Dry Heating of Curcumin in the Presence of Basic Salts Yields Anti-inflammatory Dimerization Products.

Curcumin exerts some of its biological effects via degradation products formed by spontaneous oxidation at physiological, i.e., weakly basic, pH. Here, we analyzed products formed by dry heating of curcumin in the presence of a basic salt (sodium bicarbonate and others). Under the dry heating conditions employed, curcumin was completely consumed, yielding products entirely different from those obtained by autoxidative degradation in buffer. Bioassay-guided fractionation of the reaction mixture was used to identify and isolate compounds with anti-inflammatory activity in a cell-based assay. This provided two dimers of curcumin, dicurmins A and B, featuring a partly saturated naphthalene core that inhibited lipopolysaccharide-induced activation of NF-κB in RAW264.7 cells. Dicurmin A and B are unusual derivatives of curcumin lacking key functional moieties yet exhibit increased anti-inflammatory activity. The process of dry heating of polyphenols in the presence of a basic salt can serve as a novel approach to generating bioactive compounds.

Neutron-activated, plasmonically excitable Fe-Pt-Yb2O3 nanoparticles delivering anti-cancer radiation against human glioblastoma cells.

Magnetic nanoparticles can be functionalized in many ways for biomedical applications. Here, we combine four advantageous features in a novel Fe-Pt-Yb2O3 core-shell nanoparticle. (a) The nanoparticles have a size of 10 nm allowing them to diffuse through neuronal tissue. (b) The particles are superparamagnetic after synthesis and ferromagnetic after annealing, enabling directional control by magnetic fields, enhance NMRI contrast, and hyperthermia treatment. (c) After neutron-activation of the shell, they carry low-energetic, short half-life ß-radiation from 175Yb, 177Yb, and 177Lu. (d) Additionally, the particles can be optically visualized by plasmonic excitation and luminescence. To demonstrate the potential of the particles for cancer treatment, we exposed cultured human glioblastoma cells (LN-18) to non-activated and activated particles to confirm that the particles are internalized, and that the ß-radiation of the radioisotopes incorporated in the neutron-activated shell of the nanoparticles kills more than 98% of the LN-18 cancer cells, promising for future anti-cancer applications.

Long-Term Total Neoadjuvant Therapy Leads to Impressive Response Rates in Rectal Cancer: Results of a German Single-Center Cohort.

Intensified preoperative chemotherapy after (chemo)radiotherapy, (Total Neoadjuvant Therapy-TNT), increases pathological complete response (pCR) rates and local control. In cases of clinically complete response (cCR) and close follow-up, non-operative management (NOM) is feasible. We report early outcomes and toxicities of a long-term TNT regime in a single-center cohort. Fifteen consecutive patients with distal or middle-third locally advanced rectal cancer (UICC stage II-III) were investigated, who received neoadjuvant chemoradiotherapy (total adsorbed dose: 50.4 Gy in 28 fractions and two concomitant courses 5-fluorouracil (250 mg/m2/d)/oxaliplatin (50 mg/m2), followed by consolidating chemotherapy (nine courses of FOLFOX4). NOM was offered if staging revealed cCR 2 months after TNT, with resection performed otherwise. The primary endpoint was complete response (pCR + cCR). Treatment-related side effects were quantified for up two years after TNT. Ten patients achieved cCR, of whom five opted for NOM. Ten patients (five cCR and five non-cCR) underwent surgery, with pCR confirmed in the five patients with cCR. The main toxicities comprised leukocytopenia (13/15), fatigue (12/15) and polyneuropathy (11/15). The most relevant CTC °III + IV events were leukocytopenia (4/15), neutropenia (2/15) and diarrhea (1/15). The long-term TNT regime resulted in promising response rates that are higher than the response rates of short TNT regimes. Overall tolerability and toxicity were comparable with the results of prospective trials.

The 5-lipoxygenase/cyclooxygenase-2 cross-over metabolite, hemiketal E2, enhances VEGFR2 activation and promotes angiogenesis.

