Reductive metabolites of curcumin and their therapeutic effects.

Curcumin, a secondary metabolite from the turmeric plant is one of the most promising natural products, which has been studied extensively for decades. It has demonstrated several pharmacological activities in vitro and in vivo. Various studies have indicated that the pharmacological activity of curcumin is contributed by its metabolites. The aim of this review is to present an overview of metabolic products of curcumin produced upon its reduction like di, tetra, hexa and octa-hydrocurcumin. In addition, this paper has systematically analyzed the current information regarding medicinal use of reduced metabolites of curcumin and identified the limitations which have hindered its widespread usage in the medical world. Several diverse therapeutic effects have shown to be exhibited by reduced metabolites of curcumin such as antioxidant, anti-cancerous, anti-inflammatory and immunoregulatory activities. The potential underlying molecular mechanisms of the biological activities of reduced metabolites of curcumin have also been highlighted, which may provide insight into the principle of effectiveness of curcumin.

The anti-inflammatory activity of curcumin is mediated by its oxidative metabolites.

The spice turmeric, with its active polyphenol curcumin, has been used as anti-inflammatory remedy in traditional Asian medicine for centuries. Many cellular targets of curcumin have been identified, but how such a wide range of targets can be affected by a single compound is unclear. Here, we identified curcumin as a pro-drug that requires oxidative activation into reactive metabolites to exert anti-inflammatory activities. Synthetic curcumin analogs that undergo oxidative transformation potently inhibited the pro-inflammatory transcription factor nuclear factor κB (NF-κB), whereas stable, non-oxidizable analogs were less active, with a correlation coefficient (R2) of IC50versus log of autoxidation rate of 0.75. Inhibition of glutathione biosynthesis, which protects cells from reactive metabolites, increased the potency of curcumin and decreased the amount of curcumin-glutathione adducts in cells. Oxidative metabolites of curcumin adducted to and inhibited the inhibitor of NF-κB kinase subunit ß (IKKß), an activating kinase upstream of NF-κB. An unstable, alkynyl-tagged curcumin analog yielded abundant adducts with cellular protein that were decreased by pretreatment with curcumin or an unstable analog but not by a stable analog. Bioactivation of curcumin occurred readily in vitro, which may explain the wide range of cellular targets, but if bioactivation is insufficient in vivo, it may also help explain the inconclusive results in human studies with curcumin so far. We conclude that the paradigm of metabolic bioactivation uncovered here should be considered for the evaluation and design of clinical trials of curcumin and other polyphenols of medicinal interest.

Designing a broad-spectrum integrative approach for cancer prevention and treatment.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.

A multi-targeted approach to suppress tumor-promoting inflammation.

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes.

Dietary selenium deficiency exacerbates DSS-induced epithelial injury and AOM/DSS-induced tumorigenesis.

Selenium (Se) is an essential micronutrient that exerts its functions via selenoproteins. Little is known about the role of Se in inflammatory bowel disease (IBD). Epidemiological studies have inversely correlated nutritional Se status with IBD severity and colon cancer risk. Moreover, molecular studies have revealed that Se deficiency activates WNT signaling, a pathway essential to intestinal stem cell programs and pivotal to injury recovery processes in IBD that is also activated in inflammatory neoplastic transformation. In order to better understand the role of Se in epithelial injury and tumorigenesis resulting from inflammatory stimuli, we examined colonic phenotypes in Se-deficient or -sufficient mice in response to dextran sodium sulfate (DSS)-induced colitis, and azoxymethane (AOM) followed by cyclical administration of DSS, respectively. In response to DSS alone, Se-deficient mice demonstrated increased morbidity, weight loss, stool scores, and colonic injury with a concomitant increase in DNA damage and increases in inflammation-related cytokines. As there was an increase in DNA damage as well as expression of several EGF and TGF-ß pathway genes in response to inflammatory injury, we sought to determine if tumorigenesis was altered in the setting of inflammatory carcinogenesis. Se-deficient mice subjected to AOM/DSS treatment to model colitis-associated cancer (CAC) had increased tumor number, though not size, as well as increased incidence of high grade dysplasia. This increase in tumor initiation was likely due to a general increase in colonic DNA damage, as increased 8-OHdG staining was seen in Se-deficient tumors and adjacent, non-tumor mucosa. Taken together, our results indicate that Se deficiency worsens experimental colitis and promotes tumor development and progression in inflammatory carcinogenesis.

