Antiproliferative and proapoptotic effects of DODAC/synthetic phosphoethanolamine on hepatocellular carcinoma cells.

BACKGROUND: Current studies have demonstrated that DODAC/PHO-S (Dioctadecyldimethylammonium Chloride/Synthetic phosphoethanolamine) liposomes induces cytotoxicity in Hepa1c1c7 and B16F10 murine tumor cells, with a higher proportion than PHO-S. Therefore, our aim was to evaluate the potential of DODAC/PHO-S to elucidate the mechanism of cell death whereby the liposomes induces cytotoxicity in hepatocellular carcinoma Hepa1c1c7, compared to the PHO-S alone. METHODS: Liposomes (DODAC/PHO-S) were prepared by ultrasonication. The cell cycle phases, protein expression and types of cell's death on Hepa1c1c7 were analyzed by flow cytometry. The internalisation of liposomes, mitochondrial electrical potential and lysosomal stability were also evaluated by confocal laser scanning microscopy. RESULTS: After treatment with liposomes (DODAC/PHO-S), we observed a significant increase in the population of Hepa1c1c7 cells experiencing cell cycle arrest in the S and G2/M phases, and this treatment was significantly more effective to promote cell death by apoptosis. There also was a decrease in the mitochondrial electrical potential; changes in the lysosomes; nuclear fragmentation and catastrophic changes in Hepa1c1c7 cells. The liposomes additionally promoted increases in the expression of DR4 receptor, caspases 3 and 8, cytochrome c, p53, p21, p27 and Bax. There was also a decrease in the expression of Bcl-2, cyclin D1, CD90 and CD44 proteins. CONCLUSION: The overall results showed that DODAC/PHO-S liposomes were more effective than PHO-S alone, in promoting cytotoxicity Hepa1c1c7 tumor cells, activating the intrinsic and extrinsic pathways of programmed cell death.

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.

Caracterização histológica do desenvolvimento hepático em diferentes estágios embrionários de ratos / Histological characterization of the liver development at different embryonic stages of rats

Os ratos apresentam desenvolvimento embrionário similar aos de animais domésticos e humanos, sendo assim um modelo válido para estudos científicos. Dentre eles, o F344 se destaca por ser uma linhagem isogênica, facilitando a leitura dos resultados obtidos, devido a sua homogeneidade gênica. Devido à falta de estudos histológicos acerca do desenvolvimento hepático em ratos, o presente estudo tem como objetivo caracterizar histologicamente pela primeira vez o processo de desenvolvimento hepático nos estágios embrionários de E12,5 (12,5 dias de gestação), E13,5, E14,5, E15,5 e E16,5 em ratos F344. Cinco embriões de cada estágio embrionário foram coletados, fixados em Metacarn, incluídos em paraplast e realizadas colorações histológicas e histoquímica. Os brotos hepáticos de embriões entre 12,5-14,5 dias apresentaram-se como aglomerados de hepatoblastos, ainda desorganizados e circundados por inúmeras células precursoras sanguíneas nucleadas. Observou-se que os hepatoblastos possuem um núcleo grande basofílico com pouco citoplasma. Sinusoides com eritroblastos e células de Kupffer também foram encontrados. Com 14,5 dias, foi observada a coexistência de hepatoblastos e hepatócitos, além de megacariócitos. Nos embriões com 15,5 dias, começou a verificar-se distinção entre os cordões de hepatócitos em formação, limitados pelos capilares sinusoides. Tais cordões começavam a confluir para as presentes veias centrolobulares. Com 16,5 dias, a arquitetura parenquimal estava mais próxima da encontrada em fígados adultos, sendo a quantidade de hepatócitos superior à de hepatoblastos. Nesse prazo gestacional, o fígado ainda tinha função hematopoiética. O estudo traz histologicamente o desenvolvimento hepático entre 12,5-16,5 dias de ratos da linhagem F344, evidenciando as células que compõem cada período gestacional, gerando subsídios para futuros estudos.

The rats have embryonic development similar to other domestic animals and human beings, thus a valid model for scientific studies. Among them, the F344 stands out for be isogenic, facilitating the reading of the results obtained because of their genetic homogeneity. Due to the lack of histological studies concerning hepatic development in rats, the present study aimed to characterize histologically for the first time the process of developing liver in the stages of gestation of E12.5 (12.5 days of gestation), E13.5, E14.5, E15.5 and E16.5 in rats F344. Five embryos of each embryonic stage were collected, fixed in Metacarn, embedded in Paraplast and then histological stains and histochemistry were performed. The hepatic bud of embryo among 12.5-14.5 days presented themselves a cluster of hepatoblasts still disorganized and surrounded by numerous nucleated blood precursor cells. It was observed that the hepatoblasts have a large nucleus basophilic with little cytoplasm. Sinusoids with erythroblasts and Kupffer cells also have been found. At 14.5 days it was observed the coexistence of hepatoblasts and hepatocytes. In the embryos with 15.5 days began the verify distinction between the cords of hepatocytes in formation limited by capillary sinusoids. Such cords began to converge for the present centrilobular veins. At 16.5 days the parenchymal architecture was nearer found in the adult liver, being the quantity of hepatocytes greater than hepatoblasts. During this gestation period the liver also had hematopoietic function. The study brings histologically the rats F344 hepatic development between 12.5-16.5 days, evidencing the cells that comprise each gestational period generating subsidies for future studies.