Ludovica Gabriel

Cellular uptake and cytotoxicity of solid lipid nanospheres (SLN) incorporating doxorubicin or paclitaxel

Solid Lipid Nanospheres (SLN) are colloidal therapeutic systems proposed for several administration routes and obtained by dispersing warm microemulsions in cold water. SLN as carriers of doxorubicin and paclitaxel have been previously studied. In this study, the cellular uptake of SLN and the cytotoxicity of doxorubicin and paclitaxel incorporated into SLN were investigated on two cell-lines, human promyelocytic leukemia (HL60) and human breast carcinoma (MCF-7). Cellular uptake of SLN was determined by incorporating 6-coumarin as fluorescent marker. The cellular uptake of fluorescent SLN was clearly evidenced by fluorescence microscopy. The cytotoxicity of doxorubicin incorporated in SLN was higher compared to the conventional doxorubicin solution, even at the lower concentrations. Paclitaxel in SLN was about 100-fold more effective than free paclitaxel on MCF-7 cells, while on HL60 cells a lower sensitivity was achieved with paclitaxel in SLN. Unloaded SLN had no cytotoxic effect on HL60 and MCF-7 cells.

Phagocytic uptake of fluorescent stealth and non-stealth solid lipid nanoparticles

Abstract Fluorescent non-stealth and stealth solid lipid nanoparticles (SLN) were prepared using rhodamine B base as fluorescent marker. The steric stabilization of the nanoparticles was obtained using two lipid derivatives of monomethylpoly(ethylene) glycol 2000 (PEG 2000) as stealth agents: dipalmitoylphosphatidylethanolamine-PEG 2000 and stearic acid-PEG 2000. Stealth and non-stealth SLN were in the nanometer size range. Phagocytosis was evaluated by incubating SLN with murine macrophages and determining the extent of phagocytic uptake spectrofluorimetrically; stealth SLN inhibited phagocytosis to a greater extent than did non-stealth SLN.

Biological properties of jatrophane polyesters, new microtubule-interacting agents.

Purpose. The biological activities of macrocyclic jatrophane polyesters 1–3 from the Sardinian endemism Euphorbia semiperfoliata Viv. have not been evaluated in depth. We investigated the microtubule-interacting and antiproliferative activities of these drugs and the molecular mechanisms underlying their effects.Methods. We tested jatrophanes for their interaction with purified bovine brain tubulin by an in vitro polymerization assay and by electron microscopy. At a cellular level, the effects of jatrophanes on microtubular architecture, nuclear morphology, cell viability, cell cycle perturbations, and p53 and Raf-1/Bcl-2 involvement were investigated.Results. Jatrophanes exhibited microtubule-interacting activity. They stimulated purified tubulin assembly in vitro, and induced paclitaxel-like microtubules, as revealed by electron microscopy. In the cells, rearrangement of microtubule architecture was in contrast to the bundling produced by paclitaxel. Jatrophanes inhibited the growth of some human cancer cell lines without inducing cell cycle arrest in the G2/M phase. Moreover, they influenced p53 expression and Raf-1/Bcl-2 activation.Conclusions. Despite their structural difference from paclitaxel and other microtubule-interacting agents, jatrophanes may represent a new type of tubulin binder.

Extracellular signal-regulated kinase 1\2 and protein phosphatase 2A are involved in the antiproliferative activity of conjugated linoleic acid in MCF-7 cells

Conjugated linoleic acid (CLA) has protective properties in breast cancer. Here, we studied the mechanisms underlying the effects of CLA on MCF-7 breast cancer cell proliferation, especially in correlation with the involvement of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and protein phosphatase 2A (PP2A). CLA inhibits MCF-7 cell growth in a concentration- and time-dependent manner, without triggering apoptosis. In assessing expression levels of proteins that play obligatory roles in the ERK cascade, we evidenced that CLA down-regulated Raf-1 and decreased levels of phospho-ERK1/2, as well as c-myc expression. Increase in PP2A expression rates were additionally observed after CLA treatment of MCF-7 cells. The above effects, as well as CLA-induced inhibition of cell growth, were reversed by okadaic acid, a specific inhibitor of PP2A. Thus, PP2A likely participates in deactivation of ERK1/2, and its up-regulation may represent a novel mechanism for CLA-induced inhibition of cell proliferation.

