Benefits and risks of the hormetic effects of dietary isothiocyanates on cancer prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1-5 µM) to promote cell proliferation to 120-143% of the controls in a number of human cell lines, whilst at high levels (10-40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10-20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint

Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

<p>Abstract</p> <p>Background</p> <p>Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway.</p> <p>Methods</p> <p>The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases.</p> <p>Results</p> <p>ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected.</p> <p>Conclusion</p> <p>These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.</p

Regulatory potential for concerted modulation of Nrf2- and Nfkb1-mediated gene expression in inflammation and carcinogenesis

Many studies have implicated nuclear factor E2-related factor 2 (Nrf2) and nuclear factor-κB1 (Nfkb1) in inflammation and cancer. However, the regulatory potential for crosstalk between these two important transcription factors in inflammation and carcinogenesis has not been explored. To delineate conserved transcription factor-binding site signatures, we performed bioinformatic analyses on the promoter regions of human and murine Nrf2 and Nfkb1. We performed multiple sequence alignment of Nrf2 and Nfkb1 genes in five mammalian species – human, chimpanzee, dog, mouse and rat – to explore conserved biological features. We constructed a canonical regulatory network for concerted modulation of Nrf2 and Nfkb1 involving several members of the mitogen-activated protein kinase (MAPK) family and present a putative model for concerted modulation of Nrf2 and Nfkb1 in inflammation/carcinogenesis. Our results reflect potential for putative crosstalk between Nrf2 and Nfkb1 modulated through the MAPK cascade that may influence inflammation-associated etiopathogenesis of cancer. Taken together, the elucidation of potential relationships between Nrf2 and Nfkb1 may help to better understand transcriptional regulation, as well as transcription factor networks, associated with the etiopathogenesis of inflammation and cancer

Ethanol-Mediated Regulation of Cytochrome P450 2A6 Expression in Monocytes: Role of Oxidative Stress-Mediated PKC/MEK/Nrf2 Pathway

Cytochrome P450 2A6 (CYP2A6) is known to metabolize nicotine, the major constituent of tobacco, leading to the production of toxic metabolites and induction of oxidative stress that result in liver damage and lung cancer. Recently, we have shown that CYP2A6 is induced by ethanol and metabolizes nicotine into cotinine and other metabolites leading to generation of reactive oxygen species (ROS) in U937 monocytes. However, the mechanism by which CYP2A6 is induced by ethanol is unknown. In this study, we have examined the role of the PKC/Nrf2 pathway (protein kinase C-mediated phosphorylation and translocation of nuclear erythroid 2-related factor 2 to the nucleus) in ethanol-mediated CYP2A6 induction. Our results showed that 100 mM ethanol significantly induced CYP2A6 mRNA and protein (∼150%) and increased ROS formation, and induction of gene expression and ROS were both completely blocked by treatment with either a CYP2E1 inhibitor (diallyl sulfide) or an antioxidant (vitamin C). The results suggest the role of oxidative stress in the regulation of CYP2A6 expression. Subsequently, we investigated the role of Nrf2 pathway in oxidative stress-mediated regulation of CYP2A6 expression in U937 monocytes. Our results showed that butylated hydroxyanisole, a stabilizer of nuclear Nrf2, increased CYP2A6 levels >200%. Staurosporine, an inhibitor of PKC, completely abolished ethanol-induced CYP2A6 expression. Furthermore, our results showed that a specific inhibitor of mitogen-activated protein kinase kinase (MEK) (U0126) completely abolished ethanol-mediated CYP2A6 induction and Nrf2 translocation. Overall, these results suggest that CYP2E1-mediated oxidative stress produced as a result of ethanol metabolism translocates Nrf2 into the nucleus through PKC/MEK pathway, resulting in the induction of CYP2A6 in monocytes. An increased level of CYP2A6 in monocytes is expected to further increase oxidative stress in smokers through CYP2A6-mediated nicotine metabolism. Thus, this study has clinical relevance because of the high incidence of alcohol use among smokers, especially in HIV-infected individuals

Inhibition of metastasis, angiogenesis, and tumor growth by Chinese herbal cocktail Tien-Hsien Liquid

