Marco Andreassi

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation

AbstractWe previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, β-actin, tubulin-α-1-chain and enolase-α. Interestingly, all carbonylated proteins except calreticulin and enolase-α showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

Non-covalent inclusion of ferulic acid with α-cyclodextrin improves photo-stability and delivery: NMR and modeling studies

Ferulic acid (FA) is a highly effective antioxidant and photo-protective agent, already approved in Japan as a sunscreen, but it is poorly suited for cosmetic application because of its low physicochemical stability. We prepared the inclusion complex of FA with alpha-cyclodextrin by co-precipitation from an aqueous solution, and used (1)H NMR and molecular dynamics to investigate the most probable structure of the inclusion complex. In rotating frame nuclear Overhouser effect spectroscopy (ROESY) experiments FA penetrated the alpha-CD hydrophobic cavity with the alpha,beta-unsaturated part of the molecule and some of its aromatic skeleton. In proton chemical shift measurements of FA and alpha-cyclodextrins we determined the stoichiometry of the association complex (1:1) by Jobs method, and its stability constant (K(1:1) 1162+/-140 M(-1)) and described the molecular dynamics of the complex on the basis of theoretical studies. Encapsulation with alpha-cyclodextrin improves (i) the chemical stability of FA against UVB stress (10 MED [Minimal Erythemal Dose: 1 MED=25 mJ/cm(2) for skin phototype II: 30]), since no degradation products are formed after irradiation, and (ii) the bioavailability of FA on the skin, slowing its delivery (Strainer cell model).

Antioxidant activity of topically applied lycopene

Background  Ultraviolet (UV) rays cause depletion of the antioxidant substances contained in the epidermis. This is the rationale for the use of topical antioxidant substances.

Disease modifying anti-Alzheimer’s drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation

We recently described multifunctional tools (2a–c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aβ aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level.

Targeting Dopamine D3 and Serotonin 5-HT1A and 5-HT2A Receptors for Developing Effective Antipsychotics: Synthesis, Biological Characterization, and Behavioral Studies

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

New microencapsulated sunscreens: technology and comparative evaluation

The aim of this work is to obtain new technologically improved microencapsulated sunscreens characterised by UV-radiation stability, good substantivity, low toxicity, a better tolerability and easiness to formulation. For this purpose we prepared two different systems using semisynthetic Hyaluronic Acid (HA) benzyl ester and a synthetic polymer (patent pending). We obtained these systems using two different methodologies: emulsification/solvent evaporation and emulsification/solvent extraction. The comparison between the two formulated systems was carried out in terms of their chemical-physical and biological properties.

Lycopene phytocomplex, but not pure lycopene, is able to trigger apoptosis and improve the efficacy of photodynamic therapy in HL60 human leukemia cells.

We compared the ability of pure lycopene (Lyco) versus lycopene phytocomplex (LycoC) to induce apoptosis in vitro. We found that LycoC, but not Lyco, was able to trigger apoptosis in HL60 cells, as documented by subdiploid DNA content and phosphatidylserine exposure. LycoC-induced apoptosis was associated with reactive oxygen species (ROS) generation and loss of mitochondrial transmembrane potential, suggesting that LycoC triggered apoptosis via a mitochondrial pathway. We also verified the redox state of cells by measuring glutathione (GSH) content, but only a small percentage of cells showed GSH depletion, suggesting that the loss of GSH may be a secondary consequence of ROS generation. Moreover, LycoC pretreatment effectively increased apoptosis induced by photodynamic therapy (PDT), a mode of cancer treatment using a photosensitizer and visible light. LycoC pretreatment was even more potent in improving PDT than pretreatment with ascorbic acid or alpha-tocopherol (or the two combined). Our results demonstrate that LycoC has a stronger cytotoxic effect than Lyco and is a better source of agents able to trigger apoptosis in HL60 cells and improve the efficacy of PDT in vitro.

A new device for objective assessment of skin type in Caucasians by violet light reflectance

In the present study, we tested a new device called skin phototype diagnosis (SPD) built for the purpose of objectively determining skin phototype. We compared its performance with that of phototype determinations according to Fitzpatrick method and on tristimulus colorimetry (Minolta CR‐200). Our population consisted of 100 subjects of Caucasian race (60 female, 40 male; mean age 33 years). Skin colour was measured with both devices (SPD and Minolta CR‐200) on the medial surface of the arm (constitutional skin colour). Our study showed that the SPD gave a better representation of Fitzpatrick phototype, showing 89% concordance (evaluated by classification matrix) as against the 71% concordance of the L*a*b* and Yxy colorimetric systems. The present results are important because evaluation of phototype with the SPD device is easy, fast, objective and reliable. Moreover, this instrument has potential applications in cosmetology and in photodermatology.

Topical pimecrolimus in the treatment of genital lichen sclerosus

Pimecrolimus is a drug belonging to the class of macrolactamic immunosuppressants, similar to tacrolimus, and has been employed mainly in the treatment of atopic dermatitis, but is also active in other skin diseases, including genital lichen sclerosus (LS). The safety profile of pimecrolimus was the subject of special attention by the US FDA, which in 2005 issued a Public Health Advisory on the potential risk of cancer associated with the use of topical calcineurin inhibitors. However, many scientific societies have criticized the FDA note, referring to the vast literature on the subject in which there is no evidence available that topical use of the drug is dangerous. Patients with LS, of both sexes, may develop genital squamous cell carcinoma. This event, however, is not a contraindication to the use of pimecrolimus.