Angela Maria Rizzo

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.

Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane.

BackgroundPUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells.MethodsTwo lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane.ResultsWe have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR.N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa.ConclusionsOur results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention.

Effect of omega-3 fatty acids supplementation on depressive symptoms and on health-related quality of life in the treatment of elderly women with depression: a double-blind, placebo-controlled, randomized clinical trial.

Objective: In elderly individuals, depression is one of the most frequently missed diagnoses with negative effects on quality of life. The authors investigated whether a supplement containing long-chain omega-3 polyunsaturated fatty acids (n-3 LCPUFA) improves depressive symptoms and health-related quality of life (HRQoL) in depressed elderly patients. Design: Eight-week, randomized, double-blind, placebo-controlled trial. Setting: Nursing home in Pavia, Italy. Participants: Forty-six depressed women, aged 66–95 years. Intervention: Twenty-two depressed women were included in the intervention group (n-3 group, which received 2.5 g/d of n-3 LCPUFA, with 1.67 g of eicosapentaenoic acid [EPA] and 0.83 g of docosahesaenoic acid [DHA]), and 24 patients were included in the placebo group. The primary endpoint was the improvement of depressive symptoms, as evaluated by the Geriatric Depression Scale (GDS). Secondary endpoints were the evaluation of HRQoL, by using the Short-Form 36-Item Health Survey (SF-36), and modifications of erythrocyte membrane phospholipids fatty acid profile. All variables were assessed before and after the treatment period of 8 weeks. Results: The mean GDS at 8 weeks was significantly lower compared with the n-3 group. The SF-36 physical and mental components were significantly increased in the intervention group. Compliance was good, as confirmed by erythrocyte membrane phospholipid FA concentrations, with a significant increase of EPA and DHA in the intervention group. Conclusion: Supplementation with n-3 LCPUFA is efficacious in the amelioration of depressive symptoms and quality of life in the treatment of depressed elderly female patients.

G-Quadruplex Ligand RHPS4 Potentiates the Antitumor Activity of Camptothecins in Preclinical Models of Solid Tumors

Purpose: The formation of G-quadruplex structures at telomeric DNA sequences blocks telomerase activity, offering an original strategy to design and develop new antitumor agents. The pentacyclic acridinium salt RHPS4 is one of the most effective and selective G4 ligands able to rapidly disrupt telomere architecture, resulting in apoptosis of cancer cells. Here, we studied the therapeutic index of RHPS4 and its integration with chemotherapeutics in preclinical model of solid tumors. Experimental Design: The antitumoral activity of RHPS4 was evaluated on human xenografts of different histotypes and compared with that of standard antineoplastic agents. Moreover, the effect of RHPS4/chemotherapeutics combinations on cell survival was studied and the most favorable combination was evaluated on tumor-bearing mice. Results: RHPS4 was active in vivo as single agent and showed a high therapeutic efficacy when compared with conventional drugs. Moreover, RHPS4 had antitumoral activity in human melanoma xenografts inherently resistant to chemotherapy and exhibited antimetastatic activity. RHPS4 also showed a strong synergistic interaction with camptothecins and this effect was strictly dependent on the drug sequence employed. Treatment of mice with irinotecan followed by RHPS4 was able to inhibit and delay tumor growth and to increase mice survival. Conclusions: Our data show that RHPS4 has a good pharmacodynamic profile and in combination therapy produces a strong antitumoral activity, identifying this drug as promising agent for clinical development.

Endogenous Antioxidants and Radical Scavengers

All living organisms are constantly exposed to oxidant agents deriving from both endogenous and exogenous sources capable to modify biomolecules and induce damages. Free radicals generated by oxidative stress exert an important role in the development of tissue damage and aging. Reactive species (RS) derived from oxygen (ROS) and nitrogen (RNS) pertain to free radicals family and are constituted by various forms of activated oxygen or nitrogen. RS are continuosly produced during normal physiological events but can be removed by antioxidant defence mechanism: the imbalance between RS and antioxidant defence mechanism leads to modifications in cellular membrane or intracellular molecules. In this chapter only endogenous antioxidant molecules will be critically discussed, such as Glutathione, Alpha-lipoic acid, Coenzyme Q, Ferritin, Uric acid, Bilirubin, Metallothioneine, L-carnitine and Melatonin.

Plasma, red blood cells phospholipids and clinical evaluation after long chain omega-3 supplementation in children with attention deficit hyperactivity disorder (ADHD)

Abstract Omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFAs), are crucial to brain development and function. Increasing evidence indicates that deficiencies or metabolic imbalances of these fatty acids might be associated with childhood developmental and psychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). Omega-3 are often lacking on modern diets. Moreover preliminary evidences suggest that supplementation with omega-3 LCPUFAs, might help in the management of the ADHD linked behavioural and learning difficulties. However, few studies published to date have involved different populations, study designs, treatments and outcome results. Thus, further researches are required to assess the durability of the treatment effects, to determine optimal composition and dosages of the supplement and to develop reliable ways to identify patients that might have some benefits from this kind of treatment, also because the study of LCPUFAs and their metabolism might offer new approaches to the early identification and management of ADHD. In this paper, we provide new insight on the lipid pattern in plasma and red blood cells (RBC) phospholipids, together with evaluation of the arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio which seems to correlate with the improvement of the patients both from a biochemical and clinical point of view.

Tardigrade Resistance to Space Effects: First Results of Experiments on the LIFE-TARSE Mission on FOTON-M3 (September 2007)

The Tardigrade Resistance to Space Effects (TARSE) project, part of the mission LIFE on FOTON-M3, analyzed the effects of the space environment on desiccated and active tardigrades. Four experiments were conducted in which the eutardigrade Macrobiotus richtersi was used as a model species. Desiccated (in leaf litter or on paper) and hydrated tardigrades (fed or starved) were flown on FOTON-M3 for 12 days in September 2007, which, for the first time, allowed for a comparison of the effects of the space environment on desiccated and on active animals. In this paper, we report the experimental design of the TARSE project and data on tardigrade survival. In addition, data on survival, genomic DNA integrity, Hsp70 and Hsp90 expressions, antioxidant enzyme contents and activities, and life history traits were compared between hydrated starved tardigrades flown in space and those maintained on Earth as a control. Microgravity and radiation had no effect on survival or DNA integrity of active tardigrades. Hsp expressions between the animals in space and the control animals on Earth were similar. Spaceflight induced an increase of glutathione content and its related enzymatic activities. Catalase and superoxide dismutase decreased with spaceflight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades molted, and females laid eggs. Several eggs hatched, and the newborns exhibited normal morphology and behavior.

Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation-tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs. hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. Both glutathione peroxidase and glutathione were induced by desiccation. The percentage of fatty acid composition of polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances are higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that the possession of antioxidant metabolism could represent a crucial strategy to avoid damages during desiccation in anhydrobiotic tardigrades.

Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition

BackgroundThe activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors.MethodsWe studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells.ResultsMDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.ConclusionsOur work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

Effects of Long-Term Space Flight on Erythrocytes and Oxidative Stress of Rodents

Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions; these observations have been designated as “space anemia”. Erythrocytes exposed to microgravity have a modified rheology and undergo hemolysis to a greater extent. Cell membrane composition plays an important role in determining erythrocyte resistance to mechanical stress and it is well known that membrane composition might be influenced by external events, such as hypothermia, hypoxia or gravitational strength variations. Moreover, an altered cell membrane composition, in particular in fatty acids, can cause a greater sensitivity to peroxidative stress, with increase in membrane fragility. Solar radiation or low wavelength electromagnetic radiations (such as gamma rays) from the Earth or the space environment can split water to generate the hydroxyl radical, very reactive at the site of its formation, which can initiate chain reactions leading to lipid peroxidation. These reactive free radicals can react with the non-radical molecules, leading to oxidative damage of lipids, proteins and DNA, etiologically associated with various diseases and morbidities such as cancer, cell degeneration, and inflammation. Indeed, radiation constitutes on of the most important hazard for humans during long-term space flights. With this background, we participated to the MDS tissue-sharing program performing analyses on mice erythrocytes flown on the ISS from August to November 2009. Our results indicate that space flight induced modifications in cell membrane composition and increase of lipid peroxidation products, in mouse erythrocytes. Moreover, antioxidant defenses in the flight erythrocytes were induced, with a significant increase of glutathione content as compared to both vivarium and ground control erythrocytes. Nonetheless, this induction was not sufficient to prevent damages caused by oxidative stress. Future experiments should provide information helpful to reduce the effects of oxidative stress exposure and space anemia, possibly by integrating appropriate dietary elements and natural compounds that could act as antioxidants.