Novel Anticancer Drug 5H-pyro[3,2-a] Phenoxazin-5-one (PPH) Regulates lncRNA HOTAIR and HOXC genes in Human MCF-7 Cells

Breast cancer in women is the second most commonly cancer, after skin cancer. The percentage of mortalityrisk for breast cancer is 1 in 37 women (2.7%), which makes breast cancer represent the second cause of cancerdeath in women. Recently, new research based on previously published work in systemic chemotherapy andendocrine therapy field, have improved the incidence rates. The quinonic nucleus is common to many naturaland synthetic products associated with anticancer and antibacterial activities, these compounds are typicallyDNA-intercalating agents. The Class I Homeobox genes (HOX in human and hox in mouse) control embryonicdevelopment and specific determination of positional identity anteroposterior axis of the human body. The HOXgenes, are 39 transcription factors related to morphological, physiological disease. It has been demonstratedthat any deregulation into the network is able to induce neoplastic transformation. Particularly, HOXC locuscollaborating with lncRNA HOTAIR play a key role in breast cancer. In this study, our group evaluated the chemical and metabolic stability of new anticancer molecule 5H-pyro[3,2-a] phenoxazin-5-one (PPH). In a recent paper, we have already demonstrated that a new and potent anticancersynthetic iminoquinone, the 5H-pyrido[3,2-a]phenoxazin-5-one (PPH), is able to inhibit a large number oflymphoblastoid and solid-tumor-derived cells at submicromolar concentrations. Based on our previous research, we decided to analyze the cytotoxic effect and capability of PPH to control thelncRNA HOTAIR and HOXC locus gene expression in human breast cancer cells MCF-7, in order to demonstrateits role like potential new breast cancer antitumor drug

Novel anticancer drug 5h-pyro[3,2-a] phenoxazin-5-one (PPH) regulates lncRNA HOTAIR and HOXC genes in human MCF-7 cells

Breast cancer in women is the second most commonly cancer, after skin cancer. The percentage of mortality risk for breast cancer is 1 in 37 women (2.7%), which makes breast cancer represent the second cause of cancer death in women. Recently, new research based on previously published work in systemic chemotherapy and endocrine therapy field, have improved the incidence rates. The quinonic nucleus is common to many natural and synthetic products associated with anticancer and antibacterial activities, these compounds are typically DNA-intercalating agents. The Class I Homeobox genes (HOX in human and hox in mouse) control embryonic development and specific determination of positional identity anteroposterior axis of the human body. The HOX genes, are 39 transcription factors related to morphological, physiological disease. It has been demonstrated that any deregulation into the network is able to induce neoplastic transformation. Particularly, HOXC locus collaborating with lncRNA HOTAIR play a key role in breast cancer. In this study, our group evaluated the chemical and metabolic stability of new anticancer molecule 5H-pyro[3,2-a] phenoxazin-5-one (PPH). In a recent paper, we have already demonstrated that a new and potent anticancer synthetic iminoquinone, the 5H-pyrido[3,2-a]phenoxazin-5-one (PPH), is able to inhibit a large number of lymphoblastoid and solid-tumor-derived cells at submicromolar concentrations. Based on our previous research, we decided to analyze the cytotoxic effect and capability of PPH to control the lncRNA HOTAIR and HOXC locus gene expression in human breast cancer cells MCF-7, in order to demonstrate its role like potential new breast cancer antitumor drug

A novel formulation of glucose‐sparing peritoneal dialysis solutions with l‐carnitine improves biocompatibility on human mesothelial cells

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end‐stage renal disease (ESRD) lies in the fact that the currently used glucose‐based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and L‐carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro‐inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro‐inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility

Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells

BACKGROUND: Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. METHODS: AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. RESULTS: We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+. CONCLUSION: Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation

NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells.

The gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na(+) and Cl(-) ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na(+):K(+):2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL. RESULTS: The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl(-)-dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 ω-hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites. CONCLUSIONS: The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5

Data on Manuka Honey/Gellan Gum composite hydrogels for cartilage repair

This work contains original data supporting our research paper “Antibacterial effectiveness meets improved mechanical properties: Manuka Honey/Gellan Gum composite hydrogels for cartilage repair”, Bonifacio et al., in press [1], in which innovative composite hydrogels, based on Gellan Gum/Manuka honey/Halloysite nanotubes were described as biomaterials for cartilage regeneration. Here the composites were further examined by means of Fourier Transform Infrared Spectroscopy, in Attenuated Total Reflectance mode (FT-IR/ATR). Materials devoted to cartilage replacement must possess adequate fluid permeability and lubricating capability, therefore, a deeper investigation on water uptake kinetics of freeze-dried specimens up to 21 days in PBS was carried out. Moreover, since the degradation rate of a biomaterial plays a pivotal role in tissue engineering, weight loss measurements of the prepared hydrogels were performed in simulated synovial fluid, in phosphate buffer solution (PBS) and in lysozyme. Scanning Electron Microscopy images provide insight into the morphology of the freeze-dried samples. Finally, additional information on Staphylococcus aureus and Staphylococcus epidermidis ability to adhere onto the prepared hydrogel composites in short times were obtained, as well as the chondrogenic potential of the composites assessed by SDS-PAGE followed by Coomassie blue gel staining

Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome

The K+ voltage-gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K+ current. The aim of this study was to characterize the biophysical properties of a C-terminus-truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12-lead electrocardiogram, transthoracic echocardiography and 24-hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co-expressed with KCNH2, (b) G1006fs/49 exerts a dominant-negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage-sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy