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1.  Interferon Tau Alleviates Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Regulating Macrophage Polarization 
PLoS ONE  2014;9(6):e98835.
Chronic adipose tissue inflammation is a hallmark of obesity-induced insulin resistance and anti-inflammatory agents can benefit patients with obesity-associated syndromes. Currently available type I interferons for therapeutic immunomodulation are accompanied by high cytotoxicity and therefore in this study we have examined anti-inflammatory effects of interferon tau (IFNT), a member of the type I interferon family with low cellular toxicity even at high doses. Using a diet-induced obesity mouse model, we observed enhanced insulin sensitivity in obese mice administered IFNT compared to control mice, which was accompanied by a significant decrease in secretion of proinflammatory cytokines and elevated anti-inflammatory macrophages (M2) in adipose tissue. Further investigations revealed that IFNT is a potent regulator of macrophage activation that favors anti-inflammatory responses as evidenced by activation of associated surface antigens, production of anti-inflammatory cytokines, and activation of selective cell signaling pathways. Thus, our study demonstrates, for the first time, that IFNT can significantly mitigate obesity-associated systemic insulin resistance and tissue inflammation by controlling macrophage polarization, and thus IFNT can be a novel bio-therapeutic agent for treating obesity-associated syndromes and type 2 diabetes.
doi:10.1371/journal.pone.0098835
PMCID: PMC4048269  PMID: 24905566
2.  Polybrominated Dibenzo-p-Dioxins, Dibenzofurans, and Biphenyls: Inclusion in the Toxicity Equivalency Factor Concept for Dioxin-Like Compounds 
Toxicological Sciences  2013;133(2):197-208.
In 2011, a joint World Health Organization (WHO) and United Nations Environment Programme (UNEP) expert consultation took place, during which the possible inclusion of brominated analogues of the dioxin-like compounds in the WHO Toxicity Equivalency Factor (TEF) scheme was evaluated. The expert panel concluded that polybrominated dibenzo-p-dioxins (PBDDs), dibenzofurans (PBDFs), and some dioxin-like biphenyls (dl-PBBs) may contribute significantly in daily human background exposure to the total dioxin toxic equivalencies (TEQs). These compounds are also commonly found in the aquatic environment. Available data for fish toxicity were evaluated for possible inclusion in the WHO-UNEP TEF scheme (van den Berg et al., 1998). Because of the limited database, it was decided not to derive specific WHO-UNEP TEFs for fish, but for ecotoxicological risk assessment, the use of specific relative effect potencies (REPs) from fish embryo assays is recommended. Based on the limited mammalian REP database for these brominated compounds, it was concluded that sufficient differentiation from the present TEF values of the chlorinated analogues (van den Berg et al., 2006) was not possible. However, the REPs for PBDDs, PBDFs, and non-ortho dl-PBBs in mammals closely follow those of the chlorinated analogues, at least within one order of magnitude. Therefore, the use of similar interim TEF values for brominated and chlorinated congeners for human risk assessment is recommended, pending more detailed information in the future.
doi:10.1093/toxsci/kft070
PMCID: PMC3663561  PMID: 23492812
dioxin; halogenated hydrocarbon; persistent organic chemicals; polychlorinated biphenyls; regulatory/policy; biomarkers.
3.  INHIBITION OF RHABDOMYOSARCOMA CELL AND TUMOR GROWTH BY TARGETING SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS 
Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are highly expressed in rhabdomyosarcoma (RMS) cells. In tissue arrays of RMS tumor cores from 71 patients, 80% of RMS patients expressed high levels of Sp1 protein, whereas low expression of Sp1 was detected in normal muscle tissue. The non-steroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA) inhibited growth and migration of RD and RH30 RMS cell lines and also inhibited tumor growth in vivo using a mouse xenograft (RH30 cells) model. The effects of TA were accompanied by downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes in RMS cells and tumors, and the role of Sp protein downregulation in mediating inhibition of RD and RH30 cell growth and migration was confirmed by individual and combined knockdown of Sp1, Sp3 and Sp4 proteins by RNA interference. TA treatment and Sp knockdown in RD and RH30 cells also showed that four genes that are emerging as individual drug targets for treating RMS, namely c-MET, insulin-like growth factor receptor (IGFR), PDGFRα and CXCR4, are also Sp-regulated genes. These results suggest that NSAIDs such as TA may have potential clinical efficacy in drug combinations for treating RMS patients.
