Sara Staffolani

Clinical significance of circulating miR-126 quantification in malignant mesothelioma patients.

OBJECTIVES Aim of this study was to evaluate the accuracy and precision of the detection of individual miRNA as clinical biomarkers in the serum. DESIGN AND METHODS miRNA-126 was quantified in serum using endogenous and exogenous controls for normalization and the accuracy and precision of the method evaluated. The diagnostic value of serum miRNA-126 was evaluated in malignant mesothelioma (MM) and non-small-cell lung cancer (NSCLC) patients using both relative and absolute qRT-PCR methods. RESULTS The use of endogenous invariant and exogenous synthetic controls as well sample dilution markedly improves the accuracy and precision of the assay. The inter- and intra-assay analyses revealed that relative qRT-PCR is a more reliable method. Circulating miR-126 detected in the serum by relative qRT-PCRs was found low-expressed in both malignancies, significantly differentiated MM patients from healthy controls and NSCLC from MM, but do not discriminate NSCLC patients from control subjects. Kaplan-Meier analysis revealed that low level of circulating miR-126 in MM patients was strongly associated with worse prognosis. CONCLUSIONS We propose that this approach can be adopted for accurate analysis of other suitable circulating miRNA markers of different types of cancer.

Rotating-shift nurses after a day off: peripheral clock gene expression, urinary melatonin, and serum 17-β-estradiol levels.

OBJECTIVE Impairment of clock gene expression and changes in melatonin and 17-β-estradiol levels may constitute biological alterations underlying the increased risk of breast cancer among shift workers. The aim of this study was to compare levels of selected core clock gene expression, 6-sulfatoxymelatonin (aMT6s), and 17-β-estradiol between rotational shift work (SW) and daytime (DT) workers after a day off. METHODS The cross-sectional study comprised 60 nurses with ≥2 years of SW and 56 permanent DT nurses. Transcript levels of circadian genes BMAL1, CLOCK, NPAS2, CRY1, CRY2, PER1, PER2, PER3, and REVERBα were determined by quantitative real-time polymerase chain reaction (PCR) in lymphocytes. All participants were tested in the early follicular phase of the menstrual cycle. Samples were collected at the beginning of the morning-shift after a regular nights sleep on a day off. Chronotype and sociodemographic characteristics were also evaluated. RESULTS We found a significantly higher expression of BMAL1, CLOCK, NPAS2, PER1, PER2, and REVERBα and a lower expression of PER3, CRY1 and CRY2 among SW compared to DT nurses. SW participants did not demonstrate a significant difference in aMT6s levels, but they did show significantly higher 17-β-estradiol levels compared to DT nurses. Multiple linear regression analysis confirmed the role of SW on expression of BMAL1 (β 0.21, P=0.040), CLOCK (β 0.35, P=0.008), NPAS2 (β 0.30, P=0.012), PER1 (β 0.33, P=0.008), PER2 (β 0.19, P=0.047), PER3 (β -0.27, P=0.012), CRY1 (β -0.33, P=0.002), CRY2 (β -0.31, P=0.005), REVERBα (β 0.19, P=0.045), and on 17-β-estradiol levels (β 0.32, P=0.003). The analysis also confirmed the role of chronotype as an independent factor for PER1 (β 0.48, P=0.001) and PER2 (β -0.22, P=0.022) expression, and 17-β-estradiol levels (β 0.26, P=0.011). CONCLUSIONS Rotating SW nurses show alterations in peripheral clock gene expression and 17-β-estradiol levels at the beginning of the morning shift after a day off.

MicroRNA-126 Suppresses Mesothelioma Malignancy by Targeting IRS1 and Interfering with the Mitochondrial Function

AIMS MiR126 was found to be frequently lost in many types of cancer, including malignant mesothelioma (MM), which represents one of the most challenging neoplastic diseases. In this study, we investigated the potential tumor suppressor function of MiR126 in MM cells. The effect of MiR126 was examined in response to oxidative stress, aberrant mitochondrial function induced by inhibition of complex I, mitochondrial DNA (mtDNA) depletion, and hypoxia. RESULTS MiR126 was up-regulated by oxidative stress in nonmalignant mesothelial (Met5A) and MM (H28) cell lines. In Met5A cells, rotenone inhibited MiR126 expression, but mtDNA depletion and hypoxia up-regulated MiR126. However, these various stimuli suppressed the levels of MiR126 in H28 cells. MiR126 affected mitochondrial energy metabolism, reduced mitochondrial respiration, and promoted glycolysis in H28 cells. This metabolic shift, associated with insulin receptor substrate-1 (IRS1)-modulated ATP-citrate lyase deregulation, resulted in higher ATP and citrate production. These changes were linked to the down-regulation of IRS1 by ectopic MiR126, reducing Akt signaling and inhibiting cytosolic sequestration of Forkhead box O1 (FoxO1), which promoted the expression of genes involved in gluconeogenesis and oxidative stress defense. These metabolic changes induced hypoxia-inducible factor-1α (HIF1α) stabilization. Consequently, MiR126 suppressed the malignancy of MM cells in vitro, a notion corroborated by the failure of H28(MiR126) cells to form tumors in nude mice. INNOVATION AND CONCLUSION MiR126 affects mitochondrial energy metabolism, resulting in MM tumor suppression. Since MM is a fatal neoplastic disease with a few therapeutic options, this finding is of potential translational importance.

Circadian Modulation of 8-Oxoguanine DNA Damage Repair

The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours.

α-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate

Background:A strategy to reduce the secondary effects of anti-cancer agents is to potentiate the therapeutic effect by their combination. A combination of vitamin K3 (VK3) and ascorbic acid (AA) exhibited an anti-cancer synergistic effect, associated with extracellular production of H2O2 that promoted cell death.Methods:The redox-silent vitamin E analogue α-tocopheryl succinate (α-TOS) was used in combination with VK3 and AA to evaluate their effect on prostate cancer cells.Results:Prostate cancer cells were sensitive to α-TOS and VK3 treatment, but resistant to AA upto 3.2 mM. When combined, a synergistic effect was found for VK3–AA, whereas α-TOS–VK3 and α-TOS–AA combination showed an antagonist and additive effect, respectively. However, sub-lethal doses of AA–VK3 combination combined with a sub-toxic dose of α-TOS showed to induce efficient cell death that resembles autoschizis. Associated with this cell demise, lipid peroxidation, DNA damage, cytoskeleton alteration, lysosomal–mitochondrial perturbation, and release of cytochrome c without caspase activation were observed. Inhibition of lysosomal proteases did not attenuate cell death induced by the combined agents. Furthermore, cell deaths by apoptosis and autoschizis were detected.Conclusion:These finding support the emerging idea that synergistic combinations of some agents can overcome toxicity and other side-effects associated with high doses of single drugs creating the opportunity for therapeutically relevant selectivity.

Alpha-Tocopheryl Succinate Inhibits Autophagic Survival of Prostate Cancer Cells Induced by Vitamin K3 and Ascorbate to Trigger Cell Death

Background The redox-silent vitamin E analog α-tocopheryl succinate (α-TOS) was found to synergistically cooperate with vitamin K3 (VK3) plus ascorbic acid (AA) in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s) underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer. Methodology/Principal Findings The generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF) release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects. Conclusions/Significance α-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance.

Effect of ascorbic acid-rich diet on in vivo-induced oxidative stress.

Using hyperbaric oxygen (HBO) therapy as an in vivo oxidation model, we investigated the effect of a diet enriched in ascorbic acid (AA) on HBO-induced oxidative stress. Volunteers (n 46) were allocated to the AA-rich diet group or the control group. Blood samples were collected at the basal time, after the 1-week diet before and immediately after the HBO treatment, and 1 week after the HBO treatment. AA level, total antioxidant status (TAS), hydroperoxides (HP), lymphocyte DNA oxidation and DNA repair capacity were assessed. The expression of genes involved in oxidative stress was evaluated in lymphocytes and the protein activity of the modulated genes was determined in the plasma. The AA level and the antioxidant status of plasma were increased by AA-rich food consumption. HBO exposure did not affect the AA levels or TAS, but induced HP formation in the control group. The lymphocytes isolated from dietary-supplemented subjects were resistant to ex vivo DNA oxidation, showing an increased DNA repair capacity compared with controls. A difference in gene expression pattern was observed between the groups. AA-rich foods provide dual protection against oxidative stress, enhancing plasma antioxidant levels and stimulating genes involved in cell detoxification.

MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma

Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.

Circadian gene expression and extremely low-frequency magnetic fields: an in vitro study.

It is well known that circadian clocks are mainly regulated by light targeting signaling pathways in the hypothalamic suprachiasmatic nucleus. However, an entrainment mediated by non-photic sensory stimuli was also suggested for peripheral clocks. Exposure to extremely low frequency (ELF) electromagnetic fields might affect circadian rhythmicity. The goal of this research was to investigate effects of ELF magnetic fields (ELF-MF) on circadian clock genes in a human fibroblast cell line. We found that an ELF-MF (0.1 mT, 50 Hz) exposure was capable of entraining expression of clock genes BMAL1, PER2, PER3, CRY1, and CRY2. Moreover, ELF-MF treatment induced an alteration in circadian clock gene expression previously entrained by serum shock stimulation. These results support the hypothesis that ELF-MF may be able to drive circadian physiologic processes by modulating peripheral clock gene expression.

Angiogenic effect induced by mineral fibres

Due to the toxic effect of asbestos, other materials with similar chemical-physical characteristics have been introduced to substitute it. We evaluate the angiogenic effect of certain asbestos substitute fibres such as glass fibres (GFs), ceramic fibres (CFs) and wollastonite fibres (WFs) and then compare angiogenic responses to those induced by crocidolite asbestos fibres (AFs). An in vitro model using human endothelial cells in small islands within a culture matrix of fibroblasts (Angio-Kit) was used to evaluate vessel formation. The release of IL-6, sIL-R6, IL-8, VEGF-A and their soluble receptors, sVEGFR-1, sVEGFR-2, was determined in the conditioning medium of Angio-Kit system after fibre treatment. ROS formation and cell viability were evaluated in cultured endothelial cells (HUVEC). To evaluate the involvement of intracellular mechanisms, EGFR signalling, ROS formation and nuclear factor-κB (NFκB) pathway were then inhibited by incubating HUVEC cells with AG1478, NAC and PDTC respectively, and the cytokine and growth factor release was analyzed in the culture medium after 7 days of fibre incubation. Among the mineral fibres tested, WFs markedly induced blood vessel formation which was associated with release of IL-6 and IL-8, VEGF-A and their soluble receptors. ROS production was observed in HUVEC after WFs treatment which was associated with cell cytotoxicity. The EGFR-induced ERK phosphorylation and ROS-mediated NFκB activation were involved in the cytokine and angiogenic factor release. However, only the EGFR activation was able to induce angiogenesis. The WFs are potential angiogenic agents that can induce regenerative cytokine and angiogenic factor production resulting in the formation of new blood vessels.