Punicic acid, a trojan horse to induce ferroptosis in prostate cancer cells

Ferroptosis is a recently discovered form of regulated cell death that is executed through extensive polyunsaturated fatty acid (PUFA) peroxidation, the presence of redox-active iron and a defective lipid peroxide repair. Multiple drugs have already been identified as ferroptosis inducers in cancer cells. Another strategy, yet largely unexplored, may lie in the promotion of lipid peroxidation by preferentially introducing high amounts of peroxidable PUFA in cancer cells. We propose here that plant-derived conjugated linolenic acids (CLnA) represent an attractive PUFA source to promote ferroptosis. Due to the conjugated configuration of their three double bonds, CLnA are much more peroxidation-prone than non-conjugated PUFA. Recently, our team has shown that punicic acid (PunA), a CLnA isomer, acts as a strong ferroptosis inducer in carcinoma cells. Taking advantage of the high dependency of prostate cancer cells on fatty acid metabolism, we have compared the ferroptosis-sensitizing potential of PunA in prostate cancer cells. Androgen-negative therapy-resistant prostate cancer cells were more sensitive than androgen-positive cells (i.e. IC50 of 1.5 µM versus 14 µM, respectively). PunA toxicity was further enhanced by inhibition of either ß-oxidation or lipid droplet incorporation, two protective mechanisms diverting PUFA from peroxidation-prone species. PunA differential effects were further supported by reduced expression in androgen-negative cells, of enzymes involved in PUFA incorporation into phospholipids and lipid peroxide cellular detoxification. Lipidomic analysis also revealed that PunA uptake and incorporation within cellular lipid species are increased by two-fold in the most sensitive cells. Altogether, our findings reveal critical redox metabolic differences in prostate cancer cell subtypes that may be used to select the most ferroptosis-sensitive ones

Resistance to CLnA-induced ferroptosis is acquired in Caco-2 cells upon differentiation

In contrast to canonical ferroptosis inducers, highly peroxidable conjugated linolenic acids (CLnA) directly fuel the lipid peroxidation cascade upon their incorporation into membrane phospholipids. Little is known, however, about the cytotoxicity level of CLnAs to normal epithelial cells. Caco-2 cells, derived from colorectal adenocarcinoma, spontaneously differentiate into enterocyte-like cells over a period of 21 days of cell culturing, allowing for graduated phenotypic shift from proliferative, undifferentiated cells to a functional intestinal barrier. We exploited this property to assess the sensitivity of Caco-2 cells to CLnAs at different stages of differentiation. Our results show a significant decrease in CLnA-induced ferroptotic cell death over time. The acquired resistance aligned with decreases in cell proliferation and in the extent of lipid peroxidation, as well as with an increase in the expression of GPX4 upon differentiation. These results highlight that while CLnAs are highly toxic for proliferating cancer cells, differentiated epithelial cells are resistant to CLnA-induced ferroptosis. Therefore, this study gives credential to the therapeutic use of CLnAs as an anticancer strategy and offers a new model study to further investigate the safety of peroxidable fatty acids in differentiated cells

Punicic Acid Triggers Ferroptotic Cell Death in Carcinoma Cells

Plant-derived conjugated linolenic acids (CLnA) have been widely studied for their preventive and therapeutic properties against diverse diseases such as cancer. In particular, punicic acid (PunA), a conjugated linolenic acid isomer (C18:3 c9t11c13) present at up to 83% in pomegranate seed oil, has been shown to exert anti-cancer effects, although the mechanism behind its cytotoxicity remains unclear. Ferroptosis, a cell death triggered by an overwhelming accumulation of lipid peroxides, has recently arisen as a potential mechanism underlying CLnA cytotoxicity. In the present study, we show that PunA is highly cytotoxic to HCT-116 colorectal and FaDu hypopharyngeal carcinoma cells grown either in monolayers or as three-dimensional spheroids. Moreover, our data indicate that PunA triggers ferroptosis in carcinoma cells. It induces significant lipid peroxidation and its effects are prevented by the addition of ferroptosis inhibitors. A combination with docosahexaenoic acid (DHA), a known polyunsaturated fatty acid with anticancer properties, synergistically increases PunA cytotoxicity. Our findings highlight the potential of using PunA as a ferroptosis-sensitizing phytochemical for the prevention and treatment of cancer