doi:10.1002/ijc.27730
PMCID: PMC3527649  PMID: 22815231
Tolfenamic acid; Sp proteins; downregulation; RMS cells
4.  Unifying Mechanisms of Action of the Anticancer Activities of Triterpenoids and Synthetic Analogs 
Triterpenoids such as betulinic acid (BA) and synthetic analogs of oleanolic acid [2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)] and glycyrrhetinic acid [2-cyano-3,11-dioxo-18β-oleana-1,12-dien-30-oc acid (CDODA)] are potent anticancer agents that exhibit antiproliferative, antiangiogenic, anti-inflammatory and pro-apoptotic activities. Although their effects on multiple pathways have been reported, unifying mechanisms of action have not been reported. Studies in this laboratory have now demonstrated that several triterpenoids including BA and some derivatives, celastrol, methyl ursolatee, β-boswellic acid derivatives, and the synthetic analogs CDDO, CDODA and their esters decreased expression of specificity protein (Sp) transcription factors and several pro-oncogenic Sp-regulated genes in multiple cancer cell lines. The mechanisms of this response are both compound- and cell context-dependent and include activation of both proteasome-dependent and -independent pathways. Triterpenoid-mediated induction of reactive oxygen species (ROS) has now been characterized as an important proteasome-independent pathway for downregulation of Sp transcription factors. ROS decreases expression of microRNA-27a (miR-27a) and miR-20a/miR-17-5p and this results in the induction of the transcriptional “Sp-repressors” ZBTB10 and ZBTB4, respectively, which in turn downregulate Sp and Sp-regulated genes. Triterpenoids also activate or deactive nuclear receptors and G-protein coupled receptors, and these pathways contribute to their antitumorigenic activity and may also play a role in targeting Sp1, Sp3 and Sp4 which are highly overexpressed in multiple cancers and appear to be important for maintaining the cancer phenotype.
PMCID: PMC3532564  PMID: 22583404
Sp transcription factors; downregulation; reactive oxygen species
5.  Cytotoxicity of Pomegranate Polyphenolics in Breast Cancer Cells in Vitro and Vivo - Potential Role of miRNA-27a and miRNA-155 in Cell Survival and Inflammation 
Several studies have demonstrated that polyphenolics from pomegranate (Punica granatum L.) are potent inhibitors of cancer cell proliferation and induce apoptosis, cell cycle arrest, and also decrease inflammation in vitro and vivo. There is growing evidence that botanicals exert their cytotoxic and anti-inflammatory activities, at least in part, by decreasing specificity protein (Sp) transcription factors. These are overexpressed in breast-tumors and regulate genes important for cancer cell survival and inflammation such as the p65 unit of NF-κB. Moreover, previous studies have shown that Pg extracts decrease inflammation in lung cancer cell lines by inhibiting phosphatidylinositol 3,4,5-trisphosphate (PI3K)-dependent phosphorylation of AKT in vitro and inhibiting the activation of NF-kB in vivo. The objective of this study was to investigate the roles of miR-27a-ZBTB10-Sp and miR-155-SHIP-1-PI3K on the anti-inflammatory and cytotoxic activity of pomegranate extract.
Pg extract (2.5–50 µg/ml) inhibited growth of BT-474 and MDA-MB-231 cells but not the non-cancer MCF-10F and MCF-12F cells. Pg extract significantly decreased Sp1, Sp3, and Sp4 as well as miR-27a in BT474 and MDA-MB-231 cells and increased expression of the transcriptional repressor ZBTB10. A significant decrease in Sp proteins and Sp-regulated genes was also observed. Pg extract also induced SHIP-1 expression and this was accompanied by downregulation of miRNA-155 and inhibition of PI3K-dependent phosphorylation of AKT. Similar results were observed in tumors from nude mice bearing BT474 cells as xenografts and treated with Pg extract. The effects of antagomirs and knockdown of SHIP-1 by RNA interference confirmed that the anti-inflammatory and cytotoxic effects of Pg extract were partly due to the disruption of both miR-27a-ZBTB10 and miR-155-SHIP-1.
In summary the anticancer activities of Pg extract in breast cancer cells were due in part to targeting microRNAs155 and 27a. Both pathways play an important role in the proliferative/inflammatory phenotype exhibited by these cell lines
doi:10.1007/s10549-012-2224-0
PMCID: PMC3488590  PMID: 22941571
Breast Cancer; Polyphenolics; Pomegranate; Xenografts; Inflammation; Cytotoxicity
6.  HOTAIR IS A NEGATIVE PROGNOSTIC FACTOR AND EXHIBITS PRO-ONCOGENIC ACTIVITY IN PANCREATIC CANCER 
Oncogene  2012;32(13):1616-1625.
HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared to non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR plays an important role in pancreatic cancer cell invasion and as reported in other cancer cell lines. In contrast, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression, and induced apoptosis, demonstrating an expanded function for HOTAIR in pancreatic cancer cells compared to other cancer cell lines. Results of gene array studies showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic vs. breast cancer cells and HOTAIR uniquely suppressed several interferon-related genes and gene sets related to cell cycle progression in pancreatic cancer cells and tumors. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6 pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC2-dependent and -independent. HOTAIR knockdown in L3.6pL cells inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer.
doi:10.1038/onc.2012.193
PMCID: PMC3484248  PMID: 22614017
HOTAIR; invasion; cell cycle progression; pro-oncogenic; prognostic
7.  Induction of the Transcriptional Repressor ZBTB4 in Prostate Cancer Cells by Drug-induced Targeting of microRNA-17-92/106b-25 Clusters 
Molecular cancer therapeutics  2012;11(9):1852-1862.
Androgen-insensitive DU145 and PC3 human prostate cancer cells express high levels of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and treatment of cells with methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) inhibited cell growth and downregulated Sp1, Sp3 and Sp4 expression. CDODA-Me (15 mg/kg/d) was a potent inhibitor of tumor growth in a mouse xenograft model (PC3 cells) and also decreased expression of Sp transcription factors in tumors. CDODA-Me-mediated downregulation of Sp1, Sp3 and Sp4 was due to induction of the transcriptional repressor ZBTB4 which competitively binds and displaces Sp transcription factors from GC-rich sites in Sp1, Sp3, Sp4 and Sp-regulated gene promoters. ZBTB4 levels are relatively low in DU145 and PC3 cells due to suppression by microRNA (miR) paralogs that are members of the miR-17-92 (miR-20a/17-5p) and miR-106b-25 (miR-106b/93) clusters. Examination of publically available prostate cancer patient array data showed an inverse relationship between ZBTB4 and miRs-20a/17-5p/106b/93 expression, and increased ZBTB4 in prostate cancer patients was a prognostic factor for increased survival. CDODA-Me induces ZBTB4 in prostate cancer cells through disruption of miR-ZBTB4 interactions and this results in downregulation of pro-oncogenic Sp transcription factors and Sp-regulated genes.
doi:10.1158/1535-7163.MCT-12-0181
PMCID: PMC3632183  PMID: 22752225
ZBTB4; CDODA-Me; Sp; miR-17-92; miR-106b
8.  Chemoprevention of Skin Cancer with 1,1-Bis (3′-Indolyl)-1-(Aromatic) Methane Analog through Induction of the Orphan Nuclear Receptor, NR4A2 (Nurr1) 
PLoS ONE  2013;8(8):e69519.
Background
The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.
Methods
In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.
Results
The IC50 values of DIM-D were 68.7±7.3, 48.3±10.1 and 11.5±3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1±8.3, 186.1±5.2 and 56.7±3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.
Conclusion
Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.
doi:10.1371/journal.pone.0069519
PMCID: PMC3737220  PMID: 23950896
9.  Role of peroxisome proliferator-activated receptor-γ and its co-activator DRIP205 in cellular responses to CDDO (RTA-401) in AML 
Cancer research  2010;70(12):4949-4960.
Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor family of transcription factors with important regulatory roles in cellular growth, differentiation and apoptosis. Using proteomic analysis, we demonstrated expression of PPARγ protein in a series of 260 newly diagnosed primary AML samples. Forced expression of PPARγ enhanced the sensitivity of myeloid leukemic cells to PPARγ agonists CDDO- or 15d15DPGJ2-induced apoptosis, through preferential cleavage of caspase-8. No effects on cell cycle distribution or differentiation were noted, despite prominent induction of p21 in PPARγ-transfected cells. In turn, antagonizing PPARγ function by siRNA or pharmacological PPARγ inhibitor significantly diminished apoptosis induction by CDDO. Overexpression of co-activator protein DRIP205 resulted in enhanced differentiation induction by CDDO in AML cells through PPARγ activation. Studies with DRIP205 deletion constructs demonstrated that the NR boxes of DRIP205 are not required for this co-activation. In a Phase I clinical trial of CDDO (RTA-401) in leukemia, CDDO induced an increase in PPARγ mRNA expression in 6 of 9 patient samples; of those, induction of differentiation was documented in 4, and of p21 in 3 patients, all expressing DRIP205 protein. In summary, these findings suggest that cellular levels of PPARγ regulate induction of apoptosis via caspase-8 activation, while the co-activator DRIP205 is a determinant of induction of differentiation, in response to PPARγ agonists in leukemic cells.
doi:10.1158/0008-5472.CAN-09-1962
PMCID: PMC3727426  PMID: 20501850
PPARgamma; DRIP205; AML; CDDO; differentiation; apoptosis
10.  The Toxicology Education Summit: Building the Future of Toxicology Through Education 
Toxicological Sciences  2012;127(2):331-338.
Toxicology and careers in toxicology, as well as many other scientific disciplines, are undergoing rapid and dramatic changes as new discoveries, technologies, and hazards advance at a blinding rate. There are new and ever increasing demands on toxicologists to keep pace with expanding global economies, highly fluid policy debates, and increasingly complex global threats to public health. These demands must be met with new paradigms for multidisciplinary, technologically complex, and collaborative approaches that require advanced and continuing education in toxicology and associated disciplines. This requires paradigm shifts in educational programs that support recruitment, development, and training of the modern toxicologist, as well as continued education and retraining of the midcareer professional to keep pace and sustain careers in industry, government, and academia. The Society of Toxicology convened the Toxicology Educational Summit to discuss the state of toxicology education and to strategically address educational needs and the sustained advancement of toxicology as a profession. The Summit focused on core issues of: building for the future of toxicology through educational programs; defining education and training needs; developing the “Total Toxicologist”; continued training and retraining toxicologists to sustain their careers; and, finally, supporting toxicology education and professional development. This report summarizes the outcomes of the Summit, presents examples of successful programs that advance toxicology education, and concludes with strategies that will insure the future of toxicology through advanced educational initiatives.
doi:10.1093/toxsci/kfs111
PMCID: PMC3355314  PMID: 22461448
toxicology; education
11.  STRUCTURE-DEPENDENT ACTIVATION OF NR4A2 (Nurr1) BY 1,1-BIS(3′-INDOLYL)-1-(AROMATIC)METHANE ANALOGS IN PANCREATIC CANCER CELLS 
Biochemical pharmacology  2012;83(10):1445-1455.
NR4A2 (Nurr 1) is an orphan nuclear receptor with no known endogenous ligands and is highly expressed in many cancer cell lines including Panc1 and Panc28 pancreatic cancer cells. Structure-dependent activation of NR4A2 by a series of 1,1-bis(3′-indolyl)-1-(aromatic)methane (C-DIM) analogs was determined in pancreatic cancer cells transfected with yeast GAL4-Nurr1 chimeras and a UASx5-luc reporter gene or constructs containing response elements that bind NR4A2. Among 23 different structural analogs, phenyl groups containing p-substituted trifluoromethyl, t-butyl, cyano, bromo, iodo and trifluoromethoxy groups were the most active compounds in transactivation assay. The p-bromophenyl analog (DIM-C-pPhBr) was used as a model for structure-activity studies among a series of ortho-, meta- and para-bromophenyl isomers and the corresponding indole 2- and N-methyl analogs. Results show that NR4A2 activation was maximal with the p-bromophenyl analog and methylation of the indole NH group abrogated activity. Moreover, using GAL4-Nurr1 (full length) or GAL-Nurr1-A/B and GAL4-Nurr1-(C-F) chimeras expressing N- and C-terminal domains of Nurr1, respectively, DIM-C-pPhBr activated all three constructs and these responses were differentially affected by kinase inhibitors. DIM-C-pPhBr also modulated expression of several Nurr1-regulated genes in pancreatic cancer cells including vasoactive intestinal peptide (VIP), and the immunohistochemical and western blot analyses indicated that DIM-C-pPhBr activates nuclear NR4A2.
