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

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

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT