Abstract
Aberrant fatty acid (FA) metabolism has long been recognized in colorectal cancer (CRC) cells. Since de novo lipogenesis is required for CRC tumour growth and survival, the inhibition of FA metabolism is a promising potential therapeutic target. Inhibition of the opposite process, β-oxidation of FAs, has also showed promising results in many CRC models. For patients with CRC, both FA synthesis and β-oxidation inhibitors are promising potential therapeutic options as monotherapies or as combination therapies with other anticancer agents. In this review, we discuss recent reports concerning inhibitors of FA synthesis and β-oxidation in various CRC models. The exact mechanisms of action of the selected compounds described in this review remain unknown and require precise evaluation before the development of new successful therapies for CRC is possible.
Colorectal cancer (CRC) is among the most common types of cancer, accounting for approximately 10% of cancer incidence and mortality in both sexes (1, 2). Early detection owing to advanced screening methods (3, 4) has led to a reduction in death rates over the past several decades (5). However, some modifiable risk factors, such as poor diet and inactive lifestyle, are projected to contribute to increased incidence rates globally (5), especially affecting people in developing nations and underprivileged social classes (6, 7). Advances in understanding the pathophysiological alterations underlying this disease have led to a wide array of treatment options being available for the management of CRC. In addition to endoscopic and surgical excision, radiotherapy, and chemotherapy, drugs are used for the treatment of CRC and include biologicals and immunotherapeutics (7).
Recently, for the development of successful therapeutic strategies in the treatment of cancer, lipid metabolism has been considered an avenue worthy of pursuit (8, 9). Metabolic reprogramming of cancer cells facilitates enhanced proliferation and, in later stages cell dissemination, processes in which alterations of lipid metabolism play pivotal roles. Lipids serve as structural components of membranes, fuel sources for rapidly growing and dividing cells, and secondary messengers (9, 10). In particular, researchers focus on fatty acid (FA) metabolism because FAs are the main building blocks for other, more complex classes of lipids, such as phospholipids, sphingolipids, glycosphingolipids or triglycerides, and therefore contribute to the regulation of many different biochemical processes.
Aberrant FA metabolism has long been recognized in CRC cells (11), thus providing potential targets for therapeutics. A considerable body of work shows that de novo lipogenesis is required for CRC tumour growth and survival (12-15). FA synthase (FASN) provides palmitate for cancer cells for intensive membrane formation, enhancing cell resistance to oxidative damage and to chemotherapeutics due to increased lipid saturation (16), provides fuel for FA oxidation, and promotes the metastatic capacity of CRC (12, 17, 18). The palmitate from FASN-driven lipogenesis can also be modified via elongases (ELOVLs) or desaturated by stearoyl-CoA desaturase (SCD1), and these modifications may be targets of new therapy design (9). Indeed, CRC exhibits both the enhanced elongation of FAs (19, 20) and up-regulation of SCD1 activity (21, 22). Upon activation by an enzyme from the long-chain acyl-CoA synthetase family (ACSL), endogenously synthesized or exogenous FAs can be incorporated into triglycerides and cholesterol esters, which can be stored in lipid droplets as energy depots that are available to generate ATP via β-oxidation of FA (9). In CRC cells, a decrease in triglycerides and an up-regulation of enzymes has been observed in association with β-oxidation (23), conferring unique tumour advantages, such as evasion of anoikis, which is imperative for cancer metastasis (24). Although some changes in the lipid profiles of CRC samples are contradictory (11) and not straightforward because they are affected by many whole-body relationships between different tissues and by diet (8, 25), the contribution of altered lipid metabolism to the development and progression of CRC warrants the effort to pursue inhibitors of FA metabolism as potential drugs. In this review, we cover inhibitors associated with FA synthesis and oxidation. The potential targets for the inhibitors described below are presented in Figure 1.
FA Synthesis
FA synthesis is a multistep process leading to the formation of palmitic acid. The simplest way to reduce FA levels in cancer cells is to block FA synthesis. By inhibiting enzymes and reducing FA availability from the diet, cancer cell proliferation can be limited. Inhibitors of de novo FA synthesis may slow cancer cell proliferation, since they rely mostly on the de novo synthesis of FA, and they have minimal effect on healthy cells, which access FA mostly from dietary sources (26).
