**Correction of Fatty Acids Metabolism as Treatment Strategy of Autism**

Afaf El‐Ansary and Hanan Qasem

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67488

#### **Abstract**

[36] Titz B, Luettich K, Leroy P, Boue S, Vuillaume G, Vihervaara T, et al. Alterations in serum polyunsaturated fatty acids and eicosanoids in patients with mild to moderate chronic obstructive pulmonary disease (COPD). Int J Mol Sci. 2016;17(9):1-21. DOI: 10.3390/

[37] Miyata J, Arita M. Role of omega-3 fatty acids and their metabolites in asthma and aller-

[38] McNamara RK, Strimpfel J, Jandacek R, Rider T, Tso P, Welge JA, et al. Detection and treatment of long-chain omega-3 fatty acid deficiency in adolescents with SSRIresistant major depressive disorder. PharmaNutrition. 2014;2(2):38-46. DOI: 10.1016/j.

gic diseases. Allergol Int. 2015;64(1):27-34. DOI: 10.1016/j.alit.2014.08.003

ijms17091583

22 Fatty Acids

phanu.2014.02.002

Autism is a neurodevelopmental disorder clinically presented as abnormalities in social interaction and communication, repetitive behaviors, usually accompanied by various neurobehavioral disorders, such as learning disability, hyperactivity and anxiety.

It is well known that more than 50% of human brain weight is composed of lipids with a remarkably high content of long-chain polyunsaturated fatty acids (LCPUFA). Adequate supply of different fatty acids and lipids is critically needed by developing brain to achieve normal growth. Essential polyunsaturated fatty acids (PUFAs) are critical for normal pre‐ natal brain development. There has been increasing evidence that impairment of PUFAs metabolic pathway could affect the normal function of nervous system which is related to pathogenesis of autism.

Studies have demonstrate that autistic patients may exhibit abnormal PUFAs metabolism, which manifests as varying impaired levels of lipid mediators such as prostaglandins, eico‐ sanoids, and isoprostanes in serum and plasma of autistic patients.

Consequently, interventions related to metabolic correction of fatty acids, phospholipids, prostaglandins, eicosanoids, and isoprostanes as fatty acids–derived signaling molecules were discussed in details with special reference to Omega-3 Fatty Acids supplementation and its recognized role in the correction of oxidative stress, neuroinflammation, glutamate excitotoxicity as ascertained etiological mechanisms in autism.

**Keywords:** fatty acids, omega-3, omega-6, prostaglandins, eicosanoids, isoprostanes

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

In the last decades, researchers have been focused on lipids to make clear idea about it in both physiological and disease sides. Until now, 600 molecular species have been discovered from human plasma described as lipidome [1, 2]. Lipidome provides a comprehensive classifica‐ tion of lipids with their structure and function. About 60% of dry human brain is composed approximately from lipids with over 20% polyunsaturated fatty acids [3, 4].

From biochemistry point, PUFAs are type of simple lipid that contain one or more double bonds in *Cis* configuration. PUFAs are two classes omega-3 and omega-6 and these classes do not convert to other forms and play important roles in biochemical changes in the body. Omega-3 and omega-6 are dietary essential fatty acids because they cannot be synthesized by human body beside they can prevent deficiency symptoms [5]. The main sources of Omega‐3 fatty acids are vegetable oil and fishes. Docosahexaenoic acid (DHA, 22:6ω3) and eicosa‐ pentaenoic acid (EPA, 20:5ω3) are omega-3 fatty acid with 22 carbons and 6 double bonds (22:6n-3). While vegetables are the main sources of omega-6, arachidonic acid (AA; 20:4ω6) is an omega-6 fatty acid with 20 carbons and 4 double bonds. These two fatty acids are the predominant long-chain (20 and 22 carbons) PUFAs in human brain [6].

In the last years, the interest in the health consumption for Omega‐3 has led to more researches and manufacturing of these fatty acid as supplement foods. The European committee has been suggested that the minimum requirement of omega-3 and omega-6 is approximately 0.5 and 1% of energy intake, respectively. PUFAs are now regarded as nutritionally essential fatty acids [7]. Deficiency in these fatty acids causes dermatitis, growth retardation and infertility. They play critical role as second messengers in the pro‐ cess of signal transductions, structural component of ceramide and specific role in mem‐ brane function. These essential fatty acids found in the diet in the form of α-linolenic acid LA (n-3) and linoleic acid LNA (n-6). These fatty acids contain 18 carbon atoms which can be metabolized to more highly unsaturated members of their family mainly arachi‐ donic [8] and docosahexaenoic acid [1]. The pathway takes place mainly in the liver and may be occur in the other tissues as well. In endoplasmic reticulum, the conversion of LA to AA occurs, this step consists of sequential alternating elongation and desaturation reactions catalyzed by fatty acid elongase and desaturase. DHA metabolic process occurs via separate channeled carnitine‐dependent mitochondrial pathway. The outer mitochon‐ drial membrane could well be the sole site for DHA. PUFAs accumulate in brain during myelination process. The turnover of PUFAs is unknown, but studies suggest that it is high because the huge demand of them especially in developmental stage of brain. The most important PUFA for infants is DHA. Clinical studies have shown that infants who feed milk-containing DHA in it have higher neurodevelopmental scores compare to other who do not have DHA in their feed. AA and DHA do not accrete in adult brain and plasma AA and DHA only need to replace brain consumption. About 18 and 4 mg/day are the estimate AA and DHA that up taken by brain from plasma unesterified form, respectively. Phospholipases family is responsible for releasing of AA and DHA from brain phospho‐ lipid membrane.

### **2. The importance of the omega‐6 and omega‐3 ratio**

**1. Introduction**

24 Fatty Acids

lipid membrane.

In the last decades, researchers have been focused on lipids to make clear idea about it in both physiological and disease sides. Until now, 600 molecular species have been discovered from human plasma described as lipidome [1, 2]. Lipidome provides a comprehensive classifica‐ tion of lipids with their structure and function. About 60% of dry human brain is composed

From biochemistry point, PUFAs are type of simple lipid that contain one or more double bonds in *Cis* configuration. PUFAs are two classes omega-3 and omega-6 and these classes do not convert to other forms and play important roles in biochemical changes in the body. Omega-3 and omega-6 are dietary essential fatty acids because they cannot be synthesized by human body beside they can prevent deficiency symptoms [5]. The main sources of Omega‐3 fatty acids are vegetable oil and fishes. Docosahexaenoic acid (DHA, 22:6ω3) and eicosa‐ pentaenoic acid (EPA, 20:5ω3) are omega-3 fatty acid with 22 carbons and 6 double bonds (22:6n-3). While vegetables are the main sources of omega-6, arachidonic acid (AA; 20:4ω6) is an omega-6 fatty acid with 20 carbons and 4 double bonds. These two fatty acids are the

In the last years, the interest in the health consumption for Omega‐3 has led to more researches and manufacturing of these fatty acid as supplement foods. The European committee has been suggested that the minimum requirement of omega-3 and omega-6 is approximately 0.5 and 1% of energy intake, respectively. PUFAs are now regarded as nutritionally essential fatty acids [7]. Deficiency in these fatty acids causes dermatitis, growth retardation and infertility. They play critical role as second messengers in the pro‐ cess of signal transductions, structural component of ceramide and specific role in mem‐ brane function. These essential fatty acids found in the diet in the form of α-linolenic acid LA (n-3) and linoleic acid LNA (n-6). These fatty acids contain 18 carbon atoms which can be metabolized to more highly unsaturated members of their family mainly arachi‐ donic [8] and docosahexaenoic acid [1]. The pathway takes place mainly in the liver and may be occur in the other tissues as well. In endoplasmic reticulum, the conversion of LA to AA occurs, this step consists of sequential alternating elongation and desaturation reactions catalyzed by fatty acid elongase and desaturase. DHA metabolic process occurs via separate channeled carnitine‐dependent mitochondrial pathway. The outer mitochon‐ drial membrane could well be the sole site for DHA. PUFAs accumulate in brain during myelination process. The turnover of PUFAs is unknown, but studies suggest that it is high because the huge demand of them especially in developmental stage of brain. The most important PUFA for infants is DHA. Clinical studies have shown that infants who feed milk-containing DHA in it have higher neurodevelopmental scores compare to other who do not have DHA in their feed. AA and DHA do not accrete in adult brain and plasma AA and DHA only need to replace brain consumption. About 18 and 4 mg/day are the estimate AA and DHA that up taken by brain from plasma unesterified form, respectively. Phospholipases family is responsible for releasing of AA and DHA from brain phospho‐

approximately from lipids with over 20% polyunsaturated fatty acids [3, 4].

predominant long-chain (20 and 22 carbons) PUFAs in human brain [6].

The differences between omega-6 and omega-3 acids are very small and may be insignifi‐ cant. They exert opposite effects, ω-3 PUFAs work as anti-inflammatory agent and ω-6 PUFAs as pro-inflammatory agent. These opposing effects are not easily explained. It was suggested that the variation between ω-6 and ω-3 PUFA is based on the molecular basis in particular, to recognize various PUFAs [9]. The dietary deficiency of ω-3 fatty acids, as well as the particular roles of ω-6 and ω-3, becomes an important subject, and their ratio takes a deeper look into the disease issues. The optimal recommended ratio between ω-6 and ω-3 fatty acids has many aspects. One aspect is the recommendation for total daily dietary intake in various phases of life (e.g., infancy, pregnancy, adulthood and old age). Another aspect is the optimal ratio of PUFAs as a food supplement or treatment [10]. PUFAs are used in the body in a variety of conditions, such as in dermatological diseases and in car‐ diovascular disorders. One particular area is the role of PUFAs in the brain and the utility of PUFA to protect and stabilize the neuronal membrane in health and in disease. PUFAs play a critical role in the central nervous system (CNS) and CNS conditions. Many researchers have demonstrated that various PUFAs mediate a lot of process in brain. Some studies examine the best ratio between ω-6 and ω-3 PUFAs to help body to do its role in good way. The required ratio of ω-6 and ω-3 may differ when used for different tissues or functions. 1:1 is the optimal ratio for preventing cardiovascular diseases. 4:1 is the optimal ratio for brain-mediated functions and has protective and stabilizing effects on the neuronal mem‐ brane. The ratio between those PUFAs should be stable to maintain human health [11] (**Figure 1**).

**Figure 1.** The optimal omega-6 to omega-3 PUFAs balance in the body.

Recently, it is well accepted that early alterations of the intestinal microbiota composition with environmental factors such as Cesarean delivery, bottle feeding, diet and abuse of antibiotic, can induce gut dysbiosis that might be linked to abnormal neurodevelopment and brain dysfunction [12]. The role of gut‐brain axis in the etiology of autism is ascer‐ tained and related to intestinal dysbiosis as autistic feature [13]. Based on the fact that gut microbiota are greatly affected with diet, it was interested to discuss the role of ω-3/ω-6 PUFAs on microbial composition of the gut. While some studies demonstrate that ω-6 rich diet shows negative impact on gut microbiota through the induction of overgrowth of Bacteroidetes and Firmicutes as bacterial species related to gastrointestinal inflamma‐ tion frequently occurs in autistic patients [14, 15], ω-3 was proved to induce the growth of bifidobacteria and Lactobacillus as bacterial species that dampening inflammatory responses [16].

### **3. Lipid mediators**

The releasing of AA happens in response to inflammation, ischemia and excitotoxicity, while DHA release occurs in response to ATP, Bardykinin and cholinergic and serotonergic recep‐ tors. These 20 carbon atoms are precursor of lipid mediators that regulate inflammation and immune system. These mediators include eicosanoids and docosanoid and synthesized by many different enzymes and contribute or protect from the risk of inflammation [17, 18]. Cyclooxygenase [19], lipooxygenase (LOX) and cytochrome P450 are the main enzymes involved in lipid mediator's synthesis [17]. COX facilitates conversion of AA to prostaglandin E2 (PGE2). There are two types of COX: COX-1 and COX-2 and their expression differ accord‐ ing to tissues and body situation. Expression of COX-1 occurs in all tissues, while basal COX-2 expression in neurons or in response to inflammation [20, 21] (**Figure 2**).

**Figure 2.** Polyunsaturated fatty acid and their metabolites.

Fatty acids and their mediators have numerous functions in the central nervous system (CNS), including a role in inflammation, glucose production, food intake and in analge‐ sia, beside synaptic function; they activate or suppress neurotransmitter release includ‐ ing glutamate, GABA, monoamine neurotransmitters, opioids and acetylcholine. They also lead to microglia activation and the production of pro-inflammatory cytokines in the hippocampus.

Recently, it is well accepted that early alterations of the intestinal microbiota composition with environmental factors such as Cesarean delivery, bottle feeding, diet and abuse of antibiotic, can induce gut dysbiosis that might be linked to abnormal neurodevelopment and brain dysfunction [12]. The role of gut‐brain axis in the etiology of autism is ascer‐ tained and related to intestinal dysbiosis as autistic feature [13]. Based on the fact that gut microbiota are greatly affected with diet, it was interested to discuss the role of ω-3/ω-6 PUFAs on microbial composition of the gut. While some studies demonstrate that ω-6 rich diet shows negative impact on gut microbiota through the induction of overgrowth of Bacteroidetes and Firmicutes as bacterial species related to gastrointestinal inflamma‐ tion frequently occurs in autistic patients [14, 15], ω-3 was proved to induce the growth of bifidobacteria and Lactobacillus as bacterial species that dampening inflammatory

The releasing of AA happens in response to inflammation, ischemia and excitotoxicity, while DHA release occurs in response to ATP, Bardykinin and cholinergic and serotonergic recep‐ tors. These 20 carbon atoms are precursor of lipid mediators that regulate inflammation and immune system. These mediators include eicosanoids and docosanoid and synthesized by many different enzymes and contribute or protect from the risk of inflammation [17, 18]. Cyclooxygenase [19], lipooxygenase (LOX) and cytochrome P450 are the main enzymes involved in lipid mediator's synthesis [17]. COX facilitates conversion of AA to prostaglandin E2 (PGE2). There are two types of COX: COX-1 and COX-2 and their expression differ accord‐ ing to tissues and body situation. Expression of COX-1 occurs in all tissues, while basal COX-2

expression in neurons or in response to inflammation [20, 21] (**Figure 2**).

**Figure 2.** Polyunsaturated fatty acid and their metabolites.

responses [16].

26 Fatty Acids

**3. Lipid mediators**

Experimental studies have indicated that DHA is involved in learning and memory, but the real mechanisms underlying these effects are not well studied. It has protection effect by enhancing neuronal survival neurogenesis. DHA is the main PUFA in phosphatidylserine. It plays role in suppression of inflammatory cytokine expression and can invade macrophage and microglia. It also blocks macrophage and microglia-induced activation of NF-κB in the CNS of rodents with neuroinflammation [22].

AA and DHA are rapidly incorporated in the nervous tissue of retina and brain during the brain's growth spurt, which mainly takes place from the last trimester of pregnancy up to 2 years of age. Beyond development of the central nervous system, AA and DHA fatty acids may influence brain function throughout life by modifications of neuronal mem‐ brane fluidity, membrane activity-bound enzymes, number and affinity of receptors, func‐ tion of neuronal membrane ionic channels, and production of neurotransmitters and brain peptides.

### **4. Abnormal fatty acid metabolism as etiological mechanism in autism**

To understand the effect of DHA and AA on brain development and cognition, a lot of inter‐ ests have been given to the role of PUFAs in infancy and early childhood life. Brain develop‐ ment in infants and children occurs in specific stages during early life. Unesterified ω-3 and ω-6 fatty acid content of the brain increase considerably during development. For proper CNS function high demand, sufficient supply of the essential PUFAs and proper ratio of AA to DHA are needed as critical process in the early life.

Many studies have observed a relationship between plasma or serum n-3 and n-6 PUFAs imbalances and neurodevelopmental disorders such as autism [23]. As mentioned above, DHA and AA play an important role in the nervous system, including retinal development and vision, neurogenesis and neuronal differentiation, neural plasticity, signal transduction, inflammation, learning and memory. These functions may be regulated by a number of gene products activated by PUFAs during development. Some clinical trials have been conducted on the beneficial effect of dietary ω-3 PUFA supplementation on behavior in various neurode‐ velopmental disorders, including autism [24], but trials with larger sample size are critically requested [3].

There is emerging evidence that fatty acid metabolism and homeostasis are altered in autism due to genetic defects, dietary insufficiency and abnormality in the fatty acid metabolizing enzymes [25–27]. It is well known that alterations of fatty acid metabolism can affect the normal brain function especially during the development. A direct relationship between impaired fatty acid metabolism at various sites and pathophysiology of autism was repeat‐ edly documented.

PLA 2 is an important enzyme that maintains the membrane phospholipids. It catalyzes the release of AA, a precursor of key lipid mediators such as PGs from the *sn‐2* position of phospholipids [28, 29], and it has been shown to play a critical role in neuronal plasticity [30]. Activation of PLA 2 with the excitatory neurotransmitter glutamate usually resulted in a remarkable increase of AA with concomitant impairment of membrane phospholipids [31]. Additionally, both DHA and AA can be released in the presence increased levels inflammatory cytokines [32]. ω-3 PUFA supplementation appears to provide a promising neuroprotective treatment strategy related to the reduction of neuro‐progression mediated by excitotoxicity and oxidative damage (PLA<sup>2</sup> and PUFA supplementation in UHR individuals) [33].

COX-2 has been widely studied as important enzyme that plays critical role in the body. COX-2 is highly expressed in tissues that under stress of inflammation or neurotoxicity. In study done by Boudrault et al. [34], COX-2 was shown to be modulated by ω-3 PUFA in mice brains beside its ability to control ω-6 PUFA level. These results suggest a potential mechanism by which ω-3 PUFA mediates its biological effects on inflammation or neurotransmission. ω3 PUFA suppresses the production of interleukin 1 (IL-1β) by suppressing the IL-1β mRNA, as well as the expression of Cox2 (cytooxygenase) mRNA that is induced by IL-1β [10].

LOX is a group of iron-containing dioxygenases that catalyze the addition of oxygen to AA, DHA and other PUFAs [35]. LOXs have different isoforms according to the type of tissue where they are located. 5-LOX has been shown to play important roles in human pathology by virtue of its central role in leukotriene biosynthesis. Leukotrienes have attracted much attention because of their powerful biological effects in vitro and in vivo. These lipid media‐ tors are active in the low level and elicit a cellular proinflammatory and immune modula‐ tory responses. 5-LOX and leukotrienes have been proved to play role in the pathogenesis of many human acute and chronic inflammatory diseases such as asthma, rheumatoid arthri‐ tis, inflammatory bowel disease, psoriasis, dermatitis, nephritis, atherosclerosis, autism and cancer [36–39]. The anti-inflammatory properties of ω3 PUFAs, especially EPA, are due to competition with AA as a substrate for 5‐lipoxygenase. The eicosanoids are considered a link between PUFA, inflammation and immunity. In addition, ω3 PUFAs have effect on reduce leukotrienes level [10]. From molecular genetic studies of the Icelandic population, variant 5-LOX genotypes were found to be associated with increased atherosclerosis, and dietary ω6 PUFAs promoted, whereas marine ω3 PUFAs inhibited, this effect [40].