doi:10.1016/j.bcp.2012.02.021
PMCID: PMC3408083  PMID: 22405837
DIM analogs; NR4A2/Nurr1; structure-activity
12.  Pharmacokinetic evaluation and In Vitro–In Vivo Correlation (IVIVC) of novel methylene-substituted 3,3′ diindolylmethane (DIM) 
Purpose
3,3′-Diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed oligomerization of indole-3-carbinol present in cruciferous vegetables. 1, 1-bis (3′-indolyl)-1-(p-substituted phenyl) methanes [C-substituted diindolylmethanes (C-DIMs)] are a new class of anticancer compounds derived from indole 3-carbinol. Despite rapidly increasing knowledge regarding mechanisms responsible for the chemopreventive properties of DIM-C-pPhC6H5, there have been relatively few studies determining the absorption and pharmacokinetic properties of DIM-C-pPhC6H5 to explore its clinical utility.
Methods
In this study, we assessed the solubility, lipophilicity and Caco-2 cell permeability of methylene-substituted DIM. Pharmacokinetic properties in rats were determined following i.v. and oral administration of a novel analog of DIM. Pharmacokinetic parameters were determined using noncompartmental and compartmental techniques with WinNonlin® 5.0 software. To explore potential In Vitro–In Vivo Correlation (IVIVC) between the in vitro permeability values, and the oral absorption pharmacokinetics, we employed deconvolution of i.v. and oral data using a three compartment Exact Loo–Riegelman method.
Results
The oral absorption and disposition were described by a three compartment model with combined zero-order/Michaelis–Menten limited systemic uptake using differential equations, at physiologically relevant doses. The saturation model obtained accounts for a nonlinear change in Cmax/Dose, and the absolute bioavailability (0.13 ± 0.06) was also dose dependent. The absorption rate profile of DIMC-pPhC6H5 across Caco-2 cells was significantly different than in vivo. Conclusions: The pharmacokinetic absorption model presented represents a useful basis for obtaining plasma level predictability for poorly bioavailable, highly lipophilic drugs, such as the DIM analog DIM-C-pPhC6H5.
doi:10.1016/j.ejps.2012.01.012
PMCID: PMC3374645  PMID: 22342559
Anticancer; Pharmacokinetic model; Chemoprevention; DIM; Anticarcinogen
13.  Celastrol decreases specificity proteins (Sp) and fibroblast growth factor receptor-3 (FGFR3) in bladder cancer cells 
Carcinogenesis  2012;33(4):886-894.
Celastrol (CSL) is a naturally occurring triterpenoid acid that exhibits anticancer activity, and in KU7 and 253JB-V bladder cells, CSL induced apoptosis, inhibited growth, colony formation and migration and CSL decreased bladder tumor growth in vivo. CSL also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-regulated genes/proteins including vascular endothelial growth factor, survivin and cyclin D1 and fibroblast growth factor receptor-3, a potential drug target for bladder cancer therapy, has now been characterized as an Sp-regulated gene downregulated by CSL. The mechanism of Sp downregulation by CSL was cell context-dependent due to activation of proteosome-dependent (KU7) and -independent (253JB-V) pathways. In 253JB-V cells, CSL induced reactive oxygen species (ROS) and inhibitors of ROS blocked CSL-induced growth inhibition and repression of Sp1, Sp3 and Sp4. This response was due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of miR-27a and miR-20a/17-5p, respectively, which regulate expression of these transcriptional repressors. Thus, the anticancer activity of CSL in 253JB-V cells is due to induction of ROS and ROS-mediated induction of Sp repressors (ZBTB4/ZBTB10) through downregulation of miR-27a and miR-20a/17-5p.
doi:10.1093/carcin/bgs102
PMCID: PMC3324448  PMID: 22334592
14.  Endocrine disruptors and falling sperm counts: lessons learned or not! 
Asian Journal of Andrology  2012;15(2):191-194.
doi:10.1038/aja.2012.87
PMCID: PMC3739165  PMID: 23001442
15.  The Nuclear Receptor TR3 Regulates mTORC1 Signaling in Lung Cancer Cells Expressing Wild-type p53 
Oncogene  2011;31(27):3265-3276.
The orphan nuclear receptor TR3 (NR41A, Nur77) is overexpressed in most lung cancer patients and is a negative prognostic factor for patient survival. The function of TR3 was investigated in non-small cell lung cancer A549 and H460 cells, and knockdown of TR3 by RNA interference (siTR3) inhibited cancer cell growth and induced apoptosis. The prosurvival activity of TR3 was due, in part, to formation of a p300/TR3/Sp1 complex bound to GC-rich promoter regions of survivin and other Sp-regulated genes (mechanism 1). However, in p53 wild-type A549 and H460 cells, siTR3 inhibited the mTORC1 pathway and this was due to activation of p53 and induction of the p53-responsive gene sestrin 2 which subsequently activated the mTORC1 inhibitor AMPKα (mechanism 2). This demonstrates that the pro-oncogenic activity of TR3 in lung cancer cells was due to inhibition of p53 and activation of mTORC1. 1,1-Bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) is a recently discovered inhibitor of TR3 which mimics the effects of siTR3. DIM-C-pPhOH inhibited growth and induced apoptosis in lung cancer cells and lung tumors in murine orthotopic and metastatic models, and this was accompanied by decreased expression of survivin and inhibition of mTORC1 signaling, demonstrating that inactivators of TR3 represent a novel class of mTORC1 inhibitors.
doi:10.1038/onc.2011.504
PMCID: PMC3299891  PMID: 22081070
TR3; Nur77; NR4A1; mTORC1; c-DIMs; lung cancer
16.  PAH Particles Perturb Prenatal Processes and Phenotypes: Protection from Deficits in Object Discrimination Afforded by Dampening of Brain Oxidoreductase Following In Utero Exposure to Inhaled Benzo(a)pyrene 
Toxicological Sciences  2011;125(1):233-247.
The wild-type (WT) Cprlox/lox (cytochrome P450 oxidoreductase, Cpr) mouse is an ideal model to assess the contribution of P450 enzymes to the metabolic activation and disposition of environmental xenobiotics. In the present study, we examined the effect of in utero exposure to benzo(a)pyrene [B(a)P] aerosol on Sp4 and N-methyl-D-aspartate (NMDA)–dependent systems as well as a resulting behavioral phenotype (object discrimination) in Cpr offspring. Results from in utero exposure of WT Cprlox/lox mice were compared with in utero exposed brain-Cpr-null offspring mice. Null mice were used as they do not express brain cytochrome P4501B1–associated NADPH oxidoreductase (CYP1B1-associated NADPH oxidoreductase), thus reducing their capacity to produce neural B(a)P metabolites. Subsequent to in utero (E14–E17) exposure to B(a)P (100 μg/m3), Cprlox/lox offspring exhibited: (1) elevated B(a)P metabolite and F2-isoprostane neocortical tissue burdens, (2) elevated concentrations of cortical glutamate, (3) premature developmental expression of Sp4, (4) decreased subunit ratios of NR2B:NR2A, and (5) deficits in a novelty discrimination phenotype monitored to in utero exposed brain-Cpr-null offspring. Collectively, these findings suggest that in situ generation of metabolites by CYP1B1-associated NADPH oxidoreductase promotes negative effects on NMDA-mediated signaling processes during the period when synapses are first forming as well as effects on a subsequent behavioral phenotype.
doi:10.1093/toxsci/kfr261
PMCID: PMC3243744  PMID: 21987461
NMDA receptor; neuronal activity; neurogenesis; synaptogenesis; polycyclic aromatic hydrocarbon; benzo(a)pyrene; susceptibility-exposure paradigm; B(a)P metabolites; object discrimination task
17.  Aryl Hydrocarbon Receptor (AHR) Agonists Induce MicroRNA-335 Expression And Inhibit Lung Metastasis of Estrogen Receptor Negative Breast Cancer Cells 
Molecular Cancer Therapeutics  2011;11(1):108-118.
