Composti di origine naturale e xenobiotici, caratterizzazione chimica, valutazione dell'attività biologica su linee cellulari e potenziali utilizzi chemioterapici
Author(s)
Triggiani, Doriana
Date Issued
February 28, 2011
Type
Doctoral Thesis
Abstract
Natural products derived from plants and other biological organisms are a continuous source for new drugs. A recent study shows that of 847 drug molecules, 5% are natural products, about 27% derives from natural products and the remaining 572 are synthetic molecules (Newman and Cragg 2007), of which 262 can be considered analogous to natural products. It is interesting to note that these dates do not include biological products and vaccines, which by definition come from nature.
It’ s called “xenobiotic” any type of molecule, having natural or synthetic origin, foreign to
an organism, that can exert the function both of drug than toxic poison.
The research activity,during the PhD, was pointed out on the study of natural and xenobiotic compounds, their chemical characterization, evaluation of biological activity and consequently their potential uses as drugs. The researches were focused on two different topics:
A) Identification and characterization of biologically active compounds from Rubus ulmifolius extracts.
B) Evaluation and elucidation of the biological chemistry of organotin compounds.
A) Identification and characterization of biologically active compounds from Rubus ulmifolius extracts
Rubus ulmifolius Schott is a perennial plant widely diffused in Italy and well known as “rovo” (Pignatti 1982). Rubus leaf extract contains triterpenes, fatty acids, tannins, flavonoids (Panizzi et al. 2001) and other molecules having pharmaceutical properties not completely studied. Outline. First, we investigated the biological activity (cytotoxic or antiproliferative) of alcoholic extract, with particular attention to the effects on cell proliferation and morphological changes.
The evaluation of the effect of Rubus ulmifolius extract was evaluated on human tumor cell lines HeLa, HepG2, HL60 and HL60/MX2.
In order to detect the cytotoxic or antiproliferative effects, we evaluated the cell countings and the growth curves upon treatment with different extracts..
Subsequently, the morphological and cytoskeletal alterations at cellular level were studied by microscopy techniques.
Further, by thin layer chromatography (TLC) we isolated a spot, named "R", showing the highest biological activity.
Then we identified the molecules contained in the active spot from Rubus ulmifolius leaves extract.
On the basis of these results and seen the similarity between the chemical structure of the molecules identified and tacrine, an inhibitor of the acetylcholinesterase previously studied in our lab, we tested these compounds as inhibitors of acetylcholinesterase.
The results obtained suggest that molecules from spot “R” have some pharmacological interest.
Interestingly the inhibition activity of R. ulmifolius extracts on cell proliferation and on Acetylcholinesterase is not only due to the molecules already known and studied, the flavonoids quercetin and kaempferol, but also to their glucuronide derivatives.
B) Evaluation and elucidation of the biological chemistry of organotin compounds.
Organotin compounds are xenobiotic compounds ubiquitous in the environment. They represent the most toxic pollutants known for aquatic life; environmental pollution caused by OT compounds extended widely as a result of large industrial and agricultural applications. Some OT compounds showed an anti-neoplastic action while others were cytotoxic, genotoxic, mutagenic and neurotoxic.
Although the site of interaction of the OT compounds, at mitochondrial membrane level, is well known, the mechanisms of toxicity and biological degradation remain unclear (Tobin et al. 1999). Previous studies have shown that a synthetic peptide (SNN-PEP, I1LGCWCYLR9), derived from the primary sequence of stannin, a protein involved in the detoxification of OT, and containing two vicinal cysteine residues (CXC motif), is able to dealkylate TMT to DMT suggesting that this reaction is also involved in triggering of cell death by apoptosis.
Starting from this point, studies were conducted with the aim of verified whether the SNN-peptide was able to dealkylate organotin compounds in species less substituted, thus altering their cytotoxicity and biological activity.
Therefore, we investigated the kinetics of the dealkylation TetraMT and two of the most common OT trisubstituted, TMT and TET by fluorescence spectroscopy,. The reaction mechanism cna be described as a pseudoenzymatic reaction that ends in a covalent complex formed by the di-alkyltin compound bound to SNN-PEP.
Tetra-and tri-alkyltin compounds show a marked delay in their toxic action when compared with the corresponding molecules di- and mono-alkylated. So, it was hypothesized that the delayed
toxicity is a consequence of progressive dealkylation of compounds with production of more toxic di- and mono-derivatives.
In previous studies it was suggested that organotin compounds, interacting with stannin or directly with the DNA, could initiate a cascade of events necessary to induce cell death or else, to control the stannin expression . Thus we study the biological activity of different organotin compounds on two cell lines: HeLa and NB2a by MTT assays, morphological analysis (light microscopy) and cytoskeletal observations (fluorescence microscopy). Organotin compounds used in treatments were: Triphenyltin chloride (TPT), Tetramethyltin (4MT), in particular Dimethyltin dichloride (DMT), Trimethyltin chloride (TMT), Triethyltin bromide (TET) at the concentrations ranging from 1 to 100 μM .
Investigations by immunofluorescence were carried out to verify changes in the SNN expression after treatment of Hela (human cervical adenocarcinoma) and NB2a cells (murine neuroblastoma) with increasing concentrations of DMT, TMT and TET.
Additional information
Dottorato di ricerca in Scienze ambientali
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