Inositols in the ovaries: activities and potential therapeutic applications.

INTRODUCTION: Myo-inositol (MI) and d-chiro-inositol (DCI) play a key role in ovarian physiology, as they are second messengers of insulin and gonadotropins. Ex-vivo and in-vitro experiments demonstrate that both isomers are deeply involved in steroid biosynthesis, and that reduced MI-to-DCI ratios are associated with pathological imbalance of sex hormones. AREAS COVERED: This expert opinion provides an overview of the physiological distribution of MI and DCI in the ovarian tissues, and a thorough insight of their involvement into ovarian steroidogenesis. Insulin resistance and compensatory hyperinsulinemia dramatically reduce the MI-to-DCI ratio in the ovaries, leading to gynecological disorders characterized by hyperandrogenism, altered menstrual cycle and infertility. EXPERT OPINION: Available evidence indicates that MI and DCI have very specific physiological roles and, seemingly, physiological MI-to-DCI ratios in the ovaries are crucial to maintain the correct homeostasis of steroids. Inositol treatments should be evaluated on the patients' specific conditions and needs, as long-term supplementation of high doses of DCI may cause detrimental effects on the ovarian functionality. In addition, the effects of inositol therapy on the different PCOS phenotypes should be further investigated in order to better tailor the supplementation.

Inositols: From Established Knowledge to Novel Approaches.

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

Role of inositol to improve surfactant functions and reduce IL-6 levels: A potential adjuvant strategy for SARS-CoV-2 pneumonia?

To date, the spread of SARS-CoV-2 infection is increasing worldwide and represents a primary healthcare emergency. Although the infection can be asymptomatic, several cases develop severe pneumonia and acute respiratory distress syndrome (ARDS) characterized by high levels of pro-inflammatory cytokines, primarily interleukin (IL)-6. Based on available data, the severity of ARDS and serum levels of IL-6 are key determinants for the prognosis. In this scenario, available in vitro and in vivo data suggested that myo-inositol is able to increase the synthesis and function of the surfactant phosphatidylinositol, acting on the phosphoinositide 3-kinase (PI3K)-regulated signaling, with amelioration of both immune system and oxygenation at the bronchoalveolar level. In addition, myo-inositol has been found able to decrease the levels of IL-6 in several experimental settings, due to an effect on the inositol-requiring enzyme 1 (IRE1)-X-box-binding protein 1 (XBP1) and on the signal transducer and activator of transcription 3 (STAT3) pathways. In this scenario, treatment with myo-inositol may be able to reduce IL-6 dependent inflammatory response and improve oxygenation in patients with severe ARDS by SARS-CoV-2. In addition, the action of myo-inositol on IRE1 endonuclease activity may also inhibit the replication of SARS-CoV-2, as was reported for the respiratory syncytial virus. Since the available data are extremely limited, if this potential therapeutic approach will be considered valid in the clinical practice, the necessary future investigations should aim to identify the best dose, administration route (oral, intravenous and/or aerosol nebulization), and cluster(s) of patients which may get beneficial effects from this treatment.

Experts' opinion on inositols in treating polycystic ovary syndrome and non-insulin dependent diabetes mellitus: a further help for human reproduction and beyond.

Introduction: This Experts' opinion provides an updated scientific support to gynecologists, obstetricians, endocrinologists, nutritionists, neurologists and general practitioners on the use of Inositols in the therapy of Polycystic Ovary Syndrome (PCOS) and non-insulin dependent (type 2) diabetes mellitus (NIDDM).Areas covered: This paper summarizes the physiology of Myo-Inositol (MI) and D-Chiro-Inositol (DCI), two important molecules present in human organisms, and their therapeutic role, also for treating infertility. Some deep differences between the physiological functions of MI and DCI, as well as their safety and intestinal absorption are discussed. Updates include new evidence on the efficacy exerted in PCOS by the 40:1 MI/DCI ratio, and the innovative approach based on alpha-lactalbumin to overcome the decreased therapeutic efficacy of Inositols in some patients.Expert opinion: The evidence suggests that MI, alone or with DCI in the 40:1 ratio, offers a promising treatment for PCOS and NIDDM. However, additional studies need to evaluate some still unresolved issues, such as the best MI/DCI ratio for treating NIDDM, the potential cost-effectiveness of reduced gonadotropins administration in IVF due to MI treatment, or the benefit of MI supplementation in ovulation induction with clomiphene citrate in PCOS patients.