ATP-citrate lyase (ACLY) inhibitors. The first synthesis step links the tricarboxylic cycle (TCA) to lipogenesis by transforming citrate originating in the mitochondria into oxaloacetate and acetyl-CoA through the cytosolic enzyme ATP-citrate lyase (ACLY). Inhibition of ACLY was shown to reduce the ability of cells to metabolize the glucose needed to generate lipids (27). The up-regulation of ACLY, the first-step enzyme of de novo lipogenesis, has been found in several types of cancer, including CRC (28). GSK165 is a novel small-molecule compound that inhibits ACLY activity in a concentration-dependent manner. It had an antiproliferative effect on HT29 CRC cells. Furthermore, GSK165 sensitized these cells to the antineoplastic drug SN38 (28).
Acetyl-CoA carboxylase (ACC) inhibitors. The second step of FA synthesis, the carboxylation of acetyl-CoA to generate malonyl-CoA, is catalysed by ACC. ACC inhibitors have recently gained increased interest as potential anticancer drugs. The effect of 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an allosteric inhibitor of ACC, has been examined in CRC cells. The results showed that TOFA is cytotoxic to HCT-8 and HCT-15 CRC cells and induced apoptosis in a dose-dependent manner. Supplementing cells with palmitic acid prevented the cell death induced by TOFA (29). These results suggest that TOFA should be further analysed in the context of CRC; however, it is noteworthy that TOFA did not affect cancer cell survival in some cancer types, such as breast and ovarian cancer (30, 31). Citrus limon-derived nanovesicles are naturally formed vesicles that exhibit ACC inhibitory properties. They inhibited SW480 cell proliferation without affecting normal cells; however, more studies are needed to verify whether C. limon nanovesicles can serve as supplementary compounds in CRC treatment (32, 33). To the best of our knowledge, other ACC inhibitors, such as PF-05175157, soraphen A and ND-646, were not tested in any CRC model. Altogether, novel, more-sensitive ACCA inhibitors for use as antitumour agents still need to be developed.
FASN inhibitors. FASN converts one acetyl-CoA and seven malonyl-CoA molecules into palmitic acid through a series of reactions. FASN is the key enzyme in de novo lipogenesis, which is up-regulated in CRC cells compared to normal colonic mucosal cells (34). It has been shown that a higher expression of FASN is correlated with a worse CRC prognosis; however, some reports show that this correlation is more complex and that the influence of FASN expression on patient outcome depends on body mass index (nonobese patients with CRC and tumoural FASN overexpression have better survival than obese patients) (35, 36). FASN is a 270-kDa cytosolic enzyme composed of seven functional domains: three N-terminal domains (ketoacyl synthase, malonyl/acetyltransferase, and dehydratase), and four C-terminal domains (enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase (37). FASN seems to be a promising therapeutic because of its tissue distribution. FASN inhibitors can target only colonic cancer cells, and the normal tissue thus remains unaffected. Moreover, FASN is elevated in aberrant crypt foci; thus, it may be important in the very early stage of colorectal tumorigenesis and a worthy target for possible chemoprevention (38). Several pharmacological FASN inhibitors appear promising for clinical use in the future. Cerulenin [(2S,3R)-2,3-epoxy-4-oxo-7,10-dodecadienamide] was the first FASN inhibitor discovered. It is an antibiotic isolated from Cephalosporium caerulens that has strong noncompetitive affinity for the active site of the ketoacyl synthase domain (39). Cerulenin suppresses the proliferation of many CRC cell lines in vitro by inducing apoptosis (15, 40-42). Furthermore, cerulenin reduced the number and size of liver metastases based on Colon 26 and CMT 93 murine CRC cell lines (41). Chang et al. showed that the inhibition of FASN by cerulenin in HT-29 and LoVo cells suppressed their malignant phenotype by attenuating energy metabolism and down-regulating the mammalian target of rapamycin (mTOR) signalling pathway (15). Another study revealed the potentiated effect of a combination treatment consisting of cerulenin and oxaliplatin, which led to the reduction of oxaliplatin dosage and promoted longer-lasting chemotherapy in mice (42). Tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid (C75) is a competitive irreversible FASN inhibitor with a structure similar to that of cerulenin. It has been proposed as an antitumour and anti-obesity agent. C75 inactivates the enoyl reductase, thioesterase and ketoacyl synthase activities of FASN (43). C75 and cerulenin share several mechanisms of action and have similar side-effects. Both of these compounds are critical for the rapid and profound inhibition of DNA replication and S-phase cell-cycle progression. Study showed these effects to be detected 90 min after drug exposure of RKO colon carcinoma cells, thus