PGE 2 is a signaling molecule that diffuses rapidly through the membranes and exerts its diverse effects in the brain via four G-protein coupled EP receptors: EP1, EP2, EP3 and EP4 [41, 42]. The role of PGE 2 signaling in early brain development including formation of den‐ dritic spines and neuronal plasticity is also documented [43, 44]. Tamiji and Crawford [45] reported that expression of the four G-protein coupled EP receptors was found to be signifi‐ cantly increases in the mouse brain during early neurogenesis (11–15 embryonic day). This might indicate that the PGE 2 signaling pathway may have an important role during early brain development. Early brain pathology demonstrates abnormality of certain brain regions in autism [46–48]. Among these regions are cerebellum, medulla and pons which start to develop at the early stages of the neurogenesis (embryonic day 12), in addition to thalamus, hypothalamus, hippocampus and entorhinal cortex that begin developing at around day 15 [49]. A direct involvement of COX-2/PGE 2 signaling pathway in the development of these structures still remains to be ascertained.

enzymes [25–27]. It is well known that alterations of fatty acid metabolism can affect the normal brain function especially during the development. A direct relationship between impaired fatty acid metabolism at various sites and pathophysiology of autism was repeat‐

PLA 2 is an important enzyme that maintains the membrane phospholipids. It catalyzes the release of AA, a precursor of key lipid mediators such as PGs from the *sn‐2* position of phospholipids [28, 29], and it has been shown to play a critical role in neuronal plasticity [30]. Activation of PLA 2 with the excitatory neurotransmitter glutamate usually resulted in a remarkable increase of AA with concomitant impairment of membrane phospholipids [31]. Additionally, both DHA and AA can be released in the presence increased levels inflammatory cytokines [32]. ω-3 PUFA supplementation appears to provide a promising neuroprotective treatment strategy related to the reduction of neuro‐progression mediated by excitotoxicity

COX-2 has been widely studied as important enzyme that plays critical role in the body. COX-2 is highly expressed in tissues that under stress of inflammation or neurotoxicity. In study done by Boudrault et al. [34], COX-2 was shown to be modulated by ω-3 PUFA in mice brains beside its ability to control ω-6 PUFA level. These results suggest a potential mechanism by which ω-3 PUFA mediates its biological effects on inflammation or neurotransmission. ω3 PUFA suppresses the production of interleukin 1 (IL-1β) by suppressing the IL-1β mRNA, as

LOX is a group of iron-containing dioxygenases that catalyze the addition of oxygen to AA, DHA and other PUFAs [35]. LOXs have different isoforms according to the type of tissue where they are located. 5-LOX has been shown to play important roles in human pathology by virtue of its central role in leukotriene biosynthesis. Leukotrienes have attracted much attention because of their powerful biological effects in vitro and in vivo. These lipid media‐ tors are active in the low level and elicit a cellular proinflammatory and immune modula‐ tory responses. 5-LOX and leukotrienes have been proved to play role in the pathogenesis of many human acute and chronic inflammatory diseases such as asthma, rheumatoid arthri‐ tis, inflammatory bowel disease, psoriasis, dermatitis, nephritis, atherosclerosis, autism and cancer [36–39]. The anti-inflammatory properties of ω3 PUFAs, especially EPA, are due to competition with AA as a substrate for 5‐lipoxygenase. The eicosanoids are considered a link between PUFA, inflammation and immunity. In addition, ω3 PUFAs have effect on reduce leukotrienes level [10]. From molecular genetic studies of the Icelandic population, variant 5-LOX genotypes were found to be associated with increased atherosclerosis, and dietary ω6

PGE 2 is a signaling molecule that diffuses rapidly through the membranes and exerts its diverse effects in the brain via four G-protein coupled EP receptors: EP1, EP2, EP3 and EP4 [41, 42]. The role of PGE 2 signaling in early brain development including formation of den‐ dritic spines and neuronal plasticity is also documented [43, 44]. Tamiji and Crawford [45] reported that expression of the four G-protein coupled EP receptors was found to be signifi‐ cantly increases in the mouse brain during early neurogenesis (11–15 embryonic day). This might indicate that the PGE 2 signaling pathway may have an important role during early

well as the expression of Cox2 (cytooxygenase) mRNA that is induced by IL-1β [10].

PUFAs promoted, whereas marine ω3 PUFAs inhibited, this effect [40].

and PUFA supplementation in UHR individuals) [33].

edly documented.

28 Fatty Acids

and oxidative damage (PLA<sup>2</sup>

The first reaction of mitochondrial fatty acid β-oxidation (FAO) in mitochondria is catalyzed by acyl-CoA dehydrogenase. Four different dehydrogenases participate in the complete deg‐ radation of fatty acids in mitochondria. They are flavin adenine dinucleotide (FAD)-containing enzymes which are structurally and functionally related only differ in their substrate speci‐ ficities. These are, short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD), reflect the acyl chain lengths of their preferred substrates. Deficiency of long-chain acyl-CoA dehydrogenase (LCAD), as one of these dehydrogenases, is suspected to have a link with the development of autism [50].

Fatty acid β-oxidation is the major pathway to produce ATP and reducing power from differ‐ ent chain lengths of fatty acids [51, 52]. Transport of fatty acids from the cytoplasm into mito‐ chondria is rate limiting step of FAO, and it requires carnitine as acyl carrier and carnitine palmitoyltransferase I (CPT1), which catalyzes the first regulatory reaction in this process. Trimethyllysine hydroxylase (TMLHE) is a second enzyme that catalyzes the first step of car‐ nitine biosynthesis [53]. It is very interesting that several studies had reported that mutation of TMLHE is present in human population with high rate [54]. There is great evidence dem‐ onstrating an association between impaired FAO and autism [50, 55, 56]. Individuals with autism show altered levels of blood or plasma carnitine and acyl‐carnitine, as a phenotype related to impaired long chain FAO. On the other hand, FAO-deficient children exhibit autis‐ tic features such as developmental delay [57]. Recently, Xie et al. [58] reported that efficient FAO is critically needed for the maintenance of neuronal stem cell (NSC) homeostasis in the mammalian embryonic neocortex. They suggested that linkage of NSC homeostatic mecha‐ nisms with inborn errors of metabolism (IEM) of developmental brain disorders has clinical implications. An increased risk of autism was found to be associated with TMLHE deficien‐ cies [54, 59]. They also recorded that enhanced oxidative stress was observed in NSC mito‐ chondria with impaired FAO activity, suggesting that impairment of NSC self-renewal occurs due to oxidative stress as an accepted etiological mechanism in autistic children [26, 27, 60].

Another evidence for fatty acids metabolic disturbances as one potential etiological mech‐ anism in autism is the remarkable increase of adipic and suberic acids, as two dicarbox‐ ylic acids produced by the omega (ω)-oxidation pathway, a minor catabolic pathway for medium-chain fatty acids that becomes more important when β-oxidation is impaired [51, 61] (**Figure 3**). Based on the previously discussed association between impaired FAO and autism [50, 56, 62, 63], it was suggested that altered β-oxidation can increase the activity of ω fatty acid oxidation, thus leading to increased production of adipic and suberic acid [58, 61]. There is a strong body of evidence between mitochondrial dysfunction and PUFAs transport and metabolism in autism. For this reason, shifting from β to ω-oxidation pathway considering as an emergency pathway that protect cell from deleterious effects of mitochondrial enzyme

**Figure 3.** Beta oxidation (up) and omega oxidation [44] of fatty acids.

dysfunctions. So, researchers those days are looking for biomarkers that help to understand the activity of this pathway. The attention has focused in adipic and suberic acid measure‐ ments and their correlation with other important determiners that defined in autism.

Increased level of adipic acid has shown to inhibit the activity of both l‐glutamate decarboxyl‐ ase [64] and GABA transaminase [65], leading to impaired glutamate/GABA ratio that might induce glutamate excitotoxicity, as consistent autistic feature in animal model and individu‐ als with autism, through the overstimulation of glutamate receptors [66–69].

### **5. Fatty acids and brain neurochemistry**

### **5.1. Serotonin**

The reported impaired profile of PUFAs and their related lipid mediators in autistic children can be related to their abnormal neurotransmitter physiology. In animals studies, feeding on essential fatty acids diet resulted in serotonin depletion in the frontal cortex of pre-adolescent but not in post-pubescent rats, suggesting a role of n-3 DHA and n-6 AA in neurotransmitter synthesis or turnover [70]. Based on this lower n-3, DHA can be related to the absence of agedependent changes of brain serotonin synthesis in autistic children and hyperserotonemia as biomarker of clinical severity of autism [71].

### **5.2. Gamma‐amino butyric acid (GABA)**

Takeuchi et al. reported that ω-3 DHA deficiency is related to the altered GABAergic activ‐ ity in autistic patients [72]. This might be through the prevention GABAA receptor blocking repeatedly reported in this disorder [73]. This provides an important link between PUFAs and pathogenesis of autism [74, 75]. A second mechanism of interaction between PUFAs and GABA neurotransmission is through the actions of phospholipase A2 (PLA2) a membrane phospholipid hydrolyzing enzyme. PLA2 is thought to inhibit GABAA receptor function by reducing chloride flux in the cerebral cortex [76]. Based on this, ω-6 AA usually induces neu‐ ronal excitability through the activation of PLA2 or phospholipase C (PLC) and inhibition of GABAA receptors [77]. This can be easily related to the imbalanced GABAergic/glutamater‐ gic in autistic patients [66].

### **5.3. Glutamate**

**Figure 3.** Beta oxidation (up) and omega oxidation [44] of fatty acids.

30 Fatty Acids

Multiple early studies demonstrated that activation of postsynaptic glutamate receptors by glutamate induces release of AA from membrane phospholipids either directly, by activation of phospholipase A2, or indirectly from degradation of diacylglycerol [78, 79]. On the other hand, AA has been shown to increase glutamate release from synaptosomes [80, 81] through the stimulation of the inositol phospholipid metabolism or activation of protein kinase C.

Elevation of AA can be easily related to glutamate excitotoxicity and glutathione depletion as etiological mechanisms of autism. Recently, elevation of PLA2 was recorded in plasma of autistic patients compared to healthy controls [66, 82]. This enzyme is involved in the selective release of AA from phospholipids such as PC, PS and PE [36, 83]. Higuchi et al. [84] proved that AA is involved and related to glutamate‐induced glutathione depletion and the subse‐ quent cell death through the accumulation of hydroperoxy eicosatetraenoic acids (HPETE) as AA reactive oxygen species (ROS) or hydroperoxides. This can be supported by the recent record of Gebremedhin [85] which reported that astrocytes of neonatal rat brain express mes‐ sage and protein for cytochrome P450 4A ω-hydroxylase CYP4A2/3 and synthesize 20-HETE when incubated with AA and this usually enhanced through the activation of metabotropic glutamate receptors.

