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1.  Theranostic tumor homing nanocarriers for the treatment of lung cancer 
The drugs/strategies to selectively inhibit tumor blood supply has generated interest in recent years for enhancement of cancer therapeutics. The objective of this study was to formulate tumor homing PEGylated CREKA peptide conjugated theranostic nanoparticles of DIM-C-pPhC6H5 (DIM-P) and investigate in vivo antitumor activity as well as evaluate the targeted efficiency to lung tumors using imaging techniques. DIM-P loaded Nanoparticles (NCs-D) were prepared using lipids, and DOGS-NTA-Ni and the surface of NCs-D was modified with PEGylated CREKA peptide (PCNCs-D). PCNCs-D showed 3 fold higher binding to clotted plasma proteins in tumor vasculature compared to NCs-D. PCNCs-D showed 26±4% and 22±5% increase in tumor reduction compare to NCs-D in metastatic and orthotopic models respectively. In-vivo imaging studies showed ~40 folds higher migration of PCNCs-Di in tumor vasculature than NCs-Di. Our studies demonstrate the role of PCNCs-D as theranostic tumor homing drug delivery and imaging systems for lung cancer diagnosis and treatment.
PMCID: PMC4061286  PMID: 24355163
Targeted Delivery; Theranostic; Nanoparticles; Tumor homing; Cancer Treatment; In-Vivo Imaging
2.  MicroRNA-26b Represses Colon Cancer Cell Proliferation by Inhibiting Lymphoid Enhancer Factor 1 (LEF-1) Expression 
Molecular cancer therapeutics  2014;13(7):1942-1951.
microRNAs (miR) can act as oncogenes and tumor suppressors and several miRs are associated with cancer development and progression through the modulation of multiple cellular processes. miR-26b is down regulated in several cancers and tumors and miR-26b directly targets the Lef-1 3'UTR and inhibits endogenous Lef-1 expression. We report that miR-26b expression is associated with human colon cancer through the regulation of LEF-1 expression in colon cancer cells. Analyses of multiple colon cancer cell lines revealed an inverse correlation between miR-26b and LEF-1 expression. Normal human colon cells express low levels of LEF-1 and high levels of miR-26b, however human colon cancer cells have decreased miR-26b expression and increased LEF-1 expression. We demonstrate that miR-26b expression is a potent inhibitor of colon cancer cell proliferation and significantly decreases LEF-1 expression. The LEF-1 regualted genes Cyclin D1 and c-Myc were indirectly repressed by miR-26b and this was consistent with decreased proliferation. miR-26b overexpression in SW480 colon cancer cells also inhibited tumor growth in nude mice and this was due to decreased tumor growth and not apoptosis. Analyses of human colon cancer databases also demonstrated a link between miR-26b and LEF-1 expression. c-Myc expression is associated with multiple cancers and we propose that miR-26b may act as a potential therapeutic agent in reducing cancer cell proliferation through repressing LEF-1 activation of c-Myc and Cyclin D1 expression.
PMCID: PMC4090280  PMID: 24785257
3.  Mechanistic relationships between hepatic genotoxicity and carcinogenicity in male B6C3F1 mice treated with polycyclic aromatic hydrocarbon mixtures 
Archives of toxicology  2014;89(6):967-977.
The genotoxicity of a complex mixture [neutral fraction (NF)] from a wood preserving waste and reconstituted mixture (RM) mimicking the NF with seven major polycyclic aromatic hydrocarbons (PAHs) and benzo(a)pyrene (BaP) was investigated by determining DNA adducts and tumor incidence in male B6C3F1 mice exposed to 3 different doses of the chemical mixtures. The peak values of DNA adducts were observed after 24 h and the highest levels of PAH-DNA adducts were exhibited in mice administered NF+BaP, and the highest tumor incidence and mortality were also observed in this group. DNA adduct levels after 1, 7, or 21 d were significantly correlated with animal mortality and incidence of total tumors including liver, lung, and forestomach. However, only hepatic DNA adducts after 7 d significantly correlated with liver tumor incidence. Most proteins involved in DNA repair including ATM, pATR, Chk1, pChk1, DNA PKcs, XRCC1, FANCD2, Ku80, Mre11 and Brca2 were significantly lower in liver tumor tissue compared to non-tumor tissue. Expression of proteins involved in apoptosis and cell cycle regulation were also significantly different in tumor vs non-tumor tissues and it is possible that PAH-induced changes in these gene products are important for tumor development and growth.
PMCID: PMC4254891  PMID: 24888377
Polycyclic aromatic hydrocarbon mixtures; DNA adducts; Tumor incidence; Reverse Phase Protein Array; 32P-postlabeling assay; Protein expression
4.  Nuclear Receptor 4A1 (NR4A1) as a Drug Target for Renal Cell Adenocarcinoma 
PLoS ONE  2015;10(6):e0128308.
The orphan nuclear receptor NR4A1 exhibits pro-oncogenic activity in cancer cell lines. NR4A1 activates mTOR signaling, regulates genes such as thioredoxin domain containing 5 and isocitrate dehydrogenase 1 that maintain low oxidative stress, and coactivates specificity protein 1 (Sp1)-regulated pro-survival and growth promoting genes. Transfection of renal cell carcinoma (RCC) ACHN and 786-O cells with oligonucleotides that target NR4A1 results in a 40–60% decrease in cell proliferation and induction of apoptosis. Moreover, knockdown of NR4A1 in RCC cells decreased bcl-2, survivin and epidermal growth factor receptor expression, inhibited of mTOR signaling, induced oxidative and endoplasmic reticulum stress, and decreased TXNDC5 and IDH1. We have recently demonstrated that selected 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds including the p-hydroxyphenyl (DIM-C-pPhOH) and p-carboxymethyl (DIM-C-pPhCO2Me) analogs bind NR4A1 and act as antagonists. Both DIM-C-pPhOH and DIM-C-pPhCO2Me inhibited growth and induced apoptosis in ACHN and 786-O cells, and the functional and genomic effects of the NR4A1 antagonists were comparable to those observed after NR4A1 knockdown. These results indicate that NR4A1 antagonists target multiple growth promoting and pro-survival pathways in RCC cells and in tumors (xenograft) and represent a novel chemotherapy for treating RCC.
PMCID: PMC4452731  PMID: 26035713
5.  The long non-coding RNA HOTTIP enhances pancreatic cancer cell proliferation, survival and migration 
Oncotarget  2015;6(13):10840-10852.
HOTTIP is a long non-coding RNA (lncRNA) transcribed from the 5′ tip of the HOXA locus and is associated with the polycomb repressor complex 2 (PRC2) and WD repeat containing protein 5 (WDR5)/mixed lineage leukemia 1 (MLL1) chromatin modifying complexes. HOTTIP is expressed in pancreatic cancer cell lines and knockdown of HOTTIP by RNA interference (siHOTTIP) in Panc1 pancreatic cancer cells decreased proliferation, induced apoptosis and decreased migration. In Panc1 cells transfected with siHOTTIP, there was a decrease in expression of 757 genes and increased expression of 514 genes, and a limited gene analysis indicated that HOTTIP regulation of genes is complex. For example, Aurora kinase A, an important regulator of cell growth, is coregulated by MLL and not WDR5 and, in contrast to previous studies in liver cancer cells, HOTTIP does not regulate HOXA13 but plays a role in regulation of several other HOX genes including HOXA10, HOXB2, HOXA11, HOXA9 and HOXA1. Although HOTTIP and the HOX-associated lncRNA HOTAIR have similar pro-oncogenic functions, they regulate strikingly different sets of genes in Panc1 cells and in pancreatic tumors.
PMCID: PMC4484423  PMID: 25912306
HOTTIP; lncRNA; HOX genes; pro-oncogenic; HOTAIR
6.  3,3′-Diindolylmethane (DIM) and its ring-substituted halogenated analogs (ring-DIMs) induce differential mechanisms of survival and death in androgen-dependent and –independent prostate cancer cells 
Genes & Cancer  2015;6(5-6):265-280.