they preceded the apoptotic changes observed 6 h after drug exposure (44). C75 and cerulenin both directly or indirectly activate carnitine palmitoyltransferase-1 (CPT1), which is the rate-limiting enzyme for β-oxidation (45, 46). CPT1 activation causes anorexia and weight loss in mice, which is one of the main obstacles in the clinical use of these compounds (47, 48). To address issues of weight loss, another FASN inhibitor, C93, has been designed to avoid activating CPT1. Cancer cell proliferation was significantly inhibited in mice bearing xenografts of the Colo680N oesophageal squamous cell carcinoma cell line when were treated with C93, without anorexia or weight loss (49). Orlistat is an anti-obesity drug with good patient tolerance (50). It inhibits FASN and exhibits antiproliferative effects on CRC cancer lines in vitro (Caco-2 and SW480 cells) (51) and in HT-29/tk-luc human colorectal carcinoma-bearing animals in vivo (52). Orlistat induces cell-cycle arrest at the G1 phase in a dose- and time-dependent manner and triggers apoptosis via the activation of caspase-3 (52). Taken together, these results allowed us to formulate the hypothesis that the oral formulation of orlistat may be beneficial in treating tumours such as colon cancer; however, our recent study showed that exogenous palmitate can reverse the antiproliferative effects of orlistat in HT-29 CRC cells (53). Novel potent FASN inhibitors (TVBs) were developed by 3-V Biosciences, and they demonstrate antitumour activity in vitro and in vivo and are being evaluated in phase I/II clinical trials. TVB-3166 inhibits the ketoacyl reductase enzymatic domain of FASN. TVB-3166 is a reversible, potent, and selective FASN inhibitor that interferes with cell proliferation, and viability by inducing apoptosis. TVB-3166 disrupted lipid raft architecture and inhibited signal transduction in the protein kinase B (AKT)–mTOR and β-catenin pathways in COLO-205 and HT-29 cells (54). Another study evaluated the effect of TVB-3664 on tumour growth in nine CRC patient-derived xenografts (55). More than 90% of the tumours in that study were positive for FASN expression; however, treatment with TVB-3664 led to a significant decrease in tumour volume in only one-third of the cases. The authors suggested that the presence of FASN expression does not predict the response to FASN inhibitors. Moreover, it has been proposed that the antitumour activity of TVB-3664 is associated with reduced activation of the AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) oncogenic pathways and significant alteration of the lipid composition of tumours. The authors also noted that PI3K mutational status and activation of AKT downstream signalling may affect the response of CRC cells to FASN inhibition and suggested that the combination of FASN inhibitors with inhibitors of the AKT or mitogen-activated protein kinase (MAPK) pathway may be a potential therapeutic strategy for CRC (55). Currently, TVB-3664 and TVB-3166 are undergoing preclinical evaluation for use in patients with CRC (54, 55), and TVB-2640 is in phase I clinical trials for the treatment of resectable colon cancer (56). Luteolin (3’,4’,5,7-tetrahydroxyflavone) is a potent FASN inhibitor that has been shown to reduce DNA synthesis and inhibit HT-29 cell proliferation by inducing G1 and G2/M cell-cycle arrest and apoptosis in a concentration-dependent manner (57). These findings were confirmed by the increased percentage of G2/M-phase SW480 colon cancer cells when treated with luteolin (58). Moreover, luteolin displayed a dose-dependent apoptotic effect on Caco-2 cells with negligible or no effect on normal cells. It has been suggested that the mechanisms accounting for this are AKT phosphorylation and sphingosine kinase 2 (SPHK2) inhibition. As a consequence, the endoplasmic reticulum–Golgi trafficking of ceramide is impaired, and finally, the sphingolipid rheostat is unbalanced. An observation of these reports inspired the suggestion that supplementing the diet with luteolin may be a potential clinical strategy for treating CRC (59). This hypothesis is supported by the observation that supplementation with luteolin in addition to aspirin showed a better effect than aspirin alone in rats with dimethylhydrazine-induced carcinogenesis (60). Resveratrol (3,5,4’-trihydroxystilbene) is a potent FASN inhibitor that binds reversibly to the ketoacyl reductase domain (61). A number of studies have reported that resveratrol suppresses CRC cell proliferation and elevates the apoptosis rate. CaCo-2 cells treated with resveratrol exhibit 70% inhibition of proliferation with no signs of cytotoxicity or apoptosis (62). The data suggest that resveratrol suppresses cell proliferation by inhibiting the insulin-like growth factor type 1 receptor and its downstream signalling pathways and enhances apoptosis via the activation of the p53 pathway in HT-29 and SW480 cells (63). Resveratrol has been shown to have ameliorating effects on