#### **5.4. Dopamine**

Omega-3 intake has shown therapeutic effects through dopamine neurotransmission in major depression. The antidepressant efficacy of ω-3 supplementation may raise the possibility that they may have specific value for major depressive disorder with a dopaminergic system defi‐ cit [86]. This finding may have important implications for therapeutic strategies involving augmentation of standard antidepressant medications with fish oil. ω-3 has beneficial effect as detoxification agent that remove bad effect of reactive oxygen species in Parkinson disease [87]. PUFAs have been specially associated with dopamine activity in frontal lobe of brain. In adolescents, dietary n-3 PUFA deficiency produced a modality selective and task-dependent impairment in cognitive and motivated behavior distinct from the deficits observed in adults. This deficiency affected expression of dopamine-related proteins. Adolescent behavior and dopamine availability are uniquely sensitive to dietary omega-3 fatty acid deficiency [88]**.**

### **6. PUFAs and BDNF interact with each other**

Brain-derived neurotrophic factor (BDNF) showed alteration levels in sample of autistic patients, and it is involved in the regulation of neuronal development and plasticity and has a role in learning and memory. In first several years, serum BDNF concentrations increased in healthy children and then slightly decreased after reaching the adult level. In the patients with autism, mean levels were significantly lower in children compared with healthy adults [89]. Many researches [90–92] indicated that BDNF plays a critical role in the diagnosis of autism. PUFAs and BDNF interact with each other since PUFAs are known to augment the levels of BDNF in the brain [93]. PGE2 derived from AA, induced release of BDNF from glial cells and astrocytes through a G-protein-coupled receptor and then affect on the whole signaling pathway inside cell [94]. PGE2 contributes to BDNF upregulation in neurons following nerve injury in animal models, which facilitates the synthesis of BDNF in primary sensory neurons to initiate repair of damaged neurons and neuronal regeneration [95]. Other PUFA metabolites especially lipoxin A4 (LXA4), resolvins and protectins interact with BDNF. These interactions provide anti-inflammatory effect when the body needs it [96]. Deficiency in ω-3 PUFA intake is linked to decreased BDNF content, and low BDNF levels have been described after prenatal stress [97] (**Figure 4**). Glucocorticoids have been related to such an effect, since corticosterone is able to down-regulate both mRNA and protein BDNF [98]. Over‐expressing of glucocorti‐ coids showed an increased anxiety‐like behavior [99]. Larrieu and colleagues have clarified that n-3 PUFA deficiency can influence neuronal cortical morphology and depressive-like

**Figure 4.** Interaction between PUFAs metabolites and BDNF.

that AA is involved and related to glutamate‐induced glutathione depletion and the subse‐ quent cell death through the accumulation of hydroperoxy eicosatetraenoic acids (HPETE) as AA reactive oxygen species (ROS) or hydroperoxides. This can be supported by the recent record of Gebremedhin [85] which reported that astrocytes of neonatal rat brain express mes‐ sage and protein for cytochrome P450 4A ω-hydroxylase CYP4A2/3 and synthesize 20-HETE when incubated with AA and this usually enhanced through the activation of metabotropic

Omega-3 intake has shown therapeutic effects through dopamine neurotransmission in major depression. The antidepressant efficacy of ω-3 supplementation may raise the possibility that they may have specific value for major depressive disorder with a dopaminergic system defi‐ cit [86]. This finding may have important implications for therapeutic strategies involving augmentation of standard antidepressant medications with fish oil. ω-3 has beneficial effect as detoxification agent that remove bad effect of reactive oxygen species in Parkinson disease [87]. PUFAs have been specially associated with dopamine activity in frontal lobe of brain. In adolescents, dietary n-3 PUFA deficiency produced a modality selective and task-dependent impairment in cognitive and motivated behavior distinct from the deficits observed in adults. This deficiency affected expression of dopamine-related proteins. Adolescent behavior and dopamine availability are uniquely sensitive to dietary omega-3 fatty acid deficiency [88]**.**

Brain-derived neurotrophic factor (BDNF) showed alteration levels in sample of autistic patients, and it is involved in the regulation of neuronal development and plasticity and has a role in learning and memory. In first several years, serum BDNF concentrations increased in healthy children and then slightly decreased after reaching the adult level. In the patients with autism, mean levels were significantly lower in children compared with healthy adults [89]. Many researches [90–92] indicated that BDNF plays a critical role in the diagnosis of autism. PUFAs and BDNF interact with each other since PUFAs are known to augment the levels of BDNF in the brain [93]. PGE2 derived from AA, induced release of BDNF from glial cells and astrocytes through a G-protein-coupled receptor and then affect on the whole signaling pathway inside cell [94]. PGE2 contributes to BDNF upregulation in neurons following nerve injury in animal models, which facilitates the synthesis of BDNF in primary sensory neurons to initiate repair of damaged neurons and neuronal regeneration [95]. Other PUFA metabolites especially lipoxin A4 (LXA4), resolvins and protectins interact with BDNF. These interactions provide anti-inflammatory effect when the body needs it [96]. Deficiency in ω-3 PUFA intake is linked to decreased BDNF content, and low BDNF levels have been described after prenatal stress [97] (**Figure 4**). Glucocorticoids have been related to such an effect, since corticosterone is able to down-regulate both mRNA and protein BDNF [98]. Over‐expressing of glucocorti‐ coids showed an increased anxiety‐like behavior [99]. Larrieu and colleagues have clarified that n-3 PUFA deficiency can influence neuronal cortical morphology and depressive-like

**6. PUFAs and BDNF interact with each other**

glutamate receptors.

**5.4. Dopamine**

32 Fatty Acids

behavior through corticosterone secretion. Furthermore, they showed that diet with low ω-3 induces a phenotype of social deficits and emotional behavior which is observed in autistic patients [100].

### **7. Amelioration of impaired lipid metabolism as treatment strategy of autism**

It is well accepted that imbalances in ω-3 and ω-6 fatty acids are one of the etiological mecha‐ nisms in autism and are directly related to the abnormal behavioral severity of these patients. Interestingly, omega-3 and omega-6 fatty acids supplementation resulted in increased level of these fatty acids in the blood, reduced the elevated AA:DHA ratio ameliorates some behav‐ ioral deficits such as eye contact, hyperactivity, concentration and motor skills in autistic patients [101]. This can find support in the more recent study of Yui et al. [102, 103] which proved that large doses of AA added to DHA may improve impaired social interaction in individuals with autism, and Amminger et al. [104] who suggest that the use of pure omega‐3 PUFAs (without any AA) may be beneficial in autism.

In a recent report of Klein and Kemper [105], supplementation with ω-3 fatty acids is more effective than risperidone as pharmacological drug with side effects. ω-3 fatty acids demon‐ strate many ameliorating effects presented as more social interaction, less irritability and more flexibility [106]. Due to the lack of evidence of effectiveness from large randomized clinical trials, the safety, and low cost of ω-3 fatty acids, clinicians can encourage families' use of sup‐ plemental ω-3, but more frequent and completely blind trials are requested to move ω-3 fatty acids from tolerated to recommended supplement for the treatment of autistic patients [105].

Due to the strong interaction between diet and the gut microbiota, it has been suggested that the role of dietary changes in influencing brain biochemistry and behavior may be mediated through changes in gut microbiota composition and function [107]. In addition to improving brain function, n-3 PUFA can be used as treatment strategy of autistic patients through its beneficial impact on restoring healthy gut-microbiota by inducing bifidobacteria, and lac‐ tobacillus growth, and inhibiting enterobacteria growth with subsequent anti-inflammatory responses [16].

Mediterranean diet as good source of ω-3 usually recommended as a healthy diet [108]. It consists mainly of cereals, vegetables, nuts and fruits, with moderate amount of fish and poul‐ try and low amount of red meat. Polyphenols as major ingredients of olive oil, a common component of Mediterranean diet is known to promo its protective effect by modulating dif‐ ferent signaling cascades among which is nuclear factor-kappaB (NF-kB), pro-inflammatory response and oxidative stress response as three etiological mechanisms repeatedly recorded in autism [109].

Moreover, carnitine supplements, as a compound normally required for fatty acids metabo‐ lism, and significantly reduced in some children with autism [55], it was effective in improv‐ ing the remarkably reduced DHA and very long-chain fatty acid level of autistic subjects [110]. Unlike autistic children, ω-3 supplementation showed no beneficial effect on severe autistic adults [24, 111].

### **Author details**

Afaf El‐Ansary1,2\* and Hanan Qasem<sup>2</sup>

\*Address all correspondence to: afafkelansary@gmail.com

1 Central Laboratory, King Saud University, Riyadh, Saudi Arabia

2 Autism Research and Treatment Center, King Saud University, Riyadh, Saudi Arabia

### **References**


[3] Schuchardt J.P., et al., Significance of long-chain polyunsaturated fatty acids (PUFAs) for the development and behaviour of children. European Journal of Pediatrics. 2010;**169**(2):149-164

trials, the safety, and low cost of ω-3 fatty acids, clinicians can encourage families' use of sup‐ plemental ω-3, but more frequent and completely blind trials are requested to move ω-3 fatty acids from tolerated to recommended supplement for the treatment of autistic patients [105]. Due to the strong interaction between diet and the gut microbiota, it has been suggested that the role of dietary changes in influencing brain biochemistry and behavior may be mediated through changes in gut microbiota composition and function [107]. In addition to improving brain function, n-3 PUFA can be used as treatment strategy of autistic patients through its beneficial impact on restoring healthy gut-microbiota by inducing bifidobacteria, and lac‐ tobacillus growth, and inhibiting enterobacteria growth with subsequent anti-inflammatory

Mediterranean diet as good source of ω-3 usually recommended as a healthy diet [108]. It consists mainly of cereals, vegetables, nuts and fruits, with moderate amount of fish and poul‐ try and low amount of red meat. Polyphenols as major ingredients of olive oil, a common component of Mediterranean diet is known to promo its protective effect by modulating dif‐ ferent signaling cascades among which is nuclear factor-kappaB (NF-kB), pro-inflammatory response and oxidative stress response as three etiological mechanisms repeatedly recorded

Moreover, carnitine supplements, as a compound normally required for fatty acids metabo‐ lism, and significantly reduced in some children with autism [55], it was effective in improv‐ ing the remarkably reduced DHA and very long-chain fatty acid level of autistic subjects [110]. Unlike autistic children, ω-3 supplementation showed no beneficial effect on severe

responses [16].

34 Fatty Acids

in autism [109].

autistic adults [24, 111].

Afaf El‐Ansary1,2\* and Hanan Qasem<sup>2</sup>

Medicine. 2011;**365**(19):1812-1823

\*Address all correspondence to: afafkelansary@gmail.com

1 Central Laboratory, King Saud University, Riyadh, Saudi Arabia

2 Autism Research and Treatment Center, King Saud University, Riyadh, Saudi Arabia

related to inflammation, and disease risk. Eur Psychiatry. 2017.**43**:44‐50.

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40 Fatty Acids


**Chapter 3**

## **Fatty Acids on Osteoclastogenesis**

Sergio Montserrat‐de la Paz, Rocio Abia, Beatriz Bermudez, Sergio Lopez and Francisco JG Muriana

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68117

#### **Abstract**

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42 Fatty Acids

646-649

axis. Clinical Nutrition Experimental. 2016;6:25-38

ment in autism: a naturalistic case–control study. Journal of Child and Adolescent

Excessive bone resorption is a hallmark on the onset and development of bone diseases, including osteoporosis, periodontitis, and rheumatoid arthritis. Osteoclasts are bone‐resorbing multinucleated cells that differentiate from hematopoietic progenitors of the myeloid lineage. The regulation of this differentiation process is considered an effective therapeutic interven‐ tion to the treatment of pathological bone loss. Dietary fatty acids (FAs), transported in the form of postprandial triglyceride‐rich lipoproteins, have been linked with inflammation and oxidative stress associated to the overactivation of circulating leukocytes. Monocyte differ‐ entiation by soluble cytokines is known to up‐regulate osteoclast maturation via increased expression levels of receptor activator for nuclear factor‐κB ligand relative to osteoprotegerin. This review summarizes the effects of dietary omega‐3 long‐chain polyunsaturated fatty acids, monounsaturated fatty acids, and saturated fatty acids on plasticity during osteoclast forma‐ tion and function.

**Keywords:** bone marrow, bone metabolism, fatty acids, osteoclasts, osteoporosis

#### **1. Introduction**

The links among bone and nutrition focus on considerable public health and research inter‐ ests. Over the past 20 years, the fact that there is an inverse relationship between bone mass and marrow adiposity, observed under physiological and pathological conditions, has led to increased recent interest in bone lipids [1, 2]. Under different pathologies, for example, osteoporosis, an increase of bone marrow fat that was associated with osteoclast (OC) over‐ abundance and a low bone mass [3]. Cholesterol, phospholipids, and fatty acids (FAs) either free or in the form of triglycerides, have been demonstrated to act on bone metabolism and

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

bone cell development and functions. Thus, they can be regarded as regulatory molecules important in bone health. A growing body of evidence, including the recognition that specific FA receptors are expressed in bone‐related cells, suggests that FAs both circulating and inside bone marrow, could be an active determinant role as messengers on metabolic activity and remodeling rate of bone [4]. This review will provide a current overview on the effects of FAs on OC maturation and function.

### **2. Osteoclast biology**

Bone is a specialized, hard tissue consisting of a soft part (the bone marrow), and the mineralized osseous tissue itself. To ensure bone integrity during childhood and adult‐ hood, bone undergoes a continuous remodeling process that consists of multiple cycles of bone digestion and rebuilding steps [5]. Two cell types mainly determine this remodeling process, the bone‐forming osteoblasts (OBs) and the bone‐resorbing OCs. A dysregulation of the bone remodeling balance is linked with several skeletal disorders such as osteope‐ trosis and osteoporosis. Osteopetrosis is characterized by an increase in bone mass due to a lower OC number or activity, whereas osteoporosis is characterized by the loss of bone mass due to an elevated OC activity [6]. Moreover, bone contains interconnected and embedded OBs, called osteocytes, which might respond to the mechanical pressure applied onto bone [7].

During initial bone formation, OBs produce organic bone matrix and promote its mineraliza‐ tion. At the same time, OBs indirectly affect bone resorption by the expression of ligands, including the receptor activator of NF‐κB ligand (RANKL), which is important for OC dif‐ ferentiation [5]. In contact with bone, OCs change their plasma membrane to form differ‐ ent domains, including the ruffled border that faces the bone surface. This specialized cell membrane is provided with many lysosomal integral membrane proteins, mainly the V‐type H+ ‐ATPase, ensuring the acidification of the resorption environment that is required to dis‐ solve the bone inorganic matrix. OCs also release lysosomal hydrolases such as cathepsin K to digest the organic bone matrix [8]. Furthermore, the ruffled border is composed by actin‐rich podosomes that ensure the attachment of OCs onto the bone. Bone degradation products are endocytosed through the ruffled border, transcytosed, and secreted into the extracellular space [9]. For efficient resorption, OCs undergo several cycles of adhesion, resorption, and migration along bone surfaces.

Bone biology has greatly benefited from studies using animal models. For example, silenc‐ ing Src tyrosine kinase, receptor–activator of NF‐κB (RANK), tartrate‐resistant acid phos‐ phatase (TRAP), and cathepsin K in mice result in an osteopetrotic mouse model due to the lack of OC precursor differentiation or a lack of mature OC activity [10, 11]. However, these mutant animal models do not provide an integrated view on the function of a par‐ ticular gene on OC differentiation and function and its modulation by certain cytokines, nutrients, and drugs, which could provide a better understanding of their effects on OC biology.

### **3. Osteoclastogenesis in the bone marrow**

bone cell development and functions. Thus, they can be regarded as regulatory molecules important in bone health. A growing body of evidence, including the recognition that specific FA receptors are expressed in bone‐related cells, suggests that FAs both circulating and inside bone marrow, could be an active determinant role as messengers on metabolic activity and remodeling rate of bone [4]. This review will provide a current overview on the effects of FAs

Bone is a specialized, hard tissue consisting of a soft part (the bone marrow), and the mineralized osseous tissue itself. To ensure bone integrity during childhood and adult‐ hood, bone undergoes a continuous remodeling process that consists of multiple cycles of bone digestion and rebuilding steps [5]. Two cell types mainly determine this remodeling process, the bone‐forming osteoblasts (OBs) and the bone‐resorbing OCs. A dysregulation of the bone remodeling balance is linked with several skeletal disorders such as osteope‐ trosis and osteoporosis. Osteopetrosis is characterized by an increase in bone mass due to a lower OC number or activity, whereas osteoporosis is characterized by the loss of bone mass due to an elevated OC activity [6]. Moreover, bone contains interconnected and embedded OBs, called osteocytes, which might respond to the mechanical pressure

During initial bone formation, OBs produce organic bone matrix and promote its mineraliza‐ tion. At the same time, OBs indirectly affect bone resorption by the expression of ligands, including the receptor activator of NF‐κB ligand (RANKL), which is important for OC dif‐ ferentiation [5]. In contact with bone, OCs change their plasma membrane to form differ‐ ent domains, including the ruffled border that faces the bone surface. This specialized cell membrane is provided with many lysosomal integral membrane proteins, mainly the V‐type

‐ATPase, ensuring the acidification of the resorption environment that is required to dis‐ solve the bone inorganic matrix. OCs also release lysosomal hydrolases such as cathepsin K to digest the organic bone matrix [8]. Furthermore, the ruffled border is composed by actin‐rich podosomes that ensure the attachment of OCs onto the bone. Bone degradation products are endocytosed through the ruffled border, transcytosed, and secreted into the extracellular space [9]. For efficient resorption, OCs undergo several cycles of adhesion, resorption, and

Bone biology has greatly benefited from studies using animal models. For example, silenc‐ ing Src tyrosine kinase, receptor–activator of NF‐κB (RANK), tartrate‐resistant acid phos‐ phatase (TRAP), and cathepsin K in mice result in an osteopetrotic mouse model due to the lack of OC precursor differentiation or a lack of mature OC activity [10, 11]. However, these mutant animal models do not provide an integrated view on the function of a par‐ ticular gene on OC differentiation and function and its modulation by certain cytokines, nutrients, and drugs, which could provide a better understanding of their effects on OC

on OC maturation and function.

**2. Osteoclast biology**

44 Fatty Acids

applied onto bone [7].

migration along bone surfaces.

H+

biology.

The bone‐resorbing OCs are originated from the differentiation of hematopoietic mononucle‐ ated precursors and their subsequent fusion to form multinucleated mature OCs (**Figure 1**). Physiologically, osteoclastogenesis requires two essential hematopoietic factors in the bone marrow: macrophage colony‐stimulating factor (M‐CSF/CSF‐1) and RANKL. M‐CSF/CSF‐1 is a survival and proliferation factor that induces RANK expression in OC precursor cells [12]. The role of M‐CSF/CSF‐1 in osteoclastogenesis is highlighted by the osteopetrotic phenotype M‐CSF−/− mouse model, in which mutant animals had a deficiency in OCs and circulating monocytes [13]. The second key factor in osteoclastogenesis, RANKL, is a membrane‐residing protein found on OBs and their precursors, and is recognized by its cognate receptor RANK expressed in the bone marrow macrophage/OC lineage, promoting their differentiation into OCs [14]. In mice, genetic experiments have shown the importance of RANK/RANKL axis for osteoclastogenesis, as targeted inhibition of RANK or RANKL gene results in a complete absence of OC maturation and osteopetrosis [15]. In humans *in vitro* studies, these two fac‐ tors are able to generate OCs from circulating monocytes, dendritic cells, and bone marrow– derived macrophages [16]. In addition to RANKL, osteoprotegerin (OPG) is secreted by OBs, which acts as a soluble RANKL decoy receptor; therefore, OPG negatively regulates RANKL activity (**Figure 2**) [17]. From these observations, the RANKL/OPG ratio indicates the rate of osteoclastic bone resorption [18].

The binding of RANK receptor to RANKL triggers signaling cascades that terminally dif‐ ferentiate the hematopoietic precursor cells into OCs. The initial step in RANKL signaling is the binding of RANK receptor to the cytoplasmatic tumor necrosis factor receptor‐associ‐ ated factors (TRAF), mainly to TRAF6 [19]. The Src tyrosine kinase binds to TRAF6, regu‐ lating the aspects of OC function such as cytoskeletal reorganization. In addition, RANKL signaling leads to the OC specific gene expression such as β3 integrins, TRAP, cathepsin K, and calcitonin receptor. It also leads to the morphological conversion of mononucleated cells into large multinucleated cells that are able to efficiently resorb large bone surface areas.

**Figure 1.** Diagram illustrating the differentiation of hematopoietic mononucleated precursors and their subsequent fusion to form multinucleated mature osteoclasts.

