Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control

Metastasis is the primary cause of death in prostate cancer (PCa) patients. Effective therapeutic intervention in metastatic PCa is undermined by our poor understanding of its molecular aetiology. Defining the mechanisms underlying PCa metastasis may lead to insights into how to decrease morbidity and mortality in this disease. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). Hydroimidazolone (MG‐H1) and argpyrimidine (AP) are AGEs originating from MG‐mediated post‐translational modification of proteins at arginine residues. AP is involved in the control of epithelial to mesenchymal transition (EMT), a crucial determinant of cancer metastasis and invasion, whose regulation mechanisms in malignant cells are still emerging. Here, we uncover a novel mechanism linking Glo1 to the maintenance of the metastatic phenotype of PCa cells by controlling EMT by engaging the tumour suppressor miR‐101, MG‐H1‐AP and TGF‐β1/Smad signalling. Moreover, circulating levels of Glo1, miR‐101, MG‐H1‐AP and TGF‐β1 in patients with metastatic compared with non‐metastatic PCa support our in vitro results, demonstrating their clinical relevance. We suggest that Glo1, together with miR‐101, might be potential therapeutic targets for metastatic PCa, possibly by metformin administration.     

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC₅₀ value between 70 and 110 μM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.     

Anti-herpes simplex virus 1 and immunomodulatory activities of a poly-γ- glutamic acid from Bacillus horneckiae strain APA of shallow vent origin

Applied Microbiology and Biotechnology - Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe,...     

A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology

The clinically used sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor dorzolamide (DRZ), a new sulfonamide CA inhibitor also incorporating NO-donating moieties, NCX250, and isosorbide mononitrate (ISMN) (an NO-donating compound with no CA inhibitory properties) were investigated for their intraocular pressure (IOP) lowering effects in rabbits with carbomer-induced glaucoma. NCX250 was more effective than DRZ or ISMN on lowering IOP, increasing ocular hemodynamics, decreasing the inflammatory processes and ocular apoptosis in this animal model of glaucoma. NO participate to the regulation of IOP in glaucoma, having also antiapoptotic and anti-inflammatory effects. The ophthalmic artery, both systolic and diastolic velocities, were significantly reduced in NCX250-treated eyes in comparison to DRZ treated ones, suggesting thus a beneficial effect of NCX250 on the blood supply to the optic nerve. Combining CA inhibition with NO-donating moieties in the same compound offers an excellent approach for the management of glaucoma.     

Cytotoxicity,apoptosis, DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A

This study aimed to investigate the in vitro damage induced by ochratoxin A (OTA) in BME-UV1 and MDCK epithelial cells. Both cells lines were treated with OTA (0 up to 10 μg/mL), and cell viability (MTT assay), membrane stability (lactate dehydrogenase (LDH) release assay) and apoptotic cell rate (Tunel assay) were investigated. Further, the effect of the incubation with OTA has been evaluated at DNA level by the determination of DNA integrity, by the quantification of DNA adduct formation (8-hydroxy-2′-deoxyguanosine (8-OHdG)) and by the assessment of the global DNA methylation status (5-methyl-cytosine (5-mC)). The obtained results showed that after 24 h of OTA treatment, BME-UV1 cell viability was reduced in a dose-dependent way. OTA significantly (P?<?0.05) increased LDH release in BME-UV1 cells at all concentrations tested. OTA (1.25 μg/mL) induced 35 % LDH release in MDCK cells (P?<?0.05). A significant (P?<?0.05) change in percentages of apoptotic BME-UV1 (10?±?0.86) and MDCK (25?±?0.88) cells was calculated when the cells were co-incubated with OTA. The level of 8-OHdG adduct formation was significantly (P?<?0.05) increased in BME-UV1 cells treated with 1.25 μg/mL of OTA. The results of the present study suggest that a different mechanism of action may occur in these cell lines.

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