The aryl hydrocarbon receptor (AHR) was initially identified as a receptor that bound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental toxicants; however, there is increasing evidence that the AHR is an important new drug target for treating multiple diseases including breast cancer. Treatment of estrogen receptor (ER)-negative MDA-MB-231 and BT474 breast cancer cells with TCDD or the selective AHR modulator 6-methyl-1,3,-trichlorodibenzofuran (MCDF) inhibited breast cancer cell invasion in a Boyden chamber assay. These results were similar to those previously reported for the antimetastic microRNA-335 (miR-335). Both TCDD and MCDF induced miR-335 in MDA-MB-231 and BT474 cells and this was accompanied by downregulation of SOX4, a miR-335-regulated (inhibited) gene. The effects of TCDD and MCDF on miR-335 and SOX4 expression and interactions of miR-335 with the 3'-UTR target sequence in the SOX4 gene were all inhibited in cells transfected with an oligonucleotide (iAHR) that knocks down the AHR, thus confirming AHR-miR-335 interactions. MCDF (40 mg/kg/day) also inhibited lung metastasis of MDA-MB-231 cells in a tail vein injection model, demonstrating that the AHR is a potential new target for treating patients with ER-negative breast cancer, a disease where treatment options and their effectiveness are limited.
doi:10.1158/1535-7163.MCT-11-0548
PMCID: PMC3256275  PMID: 22034498
Ah receptor; miR-335; antimetastasis
18.  Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs 
BMC Cancer  2012;12:564.
Background
Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells.
Methods
The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression.
Results
The IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors.
Conclusions
These results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.
doi:10.1186/1471-2407-12-564
PMCID: PMC3522018  PMID: 23194063
Curcumin; ROS induction; Sp transcription factors; MicroRNAs
19.  Induction of Apoptosis by Cannabinoids in Prostate and Colon Cancer Cells Is Phosphatase Dependent 
Anticancer Research  2011;31(11):3799-3807.
Aim
We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases.
Materials and Methods
The effects of cannabidiol (CBD) and the synthetic cannabinoid WIN-55,212 (WIN) on LNCaP (prostate) and SW480 (colon) cancer cell proliferation were determined by cell counting; apoptosis was determined by cleavage of poly(ADP)ribose polymerase (PARP) and caspase-3 (Western blots); and phosphatase mRNAs were determined by real-time PCR. The role of phosphatases and cannabinoid receptors in mediating CBD- and WIN-induced apoptosis was determined by inhibition and receptor knockdown.
Results
CBD and WIN inhibited LNCaP and SW480 cell growth and induced mRNA expression of several phosphatases, and the phosphatase inhibitor sodium orthovanadate significantly inhibited cannabinoid-induced PARP cleavage in both cell lines, whereas only CBD-induced apoptosis was CB1 and CB2 receptor-dependent.
Conclusion
Cannabinoid receptor agonists induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines; however, the role of the CB receptor in mediating this response is ligand-dependent.
PMCID: PMC3280884  PMID: 22110202
Cannabinoids; apoptosis; protein tyrosine phosphatases; dual-specificity phosphatases
20.  Aspirin Inhibits Colon Cancer Cell and Tumor Growth and Downregulates Specificity Protein (Sp) Transcription Factors 
PLoS ONE  2012;7(10):e48208.
Acetylsalicylic acid (aspirin) is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB). Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.
doi:10.1371/journal.pone.0048208
PMCID: PMC3482208  PMID: 23110215
21.  PHARMACOLOGIC DOSES OF ASCORBIC ACID REPRESS SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS AND Sp-REGULATED GENES IN COLON CANCER CELLS 
Nutrition and Cancer  2011;63(7):1133-1142.
Ascorbic acid (vitamin C) inhibits cancer cell growth and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound which exhibits pro-oxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (< 5 mM) is due, in part, to reactive oxygen species (ROS)-dependent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes. In this study, ascorbic acid (1 – 3 mM) decreased RKO and SW480 colon cancer cell proliferation and induced apoptosis and necrosis and this was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR) and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other pro-oxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.
doi:10.1080/01635581.2011.605984
PMCID: PMC3359146  PMID: 21919647
Ascorbic acid; colon cancer; ROS; peroxide; Sp downregulation
22.  INHIBITION OF PITUITARY TUMOR-TRANSFORMING GENE-1 IN THYROID CANCER CELLS BY DRUGS THAT DECREASE SPECIFICITY PROTEINS 
Molecular carcinogenesis  2011;50(9):655-667.
Methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) and the corresponding 2-trifluoromethyl analog (CF3DODA-Me) are derived synthetically from the triterpenoid glycyrrhetinic acid, a major component of licorice. CDODA-Me and CF3DODA-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 CF3DODA-Me also decreased expression of Sp-dependent genes, such as survivin and vascular endothelial growth factor, 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 CF3DODA-Me and this was accompanied by decreased expression of PTTG-1-dependent c-Myc and fibroblast growth factor 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 CF3DODA-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.
doi:10.1002/mc.20738
PMCID: PMC3128656  PMID: 21268135
PTTG-1; Sp proteins; thyroid cancer; anticancer agents
23.  IDENTIFICATION OF ONCOGENIC MicroRNA-17-92/ZBTB4/SPECIFICITY PROTEIN AXIS IN BREAST CANCER 
Oncogene  2011;31(8):1034-1044.
The human POK family members are transcription factors with a POZ domain and zinc fingers that act primarily as transcriptional repressors. Several members of this family are involved in oncogenesis and this prompted us to assess whether expression levels of individual POK family members are associated with clinical outcomes in cancer. We have observed that ZBTB4 is downregulated in breast cancer patients, and that its expression is significantly correlated with relapse-free survival. Further integrative analysis of mRNA and microRNA (miR) expression data from the NCI-60 cell lines revealed an inverse correlation between ZBTB4 and oncogenic miRs derived from the miR-17-92 cluster and its paralogues. The experimental results using MDA-MB-231 and MCF-7 human breast cancer cells confirm that miRNAs derived from these clusters, containing miR-17-5p, miR-20a, miR-106a, miR-106b and miR-93, negatively regulate ZBTB4 expression. Overexpression of ZBTB4 or restoration of ZBTB4 by using an antagomir inhibit growth and invasion of breast cancer cells, and this effect is due, in part, to ZBTB4-dependent repression of the specificity protein 1 (Sp1), Sp3, and Sp4 genes, and subsequent downregulation of several Sp-dependent oncogenes, in part, through competition between ZBTB4 and Sp transcription factors for GC-rich promoter sequences. These results confirm that ZBTB4 functions as a novel tumor suppressor gene with prognostic significance for breast cancer survival, and the oncogenic miR-17-92/ZBTB4/Sp axis may be a potential therapeutic target.
doi:10.1038/onc.2011.296
PMCID: PMC3288192  PMID: 21765466
ZBTB4; miR-17-92 cluster; breast cancer; prognostic; Sp transcription factors
24.  Activation of nuclear TR3 (NR4A1) by a diindolylmethane analog induces apoptosis and proapoptotic genes in pancreatic cancer cells and tumors 
Carcinogenesis  2011;32(6):836-842.
NR4A1 (Nur77, TR3) is overexpressed in pancreatic tumors and activation of TR3 by 1,1-bis(3′-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH3) inhibits cell and tumor growth and induces apoptosis. Microarray analysis demonstrates that in L3.6pL pancreatic cancer cells DIM-C-pPhOCH3 induces genes associated with metabolism, homeostasis, signal transduction, transcription, stress, transport, immune responses, growth inhibition and apoptosis. Among the most highly induced growth inhibitory and proapoptotic genes including activating transcription factor 3 (ATF3), p21, cystathionase, dual specificity phosphatase 1 and growth differentiation factor 15, RNA interference studies demonstrated that induction of all but the later gene by DIM-C-pPhOCH3 were TR3-dependent. We also observed that DIM-C-pPhOCH3 induced Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and induction of TRAIL was ATF3 dependent. Results of this and previous studies demonstrate that TR3 is unique among nuclear receptors since nuclear TR3 is activated or deactivated by diindolylmethane derivatives to induce different apoptotic and growth inhibitory pathways that inhibit pancreatic cancer cell and tumor growth.
doi:10.1093/carcin/bgr040
PMCID: PMC3106434  PMID: 21362629
25.  Multiphoton Spectral Analysis of Benzo[a]pyrene Uptake and Metabolism in a Rat Liver Cell Line 
Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 hr. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.
doi:10.1016/j.taap.2011.03.009
PMCID: PMC3081912  PMID: 21420996
Benzo[a]pyrene; microsomal epoxide hydrolase inhibitor; aryl hydrocarbon receptor; EROD activity; BaP metabolites; multiphoton spectral analysis

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