Deletion of cationic amino acid transporter 2 exacerbates dextran sulfate sodium colitis and leads to an IL-17-predominant T cell response.

L-Arginine (L-Arg) is a semiessential amino acid that has altered availability in human ulcerative colitis (UC), a form of inflammatory bowel disease, and is beneficial in murine colitis induced by dextran sulfate sodium (DSS), a model with similarity to UC. We assessed the role of cationic amino acid transporter 2 (CAT2), the inducible transporter of L-Arg, in DSS colitis. Expression of CAT2 was upregulated in tissues from colitic mice and localized predominantly to colonic macrophages. CAT2-deficient (CAT2-/-) mice exposed to DSS exhibited worsening of survival, body weight loss, colon weight, and histological injury. These effects were associated with increased serum L-Arg and decreased tissue L-Arg uptake and inducible nitric oxide synthase protein expression. Clinical benefits of L-Arg supplementation in wild-type mice were lost in CAT2-/- mice. There was increased infiltration of macrophages, dendritic cells, granulocytes, and T cells in colitic CAT2-/- compared with wild-type mice. Cytokine profiling revealed increases in proinflammatory granulocyte colony-stimulating factor, macrophage inflammatory protein-1α, IL-15, and regulated and normal T cell-expressed and -secreted and a shift from an IFN-γ- to an IL-17-predominant T cell response, as well as an increase in IL-13, in tissues from colitic CAT2-/- mice. However, there were no increases in other T helper cell type 2 cytokines, nor was there a global increase in macrophage-derived proinflammatory cytokines. The increase in IL-17 derived from both CD4 and γδ T cells and was associated with colonic IL-6 expression. Thus CAT2 plays an important role in controlling inflammation and IL-17 activation in an injury model of colitis, and impaired L-Arg availability may contribute to UC pathogenesis.

Berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor.

Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apc(min) mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth.

L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis.

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice.

Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immune responses to microflora in genetically susceptible hosts. A major challenge for IBD research is to develop new strategies for treating this disease. Berberine, an alkaloid derived from plants, is an alternative medicine for treating bacterial diarrhea and intestinal parasite infections. Recent studies suggest that berberine exerts several other beneficial effects, including inducing anti-inflammatory responses. This study determined the effect of berberine on treating dextran sulfate sodium (DSS)-induced intestinal injury and colitis in mice. Berberine was administered through gavage to mice with established DSS-induced intestinal injury and colitis. Clinical parameters, intestinal integrity, proinflammatory cytokine production, and signaling pathways in colonic macrophages and epithelial cells were determined. Berberine ameliorated DSS-induced body weight loss, myeloperoxidase activity, shortening of the colon, injury, and inflammation scores. DSS-upregulated proinflammatory cytokine levels in the colon, including TNF, IFN-γ, KC, and IL-17 were reduced by berberine. Berberine decreased DSS-induced disruption of barrier function and apoptosis in the colon epithelium. Furthermore, berberine inhibited proinflammatory cytokine production in colonic macrophages and epithelial cells in DSS-treated mice and promoted apoptosis of colonic macrophages. Activation of signaling pathways involved in stimulation of proinflammatory cytokine production, including MAPK and NF-κB, in colonic macrophages and epithelial cells from DSS-treated mice was decreased by berberine. In summary, berberine promotes recovery of DSS-induced colitis and exerts inhibitory effects on proinflammatory responses in colonic macrophages and epithelial cells. Thus berberine may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism.

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria-derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40's effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.