Antonella Miglietta

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.

An overview of the effect of linoleic and conjugated-linoleic acids on the growth of several human tumor cell lines.

Both n‐6 and n‐3 polyunsaturated fatty acids are dietary fats important for cell function, being involved in several physiologic and pathologic processes, such as tumorigenesis. Linoleic acid and conjugated linoleic acid, its geometrical and positional stereoisomer, were tested on several human tumor cell lines originating from different tissues and with different degrees of malignancy. This was to provide the widest possible view of the impact of dietary lipids on tumor development. While linoleic acid exerted different effects, ranging from inhibitory to neutral, even promoting growth, conjugated linoleic acid inhibited growth in all lines tested and was particularly effective against the more malignant cells, with the exception of mammary tumor cells, in which behavior was the opposite, the more malignant cell line being less affected. The inhibitory effect of conjugated linoleic acid on growth may be accompanied by different contributions from apoptosis and necrosis. The effects of conjugated linoleic acid on growth or death involved positive or negative variations in PPARs. The important observation is that a big increase of PPARα protein occurred in cells undergoing strong induction of apoptosis, whereas PPARβ/δ protein decreased. Although PPARα and PPARβ/δ seem to be correlated to execution of the apoptotic program, the modulation of PPARγ appears to depend on the type of tumor cell, increasing as protein content, when inhibition of cell proliferation occurred. In conclusion, CLA may be regarded as a component of the diet that exerts antineoplastic activity and its effect may be antiproliferative or pro‐apoptotic.

Expression of Cox-2 in human breast cancer cells as a critical determinant of epithelial-to-mesenchymal transition and invasiveness

Introduction: Cyclooxygenase-2 (COX-2) is overexpressed in several malignancies and is implicated in breast cancer progression. Objectives: We investigated whether changes in COX-2 expression may affect epithelial-to-mesenchymal transition (EMT) and then invasive potential of human breast cancer cells, in relationship with hypoxia. COX-2-null MCF-7 human breast cancer cells, MCF-7 cells transiently expressing COX-2 and COX-2-expressing MDA-MB-231 cells were employed. Results: COX-2 overexpression resulted in downregulation of E-cadherin and β-catenin, upregulation of vimentin, N-cadherin and SNAI1, suggesting EMT occurrence. COX-2-overexpressing MCF-7 cells were also characterized by increased invasiveness and release of matrix-metalloproteinase-9. The above-mentioned characteristics, homologous to those detected in highly invasive MDA-MB-231 cells, were reverted by treatment of COX-2-overexpressing MCF-7 cells with celecoxib, a COX-2-specific inhibitor, partly through the inhibition of COX-2-related intracellular generation of reactive oxygen species. Hypoxia further exacerbated COX-2 expression, EMT changes and invasive ability in both COX-2-overexpressing MCF-7 cells and MDA-MB-231 cells. Finally, immunohistochemistry performed on samples from normal and neoplastic human breast tissues revealed that COX-2-positive malignant cells were also positive for EMT-related antigens, hypoxia-inducible factor (HIF)-2α and the oxidative stress marker heme oxygenase. Conclusions: These findings support the existence of a direct link between COX-2 overexpression, EMT and invasiveness in human breast cancer cells, emphasizing the role of hypoxic microenvironment.

Involvement of PPARγ and E‐cadherin/β‐catenin pathway in the antiproliferative effect of conjugated linoleic acid in MCF‐7 cells