We recently reported that novel ring-substituted analogs of 3,3′-diindolylmethane (ring-DIMs) induce apoptosis and necrosis in androgen-dependent and –independent prostate cancer cells. In this paper, we have focused on the mechanism(s) associated with ring-DIM-mediated cell death, and on identifying the specific intracellular target(s) of these compounds. The 4,4′- and 7,7′-dichloroDIMs and 4,4′- and 7,7′-dibromoDIMs induced the death of LNCaP, C42B and DU145 prostate cancer cells, but not that of immortalized normal human prostate epithelial (RWPE-1) cells. Ring-DIMs caused the early loss of mitochondrial membrane potential (MMP) and decreased mitochondrial ATP generation in prostate cancer cells. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore, inhibited ring-DIM-mediated cell death, and salubrinal, an inhibitor of ER stress, inhibited cell death mediated only by 4,4′-dihaloDIMs. We found that although salubrinal did not inhibit the onset of ER stress, it prevented 4,4′-dibromoDIM mediated loss of MMP. Salubrinal potentiated cell death in response to 7,7′-dihaloDIMs and DIM, and this effect concurred with increased loss of MMP. Using in silico 3-D docking affinity analysis, we identified Ca2+/calmodulin-dependent kinase II (CaMKII) as a potential direct target for the most toxic ring-DIM, 4,4′-dibromoDIM. An inhibitor of CaMKII, KN93, but not its inactive analog KN92, abrogated cell death mediated by 4,4′-dibromoDIM. The ring-DIMs induced ER stress and autophagy, but these processes were not necessary for ring-DIM-mediated cell death. Inhibition of autophagy with bafilomycin A1, 3-methyladenine or by LC3B gene silencing sensitized LNCaP and C42B, but not ATG5-deficient DU145 cells to ring-DIM- and DIM-mediated cell death. We propose that autophagy induced by the ring-DIMs and DIM has a cytoprotective function in prostate cancer cells.
PMCID: PMC4482247  PMID: 26124925
prostate cancer; LNCaP; C42B; DU145; mitochondrial function
7.  Targeting NR4A1 (TR3) in Cancer Cells and Tumors 
NR4A1 (TR3, Nur77) is a member of the nuclear receptor superfamily of transcription factors and there is evidence that this receptor is highly expressed in multiple tumor types. Moreover, RNA interference studies indicate that NR4A1 exhibits growth promoting, angiogenic and prosurvival activity in most cancers.
Areas Covered
This review summarizes studies on several apoptosis-inducing agents that activate nuclear export of NR4A1 which subsequently forms a mitochondrial NR4A1-bcl-2 complex that induces the intrinsic pathway for apoptosis. Cytosporone B and related compounds that induce NR4A1-dependent apoptosis in cancer cells through both modulation of nuclear NR4A1 and nuclear export are also discussed. A relatively new class of diindolylmethane analogs (C-DIMs) including 1,1-bis(3′-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH3) (NR4A1 activator) and 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) (NR4A1 deactivator) are discussed in more detail. These anticancer drugs (C-DIMs) act strictly through nuclear NR4A1 and induce apoptosis in cancer cells and tumors.
Expert Opinion
It is clear that NR4A1 plays an important pro-oncogenic role in cancer cells and tumors, and there is increasing evidence that this receptor can be targeted by anticancer drugs that induce cell death via NR4A1-dependent and -independent pathways. Moreover, since many of these compounds exhibit relatively low toxicity, they represent an important class of mechanism-based anticancer drugs with excellent potential for clinical applications.
PMCID: PMC4407471  PMID: 21204731
8.  The Orphan Nuclear Receptor NR4A1 (Nur77) Regulates Oxidative and Endoplasmic Reticulum Stress in Pancreatic Cancer Cells 
Molecular cancer research : MCR  2014;12(4):527-538.
NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNAi interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum (ER) stress including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and ER stress by these agents was attenuated after co-treatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and ER stress; thus, demonstrating that NR4A1 regulates levels of ER stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of ROS/ER stress and pro-apoptotic pathways.
PMCID: PMC4407472  PMID: 24515801
NR4A1; TR3; Nur77; pancreatic cancer; oxidative stress; endoplasmic stress; apoptosis; TXNDC5
Several agents used for treatment of colon and other cancers induce reactive oxygen species (ROS) and this plays an important role in their anticancer activities. In addition to the well-known proapoptotic effects of ROS inducers, these compounds also decrease expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several pro-oncogenic Spregulated genes important for cancer cell proliferation, survival and metastasis. The mechanism of these responses involve ROS-dependent downregulation of microRNA-27a (miR-27a) or miR-20a (and paralogs) and induction of two Sp-repressors, ZBTB10 and ZBTB4 respectively. This pathway significantly contributes to the anticancer activity of ROS inducers and should be considered in development of drug combinations for cancer chemotherapy.
PMCID: PMC4288935  PMID: 25584043
Colon cancer; reactive oxygen Species; antioxidants; specificity transcription factors; targeted therapy; mechanism based drugs; zinc finger DNA binding proteins; microRNA
Molecular cancer therapeutics  2013;12(11):2483-2493.
2,3-Dihydro-5-methyl-3-([morpholinyl]methyl)pyrollo(1,2,3-de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone [WIN 55,212-2 (WIN)] is a synthetic cannabinoid that inhibits RKO, HT-29 and SW480 cell growth, induced apoptosis, and downregulated expression of survivin, cyclin D1, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and its receptor (VEGFR1). WIN also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and this is consistent with the observed downregulation of the aforementioned Sp-regulated genes. In addition, we also observed by RNA interference (RNAi) that the oncogenic cap protein eIF4E was an Sp-regulated gene also downregulated by WIN in colon cancer cells. WIN-mediated repression of Sp proteins was not affected by CB receptor antagonists or by knockdown of the receptor but was attenuated by the phosphatase inhibitor sodium orthovanadate or by knockdown of protein phosphatase 2A (PP2A). WIN-mediated repression of Sp1, Sp3 and Sp4 was due to PP2A-dependent downregulation of microRNA-27a (miR-27a) and induction of miR-27a-regulated ZBTB10 which has previously been characterized as an “Sp repressor”. The results demonstrate that the anticancer activity of WIN is due, in part, to PP2A-dependent disruption of miR-27a:ZBTB10 and ZBTB10-mediated repression of Sp transcription factors and Sp-regulated genes including eIF4E.
PMCID: PMC4288937  PMID: 24030632
Cannabinoids; WIN 55,212-2; downregulation; Sp; eIF4E
11.  Mechanism of Action of Phenethylisothiocyanate and Other Reactive Oxygen Species-Inducing Anticancer Agents 
Molecular and Cellular Biology  2014;34(13):2382-2395.
Reactive oxygen species (ROS)-inducing anticancer agents such as phenethylisothiocyanate (PEITC) activate stress pathways for killing cancer cells. Here we demonstrate that PEITC-induced ROS decreased expression of microRNA 27a (miR-27a)/miR-20a:miR-17-5p and induced miR-regulated ZBTB10/ZBTB4 and ZBTB34 transcriptional repressors, which, in turn, downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells. Decreased expression of miR-27a/miR-20a:miR-17-5p by PEITC-induced ROS is a key step in triggering the miR-ZBTB Sp cascade leading to downregulation of Sp TFs, and this is due to ROS-dependent epigenetic effects associated with genome-wide shifts in repressor complexes, resulting in decreased expression of Myc and the Myc-regulated miRs. Knockdown of Sp1 alone by RNA interference also induced apoptosis and decreased pancreatic cancer cell growth and invasion, indicating that downregulation of Sp transcription factors is an important common mechanism of action for PEITC and other ROS-inducing anticancer agents.