**Figure 2.** Key factors affecting osteoblast and osteoclast survival and functions.

### **4. Fatty acids in the bone marrow**

FAs are carboxylic acids and often contain a long, unbranched aliphatic chain. FAs are catego‐ rized as saturated (SFAs), monounsaturated (MUFAs), and polyunsaturated (PUFAs) based on their structural and chemical properties. SFAs do not contain any double bonds or other functional groups along the chain, which is fully saturated with hydrogen atoms. The prin‐ cipal dietary SFAs are palmitic acid (16:0) and stearic acid (18:0), which are composed of 16 and 18 carbon atoms, respectively. MUFAs contain one pair of carbon atoms linked by a *cis* double bond. Oleic acid (18:1n−9), which contains 18 carbon atoms with a double bond at the 9th carbon from the methyl end of the FA molecule, is the major dietary MUFA and repre‐ sents 55–83% of the total FAs in virgin olive oil [20]. Carbon chains containing 2 or more *cis* double bonds, with the first double bond located between either the 3rd and 4th or the 6th and 7th carbon atoms from the methyl end of the FA molecule, that belong to the n−3 or n−6, respectively, PUFA families. These families cannot be synthesized by the human body (dou‐ ble bonds can be introduced into all positions of the FA chain with the exception of the n−3 and n−6 positions); and therefore, must be obtained from the diet as α‐linolenic acid (18:3n−3) and linoleic acid (18:2n−6) or their long‐chain PUFA derivatives. Of these FAs, eicosapen‐ taenoic acid (EPA, 20:5n−3), docosahexaenoic acid (DHA, 22:6n−3), dihomo‐γ–linolenic acid (20:3n−6), and arachidonic acid (AA, 20:4n−6) are the most metabolically significant [21].

FA compositions of total lipids present in bone marrows change with the species studied. Thus, palmitic acid, stearic acid, and oleic acid are predominant in rats and cows [22], whereas palmitic acid, oleic acid, and linoleic acid are the main FAs in bone marrows of humans, dogs, guinea pigs, and rabbits [23]. Bone FA profile usually reflects the FA composition of the diet. For example, when animals were fed diet supplemented with linoleic acid or α‐linolenic acid, concentrations of these two FAs were higher in femoral cortical bone and marrow [24]. Recent animal and human intervention studies reported that dietary FAs affect bone health. In gen‐ eral, high intakes of long‐chain omega‐3 PUFAs rather than long‐chain omega‐6 PUFAs are beneficial for bone mass [25], whereas SFAs intake is harmful [26].

### **5. Direct action of exogenous fatty acids on bone cells**

**4. Fatty acids in the bone marrow**

46 Fatty Acids

**Figure 2.** Key factors affecting osteoblast and osteoclast survival and functions.

FAs are carboxylic acids and often contain a long, unbranched aliphatic chain. FAs are catego‐ rized as saturated (SFAs), monounsaturated (MUFAs), and polyunsaturated (PUFAs) based on their structural and chemical properties. SFAs do not contain any double bonds or other functional groups along the chain, which is fully saturated with hydrogen atoms. The prin‐ cipal dietary SFAs are palmitic acid (16:0) and stearic acid (18:0), which are composed of 16 and 18 carbon atoms, respectively. MUFAs contain one pair of carbon atoms linked by a *cis* double bond. Oleic acid (18:1n−9), which contains 18 carbon atoms with a double bond at the 9th carbon from the methyl end of the FA molecule, is the major dietary MUFA and repre‐ sents 55–83% of the total FAs in virgin olive oil [20]. Carbon chains containing 2 or more *cis* double bonds, with the first double bond located between either the 3rd and 4th or the 6th and 7th carbon atoms from the methyl end of the FA molecule, that belong to the n−3 or n−6, respectively, PUFA families. These families cannot be synthesized by the human body (dou‐ ble bonds can be introduced into all positions of the FA chain with the exception of the n−3 and n−6 positions); and therefore, must be obtained from the diet as α‐linolenic acid (18:3n−3) and linoleic acid (18:2n−6) or their long‐chain PUFA derivatives. Of these FAs, eicosapen‐ taenoic acid (EPA, 20:5n−3), docosahexaenoic acid (DHA, 22:6n−3), dihomo‐γ–linolenic acid (20:3n−6), and arachidonic acid (AA, 20:4n−6) are the most metabolically significant [21].

FA compositions of total lipids present in bone marrows change with the species studied. Thus, palmitic acid, stearic acid, and oleic acid are predominant in rats and cows [22], whereas palmitic acid, oleic acid, and linoleic acid are the main FAs in bone marrows of humans, dogs, At the level of bone cell biology, *de novo* biosynthesized FAs or FAs taken up by cells are mostly incorporated into both phospholipids located in cell membranes and triglycerides in cytoplasmic lipid droplets. On the other hand, membrane FA composition has demonstrated to modulate intracellular signaling pathways and many cell functions such as membrane flu‐ idity and permeability [20]. Thus, FAs may influence the bone formation/resorption balance by affecting the functionality of OBs and OCs.

*In vitro* studies have demonstrated that exogenous FAs supplemented to the OBs or OCs cul‐ ture media can affect their survival and functions. Data indicate that SFAs, mainly palmitic and stearic acids, are pivotal for OBs by inducing both autophagy and apoptosis [27, 28]. PUFAs also alter OB proliferation and functions [29, 30], while oleic acid seems to be neutral in OBs [31].

Few studies, summarized in **Table 1**, have focused at exogenous FA effects on OCs and the data are partially contradictory, at least for SFAs. Indeed, SFAs, mostly myristic, palmitic, and stearic acids 16:0 were first reported to inhibit osteoclastogenesis [32], and recently, to enhance it by inhibiting apoptosis of mature OCs [33]. The actions of exogenous FAs on bone cells include their ability to modulate different signaling pathways that are involved in gen‐ eral cell growth, differentiation, inflammation, and apoptosis processes. FAs can also alter expression/activation of different nuclear transcription factors which play an important role in bone metabolism, such as nuclear factor κB (NF‐κB, crucial for many bone cell processes and for OC activity), and peroxisome proliferator–activated receptor γ (PPARγ, role in bone‐ fat relationship) [35]. To start cell signaling, FAs play via protein sensors located either in cytosol (i.e., FA‐binding proteins (FABPs) and PPARs) or at cell surface (i.e., specific receptors that belong to the family of G‐protein–coupled receptors (GPCs)). These extracellular recep‐ tors are likely to play an important role in bone physiology since they are expressed at the surface of OBs and OCs [32]. As outlined in **Table 2**, there are currently six receptors known to be linked by FAs of different carbon chain length and degree of saturation. GPR120 has been reported to be expressed in OBs; however, these cells do not express GPR40, 41, or 43 [32]. In a review of the effects of exogenous FAs on osteoclast OC development at concentrations of 0.1–10 μg/ml, the most potent effects were observed in response to palmitic and stearic acids, implying that signaling through GPR120 mediates, at least in part, the direct osteoclastogenic actions of medium and long‐chain SFAs [32].

On the other hand, limited evidence exists as to the actions of PUFAs on OC development. Two studies have reported inhibitory actions of linoleic acid on osteoclastogenesis in bone


**Table 1.** Effect of exogenous free fatty acids (FAs) on osteoclast functions and survival (Adapted from Ref. [4]).


**Table 2.** Fatty acid (FA) receptors (Adapted from Ref. [36]).

marrow cultures and RAW264.7 cells [32, 34]. A subsequent report found that DHA, but not EPA, substantially decreased OC development in RANKL‐treated in bone marrow cultures and RAW264.7 cells [37, 38]. The mechanism(s) by which PUFAs modulate bone cell function are uncertain, but may include direct incorporation into cell membranes, with subsequent alteration of levels of intracellular prostanoids and eicosanoids [37].

### **6. Effect of postprandial triglyceride–rich lipoproteins on bone cells**

The postprandial state, the period that comprises and follows a meal, plays an important, yet underappreciated role in the genesis of numerous pathological conditions. After fatty food consumption, dietary FAs are largely incorporated into nascent triglyceride‐rich lipoproteins (TRLs), which are released from the small intestine into the blood. It has been previously shown that SFAs, MUFAs, and PUFAs have dissimilar postprandial effects on risk factors for chronic diseases [39], suggesting that short‐term outcomes in response to dietary FA adjustment could be useful to finely tune fat consumption, even for preventing diet‐related chronic diseases [40]. However, *in vivo* studies on markers of osteoclastogenesis during the postprandial state in humans or *in vitro* studies on interaction of human postprandial TRLs with monocyte‐derived OCs were unknown. In fact, there are only a few labs studying the link between the postprandial state and osteoclastogenesis. One of them has demonstrated that serum obtained from healthy subjects following the consumption of a meal containing almonds may inhibit OC maturation and function in primary human OC precursor cells, providing direct evidence to support the association between regular almond consump‐ tion and a reduced risk of osteoporosis [41]. Inspired in these findings, our group demon‐ strated for the first time in 2016 that the RANKL/OPG ratio is postprandially modulated by the predominant FAs in dietary fats, being particularly increased after the ingestion of an SFA‐enriched meal when compared to the ingestion of MUFA‐enriched meals [42]. *In vitro*, we also observed an increase of OC marker gene expression and a decrease of OPG gene expression in human monocyte‐derived OCs in response to postprandial TRL‐SFAs, further supporting the notion that dietary saturated fats may promote osteoclastogenesis through pathways involving the metabolism of intestinal lipoproteins. Importantly, TRL‐MUFAs and TRL‐PUFAs did not alter these osteoclastogenic markers or OPG, suggesting that the sub‐ stitution of dietary saturated fats by monounsaturated fats (in combination with omega‐3 PUFAs) may be useful to prevent excessive osteoclastogenesis associated to postprandial events.

In spite of the increasing evidence of the pivotal role of FAs on bone physiology as biological modulators of osteoclastogenesis, nutritional interventions might be a reliable therapeutic target to induce positive effects on skeletal health. Further, careful preclinical and clinical studies are likely to shed additional light on this important area of bone biology.

### **Conflicts of interest**

**Receptor Ligand(s) Sites of expression Function**

Pancreatic islets

Bone marrow Spleen Lymph node PBMCs **Osteoclasts**

Colon PBMCs **Osteoclasts**

Lung

L cells Pancreas

L cells **Osteoclasts Osteoblasts** Glucose‐stimulated insulin

**Main outcomes**

osteoclastogenesis, probably via receptors expressed at the

SFAs enhance cell survival in

Linoleic acid inhibit OC differentiation, possibly by modulating the downstream molecules of RANKL

SFAs inhibit

surface of OCs

mature OCs

signaling

secretion

Leptin production

Adipogenesis lipolysis

Regulation of inflammatory

GLP‐1 and insulin secretion

inhibition

response

GLP‐1 secretion

Gut Brain Monocytes **Osteoclasts**

**Table 1.** Effect of exogenous free fatty acids (FAs) on osteoclast functions and survival (Adapted from Ref. [4]).

**Cell model Effects on the studied** 

1,25‐Dihydroxyvitamin D3‐stimulated murine bone marrow–derived macrophages and RANKL‐stimulated murine macrophage cell line RAW264.7

RANKL/M‐CSF‐stimulated murine bone marrow– derived macrophages

RANKL‐stimulated murine macrophage cell line RAW264.7

**markers**

(SFAs)

(SFAs)

(PUFAs)

↓TRAP positive cells by SFAs and no clear‐cut differences between n‐3 and n‐6 PUFAs

↓ Annexin V staining ↑ TRAP positive cells

↑ MIP‐1α production

↑ NF‐κB, TLR4, and MyD88 activation (SFAs) ↓TRAP positive cells

↓TRAP positive cells ↓Bone resorption area

**GPR40** C6–C22 FAs, saturated and unsaturated

**FAs and concentration used** 

4:0, 8:0, 12:0, 14:0, 16:0, 18:0, 18:1, 18:2n−6, 18:3n−3, 20:4n−6, 20:5n−3, and

**[Reference]**

48 Fatty Acids

22:6n−3 (0.3–115 μM) [32]

18:2n−6 (1–100 μM) [34]

12:0 and 16:0 (20–100 μM) [33]

**GPR41** C1–C6 FAs Adipocytes

**GPR43** C1–C6 FAs Adipocytes

**GPR84** C9–C14 FAs PBMCs

**GPR119** Lysophosphatidylcholine and oleoylethanolamide

**GPR120** C14–C18 Saturated and C16– C22 unsaturated

Boldface, the main bone cells (Osteoclasts and osteoblasts)

**Table 2.** Fatty acid (FA) receptors (Adapted from Ref. [36]).

The authors state no conflict of interest.

### **Acknowledgements**

This study was supported by the research Grant AGL2016‐80852 (Spanish Ministry of Economy, Industry, and Competitiveness). BB and SL acknowledge financial support from "V Own Research Plan" (University of Seville) and the Spanish Research Council (CSIC)/Juan de la Cierva, respectively. A major portion of Sections 4 and 6 in this chapter borrows from authors' previous publications [20, 42].

### **Author details**

Sergio Montserrat‐de la Paz<sup>1</sup> \*† , Rocio Abia<sup>1</sup> , Beatriz Bermudez<sup>2</sup> , Sergio Lopez<sup>1</sup> and Francisco JG Muriana1†

\*Address all correspondence to: delapaz@us.es

1 Laboratory of Cellular and Molecular Nutrition, Spanish National Research Council (CSIC), Seville, Spain

2 Department of Cell Biology, University of Seville, Seville, Spain

†These authors contributed equally to this work.

### **References**


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50 Fatty Acids

**Author details**

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Seville, Spain

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**Fatty Acids and Cancer**

### **Short-Chain Fatty Acids Are Antineoplastic Agents**

Mohammad Salah Abaza, Aneela Afzal and

Mohammad Afzal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68441

#### **Abstract**

Human diet contains a mixture of saturated and unsaturated fatty acids. These are either long, medium or short chain fatty acids. As commonly believed, all fatty acids are not detrimental to human health. In addition to energy reserves, long chain fatty acids are known as acylating agents for many biomolecules such as cholesterol, terpenoids as well as steroid hormones. They are also involved in acylation of polyphenols such as flavonoids making them palatable for better absorption and biological activities. Polyunsaturated fatty acids (PUFAs) are known for their numerous beneficial health effects including cancer and inflammation. PUFA, particularly ω3 fatty acids, have attracted attention as anticancer agents and particularly for colorectal cancer. PUFAs exhibit immunomodulatory activities controlling inflammosome and are used as adjuvants together with standard anticancer drugs. A reciprocal interaction of short chain fatty acids with PUFAs has been suggested for their anticancer activities. Thus, in colon cancer cells, sodium butyrate (NaB) interacts with docosahexaenoic acid inducing cell differentiation or catalyze apoptosis. These results encouraged us to investigate NaB, a C4 acid, as an adjuvant to standard proteasome inhibitors. Our results show that NaB sensitizes colon cancer cell lines for treatment with proteasome inhibitors.

**Keywords:**histone deacylating agents, proteasome inhibitors, short‐chain fatty acids, sodium butyrate, polyunsaturated fatty acids

### **1. Background**

Cancer appears when the cellular growth network is disturbed and tumor cells resist apoptosis, resulting in uncontrolled growth and progression of tumor cells. Nucleosome acylation

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and deacylation of histones play a critical role in the tumorigenesis progression by regulating chromatin structure and function. The histone acetyltransferases (HATs) and deacylases (HDACs) create a fine equilibrium between acylation and deacylation of histones (**Figure 1**). Once this equilibrium is disturbed, it leads to cancer promotion and progression. A number of synthetic compounds, such as cyclic tetrapeptides, benzamides, suberoylanilide hydroxamic acid, and associated branched hydroxamic acid derivatives, are expended as inhibitors of HDAC. Short‐chain fatty acids, cogitated as novel drugs, have also been used as HDAC inhibitors. These compounds lead to an accumulation of acylated histones in healthy and tumor cells, arresting the cell cycle in the G1 and/or G2 phases, and provoking apoptosis in cancer cells. Therefore, HDAC inhibitors in controlled doses are recognized as innovative antitumor and anti‐inflammatory drugs, and sodium butyrate and sodium valproate, a C8 FA, have been used as HDAC inhibitors.

Fatty acids, contingent to the number of carbons, can be classified into three groups:


**Figure 1.** Acylation and deacylation of histones.

Several studies have shown that a combined treatment with MCFA and SCFA is more effective for inducing cell death through apoptosis. Additionally, numerous studies have reported the use of sodium butyrate and propionate as antineoplastic agents. In this chapter, the anticancer effect of water soluble NaB and its conceivable potential to augment the anticancer effect of proteasome inhibitors, as well as the principal mechanism of action of butyrate and/ or proteasome inhibitors on human colorectal tumor cells, will be discussed.

and deacylation of histones play a critical role in the tumorigenesis progression by regulating chromatin structure and function. The histone acetyltransferases (HATs) and deacylases (HDACs) create a fine equilibrium between acylation and deacylation of histones (**Figure 1**). Once this equilibrium is disturbed, it leads to cancer promotion and progression. A number of synthetic compounds, such as cyclic tetrapeptides, benzamides, suberoylanilide hydroxamic acid, and associated branched hydroxamic acid derivatives, are expended as inhibitors of HDAC. Short‐chain fatty acids, cogitated as novel drugs, have also been used as HDAC inhibitors. These compounds lead to an accumulation of acylated histones in healthy and tumor cells, arresting the cell cycle in the G1 and/or G2 phases, and provoking apoptosis in cancer cells. Therefore, HDAC inhibitors in controlled doses are recognized as innovative antitumor and anti‐inflammatory drugs, and sodium butyrate and sodium valproate, a C8 FA, have been

Fatty acids, contingent to the number of carbons, can be classified into three groups:

SCFAs have promise as antitumor agents in numerous types of cancer cells.

but some reports have also recognized them as antineoplastic mediators.

(a) C2:0–C6:0, short‐chain fatty acids (SCFAs) are dietetic and colonic fermentation products.

(b) C8:0–C14:0, intermediate chain fatty acids (ICFAs) have antimicrobial physiognomies,

(c) C16:0–C24:0, classified as long‐chain fatty acids (LCFA), provoke oxidative stress com-

used as HDAC inhibitors.

58 Fatty Acids

manding apoptosis in tumor cells.

**Figure 1.** Acylation and deacylation of histones.

The human diet is commonly deficient in ω3‐fatty acids that are common components of fish and fish products. Fish oil, high in ω3‐fatty acids, is known to have anticancer activities through apoptosis of cancer cells, and numerous reports have appeared to support this claim [1–8]. Thus, docosahexaenoic (22:6, *n*‐3) and eicosapentaenoic (20:5, *n*‐3) acids are effective antitumor adjuvants that provoke apoptosis in several types of tumor cells without an injury to natural cells [9–11]. However, most unsaturated fatty acids can undergo oxidative stress (OS), which has been implicated in many pathological conditions. OS modifies many biological molecules/pathways, often resulting in serious consequences. Lipid peroxidation, in addition to DNA and protein oxidation, is one such modification of lipids that involves unsaturated fatty acids, resulting in the formation of fatal peroxyl radicals and activating many transcription factors. These include NF‐κB, AP‐1, p53, HIF‐1α, PPAR‐γ, β‐catenin/Wnt, and Nrf2, which lead to cancer progression. In turn, a stimulation of these transcription factors activates over 500 genes, including cytokines and growth factor. This generates a strong relationship between OS, inflammation, and tumorigenesis. The fatty acid peroxyl radicals are a source of reactive hydroxyl‐aldehydes, such as 4‐hydroxynonenal, 4‐oxononenal, and malondialdehyde. These radicals elaborate Millard reactions with proteins and other N‐biomolecules, triggering mutations with an outbreak of cancer. In many ways, the metabolism of tumor cells differs from normal cells. One of the main differences between tumor cells and normal cells is the lower level of natural antioxidants desirable for defense from OS. Certain saturated fatty acids, such as odd carbon and branched carbon fatty acids, are also known to have anticancer activities [12].

Tumor cells adjust to an array of nutrient stocks for their subsistence. During an impaired glucose metabolism, tumor cells, for nutrients, switch to lipid metabolism. Cancer cells with their multiple metabolic compartments, depletion of sugars, amino acids, and lipids are considerably higher compared with their counterpart normal cells. Channeling their biosynthetic nutrients between and within cells, tumor cells contribute to their endurance and evolution. Therefore, one of the therapeutic targets to control the growth of cancer cells is to focus on the metabolic modifications between tumor and normal cells [13].

The involvement of phospholipase D1 (PLD1) in controlling the plasticity of cancer cell has been reported [14]. Cai et al. have reported that oxidation of fatty acids is the main basis of energy metabolism and inhibition of PLD1 results in a downregulation of lipid energy metabolism in tumor cells [14]. Numerous other inhibitors of lipid synthesis have also yielded encouraging results in limiting the proliferation of cancer cells. In this context, 5‐(tetradecyloxy)‐2‐furoic acid, an inhibitor of acetyl‐CoA carboxylase, giving malonyl‐CoA, which is an intermediary in the synthesis of fatty acids, has offered promising results for inhibiting cancer cell upturn and proliferation. Other lipid synthesis inhibitors, such as fatty acid synthase (FASN), cerulenin, and irgasan, suppress the proliferation of MiaPaCa‐2 and AsPC‐1 cells through depletion of fatty acids and apoptosis of cancer cells [15]. Leucine deficit also inhibits FASN in breast cancer cells [16]. An overexpression of FASN in neoplasms is widely reported in the literature, as its inhibition by certain synthetic imides, such as N‐phenylmaleimides [17].