CRC in different mouse and rat models (64, 65). Resveratrol has been shown to reduce tumorigenesis associated with colitis in C57BL/6 mice (64). Moreover, prolonged daily administration of resveratrol significantly reduced the number of aberrant crypt foci and crypt multiplicity in the colorectal mucosa (65). Resveratrol has moved into clinical trials; however, whether resveratrol is beneficial in CRC treatment is still debated. Because of its poor bioavailability, high doses have been used in clinical trials, with some side-effects being thus observed, and its use has not yet been translated into clinical practice (66, 67). Some hypotheses have suggested that resveratrol derivatives may be better cancer chemopreventive candidates than resveratrol itself. For example, the resveratrol analogue (E)-N-(2-(4-methoxystyryl) phenyl) furan-2-carboxamide was shown to induce G2/M cell-cycle arrest through the activation of p53-p21 in HCT116 cells (68). Nevertheless, resveratrol has a wide range of potential targets for chemoprevention. The detailed regulatory mechanisms for resveratrol-induced inhibition of CRC cellular proliferation were presented in a recent review by Honari et al. (69). Oridonin is a natural compound isolated from Rabdosia rubescens that effectively inhibits FASN and sterol regulatory element-binding protein 1 mRNA and protein expression in CRC contexts. It induces apoptosis and reduces cellular palmitic acid and stearic acid levels (70). Furthermore, oridonin inhibited the proliferation of LoVo cells partially by disrupting the transforming growth factor-β1–SMAD protein–plasminogen activator inhibitor-1 signalling pathway (71). Despite promising preclinical results, its poor solubility and bioavailability have slowed further research. HAO472 is an oridonin derivative that may be more applicable. It inhibits the proliferation and activation of T-cells and has been advanced into phase I clinical trials in China for the treatment of acute myelogenous leukaemia (72). Emodin (1,3,8-trihydroxy-6-methylanthraquinone) was shown to down-regulate the expression of FASN, inhibit intracellular FASN activity and FA biosynthesis, and induce the apoptosis of HCT116 cells in a dose- and time-dependent manner (73). Moreover, the combined treatment of emodin and cerulenin resulted in an additive effect (74). Curcumin inhibits FASN in vitro (75). Curcumin inhibited the proliferation of HT-29 and HCT-116 human colon cancer-derived cell lines. At least partially, this effect was due to peroxisome proliferator-activated receptors activation and the inhibition of epidermal growth factor receptor activation (76, 77). A clinical study on patients with adenocarcinoma of the colon or rectum showed a beneficial role of curcumin supplementation; however, no definite conclusions have been drawn to date (78). A plant polyphenol, epigallocatechin gallate (EGCG), present in green tea, has been tested on CRC cell lines with positive results. In vivo and in vitro studies showed that EGCG treatment induced apoptosis, affected the cell cycle and reduced the volume of tumours (79). EGCG has also been shown to induce apoptosis and suppress liver metastasis of CRC in SCID mice (80). Moreover, EGCG induced sensitization to 5-fluorouracil by targeting cancer stem cells in CRC and may serve as a supportive treatment for patients with CRC (81). A human study showed that 1 year of EGCG supplementation by increasing green tea consumption was safe and significantly prevented metachronous colorectal adenomas in Japanese patients (82). Promising preclinical results have led to clinical trials of EGCG supplementation as a potential therapeutic option for patients with CRC. The results support the hypothesis that EGCG may display some chemopreventive effects for patients with CRC; however, more studies with a larger group of patients and longer study periods are needed to confirm this effect (83-85).
ACSL inhibitors. To be further elongated or desaturated, a FA must be transformed into an acyl-CoA molecule by ACS, which are classified according to the length of the acyl chain of their substrates: - short-chain, medium-chain and long-chain ACSs. In this review, we focus on ACSL isoforms, as they are up-regulated in CRC (86). In mammals, five ACSL isoforms (ACSL1-6) have been identified. Four main ACSL isoforms are up-regulated in CRC: ACSL1, ACSL4, ACSL5, and ACSL6 (87-89). High levels of ACSL1 or ACSL4 expression in patient tumour samples were found to correlate with worse prognoses for patients with CRC (90). In CRC cells, ACSL1 and ACSL4 overexpression causes a shift of energy metabolism towards glucose utilization (90). Despite promising results obtained from analysing ACSL expression in CRC, there is only one chemical ACSL inhibitor, triacsin C, which inhibits ACSL1, -3, and -4 (91). Unfortunately, at high concentrations, triacsin C is toxic to cells, and achieving effective pharmacokinetic effects and cell penetration is challenging (92).
Inhibitors of SCD1. The desaturation of a FA is catalysed by SCD1, which introduces a double bond at the n-9 position. SCD1 is highly expressed in CRC tissues and has a negative correlation with patient prognosis; however, the SCD1 expression level was found to be different in five different human CRC cell lines: SW620, HCT116, Caco-2, SW116, and HT29 cells. Moreover, SCD1 enhanced the migration and invasion ability of CRC cells (93). Betulinic acid (BetA) is a pentacyclic triterpenoid extracted from birch trees, with a wide range of biological properties, including SCD inhibition. BetA inhibited the proliferation of HCT116, SW480, and DLD-1 cells in vitro in a time- and dose-dependent manner. Moreover, BetA induced cell apoptosis through the mitochondrial-mediated pathway and inhibited the metastasis of cancer cells. In vivo, BetA suppressed the growth of tumours in an HCT-116 xenograft tumour mouse model (22). In another study, BetA induced rapid CSC cell death via the elimination of cell clonogenic capacity (94). Other SCD inhibitors have some interesting properties; however, only a few reports are available. For example, T-3764518 is a novel and orally available small-molecule SCD inhibitor that successfully inhibited HCT-116 cell proliferation (95), and CAY10566, a chemical SCD1 inhibitor, significantly reduced cell viability and spheroid formation in a WiDr 3D culture system (96). Other SCD1 inhibitors, such as SSI-4 and MF-438, have not been tested in CRC models.
ELOVL inhibitors. The elongation of palmitate and other long-chain FAs requires a unique set of enzymes. Elongation takes place in the endoplasmic reticulum and is catalysed by ELOVLs. ELOVL isoforms can be divided into two groups: ELOVLs 1, 3, 6 and 7, which take part in the elongation of saturated and monounsaturated FAs; and ELOVLs 2, 4, and 5, which catalyse the elongation of polyunsaturated FAs (97). ELOVLs have not been thoroughly studied in the context of CRC; however, it is known that CRC specimens show enhanced FA elongation. The results from a study by our team showed a dramatic increase in ELOVL1 and ELOVL6 mRNA levels in CRC tissue, suggesting that these enzymes may play critical roles in the development and metabolism of CRC cells (19). Moreover, higher ELOVL6 activity was observed in patients with adenomas compared to those with no colonic polyps, and this characteristic was associated with an increased risk of colonic adenoma (98). To the best of our knowledge, no studies have investigated the consequences of ELOVL knockdown in CRC cell lines. Attempts have been made to discover ELOVL6 inhibitors; however, to date, no candidates have been thoroughly studied, and none have been tested on CRC cells (99, 100).
Since FA synthesis is important for cancer cells to survive, enzymes taking part in this process may be potential therapeutic targets for clinical application. The inhibition of many enzymes mentioned in this review reduces the proliferation and diminishes the metastatic capabilities of cancer cells in multiple CRC models. The role of the overexpression of these enzymes in CRC cells and the mechanisms by which exposure to different inhibitors induces cell death are still not fully understood due to significant variability in responses to these inhibitors in CRC. A better understanding of the mechanism(s) of by which various compounds inhibit different enzymes will advance the development of new potential therapeutic approaches to target CRC.
β-Oxidation of FAs
FAs are catabolized during β-oxidation (also known as FA oxidation) in the mitochondrial matrix by the sequential removal of two-carbon units (acetyl-CoA) from the carboxyl end of the FA chain in repetitive cycles. Before actual oxidation, FA must be activated by converting the FA into a fatty acyl-CoA ester in an irreversible reaction catalysed by ACSs on the outer mitochondrial membrane. After activation, the FAs are transported from the cytosol to the mitochondrial matrix. The mitochondrial inner membrane is impermeable to long-chain FAs. To cross the membrane, long-chain FAs must be conjugated with carnitine by carnitine palmitoyltransferase 1 (CPT1), which is present on the outer surface of the inner mitochondrial membrane. The conjugate is then transported across the membrane by translocase, and carnitine acyltransferase II (CPT2), located on the inner mitochondrial membrane on the matrix side, removes the carnitine (101). This is the rate-limiting step of FA oxidation. β-Oxidation involves repeated cycles of four reactions: Dehydrogenation (by acyl-CoA dehydrogenase), hydration (via enoyl-CoA hydratase), a second dehydrogenation (by 3-hydroxyacyl-CoA dehydrogenase), and thiolysis [by 3-ketoacyl-coenzyme A thiolase (3-KAT)]. This process is accompanied by the reduction of NAD to NADH and FAD to FADH2, which then transfer electrons to the respiratory chain, where they enable the production of cellular energy in the form of ATP. Moreover, the final product of β-oxidation – acetyl CoA – is also oxidized in mitochondria, leading to further production of ATP (102). FA oxidation plays a crucial role in cancer cells and is necessary for cell proliferation, survival, stemness, drug resistance and metastasis (103, 104). Rapidly growing cancer cells have high energy demands and require FAs to produce cell membranes. In CRC, FA oxidation may be critical to cancer development. Therefore, it seems that the inhibition of this process in CRC cells may be a possible treatment option for the inhibition of cancer growth.
CPT1 inhibitors. CPT1 is the key rate-limiting enzyme of FA oxidation. Its inhibition prevents the formation of acylcarnitines and makes the transport of FA chains from the cytosol into the mitochondria impossible. Currently, three different isoforms of CPT1 have been identified, which exhibit tissue-specific distribution: CPT1A in the liver, CPT1B in the muscle, and CPT1C expressed exclusively in the brain (105). Inhibitors are specific for the different CTP1 isoforms. Etomoxir {ethyl 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate} is an irreversible pharmacological inhibitor of CPT1 that is used in clinical studies for the treatment of heart failure (106). A study on MCA-38 tumour-bearing mice (colon adenocarcinoma) treated with etomoxir indicated that this drug caused a significant delay in tumour growth with minimal necrosis and a higher number of cells undergoing apoptosis. However, an in vitro study on MCA-38 cells treated with etomoxir did not show a delay in cell proliferation or a change in the number of cancer stem cells (107). Moreover, Hossain and co-workers noted that etomoxir blocked the immune inhibitory pathways and immunosuppressive function of myeloid-derived suppressor cells in MCA-38 tumour-bearing mice. These cells promote tumour progression through multiple mechanisms in CRC (107). Furthermore, etomoxir significantly inhibited FA uptake and ATP production and reduced oxygen consumption and extracellular acidification rates (107). In the HCT116 colon carcinoma cell line, etomoxir also reduced the cellular ATP level (108). Inhibition of FA oxidation by etomoxir enhanced the antitumour effect of low-dose chemotherapy, especially cisplatin, on HCT116 colon carcinoma cells (108). Another study showed that combining etomoxir with radiation improved its therapeutic efficacy in H460 human lung epithelial carcinoma cells and LNCaP prostate carcinoma cells. The authors hypothesized that administering etomoxir immediately after cell irradiation may be a promising alternative for solid tumour treatment, including for CRC (109). In light of the work by Wang et al. (24), the inhibition of CTP1A may be a potential strategy for preventing CRC metastasis. CPT1A was one of the top up-regulated genes in detached CRC cells, compared to its expression in attached CRC cells. The inhibition of CPT1A by etomoxir inhibited CRC cell proliferation and promoted anoikis in detached CRC cells (24).