4-Hydroxy-alkenals interaction with purified microtubular protein

4-Hydroxy-alkenals effect on microtubular system has been investigated, comparing the activity of both biogenic and non-biogenic aldehydes. As these aldehydes react essentially with sulphydryl groups, their action on titratable thiol groups microtubular protein was studied. These compounds evidenced an inhibitory effect on this parameter and on tubulin polymerization, where sulphydryls are essential. 4-Hydroxy-alkenals inhibit tubulin polymerization in a dose-dependent manner (0.1-1 mM), with the exception of 4-hydroxy-octenal, that shows an inhibitory action only in concentrations from 0.5 mM up. Its behaviour is very anomalous: in fact the tubulin-colchicine binding, is stimulated rather than inhibited, whereas such binding is inhibited by the other tested aldehydes. Our present results give then a support for an interaction between 4-hydroxy-alkenals and microtubular protein.

Inhibition of colchicine binding to rat liver tubulin by aldehydes and by linoleic acid hydroperoxide

The onset of fat accumulation within CCl4 poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated. The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [3H] colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes of LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active. The presence of thiols, added to the incubation medium, partially protects against the inhibition of [3H] colchicine binding by aldehydes. This suggests that aldehydes act by reacting with -SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CCl4 poisoning is discussed.

Cytoskeletal modifications induced by 4-hydroxynonenal

The antiproliferative action of 4-hydroxynonenal (HNE) could be related to an interaction with cytoskeletal structures. In this paper the effects exerted by HNE on microtubules and on microfilaments are examined by immunofluorescence. HNE alters cell morphology causing both the depolymerization of the microtubular structures and the dissolution of the stress-fibres. Taxol protects microtubules, preventing the depolymerizing effect of the aldehyde. The action of HNE could be attributed to its affinity for sulphydryl groups, which are essential in maintaining tubulin and actin both in the polymerized form.

Further observation on the effects of 3-methylcholanthrene and phenobarbital on carbon tetrachloride hepatotoxicity.

Abstract The behavior of PB and 3-MC, inducers of the enzymatic chain of the hepatic DMES, was studied in relation to their effects upon CCl 4 -hepatotoxicity. The secretion of triglycerides from liver into the plasma compartment was inhibited in CCl 4 -poisoned rats, as well, in the animals pretreated with PB and 3-MC. The lipid peroxidative phenomena (diene conjugation absorption), evidenced in rats intoxicated with the halogenated hydrocarbon, were enhanced by pretreatment with PB, while 3-MC, in this respect, had no effect. At 60 min after poisoning, the severe polysomal damage was not modified either by 3-MC or PB, while the recovery process (25 hr of intoxication) was at its highest in rats treated with CCl 4 alone, slower in 3-MC or PB pretreated rats. Cell necrosis, as measured by S-GOT and S-GPT, was protected in CCl 4 -intoxicated rats by pretreating the animals with 3-MC.

Protection by glutathione and propyl gallate on the impaired in vitro amino acid incorporation into liver microsomal protein of CCl4-poisoned rats

Abstract The endogenous mRNA directed amino acid incorporation into rat liver microsomes in vitro is lowered as soon as 30 min after CCl4 poisoning; at the same time a strong decrease in rat liver polysomes appears. Previous treatment with the watersoluble antioxidants glutathione and propyl gallate completely prevents these changes in polysome activity and structure. These results seem to indicate a direct relationship between lipoperoxidation and alteration in protein synthesis.

Effects of some aldehydes on brain microtubular protein

4-Hydroxynonenal is one of the main breakdown products of lipid peroxidation. It has an antiproliferative effect, which may partly be the consequence of an interaction with cytoskeletal structures. Its effects on microtubular protein are compared with those of homologous aldehydes with the same number of carbon atoms, and with that of benzaldehyde. Unlike the other aliphatic aldehydes, this latter aldehyde does not impair microtubular functions at every concentration in the range. Nonanal has the greatest effect on tubulin polymerization, whereas it only slightly impairs colchicine binding activity. 2-Nonenal and 4-hydroxynonenal have less inhibiting effect on tubulin polymerization; their effect on colchicine binding activity is dose-dependent. The targets of 4-hydroxynonenal on tubulin are -SH groups; the action mechanism of other aldehydes has not yet been identified.