Sujit Nair

Modulation of nuclear factor E2-related factor 2–mediated gene expression in mice liver and small intestine by cancer chemopreventive agent curcumin

Curcumin has been shown to prevent and inhibit carcinogen-induced tumorigenesis in different organs of rodent carcinogenesis models. Our objective is to study global gene expression profiles elicited by curcumin in mouse liver and small intestine as well as to identify curcumin-regulated nuclear factor E2-related factor 2 (Nrf2)–dependent genes. Wild-type C57BL/6J and Nrf2 knockout C57BL/6J/Nrf2(−/−) mice were given a single oral dose of curcumin at 1,000 mg/kg. Liver and small intestine were collected at 3 and 12 hours after treatments. Total RNA was extracted and analyzed using Affymetrix (Santa Clara, CA) mouse genome 430 array (45K) and GeneSpring 6.1 software (Silicon Genetics, Redwood City, CA). Genes that were induced or suppressed >2-fold by curcumin treatments compared with vehicle in wild-type mice but not in knockout mice were filtered using GeneSpring software and regarded as Nrf2-dependent genes. Among those well-defined genes, 822 (664 induced and 158 suppressed) and 222 (154 induced and 68 suppressed) were curcumin-regulated Nrf2-dependent genes identified in the liver and small intestine, respectively. Based on their biological functions, these genes can be classified into the category of ubiquitination and proteolysis, electron transport, detoxification, transport, apoptosis and cell cycle control, cell adhesion, kinase and phosphatase, and transcription factor. Many phase II detoxification/antioxidant enzyme genes, which are regulated by Nrf2, are among the identified genes. The identification of curcumin-regulated Nrf2-dependent genes not only provides potential novel insights into the biological effects of curcumin on global gene expression and chemoprevention but also points to the potential role of Nrf2 in these processes. [Mol Cancer Ther 2006;5(1):39–51]

Comparison of (-)-epigallocatechin-3-gallate elicited liver and small intestine gene expression profiles between C57BL/6J mice and C57BL/6J/Nrf2 (-/-) mice.

PurposeThis study was conducted to study global gene expression profiles elicited by (−)-epigallocatechin-3-gallate (EGCG) in mouse liver and small intestine, as well as to identify EGCG-regulated Nrf2-dependent genes.MethodsC57BL/6J and C57BL/6J/Nrf2(−/−) mice were given an oral dose of EGCG at 200xa0mg/kg or treated with vehicle. Both liver and small intestine were collected 3xa0h and 12xa0h after treatment. Total RNA was extracted from the tissues and gene expression profiles were analyzed using Affymetrix mouse genome 430 2.0 array and GeneSpring 6.1 software. Microarray data were validated using quantitative real-time reverse transcription-PCR chain reaction analysis.ResultsGenes that were either induced or suppressed more than two fold by EGCG treatment compared with vehicle treatment in the same genotype group were filtered using the GeneSpring software. Among these well-defined genes, 671 EGCG-regulated Nrf2-dependent genes and 256 EGCG-regulated Nrf2-independent genes were identified in liver, whereas 228 EGCG-regulated Nrf2-dependent genes and 98 EGCG-regulated Nrf2-independent genes were identified in the small intestine. Based on their biological functions, these genes mainly fall into the category of ubiquitination and proteolysis, electron transport, detoxification, transport, cell growth and apoptosis, cell adhesion, kinase and phosphatases, and transcription factors.ConclusionsGenes expressed in mouse liver are more responsive to oral treatment of EGCG than those expressed in small intestine. EGCG could regulate many genes in both organs in an Nrf2-dependent manner. The identification of genes related to detoxification, transport, cell growth and apoptosis, cell adhesion, kinase, and transcription regulated by EGCG not only provide potential novel insight into the effect of EGCG on global gene expression and chemopreventive effects, but also point to the potential role of Nrf2 in these processes.

Chemoprevention of Familial Adenomatous Polyposis by Natural Dietary Compounds Sulforaphane and Dibenzoylmethane Alone and in Combination in ApcMin/+ Mouse

Cancer chemopreventive agent sulforaphane (SFN) and dibenzoylmethane (DBM) showed antitumorigenesis effects in several rodent carcinogenesis models. In this study, we investigated the cancer chemopreventive effects and the underlying molecular mechanisms of dietary administration of SFN and DBM alone or in combination in the ApcMin/+ mice model. Male ApcMin/+ mice (12 per group) at age of 5 weeks were given control AIN-76A diet, diets containing 600 ppm SFN and 1.0% DBM, or a combination of 300 ppm SFN and 0.5% DBM for 10 weeks. Mice were then sacrificed, and tumor numbers and size were examined. Microarray analysis, Western blotting, ELISA, and immunohistochemical staining were done to investigate the underlying molecular mechanisms of cancer chemopreventive effects of SFN and DBM. Dietary administrations of SFN and DBM alone or in combination significantly inhibited the development of intestinal adenomas by 48% (P=0.002), 50% (P=0.001), and 57% (P<0.001), respectively. Dietary administration of 600 ppm SFN and 1.0% DBM also reduced colon tumor numbers by 80% (P=0.016) and 60% (P=0.103), respectively, whereas the combination of SFN and DBM treatment blocked the colon tumor development (P=0.002). Both SFN and DBM treatments resulted in decreased levels of prostaglandin E2 or leukotriene B4 in intestinal polyps or apparently normal mucosa. Treatments also led to the inhibition of cell survival and growth-related signaling pathways (such as Akt and extracellular signal-regulated kinase) or biomarkers (such as cyclooxygenase-2, proliferating cell nuclear antigen, cleaved caspases, cyclin D1, and p21). In conclusion, our results showed that both SFN and DBM alone as well as their combination are potent natural dietary compounds for chemoprevention of gastrointestinal cancers.

Metastasis-Associated Protein 1/Nucleosome Remodeling and Histone Deacetylase Complex in Cancer

Cancer cells frequently exhibit deregulation of coregulatory molecules to drive the process of growth and metastasis. One such group of ubiquitously expressed coregulators is the metastasis-associated protein (MTA) family, a critical component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA1 occupies a special place in cancer biology because of its dual corepressor or coactivator nature and widespread overexpression in human cancers. Here, we highlight recent advances in our understanding of the vital roles of MTA1 on transformation, epithelial-mesenchymal transition, and the functions of key cancer-relevant molecules such as a nexus of multiple oncogenes and tumor suppressors. In addition to its paramount role in oncogenesis, we reveal several new physiologic functions of MTA1 related to DNA damage, inflammatory responses, and infection, in which MTA1 functions as a permissive gate keeper for cancer-causing parasites. Further, these discoveries unraveled the versatile multidimensional modes of action of MTA1, which are independent of the NuRD complex and/or transcription. Given the emerging roles of MTA1 in DNA repair, inflammation, and parasitism, we discuss the possibility of MTA1-targeted therapy for use not only in combating cancer but also in other inflammation and pathogen-driven pathologic conditions.

Structural Influence of Isothiocyanates on the Antioxidant Response Element (ARE)-Mediated Heme Oxygenase-1 (HO-1) Expression

PurposeIsothiocyanates (ITCs), existing abundantly in cruciferous vegetables, is one class of promising dietary cancer chemopreventive agents that possess strong cancer protective effects by modulation of phase II detoxifying/antioxidant enzyme activities. However, limited studies regarding to the structure-activity relationship (SAR) of ITCs on the induction of phase II detoxifying/antioxidant enzymes are reported. In this study, the effects of ten structurally related isothiocyanates on the antioxidant response element (ARE)-mediated antioxidant enzyme heme oxygenase-1 (HO-1) induction in human hepatoma HepG2-C8 cells were evaluated.Materials and MethodsAfter exposure of HepG2-C8 cells to ITCs, cell viability, luciferase reporter assay, Western blot analysis and quantitative real-time PCR were conducted.ResultsTreatments with most ITCs significantly activated ARE-mediated luciferase activity with different maximal degree of ARE induction. In addition, ITCs caused a substantial induction of HO-1 protein, which was closely correlated with inductive level of Nrf2 protein. Real-time PCR revealed that the expression of HO-1 mRNA and protein was significantly increased after treatments with ITCs, although not directly correlated. HO-1 induction by ITCs was attenuated in HepG2-C8 cells transiently transfected with a dominant negative mutant of Nrf2 (Nrf2-M4), whereas it was totally absent in Nrf2−/− mouse embryonic fibroblasts. In addition, ARE activation by ITCs was associated with the depletion of intracellular glutathione.ConclusionCollectively, our results demonstrate that the ITC class of compounds activates ARE-mediated HO-1 gene transcription through Nrf2/ARE signaling pathway, however, their inductive effects are quite specific, depending on the chemical structure. These results suggest the possibility that some synthetic ITCs might have superior chemopreventive activity than natural ITCs.

Regulation of Nrf2- and AP-1-mediated gene expression by epigallocatechin-3-gallate and sulforaphane in prostate of Nrf2-knockout or C57BL/6J mice and PC-3 AP-1 human prostate cancer cells

AbstractAim:To examine the regulatory crosstalk between the transcription factors Nrf2 and AP-1 in prostate cancer (PCa) by dietary cancer chemopreventive compounds (−)epigallocatechin-3-gallate (EGCG) from green tea and sulforaphane (SFN) from cruciferous vegetables.Methods:We performed (i) in vitro studies including luciferase reporter gene assays, MTS cell viability assays, and quantitative real-time PCR (qRT-PCR) in PC-3 AP-1 human PCa cells, (ii) in vivo temporal (3 h and 12 h) microarray studies in the prostate of Nrf2-deficient mice that was validated by qRT-PCR, and (iii) in silico bioinformatic analyses to delineate conserved Transcription Factor Binding Sites (TFBS) in the promoter regions of Nrf2 and AP-1, as well as coregulated genes including ATF-2 and ELK-1.Results:Our study shows that AP-1 activation was attenuated by the combinations of SFN (25 μmol/L) and EGCG (20 or 100 μmol/L) in PC-3 cells. Several key Nrf2-dependent genes were down-regulated (3-fold to 35-fold) after in vivo administration of the combination of EGCG (100 mg/kg) and SFN (45 mg/kg). Conserved TFBS signatures were identified in the promoter regions of Nrf2, AP-1, ATF2, and ELK-1 suggesting a potential regulatory mechanism of crosstalk between them.Conclusion:Taken together, our present study of transcriptome profiling the gene expression changes induced by dietary phytochemicals SFN and EGCG in Nrf2-deficient mice and in PC-3 cells in vitro demonstrates that the effects of SFN+EGCG could be mediated via concerted modulation of Nrf2 and AP-1 pathways in the prostate.

Synergistic Effects of a Combination of Dietary Factors Sulforaphane and (−) Epigallocatechin-3-gallate in HT-29 AP-1 Human Colon Carcinoma Cells

PurposeThe objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20xa0μM (or 100xa0μM) and SFN (25xa0μM) in HT-29 AP-1 human colon carcinoma cells.MethodsAfter exposure of HT-29 AP-1 cells to SFN and EGCG, individually or in combination, we performed AP-1 luciferase reporter assays, cell viability assays, isobologram analyses, senescence staining, quantitative real-time PCR (qRT-PCR) assays, Western blotting, and assays for HDAC activity and hydrogen peroxide. In some experiments, we exposed cells to superoxide dismutase (SOD) or Trichostatin A (TSA) in addition to the treatment with dietary factors.ResultsThe combinations of SFN and EGCG dramatically enhanced transcriptional activation of AP-1 reporter in HT-29 cells (46-fold with 25xa0μM SFN and 20xa0μM EGCG; and 175-fold with 25xa0μM SFN and 100xa0μM EGCG). Isobologram analysis showed synergistic activation for the combinations with combination index, CIu2009<u20091. Interestingly, co-treatment with 20units/ml of SOD, a free radical scavenger, attenuated the synergism elicited by the combinations (2-fold with 25xa0μM SFN and 20xa0μM EGCG; and 15-fold with 25xa0μM SFN and 100xa0μM EGCG). Cell viability assays showed that the low-dose combination decreased cell viability to 70% whereas the high-dose combination decreased cell viability to 40% at 48xa0h, with no significant change in cell viability at 24xa0h as compared to control cells. In addition, 20xa0μM and 100xa0μM EGCG, but not 25xa0μM SFN, showed induction of senescence in the HT-29 AP-1 cells subjected to senescence staining. However, both low- and high-dose combinations of SFN and EGCG attenuated the cellular senescence induced by EGCG alone. There was no significant change in the protein levels of phosphorylated forms of ERK, JNK, p38, and Akt-Ser473 or Akt-Thr308. Besides, qRT-PCR assays corroborated the induction of the luciferase gene seen with the combinations in the reporter assay. Relative expression levels of transcripts of many other genes known to be either under the control of the AP-1 promoter or involved in cell cycle regulation or cellular influx–efflux such as cyclin D1, cMyc, ATF-2, Elk-1, SRF, CREB5, SLCO1B3, MRP1, MRP2 and MRP3 were also quantified by qRT-PCR in the presence and absence of SOD at both 6 and 10xa0h. In addition, pre-treatment with 100xa0ng/ml TSA, a potent HDAC inhibitor, potentiated (88-fold) the synergism seen with the low-dose combination on the AP-1 reporter transcriptional activation. Cytoplasmic and nuclear fractions of treated cells were tested for HDAC activity at 2 and 12xa0h both in the presence and absence of TSA, however, there was no significant change in their HDAC activity. In addition, the H2O2 produced in the cell system was about 2xa0μM for the low-dose combination which was scavenged to about 1xa0μM in the presence of SOD.ConclusionTaken together, the synergistic activation of AP-1 by the combination of SFN and EGCG that was potentiated by HDAC inhibitor TSA and attenuated by free radical scavenger SOD point to a possible multifactorial control of colon carcinoma that may involve a role for HDACs, inhibition of cellular senescence, and SOD signaling.

Architecture of Signature miRNA Regulatory Networks in Cancer Chemoprevention

With new high-throughput technologies and superior computational power available for application to current pharmacology research, biomarker discovery has probably entered its most exciting phase to date, especially with the concurrent advent of systems network biology for “big data.” Study of recurrent network motifs in network architecture can inform us better about regulatory pathways in the cellular milieu, more so in complex disease states like cancer. In this review, we focus on the architecture of miRNA networks with emphasis on chemoresistance networks in response to chemotherapeutic drugs, chemoprevention networks modulated by dietary phytochemicals, and novel bifunctional networks comprised of bifunctional miRNAs that operate in both chemoresistance and chemoprevention. Since miRNA cancer networks are very complex, the regulatory architecture in chemoresistance and/or chemoprevention may likely include added dimensions of modulation by epigenetic miRNAs and lncRNAs, which may explain, at least in part, the bifunctionality associated with signature miRNA nodes in these networks in addition to temporal dynamics, spatial localization, and stress conditions in the dynamic networks representing the complex cellular milieu. Collectively, by a perusal of our chemoresistance, chemoprevention, and bifunctional networks, we can gain deeper insights into the architecture of signature miRNA regulatory networks in cancer that will serve as the basis for future dynamic network studies and facilitate the discovery of novel miRNA/target biomarkers for preventive and/or therapeutic intervention in cancer.

Pharmacokinetics, Pharmacodynamics and Drug Metabolism

The involvement of Nrf2-a bZip transcription factor in soy isoflavones induced protection against oxidative stress and cancer has been reported. To gain better insight into the role of Nrf2 in prostate cancer chemoprevention by soy isoflavones, we examined the pharmacogenomics and gene expression profiles elicited by soy isoflavones in the prostates of C57BL/6J/Nrf2(-/-) and C57BL6J/Nrf2(+/+) wildtype. The profiles were analyzed using 45000 Affymetrix mouse genome 430-2.0 array and Genespring-7.2 software. The results obtained from microarray were further validated by real-time reverse transcription-PCR. Clusters of genes that were induced or suppressed more than twofold were identified as Nrf2 regulated soy isoflavone induced or suppressed genes. Classification based on their biological function revealed that genes mainly belonging to the categories of electron transport, phase II metabolizing enzymes, cell growth and differentiation, apoptosis, cell cycle, transcription factors, transport, mRNA processing, and carbohydrate homeostasis were either induced or suppressed by soy isoflavone and regulated by Nrf2. In addition, modulation of novel target genes such as LATS2 and GREB1 were identified to be mediated by Nrf2. Thus our current study provides a potential link between cancer chemopreventive properties of soy derived phytochemicals, the transcription factor Nrf2 and prevention of prostate cancer.

New perspectives in personalised medicine for ethnicity in cancer: population pharmacogenomics and pharmacometrics

Personalised medicine takes into consideration the interindividual differences amongst people, which may be responsible for the variability seen in response to a therapeutic intervention in any disease. When this epistemologic knowledge is applied to clinical oncology care, the exciting field is known as personalised cancer medicine – a field that has now evolved to ‘precision medicine’. This knowledge enables the clinician to tailor the right dose of oncology therapeutic to the right subpopulation of cancer patients who will likely respond to the intervention, thereby resulting in greater therapeutic success and decreased ‘financial toxicity’ [1] to cancer patients, families and caregivers. However, the road to success in personalised cancer care is beset with many difficulties in the practical realm, including the lack of reliable biomarkers, the inability to identify an accurate signature biomarker panel for each cancer type, the variability between patients diagnosed with the same cancer, etc. However, advances in modern diagnostic techniques and next-generation sequencing methodologies have provided some opportunities to mitigate the challenges in better informing the clinician regarding the best therapeutic strategies for a particular cancer type or patient. Nonetheless, significant limitations remain in clinical practice, and these adversely affect the best options available to the cancer patient who also has to bear the rising costs of cancer care.