The Involvement of Polyunsaturated Fatty Acids in Apoptosis Mechanisms and Their Implications in Cancer

Cancer is a significant global public health issue and, despite advancements in detection and treatment, the prognosis remains poor. Cancer is a complex disease characterized by various hallmarks, including dysregulation in apoptotic cell death pathways. Apoptosis is a programmed cell death process that efficiently eliminates damaged cells. Several studies have indicated the involvement of polyunsaturated fatty acids (PUFAs) in apoptosis, including omega-3 PUFAs such as alpha-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid. However, the role of omega-6 PUFAs, such as linoleic acid, gamma-linolenic acid, and arachidonic acid, in apoptosis is controversial, with some studies supporting their activation of apoptosis and others suggesting inhibition. These PUFAs are essential fatty acids, and Western populations today have a high consumption rate of omega-6 to omega-3 PUFAs. This review focuses on presenting the diverse molecular mechanisms evidence in both in vitro and in vivo models, to help clarify the controversial involvement of omega-3 and omega-6 PUFAs in apoptosis mechanisms in cancer.


Introduction
According to the World Health Organization (WHO), cancer is a significant global public health problem, with over 19,292,789 new cases and 9,958,133 deaths reported in 2020 [1]. Despite advancements in cancer therapy, conventional treatments often result in unsatisfactory survival rates. One of the primary processes affected in cancer is cell death pathways, and resistance to therapy often occurs due to the evasion of apoptosis [2]. Apoptosis is a programmed cell death process characterized by two main activation pathways: the extrinsic and intrinsic pathways [3]. There is substantial evidence suggesting that polyunsaturated fatty acids (PUFAs) play a crucial role in cancer development and can regulate various biological processes, including apoptosis [4][5][6]. Several studies have attributed a pro-apoptotic activity to omega-3 PUFAs, as they modulate the expression of key molecules involved in apoptosis induction [7,8]. For example, omega-3 PUFAs have been shown to upregulate miRNAs involved in the regulation of apoptotic genes in glioma cells [9]. On the other hand, the effect of omega-6 PUFAs remains controversial, with some authors indicating their role in inducing cell death; however, others suggest otherwise [10]. These findings are significant because PUFAs are considered essential fatty acids that are consumed through the diet [11]. Based on the first unsaturation and counting from the end of the alphatic chain, there are two types of PUFAs [12]: (1) omega-3 PUFAs, such as alpha-linolenic acid (ALA) (C18:3, n-3), eicosapentaenoic acid (EPA) (C20:5, n-3), and docosahexaenoic acid (DHA) (C22:6, n-3) and (2) omega-6 PUFAs, including linoleic acid (LA) (C18:2, n-6), gamma-linolenic acid (GLA) (C18:3, n-6), and arachidonic acid (ARA) (C20:4, n-6) [13].

Omega-3 PUFAs and Apoptosis
Omega-3 PUFAs, including ALA, EPA, and DHA, play a crucial role in regulating various cellular functions such as membrane fluidity, protein functions, eicosanoid metabolism, gene expression, and cell signaling [25]. These fatty acids have been extensively studied for their potential in inhibiting the occurrence and progression of cancer. In fact, human studies have demonstrated that a high consumption of fish oil, rich in omega-3 PUFAs, can reduce the risk of cancer [26,27]. Furthermore, numerous studies have provided evidence supporting the role of omega-3 PUFAs in the regulation of apoptotic pathways, which is considered one of the biological mechanisms responsible for their positive effects. In this section, we will examine the involvement of omega-3 PUFAs in triggering apoptotic pathways.

Alpha-Linolenic Acid
ALA is an essential polyunsaturated fatty acid (PUFA) that cannot be synthesized by the human body and must be obtained from the diet [28]. It is found in leafy green vegetables, walnuts, flaxseeds, hemp, canola, and soybean oils [29]. Previous studies have demonstrated the anticarcinogenic potential of ALA. For instance, ALA has been shown to induce apoptosis through various molecular alterations. These include the accumulation of lipid droplets in the cytoplasm, lipid peroxidation, generation of ROS, induction of superoxide dismutase (SOD) activity in gastric carcinoma cells, (MGC and SGC) [30], and a mouse papilloma model [31].
