Milena De Nicola

Ce3+ Ions Determine Redox-Dependent Anti-apoptotic Effect of Cerium Oxide Nanoparticles

Antioxidant therapy is the novel frontier to prevent and treat an impressive series of severe human diseases, and the search for adequate antioxidant drugs is fervent. Cerium oxide nanoparticles (nanoceria) are redox-active owing to the coexistence of Ce(3+) and Ce(4+) oxidation states and to the fact that Ce(3+) defects, and the compensating oxygen vacancies, are more abundant at the surface. Nanoceria particles exert outstanding antioxidant effects in vivo acting as well-tolerated anti-age and anti-inflammatory agents, potentially being innovative therapeutic tools. However, the biological antioxidant mechanisms are still unclear. Here, the analysis on two leukocyte cell lines undergoing apoptosis via redox-dependent or independent mechanisms revealed that the intracellular antioxidant effect is the direct cause of the anti-apoptotic and prosurvival effects of nanoceria. Doping with increasing concentrations of Sm(3+), which progressively decreased Ce(3+) without affecting oxygen vacancies, blunted these effects, demonstrating that Ce(3+)/Ce(4+) redox reactions are responsible for the outstanding biological properties of nanoceria.

Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2

Abstract:  We have recently shown that melatonin antagonizes damage‐induced apoptosis by interaction with the MT‐1/MT‐2 plasma membrane receptors. Here, we show that melatonin interferes with the intrinsic pathway of apoptosis at the mitochondrial level. In response to an apoptogenic stimulus, melatonin allows mitochondrial translocation of the pro‐apoptotic protein Bax, but it impairs its activation/dimerization The downstream apoptotic events, i.e. cytochrome c release, caspase 9 and 3 activation and nuclear vesiculation are equally impaired, indicating that melatonin interferes with Bax activation within mitochondria. Interestingly, we found that melatonin induces a strong re‐localization of Bcl‐2, the main Bax antagonist to mitochondria, suggesting that Bax activation may in fact be antagonized by Bcl‐2 at the mitochondrial level. Indeed, we inhibit the melatonin anti‐apoptotic effect (i) by silencing Bcl‐2 with small interfering RNAs, or with small‐molecular inhibitors targeted at the BH3 binding pocket in Bcl‐2 (i.e. the one interacting with Bax); and (ii) by inhibiting melatonin‐induced Bcl‐2 mitochondrial re‐localization with the MT1/MT2 receptor antagonist luzindole. This evidence provides a mechanism that may explain how melatonin through interaction with the MT1/MT2 receptors, elicits a pathway that interferes with the Bcl‐2 family, thus modulating the cell life/death balance.

Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells

Abstract:  Among the non‐neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin‐specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT1, MT2); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonins antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca2+ influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.

Rapid and transient stimulation of intracellular reactive oxygen species by melatonin in normal and tumor leukocytes

Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (<1 min) and transient (up to 5-6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal or tumor leukocytes; intriguingly, ROS production does not lead to oxidative stress, as shown by absence of protein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROS production is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed to nanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is not mimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulin inhibitor shown to prevent melatonin-calmodulin interaction, also prevents melatonin pro-radical effect, suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROS production.

Glutathione depletion up-regulates Bcl-2 in BSO-resistant cells

Glutathione depletion by inhibition of its synthesis with buthionine sulfoximine (BSO) is a focus of the current research in antitumor therapy, BSO being used as chemosensitizer. We had previously shown that two human tumor cell lines (U937 and HepG2) survive to treatment with BSO: BSO can elicit an apoptotic response, but the apoptotic process is aborted after cytochrome c release and before caspase activation, suggesting the development of an adaptive response (FASEB J., 1999, 13, 2031–2036). Here, we investigate the mechanisms of such an adaptation. We found that following BSO, U937 up‐regulate Bcl‐2 mRNA and protein levels, by a mechanism possibly involving NF‐κB transcription factor; the increase in protein level is limited by a rapid decay of Bcl‐2 in BSO‐treated cells, suggesting that redox imbalance speeds up Bcl‐2 turnover. BSO‐dependent Bcl‐2 up‐regulation is associated with the ability to survive to BSO. Indeed, 1) its abrogation by CAPE or protein synthesis inhibition sensitizes U937 to BSO; 2) in a panel of four tumor lines, BSO‐resistant (U937, HepG2, and HGB1) but not BSO‐sensitive (BL41) cells can up‐regulate Bcl‐2 following GSH depletion; remarkably, only the latter are chemosensitized by BSO.

