Punicic acid-related cytotoxicity on SiHa cancer cells at physiological pH or under chronic acidosis

In Europe, cancer accounted for a quarter of the total number deaths in 2015. Poor dietary habits increase the risk of developing cancer. However, we could take this opportunity, tip the balance and make the diet a preventive factor. Cancer is a term encompassing a group of diseases in which cells divide without control and can invade other organs in the body. During tumor development, cancer cells face different conditions in their microenvironment such as hypoxia and acidosis, under which they adapt their metabolism to survive and proliferate. The major contribution of lipids in cancer cell proliferation under an acidic environment was recently demonstrated. Indeed, fatty acid synthesis and oxidation, two exclusive pathways in normal cells, have been found to occur concomitantly in cancer cells under chronic acidosis. Of particular interest, cancer cells become addicted to exogenous fatty acids. Among these, some unusual fatty acids, such as punicic acid (PunA) have been proposed to exert a cytotoxic effect on these cells. PunA is a fatty acid with 18 carbons and 3 conjugated double bonds (c9,t11,c13-C18:3) that is found in pomegranate (Punica granatum) seed oil. PunA has been studied in vitro on SiHa cells (cervix cancer) grown at the physiological pH of 7.4 or adapted to chronic acidosis at pH 6.5. In viability experiments, PunA was confirmed to be highly cytotoxic for SiHa cells. Surprisingly, cells under chronic acidosis were more resistant to PunA than cells at physiological pH. In an effort to bring the in vitro model closer to realistic physiological conditions, some common dietary fatty acids were combined with PunA and were found to inhibit the PunA cytotoxicity in a dose-dependent manner for the cells treated with PunA 14 µM at pH 7.4, as well as for those treated with PunA 35 µM at pH 6.5. In contrast, the effects were very limited for cells at pH 7.4 treated with PunA 35 µM. Palmitic, stearic, oleic and linoleic acids displayed a similar and efficient effect while the α-linolenic acid effect was weaker or absent. Vitamin E powerfully protected the cells against the PunA-related cytotoxicity. The effect was dose-dependent from low concentrations (0.03 µM or 0.2 µM). Two fibroblast cell lines used as a model of normal cells were also highly sensitive to PunA (from 7 or 14 µM). In a second set of experiments, the fatty acid profiles of SiHa cells exposed to PunA alone or PunA + palmitic or α-linolenic acids were analyzed. The absorption rate of PunA was considerably higher when PunA was added alone. The PunA proportions in the neutral and phospho-lipids fractions were higher when cells were treated with PunA alone. PunA was proportionally more oriented towards the neutral lipids in cells under chronic acidosis. A low proportion of PunA (<10% after 24h) was metabolized into rumenic acid. The use of PunA for other biological phenomena is suspected. A yet-to-be-proven model was proposed in which lipid peroxidation would account for cell decline. The outreaching of a threshold in the PunA proportion in the phospholipid would act as the main determinant of cell death. The addition of a second fatty acid was suggested to inhibit the PunA-related cytotoxicity by a competition effect at the absorption level. This master’s thesis allows to highlight that PunA incorporation in the diet or therapeutic administration could be envisioned but that many questions are yet to be addressed to confirm or not its efficiency in a human body environment and to understand the mechanisms underlying cytotoxicity and its determinant parameters. In case the positive impacts of PunA are attested, the development of health promoting foods containing pomegranate seed oil that is extracted from by-products of juice production would be an excellent way to enhance the sustainability of the pomegranate production chains by avoiding waste, generating economic activity and providing people with healthy beneficial items.