Other studies have also suggested the participation of PUFAs as potent modulators of animal ion channels. They can form micelles that can fuse with the lipid bilayer, influencing cell membrane organization and altering channel function. PUFAs are known to increase membrane fluidity and screen surface charges, resulting in a shift in the voltagedependence of the channels [32]. Particularly, ALA can cause accumulation and increase in intracellular calcium ion (Ca 2+ ) levels, generating arrest in the sub-G1 phase and triggers the pro-apoptotic process [33,34]. Additionally, it has been observed that ALA provides a downregulation of the voltage-dependent anion channel (VDAC) present in the outer mitochondrial membrane, support the mitochondrial apoptosis [35].
Regarding the extrinsic pathway, one study reported that ALA induces an increased levels of caspase-8 in ER+ MCF-7 breast cancer cells [35]. Another study demonstrated an increase in Fas ligand (FasL), activation of caspases-8, -3, -7, and DNA damage as indicated by elevated p-H2A.X levels in human Jurkat cells [40] ( Figure 2B). In summary, all this evidence supports the notion that the consumption of ALA primarily triggers the activation of mitochondrial apoptosis and to a lesser extent the extrinsic pathway, imparting anticancer effects.  Regarding the extrinsic pathway, one study reported that ALA induces an increased levels of caspase-8 in ER+ MCF-7 breast cancer cells [35]. Another study demonstrated an increase in Fas ligand (FasL), activation of caspases-8, -3, -7, and DNA damage as indicated by elevated p-H2A.X levels in human Jurkat cells [40] ( Figure 2B). In summary, all this evidence supports the notion that the consumption of ALA primarily triggers the activation of mitochondrial apoptosis and to a lesser extent the extrinsic pathway, imparting anticancer effects.

Eicosapentaenoic Acid
EPA can be obtained from ALA metabolism by a series of desaturation and elongation reactions [41]. Moreover, EPA is found in seafood like salmon, tuna, sardine, and marine oils [42]. EPA has important anti-cancer effects, mediated by the induction of expression of anti-inflammatory mediators, inhibition of cell proliferation, and modulation of cell death pathways [43]. The proposal mechanisms in the activation of apoptosis by EPA involve changes in numerous cell pathways. For example, induction of cell cycle arrest on breast cancer cell lines (MCF-7, SKBR-3, and MDA-MB-231) [44], particularly arresting the progression of the cell cycle from S to G2-M phase on BT20 breast tumor cells [45], downregulation of Akt/NFkB cell survival pathway on human breast cancer cell line MDA-MB-231 [46], in combination with DHA and vitamin D3 promote the upregulation of Raf/MAPK pathway [47], modification of lipid rafts and sustained activation of EGFR/p38MAPK pathway on MDA-MB-231 cells [48], in combination with estrogen increase GPER1/cAMP/PKA signaling on MCF-7 and T47D cells [49], inhibition of cholesterol biosynthesis inducer (SREBP2) and cholesterol efflux channel protein (ABCA1), causing cellular accumulation of cholesterol and subsequent increase in cell membrane polarity on TNBC cells [50]. Moreover, studies have shown that EPA induces inhibition of the phosphorylation of ERK1/2, Akt, and mammalian target of rapamycin (mTOR), blocking nuclear factor κB (NF-κB) p65 translocation from the cytoplasm into the nucleus on SKOV-3 cells [51]. This agrees with other authors who presented the inhibition of NFκB activity on multiple myeloma (MM) cells model [52,53]. Additionally, another main trigger is the high production of ROS after EPA treatment, which has been reported on breast cancer cells [44], bladder cancer cells [54], colorectal cancer cells [55], human glioma cells [56], and PC3 prostate cancer cells lines [57]. Zhang et al. described that ROS formation, leads to Ca 2+ accumulation, the mitochondrial permeability transition pore opening, and JNK activation on the HepG2 cells [58]. Other less common mechanisms including Bcl-2 suppression through the p53/miR-34a axis on MM cells [59], wt-p53 accumulation on wt-p53 Molt-4 cells ALL cell line [60], inhibition of Akt/mTOR pathways through PPARγ/PTEN axis on MCF-7 cells [61], activation of acyl-CoA synthetase (ACS) on lymphoma cell line Ramos [62], and displacement of ARA from tumor-cell membranes after omega-3 PUFAs treatment on in vivo model [63].
The available data indicates that apoptosis induced by EPA, also follows the extrinsic pathway, as shown in Figure 3B. Ewashuk et al. showed that EPA increased FAS surface expression on MDA-MB-231 cells [77]. Furthermore, Giros et al. reported downregulation of FLICE inhibitory protein (FLIP) on colorectal cancer cells [75]. Additionally, Fukui et al. also described that EPA caused caspase-8 dependent apoptosis on human pancreatic cancer cells MIA-PaCa-2 and Capan-2 [78]. This same effect was observed on human promyelocytic leukemia cells) [79], and breast tumor cells [45]. Moreover, evidence has shown that a crosstalk exists between both the extrinsic and intrinsic pathways mediated by EPA mainly through tBid [79]. Complementing this idea Giros et al. indicated that the timing of caspase-8 activation, and the oligomerization of Bid with Bax, support this crosstalk. It is worth noting that none of the mechanisms investigated were found to be responsible for triggering the apoptosis cascade induced by EPA [75]. Likewise, treatment with C20E (an analog of EPA) through TNFR1 activate ASK1-MKK4/7-JNK/p38MAPK pathway, promoted tBid, leading to MOMP and the activation of the mitochondrial pathway on human triple-negative MDA-MB-231 breast cancer cells and xenograft models [80] (see Figure 3C). mechanisms, including both classical and alternative apoptotic pathways, and can regulate survival and cell growth signaling, ultimately leading to cell death in various cellular and animal models. However, further experiments are needed to explore the specific details of the extrinsic pathways, especially the upstream events. These findings suggest that EPA holds promise as a potential novel treatment for cancer, which could complement existing therapies.  In summary, the research findings indicate that EPA can activate multiple molecular mechanisms, including both classical and alternative apoptotic pathways, and can regulate survival and cell growth signaling, ultimately leading to cell death in various cellular and animal models. However, further experiments are needed to explore the specific details of the extrinsic pathways, especially the upstream events. These findings suggest that EPA holds promise as a potential novel treatment for cancer, which could complement existing therapies.

Docosahexaenoic Acid
DHA is a marine-derived PUFA, that can be obtained from the metabolism of ALA [42,81]. Numerous studies have demonstrated the significant antitumor effects of DHA, including the suppression of neoplastic transformation, angiogenesis, and proliferation, as well as the induction of apoptosis in various cancer cells [65,82].
DHA exerts its proapoptotic effect by altering various molecular mechanisms. One of the key mechanisms involves modifications of the plasma membrane environment, which result in changes in the molecular composition, fluidity, structure, and function of lipid rafts [10]. These alterations impact the localization of cell surface receptors such as G protein-coupled receptors (GPCRs), Toll-like receptors (TLRs), retinoid X receptors (RXRs), and the epidermal growth factor receptor (EGFR), which play crucial roles in regulating cell growth signaling and apoptosis [83][84][85]. Another mechanism that affects the plasma membrane is the upregulation of syndecan-1 (SDC-1), a plasma membrane molecule that is induced by DHA through peroxisome proliferator-activated receptorgamma (PPARγ) [86,87].
Another important mechanism by which DHA triggers apoptosis is through oxidative stress resulting from the accumulation of ROS and lipid peroxidation [100]. DHA has been shown to stimulate ROS generation in various cell lines, including human promyelocytic leukemia cells (HL-60) [79], A549 cells [82], HT-29 cells [100], C6 glioma, SH-SY5Y neuroblastoma cell lines [93], cisplatin-resistant gastric cancer SNU-601/cis2 cells [101], docetaxel-resistant PC3 prostate cancer cells [102], and in an in vivo model of breast cancer [103]. Tsai et al. demonstrated that increased ROS production by DHA activates the PI3K/Akt/Nrf2 signaling pathway and induces the expression of Oxidative stress-induced growth inhibitor 1 (OSGIN1) [104]. Similar, Nrf2 nuclear translocation in response to oxidative stress has been observed in AML cell lines (U937, MOLM-13, and HL-60) [105]. Furthermore, downregulation of antioxidant enzymes such as catalase (CAT) in A549 cells [82] and glutathione (GSH) in the human PaCa-44 pancreatic cancer cell line has been reported to contribute to the accumulation of intracellular ROS [106]. However, Geng et al. demonstrated that DHA treatment increased the activity levels of major antioxidant enzymes, including total superoxide dismutase (t-SOD), CAT, and glutathione peroxidase (GSH-PX), while decreasing the concentration of malondialdehyde (MDA) in human malignant breast tissues [97]. The effect of DHA on antioxidant enzymes is not completely clear, and discrepancies observed may be related to the cell models, requiring further research. Another proposed mechanism is that DHA causes inactivation of prostaglandin family genes, lipoxygenases, and alters the expression of PPARα and γ, leading to lipid peroxidation in CaCo-2 colon cancer cell lines [107].
Furthermore, recent innovative studies have focused on how DHA exerts a proapoptotic effect through the activation of ER stress. It is well-known that conditions interfering with ER function lead to the accumulation and aggregation of unfolded proteins, which can trigger apoptotic cell death if the stress is prolonged or the adaptive response fails [108]. Through RNA-seq analysis of MDA-MB-231 cells treated with DHA, Chénais et al. showed that DHA downregulates genes involved in the cholesterol biosynthesis pathway and upregulates genes associated with ER stress response, including NEF, BiP, HSP40, GADD34, ATF4, IRE1a, XBP1, CHOP, ERO1B, SEL1L, HERPUD1, and HSPA6 [109]. Similarly, Jakobsen et al. demonstrated that DHA induces the expression of various factors involved in ER stress response, such as XBP1, PERK, ATF4, ATF6, and phosphorylated EIF2a, in SW620 colon carcinoma cells [110]. This discovery has also been observed in cisplatin-resistant gastric cancer SNU-601/cis2 cells [101].
Other studies also support the activation of the extrinsic pathway by DHA ( Figure 4B), which involves an increase in death receptors such as TRAIL, death receptor 4 (DR4), and FAS in MCF 7 breast cancer cells [115]. Additionally, Ewaschuk et al. demonstrated that DHA increases FAS surface expression and induces the movement and raft clustering of FAS and FADD in the cell membrane of MDA-MB-231 cells [77]. In contrast, Giros et al. did not find any of the death receptors (FAS, TNFR1, and TRAIL-R2) to be responsible for triggering the apoptosis cascade induced by DHA. However, they proposed that the downregulation of FLIP is responsible for cell death [75]. Further research is required to elucidate the effect of DHA on death receptors. Moreover, the effect of DHA on different death ligands has also been investigated. Particularly, DHA sensitizes the apoptotic response to TNF-α and anti-FAS antibody (CH-11) in human colon adenocarcinoma HT-29 cells [100]. It has also been reported that DHA combined with TRAIL treatment triggers the extrinsic pathway, causes ER stress, decreases XIAP and cIAP1 levels, and alters sphingolipid metabolism, inducing apoptosis in human colon cancer cells [122,123]. Similarly, Fluckiger et al. proposed a novel indirect mechanism in which treatment with DHA leads to the nuclear accumulation of Foxo3a, which binds to the microRNA-21 (miR-21) promoter, triggering its transcriptional repression. This, in turn, increases TNFα mRNA levels and induces apoptosis in an autocrine manner in human colorectal cancer cells [124]. These events can activate caspase-9 of 24 8 and executioner caspases, thereby promoting apoptosis [79,103]. Similar to EPA, DHA can also initiate a crosstalk between the extrinsic and intrinsic apoptotic pathways through Bid cleavage ( Figure 4C) [75,79,122,123]. moter, triggering its transcriptional repression. This, in turn, increases TNFα mRNA levels and induces apoptosis in an autocrine manner in human colorectal cancer cells [124]. These events can activate caspase-8 and executioner caspases, thereby promoting apoptosis [79,103]. Similar to EPA, DHA can also initiate a crosstalk between the extrinsic and intrinsic apoptotic pathways through Bid cleavage ( Figure 4C) [75,79,122,123].
In conclusion, it is important to highlight that DHA induces apoptosis through direct or indirect pathways, involving the suppression of survival pathways and the activation of extrinsic and intrinsic death pathways. These findings demonstrate that DHA has the ability to induce cell apoptosis, offering promising and safe options for cancer treatment in the future.  In conclusion, it is important to highlight that DHA induces apoptosis through direct or indirect pathways, involving the suppression of survival pathways and the activation of extrinsic and intrinsic death pathways. These findings demonstrate that DHA has the ability to induce cell apoptosis, offering promising and safe options for cancer treatment in the future.

Omega-6 PUFAs and Apoptosis
In Western populations, there has been an observed increase in the consumption of omega-6 PUFAs, resulting in a skewed omega-6/omega-3 PUFA ratio of 20:1 [125]. This imbalance has been suggested to contribute to the rising incidence of cardiovascular, inflammatory, and oncological diseases [14]. Mounting evidence indicates that the adverse effects associated with omega-6 PUFAs may be attributed to arachidonic acid (ARA). On the other hand, linoleic acid (LA) and γ-linolenic acid (GLA) have demonstrated certain anti-cancer activities, including the induction of apoptosis [126]. In this section, we will present and discuss recent findings on the potential effects of omega-6 PUFAs in apoptotic processes.

Linoleic Acid
Like ALA, LA is an essential polyunsaturated fatty acid that humans can only obtain from their diet [28]. LA is the most abundant PUFA in nature, comprising 50% to 80% of the fatty acids found in vegetable oils such as soybean, sunflower, safflower, and corn oils [127,128]. In the Western diet, LA is the primary PUFA, accounting for over 85% of PUFA intake [129]. Numerous pieces of evidence suggest that LA may be involved in various pathological processes, including cancer. Specifically focusing on cell death processes, the data indicates that LA has a proapoptotic effect.
The proposed mechanisms for LA-induced apoptosis involve the accumulation of lipid droplets and increased lipid peroxidation in gastric carcinoma cells [30], and colon cancer cells [130]. Additionally, an LA isomer called alpha-eleostearic acid (α-ESA), which possesses a conjugated triene system, has been associated with the inhibition of survival pathways. For example, it downregulates ERK1/2 through PPARγ in MCF-7 breast cancer cells [131], and inhibits the Akt/GSK-3β survival pathway by activating PTEN in SKBR3 and T47D breast cancer cell lines [132]. Another LA isomer, beta-eleostearic acid (β-ESA, 9E11E13E-18:3), induces apoptosis through oxidative stress, leading to the accumulation of ROS and a decrease in GSH in T24 human bladder cancer cells [128], as well as in gastric carcinoma cells [30]. Moreover, LA induces ROS production on HepG2 Cells [133]. The involvement of ER stress has also been investigated, with studies reporting intracellular calcium (CA 2+ ) accumulation and the expression of unfolded protein response (UPR)associated genes (CHOP, GRP78, and GRP94) in hepatoma H4IIE cell lines [134]. Another reported mechanism is the downregulation of prostaglandin E2 (PGE2) production and telomerase activity through the suppression of cyclooxygenase 2 (COX-2) expression in gastric adenocarcinoma AGS cells [135].
Through the aforementioned mechanisms, LA triggers the mitochondrial pathway by causing a loss of mitochondrial membrane potential, upregulating the expression of Bax and Bad, and downregulating the expression of Bcl-2. This leads to MOMP and the release of cytochrome c, subsequently activating caspase-9 and caspase-3 and reducing ATP levels in various cancer cell lines ( Figure 5A) [130][131][132][136][137][138][139]. Regarding the extrinsic pathway, Muzio et al. reported that conjugated linoleic acid (CLA), a term used for a series of isomers of linoleic acid, increases caspase-8 levels in human hepatoma SK-HEP-1 cells [140]. Liu et al. also demonstrated enhanced expression of FAS, a death receptor, in the SGC-7901 gastric adenocarcinoma cell line when exposed to the c9,t11 isomer of CLA [141], suggesting the induction of the extrinsic apoptotic pathway, as shown in Figure 5B. However, further research is needed to elucidate the detailed mechanisms of activation. telomerase activity through the suppression of cyclooxygenase 2 (COX-2) expression in gastric adenocarcinoma AGS cells [135]. Through the aforementioned mechanisms, LA triggers the mitochondrial pathway by causing a loss of mitochondrial membrane potential, upregulating the expression of Bax and Bad, and downregulating the expression of Bcl-2. This leads to MOMP and the release of cytochrome c, subsequently activating caspase-9 and caspase-3 and reducing ATP levels in various cancer cell lines ( Figure 5A) [130][131][132][136][137][138][139]. Regarding the extrinsic pathway, Muzio et al. reported that conjugated linoleic acid (CLA), a term used for a series of isomers of linoleic acid, increases caspase-8 levels in human hepatoma SK-HEP-1 cells [140]. Liu et al. also demonstrated enhanced expression of FAS, a death receptor, in the SGC-7901 gastric adenocarcinoma cell line when exposed to the c9,t11 isomer of CLA [141], suggesting the induction of the extrinsic apoptotic pathway, as shown in Figure 5B. However, further research is needed to elucidate the detailed mechanisms of activation.
All this evidence indicates that LA plays a crucial role in inducing apoptosis. Several lines of evidence suggest that the effect is primarily mediated through the intrinsic pathway, while more research is needed on this subject.  All this evidence indicates that LA plays a crucial role in inducing apoptosis. Several lines of evidence suggest that the effect is primarily mediated through the intrinsic pathway, while more research is needed on this subject.

Gamma-Linolenic Acid
GLA is synthesized in the body from LA through the action of the enzyme D-6desaturase. While small amounts of GLA can be obtained from green leafy vegetables and nuts, it is primarily produced endogenously [10]. Numerous pieces of evidence suggest that GLA exhibits anti-cancer activities both in laboratory studies and animal models [126].
Additionally, several studies have demonstrated the influence of GLA and its metabolites on the expression of various genes and proteins involved in the apoptotic process [10].
The primary mechanism proposed for the induction of apoptosis by GLA involves oxidative stress resulting from the accumulation of ROS and lipid peroxidation. Colquhoun et al. showed that GLA increases ROS and lipid peroxide production, while decreasing the activity of mitochondrial respiratory chain complexes I+III and IV and mitochondrial membrane potential in carcinosarcoma cells [142]. Similarly, high levels of MDA, a marker of lipid peroxidation, have been observed in leukemia cells treated with GLA, and the cytotoxic effects of GLA were blocked by the antioxidant BHT [143,144]. GLA has also been found to inhibit mitochondrial carnitine palmitoyltransferase I (CPT I) in Hep2 human larynx tumor cells, leading to fatty acid oxidation [145]. Another pathway implicated in GLA-induced apoptosis is the ROS/ASK1/JNK/p38 MAPK axis in KF28 ovarian cancer cells [146]. Furthermore, GLA has been shown to induce cell cycle arrest and sub-G1 accumulation in ER+ MCF-7 cells and lymphoblast cell lines (TK6 and WTK1) [35,147].
All of the aforementioned mechanisms contribute to intrinsic apoptosis by downregulating the expression of Bcl-xL and Bcl-2, upregulating Bad, leading to the loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase-9 and -3, cleavage of PARP, DNA fragmentation in various cell lines [142,[148][149][150], as well as in in vivo models [56,66,149,151]. The involvement of caspases in GLA-induced apoptosis has been confirmed by the inhibition of apoptosis through the use of a pan-caspase inhibitor (z-VAD-fmk) in leukemic cells [144]. Morphological and physiological changes indicative of apoptosis, such as nuclear staining with DNA-binding fluorochrome Hoechst 33342, chromatin condensation, nuclear fragmentation [143], and TUNEL-positive cells [56,148,150], have further supported the apoptotic effect of GLA ( Figure 6).
Taken together, these findings suggest that GLA predominantly induces intrinsic apoptotic pathway through mechanisms associated with the generation of oxidative stress. However, information regarding the involvement of GLA in the extrinsic pathway is limited, and further investigations are required to determine its role in the pathways that trigger extrinsic apoptosis.

Arachidonic Acid
ARA is typically esterified to membrane phospholipids and is one of the most abundant PUFA present in human body [152]. ARA consumption is very abundant in our daily diet, particularly in the Western diet, and various foods contain high concentrations of ARA, like eggs, lean meat and meat fats of beef, lamb, pork, chicken, duck, and turkey [153,154]. Additionally, ARA is synthesized by a series of desaturases and elongases from LA [155]. Plenty of studies have associated the high ARA intake with many adverse effects on the human body, including cancer promotion, mainly attributed to its metabolites like PGE2 [126]. However, increasing evidence also suggests that ARA has ability to enhance the cytotoxic action of various anti-cancer drugs and possess certain antitumoral activities, including proapoptotic effect [10]. In this context, it is noteworthy that ARA presents a contradictory role in cancer progression.
To understand this controversial effect, it is crucial to examine the molecular apoptotic pathways regulated by ARA. As shown in Figure 7A, the mechanisms proposed in the activation of apoptosis by ARA included the increased oxidative stress generated by ROS production and lipid peroxidation, high levels of MDA, 4-hydroxy-2-nonenal (4-HNE), high activity of SOD, and GSH-PX, as well as downregulation of GSH on several cancer cell lines [156][157][158][159][160]. In the same way, elevation of ROS levels and intracellular Ca 2+ concentration trigger activation of p38α MAPK and JNK1 pathways on human

Arachidonic Acid
ARA is typically esterified to membrane phospholipids and is one of the most abundant PUFA present in human body [152]. ARA consumption is very abundant in our daily diet, particularly in the Western diet, and various foods contain high concentrations of ARA, like eggs, lean meat and meat fats of beef, lamb, pork, chicken, duck, and turkey [153,154]. Additionally, ARA is synthesized by a series of desaturases and elongases from LA [155]. Plenty of studies have associated the high ARA intake with many adverse effects on the human body, including cancer promotion, mainly attributed to its metabolites like PGE2 [126]. However, increasing evidence also suggests that ARA has ability to enhance the cytotoxic action of various anti-cancer drugs and possess certain antitumoral activities, including proapoptotic effect [10]. In this context, it is noteworthy that ARA presents a contradictory role in cancer progression.