Consecutive oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2 yields the hemiketal eicosanoids, HKE2 and HKD2. Hemiketals stimulate angiogenesis by inducing endothelial cell tubulogenesis in culture; however, how this process is regulated has not been determined. Here, we identify vascular endothelial growth factor receptor 2 (VEGFR2) as a mediator of HKE2-induced angiogenesis in vitro and in vivo. We found that HKE2 treatment of human umbilical vein endothelial cells dose-dependently increased the phosphorylation of VEGFR2 and the downstream kinases ERK and Akt that mediated endothelial cell tubulogenesis. In vivo, HKE2 induced the growth of blood vessels into polyacetal sponges implanted in mice. HKE2-mediated effects in vitro and in vivo were blocked by the VEGFR2 inhibitor vatalanib, indicating that the pro-angiogenic effect of HKE2 was mediated by VEGFR2. HKE2 covalently bound and inhibited PTP1B, a protein tyrosine phosphatase that dephosphorylates VEGFR2, thereby providing a possible molecular mechanism for how HKE2 induced pro-angiogenic signaling. In summary, our studies indicate that biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways gives rise to a potent lipid autacoid that regulates endothelial cell function in vitro and in vivo. These findings suggest that common drugs targeting the arachidonic acid pathway could prove useful in antiangiogenic therapy.

Conservation of Nickel Ion Single-Active Site Character in a Bottom-Up Constructed π-Conjugated Molecular Network.

On-surface chemistry holds the potential for ultimate miniaturization of functional devices. Porphyrins are promising building-blocks in exploring advanced nanoarchitecture concepts. More stable molecular materials of practical interest with improved charge transfer properties can be achieved by covalently interconnecting molecular units. On-surface synthesis allows to construct extended covalent nanostructures at interfaces not conventionally available. Here, we address the synthesis and properties of covalent molecular network composed of interconnected constituents derived from halogenated nickel tetraphenylporphyrin on Au(111). We report that the π-extended two-dimensional material exhibits dispersive electronic features. Concomitantly, the functional Ni cores retain the same single-active site character of their single-molecule counterparts. This opens new pathways when exploiting the high robustness of transition metal cores provided by bottom-up constructed covalent nanomeshes.

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection.

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Cystathionine γ-lyase exacerbates Helicobacter pylori immunopathogenesis by promoting macrophage metabolic remodeling and activation.

Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world's population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme cystathionine γ-lyase (CTH) is upregulated in humans and mice with H. pylori infection. Here, we show that induction of CTH in macrophages by H. pylori promoted persistent inflammation. Cth-/- mice had reduced macrophage and T cell activation in H. pylori-infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori-induced gastritis. CTH is downstream of the proposed antiinflammatory molecule, S-adenosylmethionine (SAM). Whereas Cth-/- mice exhibited gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori-induced inflammation. Instead, we demonstrated that Cth-deficient macrophages exhibited alterations in the proteome, decreased NF-κB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation, contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori-induced gastric disease.

Disproportionation of Nitric Oxide at a Surface-Bound Nickel Porphyrinoid.

Uncommon metal oxidation states in porphyrinoid cofactors are responsible for the activity of many enzymes. The F430 and P450nor co-factors, with their reduced NiI - and FeIII -containing tetrapyrrolic cores, are prototypical examples of biological systems involved in methane formation and in the reduction of nitric oxide, respectively. Herein, using a comprehensive range of experimental and theoretical methods, we raise evidence that nickel tetraphenyl porphyrins deposited in vacuo on a copper surface are reactive towards nitric oxide disproportionation at room temperature. The interpretation of the measurements is far from being straightforward due to the high reactivity of the different nitrogen oxides species (eventually present in the residual gas background) and of the possible reaction intermediates. The picture is detailed in order to disentangle the challenging complexity of the system, where even a small fraction of contamination can change the scenario.

Protective Role of Spermidine in Colitis and Colon Carcinogenesis.

BACKGROUND & AIMS: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis. METHODS: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox-/- mice and was reversed with Spd. CONCLUSIONS: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.