Conjugated linoleic acid (CLA) is a naturally occurring fatty acid, which has been shown to exert beneficial effects against breast carcinogenesis. It has been reported that CLA could modulate cellular proliferation and differentiation through the activation of peroxisome proliferator‐activated receptors (PPARs). Among different PPAR isotypes, PPARγ is involved in growth inhibition of transformed cells. Ligands of PPARγ are considered as potential anticancer drugs, so CLA was tested for its ability to induce PPARγ expression in MCF‐7 breast cancer cells. The effects of CLA and of a specific synthetic PPARγ antagonist were evaluated on cell growth as well as on parameters responsible for cell growth regulation. We demonstrated here that CLA stimulated the expression of PPARγ to levels up to control and caused PPARγ translocation into the nucleus. Furthermore, the overexpression of PPARγ positively correlates with the inhibition of cell proliferation and with the modulation of ERK signaling induced by CLA; in all cases the administration of the antagonist reverted CLA effects. The PPAR‐signaling pathway is connected with the β‐catenin/E‐cadherin pathway, thus we evaluated CLA effects on the expression and cellular distribution of these proteins, which are involved in cell adhesion and responsible for invasive behavior. The treatment with CLA determined the up‐regulation and the redistribution of β‐catenin and E‐cadherin and the antagonist reverted only the effect on β‐catenin. These studies indicate that CLA regulates PPARγ expression by selectively acting as an agonist and may influence cell–cell adhesion and invasiveness of MCF‐7 cells.

Antiproliferative effects of COX-2 inhibitor celecoxib on human breast cancer cell lines

The inducible COX-2 enzyme is over-expressed in human breast cancer and its over-expression generally correlates with angiogenesis, deregulation of apoptosis and worse prognosis. This observation may explain the beneficial effect of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors on breast cancer treatment. Here, we evaluated the antiproliferative activity of celecoxib, a selective COX-2 inhibitor, and its nitro-oxy derivative on human breast cancer cells characterized by low and high COX-2 expression, respectively. In ERα(+) MCF-7 cells celecoxib and its derivative induce a strong inhibition of cell growth, inhibition that is associated with the reduction of ERα expression and activation. These effects may be directly associated with ERK and Akt suppression and with PP2A and PTEN induction. In this cell line the drugs exert only weak effect on COX-2 level while they are able to reduce aromatase expression. On the contrary, in ERα(−) MDA-MB-231 cells, both drugs induce a marked inhibition of COX-2, inhibition that is associated with the reduction of aromatase expression and of cell proliferation. In both cell lines the effects of the drugs are associated with the suppression of cell invasion.

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.

CLA reduces breast cancer cell growth and invasion through ERα and PI3K/Akt pathways.

We previously reported that conjugated linoleic acid (CLA), a naturally occurring fatty acid, inhibits the growth of ERalpha(+) MCF-7 and ERalpha(-) MDA-MB-231 human breast cancer cells by negative modulation of the ERK/MAPK pathway and apoptosis induction. Here we show that in these cell lines CLA also down-regulates the PI3K/Akt cascade. In MCF-7 cells CLA also triggers ERalpha/PP2A complex formation reducing the phosphorylation state and transcriptional activity of Eralpha whereas in MDA-MB-231 cells CLA does not induce PP2A activation. Moreover, CLA induces the expression of proteins involved in cell adhesion and inhibits cell migration and MMP-2 activity. These findings suggest that CLA may induce the down-regulation of ERalpha signalling and the reduction of cell invasion through the modulation of balancing between phosphatases and kinases.

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.

Angiogenesis and Fibrogenesis in Chronic Liver Diseases

Pathologic angiogenesis appears to be intrinsically associated with the fibrogenic progression of chronic liver diseases, which eventually leads to the development of cirrhosis and related complications, including hepatocellular carcinoma. Several laboratories have suggested that this association is relevant for chronic liver disease progression, with angiogenesis proposed to sustain fibrogenesis. This minireview offers a synthesis of relevant findings and opinions that have emerged in the last few years relating liver angiogenesis to fibrogenesis. We discuss liver angiogenesis in normal and pathophysiologic conditions with a focus on the role of hypoxia and hypoxia-inducible factors and assess the evidence supporting a clear relationship between angiogenesis and fibrogenesis. A section is dedicated to the critical interactions between liver sinusoidal endothelial cells and either quiescent hepatic stellate cells or myofibroblast-like stellate cells. Finally, we introduce the unusual, dual (profibrogenic and proangiogenic) role of hepatic myofibroblasts and emerging evidence supporting a role for specific mediators like vasohibin and microparticles and microvesicles.