To understand this controversial effect, it is crucial to examine the molecular apoptotic pathways regulated by ARA. As shown in Figure 7A, the mechanisms proposed in the activation of apoptosis by ARA included the increased oxidative stress generated by ROS production and lipid peroxidation, high levels of MDA, 4-hydroxy-2-nonenal (4-HNE), high activity of SOD, and GSH-PX, as well as downregulation of GSH on several cancer cell lines [156][157][158][159][160]. In the same way, elevation of ROS levels and intracellular Ca 2+ concentration trigger activation of p38α MAPK and JNK1 pathways on human neuroblastoma SK-N-SH cells [161]. On the other hand, Bae et al. showed that ARA increases the processed form of XBP1 (Pxbp1) and phosphorylated Eif2α (p-Eif2α) triggering ER stress on HT-29 human colon cancer cells [162]. Moreover, another mechanism reported, involves the high accumulation of unmetabolized ARA which activates the enzyme sphingomyelinase (Smase), this produces elevated ceramide levels that induces apoptosis [163]. For this reason, several studies have used inhibitors of enzymes involved in ARA metabolism such as Cpla2, COX-2, CYP4A, fatty acid coenzyme-A ligase 4 (FACL-4), coenzyme-A independent transacylase (CoA-IT), 5-LOX, leading to accumulation of intracellular ARA, elevation ceramide levels, and induction of apoptotic cell death on several cancer cell lines [164][165][166][167]. ARA mediates the mitochondria-dependent death pathway through previous stimuli, causing downregulation of Bcl-Xl and Bcl-2, as well as the loss of potential in the mitochondrial membrane, release of cytochrome c, accompanied with activation of caspase-9 and -3 that trigger the breakdown of PPAR. As well of externalization of phosphatidylserine and condensation of chromatin, on AS-30D rat hepatoma cells [168], Y79 retinoblastoma cells [160], HT-29 human colon cancer cells [162], and LoVo and RKO cells [130], among others. Furthermore, studies have reported typical morphological changes of programmed cell death such as pyknosis, karyorrhexis, cell-shrinkage and -blebbing were observed [158]. The proapoptotic effect of ARA has been supported by other experiments such as mitochondrial permeability assay, TUNEL labeling, and DNA laddering experiments [149,157]. Upon analyzing all the studies on ARA, the evidence presented shows that ARA can elicit both pro-and anti-apoptotic effects. The observed differences may be related to the specific models or concentrations of this PUFA used in the studies. The conflicting results mentioned above suggest that further research is required to fully understand the correlation between ARA and the apoptotic process.  On the other hand, the evidence of ARA's participation in the extrinsic pathway is limited. In this regard, Polavarapu et al. demonstrated that the administration of ARA plus bleomycin increased the expression of FAS, caspases-8 and -3 in IMR-32 human neuroblastoma cells, suggesting the activation of the extrinsic apoptotic pathway [169]. However, more research is needed to fully elucidate this pathway ( Figure 7B).
In contrast to the aforementioned evidence, many reports propose an inhibitory role of ARA in apoptosis. It is worth mentioning that ARA can also activate survival aproliferation pathways [13] which may prevent the activation of apoptosis. For example, Yang et al. showed that ARA can activate the Akt survival pathway in ovarian cancer cells [170]. Other authors have mentioned that metabolites derived from ARA, such as PGE2, PGE4, 12-HETE, and 20-HETE, are responsible for inhibiting apoptosis. Liu et al. reported that 12-HETE inhibits cell apoptosis in ovarian carcinoma cells through the activation of the integrin-linked kinase (ILK)/NF-κB axis [171]. Additionally, Cui et al. proposed a novel mechanism in which active caspase-3 can activate cytosolic calcium-independent phospholipase A2 (ciPLA2), leading to the release of ARA and the production of PGE2. This abnormal activation of focal adhesion kinase (FAK) can eventually accelerate the proliferation of SKOV3 ovarian cancer cells, suggesting that excessive apoptosis can have a negative effect [172] (see Figure 7C).
Interestingly, our recent research has demonstrated that a high intake of omega-6 PUFAs in a lung cancer model leads to a decrease in the expression of active caspases-3, -8, and -9. This decrease in caspase expression is associated with a corresponding increase in cell proliferation, as indicated by both the mitotic index and the expression of minichromosome maintenance protein 2 (MCM2) [17]. What makes these findings particularly intriguing is that despite the high concentration of omega-6 PUFAs in the diet (with an omega-6/omega-3 ratio of 20:1), our results are consistent with studies that support the idea of ARA, an omega-6 PUFAs, having an anti-apoptotic role. It is important to note that omega-6 PUFAs are essential fatty acids that play critical roles in various biological processes. However, maintaining a balanced ratio between omega-6 and omega-3 fatty acids is essential for overall health. The optimal ratio between these two types of fatty acids is still a subject of ongoing research and debate, but most experts suggest a ratio closer to 4:1 or even lower for maximum health benefits [173]. These findings highlight the complex relationship between omega-6 PUFAs, apoptosis, and cell proliferation specifically in the context of lung cancer. Further research is needed to better understand the underlying mechanisms involved and to evaluate the implications for human health.
Upon analyzing all the studies on ARA, the evidence presented shows that ARA can elicit both pro-and anti-apoptotic effects. The observed differences may be related to the specific models or concentrations of this PUFA used in the studies. The conflicting results mentioned above suggest that further research is required to fully understand the correlation between ARA and the apoptotic process.

Concluding Remarks and Future Perspectives
In conclusion, this review has provided a comprehensive compilation of information regarding the modulation of apoptotic pathways by PUFAs, which are essential components of a daily diet. Overall, PUFAs have been shown to have a proapoptotic effect, except for ARA, which has a dual role with both anti-apoptotic and pro-apoptotic effects. The evidence supports the notion that PUFAs play a significant role in the induction and sensitization of apoptosis in various tumor cells and murine cancer models. Considering their wide availability and lack of toxic effects, PUFAs may represent a promising and novel strategy for cancer therapy.
In addition, based on the comprehensive information presented in this review, we have a strong belief that omega-3 polyunsaturated fatty acids (PUFAs) exert a clear pro-apoptotic effect. On the other hand, omega-6 PUFAs, particularly arachidonic acid (ARA), exhibit a dual effect, including both pro-apoptotic and pro-survival outcomes. However, these behaviors appear to vary depending on the specific in vitro and in vivo models studied, the concentration of fatty acids used, the duration of incubation, and the combination of treatments with other drugs.
These findings highlight the complexity of the effects of PUFAs on apoptosis, suggesting that the specific outcomes are influenced by various factors. The context-dependent nature of these effects underscores the need for further research to elucidate the precise mechanisms underlying the diverse actions of PUFAs in apoptosis regulation. By considering these factors, future studies can provide valuable insights into optimizing the therapeutic potential of PUFAs in cancer treatment.
We also emphasize the importance of maintaining a balance between omega-6 and omega-3 fatty acid consumption, as it can lead to favorable biological outcomes, including decreased inflammation and the induction of apoptosis, which have a beneficial effect in preventing cancer development. Furthermore, the use of omega-3 polyunsaturated fatty acids (PUFAs) as adjuvant therapy in cancer treatment has been suggested by various researchers. Omega-3 supplementation in the diets of cancer patients undergoing chemotherapy has shown potential benefits, such as improved treatment tolerance, decreased tumor size, and increased lean body mass [174]. For example, in a randomized trial with 11 colorectal cancer patients, the group that ingested 2 g/day of fish oil exhibited significant increases in EPA and DHA levels in blood plasma (1.8 and 1.4 times higher, respectively), along with muscle mass gain (mean of +1.2 kg). This group also demonstrated greater chemotherapy tolerance and a reduction in tumor size compared to the untreated group [175]. In another study, 128 patients with gastrointestinal cancer and cachexia were provided with a diet containing or lacking 1.1 g of EPA, 0.5 g of DHA, and 16 g of protein. The patients were monitored using bioelectrical impedance analysis, and the authors observed that the fish oil-enriched diet contributed to improved chemotherapy tolerance, tumor shrinkage, and increased lean body mass over time [176].
Importantly, the use of enzyme inhibitors to enhance the accumulation of specific oxylipins, exerting pro-apoptotic effects, has shown promise in in vitro and murine models [177,178]. These findings indicate the potential of such inhibitors as powerful tools in cancer treatment. However, further research and clinical trials are necessary to validate their efficacy and safety in human patients.
Looking ahead, future perspectives in this field should aim to further elucidate the molecular mechanisms underlying the apoptotic effects of PUFAs, including the specific pathways involved and the interplay between different PUFAs. Additionally, more studies are needed to investigate the effects of PUFAs on different types of cancer cells and animal models, as well as to evaluate their potential synergistic effects with existing cancer therapies. Furthermore, clinical trials are warranted to assess the efficacy and safety of PUFAs as adjuvant or standalone therapies for cancer treatment. Overall, continued research in this area has the potential to uncover valuable therapeutic strategies and contribute to the development of novel approaches in cancer therapy.