Curcumin Inhibition of TGFß signaling in bone metastatic breast cancer cells and the possible role of oxidative metabolites.

TGFß signaling promotes progression of bone-metastatic (BMET) breast cancer (BCa) cells by driving tumor-associated osteolysis, a hallmark of BCa BMETs, thus allowing for tumor expansion within bone. Turmeric-derived bioactive curcumin, enriched in bone via local enzymatic deconjugation of inactive circulating curcumin-glucuronides, inhibits osteolysis and BMET progression in human xenograft BCa BMET models by blocking tumoral TGFß signaling pathways mediating osteolysis. This is a unique antiosteolytic mechanism in contrast to current osteoclast-targeting therapeutics. Therefore, experiments were undertaken to elucidate the mechanism for curcumin inhibition of BCa TGFß signaling and the application of this finding across multiple BCa cell lines forming TGFß-dependent BMETs, including a possible role for bioactive curcumin metabolites in mediating these effects. Immunoblot analysis of TGFß signaling proteins in bone tropic human (MDA-SA, MDA-1833, MDA-2287) and murine (4T1) BCa cells revealed uniform curcumin blockade of TGFß-induced Smad activation due to down-regulation of plasma membrane associated TGFßR2 and cellular receptor Smad proteins that propagate Smad-mediated gene expression, resulting in downregulation of PTHrP expression, the osteolytic factor driving in vivo BMET progression. With the exception of early decreases in TGFßR2, inhibitory effects appeared to be mediated by oxidative metabolites of curcumin and involved inhibition of gene expression. Interestingly, while not contributing to changes in Smad-mediated TGFß signaling, curcumin caused early activation of MAPK signaling in all cell lines, including JNK, an effect possibly involving interactions with TGFßR2 within lipid rafts. Treatment with curcumin or oxidizable analogs of curcumin may have clinical relevancy in the management of TGFß-dependent BCa BMETs.

Disproportionation of Nitric Oxide at a Surface-Bound Nickel Porphyrinoid.

Uncommon metal oxidation states in porphyrinoid cofactors are responsible for the activity of many enzymes. The F430 and P450nor co-factors, with their reduced NiI- and FeIII-containing tetrapyrrolic cores, are prototypical examples of biological systems involved in methane formation and in the reduction of nitric oxide, respectively. Herein, using a comprehensive range of experimental and theoretical methods, we raise evidence that nickel tetraphenyl porphyrins deposited in vacuo on a copper surface are reactive towards nitric oxide disproportionation at room temperature. The interpretation of the measurements is far from being straightforward due to the high reactivity of the different nitrogen oxides species (eventually present in the residual gas background) and of the possible reaction intermediates. The picture is detailed in order to disentangle the challenging complexity of the system, where even a small fraction of contamination can change the scenario.

Targeting Mammalian 5-Lipoxygenase by Dietary Phenolics as an Anti-Inflammatory Mechanism: A Systematic Review.

5-Lipoxygenase (5-LOX) plays a key role in inflammation through the biosynthesis of leukotrienes and other lipid mediators. Current evidence suggests that dietary (poly)phenols exert a beneficial impact on human health through anti-inflammatory activities. Their mechanisms of action have mostly been associated with the modulation of pro-inflammatory cytokines (TNF-α, IL-1ß), prostaglandins (PGE2), and the interaction with NF-κB and cyclooxygenase 2 (COX-2) pathways. Much less is known about the 5-lipoxygenase (5-LOX) pathway as a target of dietary (poly)phenols. This systematic review aimed to summarize how dietary (poly)phenols target the 5-LOX pathway in preclinical and human studies. The number of studies identified is low (5, 24, and 127 human, animal, and cellular studies, respectively) compared to the thousands of studies focusing on the COX-2 pathway. Some (poly)phenolics such as caffeic acid, hydroxytyrosol, resveratrol, curcumin, nordihydroguaiaretic acid (NDGA), and quercetin have been reported to reduce the formation of 5-LOX eicosanoids in vitro. However, the in vivo evidence is inconclusive because of the low number of studies and the difficulty of attributing effects to (poly)phenols. Therefore, increasing the number of studies targeting the 5-LOX pathway would largely expand our knowledge on the anti-inflammatory mechanisms of (poly)phenols.

Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A.

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

Bone-Specific Metabolism of Dietary Polyphenols in Resorptive Bone Diseases.

SCOPE: Curcumin prevents bone loss in resorptive bone diseases and inhibits osteoclast formation, a key process driving bone loss. Curcumin circulates as an inactive glucuronide that can be deconjugated in situ by bone's high ß-glucuronidase (GUSB) content, forming the active aglycone. Because curcumin is a common remedy for musculoskeletal disease, effects of microenvironmental changes consequent to skeletal development or disease on bone curcumin metabolism are explored. METHODS AND RESULTS: Across sexual/skeletal development or between sexes in C57BL/6 mice ingesting curcumin (500 mg kg-1 ), bone curcumin metabolism and GUSB enzyme activity are unchanged, except for >twofold higher (p < 0.05) bone curcumin-glucuronide substrate levels in immature (4-6-week-old) mice. In ovariectomized (OVX) or bone metastasis-bearing female mice, bone substrate levels are also >twofold higher. Aglycone curcumin levels tend to increase proportional to substrate such that the majority of glucuronide distributing to bone is deconjugated, including OVX mice where GUSB decreases by 24% (p < 0.01). GUSB also catalyzes deconjugation of resveratrol and quercetin glucuronides by bone, and a requirement for the aglycones for anti-osteoclastogenic bioactivity, analogous to curcumin, is confirmed. CONCLUSION: Dietary polyphenols circulating as glucuronides may require in situ deconjugation for bone-protective effects, a process influenced by bone microenvironmental changes.

Mechanistic Differences in the Inhibition of NF-κB by Turmeric and Its Curcuminoid Constituents.

Turmeric extract, a mixture of curcumin and its demethoxy (DMC) and bisdemethoxy (BDMC) isomers, is used as an anti-inflammatory preparation in traditional Asian medicine. Curcumin is considered to be the major bioactive compound in turmeric but less is known about the relative anti-inflammatory potency and mechanism of the other components, their mixture, or the reduced in vivo metabolites. We quantified inhibition of the NF-κB pathway in cells, adduction to a peptide mimicking IκB kinase ß, and the role of cellular glutathione as a scavenger of electrophilic curcuminoid oxidation products, suggested to be the active metabolites. Turmeric extracts (IC50 14.5 ± 2.9 µM), DMC (IC50 12.1 ± 7.2 µM), and BDMC (IC50 8.3 ± 1.6 µM), but not reduced curcumin, inhibited NF-κB similar to curcumin (IC50 18.2 ± 3.9 µM). Peptide adduction was formed with turmeric and DMC but not with BDMC, and this correlated with their oxidative degradation. Inhibition of glutathione biosynthesis enhanced the activity of DMC but not BDMC in the cellular assay. These findings suggest that NF-κB inhibition by curcumin and DMC involves their oxidation to reactive electrophiles, whereas BDMC does not require oxidation. Because it has not been established whether curcumin undergoes oxidative transformation in vivo, oxidation-independent BDMC may be a promising alternative to test in clinical trials.

Spermine oxidase mediates Helicobacter pylori-induced gastric inflammation, DNA damage, and carcinogenic signaling.

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and ß-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and ß-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of ß-catenin signaling.

Bacterial Pathogens Hijack the Innate Immune Response by Activation of the Reverse Transsulfuration Pathway.

The reverse transsulfuration pathway is the major route for the metabolism of sulfur-containing amino acids. The role of this metabolic pathway in macrophage response and function is unknown. We show that the enzyme cystathionine γ-lyase (CTH) is induced in macrophages infected with pathogenic bacteria through signaling involving phosphatidylinositol 3-kinase (PI3K)/MTOR and the transcription factor SP1. This results in the synthesis of cystathionine, which facilitates the survival of pathogens within myeloid cells. Our data demonstrate that the expression of CTH leads to defective macrophage activation by (i) dysregulation of polyamine metabolism by depletion of S-adenosylmethionine, resulting in immunosuppressive putrescine accumulation and inhibition of spermidine and spermine synthesis, and (ii) increased histone H3K9, H3K27, and H3K36 di/trimethylation, which is associated with gene expression silencing. Thus, CTH is a pivotal enzyme of the innate immune response that disrupts host defense. The induction of the reverse transsulfuration pathway by bacterial pathogens can be considered an unrecognized mechanism for immune escape.IMPORTANCE Macrophages are professional immune cells that ingest and kill microbes. In this study, we show that different pathogenic bacteria induce the expression of cystathionine γ-lyase (CTH) in macrophages. This enzyme is involved in a metabolic pathway called the reverse transsulfuration pathway, which leads to the production of numerous metabolites, including cystathionine. Phagocytized bacteria use cystathionine to better survive in macrophages. In addition, the induction of CTH results in dysregulation of the metabolism of polyamines, which in turn dampens the proinflammatory response of macrophages. In conclusion, pathogenic bacteria can evade the host immune response by inducing CTH in macrophages.

Magnetic Field-Assisted Chemical Vapor Deposition of Iron Oxide Thin Films: Influence of Field-Matter Interactions on Phase Composition and Morphology.

Magnetic field-assisted CVD offers a direct pathway to manipulate the evolution of microstructure, phase composition, and magnetic properties of the as-prepared film. We report on the role of applied magnetic fields (0.5 T) during a cold-wall CVD deposition of iron oxide from [FeIII(OtBu)3]2 leading to higher crystallinity, larger particulates, and better out-of-plane magnetic anisotropy, if compared with zero-field depositions. Whereas selective formation of homogeneous magnetite films was observed for the field-assisted process, coexistence of hematite and amorphous iron(III) oxide was confirmed under zero-field conditions. Comparison of the coercive field (11 vs 60 mT) indicated lower defect concentration for the field-assisted process with nearly superparamagnetic behavior. X-ray photoemission electron microscopy (X-PEEM) in absorption mode at the O-K and Fe-L3,2 edges confirmed the selective formation of magnetite (field-assisted) and hematite (zero-field) with coexisting amorphous phases, respectively, emphasizing the importance of field-matter interactions in the phase-selective synthesis of magnetic thin films.

Curcumin induces secretion of glucagon-like peptide-1 through an oxidation-dependent mechanism.

Curcumin shows antiglycemic effects in animals. Curcumin is chemically unstable at physiological pH, and its oxidative degradation products were shown to contribute to its anti-inflammatory effects. Since the degradation products may also contribute to other effects, we analyzed their role in the antiglycemic activity of curcumin. We quantified curcumin-induced release of glucagon-like peptide 1 (GLP-1) from mouse STC-1 cells that represent enteroendocrine L-cells as a major source of this anti-diabetic hormone. Curcumin induced secretion of GLP-1 in a dose-dependent manner. Two chemically stable analogues of curcumin that do not readily undergo degradation, were less active while two unstable analogues were active secretagogues. Chromatographically isolated spiroepoxide, an unstable oxidative metabolite of curcumin with anti-inflammatory activity, also induced secretion of GLP-1. Stable compounds like the final oxidative metabolite bicyclopentadione, and the major plasma metabolite, curcumin-glucuronide, were inactive. GLP-1 secretion induced by curcumin and its oxidative degradation products was associated with activation of PKC, ERK, and CaM kinase II. Since activity largely correlated with instability of curcumin and the analogues, we tested the extent of covalent binding to proteins in STC-1 cells and found it occurred with similar affinity as N-ethylmaleimide, indicating covalent binding occurred with nucleophilic cysteine residues. These results suggest that oxidative metabolites of curcumin are involved in the antiglycemic effects of curcumin. Our findings support the hypothesis that curcumin functions as a pro-drug requiring oxidative activation to reveal its bioactive metabolites that act by binding to target proteins thereby causing a change in function.

α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY.

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.