Guoxiang Shen

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.

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.

Suppression of NF-κB and NF-κB-regulated gene expression by sulforaphane and PEITC through IκBα, IKK pathway in human prostate cancer PC-3 cells

Recent studies indicate that natural isothiocyanates, such as sulforaphane (SFN) and phenethyl isothiocyanate (PEITC) possess strong antitumor activities in vitro and in vivo. The nuclear factor kappa B (NF-κB) is believed to play an important role in cancer chemoprevention due to its involvement in tumor cell growth, proliferation, angiogenesis, invasion, apoptosis, and survival. In this study, we investigated the effects and the molecular mechanisms of SFN and PEITC on NF-κB transcriptional activation and NF-κB-regulated gene expression in human prostate cancer PC-3 C4 cells. Treatment with SFN (20 and 30 μM) and PEITC (5 and 7.5 μM) significantly inhibited NF-κB transcriptional activity, nuclear transloction of p65, and gene expression of NF-κB-regulated VEGF, cylcin D1, and Bcl-XL in PC-3 C4 cells. To further elucidate the mechanism, we utilized the dominant-negative mutant of inhibitor of NF-κB alpha (IκBα) (SR-IκBα). Analogous to treatments with SFN and PEITC, SR-IκBα also strongly inhibited NF-κB transcriptional activity as well as VEGF, cylcin D1, and Bcl-XL expression. Furthermore, SFN and PEITC also inhibited the basal and UVC-induced phosphorylation of IκBα and blocked UVC-induced IκBα degradation in PC-3 C4 cells. In examining the upstream signaling, we found that the dominant-negative mutant of IKKβ (dnIKKβ) possessed inhibitory effects similar to SFN and PEITC on NF-κB, VEGF, cylcin D1, Bcl-XL as well as IκBα phosphorylation. In addition, treatment with SFN and PEITC potently inhibited phosphorylation of both IKKβ and IKKα and significantly inhibited the in vitro phosphorylation of IκBα mediated by IKKβ. Taken together, these results suggest that the inhibition of SFN and PEITC on NF-κB transcriptional activation as well as NF-κB-regulated VEGF, cyclin D1, and Bcl-XL gene expression is mainly mediated through the inhibition of IKK phosphorylation, particularly IKKβ, and the inhibition of IκBα phosphorylation and degradation, as well as the decrease of nuclear translocation of p65 in PC-3 cells.

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]

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.

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]

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.

Curcumin inhibits Akt/mammalian target of rapamycin signaling through protein phosphatase-dependent mechanism

Akt/mammalian target of rapamycin (mTOR) signaling plays an important role in tumorigenesis and is dysregulated in many tumors, especially metastatic prostate cancers. Curcumin has been shown to effectively prevent or inhibit prostate cancer in vivo and inhibit Akt/mTOR signaling in vitro, but the mechanism(s) remains unclear. Here, we show that curcumin concentration- and time-dependently inhibited the phosphorylation of Akt, mTOR, and their downstream substrates in human prostate cancer PC-3 cells, and this inhibitory effect acts downstream of phosphatidylinositol 3-kinase and phosphatidylinositol-dependent kinase 1. Overexpression of constitutively activated Akt or disruption of TSC1-TSC2 complex by small interfering RNA or gene knockout only partially restored curcumin-mediated inhibition of mTOR and downstream signaling, indicating that they are not the primary effectors of curcumin-mediated inhibition of Akt/mTOR signaling. Curcumin also activated 5′-AMP-activated protein kinase and mitogen-activated protein kinases; however, inhibition of these kinases failed to rescue the inhibition by curcumin. Finally, it was shown that the inhibition of Akt/mTOR signaling by curcumin is resulted from calyculin A-sensitive protein phosphatase-dependent dephosphorylation. Our study reveals the profound effects of curcumin on the Akt/mTOR signaling network in PC-3 cells and provides new mechanisms for the anticancer effects of curcumin. [Mol Cancer Ther 2008;7(9):2609–20]

Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters.

The nuclear transcription factor E2-related factor 2 (Nrf2) has been shown to play pivotal roles in preventing xenobiotic-related toxicity and carcinogen-induced carcinogenesis. These protective roles of Nrf2 have been attributed in part to its involvement in the induction of Phase II drug conjugation/detoxification enzymes as well as antioxidant enzymes through the Nrf2-antioxidant response element (ARE) signaling pathways. This review summarizes the current research status of the identification of Nrf2-regulated drug metabolism enzymes (DMEs), especially Phase II DMEs, and Phase III drug transporters. In addition, the molecular mechanisms underlying the coordinated regulation of Phase II DMEs and Phase III transporters will also be discussed based on findings published in the literature.

Butylated hydroxyanisole regulates ARE-mediated gene expression via Nrf2 coupled with ERK and JNK signaling pathway in HepG2 cells.

Many natural and synthetic cancer chemopreventive compounds are potent inducers of phase II detoxifying and antioxidant stress responsive genes. The phase II/antioxidant gene expression plays critical role in chemoprevention of carcinogenesis. The antioxidant responsive element (ARE), located on many phase II/antioxidant genes, binds with the transcription factor Nrf2, and is required for the activation of these phase II/antioxidant gene expression induced by many natural and synthetic cancer chemopreventive compounds. In this study, we investigated the potential roles of extracellular signal‐regulated kinase (ERK) and c‐jun N‐terminal kinase (JNK) in the regulation of butylated hydroxyanisole (BHA)‐induced and Nrf2‐dependent ARE transcriptional activity and ARE‐mediated endogenous heme oxygenase‐1 (HO‐1) protein expression in HepG2 cells. ARE transcriptional activity and HO‐1 protein expression were increased dose dependently after treatment with BHA in HepG2 cells. Dose‐response and time‐course experiments showed that BHA increased the accumulation of Nrf2, and concomitantly decreased the protein level of Keap1. We next examined the phosphorylation of the MAPKs, and found that BHA significantly increased the phosphorylation levels of ERK1/2 and JNK1/2. Importantly BHA‐induced ARE transcriptional activity was attenuated by the inhibition of ERK and JNK signaling using biochemical inhibitors and their dominant‐negative mutants. Using confocal microscopy technique, treatment with BHA showed the release of Nrf2 sequestered by Keap1 in the cytosol, and that Nrf2 translocated into the nucleus. Importantly, cDNA transfections of ERK and JNK signaling pathways similarly released Nrf2 from Keap1 cytosolic sequestration and translocating Nrf2 into the nucleus. Taken together, these results strongly suggested that ERK and JNK signaling pathways played important and positive roles in BHA‐induced and Nrf2‐dependent regulation of ARE‐mediated gene expression, as well as the nuclear translocation of Nrf2 in HepG2 cells.