Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Evaluation of 30% DAS-444Ø6-6 soybean meal in a subchronic rat toxicity study.

Event DAS-444Ø6-6 soybean is genetically modified (GM) to provide tolerance to 2,4-diclorophenoxyacetic acid (2,4-D), glyphosate, and glufosinate herbicides through expression of the AAD-12, 2mEPSPS, and PAT proteins, respectively. DAS-444Ø6-6 soybeans were evaluated for safety in subchronic rat feeding studies. The results from two previous subchronic rat feeding studies evaluating diets formulated with 20% inclusion of DAS-444Ø6-6 soybean meal (the latter also containing DAS-444Ø6-6 derived hulls and oil) did not show any treatment-related adverse effects. In 2017, to comply with recent guidance from EFSA, a third 90-day rat feeding study was conducted with Sprague-Dawley rats (16/sex/group) with diets formulated either with 15% or 30% w/w of toasted DAS-444Ø6-6 soybean meal. DAS-444Ø6-6 soybean hulls and oil were also added to the transgenic test diets at 1% or 2% w/w and 1.35% or 2.7%, respectively, for the low- and high-dose groups. No toxicologically significant effects were observed under the conditions of this study.

No treatment-related effects with aryloxyalkanoate dioxygenase-12 in three 28-day mouse toxicity studies.

The aryloxyalkanoate dioxygenase-12 (AAD-12) protein is expressed in genetically modified soybean events DAS-68416-4 and DAS-444Ø6-6. Expression of the AAD-12 protein in soybeans confers tolerance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) providing an additional herbicide choice to farmers. This enzyme acts by catalyzing the degradation of 2,4-D into herbicidally inactive metabolites. To meet evolving interpretation of regulations in the European Union, three separate 28-day repeat-dose oral mouse studies were conducted at increasing doses of up to 1100 mg AAD-12 protein/kg bw/day. No treatment-related effects were seen in any of these three studies.

Safety evaluation of DAS-44406-6 soybeans in Wistar rats.

A 90-day in-country feeding trial in Wistar rats was conducted at Tianjin Laboratory in China to assess toxicity of diets containing DAS-44406-6 soybean meal. There were no treatment-related changes observed when compared with the non-GM isoline control groups but histopathologically, 2 of 10 high-dose females were reported to show kidney lesions. However, these findings contrasted with the absence of any treatment-related kidney lesions in 3 separate 90-day toxicity studies previously conducted in Sprague Dawley rats. Strain difference is not expected in the kidney response, and based on the low incidence and contrary evidence from previous studies, it is likely that these lesions were of spontaneous origin, or artefactual. To determine that the lesions observed were not treatment-related in Wistar rats, a specific follow-up confirmatory study was conducted under Good Laboratory Practices (GLP) in the Wistar strain of rats following an identical study design to the Tianjin study. To increase the power of detecting effects, twice the number of animals per group (20/sex/group) were used, and no treatment-related kidney histopathological changes were observed. Based on these results and entire weight of evidence evaluation, it is concluded that the histopathological changes previously noted in the 2 female Wistar rats of Tianjin study were not treatment-related and that DAS-44406-6 soybeans are as safe as conventional non-GM soybeans.

Food and feed safety of DAS-444Ø6-6 herbicide-tolerant soybean.

DAS-444Ø6-6 soybean was genetically engineered (GE) to withstand applications of three different herbicides. Tolerance to glufosinate and glyphosate is achieved through expression of the phosphinothricin acetyltransferase (PAT) and double-mutated maize 5-enolpyruvyl shikimate-3-phosphate synthase (2mEPSPS) enzymes, respectively. These proteins are expressed in currently commercialized crops and represent no novel risk. Tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) is achieved through expression of the aryloxyalkanoate dioxygenase 12 (AAD-12) enzyme, which is novel in crops. The safety of the AAD-12 protein and DAS-444Ø6-6 event was assessed for food and feed safety based on the weight of evidence and found to be as safe as non-GE soybean.

A predictive data-driven framework for endocrine prioritization: a triazole fungicide case study.

The US Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a tiered screening approach to determine the potential for a chemical to interact with estrogen, androgen, or thyroid hormone systems and/or perturb steroidogenesis. Use of high-throughput screening (HTS) to predict hazard and exposure is shifting the EDSP approach to (1) prioritization of chemicals for further screening; and (2) targeted use of EDSP Tier 1 assays to inform specific data needs. In this work, toxicology data for three triazole fungicides (triadimefon, propiconazole, and myclobutanil) were evaluated, including HTS results, EDSP Tier 1 screening (and other scientifically relevant information), and EPA guideline mammalian toxicology study data. The endocrine-related bioactivity predictions from HTS and information that satisfied the EDSP Tier 1 requirements were qualitatively concordant. Current limitations in the available HTS battery for thyroid and steroidogenesis pathways were mitigated by inclusion of guideline toxicology studies in this analysis. Similar margins (3-5 orders of magnitude) were observed between HTS-predicted human bioactivity and exposure values and between in vivo mammalian bioactivity and EPA chronic human exposure estimates for these products' registered uses. Combined HTS hazard and human exposure predictions suggest low priority for higher-tiered endocrine testing of these triazoles. Comparison with the mammalian toxicology database indicated that this HTS-based prioritization would have been protective for any potential in vivo effects that form the basis of current risk assessment for these chemicals. This example demonstrates an effective, human health protective roadmap for EDSP evaluation of pesticide active ingredients via prioritization using HTS and guideline toxicology information.

Assessment of potential adjuvanticity of Cry proteins.

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops.

Pronamide: Weight of evidence for potential estrogen, androgen or thyroid effects.

Based on the exposure potential to humans and environment, pronamide was one of 52 chemicals on the first list evaluated under US EPA's Endocrine Disruptor Screening Program (EDSP). The purpose of EDSP is to screen chemicals for their potential to interact with estrogen-, androgen-, or thyroid-signaling pathways. A battery of 11 Tier 1 assays was completed for pronamide in accordance with EDSP test guidelines. In addition, Other Scientifically Relevant Information, which included existing data from regulatory guideline studies and published literature, was used in a weight-of-evidence (WoE) evaluation of potential endocrine activity. The WoE conclusion is that pronamide does not interact directly with estrogen, androgen, or thyroid receptors or post-receptor events. Across in vivo studies, the liver is consistently and reproducibly the target organ for pronamide's effects. Pronamide activates hepatocytic nuclear receptors (including constitutive androstane receptor), induces hepatic enzymes, produces hepatocellular hypertrophy and increases liver weights. These changes are coupled with increased metabolic activity and a subsequent increased metabolism and/or clearance of both steroid and thyroid hormones. Thus, while pronamide alters some endocrine-sensitive endpoints in EDSP Tier 1 assays, effects on liver metabolism likely explain altered hormone levels and indirect endocrine changes.

Mode of action and human relevance of pronamide-induced rat thyroid tumors.

Pronamide, a selective, systemic, pre- and post-emergence herbicide, caused an increased incidence of thyroid follicular cell adenomas in a rat carcinogenicity study. Thyroid tumors, as well as liver and pituitary changes, were limited only to the high-dose group. The evidence for and against specific potential modes of action (MoAs) for rat thyroid follicular cell adenomas and their relevance to humans is discussed. Pronamide is not mutagenic and therefore, direct DNA reactivity is not relevant as a MoA. The hypothesized MoA for this effect is altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis mediated by the induction of hepatic enzymes, including uridine diphosphate glucuronosyltransferase (UGT). Evaluation of data from a series of regulatory guideline and MoA studies aimed at identifying the causative and associated key events supported a UGT-mediated MoA in the development of thyroid follicular tumors. This MoA for pronamide-induced thyroid tumors in rats, which involves increased thyroid hormone metabolism/clearance, altered thyroid hormone homeostasis and HPT stimulation is not considered relevant to humans based on quantitative species differences, making rats markedly more sensitive than humans to thyroid perturbations.

Pronamide: Human relevance of liver-mediated rat leydig cell tumors.

Dietary exposure to pronamide resulted in higher incidences of Leydig cell tumors (LCT) at 1000ppm in a 2-year cancer bioassay, but there were no testes effects at 40 or 200ppm, and no testes effects at 12-months at any concentration. A 90-day mode-of-action (MoA) study was conducted at concentrations of 0, 200, 1000 and 2000ppm. Standard parameters and stereological and proliferation analyses of LCs, targeted testis and liver gene expression, in vitro metabolism of testosterone by liver microsomes, and quantification of serum hormones and testosterone metabolites were evaluated. Increased testosterone metabolism due to increases in hepatic microsomal activity, alterations in serum hormone levels, and other data suggest that LCTs were mediated through a perturbation of the HPG-axis. Data suggest that this occurs after a threshold of exposure is reached, indicating a nonlinear/threshold dose-response. Pronamide-induced rat LCTs mediated by alterations to the HPG-axis have low relevance to humans due to quantitative differences in sensitivity between rats and humans to LCTs. Pronamide displayed no genotoxicity or direct endocrine effects. A margin of exposure approach for risk assessment and derivation of the chronic reference dose based on a point of departure of 200ppm is most appropriate and protective of human health.

Inhibition of pituitary tumor-transforming gene-1 in thyroid cancer cells by drugs that decrease specificity proteins.

Methyl 2-cyano-3,11-dioxo-18ß-olean-1,12-dien-30-oate (CDODA-Me) and the corresponding 2-trifluoromethyl analog (CF(3)DODA-Me) are derived synthetically from the triterpenoid glycyrrhetinic acid, a major component of licorice. CDODA-Me and CF(3)DODA-Me inhibited growth of highly invasive ARO, DRO, K-18, and HTh-74 thyroid cancer cells and this was due, in part, to decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in these cells. CDODA-Me and CF(3)DODA-Me also decreased expression of Sp-dependent genes, such as survivin and vascular endothelial growth factor (VEGF), and induced apoptosis. In addition, pituitary tumor-transforming gene-1 (PTTG-1) protein and mRNA levels were also decreased in thyroid cancer cells treated with CDODA-Me or CF(3)DODA-Me and this was accompanied by decreased expression of PTTG-1-dependent c-Myc and fibroblast growth factor-2 (FGF-2) genes. RNA interference studies against Sp1, Sp3, and Sp4 proteins showed that in thyroid cancer cells, PTTG-1 was an Sp-dependent gene. This study demonstrates for the first time that drugs, such as CDODA-Me and CF(3)DODA-Me, that decrease Sp protein expression also downregulate PTTG-1 in thyroid cancer cells and therefore have potential for clinical treatment of thyroid cancer and other endocrine neoplasias where PTTG-1 is a major pro-oncogenic factor.

Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors.

BACKGROUND: Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells. METHODS: The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression. RESULTS: BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10. CONCLUSIONS: These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.

Tolfenamic acid inhibits esophageal cancer through repression of specificity proteins and c-Met.

The non-steroidal anti-inflammatory drug tolfenamic acid (TA) inhibits proliferation of SEG-1 and BIC-1 esophageal cancer cells with half-maximal growth inhibitory concentration values of 36 and 48 muM, respectively. TA also increased Annexin V staining in both cell lines, indicative of proapoptotic activity. Treatment of SEG-1 and BIC-1 cells with TA for up to 72 h decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and this was accompanied by decreased expression of the well-characterized Sp-regulated genes cyclin D1, vascular endothelial growth factor and survivin. TA also decreased hepatocyte growth factor receptor, (c-Met), a receptor tyrosine kinase that is overexpressed in esophageal cancer cells and tumors and is an important drug target. Knockdown of Sp1, Sp3 and Sp4 by RNA interference in SEG-1 and BIC-1 cells also decreased c-Met expression, demonstrating that c-Met is an Sp-regulated gene in esophageal cancer cells. Sp1 was overexpressed in esophageal cancer cells and tumors and increased Sp1 staining was observed in esophageal tumors from patients. TA (20 mg/kg/day) also decreased tumor growth and weight in athymic nude mice bearing SEG-1 cells as xenografts and this was accompanied by increased apoptosis and decreased Sp1 and c-Met staining in tumors from treated mice. Thus, TA-dependent downregulation of Sp transcription factors and c-Met defines a novel chemotherapeutic approach for treatment of esophageal cancer.

Oncogenic microRNA-27a is a target for anticancer agent methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate in colon cancer cells.

Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (CDODA-Me) is a synthetic derivative of glycyrrhetinic acid, a triterpenoid phytochemical found in licorice extracts. CDODA-Me inhibited growth of RKO and SW480 colon cancer cells and this was accompanied by decreased expression of Sp1, Sp3 and Sp4 protein and mRNA and several Sp-dependent genes including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1 or Flt-1). CDODA-Me also induced apoptosis, arrested RKO and SW480 cells at G(2)/M, and inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. CDODA-Me decreased expression of microRNA-27a (miR-27a), and this was accompanied by increased expression of 2 miR-27a-regulated mRNAs, namely ZBTB10 (an Sp repressor) and Myt-1 which catalyzes phosphorylation of cdc2 to inhibit progression of cells through G(2)/M. Both CDODA-Me and antisense miR-27a induced comparable responses in RKO and SW480 cells, suggesting that the potent anticarcinogenic activity of CDODA-Me is due to repression of oncogenic miR-27a.

Induction of apoptosis and nonsteroidal anti-inflammatory drug-activated gene 1 in pancreatic cancer cells by a glycyrrhetinic acid derivative.

Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid, a bioactive phytochemical in licorice, CDODA-Me inhibits growth of Panc1 and Panc28 pancreatic cancer cell lines and activates peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent transactivation in these cells. CDODA-Me also induced p21 and p27 protein expression and downregulates cyclin D1; however, these responses were receptor-independent. CDODA-Me induced apoptosis in Panc1 and Panc28 cells, and this was accompanied by receptor-independent induction of the proapoptotic proteins early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), and activating transcription factor-3 (ATF3). Induction of NAG-1 and Egr-1 by CDODA-Me was dependent on activation of phosphatidylinositol-3-kinase (PI3-K) and/or p42 and p38 mitogen-activated protein kinase (MAPK) pathways but there were differences between Panc28 and Panc1 cells. Induction of NAG-1 in Panc28 cells was p38-MAPK- and PI3-K-dependent but Egr-1-independent, whereas induction in Panc1 cells was associated with activation of p38-MAPK, PI3-K, and p42-MAPK and was only partially Egr-1-dependent. This is the first report of the induction of the proapoptotic protein NAG-1 in pancreatic cancer cells.

Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors.

Betulinic acid is a pentacyclic triterpene natural product initially identified as a melanoma-specific cytotoxic agent that exhibits low toxicity in animal models. Subsequent studies show that betulinic acid induces apoptosis and antiangiogenic responses in tumors derived from multiple tissues; however, the underlying mechanism of action is unknown. Using LNCaP prostate cancer cells as a model, we now show that betulinic acid decreases expression of vascular endothelial growth (VEGF) and the antiapoptotic protein survivin. The mechanism of these betulinic acid-induced antiangiogenic and proapoptotic responses in both LNCaP cells and in tumors is due to activation of selective proteasome-dependent degradation of the transcription factors specificity protein 1 (Sp1), Sp3, and Sp4, which regulate VEGF and survivin expression. Thus, betulinic acid acts as a novel anticancer agent through targeted degradation of Sp proteins that are highly overexpressed in tumors.

1,1-Bis(3'-indolyl)-1-(p-chlorophenyl)methane activates the orphan nuclear receptor Nurr1 and inhibits bladder cancer growth.

Nurr1 is an orphan nuclear receptor and a member of the nerve growth factor I-B subfamily of transcription factors with no known endogenous ligand or stimulator. We show, for the first time, evidence that Nurr1 is expressed in a panel of 11 human bladder cancer cell lines. A new class of methylene-substituted diindolylmethanes (C-DIM) were screened and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) activated the ligand-binding domain of Nurr1. Treatment of bladder cancer cells with Nurr1-active C-DIM resulted in decreased cell survival (MTT assay) and induction of cell death pathways, resulting in poly(ADP-ribose) polymerase cleavage and DNA fragmentation. The specificity of the Nurr1-active compound was shown using RNA interference in 253J B-V cells, whereby small interfering RNA against Nurr1 attenuated ligand-dependent activation of Nurr1 and poly(ADP-ribose) polymerase cleavage. Furthermore, activation of Nurr1 resulted in stimulation of tumor necrosis factor-related apoptosis-inducing ligand and small interfering RNA experiments attenuated tumor necrosis factor-related apoptosis-inducing ligand production. In an orthotopic model of human bladder tumors established in nude mice, administration of a Nurr1-active C-DIM suppressed bladder cancer growth. These results identify Nurr1 as a potential target for bladder cancer therapy and also identify a novel agent for activating Nurr1.

5,5'-Dibromo-bis(3'-indolyl)methane induces Kruppel-like factor 4 and p21 in colon cancer cells.

Bis(3'-indolyl)methane (DIM) is a metabolite of the phytochemical indole-3-carbinol, and both compounds exhibit a broad spectrum of anticancer activities. We have developed a series of synthetic symmetrical ring-substituted DIM analogues, including 5,5'-dibromoDIM, which are more potent than DIM as inhibitors of cancer cell and tumor growth. In colon cancer cells, 5,5'-dibromoDIM decreased cell proliferation and inhibited G(0)-G(1)- to S-phase progression, and this was accompanied by induction of the cyclin-dependent kinase inhibitor p21 in HT-29 and RKO colon cancer cells. Mechanistic studies showed that induction of p21 in both RKO (p53 wild-type) and HT-29 (p53 mutant) cells by 5,5'-dibromoDIM was Krüppel-like factor 4 (KLF4) dependent, and induction of p53 in RKO cells was also KLF4 dependent. Analysis of the p21 promoter in p53-dependent RKO cells showed that 5,5'-dibromoDIM activated p21 gene expression through the proximal GC-rich sites 1 and 2, and chromatin immunoprecipitation assays showed that KLF4 and p53 bound to this region of the promoter, whereas in HT-29 cells unidentified upstream cis-elements were required for induction of p21. 5,5'-DibromoDIM (30 mg/kg/d) also inhibited tumor growth and induced p21 in athymic nude mice bearing RKO cells as xenografts, showing that ring-substituted DIM such as 5,5'-dibromoDIM represent a novel class of mechanism-based drugs for clinical treatment of colon cancer.

Methyl 2-cyano-3,11-dioxo-18 beta-olean-1,12-dien-30-oate is a peroxisome proliferator-activated receptor-gamma agonist that induces receptor-independent apoptosis in LNCaP prostate cancer cells.

Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-diene-30-oate (beta-CDODA-Me) is a synthetic analog of the naturally occurring triterpenoid glycyrrhetinic acid, which contains a 2-cyano substituent in the A-ring. beta-CDODA-Me was a potent inhibitor of LNCaP prostate cancer cell growth (IC(50) approximately 1 muM) and activated peroxisome proliferator-activated receptor gamma (PPARgamma), whereas analogs without the cyano group were weakly cytotoxic and did not activate PPARgamma. beta-CDODA-Me induced p21 and p27, down-regulated cyclin D1 protein expression, and induced two other proapoptotic proteins, namely nonsteroidal anti-inflammatory drug-activated gene-1 and activating transcription factor-3. However, induction of these responses by beta-CDODA-Me was PPARgamma-independent and due to activation of phosphatidylinositol-3-kinase, mitogen-activated protein kinase, and jun N-terminal kinase pathways by this compound. In contrast, beta-CDODA-Me also decreased androgen receptor (AR) and prostate-specific antigen (PSA) mRNA and protein levels through kinase-independent pathways. beta-CDODA-Me repressed AR mRNA transcription, whereas decreased PSA mRNA levels were dependent on protein synthesis and were reversed by cycloheximide. Thus, potent inhibition of LNCaP cell survival by beta-CDODA-Me is due to PPARgamma-independent activation of multiple pathways that selectively activate growth-inhibitory and proapoptotic responses.

1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane activates Nur77-independent proapoptotic responses in colon cancer cells.

1,1-Bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) is a methylene-substituted diindolylmethane (C-DIM) analog that activates the orphan receptor nerve growth factor-induced-Balpha (NGFI-Balpha, Nur77). RNA interference studies with small inhibitory RNA for Nur77 demonstrate that DIM-C-pPhOCH(3) induces Nur77-dependent and -independent apoptosis, and this study has focused on delineating the Nur77-independent proapoptotic pathways induced by the C-DIM analog. DIM-C-pPhOCH(3) induced caspase-dependent apoptosis in RKO colon cancer cells through decreased mitochondrial membrane potential which is accompanied by increased mitochondrial bax/bcl-2 ratios and release of cytochrome c into the cytosol. DIM-C-pPhOCH(3) also induced phosphatidylinositol-3-kinase-dependent activation of early growth response gene-1 which, in turn, induced expression of the proapoptotic nonsteroidal anti-inflammatory drug-activated gene-1 (NAG1) in RKO and SW480 colon cancer cells. Moreover, DIM-C-pPhOCH(3) also induced NAG-1 expression in colon tumors in athymic nude mice bearing RKO cells as xenografts. DIM-C-pPhOCH(3) also activated the extrinsic apoptosis pathway through increased phosphorylation of c-jun N-terminal kinase which, in turn, activated C/EBP homologous transcription factor (CHOP) and death receptor 5 (DR5). Thus, the effectiveness of DIM-C-pPhOCH(3) as a tumor growth inhibitor is through activation of Nur77-dependent and -independent pathways.