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

Abstract

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.

Keywords: Antitumoral alkylphospholipids; Hepatocellular carcinoma; Liposomes; Nanomedicine.

Fig. 1

Analysis of cell cycle phases of Hepa1c1c7 cells treated with PHO-S, DODAC and DODAC/PHO-S. The cells were treated with different concentrations of the formulations for a period of 12 h. The graph shows the correlation of the effect expressed as a mean ± SD of three independent experiments. a Representative histograms of cell cycle analysis. Y-axis represent number of cells; X-axis represents DNA content (PI intensity). b Population of cells in sub-G1, G₀/G₁, S and G₂/M  phase. Level of statistical significance * P < 0.05, ** p < 0.01 and *** p < 0.001

Fig. 2

Evaluation of cell death mechanism by quantifying the percentage of Hepa1c1c7 cells undergoing apoptosis, late apoptosis and necrosis, by flow cytometry using Annexin V / PI kit. The Hepa1c1c7 cells were treated with different concentrations of PHO-S, DODAC/PHO-S and DODAC, for a period of 12 h. a representative dot plots of fluorescence levels obtained by staining the cells for Annexin V/PI kit. b The bar chart shows the correlation of the effect expressed as a mean ± SD of three independent experiments. Level of statistical significance * P < 0.05, ** p < 0.01 and *** p < 0.001

Fig. 3

Photomicrographs of liposomes DODAC/PHO-S/Dil in Hepa1c1c7 cells analyzed by confocal laser microscopy. Cells were treated with the liposomal formulation (0.3 and 2.0 mM) with a fluorescent marker for a period of 3 to 6 h. Labeled liposomes can be observed and distinguished by the colour green in the cytoplasm of the cells. Cells treated with a and b DODAC/PHO-S 0.3 mM; c and d DODAC/PHO-S  2.0 mM, for a period of 3 h. Cells treated with e and f DODAC/PHO-S 0.3 mM; g, e, h DODAC/PHO-S 2.0 mM, for a period of 6 h

Fig. 4

Photomicrographs of the Hepa1c1c7 cells mitochondria labeled with rhodamine 123 (green) and nucleus with DAPI (blue) and analyzed by confocal laser microscopy. Cells were treated with PHO-S, DODAC and DODAC/PHO-S 1: 1, for a period of 6 h and mitochondria were labeled with rhodamine 123. a and b control; c and d PHO-S -0.3 mM; e and f PHO-S 2.0 mM; g and h DODAC/PHO-S 0.3 mM; i and j DODAC/PHO-S 2.0 mM; k and l DODAC 0.3 mM; m and n DODAC 2.0 mM. Scale bar = 10 and 20 μm

Fig. 5

Photomicrographs of the cells of murine Hepa1c1c7 hepatocarcinoma lysosomes labeled with acridine orange (green) and nucleus with DAPI (blue) and analyzed by confocal laser microscopy. Cells were treated with PHO-S, DODAC and DODAC/PHO-S 1: 1, for a period of 6 h and lysosomes and nucleus were labeled. a control; b and c  PHO-S 2.0 mM; d and e DODAC/PHO-S 2.0 mM; f DODAC 2.0 mM. Scale bar = 20 μm

Fig. 6

Analysis of protein expression in Hepa1c1c7 cells. The expression of proteins was quantified by flow cytometry after 24 h of treatment with PHO-S, DODAC and DODAC/PHO-S. The bar graphs show the level of expression of TRAIL - DR4 receptor (a); protein caspase 3 (b); caspase-8 protein (c); Free cytochrome c (d); protein Bax (e); protein Bcl-2 (f). Values are expressed as a mean ± SD standard deviation of three independent experiments. Level of significance * P < 0.05, ** p < 0.01 and *** p < 0.001

Fig. 7

Analysis of expression of proteins involved in the proliferation and metastatic process in Hepa1c1c7 cells. The expression of the proteins was quantified by flow cytometry after 24 h of treatment with PHO-S, DODAC and DODAC/PHO-S. The bar graphs show the expression level of the protein p21 protein (a); p27 protein (b); p53 protein (c); cyclin D1 protein (d); CD90 marker (e) and CD44 marker (f). Values are expressed as a mean ± SD standard deviation of three independent experiments. Level of significance ** p < 0.01 and *** p < 0.001