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.

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.

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.

The aryloxyalkanoate dioxygenase-12 (AAD-12) protein is not acutely toxic in mice.

Typically, when a protein is orally toxic, it acts via acute mechanisms, especially at high doses. Therefore, an acute oral toxicity study is considered appropriate for evaluating the safety of transgenic proteins. Soybean plants (events DAS-68416-4 and DAS-444Ø6-6) have been genetically modified to express the aryloxyalkanoate dioxygenase-12 (AAD-12) protein. The AAD-12 protein provides tolerance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). This paper summarizes the study designs of two acute oral toxicity studies evaluating the AAD-12 protein and reports the results of these studies. No mortalities or adverse effects were observed in mice when AAD-12 was tested up to a limit dose of 5000 mg/kg body weight. Based on the results of these studies, it can be concluded that AAD-12 protein, as expressed in genetically modified DAS-68416-4 and DAS-444Ø6-6 soybeans, lacks acute toxicity via the oral route.

Functional classification of protein toxins as a basis for bioinformatic screening.

Proteins are fundamental to life and exhibit a wide diversity of activities, some of which are toxic. Therefore, assessing whether a specific protein is safe for consumption in foods and feeds is critical. Simple BLAST searches may reveal homology to a known toxin, when in fact the protein may pose no real danger. Another challenge to answer this question is the lack of curated databases with a representative set of experimentally validated toxins. Here we have systematically analyzed over 10,000 manually curated toxin sequences using sequence clustering, network analysis, and protein domain classification. We also developed a functional sequence signature method to distinguish toxic from non-toxic proteins. The current database, combined with motif analysis, can be used by researchers and regulators in a hazard screening capacity to assess the potential of a protein to be toxic at early stages of development. Identifying key signatures of toxicity can also aid in redesigning proteins, so as to maintain their desirable functions while reducing the risk of potential health hazards.

Evaluation of the safety of a genetically modified DAS-444Ø6-6 soybean meal and hulls in a 90-day dietary toxicity study in rats.

A 90-day sub chronic toxicity study was conducted in rats to evaluate the safety of genetically modified DAS-444Ø6-6 soybeans expressing herbicide tolerant proteins when compared with its conventional comparators (non-transgenic near isoline control soybean and three commercially available non-transgenic line control soybeans). Rats were given diets formulated with either 10% or 20% w/w of soybean meal and 1% or 2% hulls of DAS-444Ø6-6 soybean with an equivalent amount of hulls from an isoline non-transgenic control soybean for at least 90 days. In addition, three separate 20% w/w non-transgenic commercially available soybean varieties were also given to groups of rats to serve as reference controls. Animals were evaluated by cage-side and hand-held detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, selected organ weights, and gross and histopathologic examinations. Under the conditions of this study, the genetically modified DAS-444Ø6-6 diets did not cause any treatment-related effects in rats following 90 days of dietary administration as compared with rats fed diets with soybean of isoline control or commercial reference controls and are considered equivalent to the diets prepared from conventional comparators.

Comparative performance of broilers fed diets containing DAS-44406-6 and non-transgenic soybean meal.

Broiler chickens are fast growing monogastric animals considered a sensitive test species to evaluate the safety and nutritional equivalence between transgenic and non-transgenic grains as part of the human safety evaluation process. DAS-44406-6 soybean expresses three herbicide-tolerant proteins: the aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme which provides tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) herbicides, the double-mutant 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS) enzyme encoded by a modified version of the epsps gene from maize (Zea mays), which provides tolerance to glyphosate herbicides, and the phosphinothricin acetyltransferase (PAT) enzyme from Streptomyces viridochromogenes, which provides tolerance to glufosinate herbicides. A 42-day broiler study was conducted with diets containing toasted DAS-44406-6 soybean meal to evaluate nutritional wholesomeness and safety compared with non-transgenic, near-isoline soybean and conventional comparators. The study used a randomized complete block design with five dietary treatments assigned randomly within six blocks of 12 pens per treatment, with 10 birds per pen. Broiler performance and carcass parameters were measured over a 6-week period of exposure to diets containing different sources of toasted soybean meal and hulls. Among the 13 endpoints measured, only thigh weight was numerically different between birds fed DAS-44406-6 soybean meal and those fed isoline soybean meal. However, no significant differences to thigh weight were observed between birds fed DAS-44406-6 soybean and any of the non-transgenic reference varieties. Results indicate that DAS-44406-6 soybeans are nutritionally equivalent to conventional varieties.

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: 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.

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.

Characterization of nuclear receptor-mediated murine hepatocarcinogenesis of the herbicide pronamide and its human relevance.

The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans.

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.

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.

Inactivation of the orphan nuclear receptor TR3/Nur77 inhibits pancreatic cancer cell and tumor growth.

Activation of the orphan nuclear receptor TR3/Nur77 (NR4A1) promotes apoptosis and inhibits pancreatic tumor growth, but its endogenous function and the effects of its inactivation have yet to be determined. TR3 was overexpressed in human pancreatic tumors compared with nontumor tissue. Small interfering RNA-mediated knockdown of TR3 or cell treatment with the TR3 antagonist 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) decreased proliferation, induced apoptosis, and decreased expression of antiapoptotic genes including Bcl-2 and survivin in pancreatic cancer cells. Survivin suppression was mediated by formation of a TR3-Sp1-p300 DNA binding complex on the proximal GC-rich region of the survivin promoter. When administered in vivo, DIM-C-pPhOH induced apoptosis and inhibited tumor growth in an orthotopic model of pancreatic cancer, associated with inhibition of the same antiapoptotic markers observed in vitro. Our results offer preclinical validation of TR3 as a drug target for pancreatic cancer chemotherapy, based on the ability of TR3 inhibitors to block the growth of pancreatic tumors.

Peroxisome proliferator-activated receptor gamma-dependent activity of indole ring-substituted 1,1-bis(3'-indolyl)-1-(p-biphenyl)methanes in cancer cells.

PURPOSE: 1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) substituted in the phenyl ring with a para-, t-butyl, trifluoromethyl (DIM-C-pPhCF(3)) or phenyl (DIM-C-pPhC(6)H(5)) group activate peroxisome proliferator-activated receptor gamma (PPARgamma) in several cancer cell lines, and DIM-C-pPhCF(3) also activates the orphan receptor Nur77. In this study, we have examined the effects of 5,5'-dihydroxy, 5,5'-dimethyl, 5,5'-dibromo, 5,5'-dinitro and 5,5'-dimethoxyindole ring-substituted analogs of DIM-C-pPhC(6)H(5) on their activity as PPARgamma agonists. METHODS: Various substituted C-DIM analogs were used to investigate their growth-inhibitory activities and activation of PPARgamma-mediated transactivation in colon and pancreatic cancer cells. Their structure-dependent induction of putative PPARgamma-responsive genes/proteins including p21, KLF-4 and caveolin1 were also determined by Western and Northern blot analysis. RESULTS: Introduction of the 5,5'-dihydroxy and 5,5'-dimethyl substituents enhanced activation of PPARgamma in colon and pancreatic cancer cells. However, activation of p21 in Panc28 pancreatic cancer cells and induction of caveolin-1 and KLF4 in colon cancer cells by the C-DIM compounds were structure- and cell context-dependent. CONCLUSIONS: The results demonstrate that DIM-C-pPhC(6)H(5) and indole ring-substituted analogs are selective PPARgamma modulators.

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.