Lanfranco Masotti

New antitumor imidazo[2,1-b]thiazole guanylhydrazones and analogues.

The synthesis of new antitumor 6-substituted imidazothiazole guanylhydrazones is described. Moreover, a series of compounds with a different basic chain at the 5 position were prepared. Finally, the replacement of the thiazole ring in the imidazothiazole system was also considered. All the new compounds prepared were submitted to the NCI cell line screen for evaluation of their antitumor activity. A few selected compounds were submitted to additional biological studies concerning effects on the cell cycle, apoptosis, and mitochondria.

Lipid-protein interactions in mitochondria. I. Conditions affecting binding of phospholipids to lipid-depleted mitochondria.

Abstract The interaction of lipid-depleted mitochondria with mixed micellar phospholipids is not inhibited by increasing the ionic strength of the medium with NaCl or certain other salts. The graph describing binding as function of ionic strength has a diphasic character. The extent of the binding increases with temperature. Mitochondrial membranes freed of soluble and detachable proteins can also be depleted of their lipids and reconstituted by readdition of phospholipid micelles: this interaction also is not impaired by NaCl. The interaction is, however, inhibited by alcohols and by lyotropic agents inducing disorder in the structure of water (such as certain salts at high concentrations). From these data and other characteristics of the binding we conclude that the binding of phospholipids to the proteins of the mitochondrial membranes is largely hydrophobic in nature.

Antitumor Activity of New Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-Imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones : Selectivity against Colon Tumor Cells and Effect on Cell Cycle-Related Events

The synthesis of new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones is reported. The antitumor activity was evaluated according to the protocols available at the National Cancer Institute (NCI), Bethesda, MD. To investigate the mechanism of action of the most potent antitumor agent of this series, its effect on growth of HT-29 colon carcinoma cells was studied. Its ability to inhibit cellular proliferation was mediated by cell cycle arrest at the G2/M phase, accompanied by inhibition of ornithine decarboxylase (ODC), the limiting enzyme of polyamine synthesis, and followed by induction of apoptosis.

Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and Analogues: Synthesis, Cytotoxic Activity and Study of the Mechanism of Action

The synthesis of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and analogues is reported. Their cytotoxic activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of selected compounds was examined for potential inhibition of tubulin assembly in comparison with the potent colchicine site agent combretastatin A-4. The most potent compounds also strongly and selectively inhibited the phosphorylation of the oncoprotein kinase Akt in cancer cells. The effect of the most interesting compounds was examined on the growth of HT-29 colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for inhibitors of tubulin assembly.

Determination of 4-hydroxy-2-nonenal at cellular levels by means of electrospray mass spectrometry

trans-4-Hydroxy-2-nonenal (HNE) is an end-product of lipid peroxidation in biological systems which produces a variety of powerful biological effects. A method based on electrospray mass spectrometry was developed for the determination of 4-HNE at cellular levels. Quantification was carried out by using HNE-d(11) as internal standard; the mass chromatograms were acquired in the single ion monitoring mode (SIM) on the [M + H](+) monoisotopic species for HNE and HNE-d(11). With this approach a higher precision and lower detection limit and biological sample size than those typical of the methods so far employed are achieved. Furthermore the determination of the analyte from the cell extract is directly performed without the need of any HNE derivatization. As a first application the method was used to identify and quantify HNE in human T cell leukemia extracts.

Effect of phospholipids on the protein conformation in the inner mitochondrial membranes

Summary Lipid extracted inner mitochondrial membranes, studied by circular dichroism, exhibit a mean protein conformation which differs from that of intact mitochondria. Reconstitution of the membranes by addition of different phospolipid preparations restores to varying extents the original mean protein conformation. The results demonstrate the effect of lipid in modulating protein conformation and of the lipids studied cardiolipin is the most effective in restoring ellipticity values to those of the intact mitochondrial membrane.

Parallel synthesis and cytotoxicity evaluation of a polyamine-quinone conjugates library.

A library of 24 derivatives designed by combining two natural products-derived fragments was prepared and tested to determine their anticancer potential in HT29 colon cancer cells. All library members inhibit cell proliferation as measured by MTT mitochondrial functional assay, with IC50 values in the 1-100 microM range. Entry 1b caused apoptotic EGFR-mediated intracellular signaling. Thus, polyamino-quinones emerged as readily accessible and easily diversified scaffolds for anticancer lead discovery.

Lipid-protein interactions in mitochondria: II. On the nature and biochemical significance of the interaction between phospholipids and lipid-depleted mitochondria

Abstract The binding of phospholipid micelles to lipid-depleted mitochondrial membranes has been studied in further detail. The diphasic character of the curve of binding plotted against the concentration of salt has been explained on the basis of an enhancement of hydrophobic binding by increasing ionic strengths and subsequent impairment of the binding due to the lyotropic effects of high salt concentrations. The amount of phospholipid bound in certain conditions is above the natural levels in mitochondrial membranes, because sites normally buried become exposed and available for interaction with phospholipid. The composition of the bound phospholipids is the same when the interaction is done in the absence or in the presence of 1 m NaCl. Succinoxidase activity is restored in lipid-depleted mitochondria by phospholipid addition either in salt-free media or at low ionic strengths, but is decreased when the incubation is carried out in 1 m NaCl, in 0.5 m NaSCN, and also in 3 m urea. From these results we conclude that the interactions we have studied appear to be relevant to the interactions in the native membranes and that the bonds formed between protein and lipids must be primarily hydrophobic, although a contribution of polar forces to the binding cannot be excluded.

Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.

Lipid-protein interactions in mitochondria: III. Solvent extraction of phospholipids from mitochondrial preparations☆

Abstract Monohydric alcohols extract phospholipids from beef heart mitochondria and submitochondrial particles with an efficacy which depends on the chain length of the alcohol. MgCl2 and CaCl2 prevent alcohol extraction of part of the phospholipids. All particles used are affected by alcohols to similar extents. Diethyl ether extracts phospholipids from sonicated phospholipid vesicles in presence of NaCl or at acid pH even when the vesicles were bound to lipid-depleted mitochondria; ether extracts little phospholipid from beef heart mitochondria or submitochondrial particles under similar conditions; NaSCN is however much more effective than NaCl in increasing the extractability of phospholipids by ether. Mitochondrial membranes treated with 10 m m HCl are freed of the soluble proteins and of the proteins of the ATPase complex; phospholipids can be extracted in large amounts from such membranes by ether in presence of salt. Detachment of the ATPase complex from mitochondria by other means also results in higher phospholipid extractability by ether. Lyophilized mitochondria or submitochondrial particles can be extracted of phospholipids by means of ether or pentane only after HCl treatment. Chloroform-methanol 2:1 extracts phospholipids and a small but significant portion of the protein from mitochondria and various mitochondrial preparations. The possible presence of proteolipids is discussed. The results of this investigation are discussed in relation to the importance of hydrophobic interactions between phospholipids and proteins in mitochondria; hydrophobic bonds however are not unique and the hypothesis is advanced that superficial proteins are linked to the mitochondrial membrane by other types of bonds.