It is noteworthy that some data suggest that etomoxir has off-target effects at high concentrations. For example, this CPT1 inhibitor may also inhibit complex I of the electron transport chain and reduce cell proliferation independently of FA oxidation (110). Referenced data suggest that etomoxir may be a promising treatment for many cancer types, including CRC as a monotherapy or in combination with other anticancer agents, such as cisplatin or radiation; however, cancer cachexia and possible off-target effects must be taken into account during further studies. Because of the possible toxicity induced by etomoxir, a more selective CPT1 inhibitor ST1326 (teglicar) was developed. This aminocarnitine derivative is a liver-specific reversible inhibitor and probably does not share a toxicity profile similar to that of etomoxir (111). Its efficiency in CRC has yet to be investigated. Perhexiline 2-(2,2-dicyclohexylethyl)piperidine is another partial inhibitor of the carnitine shuttle of long-chain FAs into the mitochondria. Perhexiline is a potential dual CPT1/CPT2 inhibitor. In vitro studies demonstrated that the cardiac isoform of CPT1 is more sensitive than the hepatic isoform to inhibition by perhexiline (112). Perhexiline has been approved for the treatment of heart disease in some countries; however, it may induce toxicity, especially in long-term therapy, and recently, its use has thus declined (113, 114). The results of studies by Wang and co-workers from 2020 indicate that using perhexiline to block FA catabolism sensitizes CRC cells to the antitumour effect of oxaliplatin, as demonstrated by the enhanced apoptosis of treated HCT116 and DLD-1 CRC cells (115). Similar to other CPT inhibitors, such as etomoxir, perhexiline-induced inhibition of FA oxidation and intensified ROS accumulation enables classic chemotherapeutic drugs to kill more CRC cells (115). Oxfenicine (S-2-(4-hydroxyphenyl)glycine) is an effective inhibitor of carnitine-palmitoyl transferase 1B (CPT1B) in the heart but not in the liver. It is still in a preclinical stage, and no data concerning its effect on CRC are available (116).
3-KAT inhibitors. 3-KAT is the final enzyme activated in the FA β-oxidation process, and it releases acetyl-CoA. Trimetazidine and ranolazine are 3-KAT inhibitors that directly inhibit FA oxidation. Trimetazidine is used as an anti-anginal therapy in Europe and Asia. Ranolazine suppresses FA oxidation in rat cardiac and skeletal muscle cells and serves as a therapeutic agent in heart disease in Europe and the United States (113). Ranolazine is a clinically used inhibitor of voltage-gated Na+ channels, and has also been shown to inhibit breast cancer metastasis in vivo (117, 118). Sodium channel protein type 5 subunit alpha is a voltage-gated Na+ channel isoform that is expressed in colon cancer. It is a key regulator of a gene transcriptional network that controls the invasion of CRC, although the mechanisms are still not fully clear (119). The most recent study placed some inhibitors of FA oxidation mentioned in this review in a completely different light. Ma and co-workers pointed out that ranolazine and trimetazidine did not show any inhibitory effects on FA oxidation in established cell lines, primary cells, or mice; therefore, they formulated a hypothesis that these two molecules may not be adequate FAO inhibitors of β-oxidation at the cellular level. Similarly, they did not detect anti-FAO activity of perhexiline, an outcome that the authors explained may be related to its poor specificity as a dual CPT1/CPT2 inhibitor. Only etomoxir and oxfenicine at higher concentrations were able to significantly inhibit FA oxidation in MCF-7 and T47D cells (120).
α-Methylacyl CoA racemase (AMACR) inhibitors. The enzyme AMACR, also known as P504S, is involved in converting an R-configurated branched FA to an S-configuration before β-oxidation (121). AMACR protein overexpression was found in a number of types of cancer, including CRC (122-124). Several inhibitors of AMACR have been indicated, for instance ebselen, the seleno-organic compound ebselen oxide, pyrazoloquinolines, pyrazolopyrimidines, 2-trifluoromethyl tetradecanoyl CoA and both the R and S isomers of ibuprofenoyl CoA (125-127); however, due to their modest affinity, there is still a need to discover more potent inhibitors in order to evaluate the possibility of targeting AMACR for inhibition in CRC treatment.
