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.

Ferroptosis has recently emerged as a promising strategy to combat therapy-resistant cancers. As lipid peroxidation is a key trigger of ferroptotic cell death, enhancing cancer cell susceptibility through the supply of highly peroxidisable fatty acids represents a novel therapeutic approach. Conjugated linolenic acids (CLnAs) fulfill this requirement, exhibiting a peroxidation propagation rate eight times higher than their non-conjugated counterpart, α-linolenic acid. This study evaluates jacaric acid (JA), a plant-derived CLnA, as a ferroptotic inducer, both as a monotherapy and in combination with RAS-selective lethal 3 (RSL3), a canonical ferroptosis inducer, in 2D and 3D breast cancer cell models. JA treatment significantly reduced cell viability across all models, primarily through lipid peroxidation driven by JA incorporation into cellular lipids rather than alterations in anti-ferroptotic gene expression. Moreover, JA synergistically enhanced RSL3 cytotoxicity under 2D and several 3D conditions. Similar effects were observed with punicic acid, another plant-derived CLnA isomer. Our study exploits a common feature of cancer metabolism, increased fatty acid uptake, to turn it into a vulnerability. The incorporation of JA into breast cancer cells creates a highly peroxidisable environment that increases cancer cell sensitivity to RSL3, potentially reducing required doses and minimising side effects.