Modulation of pro-apoptotic effects and mitochondrial potential on B16F10 cells by DODAC/PHO-S liposomes.

OBJECTIVE: We aimed to evaluate the potential of DODAC/PHO-S liposomes on the modulation of the expression of pro-apoptotic proteins, loss of lysosomal integrity and the mitochondrial electrical potential, compared with phosphoethanolamine. RESULTS: The results of this study demonstrate that DODAC/PHO-S liposomes have exhibited broad cytotoxic potential in B16F10 murine melanoma cells, with significantly greater proportions than treatment with PHO-S. The treatment with the DODAC/PHO-S 2.0 mM liposomal formulation was more efficient in decreasing mitochondrial electrical potential at the same concentrations and treatment time than PHO-S The liposomal formulation DODAC/PHO-S (2.0 mM) was more efficient to promote morphological changes in the cells, without presenting intact lysosomes, at the same time of treatment and concentration as PHO-S Our results demonstrated that the liposomal formulation increased DR4 receptor expression and activated caspases 8 and 3, resulting in the release of cytochrome c in B16F10 tumour cells, when compared to treatment with PHO-S The data obtained prove that the use of DODAC as carrier can maximize the cytotoxic effects of PHO-S This was demonstrated by the translocation of cytochrome c to the cytoplasm and activation of caspase-3 and 8, decreasing the mitochondrial electrical potential and generating morphological changes, in B16F10 cells.

Modulation of pro-apoptotic effects and mitochondrial potential on B16F10 cells by DODAC/PHO-S liposomes

Objective We aimed to evaluate the potential of DODAC/PHO-S liposomes on the modulation of the expression of pro-apoptotic proteins, loss of lysosomal integrity and the mitochondrial electrical potential, compared with phosphoethanolamine. Results The results of this study demonstrate that DODAC/PHO-S liposomes have exhibited broad cytotoxic potential in B16F10 murine melanoma cells, with significantly greater proportions than treatment with PHO-S. The treatment with the DODAC/PHO-S 2.0 mM liposomal formulation was more efficient in decreasing mitochondrial electrical potential at the same concentrations and treatment time than PHO-S The liposomal formulation DODAC/PHO-S (2.0 mM) was more efficient to promote morphological changes in the cells, without presenting intact lysosomes, at the same time of treatment and concentration as PHO-S Our results demonstrated that the liposomal formulation increased DR4 receptor expression and activated caspases 8 and 3, resulting in the release of cytochrome c in B16F10 tumour cells, when compared to treatment with PHO-S The data obtained prove that the use of DODAC as carrier can maximize the cytotoxic effects of PHO-S This was demonstrated by the translocation of cytochrome c to the cytoplasm and activation of caspase-3 and 8, decreasing the mitochondrial electrical potential and generating morphological changes, in B16F10 cells.

Modulation of pro-apoptotic effects and mitochondrial potential on B16F10 cells by DODAC/PHO-S liposomes

Objective We aimed to evaluate the potential of DODAC/PHO-S liposomes on the modulation of the expression of pro-apoptotic proteins, loss of lysosomal integrity and the mitochondrial electrical potential, compared with phosphoethanolamine. Results The results of this study demonstrate that DODAC/PHO-S liposomes have exhibited broad cytotoxic potential in B16F10 murine melanoma cells, with significantly greater proportions than treatment with PHO-S. The treatment with the DODAC/PHO-S 2.0 mM liposomal formulation was more efficient in decreasing mitochondrial electrical potential at the same concentrations and treatment time than PHO-S The liposomal formulation DODAC/PHO-S (2.0 mM) was more efficient to promote morphological changes in the cells, without presenting intact lysosomes, at the same time of treatment and concentration as PHO-S Our results demonstrated that the liposomal formulation increased DR4 receptor expression and activated caspases 8 and 3, resulting in the release of cytochrome c in B16F10 tumour cells, when compared to treatment with PHO-S The data obtained prove that the use of DODAC as carrier can maximize the cytotoxic effects of PHO-S This was demonstrated by the translocation of cytochrome c to the cytoplasm and activation of caspase-3 and 8, decreasing the mitochondrial electrical potential and generating morphological changes, in B16F10 cells.

Potential antitumor activity of novel DODAC/PHO-S liposomes.

In recent studies, we showed that synthetic phosphoethanolamine (PHO-S) has a great potential for inducing cell death in several tumor cell lines without damage to normal cells. However, its cytotoxic effect and selectivity against tumor cells could increase with encapsulation in cationic liposomes, such as dioctadecyldimethylammonium chloride (DODAC), due to electrostatic interactions between these liposomes and tumor cell membranes. Our aim was to use cationic liposomes to deliver PHO-S and to furthermore maximize the therapeutic effect of this compound. DODAC liposomes containing PHO-S (DODAC/PHO-S), at concentrations of 0.3-2.0 mM, prepared by ultrasonication, were analyzed by scanning electron microscopy (SEM) and dynamic light scattering. The cytotoxic effect of DODAC/PHO-S on B16F10 cells, Hepa1c1c7 cells, and human umbilical vein endothelial cells (HUVECs) was assessed by MTT assay. Cell cycle phases of B16F10 cells were analyzed by flow cytometry and the morphological changes by SEM, after treatment. The liposomes were spherical and polydisperse in solution. The liposomes were stable, presenting an average of ∼ 50% of PHO-S encapsulation, with a small reduction after 40 days. DODAC demonstrated efficient PHO-S delivery, with the lowest values of IC50% (concentration that inhibits 50% of the growth of cells) for tumor cells, compared with PHO-S alone, with an IC50% value of 0.8 mM for B16F10 cells and 0.2 mM for Hepa1c1c7 cells, and without significant effects on endothelial cells. The Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation when compared to B16F10 cells and HUVECs. The use of DODAC/PHO-S on B16F10 cells induced G2/M-phase cell cycle arrest, with the proportion significantly greater than that treated with PHO-S alone. The morphological analysis of B16F10 cells by SEM showed changes such as "bleb" formation, cell detachment, cytoplasmic retraction, and apoptotic bodies after DODAC/PHO-S treatment. Cationic liposomal formulation for PHO-S delivery promoted cytotoxicity more selectively and effectively against B16F10 and Hepa1c1c7 cells. Thus, the DODAC/PHO-S liposomal formulation presents great potential for preclinical studies.

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase.

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Pho-s has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma.

Effects of Ricinus communis oil esters on salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

This study showed the interference of esters extracted from Ricinus communis in the secretory cycle of salivary glands of Rhipicephalus sanguineus ticks, which consequently caused collateral effects on their feeding process. Ticks attached on hosts which were fed with commercial feed containing different concentrations of R. communis oil esters suffered damages such as cytoplasmic changes in their salivary glands, notably in the acinar cells, impairing the functioning of the acini and accelerating the organs degeneration as a whole. It was found that esters interfered with the activity of cellular secretion by changing the glycoprotein of salivary composition especially in acini II cells. It was also shown that the damages caused by esters in the salivary glands cells of these ectoparasites increased in higher concentrations of the product and degenerative glandular changes were more pronounced.

Synthetic Phosphoethanolamine Induces Apoptosis Through Caspase-3Pathway by Decreasing Expression of Bax/Bad Protein and Changes CellCycle in Melanoma

Phospholipids are potential antineoplastic agents that are abundant constituents of the cell membrane ofeukaryotes and are supposed to be involved in specific intracellular signaling such as cell death. The aim of this study was to assess the in vitro and in vivo antitumor effects of synthetic phosphoethalomanine (PHO-S) on B16F10 murine melanoma cells and normal human fibroblasts. The cytotoxicty was evaluated by MTT assay and PHO-S was cytotoxic in melanoma cells but not in fibroblasts with IC50% of 1.4 mg/ml to melanoma cells. In vivo antitumor activity was evaluated in a mice model subcutaneously injected with B16F10 melanoma cells. The mice treated with PHO-S in all concentrations showed a decrease of the tumor growth and metastasis. Cytometry analysis showed that the PHO-S blocked DNA synthesis, decreased number of melanoma cells in S phase and G2/M, besides increasing number of apoptotic cells, inducing caspase-3 activity and decreasing Bad/Bax protein expression. Histologically, the dorsal tumors in the control group showed pigmented nodular masses with high vascularization and pleomorphictumor cells. In the treated group, PHO-S reduction vascularization intratumoral with increased of collagen fibersand infiltrates neutrophils. The data indicate that PHO-S is a lipid compound potential with proapoptotic andantiproliferative effects but further work will be necessary to elucidate the antitumor mechanisms.

Polyurethane from biosource as a new material for fabrication of microfluidic devices by rapid prototyping.

This paper presents the use of elastomeric polyurethane (PU), derived from castor oil (CO) biosource, as a new material for fabrication of microfluidic devices by rapid prototyping. Including the irreversible sealing step, PU microchips were fabricated in less than 1h by casting PU resin directly on the positive high-relief molds fabricated by standard photolithography and nickel electrodeposition. Physical characterization of microchannels was performed by scanning electron microscopy (SEM) and profilometry. Polymer surface was characterized using contact angle measurements and the results showed that the hydrophilicity of the PU surface increases after oxygen plasma treatment. The polymer surface demonstrated the capability of generating an electroosmotic flow (EOF) of 2.6 x 10(-4)cm(2)V(-1)s(-1) at pH 7 in the cathode direction, which was characterized by current monitoring method at different pH values. The compatibility of PU with a wide range of solvents and electrolytes was tested by determining its degree of swelling over a 24h period of contact. The performance of microfluidic systems fabricated using this new material was evaluated by fabricating miniaturized capillary electrophoresis systems. Epinephrine and l-DOPA, as model analytes, were separated in aqueous solutions and detected with end-channel amperometric detection.