O3FAs can modulate cyclooxygenase (COX) metabolism and reduce production of several prostanoids including prostaglandin (PG) E
2 in tumours [
21,
22], whilst possibly increasing the production of lipid mediators involved in the resolution of inflammation such as lipoxins and resolvins [
21,
22], which may have anti-cancer properties [
23‐
25]. Elevated COX-2 expression is found in greater than 90% of CRCs [
26‐
28], associated with high levels of PGE
2, which drives pro-tumorigenic proliferation, migration, and invasion, but also promotes an immune-suppressive tumour microenvironment beneficial for tumour growth [
29,
30].
Proliferation and survival of cancer cells is linked to the activation of signalling pathways from surface molecules, such as cytokine or growth factor receptors [e.g. epidermal growth factor receptor (EGFR)], which transduce signals upon activation
via protein linked the cytoplasmic membrane and kinase signalling cascades [
31]. O3FAs have been shown to incorporate into the plasma membrane of cancer cells, where they alter lipid raft composition and fluidity. This can result in an inhibition of signal transduction, limiting cancer cell survival and promoting apoptosis [
32]. O3FAs will also incorporate into non-cancer cell membranes within the tumour microenvironment and potentially alter their phenotype.
O3FAs have also been shown to downregulate other CRC promoting signalling pathways such as the Wnt/ß-catenin pathway [
33], the MAPK/ERK pathway [
34], and PI3K-PTEN pathway [
35,
36].
O3FAs can exert anti-CRC activity following their interaction with surface free fatty acid (FFA) G protein-coupled receptors (GPCRs), thereby activating pro-apoptotic signalling [
17]. These GPCRs have been shown to be expressed on non-epithelial cells such as adipocytes [
38,
39] and macrophages [
40], on which activation can alter macrophage polarisation and reduce inflammation that is potentially important for anti-cancer activity of O3FAs.
The respective contribution of these putative diverse mechanisms of action described in in vitro and in vivo models to potential anti-CRC activity in man is not known and will likely be context-dependent, e.g. tumour type and composition of the microenvironment. Due to the diversity of likely molecular targets of O3FAs, preclinical studies on the potential activity of O3FAs against established CRC, rather than prevention, have focussed on pharmacodynamic endpoints relevant to the hallmarks of cancer such as cell proliferation, apoptosis, and migration.
3.1 O3FAs exert anti-proliferative and pro-apoptotic effects in CRC models
High doses of LNA (over 1 mM) have been shown to reduce cell proliferation, cell adhesion, and the ability of both human (HCT116 and HT29) and mouse (MC38) CRC cell lines to invade matrigel [
41]. This study did not indicate the molecular basis of this effect. However, it is unlikely to be COX-2-dependent as mammalian cells are inefficient at converting LNA into EPA or DHA [
2]. Similarly, another group investigated the impact of DHA on migration in CRC cell lines and reported that 100 μM DHA could inhibit Granzyme B expression in three human CRC cell lines (HCT116, CSC4, and HT-8), thus reducing their ability to undergo epithelial-mesenchymal transition (EMT) and invade matrigel [
18]. The same group has also published similar data in the context of bladder and pancreatic cancer models, suggesting that this mechanism is not tissue-specific [
42]. Downregulation of genes related to metastatic behaviour was also highlighted from a list of differentially expressed genes in HT15 CRC xenografts grown in nude mice treated with a DHA-rich diet compared to a control diet (corn oil) for 30 days [
43].
In terms of COX-independent activity, both DHA and EPA have been shown to act as ligands for and inhibit cell proliferation
via GPCRs such as GPR120 [
44]. More recently, it has been reported that this interaction leads to the activation of the Hippo signalling pathway in LoVo and HT29 cells
in vitro [
17].
In vivo, GPCR activation resulted in a reduction in tumour burden in the azoxymethane (AOM)/dextran-sulphate model in
Balb/
c mice fed a 10% fish oil diet [
17]. Another study reinforced the link between the anti-cancer effects of O3FAs and oxidative stress, showing that polyunsaturated fatty acids induced apoptosis in human CRC cells (LoVo and RKO cells)
via the generation of reactive oxygen species and the induction of the caspase cascade [
14]. It is notable that this effect was not O3FA-specific as the study reported similar results for both omega-3 (DHA and EPA) and omega-6 (arachidonic acid) FAs at the same concentration of 150 μM
in vitro [
14]. The same authors published a parallel study using the same models showing that the effect of polyunsaturated fatty acids on cell proliferation and lipid mediators [
12]. The data highlight the context-dependent manner of the mechanisms of action of EPA and DHA as both reduced cell proliferation in each cell line, but 150 μM DHA induced an increase in PGE
2 and lipoxin A4 (LXA4) in levels in LoVo cells, but not in RKO cells, with the opposite result obtained when treating cells with 150 μM EPA [
12].
