Evaluation of the cytotoxic effect of conjugated linolenic acids on the in vitro ZMEL1 model, comparison between different isomers and with other fatty acids

Details

Supervisors

Larondelle, Yvan

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

Cancer remains a major challenge for the scientific community. According to WHO estimates, one in six deaths is due to cancer. Polyunsaturated fatty acids have long been known for their numerous health benefits. Among them, conjugated linolenic acids (CLnA), naturally present in large quantities in specific vegetal oils, have recently been highlighted for their potential strong anti-cancer effects. These effects have already been widely demonstrated in vitro on human cancer cells, but few solid in vivo studies have been conducted to date. Recently, a very promising in vivo model for the analysis of tumor development has been developed. This model is composed of Casper zebrafish, a strain created to have no skin pigmentation and reduced immunity, grafted with fluorescent melanoma cells (ZMEL1). In preparation for using this model to determine the anti-carcinogenic effects of CLnA, this master's thesis aimed to first test the cytotoxicity of CLnA on ZMEL1 cells in an in vitro approach. Four different CLnA isomers namely Punicic acid (PunA), α-eleostearic acid (α-ESA), β-eleostearic acid (β-ESA) and Jacaric acid (JA) were tested for their cytotoxic effect. Their effect on the viability of ZMEL1 cells were also compared to different other type of fatty acids including saturated, monounsaturated and omega 3 polyunsaturated fatty acids. PrestoBlue viability assays were performed after applying different concentrations of fatty acids for a treatment time of 72 h. Preliminary experiments were performed to optimize the parameters of the viability assay, initially designed for mammalian cells. As expected, the results concerning CLnA showed significant toxicity for all four isomers, with the strongest toxic effect for JA. Experiments were performed on different subclones and passage numbers. The results showed that the sensitivity of ZMEL1 cells to CLnA varies strongly with subclone origin and increase for high passage ZMEL1 cells (passages 28-29-30). All other fatty acids showed no important toxicity compared to CLnA with the exception of saturated fatty acids (palmitic acid and stearic acid) which showed a significant toxicity. In order to investigate how CLnA are handled by ZMEL1 cells, the fatty acid profiles of the cells and culture solutions were analysed after a treatment with 40 µM of PunA for 6 and 8 h. The analyses were performed by gas chromatography after fatty acid extraction by the Bligh and Dyer method. The results showed that PunA is very well taken up by ZMEL1 cells and that a relevant part of PunA is integrated into the phospholipid membrane.