Resveratrol depolarizes the membrane potential in human granulosa cells and promotes mitochondrial biogenesis.

OBJECTIVE: To study the biological effects of resveratrol on the growth, electrophysiology, and mitochondrial function of human granulosa cells (h-GCs). DESIGN: Preclinical study. SETTING: Electrophysiology laboratory and in vitro fertilization unit. PATIENT(S): This study included h-GCs from seven infertile women undergoing assisted reproductive techniques. INTERVENTION(S): Human ovarian Granulosa Cell Tumor (GCT) cell line COV434 and h-GCs obtained after oocyte retrieval were cultured in the absence or presence of resveratrol. MAIN OUTCOME MEASURE(S): Granulosa cells were evaluated for cell viability and mitochondrial activity. Electrophysiological recordings and evaluation of potassium current (IKur) and Ca2+ concentration were also performed. RESULT(S): Resveratrol induced mitochondrial activity in a bell-shaped, dose-effect-dependent manner. Specifically, resveratrol treatment (3 µM, 48 hours) increased ATP production and cell viability and promoted the induction of cellular differentiation. These biological changes were associated with mitochondrial biogenesis. Electrophysiological recordings showed that resveratrol reduced the functional expression of an ultra rapid activating, slow inactivating, delayed rectifier potassium current (IKur) that is associated with a plasma membrane depolarization and that promotes an increase in intracellular Ca2+. CONCLUSION(S): The effects of resveratrol on potassium current and mitochondrial biogenesis in h-GCs could explain the beneficial effects of this polyphenol on the physiology of the female reproductive system. These findings suggest there are therapeutic implications of resveratrol in a clinical setting.

New Insights on KCa3.1 Channel Modulation.

The human intermediate conductance calcium-activated potassium channel, KCa3.1, is involved in several pathophysiological conditions playing a critical role in cell secretory machinery and calcium signalling. The recent cryo-EM analysis provides new insights for understanding the modulation by both endogenous and pharmacological agents. A typical feature of this channel is the low open probability in saturating calcium concentrations and its modulation by potassium channel openers (KCOs), such as benzo imidazolone 1-EBIO, without changing calcium-dependent activation. In this paper, we proposed a model of KCOs action in the modulation of channel activity. The KCa3.1 channel has a very rich pharmacological profile with several classes of molecules that selectively interact with different binding sites of the channel. Among them, benzo imidazolones can be openers (positive modulators such as 1-EBIO, DC-EBIO) or blockers (negative modulators such as NS1619). Through computation modelling techniques, we identified the 1,4-benzothiazin-3-one as a promising scaffold to develop new KCa3.1 channel modulators. Further studies are needed to explore the potential use of 1-4 benzothiazine- 3-one in KCa3.1 modulation and its pharmacological application.

A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells.

BACKGROUND: Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. RESULTS: We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. CONCLUSION: The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

Silver ions promote blebs growth in U251 glioblastoma cell by activating nonselective cationic currents.

Glioblastoma (GBM) is the most common and aggressive human brain cancer with low prognosis and therefore the discovery of new anticancer agents is needful. Sulfydryl reagents, such as silver, have been shown to induce membrane vesiculation in several cellular models through a mechanism that has not been yet completely clarified. Using U251 glioblastoma cells, we observed that silver induced irreversible bleb formation of the plasma membrane. This morphological event was anticipated by an increase of intracellular Ca2+ associated to extracellular Ca2+ influx. Accordingly, using patch-clamp whole cell recording during silver ion application, inward current/s (IAg) at -90 mV were detected and cells were permeable to Ca2+ and monovalent ions such as Na+. IAg activation and the intracellular Ca2+ increase promoted by silver ions (Ag+) were prevented by co-application of 20 µM cysteine and 300 µM DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid), suggesting a critical role of thiol groups in the biological effects of silver ions. IAg was partially inhibited by 1 mM Gd3+, an unspecific inhibitor of cationic currents. Cysteine, Gd3+ and extracellular free Ca2+ solution completely abolished blebbing formation promoted by Ag+. Furthermore, extracellular Na+ ion replacement with TEA or an increase of extracellular tonicity by sucrose (100 mM) reduced both size and growth of membrane blebbing. Our data suggest that Ag+ promotes the formation necrotic blebs as consequence of the increase of intracellular Ca2+ and intracellular hydrostatic pressure associated to the activation of cationic currents. Since silver-induced blebs were less evident in benign glial human Müller MIO-M1 cells, silver compounds could represent new adjuvant to anticancer agents to improve GBM therapies.

Solid Dispersion of Resveratrol Supported on Magnesium DiHydroxide (Resv@MDH) Microparticles Improves Oral Bioavailability.

Resveratrol, because of its low solubility in water and its high membrane permeability, is collocated in the second class of the biopharmaceutical classification system, with limited bioavailability due to its dissolution rate. Solid dispersion of resveratrol supported on Magnesium DiHydroxide (Resv@MDH) was evaluated to improve solubility and increase bioavailability of resveratrol. Fluorimetric microscopy analysis displays three types of microparticles with similar size: Type 1 that emitted preferably fluorescence at 445 nm with bandwidth of 50 nm, type 2 that emitted preferably fluorescence at 605 nm with bandwidth of 70 nm and type 3 that is non-fluorescent. Micronized pure resveratrol displays only microparticles type 1 whereas type 3 are associated to pure magnesium dihydroxide. Dissolution test in simulated gastric environment resveratrol derived from Resv@MDH in comparison to resveratrol alone displayed better solubility. A 3-fold increase of resveratrol bioavailability was observed after oral administration of 50 mg/kg of resveratrol from Resv@MDH in rabbits. We hypothesize that type 2 microparticles represent magnesium dihydroxide microparticles with a resveratrol shell and that they are responsible for the improved resveratrol solubility and bioavailability of Resv@MDH.

A pool of peptides extracted from wheat bud chromatin inhibits tumor cell growth by causing defective DNA synthesis.

BACKGROUND: We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis. METHODS: BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively. RESULTS: BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected. CONCLUSION: The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.

Aging reversibility: from thymus graft to vegetable extract treatment-- application to cure an age-associated pathology.

Neonatal thymus graft and thymus calf extract (TME) in vivo treatment exert similar corrective actions on different mouse age-related alterations. The aim of the present paper is to investigate whether a vegetal extract, wheat sprout extract (WESPRE), could mimic the thymus action on recovering age-related alterations and if this extract can cure an age-associated pathology, the cataract in dogs. Present experiments were carried out by using WESPRE and TME in vivo in old mice to check their ability to recover the altered DNA synthesis in hepatocyte primary cultures. Old mice treated with WESPRE and TME showed a recovery of hepatocyte DNA synthesis levels when compared with the old untreated ones. The increase of DNA and protein contents observed in aged animals is reduced by WESPRE treatments to levels observed in young mice hepatocytes. We measured also WESPRE phosphorylation activity by endogenous kinase: it was from 10 to 40 times higher with respect to wheat seeds. Old dogs were orally treated for a month and the lens opacity analysed before and after the treatment. Results showed a reduction from 25 to 40% of lens opacity. The efficacy of wheat sprouts in the recovery of age-related alterations and in treating age-associated pathologies could be due to the contemporary presence of small regulatory acid peptides, a remarkable level of highly energetic phosphoric radicals and antioxidant molecules, peculiarities that may be, to some extent, related to the aging process regulation.