## 3 Molecular Mechanisms of Omega-3 Fatty Acids in Cancer
### DHA and Autophagy in Crosstalk with Cell Cycle Modulation  
Other studies have demonstrated an alternative mechanism through which PUFAs, particularly DHA, induce cell cycle arrest in cancer cell lines concomitant with autophagy. An investigation examined the interplay between these two biological processes: cell cycle modulators, such as CKDIs--notably p16, p21, p27 and p57--and cyclin-dependent kinases were revealed to additionally regulate autophagy in addition to their primary roles in cell cycle regulation [60]. Similarly, autophagy has been demonstrated to regulate cell cycle progression and play a role in cancer development, adaptation and survival, exhibiting context-dependent effects. Figure 4: General mechanism by which n-3 affects the cell cycle and cell death. \(\downarrow\), decrease; \(\uparrow\), increase; CDK1, Cyclin-dependent kinase 1; CDK2, Cyclin-dependent kinase 2; CDK4, Cyclin-dependent kinase 4; CDK6, Cyclin-dependent kinase 6; LC3, Microtubule-associated protein 1 light chain 3; n-3 PUFA, n-3 polyunsaturated fatty acids. Numerous studies have demonstrated that n-3 PUFAs activate both endoplasmic reticulum (ER) and mitochondrial-dependent pathways of apoptosis. [PERSON] et al. showed that DHA produced ER stress and also induced unfolded protein response [56]. In SW620 cells, this process is initiated by the dissociation of phosphorylated extracellular signal-regulated kinases (p-ERK) from the ER-resident chaperone BiP/GRP78, which participates in various complexes [57]. The combined application of DHA and EPA proved to be more efficacious in inducing apoptosis in cancer stem-like cells SW620 than either DHA or EPA administered individually [58]. This dual role of autophagy in cancer was also demonstrated by [PERSON] et al., wherein it was implicated in both the development of multidrug-resistant (MDR) cancer and the activation of the apoptosis pathway in MDR cancer, contingent upon contextual factors [61]. Continuing along this line of investigation, a study revealed the inhibitory effects of DHA in a human esophageal cancer cell line, leading to a G2/M cell cycle arrest concomitant with a significant upregulation of microtubule-associated protein light chain (LC3) and a slight downregulation of p62, both recognized as autophagy protein markers (Figure 3). These findings suggest that DHA may have induced autophagy in this particular cell line [62]. Additionally, this phenomenon was accompanied by the production of ROS. It was observed that the induction of autophagy by DHA was diminished upon the addition of N-acetylcysteine NAC, an antioxidant, suggesting that ROS was primarily responsible for this effect. A study conducted in human epithelial ovarian cancer cells yielded comparable findings, demonstrating growth-inhibitory effects of DHA accompanied by induction of cell cycle arrest at the G2/M phase, alongside upregulation of LC3 levels (Figure 4), with effects observed in a dose-dependent manner. Notably, this effect was markedly attenuated when cells were co-treated with CQ and Bafilomycin A1, both autophagy inhibitors. Additionally, DHA exhibited anti-inflammatory properties, mechanistically attributed to its inhibition of the NF-kB signaling pathway [63]. Regarding the mechanism by which DHA induces autophagy in cancer cells, [PERSON] et al. investigated this phenomenon in the HeLa cell line. They observed upregulation of phosphorylation of Beclin (Bcl)-2 at Ser70 (p-Bcl-2) without altering its expression level within the cell [63]. Usually, Bcl-2 is bound to Bcl-1, whose function is linked to the initiation of autophagy in mammalian cells and is hindered when bound to Bcl-2. However, p-Bcl-2 may dissociate from Bcl-1. This dissociation occurs independently of c-Jun N-terminal kinase (JNK) 1/2, a kinase protein involved in Bcl-2 phosphorylation at Ser70 in response to increased levels of ROS, which is one of the reported effects of DHA.