PMCID: PMC4054319  PMID: 24732804
12.  The Transcriptional Repressor ZBTB4 Regulates EZH2 Through a MicroRNA-ZBTB4-Specificity Protein Signaling Axis12 
Neoplasia (New York, N.Y.)  2014;16(12):1059-1069.
ZBTB4 is a transcriptional repressor and examination of publically-available microarray data sets demonstrated an inverse relationship in the prognostic value and expression of ZBTB4 and the histone methyltransferase EZH2 in tumors from breast cancer patients. The possibility of functional interactions between EZH2 and ZBTB4 was investigated in breast cancer cells and the results showed that EZH2 is directly suppressed by ZBTB4 which in turn is regulated (suppressed) by miR-106b and other paralogues from the miR-17-92, miR-106b-25 and miR-106a-363 clusters that are highly expressed in breast and other tumors. ZBTB4 also acts a suppressor of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and RNA interference studies show that Sp proteins are required for EZH2 expression. The prediction analysis results from breast cancer patient array data sets confirm an association of Sp1-dependent EZH2 gene signature with decreased survival of breast cancer patients. Disruption of oncogenic miR-ZBTB4 signaling axis by anticancer agent such as betulinic acid that induce down-regulation of Sp proteins in breast cancer cells resulted in inhibition of tumor growth and colonization of breast cancer cells in a mouse model. Thus, EZH2 is reciprocally regulated by a novel signaling network consisting of Sp proteins, oncogenic miRs and ZBTB4, and modulation of this gene network is a novel therapeutic approach for treatment of breast cancer and possibly other cancers.
PMCID: PMC4309261  PMID: 25499219
Sp, Specificity protein; ZBTB4, Zinc finger BTB domain protein 4; miR, microRNA; BA, Betulinic acid; EMSA, Electrophoretic mobility shift assay; Chip, Chromatin immunoprecipitation assay
13.  Metformin inhibits pancreatic cancer cell and tumor growth and downregulates Sp transcription factors 
Carcinogenesis  2013;34(12):2870-2879.
Metformin is a widely used antidiabetic drug, and epidemiology studies for pancreatic and other cancers indicate that metformin exhibits both chemopreventive and chemotherapeutic activities. Several metformin-induced responses and genes are similar to those observed after knockdown of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 by RNA interference, and we hypothesized that the mechanism of action of metformin in pancreatic cancer cells was due, in part, to downregulation of Sp transcription factors. Treatment of Panc1, L3.6pL and Panc28 pancreatic cancer cells with metformin downregulated Sp1, Sp3 and Sp4 proteins and several pro-oncogenic Sp-regulated genes including bcl-2, survivin, cyclin D1, vascular endothelial growth factor and its receptor, and fatty acid synthase. Metformin induced proteasome-dependent degradation of Sps in L3.6pL and Panc28 cells, whereas in Panc1 cells metformin decreased microRNA-27a and induced the Sp repressor, ZBTB10, and disruption of miR-27a:ZBTB10 by metformin was phosphatase dependent. Metformin also inhibited pancreatic tumor growth and downregulated Sp1, Sp3 and Sp4 in tumors in an orthotopic model where L3.6pL cells were injected directly into the pancreas. The results demonstrate for the first time that the anticancer activities of metformin are also due, in part, to downregulation of Sp transcription factors and Sp-regulated genes.
PMCID: PMC3845888  PMID: 23803693
14.  1, 1-Bis (3′-indolyl)-1-(p-substituted phenyl) methane compounds inhibit lung cancer cell and tumor growth in a metastasis model 
1,1-Bis(3-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds exhibit remarkable antitumor activity with low toxicity in various cancer cells including lung tumors. Two C-DIM analogs, DIM-C-pPhOCH3 (C-DIM-5) and DIM-C-pPhOH (C-DIM-8) while acting differentially on the orphan nuclear receptor, TR3/Nur77 inhibited cell cycle progression from G0/G1 to S-phase and induced apoptosis in A549 cells. Combinations of docetaxel (doc) with C-DIM-5 or C-DIM-8 showed synergistic anticancer activity in vitro and these results were consistent with their enhanced antitumor activities in vivo. Respirable aqueous formulations of C-DIM-5 (mass median aerodynamic diameter of 1.92 ± 0.22 um and geometric standard deviation of 2.31 ± 0.12) and C-DIM-8 (mass median aerodynamic diameter of 1.84 ± 0.31 um and geometric standard deviation of 2.11 ± 0.15) were successfully delivered by inhalation to athymic nude mice bearing A549 cells as metastatic tumors. This resulted in significant (p<0.05) lung tumor regression and an overall reduction in tumor burden. Analysis of lung tumors from mice treated with inhalational formulations of C-DIM-5 and C-DIM-8 showed decreased mRNA and protein expression of mediators of tumor initiation, metastasis, and angiogenesis including MMP2, MMP9, c-Myc, β-catenin, c-Met, c-Myc, and EGFR. Microvessel density assessment of lung tissue sections showed significant reduction (p<0.05) in angiogenesis and metastasis as evidenced by decreased distribution of immunohistochemical staining of VEGF, and CD31. Our studies demonstrate both C-DIM-5 and C-DIM-8 have similar anticancer profiles in treating metastatic lung cancer and possibly work as TR3 inactivators.
PMCID: PMC3838903  PMID: 23892137
Nur77/TR3; metastatic; nebulizer; angiogenesis; inhalation
15.  Role of the Aryl Hydrocarbon Receptor in Carcinogenesis and Potential as a Drug Target 
Toxicological Sciences  2013;135(1):1-16.
The aryl hydrocarbon receptor (AHR) is highly expressed in multiple organs and tissues, and there is increasing evidence that the AHR plays an important role in cellular homeostasis and disease. The AHR is expressed in multiple tumor types, in cancer cell lines, and in tumors from animal models, and the function of the AHR has been determined by RNA interference, overexpression, and inhibition studies. With few exceptions, knockdown of the AHR resulted in decreased proliferation and/or invasion and migration of cancer cell lines, and in vivo studies in mice overexpressing the constitutively active AHR exhibited enhanced stomach and liver cancers, suggesting a pro-oncogenic role for the AHR. In contrast, loss of the AHR in transgenic mice that spontaneously develop colonic tumors and in carcinogen-induced liver tumors resulted in increased carcinogenesis, suggesting that the receptor may exhibit antitumorigenic activity prior to tumor formation. AHR ligands also either enhanced or inhibited tumorigenesis, and these effects were highly tumor specific, demonstrating that selective AHR modulators that exhibit agonist or antagonist activities represent an important new class of anticancer agents that can be directed against multiple tumors.
PMCID: PMC3748760  PMID: 23771949
Ah receptor; agonist activity; antagonist activity; drug target.
16.  Betulinic acid decreases ER-negative breast cancer cell growth in vitro and in vivo: role of Sp transcription factors and microRNA-27a:ZBTB10 
Molecular carcinogenesis  2012;52(8):591-602.
Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark is cytotoxic to cancer cells. There is evidence that specificity proteins (Sps), such as Sp1, Sp3 and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. The objective of this study was to determine the efficacy of BA in decreasing the Sps expression and underlying mechanisms. Results show that BA decreased proliferation and induced apoptosis of estrogen-receptor-negative breast cancer MDA-MB-231 cells. The BA-induced Sp1, Sp3, and Sp4 downregulation was accompanied by increased zinc finger ZBTB10 expression, a putative Sp-repressor (ZBTB10) and decreased microRNA-27a levels, a microRNA involved in the regulation of ZBTB10. Similar results were observed in MDA-MB-231 cells transfected with ZBTB10 expression plasmid. BA induced cell cycle arrest in the G2/M phase and increased Myt-1 mRNA (a microRNA-27a target gene), which causes inhibition in G2/M by phosphorylation of cdc2. The effects of BA were reversed by transient transfection with a mimic of microRNA-27a. In nude mice with xenografted MDA-MB-231 cells, tumor size and weight were significantly decreased by BA treatment. In tumor tissue, ZBTB10 mRNA was increased while mRNA and protein of Sp1, Sp3 and Sp4, as well as mRNA of vascular endothelial growth factor receptor (VEGFR), survivin and microRNA-27a were decreased by BA. In lungs of xenografted mice, human β2-microglobulin mRNA was decreased in BA-treated animals. These results show that the anti-cancer effects of BA are at least in part based on interactions with the microRNA-27a-ZBTB10-Sp1-axis causing increased cell death.
PMCID: PMC3418350  PMID: 22407812
MDA-MB-231-breast cancer; ZBTB10; Sp-transcription factors
17.  The aryl hydrocarbon receptor ligand omeprazole inhibits breast cancer cell invasion and metastasis 
BMC Cancer  2014;14:498.
Patients with ER-negative breast tumors are among the most difficult to treat and exhibit low survival rates due, in part, to metastasis from the breast to various distal sites. Aryl hydrocarbon receptor (AHR) ligands show promise as antimetastatic drugs for estrogen receptor (ER)-negative breast cancer.
Triple negative MDA-MB-231 breast cancer cells were treated with eight AHR-active pharmaceuticals including 4-hydroxtamoxifen, flutamide leflunomide, mexiletine, nimodipine, omeprazole, sulindac and tranilast, and the effects of these compounds on cell proliferation (MTT assay) and cell migration (Boyden chamber assay) were examined. The role of the AHR in mediating inhibition of MDA-MB-231 cell invasion was investigated by RNA interference (RNAi) and knockdown of AHR or cotreatment with AHR agonists. Lung metastasis of MDA-MB-231 cells was evaluated in mice administered cells by tail vein injection and prometastatic gene expression was examined by immunohistochemistry.
We showed that only the proton pump inhibitor omeprazole decreased MDA-MB-231 breast cancer cell invasion in vitro. Omeprazole also significantly decreased MDA-MB-231 cancer cell metastasis to the lung in a mouse model (tail vein injection), and in vitro studies showed that omeprazole decreased expression of at least two prometastatic genes, namely matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4). Results of RNA interference studies confirmed that omeprazole-mediated downregulation of CXCR4 (but not MMP-9) was AHR-dependent. Chromatin immunoprecipitation assays demonstrated that omeprazole recruited the AHR to regions in the CXCR4 promoter that contain dioxin response elements (DREs) and this was accompanied by the loss of pol II on the promoter and decreased expression of CXCR4.
AHR-active pharmaceuticals such as omeprazole that decrease breast cancer cell invasion and metastasis may have important clinical applications for late stage breast cancer chemotherapy.
PMCID: PMC4226953  PMID: 25011475
Omeprazole; Ah receptor; Metastasis; Inhibition; CXCR4
18.  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.
PMCID: PMC4048269  PMID: 24905566
19.  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.
PMCID: PMC3663561  PMID: 23492812
dioxin; halogenated hydrocarbon; persistent organic chemicals; polychlorinated biphenyls; regulatory/policy; biomarkers.
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.
PMCID: PMC3527649  PMID: 22815231
Tolfenamic acid; Sp proteins; downregulation; RMS cells
21.  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
22.  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
PMCID: PMC3488590  PMID: 22941571
Breast Cancer; Polyphenolics; Pomegranate; Xenografts; Inflammation; Cytotoxicity
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.
PMCID: PMC3484248  PMID: 22614017
HOTAIR; invasion; cell cycle progression; pro-oncogenic; prognostic
24.  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.
PMCID: PMC3632183  PMID: 22752225
ZBTB4; CDODA-Me; Sp; miR-17-92; miR-106b
25.  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.
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.
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.
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.
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.
PMCID: PMC3737220  PMID: 23950896

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