Oxidative Stress-Related Signaling Pathways Predict Oocytes' Fertilization In Vitro and Embryo Quality.

Oocyte development and fertilization are largely influenced by the microenvironment of the follicular fluid (FF), and the exploration of its molecular/metabolic composition may help in improving in vitro fertilization (IVF) outcomes. Here, the concentrations of molecules related to oxidative stress/inflammation were measured in FF from follicles at oocyte retrieval during IVF. Here, the FF antioxidant potential was correlated with the number of retrieved/mature oocytes and the number of fertilized ones. FF collected from the follicles of normal fertilized oocytes presented an elevated antioxidant capability, lower levels of pro-inflammatory molecules (i.e., IL-6, IL-8, IL-12, TGF-ß, and HIF-1α), and a higher IL-10 concentration. FF samples from follicles at oocyte retrieval that resulted in top-quality embryos displayed a peculiar antioxidant capability and a further decrease in proinflammatory molecules when compared with FF, giving rise to poor-quality embryos. Finally, pro-inflammatory molecules were lower and accompanied by a high antioxidant capability in samples giving rise to successful embryo implantation. The antioxidant capability and IL-10 displayed a good predictive ability for fertilization and embryo quality. Overall, our data showed the great influence of oxidative stress on the oocytes' fertilization, and shed light on the importance of controlling the inflammatory and oxidative status of FF to obtain good-quality embryos with significant implantation potential.

Endometrial Dysbiosis Is Related to Inflammatory Factors in Women with Repeated Implantation Failure: A Pilot Study.

An abnormal endometrial microbiota has been suggested to impair the process of embryo implantation, thus leading to repeated implantation failure (RIF) in women undergoing in vitro fertilization (IVF). However, the molecular mechanisms linking uterine microbiota and IVF out-comes are still an open question. The aim of this cohort study was to outline the relationship between endometrial microbiota, inflammation and IVF outcomes. To this purpose, endometrial microbiota and selected components of the "cytokine network" were analyzed in women presenting RIF and divided between eubiosis and dysbiosis groups, according to the percentage of endometrial lactobacilli (≥90% or <90%, respectively). The Dysbiosis group presented significantly higher tissue concentrations of the inflammatory markers (IL-6, IL-1ß, HIF-1α and COX-2) and significantly lower levels of the anti-inflammatory/well-being factors, IL-10 and IGF-1, with respect to women with eubiosis. Moreover, the Lactobacillus percentage was negatively related to the concentrations of the inflammatory molecules and positively related to IL-10/IGF-1. Interestingly, the number of IVF attempts was directly related to the levels of the inflammatory factors COX-2, IL-1ß and HIF-1α in the eubiosis group. Overall, endometrial dysbiosis was demonstrated to be associated with inflammation-related endometrial changes affecting the process of embryo implantation, underlining the importance of assessing uterine microbiota in patients undergoing IVF.

Exploring Epithelial-Mesenchymal Transition Signals in Endometriosis Diagnosis and In Vitro Fertilization Outcomes.

Endometriosis (EMS) pathogenesis has been related to the release of inflammatory mediators in peritoneal fluid, creating an altered microenvironment that leads to low-grade oocyte/embryos and to the reduction of implantation rates. The Epithelial-Mesenchymal Transition (EMT), an inflammation-related process, can be a further contributing factor to EMS. This study aimed to investigate, among various cytokines and EMT markers (Cadherins, TGF-ß, HIF-1α), diagnostic markers of EMS and prognostic factors of in vitro fertilization (IVF) outcomes. Herein, EMS patients manifested higher serum levels of the inflammatory molecules IL-6, IL-8, and IL-12 and a decrease in the concentrations of the anti-inflammatory IL-10. Moreover, biochemical markers associated with the EMT process were more elevated in serum and follicular fluid (FF) of EMS patients than in controls. At the end, the number of good-quality embryos was inversely related to serum IL-6 and EMT markers. Interestingly, serum IL-6 and FF IL-10 concentrations differentiated EMS patients from controls. Finally, serum IL-8 and E-Cadherin levels, as well as FF IL-10, predicted positive IVF outcome with great accuracy. Our data confirm the pivotal role of inflammatory mediators (i.e., IL-6 and IL-10) in EMS pathogenesis and suggest that EMT-related markers are elevated in EMS patients and can be predictive of IVF outcome.

D-Chiro-Inositol Regulates Insulin Signaling in Human Adipocytes.

D-Chiro-Inositol (D-Chiro-Ins) is a secondary messenger in the insulin signaling pathway. D-Chiro-Ins modulates insulin secretion, the mitochondrial respiratory chain, and glycogen storage. Due to these actions D-Chiro-Ins has been proposed to correct defective insulin function in a variety of conditions characterized by metabolic dysfunction, such as polycystic ovary syndrome (PCOS), obesity, gestational diabetes and fat accumulation at menopause. Since it is unclear whether D-Chiro-Ins directly acts on adipocytes, we aimed to study D-Chiro-Ins's actions on adipocyte viability, proliferation, differentiation, and insulin-related protein expression using a human adipocyte cell line derived from Simpson-Golabi-Behmel Syndrome (SGBS) which fully differentiates to mature adipocytes. Throughout differentiation, cells were treated with D-Chiro-Ins, 17ß-estradiol (E2) or Insulin. Cell viability and proliferation were not affected by D-Chiro-Ins, then D-Chiro-Ins promoted cell differentiation only during the final days of the process, while E2 enhanced it from the first phases. D-Chiro-Ins stimulated lipid storage and the production of big lipid droplets, thus reducing the content of free fatty acids. We also found that D-Chiro-Ins, either alone or in combination with insulin and E2 increased the expression and activation of insulin receptor substrate-1 (IRS1) and glucose transporter type 4 (GLUT4). In conclusion, this work shows that D-Chiro-Ins plays a direct role in the differentiation and in the function of human adipocytes, where it synergizes with insulin and estrogen through the recruitment of signal transduction pathways involved in lipid and glucose storage. These findings give clear insights to better understand the actions of D-Chiro-Ins on fat metabolism in women in physiology and in a variety of diseases.

Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment.

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.

The unveiling of the Warburg effect and the inscribed innovative approach to a radical non toxic anticancer therapy.

The purpose of this research has been deciphering the Warburg paradox, the biochemical enigma unsolved since 1923. We solved it by demonstrating that its specific character, i.e. the forced aerobic lactate exportation, represents a crucial metabolic device to counteract the cytotoxic effect produced by an excess of pyruvate at the connection of glycolysis with the Krebs cycle. This solution was verified by exposing cancer cells of different histogenesis to pyruvate concentrations higher than the physiological ones, after showing that these concentrations are totally innocuous when injected into mice. The mechanism of the pyruvate cytotoxicity relies on the saturation of the respiratory chain, leading to a negative shift of the cytosolic NADP/NADPH ratio and the consequent restriction of the purine synthesis and the related cell apoptosis. The reducing equivalents generated by glycolysis and by cytosolic metabolism compete each other for their disposal trough the respiratory chain; this makes it that the cytotoxicity of pyruvate is inversely related to the mitochondrial number and efficiency of various cell types. Thus, the cytotoxicity is high in anaplastic cancer stem cells, whose mitochondria are extremely few and immature (cristae-poor); on the contrary, no inhibition is brought about in adult differentiated cells, physiologically rich of mature mitochondria. All this generates the pyruvate anticancer selectivity, together with the lack of a general toxicity, making pyruvate represent an ideal candidate for a radical non toxical anticancer treatment.

Low-dose methotrexate enhances cycling of highly anaplastic cancer cells.

We previously showed that cellular RedOx state governs the G1-S transition of AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem cell stage. This transition is impaired when the mithocondrial electron transport system is blocked by specific inhibitors (antimycin A) or the respiratory chain is saturated by adding to the cells high concentrations of pyruvate. The antimycin A or pyruvate block is removed by the addition of adequate concentrations of folate (F). This suggests that the G1-S transition of AH130 cells depends on a respiration-linked step of DNA synthesis related to folate metabolism. In the study reported here, we characterized the effects of methotrexate (MTX), an inhibitor of dihydofolate-reductase, on the G1-S transition of hepatoma cells, in the absence or the presence of exogenously added F, dihydrofolate (FH2) or tetrahydrofolate (FH4). MTX, at 1 µM or higher concentrations, inhibited G1-S transition. This inhibition was completely removed by exogenous folates. Surprisingly, 10 nM MTX stimulated G1-S transition. The addition of F, but not FH2 or FH4, significantly increased this effect. Furthermore, 10 nM MTX removed the block of the G1-S transition operated by antimycin A or pyruvate, an effect which was enhanced in the presence of F. Finally, the stimulatory effect of 10 nM MTX was inhibited in the presence of serine. Our findings indicated that, under certain conditions, MTX may stimulate, rather than inhibiting, the cycling of cancer cells exhibiting a stem cell-like phenotype, such as AH130 cells. This may impact the therapeutic use of MTX and of folates as supportive care.

Chronic Myeloid Leukemia and Hepatoblastoma: Two Cancer Models to Link Metabolism to Stem Cells.

Low oxygen tension is a critical aspect of the stem cell niche where stem cells are long-term maintained. In "physiologically hypoxic" stem cell niches, low oxygen tension restrains the clonal expansion of stem cells without blocking their cycling, thereby contributing substantially to favor their self-renewal. The capacity of stem cells, hematopoietic stem cells in particular, to reside in low oxygen is likely due to their specific metabolic profile. A strong drive to the characterization of this profile emerges from the notion that cancer stem cells (CSC), like normal stem cells, most likely rely on metabolic cues for the balance between self-renewal/maintenance and clonal expansion/differentiation. Accordingly, CSC homing to low oxygen stem cell niches is the best candidate mechanism to sustain the so-called minimal residual disease. Thus, the metabolic profile of CSC impacts long-term cancer response to therapy. On that basis, strategies to target CSC are intensely sought as a means to eradicate neoplastic diseases. Our "metabolic" approach to this challenge was based on two different experimental models: (A) the Yoshida's ascites hepatoma AH130 cells, a highly homogeneous cancer cell population expressing stem cell features, used to identify, in CSC adapted to oxygen and/or nutrient shortage, metabolic features of potential therapeutic interest; (B) chronic myeloid leukemia, used to evaluate the impact of oxygen and/or nutrient shortage on the expression of an oncogenetic protein, the loss of which determines the refractoriness of CSC to oncogene-targeting therapies.

The complex metabolic network gearing the G1/S transition in leukemic stem cells: Hints to a rational use of antineoplastic agents.

We defined the stem cell profile of K562 line, demonstrating the expression of the Embryonic Transcription Factors Oct3/4, Sox2, Klf4 and Nanog. This profile was associated with a high vulnerability to the physiological oxidizable substrate pyruvate. remarkably, this substrate was shown to be innocuous, even at the highest doses, to normal differentiated cells. This vulnerability is based on a complex metabolic trim centered on the cellular redox state expressed by the NADP/NADPH ratio geared by the mitochondrial respiratory chain. Flow cytometry revealed that the inhibition of this chain by antimycin A produced cell accumulation in the S phase of cell cycle and apoptosis. This block negatively interferes with the aerobic synthesis of purines, without affecting the anaerobic synthesis of pyrimidines. This imbalance was reproduced by using two antifolate agents, LY309887 and raltitrexed (TDX), inhibitors of purine or pyrimidine synthesis, respectively. All this revealed the apparent paradox that low doses of TDX stimulated, instead of inhibiting, leukemia cell growth. This paradox might have significant impact on therapy with regard to the effects of TDX during the intervals of administration, when the drug concentrations become so low as to promote maintenance of dormant cancer cells in hypoxic tissue niches.

The metabolically-modulated stem cell niche: a dynamic scenario regulating cancer cell phenotype and resistance to therapy.

This Perspective addresses the interactions of cancer stem cells (CSC) with environment which result in the modulation of CSC metabolism, and thereby of CSC phenotype and resistance to therapy. We considered first as a model disease chronic myeloid leukemia (CML), which is triggered by a well-identified oncogenetic protein (BCR/Abl) and brilliantly treated with tyrosine kinase inhibitors (TKi). However, TKi are extremely effective in inducing remission of disease, but unable, in most cases, to prevent relapse. We demonstrated that the interference with cell metabolism (oxygen/glucose shortage) enriches cells exhibiting the leukemia stem cell (LSC) phenotype and, at the same time, suppresses BCR/Abl protein expression. These LSC are therefore refractory to the TKi Imatinib-mesylate, pointing to cell metabolism as an important factor controlling the onset of TKi-resistant minimal residual disease (MRD) of CML and the related relapse. Studies of solid neoplasias brought another player into the control of MRD, low tissue pH, which often parallels cancer growth and progression. Thus, a 3-party scenario emerged for the regulation of CSC/LSC maintenance, MRD induction and disease relapse: the "hypoxic" versus the "ischemic" vs. the "acidic" environment. As these environments are unlikely constrained within rigid borders, we named this model the "metabolically-modulated stem cell niche."

One more stem cell niche: how the sensitivity of chronic myeloid leukemia cells to imatinib mesylate is modulated within a "hypoxic" environment.

This is a review (by no means comprehensive) of how the stem cell niche evolved from an abstract concept to a complex system, implemented with a number of experimental data at the cellular and molecular levels, including metabolic cues, on which we focused in particular. The concept was introduced in 1978 to model bone marrow sites suited to host hematopoietic stem cells (HSCs) and favor their self-renewal, while restraining clonal expansion and commitment to differentiation. Studies of the effects of low oxygen tension on HSC maintenance in vitro led us to hypothesize niches were located within bone marrow areas where oxygen tension is lower than elsewhere. We named these areas hypoxic stem cell niches, although a low oxygen tension is to be considered physiological for the environment where HSCs are maintained. HSCs were later shown to have the option of cycling in low oxygen, which steers this cycling to the maintenance of stem cell potential. Cell subsets capable of withstanding incubation in very low oxygen were also detected within leukemia cell populations, including chronic myeloid leukemia (CML). The oncogenetic Bcr/Abl protein is completely suppressed in these subsets, whereas Bcr/Abl messenger ribonucleic acid is not, indicating that CML cells resistant to low oxygen are independent of Bcr/Abl for persistence in culture but remain genetically leukemic. Accordingly, leukemia stem cells of CML selected in low oxygen are refractory to the Bcr/Abl inhibitor imatinib mesylate. Bcr/Abl protein suppression turned out to be actually determined when glucose shortage complicated the effects of low oxygen, indicating that ischemia-like conditions are the driving force of leukemia stem cell refractoriness to imatinib mesylate. These studies pointed to "ischemic" stem cell niches as a novel scenario for the maintenance of minimal residual disease of CML. A possible functional relationship of the "ischemic" with the "hypoxic" stem cell niche is discussed.

Hypoxia-resistant profile implies vulnerability of cancer stem cells to physiological agents, which suggests new therapeutic targets.

We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH 4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.

The involvement of a Nanog, Klf4 and c-Myc transcriptional circuitry in the intertwining between neoplastic progression and reprogramming.

One undisputed milestone of traditional oncology is neoplastic progression, which consists of a progressive selection of dedifferentiated cells driven by a chance sequence of genetic mutations. Recently it has been demonstrated that the overexpression of well-defined transcription factors reprograms somatic cells to the pluripotent stem status. The demonstration raises crucial questions as to whether and to what extent this reprogramming contributes to tumorigenesis, and whether the epigenetic changes involved in it are reversible. Here, we show for the first time that a tumor produced in vivo by a chemical carcinogen is the product of the interaction between neoplastic progression and reprogramming. The experimental model employed the prototype of ascites tumors, the Yoshida AH130 hepatoma and other neoplasias, including human melanoma. AH130 hepatoma was started in the liver by the carcinogen o-aminoazotoluene. This compound binds to and abolishes the p53 protein, producing a genomic instability that promotes both the neoplastic progression and the hepatoma reprogramming. Eventually this tumor contained 100% CD133(+) elements and pO(2)-dependent percentages of the three embryonic transcription factors Nanog, Klf4 and c-Myc. Once transferred into aerobic cultures, the minor cellular fraction expressing this triad generates various types of adherent cells, which are progressively substituted by non-tumorigenic elements committed to fibromuscular, neuronal and glial differentiation. This reprogramming appears to be accomplished stepwise, with the assembly of the triad into a sophisticated transcriptional, oxygen-dependent circuitry, in which Nanog and Klf4 antagonistically regulate c-Myc, and hence, cell hypoxia survival and cell cycle activation.

Time- and residue-specific differences in histone acetylation induced by VPA and SAHA in AML1/ETO-positive leukemia cells.

We analyzed the activity of the histone deacetylase inhibitor (HDACi) suberoyl-anilide hydroxamic acid (SAHA) on Kasumi-1 acute myeloid leukemia (AML) cells expressing AML1/ETO. We also compared the effects of SAHA to those of valproic acid (VPA), a short-chain fatty acid HDACi. SAHA and VPA induced histone H3 and H4 acetylation, myeloid differentiation and massive early apoptosis. The latter effects were not determined by either drug in AML cell lines, such as NB4 or THP-1, not expressing AML1/ETO. SAHA was more rapid and effective than VPA in increasing H3 and H4 acetylation in total Kasumi-1 cell lysates and more effective than VPA in inducing acetylation of H4K8, H4K12, H4K16 residues. At the promoter of IL3, a transcriptionally-silenced target of AML1/ETO, SAHA was also more rapid than VPA in inducing total H4, H4K5, H4K8 and H3K27 acetylation, while VPA was more effective than SAHA at later times in inducing acetylation of total H4, H4K12, H4K16, as well as total H3. Consistent with these differences, SAHA induced the expression of IL3 mRNA more rapidly than VPA, while the effect of VPA was delayed. These differences might be exploited to design clinical trials specifically directed to AML subtypes characterized by constitutive HDAC activation. Our results led to include SAHA, an FDA-approved drug, among the HDACi active in the AML1/ETO-expressing AML cells.

Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment.

We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neuroblastoma clone. This protocol was based on the prolonged treatment of the wild-type cell population with either hypoxia or the antiblastic etoposide. Cell fate was monitored by immunocytochemical and electrophysiologic (patch-clamp) techniques. Both treatments produced the progressive disappearance of neuronal type (N) cells (which constitute the bulk of the tumor), leaving space for a special category of epithelial-like substrate-adherent cells (S(0)). The latter represent a minimal cell component of the untreated population and are endowed with immunocytochemical markers (p75, c-kit, and CD133) and the electrophysiologic "nude" profile, typical of the neural crest stem cells. S(0) cells displayed a highly clonogenic potency and a substantial plasticity, generating both the N component and an alternative subpopulation terminally committed to the fibromuscular lineage. Unlike the N component, this lineage was highly insensitive to the apoptotic activity of hypoxia and etoposide and developed only when the neuronal option was abolished. Under these conditions, the fibromuscular progeny of S(0) expanded and progressed up to the exhaustion of the staminal compartment and to the extinction of the tumor. When combined, hypoxia and etoposide cooperated in abolishing the N cell generation and promoting the conversion of the tumor described. This synergy might mirror a natural condition in the ischemic areas occurring in cancer. These results have relevant implications for the understanding of the documented tendency of neuroblastomas to regress from a malignant to a benign phenotype, either spontaneously or on antiblastic treatment.

Cell renewing in neuroblastoma: electrophysiological and immunocytochemical characterization of stem cells and derivatives.

We explored the stem cell compartment of the SH-SY5Y neuroblastoma (NB) clone and its development by a novel approach, integrating clonal and immunocytochemical investigations with patch-clamp measurements of ion currents simultaneously expressed on single cells. The currents selected were the triad IHERG, IKDR, INa, normally expressed at varying mutual ratios during development of neural crest stem cells, from which NB derives upon neoplastic transformation. These ratios could be used as electrophysiological clusters of differentiation (ECDs), identifying otherwise indistinguishable stages in maturation. Subcloning procedures allowed the isolation of highly clonogenic substrate-adherent (S-type) cells that proved to be p75- and nestinpositive and were characterized by a nude electrophysiological profile (ECDS0). These cells expressed negligible levels of the triad and manifested the capacity of generating the two following lineages: first, a terminally differentiating, smooth muscular lineage, positive for calponin and smooth muscle actin, whose electrophysiological profile is characterized by a progressive diminution of IHERG, the increase of IKDR and INa, and the acquisition of IKIR (ECDS2); second, a neuronal abortive pathway (NF-68 positive), characterized by a variable expression of IHERG and IKDR and a low expression of INa (ECDNS). This population manifested a vigorous amplification, monopolizing the stem cell compartment at the expense of the smooth muscular lineage to such an extent that neuronal-like (N-type) cells must be continuously removed if the latter are to develop.