Myo-inositol and D-chiro-inositol (40:1) reverse histological and functional features of polycystic ovary syndrome in a mouse model.

Mice exposed to continuous light undergo functional and histological changes that mimic those of human Polycystic Ovary Syndrome (PCOS). We herein induced the syndrome by exposing 30-day-old females to 10 weeks of permanent light. Ovarian morphology and histology, as well as reproductive parameters (time of observed pregnancy/delivery) were investigated. Ovaries of PCOS-modeled mice showed lack of tertiary follicles and corpora lutea, altered ovarian architecture, and increased thickness of the theca layer. When mice were returned to a normal light-dark regimen for 10 days, a slight, spontaneous improvement occurred, whereas a quick and almost complete recovery from PCOS signs and symptoms was obtained by treating animals with a daily supplementation of 420 mg/kg myo-inositol and D-chiro-inositol (MyoIns/DCIns) in a 40:1 molar ratio. Namely, ovaries from mice treated by this protocol recovered normal histological features and a proper ratio of theca/granulosa cell layer thickness (TGR), suggesting that the androgenic phenotype was efficiently reversed. Indeed, we identified TGR as a useful index of PCOS, as its increase in PCOS-modeled mice correlated linearly with reduced reproductive capability ( r = 0.75, p < 0.0001). Mice treated with a 40:1 formula regained low TGR values and faster recovery of their fertility, with a physiological delivery time after mating. On the other hand, a higher D-chiro-inositol treatment formula, such as MyoIns versus DCIns 5:1, was ineffective or even had a negative effect on clinical-pathological outcomes.

Alpha-Lipoic Acid Downregulates IL-1ß and IL-6 by DNA Hypermethylation in SK-N-BE Neuroblastoma Cells.

Alpha-lipoic acid (ALA) is a pleiotropic molecule with antioxidant and anti-inflammatory properties, of which the effects are exerted through the modulation of NF-kB. This nuclear factor, in fact, modulates different inflammatory cytokines, including IL-1b and IL-6, in different tissues and cell types. We recently showed that IL-1b and IL-6 DNA methylation is modulated in the brain of Alzheimer's disease patients, and that IL-1b expression is associated to DNA methylation in the brain of patients with tuberous sclerosis complex. These results prompted us to ask whether ALA-induced repression of IL-1b and IL-6 was dependent on DNA methylation. Therefore, we profiled DNA methylation in the 5'-flanking region of the two aforementioned genes in SK-N-BE human neuroblastoma cells cultured in presence of ALA 0.5 mM. Our experimental data pointed out that the two promoters are hypermethylated in cells supplemented with ALA, both at CpG and non-CpG sites. Moreover, the observed hypermethylation is associated with decreased mRNA expression and decreased cytokine release. These results reinforce previous findings indicating that IL-1b and IL-6 undergo DNA methylation-dependent modulation in neural models and pave the road to study the epigenetic mechanisms triggered by ALA.

Myo-Inositol Safety in Pregnancy: From Preimplantation Development to Newborn Animals.

Myo-inositol (myo-Ins) has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction. We have previously shown that mouse embryo exposure to myo-Ins through preimplantation development in vitro increases proliferation activity and blastocyst production, representing an improvement in culture conditions. We have herein investigated biochemical mechanisms elicited by myo-Ins in preimplantation embryos and evaluated myo-Ins effects on postimplantation/postnatal development. To this end naturally fertilized embryos were cultured in vitro to blastocyst in the presence or absence of myo-Ins and analyzed for activation of the PKB/Akt pathway, known to modulate proliferation/survival cellular processes. In parallel, blastocyst-stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning. Results obtained provide evidence that myo-Ins induces cellular pathways involving Akt and show that (a) exposure of preimplantation embryos to myo-Ins increases the number of blastocysts available for uterine transfer and of delivered animals and (b) the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo-Ins, up to three weeks of age, overlap. These data further identify myo-Ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology.

Results from the International Consensus Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS.

In recent years, interest has been focused to the study of the two major inositol stereoisomers: myo-inositol (MI) and d-chiro-inositol (DCI), because of their involvement, as second messengers of insulin, in several insulin-dependent processes, such as metabolic syndrome and polycystic ovary syndrome. Although these molecules have different functions, very often their roles have been confused, while the meaning of several observations still needs to be interpreted under a more rigorous physiological framework. With the aim of clarifying this issue, the 2013 International Consensus Conference on MI and DCI in Obstetrics and Gynecology identified opinion leaders in all fields related to this area of research. They examined seminal experimental papers and randomized clinical trials reporting the role and the use of inositol(s) in clinical practice. The main topics were the relation between inositol(s) and metabolic syndrome, polycystic ovary syndrome (with a focus on both metabolic and reproductive aspects), congenital anomalies, gestational diabetes. Clinical trials demonstrated that inositol(s) supplementation could fruitfully affect different pathophysiological aspects of disorders pertaining Obstetrics and Gynecology. The treatment of PCOS women as well as the prevention of GDM seem those clinical conditions which take more advantages from MI supplementation, when used at a dose of 2g twice/day. The clinical experience with MI is largely superior to the one with DCI. However, the existence of tissue-specific ratios, namely in the ovary, has prompted researchers to recently develop a treatment based on both molecules in the proportion of 40 (MI) to 1 (DCI).

Quercetin affects Hsp70/IRE1α mediated protection from death induced by endoplasmic reticulum stress.

Relative to their normal counterparts, tumor cells generally exhibit a greater "stress phenotype" and express heat shock proteins (Hsp) that represent candidate targets for anticancer therapy. Here we investigated the role of Hsp70 in survival induced by endoplasmic reticulum (ER) stressors in human leukemia U937 cells. Quercetin, a major dietary flavonoid, or specific silencing affected the expression level of Hsp70 and did not allow the upregulation of inositol-requiring kinase 1α (IRE1α), the prototype ER stress sensor regulating the unfolded protein response (UPR), that protects the cells against the stress of misfolded proteins in the ER. The reduction of Hsp70 prevented the upregulation of immunoglobulin heavy-chain binding protein (BiP), but not of CCAAT/enhancer-binding protein-homologous protein (CHOP), and induced apoptosis. Also specific silencing of IRE1α or inhibition of its endoribonuclease activity by 4µ8c hampered the upregulation of BiP, but not of CHOP, and induced apoptosis. These results suggest that drugs affecting the Hsp70-IRE1α axis, like quercetin, or affecting directly IRE1α may represent an effective adjuvant antileukemia therapy.

Soft gel capsules improve melatonin's bioavailability in humans.

OBJECTIVE: Oral bioavailability is one of the most important properties in drug design and development. A poor oral bioavailability can result in low efficacy and unpredictable response to a drug. Several dosages of melatonin have been used for various investigations to clarify its bioavailability in humans. Aiming to search for a pharmaceutical form, which is better absorbed, the pharmacokinetic (PK) profile of the new manufactured melatonin soft gelatin (soft gel) capsule form has been evaluated and compared with the commercially available melatonin powder. RESEARCH DESIGN AND METHODS: A total of 60 healthy volunteers received 1, 3 mg of melatonin powder and 1 mg of melatonin in soft gel capsules. PK profiles were obtained by analysis of melatonin plasma concentration, and the respective melatonin bioavailability was compared. RESULTS: Melatonin soft gel capsule form showed similar PK parameters compared with the highest doses of melatonin in powder form, but its bioavailability was improved. CONCLUSIONS: Soft gel capsules improved the bioavailability of melatonin in humans even when administered dose was reduced. Considering the number of conditions in which melatonin supplementation is recommended, this evidence could support a broader use of melatonin in clinical practice.

The rationale of the myo-inositol and D-chiro-inositol combined treatment for polycystic ovary syndrome.

PCOS is one of the most common endocrine disorders affecting women and it is characterized by a combination of hyper-androgenism, chronic anovulation, and insulin resistance. While a significant progress has recently been made in the diagnosis for PCOS, the optimal infertility treatment remains to be determined. Two inositol isomers, myo-inositol (MI) and D-chiro-inositol (DCI) have been proven to be effective in PCOS treatment, by improving insulin resistance, serum androgen levels and many features of the metabolic syndrome. However, DCI alone, mostly when it is administered at high dosage, negatively affects oocyte quality, whereas the association MI/DCI, in a combination reproducing the plasma physiological ratio (40:1), represents a promising alternative in achieving better clinical results, by counteracting PCOS at both systemic and ovary level.

Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion.

BACKGROUND AND AIM: Breast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion. METHODS: After MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, ß-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. ß-Catenin localization was observed by confocal microscopy. RESULTS: We observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering ß-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9. CONCLUSIONS: These results make GSE a powerful candidate for developing preventive agents against cancer metastasis.

Nicotine increases survival in human colon cancer cells treated with chemotherapeutic drugs.

Cigarette smoking is implicated in the development of colon cancer. Furthermore, nicotine increases cell proliferation and inhibits apoptosis through α7-nicotinic acetylcholine receptor (α7-nAChR) activation in human colon carcinoma cells. An open issue is whether nicotine interfere with colorectal cancer pharmacological treatment, by inhibiting drug-mediated apoptosis. To assess this hypothesis, we evaluated nicotine effect on Caco-2 and HCT-8 colon cancer cells, treated with 5-Fluorouracil (5-FU) and Camptothecin (CPT), chemotherapeutics commonly utilized as adjuvant treatment of colon cancer. Nicotine decreased anti-proliferative and pro-apoptotic effects exerted by chemotherapeutics on both cell lines. These effects partially reverted by exposure to α-bungarotoxin (α-BTX), an inhibitor of α7-nAChR. Nicotine addition to Caco-2 and HCT-8, treated with 5-FU or CPT, decreased the cleavage of substrate of caspase 3 and 7, poly-ADP-ribose polymerase (PARP). Moreover, P-ERK/ERK ratio was modified by nicotine addition to 5-FU and CPT treated cells in an opposite manner. However, when co-administrating PD98059, an ERK phosphorylation inhibitor, an increased apoptosis was observed. In Caco-2 and HCT-8 nicotine reverted 5-FU and CPT apoptotic effects through AKT phosphorylation, as demonstrated by apoptotic increase in presence of LY294002, an AKT phosphorylation inhibitor. Nicotine interfered with colorectal cancer pharmacological treatment in vitro by inhibiting apoptosis induced by chemotherapeutic drugs. Nicotine anti-apoptotic effects were exerted through ERK and AKT pathway activation.

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

Antiproliferative and apoptotic effects triggered by Grape Seed Extract (GSE) versus epigallocatechin and procyanidins on colon cancer cell lines.

Grape seed extract has been proven to exert anticancer effects on different tumors. These effects are mainly ascribed to catechin and procyanidin content. Analytical studies demonstrated that grape seed extract composition is complex and it is likely other components could exert biological activities. Using cell count and flow cytometry assays, we evaluated the cytostatic and apoptotic effects produced by three different grape seed extracts from Italia, Palieri and Red Globe cultivars, on Caco2 and HCT-8 colon cancer cells. These effects were compared to those induced by epigallocatechin and procyanidins, alone or in association, on the same cell lines. All the extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells, along the intrinsic apoptotic pathway. On both cell lines, growth inhibition induced by Italia and Palieri grape seed extracts was significantly higher than that it has been recorded with epigallocatechin, procyanidins and their association. In Caco2 cells, the extract from Red Globe cultivar was less effective in inducing growth inhibition than procyanidins alone and in association with epigallocatechin, whereas, in HCT-8 cells, only the association of epigallocatechin and procyanidins triggers a significant proliferation decrease. On both cell lines, apoptosis induced by Italia, Palieri and Red Globe grape seed extracts was considerably higher than has been recorded with epigallocatechin, procyanidins and their association. These data support the hypothesis by which other compounds, present in the grape seed extracts, are likely to enhance the anticancer effects.

Apoptosis-inducing factor and caspase-dependent apoptotic pathways triggered by different grape seed extracts on human colon cancer cell line Caco-2.

Consumption of grape seed extract (GSE) is widely marketed as a dietary supplement and is considered safe for human health. Nevertheless, the analytical composition of GSE from different grape cultivars, growing in special agronomic constraints, differs greatly in flavan-3-ols content. The major concern with GSE studies is a lack of availability of uniformly standardised preparations, which raises an important question whether different GSE samples have comparable activity and trigger the same mechanisms of action on a given biological system. Therefore, it is tempting to speculate that GSE, obtained from different cultivars, could exert differentiated anticancer effects. The focus of the present study is to determine the selective biological efficacy of GSE obtained from three different sources on the human colon cancer cell line Caco-2. Irrespective of its source, high doses of GSE induced a significant inhibition on Caco-2 cell growth. Moreover, apoptosis was enhanced through both caspase-dependent and caspase-independent mechanisms, leading to an early apoptosis-inducing factor release and, further, to a dramatic increase in caspase 7 and 3 activity. However, a significant difference in apoptotic rates induced by the three grape sources clearly emerged when treating cancer cells with low and intermediate GSE concentrations (25 and 50 microg/ml).

Melatonin and vitamin D3 increase TGF-beta1 release and induce growth inhibition in breast cancer cell cultures.

BACKGROUND: Evidence has accumulated that 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is involved in the regulation of the proliferation of breast tumor cells. For complete tumor suppression high hypercalcemic doses of 1,25-(OH)(2)D(3) are needed. The aim of this study was to assess the effect of combined treatment of 1,25-(OH)(2)D(3) at low doses and melatonin (MEL) on the proliferation of estrogen-responsive rat breast cancer cell line RM4. MATERIALS AND METHODS: RM4 cell proliferation was assessed by [3H]thymidine uptake. The presence of TGF-beta(1) in serum-free conditioned medium was determined by inhibition antibody binding assay. RESULTS: In 17-betaE cultured RM4 cells both MEL and 1,25-(OH)(2)D(3) alone and in combination significantly reduced [3H]thymidine incorporation in a dose-related fashion. MEL by itself was ineffective in inhibiting the FCS-cultured RM4 cells, while 1,25-(OH)(2)D(3) strongly inhibited [3H]thymidine incorporation. Meanwhile, MEL increased the sensitivity of the FCS-cultured RM4 cells to 1,25-(OH)(2)D(3) in the combined regimen, from 20- to 100-fold. MEL significantly enhanced the TGF-beta(1) secretion from RM4 cells and vitamin D(3) increased the TGF-beta(1) secretion in a dose-dependent manner, from 2- to 7-fold. Moreover, a further enhancement of the TGF-beta(1) release was obtained with the combined treatment, but only for low 1,25-(OH)(2)D(3) concentrations. The addition of monoclonal anti-TGF-beta(1) antibody to the medium of RM4 cells exposed to vitamin D(3) alone or in combination with MEL increased the [3H]thymidine uptake compared to the correspondent cells cultured without antibody. CONCLUSIONS: Our data point to a potential benefit of combination therapy with 1,25-(OH)(2)D(3) and MEL in the treatment of breast cancer and suggest that the growth inhibition could be related, at least in part, to the enhanced TGF-beta(1) secretion.