Pharmacological potential of bioactive engineered nanomaterials

In this study we present an overview of the recent results of a novel approach to antioxidant and anticancer therapies, consisting in the administration of intrinsically active nano-structured particles. Their particulate (as opposed to molecular) nature allows designing multifunctional platforms via the binding of molecular determinants, including targeting molecules and chemotherapy drugs, thereby facilitating their localization at the desired site. The intrinsic activity of nanomaterials with pharmacological potential include peculiar trans-excitation reactions that render them able to transform radiofrequency, UV, visible or infrared radiations into cytocidal reactive oxygen species or heat, thereby inducing local cytotoxity in selected areas. The use of such devices has been shown to improve the efficacy of antitumor chemo- and radio-therapies, increasing the selectivity of the cytocidal effects, and reducing systemic side effects. In addition, catalytic nanomaterials such as cerium oxide nanoparticles can perform energy-free antioxidant cycles that scavenge the most noxious reactive oxygen species via SOD- and catalase-mimetic activities. A vast body of in vivo and in vitro studies has demonstrated that they reduce the damage induced by environmental stress and ameliorate an impressive series of clinically relevant oxidation-related pathologies. Similar effects are reported for carbon-based materials such as fullerenes. Overall, great improvements are expected by this novel approach. However, caution must be posed due to the poor knowledge of possible adverse body reactions against these novel devices, thoroughly analyzing the biocompatibility of these nanomaterials, especially concerning the biokinetics and the problems potentially caused by long term retention of non-biodegradable inorganic nanomaterials.

Cerium oxide nanoparticles, combining antioxidant and UV shielding properties, prevent UV-induced cell damage and mutagenesis

Efficient inorganic UV shields, mostly based on refracting TiO2 particles, have dramatically changed the sun exposure habits. Unfortunately, health concerns have emerged from the pro-oxidant photocatalytic effect of UV-irradiated TiO2, which mediates toxic effects on cells. Therefore, improvements in cosmetic solar shield technology are a strong priority. CeO2 nanoparticles are not only UV refractors but also potent biological antioxidants due to the surface 3+/4+ valency switch, which confers anti-inflammatory, anti-ageing and therapeutic properties. Herein, UV irradiation protocols were set up, allowing selective study of the extra-shielding effects of CeO2vs. TiO2 nanoparticles on reporter cells. TiO2 irradiated with UV (especially UVA) exerted strong photocatalytic effects, superimposing their pro-oxidant, cell-damaging and mutagenic action when induced by UV, thereby worsening the UV toxicity. On the contrary, irradiated CeO2 nanoparticles, via their Ce(3+)/Ce(4+) redox couple, exerted impressive protection on UV-treated cells, by buffering oxidation, preserving viability and proliferation, reducing DNA damage and accelerating repair; strikingly, they almost eliminated mutagenesis, thus acting as an important tool to prevent skin cancer. Interestingly, CeO2 nanoparticles also protect cells from the damage induced by irradiated TiO2, suggesting that these two particles may also complement their effects in solar lotions. CeO2 nanoparticles, which intrinsically couple UV shielding with biological and genetic protection, appear to be ideal candidates for next-generation sun shields.

Anti-apoptotic effect of HIV protease inhibitors via direct inhibition of calpain

Treatment with drugs designed to inhibit the HIV protease ameliorates immune functions in AIDS patients, reducing cell deletion by apoptosis even in the absence of inhibition of viral spread. This suggests that they interact with the intrinsic apoptotic signaling. We found that caspases, the main executioner of the apoptotic process, are not directly inhibited. In search for the mechanism responsible for their anti-apoptotic effect, we have found that indinavir and ritonavir are able to inhibit apoptosis only in those cell systems where apoptosis involves the activation of calpains. They directly inhibit a calpain-like activity expressed in lysates from apoptotic cells, to the same extent as commercially available calpain inhibitor 1. In in vitro assays with purified calpains, indinavir and ritonavir strongly inhibit m-calpain, and moderately mu-calpain. These results have great therapeutic implications, going beyond AIDS treatment, since many degenerative disorders involve abnormal calpain activation, indicating calpain as an ideal pharmacological target. Indinavir and ritonavir, potent m-calpain inhibitors, largely used since several years on humans without important negative side effects, may become powerful tools against those pathologies.

Melatonin as an apoptosis antagonist

Abstract:  The pineal hormone melatonin (Mel), in addition to having a well‐established role as a regulator of circadian rhythms, modulates nonneural compartments by acting on specific plasma membrane receptors (MT1/MT2) present in many different cell types. Mel plays immunomodulatory roles and is an oncostatic and antiproliferative agent; this led to the widespread belief that Mel may induce or potentiate apoptosis on tumor cells, even though no clear indications have been presented so far. Here we report that Mel is not apoptogenic on U937 human monocytic cells, which are known to possess MT1 receptors at the times (up to 48 h) and doses (up to 1 mM) tested. Mel does not even potentiate apoptosis, but instead, significantly reduces apoptosis induced by both cell‐damaging agents (intrinsic pathway) and physiological means (extrinsic pathway). The doses required for the antiapoptotic effect (≥100 μM) are apparently not compatible with receptor stimulation (receptor affinity <1 nM). However, receptor involvement cannot be ruled out, because we discovered that the actual Mel concentration active on cells was lower than the nominal one because of sequestration by fetal calf serum (FCS). Accordingly, in FCS‐free conditions, Mel doses required for a significant antiapoptotic effect are much lower.

Multiple mechanisms for hydrogen peroxide-induced apoptosis.

The mechanisms of cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet well clarified. Here, we show that during recovery after H2O2 treatment, apoptosis occurs in two different waves, peaking at 8 h (early) and 18 h (late) of recovery from oxidative stress. The two peaks are differentially modulated by a set of inhibitors of metabolic processes, which suggests that the first peak depends on DNA break formation, whereas the second may be correlated with H2O2‐induced mitochondrial alterations.