The presence of cancer stem cells within a tumour mass has been linked to resistance to radiotherapy and chemotherapy. More than one research group has investigated whether O3FAs could exert their anti-cancer activity cancer stem-like cells within the tumour mass. De Carlo et al. (2013) used COLO320 DM cells, which grow as a mixed population of CD133
− cells and CD133
+ cancer stem-like cells, as an
in vitro model of a mixed tumour cell population. They established that doses of EPA, which are comparable to that achieved in human plasma, reduced proliferation of “standard” cancer cells (CD133
−) but not CD133
+ stem cell-like cancer cells. However, in the presence of EPA, there was a change in the ratio of “standard” to stem cell-like markers with a reduction in CD133 expression level and an increase in epithelial marker expression such as MUC2 [
45]. Another study suggested that O3FAs could affect both CD133
+ and CD133
− cells: EPA and DHA were shown to reduce cell proliferation in SW620 monolayer and 3D cultures that display a stem cell-like phenotype [
46]. Using the LS174T cell line, which is considered a model of CRC initiating cells with stem-like properties, Sam et al. (2016) showed that both EPA and DHA (at concentrations between 50 and 150 μM) reduced LS174T cell growth in a time- and dose-dependent manner. The authors proposed that O3FAs decreased survivin expression and induced caspase-3 activation to promote cell death [
16].
Although data continue to accumulate supporting the hypothesis that O3FA are good candidate compounds for the treatment of CRC, in the era of personalised cancer therapy, there remains a lack of studies investigating predictive markers of response to O3FA in CRCs in a translational setting. One study reported that EPA exposure decreased C-C motif chemokine ligand 2 (CCL2) production and expression of its receptor C-C chemokine receptor 2 (CCR2) expression in human HCA-7 and mouse MC38 CRC cells in a dose-dependent matter
in vitro [
13]. These results were confirmed
in vivo using a MC38 xenograft model and were then translated into the clinical setting with demonstration that changes in plasma CCL2 levels in EPA-treated CRC liver metastasis patients [
47] were associated with a specific tumour gene expression profile and may predict patient CRC outcomes [
13].
O3FA are commercially available in various formulations: as the FFA, conjugated to ethyl esters (EE), as a triglyceride (TG), or as phospholipids. Only a limited number of studies have investigated novel formulations to improve the anti-cancer effect of O3FAs.
In 2013, Morin et al. reported the impact of O3FA conjugation as the mono-glyceride as opposed to FFA, EE, or TG on the O3FA incorporation and anti-cancer activity. They showed that monoglyceride-conjugated O3FAs were more efficiently incorporated in HCT116 cells than as the FFA form, but also displayed greater anti-proliferative activity in this model, potentially
via lipoxygenase and CYP450 metabolism [
48]. Docosapentaenoic acid (DPA) was especially promising in this form and was also shown to inhibit HCT116 tumour growth
in vivo [
48]. The same group went on to establish the anti-inflammatory properties of these agents in non-cancer disease models such as cystic fibrosis [
49]. More recently, they have demonstrated that DHA-monoglyceride could be used to potentiate carboplatin anti-cancer activity both
in vitro and
in vivo in A549 and H1299 lung cancer models [
50]. It remains to be seen whether these results can be translated to the CRC setting, in which patients more likely receive oxaliplatin chemotherapy.
More recently, encapsulation of LNA or DHA in liposomes with the anti-oxidant polyphenol resveratrol was shown to increase incorporation of the O3FA in HT29 cells [
51]. Serini and colleagues also suggested that this liposome formulation lead to an increased conversion of LNA into EPA/DHA [
51]. They also reported increased cell growth inhibition in HT29 and HCT116 cells treated with O3FA-loaded liposomes compared to the FFA equivalent, associated with a reduction in proliferation rate, but no increase in apoptosis induction [
51]. This formulation requires validation in
in vivo models to ascertain its bioavailability and efficacy.
3.3 Combination O3FA treatment with chemotherapies and other nutraceuticals
Cancer therapies are rarely administered as single agents. A combination of agents often allows dose reduction of one or more agents in order to mitigate the risk of cumulative side effects. In this context, O3FAs represent strong potential for adjuvant therapy given their low toxicity profile, allowing the use of other agents at more effective doses. A previous study demonstrated that EPA and DHA can modulate cholesterol synthesis and as a result downregulate the expression of the efflux pump, P glycoprotein, in a doxorubicin-resistant variant of HT29 cells. Gelsomino et al. suggested this reduction in drug efflux pump expression could be significant when using O3FAs in combination with standard chemotherapies limited by such detoxifying mechanisms [
19].
Multiple studies have analysed whether O3FAs could improve response to chemotherapeutic agents routinely used in the treatment of CRC. Vasudevan et al. demonstrated a synergistic anti-cancer effect between EPA and a regimen of 5-fluorouracil (5-FU) and oxaliplatin
in vitro and
in vivo against HT29 and HCT116 CRC models [
52]. Several other
in vivo studies have demonstrated that O3FAs can both potentiate 5-FU anti-cancer activity (reduction of tumour burden, increased apoptosis, and DNA damage) and also reduce 5-FU-related toxicity [
53‐
55]. In 2017, a study showed that 5-FU and irinotecan treatment can lead to impaired lipid storage in rat models, resulting in loss of O3FA in tissues. The authors hypothesised that combining O3FA with standard chemotherapy regimens could help restore lipid stocks, thus potentially limiting 5-FU-associated side effects [
56].
Pichard and colleagues have focussed on the potential combination of O3FAs with standard CRC treatment modalities for over a decade. They have shown that O3FAs have the potential to radio-sensitise cancer cells, demonstrating that radio-resistant HT29 cells became responsive to radiation when treated with O3FA, and DHA in particular [
57]. An additive cytotoxic effect was also observed in radio-resistant LS174T (stem cell-like) CRC cells. The proposed mechanism for this enhanced response was an increase in lipid peroxidation products within the cells [
57]. The same group has also investigated the impact of O3FAs on the anti-CRC activity of 5FU, oxaliplatin, and irinotecan on HT29 and LS174T cells. They reported an increase in apoptosis when combining these agents with a fish oil emulsion containing 20.6 g/L of EPA and 19 g/L of DHA [
15].
In the preclinical study of EPA activity on CD133
+ COLO320DM cells [
45], low-dose EPA (25 μM) exposure not only sensitised CD133
− COLO 320 DM cells to both 5-FU and oxaliplatin but also increased the sensitivity of the CD133
+ stem-like cell population to 5-FU [
45]. Likewise, an increased sensitivity to 5-FU and mitomycin C was observed in SW620 human CRC cells when combined with low-dose O3FA [
46].
Aside from standard chemotherapies, DHA has been shown to enhance TRAIL-induced apoptosis in SW620 CRC cells but not normal colorectal NCM460 epithelial cells, suggesting that a combination of O3FAs with an agent targeting this apoptosis pathway should be investigated as a novel anti-cancer therapy [
58].
Combinations with other naturally occurring compounds such as curcumin, which have been previously considered for cancer prevention strategies, are now being tested for therapeutic interventions in the advanced cancer setting. A novel potential mechanism of action was highlighted in an
in vitro study, which showed that a combination of EPA with two natural phytochemicals, grape seed extract and epigallocatechin-3-gallate (found in green tea), could inhibit mTOR as effectively as rapamycin in SW480 and HCT116 cells [
20]. Kim et al. used the AOM model to induce colonic carcinogenesis and establish the impact of curcumin and O3FA on cancer stem cell survival. They showed that the combination of curcumin and O3FA resulted in an increase in apoptosis, as well as a reduction in nuclear ß-catenin in Lgr5
+ colonic stem cells, within aberrant crypts [
59]. Another study combined DHA with butyrate, a short-chain fatty acid used by colonocytes for energy production with known histone deacetylase inhibitory properties, which enhanced apoptosis compared with butyrate alone through increased downregulation of promoter methylation of several pro-apoptotic genes such as Tnfrsf25 [
60].