<p>Abstract</p> <p>Background</p> <p>Advanced cancer is a multifactorial disease that demands treatments targeting multiple cellular pathways. Chinese herbal cocktail which contains various phytochemicals may target multiple dys-regulated pathways in cancer cells and thus may provide an alternative/complementary way to treat cancers. Previously we reported that the Chinese herbal cocktail Tien-Hsien Liguid (THL) can specifically induce apoptosis in various cancer cells and have immuno-modulating activity. In this study, we further evaluated the anti-metastatic, anti-angiogenic and anti-tumor activities of THL with a series of <it>in vitro </it>and <it>in vivo </it>experiments.</p> <p>Methods</p> <p>The migration and invasion of cancer cells and endothelial cells was determined by Boyden chamber transwell assays. The effect of THL on pulmonary metastasis was done by injecting CT-26 colon cancer cells intravenously to syngenic mice. The <it>in vitro </it>and <it>in vivo </it>microvessel formation was determined by the tube formation assay and the Matrigel plug assay, respectively. The <it>in vivo </it>anti-tumor effect of THL was determined by a human MDA-MB-231 breast cancer xenograft model. The expression of metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) was measured by gelatin zymography. The expression of HIF-1α and the phosphorylation of ERK1/2 were determined by Western blot.</p> <p>Results</p> <p>THL inhibited the migration and invasion ability of various cancer cells <it>in vitro</it>, decreased the secretion of MMP-2, MMP-9, and uPA and the activity of ERK1/2 in cancer cells, and suppressed pulmonary metastasis of CT-26 cancer cells in syngenic mice. Moreover, THL inhibited the migration, invasion, and tube formation of endothelial cells <it>in vitro</it>, decreased the secretion of MMP-2 and uPA in endothelial cells, and suppressed neovascularization in Matrigel plugs in mice. Besides its inhibitory effect on endothelial cells, THL inhibited hypoxia-induced HIF-1α and vascular endothelial growth factor-A expression in cancer cells. Finally, our results show that THL inhibited the growth of human MDA-MB-231 breast cancer xenografts in <it>NOD-SCID </it>mice. This suppression of tumor growth was associated with decreased microvessel formation and increased apoptosis caused by THL.</p> <p>Conclusion</p> <p>Our data demonstrate that THL had broad-spectra anti-cancer activities and merits further evaluation for its use in cancer therapy.</p

American ginseng suppresses Western diet-promoted tumorigenesis in model of inflammation-associated colon cancer: role of EGFR

<p>Abstract</p> <p>Background</p> <p>Western diets increase colon cancer risk. Epidemiological evidence and experimental studies suggest that ginseng can inhibit colon cancer development. In this study we asked if ginseng could inhibit Western diet (20% fat) promoted colonic tumorigenesis and if compound K, a microbial metabolite of ginseng could suppress colon cancer xenograft growth.</p> <p>Methods</p> <p>Mice were initiated with azoxymethane (AOM) and, two weeks later fed a Western diet (WD, 20% fat) alone, or WD supplemented with 250-ppm ginseng. After 1 wk, mice received 2.5% dextran sulfate sodium (DSS) for 5 days and were sacrificed 12 wks after AOM. Tumors were harvested and cell proliferation measured by Ki67 staining and apoptosis by TUNEL assay. Levels of EGF-related signaling molecules and apoptosis regulators were determined by Western blotting. Anti-tumor effects of intraperitoneal compound K were examined using a tumor xenograft model and compound K absorption measured following oral ginseng gavage by UPLC-mass spectrometry. Effects of dietary ginseng on microbial diversity were measured by analysis of bacterial 16S rRNA.</p> <p>Results</p> <p>Ginseng significantly inhibited colonic inflammation and tumorigenesis and concomitantly reduced proliferation and increased apoptosis. The EGFR cascade was up-regulated in colonic tumors and ginseng significantly reduced EGFR and ErbB2 activation and Cox-2 expression. Dietary ginseng altered colonic microbial diversity, and bacterial suppression with metronidazole reduced serum compound K following ginseng gavage. Furthermore, compound K significantly inhibited tumor xenograft growth.</p> <p>Conclusions</p> <p>Ginseng inhibited colonic inflammation and tumorigenesis promoted by Western diet. We speculate that the ginseng metabolite compound K contributes to the chemopreventive effects of this agent in colonic tumorigenesis.</p

Two Genes on A/J Chromosome 18 Are Associated with Susceptibility to Staphylococcus aureus Infection by Combined Microarray and QTL Analyses

Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N2 backcross mice (F1 [C18A]×C57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus–challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 β and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies