Antioxidant and Anti-Inflammatory Effect of Cinnamon (Cinnamomum verum J. Presl) Bark Extract after In Vitro Digestion Simulation

Cinnamon bark is widely used for its organoleptic features in the food context and growing evidence supports its beneficial effect on human health. The market offers an increasingly wide range of food products and supplements enriched with cinnamon extracts which are eliciting beneficial and health-promoting properties. Specifically, the extract of Cinnamomum spp. is rich in antioxidant, anti-inflammatory and anticancer biomolecules. These include widely reported cinnamic acid and some phenolic compounds, such asproanthocyanidins A and B, and kaempferol. These molecules are sensitive to physical-chemical properties (such as pH and temperature) and biological agents that act during gastric digestion, which could impair molecules' bioactivity. Therefore, in this study, the cinnamon's antioxidant and anti-inflammatory bioactivity after simulated digestion was evaluated by analyzing the chemical profile of the pure extract and digested one, as well as the cellular effect in vitro models, such as Caco2 and intestinal barrier. The results showed that the digestive process reduces the total content of polyphenols, especially tannins, while preserving other bioactive compounds such as cinnamic acid. At the functional level, the digested extract maintains an antioxidant and anti-inflammatory effect at the cellular level

Molecular insight into substrate recognition by human cytosolic sialidase NEU2

Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide alpha(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide alpha(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid. Proteins 2012; (c) 2011 Wiley Periodicals, Inc

Synergistic Antioxidant Effect of Prebiotic Ginseng Berries Extract and Probiotic Strains on Healthy and Tumoral Colorectal Cell Lines

Oxidative stress caused by reactive oxygen species (ROS, O2•−, HO•, and H2O2) affects the aging process and the development of several diseases. A new frontier on its prevention includes functional foods with both specific probiotics and natural extracts as antioxidants. In this work, Panax ginseng C.A. Meyer berries extract was characterized for the presence of beneficial molecules (54.3% pectin-based polysaccharides and 12% ginsenosides), able to specifically support probiotics growth (OD600nm > 5) with a prebiotic index of 0.49. The administration of the extract to a probiotic consortium induced the production of short-chain fatty acids (lactic, butyric, and propionic acids) and other secondary metabolites derived from the biotransformation of Ginseng components. Healthy and tumoral colorectal cell lines (CCD841 and HT-29) were then challenged with these metabolites at concentrations of 0.1, 0.5, and 1 mg/mL. The cell viability of HT-29 decreased in a dose-dependent manner after the exposition to the metabolites, while CCD841 vitality was not affected. Regarding ROS production, the metabolites protected CCD841 cells, while ROS levels were increased in HT-29 cells, potentially correlating with the less functionality of glutathione S-transferase, catalase, and total superoxide dismutase enzymes, and a significant increase in oxidized glutathione

Role of NEU3 Overexpression in the Prediction of Efficacy of EGFR-Targeted Therapies in Colon Cancer Cell Lines

The epidermal growth factor receptor (EGFR), through the MAP kinase and PI3K-Akt-mTOR axis, plays a pivotal role in colorectal cancer (CRC) pathogenesis. The membrane-associated NEU3 sialidase interacts with and desialylates EGFR by promoting its dimerization and downstream effectors’ activation. Among the targeted therapies against EGFR, the monoclonal antibody cetuximab is active only in a subgroup of patients not carrying mutations in the MAP kinase pathway. In order to better understand the EGFR-NEU3 interplay and the mechanisms of pharmacological resistance, we investigated the role of NEU3 deregulation in cetuximab-treated CRC cell lines transiently transfected with NEU3 using Western blot analysis. Our results indicate that NEU3 overexpression can enhance EGFR activation only if EGFR is overexpressed, indicating the existence of a threshold for NEU3-mediated EGFR activation. This enhancement mainly leads to the constitutive activation of the MAP kinase pathway. Consequently, we suggest that the evaluation of NEU3 expression cannot entirely substitute the evaluation of EGFR because EGFR-negative cases cannot be stimulated by NEU3. Furthermore, NEU3-mediated hyperactivation of EGFR is counterbalanced by the administration of cetuximab, hypothesizing that a combined treatment of NEU3- and EGFR-targeted therapies may represent a valid option for CRC patients, which must be investigated in the future

Characterization of the Biological Activities of a New Polyphenol-Rich Extract from Cinnamon Bark on a Probiotic Consortium and Its Action after Enzymatic and Microbial Fermentation on Colorectal Cell Lines

Cinnamon polyphenols are known as health-promoting agents. However, their positive impact depends on the extraction method and their bioaccessibility after digestion. In this work, cinnamon bark polyphenols were extracted in hot water and subjected to an in vitro enzymatic digestion. After a preliminary characterization of total polyphenols and flavonoids (respectively 520.05 ± 17.43 µgGAeq/mg and 294.77 ± 19.83 µgCATeq/mg powder extract), the extract antimicrobial activity was evidenced only against Staphylococcus aureus and Bacillus subtilis displaying a minimum inhibition growth concentration value of 2 and 1.3 mg/mL, respectively, although it was lost after in vitro extract digestion. The prebiotic potential was evaluated on probiotic Lactobacillus and Bifidobacterium strains highlighting a high growth on the in vitro digested cinnamon bark extract (up to 4 × 108 CFU/mL). Thus, the produced SCFAs and other secondary metabolites were extracted from the broth cultures and determined via GC-MSD analyses. The viability of healthy and tumor colorectal cell lines (CCD841 and SW480) was assayed after the exposition at two different concentrations (23 and 46 µgGAeq/mL) of the cinnamon extract, its digested, and the secondary metabolites produced in presence of cinnamon extract or its digested, showing positive protective effects against a tumorigenic condition

Synthesis and Biological Evaluation of 1,4-Diaryl-2-azetidinones as Specific Anticancer Agents: Activation of Adenosine Monophosphate Activated Protein Kinase and Induction of Apoptosis

A series of novel 1,4-diaryl-2-azetidinones were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was observed with the best compounds (±)-<i>trans</i>-<b>20</b>, (±)-<i>trans</i>-<b>21</b>, and enantiomers (+)-<i>trans</i>-<b>20</b> and (+)-<i>trans</i>-<b>21</b>, which exhibited IC₅₀ values of 3–13 nM against duodenal adenocarcinoma cells. They induced inhibition of tubulin polymerization and subsequent G2/M arrest. This effect was accompanied by activation of AMP-activated protein kinase (AMPK), activation of caspase-3, and induction of apoptosis. Additionally, the most potent compounds displayed antiproliferative activity against different colon cancer cell lines, opening the route to a new class of potential therapeutic agents against colon cancer

Deregulation of sialidases in human normal and tumor tissues

BACKGROUND: Aberrant sialylation is a characteristic feature associated with cancer. The four types of mammalian sialidases identified to date have been shown to behave in different manners during carcinogenesis. While NEU1, NEU2 and NEU4 have been observed to oppose malignant phenotypes, the membrane-bound sialidase NEU3 was revealed to promote cancer progression. OBJECTIVES: With the aim of improving the knowledge about sialidases deregulation in various cancer types, we investigated the amount of NEU1, NEU3 and NEU4 transcripts in paired normal and tumor tissues from 170 patients with 11 cancer types. METHODS: mRNA was extracted from patients tissue specimens and retrotranscribed into cDNA, which was quantified by Real-Time PCR. RESULTS: We found NEU1 and NEU3 to be up regulated, while NEU4 was down regulated in most cancer types. In particular, colorectal cancer tissues showed the highest increase in NEU3 expression. Both NEU1 and NEU3 showed a strong up-regulation in ovarian cancer. CONCLUSIONS: Our data show that human sialidases are expressed at different levels in healthy tissues and are strongly deregulated in tumors. Moreover, sialidases expression in our European cohort showed significant differences from Asian populations. Some of these peculiar features open up potential applications of sialidases in cancer diagnosis and therapy

Ability to express CRH in Neuro2A cells transiently transfected with wild-type or mutant preproCRH construct.

<p>A) CRH levels of expression detected by realtime quantitative PCR in not transfected (NT) or transfected cells (wt or p.Pro30Arg) at three different times: 24 h, 48 h and 72 h. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) of mRNA levels normalized to the basal CRH expression in Neuro2A cells (NT values) and to a housekeeping control gene (b-Actin). * t = −3.676 and p = 0.020 compared with wt at 24 h; ** t = 5.274 and p = 0.002 compared with wt at 24 h. B) Densitometric analysis of CRH immunoreactive proteins in subcellular fractions of the Neuro2A cells. Each bar represents the mean ± S.E.M. (<i>n</i> = 3) and protein content is expressed in arbitrary units. C) Levels of secreted CRH protein measured by ELISA. The ability of cells to secrete the CRH hormone was evaluated by measuring the protein level in cultured media of cells transfected either with the wild-type or the mutant construct at 24 h or 48 h after the transfection. Each bar represents the mean ± S.E.M. (<i>n</i> = 2) and protein content is expressed as % in respect to the mean value of wt 24 h (assumed equal to 100%).* t = −7.403 and p = 0.005 compared with wt at 24 h; ** t = 7.796 and p = 0.004 compared with wt at 24 h.</p

Confocal images of Neuro2A cells transfected with the wild-type or the mutant construct.

<p>To investigate intracellular distribution of CRH, cell were fixed in PFA and probed with mouse polyclonal anti-GM130 (red) for Golgi visualization and rabbit polyclonal anti-CRH (green) antibodies.</p