Tin Oo Khor

Activation of Nrf2-antioxidant signaling attenuates NFκB-inflammatory response and elicits apoptosis☆

Oxidative stress has been implicated in the etiology of neurodegenerative disease, cancer and aging. Indeed, accumulation of reactive oxygen and nitrogen species generated by inflammatory cells that created oxidative stress is thought to be one of the major factor by which chronic inflammation contributes to neoplastic transformation as well as many other diseases. We have recently reported that mice lacking nuclear factor-erythroid 2-related factor 2 (Nrf2) are more susceptible to dextran sulfate sodium (DSS)-induced colitis and colorectal carcinogenesis. Nrf2 is a basic leucine zipper redox-sensitive transcriptional factor that plays a center role in ARE (antioxidant response element)-mediated induction of phase II detoxifying and antioxidant enzymes. We found that increased susceptibility of Nrf2 deficient mice to DSS-induced colitis and colorectal cancer was associated with decreased expression of antioxidant/phase II detoxifying enzymes in parallel with upregulation of pro-inflammatory cytokines/biomarkers. These findings suggest that Nrf2 may play an important role in defense against oxidative stress possibly by activation of cellular antioxidant machinery as well as suppression of pro-inflammatory signaling pathways. In addition, in vivo and in vitro data generated from our laboratory suggest that many dietary compounds can differentially regulate Nrf2-mediated antioxidant/anti-inflammatory signaling pathways as the first line defense or induce apoptosis once the cells have been damaged. In this review, we will summarize our thoughts on the potential cross-talks between Nrf2 and NFkappaB pathways. Although the mechanisms involved in the cross-talk between these signaling pathways are still illusive, targeting Nrf2-antioxidative stress signaling is an ideal strategy to prevent or treat oxidative stress-related diseases.

Nrf2-Deficient Mice Have an Increased Susceptibility to Dextran Sulfate Sodium–Induced Colitis

Inflammatory bowel diseases, chronic inflammatory disorders, have been strongly linked with an increased risk of the development of colorectal cancer. Understanding the etiology of these diseases is pivotal for the improvement of currently available strategies to fight against inflammatory bowel disease, and more importantly, to prevent colorectal cancer. Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been known to be a transcriptional factor which plays a crucial role in cytoprotection against inflammation, as well as oxidative and electrophilic stresses. The aim of this study is to investigate the role of Nrf2 in the regulation of dextran sulfate sodium (DSS)-induced experimental colitis in mice. Nrf2-deficient mice were found to be more susceptible to DSS-induced colitis as shown by the increased severity of colitis following 1 week of oral administration of 1% DSS. The increased severity of colitis in Nrf2(-/-) mice was found to be associated with decreased expression of antioxidant/phase II detoxifying enzymes including heme-oxygenase-1, NAD(P)H-quinone reductase-1, UDP-glucurosyltransferase 1A1, and glutathione S-transferase Mu-1. In addition, proinflammatory mediators/cytokines such as COX-2, inducible nitric oxide, interleukin 1beta, interleukin 6, and tumor necrosis factor alpha were significantly increased in the colonic tissues of Nrf2(-/-) mice compared with their wild-type (Nrf2+/+) counterparts. In summary, we show for the first time that mice lacking Nrf2 are more susceptible to DSS-induced colitis. Our data suggests that Nrf2 could play an important role in protecting intestinal integrity, through regulation of proinflammatory cytokines and induction of phase II detoxifying enzymes.

Inhibition of 7,12-Dimethylbenz(a)anthracene-Induced Skin Tumorigenesis in C57BL/6 Mice by Sulforaphane Is Mediated by Nuclear Factor E2–Related Factor 2

Sulforaphane, a dietary isothiocyanate, possesses potent chemopreventive effects through the induction of cellular detoxifying/antioxidant enzymes via the transcription factor nuclear factor E2-related factor 2 (Nrf2). To investigate carcinogenesis mechanisms related to the regulation of Nrf2, we examined the tumor incidence and tumor numbers per mouse in Nrf2 wild-type (+/+) and Nrf2 knockout (-/-) mice. 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatments resulted in an increase in the incidence of skin tumors and tumor numbers per mouse in both genotypes; however, both indices were markedly higher in Nrf2(-/-) mice as compared with Nrf2(+/+) mice. Western blot analysis revealed that Nrf2 as well as heme oxygenase-1, a protein regulated by Nrf2 were not expressed in skin tumors from mice of either genotype, whereas expression of heme oxygenase-1 in Nrf2(+/+) mice was much higher than that in Nrf2(-/-) mice in nontumor skin samples. Next, we examined the chemopreventive efficacy of sulforaphane in mice with both genotypes. Topical application of 100 nmol of sulforaphane once a day for 14 days prior to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate applications decreased the incidence of skin tumor in the Nrf2(+/+) mice when compared with the vehicle-treated group. Importantly, there was no chemoprotective effect elicited by sulforaphane pretreatment in the Nrf2(-/-) mice group. Taken together, our results show for the first time that Nrf2(-/-) mice are more susceptible to skin tumorigenesis and that the chemopreventive effects of sulforaphane are mediated, at least in part, through Nrf2.

Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2.

The up-regulation of phase II detoxifying and stress-responsive genes is believed to play an important role in cancer prevention, and many natural compounds have been shown to be potent inducers of these genes. Previous studies showed that the antioxidant responsive element (ARE), found in these genes, can be bound by the transcription factor Nrf2, and is responsive to the activation by chemopreventive compounds and by oxidative stress. In the present study, we investigated the roles of extracellular signal-regulated kinase (ERK) and c-Jun-NH2-kinase (JNK) in the regulation of phenethyl isothiocyanate (PEITC)–induced and Nrf2-dependent ARE activity and ARE-driven heme oxygenase-1 (HO-1) gene expression in PC-3 cells. ARE activity and HO-1 expression were strongly increased after treatment with PEITC. PEITC also increased the phosphorylation of ERK1/2 and JNK1/2 and caused release of Nrf2 from sequestration by Keap1, and its subsequent translocation into the nucleus. Importantly, Nrf2 was also translocated into the nucleus after transfection with ERK or JNK and that these activated ERK and JNK colocalized with Nrf2 in the nucleus. Activation of ERK and JNK signaling also resulted in the elevation of ARE activity and HO-1 expression. Importantly, PEITC-induced ARE activity was attenuated by inhibition of ERK and JNK signaling. In vitro kinase assays showed that both ERK2 and JNK1 could directly phosphorylate glutathione S-transferase–Nrf2 protein. Taken together, these results strongly suggest a model in which PEITC treatment of PC-3 cells activates ERK and JNK, which, in turn, phosphorylate Nrf2 and induce its translocation to the nucleus. Nuclear Nrf2 activates ARE elements and induces expression of stress-responsive genes, including HO-1. [Mol Cancer Ther 2006;5(8):1918–26]

Increased Susceptibility of Nrf2 Knockout Mice to Colitis-Associated Colorectal Cancer

The nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting various tissues against inflammation, which is a potential risk factor for colorectal and other cancers. Our previously published mouse model work showed that Nrf2 helps protect against dextran sulfate sodium (DSS)–induced colitis/inflammation, and others have shown that Nrf2 helps protect against inflammation-associated colorectal carcinogenesis (aberrant crypt foci). The present study extended these important earlier findings by exploring the role of Nrf2 in colitis-associated colorectal cancer in a mouse model involving azoxymethane/DSS–induced colorectal carcinogenesis in Nrf2 knockout mice. Azoxymethane/DSS–treated Nrf2 knockout mice had increased incidence, multiplicity, and size of all colorectal tumors, including adenomas, versus treated wild-type (WT) mice, and the proportion of tumors that were adenocarcinoma was much higher in knockout (80%) versus WT (29%) mice. Compared with WT mice, knockout mice also had increased markers of inflammation in tumor tissue (cyclooxygenase-2 and 5-lipoxygenase expressions and prostaglandin E2 and leukotriene B4 levels) and in inflamed colonic mucosa (nitrotyrosine expression), supporting the association of knockout mouse tumor formation with inflammation. The phase II detoxifying/antioxidant enzymes NAD(P)H-quinone reductase 1 and UDP-glucurosyltransferase 1A1 were elevated in the normal mucosa of WT, but not Nrf 2 knockout, mice treated with azoxymethane/DSS. Our findings show that Nrf2 plays a critical role in protecting against inflammation-associated colorectal cancer.

Combined Inhibitory Effects of Curcumin and Phenethyl Isothiocyanate on the Growth of Human PC-3 Prostate Xenografts in Immunodeficient Mice

Earlier studies using prostate cancer cells in culture showed that phenethyl isothiocyanate (PEITC) and curcumin have significant chemopreventive and possibly chemotherapeutic effects. However, their in vivo effects are still lacking. Hence, this study was undertaken to determine the possible in vivo efficacy of prostate cancer-prevention as well as cancer-therapeutic treatment by PEITC and curcumin alone or in combination. We evaluated the effects on tumor growth in vivo, using NCr immunodeficient (nu/nu) mice bearing s.c. xenografts of PC-3 human prostate cancer cells. Molecular biomarkers representing proliferation and apoptosis were determined. Continued i.p. injection of curcumin or PEITC (6 and 5 mumol; thrice a week for 28 days), beginning a day before tumor implantation significantly retarded the growth of PC-3 xenografts. Combination of i.p. administration of PEITC (2.5 mumol) and curcumin (3 mumol) showed stronger growth-inhibitory effects. Next, we evaluated the cancer-therapeutic potential of curcumin and PEITC in mice with well-established tumors, and the results showed that PEITC or curcumin alone had little effect, whereas combination of curcumin and PEITC significantly reduced the growth of PC-3 xenografts. Immunohistochemistry staining and Western blot analysis revealed that the inhibition of Akt and nuclear factor-kappaB signaling pathways could contribute to the inhibition of cell proliferation and induction of apoptosis. Taken together, our results show that PEITC and curcumin alone or in combination possess significant cancer-preventive activities in the PC-3 prostate tumor xenografts. Furthermore, we found that combination of PEITC and curcumin could be effective in the cancer-therapeutic treatment of prostate cancers.

Phytochemicals: cancer chemoprevention and suppression of tumor onset and metastasis.

Carcinogenesis is a multi-step process which could be prevented by phytochemicals. Phytochemicals from dietary plants and other plant sources such as herbs are becoming increasingly important sources of anticancer drugs or compounds for cancer chemoprevention or adjuvant chemotherapy. Phytochemicals can prevent cancer initiation, promotion, and progression by exerting anti-inflammatory and anti-oxidative stress effects which are mediated by integrated Nrf2, NF-κB, and AP-1 signaling pathways. In addition, phytochemicals from herbal medicinal plants and/or some dietary plants developed in recent years have been shown to induce apoptosis in cancer cells and inhibition of tumor growth in vivo. In advanced tumors, a series of changes involving critical signaling molecules that would drive tumor cells undergoing epithelial–mesenchymal transition and becoming invasive. In this review, we will discuss the potential molecular targets and signaling pathways that mediate tumor onset and metastasis. In addition, we will shed light on some of the phytochemicals that are capable of targeting these signaling pathways which would make them potentially applicable to cancer chemoprevention, treatment and control of cancer progression.

Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.

Dietary phytochemicals and cancer prevention: Nrf2 signaling, epigenetics, and cell death mechanisms in blocking cancer initiation and progression.

Reactive metabolites from carcinogens and oxidative stress can drive genetic mutations, genomic instability, neoplastic transformation, and ultimately carcinogenesis. Numerous dietary phytochemicals in vegetables/fruits have been shown to possess cancer chemopreventive effects in both preclinical animal models and human epidemiological studies. These phytochemicals could prevent the initiation of carcinogenesis via either direct scavenging of reactive oxygen species/reactive nitrogen species (ROS/RNS) or, more importantly, the induction of cellular defense detoxifying/antioxidant enzymes. These defense enzymes mediated by Nrf2-antioxidative stress and anti-inflammatory signaling pathways can contribute to cellular protection against ROS/RNS and reactive metabolites of carcinogens. In addition, these compounds would kill initiated/transformed cancer cells in vitro and in in vivo xenografts via diverse anti-cancer mechanisms. These mechanisms include the activation of signaling kinases (e.g., JNK), caspases and the mitochondria damage/cytochrome c pathways. Phytochemicals may also have anti-cancer effects by inhibiting the IKK/NF-κB pathway, inhibiting STAT3, and causing cell cycle arrest. In addition, other mechanisms may include epigenetic alterations (e.g., inhibition of HDACs, miRNAs, and the modification of the CpG methylation of cancer-related genes). In this review, we will discuss: the current advances in the study of Nrf2 signaling; Nrf2-deficient tumor mouse models; the epigenetic control of Nrf2 in tumorigenesis and chemoprevention; Nrf2-mediated cancer chemoprevention by naturally occurring dietary phytochemicals; and the mutation or hyper-expression of the Nrf2-Keap1 signaling pathway in advanced tumor cells. The future development of dietary phytochemicals for chemoprevention must integrate in vitro signaling mechanisms, relevant biomarkers of human diseases, and combinations of different phytochemicals and/or non-toxic therapeutic drugs, including NSAIDs.

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.