Fausto Moroni

Melatonin provokes cell death in human B‐lymphoma cells by mitochondrial‐dependent apoptotic pathway activation

Abstract:  Apoptosis is an important cell suicide programme involved in physiological and pathological processes. Apoptosis can be induced in different ways depending on cell type and acquired signal. Melatonin, the major secretory product of the pineal gland, participates in many important physiological functions and displays a remarkable functional versatility exhibiting antioxidant, oncostatic, anti‐aging, and immunomodulatory properties. Recently, it has been shown that, in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that may be involved in the regulation of the immune system. In this work, we examine the effect of melatonin on RAMOS‐1 human leukaemic cells. Cell growth and viability, DNA fragmentation and JC‐1, and annexin V expression have been determined. To elucidate the mechanism of action of melatonin, Western blot analyses for Bcl‐2 and caspase‐3 expression, and cytochrome c release were carried out. The results suggest that the apoptotic effect of melatonin is associated with cell‐cycle arrest, downregulation of Bcl‐2, mitochondrial membrane depolarization, cytochrome c release and activation of caspase‐3. The intrinsic (mitochondrial dependent) pathway of caspase activation is the ‘point of no return’ commitment to cell death. Taken together, our study indicates that melatonin may play a role as potential therapeutic drug in specific lymphoproliferative diseases.

Food restriction in female Wistar rats: V. Lipid peroxidation and antioxidant enzymes in the liver

The activities of antioxidant enzymes as well as the levels of basal and enzyme induced peroxidation have been investigated in liver of female Wistar undernourished rats. Food restriction was applied starting from the age of 3.5 months by feeding the animals on every-other-day schedule (EOD). Diet restriction prevented the age-dependent increase of basal and enzyme induced lipid peroxidation in both mitochondrial and microsomal liver membrane preparations. The activities of antioxidant enzyme, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) of liver decreased during aging in ad libitum fed rats. In the diet conditioned animals, a small increase of SOD and a complete recovery of CAT activities were observed. Present data support that food restriction improved the protection against peroxidation, and this may be in close relationship with the life prolonging effect of such a treatment.

Interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool in very old age.

Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative burst and phagocytosis with subsequent less capability to destroy bacteria. In successful ageing (nonagenarians), neutrophil efficiency (phagocytosis) increases. After ingested microbes, aged neutrophils are less prone to undergo apoptosis favouring chronic inflammation. Moreover, the superoxide dismutase (SOD) activity, which is necessary in avoiding ROS produced by oxidative burst, is limited in ageing. The mechanisms of age-related changes in neutrophil function are not fully understood, taking also into account that nonagenarians escape infections in comparison with elderly. Zinc pool may be involved because it is pivotal for neutrophil efficiency and SOD activity. Since zinc also controls the inflammation, via IL-6 and soluble factor of gp130 (sgp130), we have assessed the possible interrelationship among oxidative burst, apoptosis, inflammation, SOD, adhesion molecule Mac-1 and zinc pool in elderly and in nonagenarians. The oxidative burst and the capacity to increase Mac-1 after PMA stimulation decrease both in elderly and nonagenarians, but the latter display a slight increased neutrophil induced apoptosis, decreased sgp130, increased SOD, and more neutrophil zinc content, as it occurs in young-adults. Significant correlation exists between sgp130 and zinc pool in very old age. These findings suggest lower chronic inflammation in nonagenarians, via more zinc available, with subsequent long-life survival. Therefore, a more correct interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool exists in successful ageing with subsequent more effectiveness to control the inflammatory response to pathogens.

Age-dependent modifications of mitochondrial trans-membrane potential and mass in rat splenic lymphocytes during proliferation

The specific fluorescent probes, Rhodamine 123 (Rh-123) and Nonyl-Acridine Orange (NAO) were, respectively, used to monitor the changes in membrane potential and mass of lymphocyte mitochondria during aging and proliferation. An age-dependent increase of the uptake of both fluorochromes was observed in resting cells; however, NAO fluorescence increased to a greater extent when compared with the Rh-123 probe. This resulted in a lower respiratory activity per unit of mitochondrial mass in old cells than in the young ones. Following mitogenic stimulation, most of the lymphocytes from young rats showed an increase in their membrane potential and mass. On the contrary about 50% of cells from old rats had depolarized mitochondria after 72 h from the stimulation. Present data support that mitochondria of lymphocytes from old rats are extremely sensitive to the stressing conditions resulting from mitogenic stimulation.

Melatonin regulates the respiratory burst of human neutrophils and their depolarization

Pieri C, Recchioni R, Moroni F, Marcheselli F, Marra M, Marinoni Silvia, Di Primio R. Melatonin regulates the respiratory burst of human neutrophils and their depolarization. J. Pineal Res. 1998; 24:43–49.

Ligand and voltage gated sodium channels may regulate electrogenic pump activity in human, mouse and rat lymphocytes

Bretylium tosylate - a sodium channel opener - resulted in an increase of membrane potential of depolarized human, rat and mouse T and B lymphocytes. Flow cytometric membrane potential measurements with bis-oxonol revealed that the above hyperpolarizing effect was amiloride, ouabain, tetrodotoxin, azide and temperature sensitive. The effect showed an absolute dependence on the extracellular sodium but it was insensitive to the extracellular Ca2+ level. The voltage gating of the effect can be eliminated by either an increase of the extracellular potassium concentration or low doses of veratrin. The existence of a voltage and ligand gated sodium channel is suggested in the plasma membrane of all kinds of lymphocytes. The hyperpolarization is explained by an increased activity of the electrogenic sodium-potassium ATP-ase. Induced opening of such sodium channels may regulate the electrogenic pump activity and indirectly cell activation.

Glutathione influences the proliferation as well as the extent of mitochondrial activation in rat splenocytes

The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augmented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization.

Food restriction in female Wistar rats. I. Survival characteristics, membrane microviscosity and proliferative response in lymphocytes.

The effect of food restriction on the survival characteristics, membrane microviscosity and proliferative response in lymphocytes of female Wistar undernourished rats has been evaluated. Diet restriction was applied starting from the age of 3.5 months by feeding the animals on an every-other-day schedule (EOD). Diet restricted animals showed an increase of both mean, median and maximal life span as compared to the rats fed ad libitum (AL). Analyzing the survival curves by a parametric model, it emerged that undernutrition increased the individual resistance to environmental insults. In particular, it could be speculated that the positive influence was more pronounced in individuals with the lowest physiological capacities. The membrane microviscosity of lymphocytes was lower in EOD animals as compared to the AL ones even if one assumes a decrease in body temperature of 1-2 degrees C in EOD groups. The improvement of membrane microviscosity due to diet restriction may in part explain the improvement of proliferative response of lymphocytes from EOD groups.

Platelet as a physiological model to investigate apoptotic mechanisms in Alzheimer β-amyloid peptide production

Although neuronal apoptosis in Alzheimers disease is generally interpreted as the consequence of the toxicity of extracellular beta-amyloid (Abeta) peptide aggregates, some experimental results provide evidence that the Abeta overproduction can be the result of a primary neuronal degeneration. As platelets are considered a good model where to study proteolytic processing of the amyloid precursor protein (APP), we exposed platelets to the proapoptotic agent ionomycin and analyzed Abeta40 and Abeta42 levels in the intracellular and extracellular compartments. The activation of apoptotic pathways in platelets has been verified by mitochondrial membrane depolarization, exposure of phosphatidylserine, protease activation and morphological changes. A significative increase in intraplatelet Abeta40, but not Abeta42, was observed after 10 min treatment with ionomycin. Thus, the activation of apoptotic pathways in platelets determines an altered processing of APP leading to elevated levels of intracellular Abeta40. The specific intracellular production of Abeta40 represents a potential threat to the cells since very high local Abeta40 concentration increases the risk of its aggregation and toxicity. As a result, Abeta40 might be dangerous even before it becomes secreted rendering neurons highly vulnerable.

Crystalline silica induces apoptosis in human endothelial cells in vitro

We investigated whether incubation of cultured human aortic endothelial cells (HAEC) with crystalline silica at the concentration 1 cm2/ml (chosen on the basis of a pilot experiment) leads to alterations typical of apoptosis. The binding of annexin V as early, and DNA fragmentation as late events of apoptosis were measured besides the number of cells with depolarized mitochondria. The generation of reactive oxygen species (ROS) by HAEC in presence of silica was determined as well as silica ability to in vitro generate hydroxyl radicals was investigated. After 18 h of silica incubation, about 30% of viable cells bound annexin V. After 24 h of silica treatment, the percentage of cells with fragmented DNA (Tunel positive) was 27% and it increased up to 50% after 48 h, whereas in untreated cells this percentage was 7% and 11% after 24 and 48 h, respectively. The presence of fragmented DNA in cells treated with silica was confirmed by agarose gel electrophoresis. In agreement with these results showing an induction of HAEC apoptosis by silica incubation, the number of cells with depolarized mitochondria was significantly higher after silica treatment as compared to the control. Apoptosis was also obtained with silica added to aliquots of anti-C5a-absorbed-medium. In the cells exposed to silica there was a significant increasing of ROS generation in comparison to the untreated cells. Apoptosis might be due to peroxidative stress since silica can generate hydroxyl radicals.