol Cancer Ther
2008
;
7
:
2367
76
.
31.
Deeb
D
,
Gao
X
,
Dulchavsky
SA
,
Gautam
SC
. 
CDDO-me induces apoptosis and inhibits Akt, mTOR and NF-kappaB signaling proteins in prostate cancer cells
.
Anticancer Res
2007
;
27
:
3035
44
.
32.
Deeb
D
,
Gao
X
,
Dulchavsky
SA
,
Gautam
SC
. 
CDDO-Me inhibits proliferation, induces apoptosis, down-regulates Akt, mTOR, NF-kappaB and NF-kappaB-regulated antiapoptotic and proangiogenic proteins in TRAMP prostate cancer cells
.
J Exp Ther Oncol
2008
;
7
:
31
9
.
33.
Gao
X
,
Deeb
D
,
Liu
Y
,
Arbab
AS
,
Divine
GW
,
Dulchavsky
SA
, et al
Prevention of prostate cancer with oleanane synthetic triterpenoid CDDO-Me in the TRAMP mouse model of prostate cancer
.
Cancers
2011
;
3
:
3353
69
.
34.
Shishodia
S
,
Sethi
G
,
Konopleva
M
,
Andreeff
M
,
Aggarwal
BB
. 
A synthetic triterpenoid, CDDO-Me, inhibits IkappaBalpha kinase and enhances apoptosis induced by TNF and chemotherapeutic agents through down-regulation of expression of nuclear factor kappaB-regulated gene products in human leukemic cells
.
Clin Cancer Res
2006
;
12
:
1828
38
.
35.
Samudio
I
,
Kurinna
S
,
Ruvolo
P
,
Korchin
B
,
Kantarjian
H
,
Beran
M
, et al
Inhibition of mitochondrial metabolism by methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate induces apoptotic or autophagic cell death in chronic myeloid leukemia cells
.
Mol Cancer Ther
2008
;
7
:
1130
9
.
36.
Stadheim
TA
,
Suh
N
,
Ganju
N
,
Sporn
MB
,
Eastman
A
. 
The novel triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) potently enhances apoptosis induced by tumor necrosis factor in human leukemia cells
.
J Biol Chem
2002
;
277
:
16448
55
.
37.
Pedersen
IM
,
Kitada
S
,
Schimmer
A
,
Kim
Y
,
Zapata
JM
,
Charboneau
L
, et al
The triterpenoid CDDO induces apoptosis in refractory CLL B cells
.
Blood
2002
;
100
:
2965
72
.
38.
Qin
Y
,
Deng
W
,
Ekmekcioglu
S
,
Grimm
EA
. 
Identification of unique sensitizing targets for anti-inflammatory CDDO-Me in metastatic melanoma by a large-scale synthetic lethal RNAi screening
.
Pigment Cell Melanoma Res
2013
;
26
:
97
112
.
39.
Konopleva
M
,
Zhang
W
,
Shi
YX
,
McQueen
T
,
Tsao
T
,
Abdelrahim
M
, et al
Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells
.
Mol Cancer Ther
2006
;
5
:
317
28
.
40.
Duan
Z
,
Ames
RY
,
Ryan
M
,
Hornicek
FJ
,
Mankin
H
,
Seiden
MV
. 
CDDO-Me, a synthetic triterpenoid, inhibits expression of IL-6 and Stat3 phosphorylation in multi-drug resistant ovarian cancer cells
.
Cancer Chemother Pharmacol
2009
;
63
:
681
9
.
41.
Gao
X
,
Deeb
D
,
Liu
P
,
Liu
Y
,
Arbab-Ali
S
,
Dulchavsky
SA
, et al
Role of reactive oxygen species (ROS) in CDDO-Me-mediated growth inhibition and apoptosis in colorectal cancer cells
.
J Exp Ther Oncol
2011
;
9
:
119
27
.
42.
Zou
W
,
Chen
S
,
Liu
X
,
Yue
P
,
Sporn
MB
,
Khuri
FR
, et al
c-FLIP downregulation contributes to apoptosis induction by the novel synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate (CDDO-Me) in human lung cancer cells
.
Cancer Biol Ther
2007
;
6
:
1614
20
.
43.
Deeb
D
,
Gao
X
,
Jiang
H
,
Janic
B
,
Arbab
AS
,
Rojanasakul
Y
, et al
Oleanane triterpenoid CDDO-Me inhibits growth and induces apoptosis in prostate cancer cells through a ROS-dependent mechanism
.
Biochem Pharmacol
2010
;
79
:
350
60
.
44.
Kim
SB
,
Pandita
RK
,
Eskiocak
U
,
Ly
P
,
Kaisani
A
,
Kumar
R
, et al
Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation
.
Proc Natl Acad Sci U S A
2012
;
109
:
E2949
55
.
45.
Reisman
SA
,
Lee
C-Y
,
Meyer
CJ
,
Proksch
JW
,
Sonis
ST
,
Ward
KW
. 
Topical application of the synthetic triterpenoid RTA 408 protects mice from radiation-induced dermatitis
.
Radiat Res
2014
;
181
:
512
20
.
46.
Xu
Y
,
Fang
F
,
Miriyala
S
,
Crooks
PA
,
Oberley
TD
,
Chaiswing
L
, et al
KEAP1 is a redox sensitive target that arbitrates the opposing radiosensitive effects of parthenolide in normal and cancer cells
.
Cancer Res
2013
;
73
:
4406
17
.
47.
Garcia-Pineres
AJ
,
Castro
V
,
Mora
G
,
Schmidt
TJ
,
Strunck
E
,
Pahl
HL
, et al
Cysteine 38 in p65/NF-kappaB plays a crucial role in DNA binding inhibition by sesquiterpene lactones
.
J Biol Chem
2001
;
276
:
39713
20
.
48.
Kwok
BH
,
Koh
B
,
Ndubuisi
MI
,
Elofsson
M
,
Crews
CM
. 
The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase
.
Chem Biol
2001
;
8
:
759
66
.
49.
Hehner
SP
,
Hofmann
TG
,
Droge
W
,
Schmitz
ML
. 
The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-kappa B by targeting the I kappa B kinase complex
.
J Immunol
1999
;
163
:
5617
23
.
50.
Nagaraj
S
,
Youn
JI
,
Weber
H
,
Iclozan
C
,
Lu
L
,
Cotter
MJ
, et al
Anti-inflammatory triterpenoid blocks immune suppressive function of MDSCs and improves immune response in cancer
.
Clin Cancer Res
2010
;
16
:
1812
23
.

Supplementary data