Non-steroidal anti-inflammatory drugs (NSAIDs) and FA oxidation. Inflammation increases the risk of CRC, thus, it is widely assumed that NSAIDs may reduce this risk (128). Substantial available evidence showed that NSAIDs reduced the proliferation of CRC cell lines in several experimental models. NSAIDs (particularly aspirin) have been reported to reduce the risk of CRC by approximately 20% in the general population or even by 50% in those with a first-degree relative with a history of colon cancer (129-131). Although NSAIDs are among the drugs most used worldwide, their mechanism of action is still not fully understood. It is known that these molecules inhibit the cyclo-oxygenases COX1 and COX2, which are involved in the synthesis of eicosanoids, such as prostaglandins, from arachidonic acid. Prostaglandins are engaged in numerous biological responses and play roles in maintaining both normal and cancer cell viability and proliferation (132). Although CRC is characterized by overexpression of COX2 but not of COX1, studies concerning the inhibitory effects of NSAIDs on CRC cells suggest that some inhibit CRC cell proliferation, cause cell-cycle arrest and induce apoptosis independently of the COX1 or COX2 pathway (131, 133). These findings led to the assumption that other inhibitory pathways must be critical for NSAID effects (134-136). Some NSAIDs may inhibit long-chain FA oxidation in mouse liver mitochondria in vivo and in vitro (137-139). The first documented evidence of this by NSAIDs in a CRC cell line was published by Yang et aI. (136). LIM 1215, LIM 1899 and HT-29 CRC cell lines were treated with aspirin, ibuprofen, indomethacin, meclofenamate, sulindac sulfide, and sulindac sulfone. These tested NSAIDs inhibited long-chain FA oxidation and thus contributed to the inhibition of cell proliferation (136). The detailed mechanism of β-oxidation inhibition by different NSAIDs remains unclear and needs to be determined. Although daily aspirin consumption may have beneficial effects on CRC risk and taking particular NSAIDs seems to be a promising therapeutic option for CRC, complications and side-effects should be taken into consideration. The inhibition of β-oxidation leads to the accumulation of long-chain FAs, some of which may induce toxicity. In further studies, a more complex analysis of the long-term intake of NSAIDs on diverse colon cancer types should be performed, taking into account other factors such as body mass index, comorbidities, and stage of the disease (140).
Other FA oxidation inhibitors. Dipropylacetic acid (VPA) is a branched-chain FA that directly or indirectly affects FA oxidation. There are many hypotheses concerning the mechanism of action of VPA in the case of inhibition of FA oxidation, such as the generation of reactive metabolites that irreversibly inactivate oxidative enzymes, but the pleiotropic effects of the drug on mitochondrial metabolism are the most likely contributors to its effects on CRC (141, 142). VPA is also considered a first-generation histone deacetylase inhibitor (143). VPA administered with fluoropyrimidine-based chemoradiotherapy had antiproliferative effects on CRC cell lines expressing P53 (wild-type or mutant) (144). Thia FAs are FAs with a sulfur atom inserted into the FA backbone. Saturated 3-thia tetradecylthioacetic acid (TTA) and 4-thia tetradecylthiopropionic acid (TTP) induce nearly opposite effects on FA metabolism in the liver. TTA increases the catabolism of FA, and TTP is a powerful inhibitor of FA oxidation (145). Thia FA with the sulfur atom in an even carbon position, such as TTP, blocked FA oxidation in rat liver and heart, probably by inducing the accumulation of alkylthioacryloyl-CoA in mitochondria (146, 147). Interestingly, it has TTA was observed to inhibit SW620 CRC cell proliferation, probably by inducing endoplasmic reticulum stress and activating the unfolded protein response pathway independently of FA oxidation (148).
FA oxidation inhibitors are promising therapeutic options for some types of cancer, including CRC, when used in monotherapy or in combination with other anticancer agents. Inhibition of FA oxidation may also be an attractive strategy for overcoming drug resistance or metastasis of tumours. Their potential for affecting these outcomes is extremely important because more than 20% of patients with CRC exhibit distant metastases at initial diagnosis (149). The exact mechanisms of action of the selected inhibitors described in this review remain unclear, and their effects on CRC growth are also unknown. Therefore, there is an urgent need to appropriately evaluate their β-oxidation-inhibitory and antiproliferative functions at the cellular level in CRC models. Understanding the specific mechanisms of action of these inhibitors is key for the development of new successful therapies of CRC.
Conclusion
The balance between de novo FA synthesis and β-oxidation is crucial for cell homeostasis, and dysregulation of these opposing processes can lead to serious consequences. In the case of cancer cells, the chemical inhibition of the synthesis or β-oxidation of FAs often leads to slower proliferation and an increased rate of apoptosis of cancer cells without affecting normal cells. These outcomes suggest that targeting enzymes engaged in FA synthesis and oxidation is a promising therapeutic approach for CRC; however, notably, more research is needed to confirm their beneficial effects in human patients with CRC.
Acknowledgements
This research was supported by the National Science Centre of Poland (grant no. 2016/22/E/NZ4/00665) and Medical University of Gdansk (grants no. ST-40).
Footnotes
Authors' Contributions
P.M., A.M. and TS conceived and designed the review; P.M., K.D. and A.P. studied the literature and wrote the article; T.S. and A.M. verified the article. All Authors agreed on the final version of the review.
Conflicts of Interest
The Authors declare that there are no conflicts of interest regarding the publication of this article.
- Received June 17, 2020.
- Revision received July 10, 2020.
- Accepted July 13, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved