Natural compounds as inhibitors of lactate dehydrogenase

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate, utilizing NADH as co-factor. It’s a tetrameric enzyme composed of two subunits, M and H, whose association can generate five isoforms. One of this, the human isoform 5, hLDH5 has the highest activity in converting pyruvate to lactate under anaerobic conditions, such as those found in hypoxic tumors and for this reason it’s up-regulated in tumor tissues where cells glycolytic rate is up to 200 times higher than that of the normal tissue. hLDH5 inhibition should cause cancer cell death by starvation, without interfering with healthy cells that normally use oxidative phosphorylation for ATP generation (1). Inhibition of LDH is so considered as a promising target in cancer treatment, and natural compounds could serve as useful scaffold to study new anticancer agents. Among the few plant derived hLDH5 inhibitors already investigated there are mainly phenolic derivatives such as gossypol, morin, and galloflavin (2,3). In the last decade our research group successfully detected a good number of compounds obtained from Mediterranean plants with anticancer effect, and for this reason start a research program aimed to discover new classes of natural products having hLDH5 inhibitory activity. In a first study, since some species of Phlomis (Lamiaceae) proved to possess anti-cancer properties, the crude extract of P. kurdica aerial parts was selected as the starting material. Two new flavonoids and one new phenylpropanoid, together with eleven known phenolic compounds, including flavonoids and phenylpropanoids were isolated and assayed for their hLDH5 inhibitory activity. Luteolin 7-O-β-D-glucopyranoside showed an IC50 value similar to that of reference compound galloflavin (4). Then, since Polygala genus (Polygalaceae) is well known to contain phenolic oligosaccharides, xanthones, lignans, and triterpenic saponins and it’s largely used in the traditional medicine, an Italian species P. flavescens subsp. flavescens was chosen. Ten new compounds were isolated from the methanol residue of the aerial parts through Sephadex and RP-HPLC separations, including four flavonol glycosides, two oligosaccharides, one α-ionone, and three triterpenoidic saponins, together with two known oligosaccharides and two flavonol glycosides. The isolates were assayed for their inhibitory activity against hLDH5 and 3,6'-di-O-sinapoylsucrose showed an inhibition potency comparable or even slightly better than reference inhibitor galloflavin. Docking studies were carried out to hypothesize the interaction mode of active compounds in the enzyme active site

Inhibitors of lactate dehydrogenase (hLDH5) from Polygala flavescens subsp. flavescens

The human isoform 5 of lactate dehydrogenase (hLDH5) is an enzyme up-regulated in tumor tissues since cancer cells depend mainly on anaerobic respiration and their glycolytic rate is up to 200 times higher than that of the normal tissue. hLDH5 inhibition should cause cancer cell death by starvation, without interfering with healthy cells that normally use oxidative phosphorylation for ATP generation. Inhibition of LDH is so considered as a promising target in cancer treatment, since it is possible to cause a starving of cancerous cells by reducing glycolysis or by inhibiting the conversion of glucose to lactate. In the course of our research program on the hLDH5 inhibitory activity of natural products [1], a chemical study of P. flavescens subsp. flavescens was carried out. Polygala L. genus (Polygalaceae) is well known to contain phenolic oligosaccharides, xanthones, lignans, and triterpenic saponins and it’s largely used in the traditional medicine [2]. Ten new compounds were isolated from the n-BuOH residue of the aerial parts through Sephadex and RP-HPLC separations, including four flavonol glycosides, two oligosaccharides, one α-ionone, and three triterpenoidic saponins, together with two known oligosaccharides and two flavonol glycosides. All structures were elucidated on the basis of their spectroscopic and spectrometric data. The isolates were assayed for their inhibitory activity against hLDH5 and 3,6'-di-O-sinapoylsucrose showed an IC50 value of 90.4 µM. Modeling studies were carried out to suggest the putative interaction mode of this compound in the enzyme active site. This analysis highlighted that 3,6'-di-O-sinapoylsucrose shows a high number of H-bonds and interacts with enzyme regions rarely explored by the known hLDH5 inhibitors

Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains

BACKGROUND: Sinorhizobium meliloti is a soil bacterium that forms nitrogen-fixing nodules on the roots of leguminous plants such as alfalfa (Medicago sativa). This species occupies different ecological niches, being present as a free-living soil bacterium and as a symbiont of plant root nodules. The genome of the type strain Rm 1021 contains one chromosome and two megaplasmids for a total genome size of 6 Mb. We applied comparative genomic hybridisation (CGH) on an oligonucleotide microarrays to estimate genetic variation at the genomic level in four natural strains, two isolated from Italian agricultural soil and two from desert soil in the Aral Sea region. RESULTS: From 4.6 to 5.7 percent of the genes showed a pattern of hybridisation concordant with deletion, nucleotide divergence or ORF duplication when compared to the type strain Rm 1021. A large number of these polymorphisms were confirmed by sequencing and Southern blot. A statistically significant fraction of these variable genes was found on the pSymA megaplasmid and grouped in clusters. These variable genes were found to be mainly transposases or genes with unknown function. CONCLUSION: The obtained results allow to conclude that the symbiosis-required megaplasmid pSymA can be considered the major hot-spot for intra-specific differentiation in S. meliloti

An integrated analysis of the effects of Esculentin 1-21 on Saccharomyces cerevisiae.

The antimicrobial peptide esculentin 1–21 (Esc 1–21) is a shorter synthetic version of the 46-residue peptide occurring in the Rana esculenta skin secretion. Here we propose an integrated proteomic and transcriptomic approach to interpret the biological effects of this peptide on Saccharomyces cerevisiae. We further investigated the response to this peptide by correlating the results of the transcriptome and proteome analysis with phenotypic effects. The results show that S. cerevisiae adapts to Esc 1–21 using the High Osmolarity Glycerol (HOG) pathway involved in osmotic tolerance and cell wall maintenance. Comparative proteomics reveals that Esc 1–21 causes downregulation of enzymes of the lower glycolytic pathway and in genes involved in spindle body formation and remodelling of cell-wall synthesis. Moreover the peptide induces downexpression of protein actin within 45 min and cells pre-treated with peptide show less sensitivity to osmotic stress and increased sensitivity to heat shock stress. The results obtained with the two different methodologies are in agreement at the cellular process levels. A combined approach may help elucidate the main aspects related to the effects of this peptide on the eukaryotic cell. The employment of different technologies may reveal the potential and limitations of each adapted approach in a prospective application for drug screening

Tannic Acid-Iron Complex-Based Nanoparticles as a Novel Tool against Oxidative Stress

: Accumulation of reactive oxygen species in cells leads to oxidative stress, with consequent damage for cellular components and activation of cell-death mechanisms. Oxidative stress is often associated with age-related conditions, as well as with several neurodegenerative diseases. For this reason, antioxidant molecules have attracted a lot of attention, especially those derived from natural sources─like polyphenols and tannins. The main issue related to the use of antioxidants is their inherent tendency to be oxidized, their quick enzymatic degradation in biological fluids, and their poor bioavailability. Nanomedicine, in this sense, has helped in finding new solutions to deliver and protect antioxidants; however, the concentration of the encapsulated molecule in conventional nanosystems could be very low and, therefore, less effective. We propose to exploit the properties of tannic acid, a known plant-derived antioxidant, to chelate iron ions, forming hydrophobic complexes that can be coated with a biocompatible and biodegradable phospholipid to improve stability in biological media. By combining nanoprecipitation and hot sonication procedures, we obtained three-dimensional networks composed of tannic acid-iron with a hydrodynamic diameter of ≈200 nm. These nanostructures show antioxidant properties and scavenging activity in cells after induction of an acute chemical pro-oxidant insult; moreover, they also demonstrated to counteract damage induced by oxidative stress both in vitro and on an in vivo model organism (planarians)

IL-13 mRNA tissue content identifies two subsets of adult ulcerative colitis patients with different clinical and mucosa-associated microbiota profiles

BACKGROUND AND AIM: A personalized approach to therapy has great promise to improve disease outcomes. To this end, the identification of different subsets of patients according with the prevalent pathogenic process might guide in the choice of therapeutic strategy. We hypothesize that UC patients might be stratified according to distinctive cytokine profiles and/or to a specific mucosa-associated microbiota. METHODS: In a cohort of clinically and endoscopic active UC patients and controls, we analyzed by qPCR the mucosal cytokine mRNA content and the mucosa-associated microbiota composition assessed by the 16SrRNA gene sequencing. RESULTS: We demonstrate, by means of data-driven approach, the existence of a specific UC patient subgroup characterized by elevated IL-13mRNA tissue content separated by patients with low IL-13 mRNA tissue content. The two subsets differ in clinical-pathological characteristics. High IL-13mRNA patients are younger at diagnosis and show higher prevalence of extensive colitis than low IL-13mRNA ones. They also show a more frequent use of steroid/immunosuppressant/anti-TNFα therapy during a one-year follow-up. The two subgroups show a differential enrichment of mucosa associated microbiota genera with prevalence of Prevotella in patients with high IL-13mRNA tissue content and Sutterella and Acidaminococcus in patients with low IL-13mRNA tissue content. CONCLUSION: Assessment of mucosal IL-13mRNA might help in the identification of the patients' subgroup that might benefit from a therapeutic approach modulating IL-13

Gut microbiota profiles and characterization of cultivable fungal isolates in IBS patients

Studies so far conducted on irritable bowel syndrome (IBS) have been focused mainly on the role of gut bacterial dysbiosis in modulating the intestinal permeability, inflammation, and motility, with consequences on the quality of life. Limited evidences showed a potential involvement of gut fungal communities. Here, the gut bacterial and fungal microbiota of a cohort of IBS patients have been characterized and compared with that of healthy subjects (HS). The IBS microbial community structure differed significantly compared to HS. In particular, we observed an enrichment of bacterial taxa involved in gut inflammation, such as Enterobacteriaceae, Streptococcus, Fusobacteria, Gemella, and Rothia, as well as depletion of health-promoting bacterial genera, such as Roseburia and Faecalibacterium. Gut microbial profiles in IBS patients differed also in accordance with constipation. Sequence analysis of the gut mycobiota showed enrichment of Saccharomycetes in IBS. Culturomics analysis of fungal isolates from feces showed enrichment of Candida spp. displaying from IBS a clonal expansion and a distinct genotypic profiles and different phenotypical features when compared to HS of Candida albicans isolates. Alongside the well-characterized gut bacterial dysbiosis in IBS, this study shed light on a yet poorly explored fungal component of the intestinal ecosystem, the gut mycobiota. Our results showed a differential fungal community in IBS compared to HS, suggesting potential for new insights on the involvement of the gut mycobiota in IBS. KEY POINTS: Comparison of gut microbiota and mycobiota between IBS and healthy subjects Investigation of cultivable fungi in IBS and healthy subjects Candida albicans isolates result more virulent in IBS subjects compared to healthy subjects