Histone deacylase (HDAC) promotes deacylation by hydrolyzing histone lysine residues and plays a significant role in the regulation of gene expression. However, HDAC is overexpressed in several forms of cancer and is a target for several anticancer drugs. HDAC inhibitors prohibit the deacylation of not only histone but also nonhistone proteins and promote cell survival and anticancer activity. The inhibitors of HADC, presently approved by the FDA, include vorinostat, romidepsin, belinostat, and panobinostat, and several other HDAC inhibitors are already in clinical trial. Manal et al. have reported that SCFAs are novel HDAC inhibitors with advanced anticancer characteristics [18]. However, carnitine palmitoyl transferase 1 (CPT1), which is involved in the transport of long‐chain fatty acids for β‐oxidation, has been reported to be a specific target for anticancer therapies that is more selective than HDAC [19].

Gonadotropin‐releasing hormone‐III, when acylated with butyric acid at lysine position four, forms the bioconjugate, GnRH‐III(4)Lys(Bu), and is reported to have significant benefits over free daunorubicin as an antitumor agent [20].

### **2. Butyrate**

Butyrate is a short 4‐carbon fatty acid and is one of the three observed in the mammalian colonic lumen [21]. It is known that anaerobic fermentation of carbohydrates and proteins in the lumen produces butyric acid [22]. Biological reaction modification, resulting in gene activation and growth control, by butyrate and its water‐soluble sodium salt has been reported [23]. An inhibition of DNA synthesis may be responsible for arresting the proliferating cells and an induction of cell differentiation [24]. These results have led to contemplate that a short‐chain fatty acid, such as butyrate, may be a useful agent with antiproliferative and antineoplastic significance for typical mucosal epithelial cells [25]. Since butyrate is a dietary short‐chain fatty acid with low toxicity and growth inhibitory consequences, we decided to investigate its potential to augment the anticancer effect of a collection of proteasome inhibitors (MG115, MG132, PSI‐1, PSI‐2, and epoxomicin) on colorectal cancer cells [26].

For ubiquitin‐dependence of cellular proteins, a proteasome of multicatalytic nature and a degradation of over 80% intracellular proteins have been proposed [27]. For the regulation of protein synthesis during cellular stress, including apoptosis, impaired DNA, hypoxia, signal transduction, and so on, ubiquitin is recognized to play a dynamic role [28]. In oncology, validation of proteasome as a clinical agent has been provided by the use of bortezomib, which is a boronic acid dipeptide [28] and is an effective agent for treating multiple myeloma and certain types of non‐Hodgkin's lymphoma [29, 30]. Nevertheless, many patients do not respond to bortezomib. This is despite the fact that a regular use of bortezomib has many serious consequences including cardiac problems, excruciating neuropathy as well as thrombocytopenia [31–34]. For proteasome recovery, the treatment with bortezomib has been restricted biweekly [35]. Furthermore, in tumorigenesis, drug resistance to proteasome inhibitors [36] is a challenge.

cancer cell upturn and proliferation. Other lipid synthesis inhibitors, such as fatty acid synthase (FASN), cerulenin, and irgasan, suppress the proliferation of MiaPaCa‐2 and AsPC‐1 cells through depletion of fatty acids and apoptosis of cancer cells [15]. Leucine deficit also inhibits FASN in breast cancer cells [16]. An overexpression of FASN in neoplasms is widely reported in the literature, as its inhibition by certain synthetic imides, such as N‐phenylma-

Histone deacylase (HDAC) promotes deacylation by hydrolyzing histone lysine residues and plays a significant role in the regulation of gene expression. However, HDAC is overexpressed in several forms of cancer and is a target for several anticancer drugs. HDAC inhibitors prohibit the deacylation of not only histone but also nonhistone proteins and promote cell survival and anticancer activity. The inhibitors of HADC, presently approved by the FDA, include vorinostat, romidepsin, belinostat, and panobinostat, and several other HDAC inhibitors are already in clinical trial. Manal et al. have reported that SCFAs are novel HDAC inhibitors with advanced anticancer characteristics [18]. However, carnitine palmitoyl transferase 1 (CPT1), which is involved in the transport of long‐chain fatty acids for β‐oxidation, has been reported to be a specific target for anticancer therapies that is more selective than

Gonadotropin‐releasing hormone‐III, when acylated with butyric acid at lysine position four, forms the bioconjugate, GnRH‐III(4)Lys(Bu), and is reported to have significant benefits over

Butyrate is a short 4‐carbon fatty acid and is one of the three observed in the mammalian colonic lumen [21]. It is known that anaerobic fermentation of carbohydrates and proteins in the lumen produces butyric acid [22]. Biological reaction modification, resulting in gene activation and growth control, by butyrate and its water‐soluble sodium salt has been reported [23]. An inhibition of DNA synthesis may be responsible for arresting the proliferating cells and an induction of cell differentiation [24]. These results have led to contemplate that a short‐chain fatty acid, such as butyrate, may be a useful agent with antiproliferative and antineoplastic significance for typical mucosal epithelial cells [25]. Since butyrate is a dietary short‐chain fatty acid with low toxicity and growth inhibitory consequences, we decided to investigate its potential to augment the anticancer effect of a collection of proteasome inhibitors (MG115, MG132, PSI‐1, PSI‐2, and epoxomicin) on

For ubiquitin‐dependence of cellular proteins, a proteasome of multicatalytic nature and a degradation of over 80% intracellular proteins have been proposed [27]. For the regulation of protein synthesis during cellular stress, including apoptosis, impaired DNA, hypoxia, signal transduction, and so on, ubiquitin is recognized to play a dynamic role [28]. In oncology, validation of proteasome as a clinical agent has been provided by the use of bortezomib, which is

leimides [17].

60 Fatty Acids

HDAC [19].

**2. Butyrate**

colorectal cancer cells [26].

free daunorubicin as an antitumor agent [20].

Numerous types of tumors have been treated by an induction of apoptosis that can be triggered by several drugs and proteasome inhibitors. Again, drug toxicity and cell resistance are the cost that the patients have to bear [37]. The dietary sodium butyrate offers a valuable treatment with minor toxicity but a high degree of apoptic strength [38] making it a substance of choice for treating various types of tumors. We hypothesized that the anticancer characteristics of the proteasome inhibitors MG115, MG132, PSI‐1, PSI‐2 and epoxomicin in human colorectal carcinoma could be potentiated by NaB [26].

Human colorectal cancer cell line SW837 treated with NaB, MG115, and a combination of the two, for 24 h, showed a minor growth inhibition of the tumor cells (mean, 6 ± 0.4%) (**Figure 2A**). A distinct inhibition of SW837 cells (mean, 85 +\_ 2%), with an increase in the treatment time to 72 h, was observed. While a modest inhibition (mean 31 ± 4%) was detected after a single treatment with MG115, in 72 hrs. A combination of two of the therapies NaB and MG115 had a vivid inhibitory effect on the growth of SW837 tumor cells (mean, 85 ± 2%) and it was comparable with NaB when applied alone. However, the combination treatment for 72 h produced a statistically significant (*P* ≤ 0.004) inhibition of SW837 tumor cells compared with a single treatment with MG115. Increasing the treatment time to 120 h with the combination therapy, SW837, exhibited an inhibition of growth (mean, 90 ± 2%) compared with a sole action of NaB (mean, 87 ± 2%). Contrarily, after 120 h of treatment, MG115 alone showed only a humble inhibition (mean, 31 ± 5%). The growth inhibition of SW837 was statistically significant (*P* ≤ 0.002) in a combination therapy of NaB and MG115 for 120 h compared with MG115 alone (**Figure 2A**).

Next, we tested the efficacy of MG132 and NaB on the growth of SW837 for 24 h. It was found that the growth of SW837 was nonsignificantly affected by the individual two therapies (**Figure 2B**). However, after 72 h of treatment, a combination of the two therapies significantly constrained the growth of SW837 (mean, 86 ± 2%). A growth inhibition (mean, 84 ± 2%) comparable to the combination treatment was observed with NaB. A solitary treatment with MG132 for 72 h resulted in nonsignificant inhibition of SW837 (mean, 40 ± 4%). The inhibition change in SW837, after a combination treatment of NaB/MG132 and a sole treatment with MG132, was statistically significant (*P* ≤ 0.022). With a prolonged treatment of SW837 for 120 h, the combination of NaB/MG132 resulted in a distinctive reticence in the cell growth (mean, 89 ± 2%). However, a treatment with MG132 alone for 120 h resulted in a minuscule growth inhibition (mean, 47 ± 5%). The change in inhibition of SW837 after a combination treatment of NaB/MG132 and a sole treatment with MG132 was statistically significant (*P* ≤ 0.037) (**Figure 2B**).

We also investigated the effect of another proteasome PSI‐1 alone and in combination with NaB. A 24 h treatment of SW837 tumor cells resulted in (mean, 12 ± 1.0%; 1.3 ± 0.4% and 3.0 ± 0.4%) for NaB, PSI‐1, and combination of the two agents, respectively (**Figure 2C**). A striking difference (mean, 96.0 ± 4.0%) was observed when SW837 was treated with a combination of NaB and PSI‐1, compared with PSI‐1 alone (mean, 54.0 ± 2.0%) for 72 h. The change in inhibition of SW837 after a combination treatment of NaB/PSI‐1 and a sole treatment with PSI‐1 was statistically significant (*P* ≤ 0.001). The change in inhibition of SW837 cells after a combination treatment of NaB/PSI‐1 and a sole treatment with PSI‐1 was statistically nonsignificant (*P* ≤ 0.32). A prolonged treatment of SW837 cells for 120 h produced comparable results **Figure 2C**. The combination action showed a higher inhibition for SW837 cells, compared

**Figure 2.** Enhancement of the anticancer effect of proteasome inhibitors MG115, MG132, and PSI‐1 with NaB on human colorectal cancer SW837 cells. SW837 cells were plated (27 × 103 cells/well) into 96‐well plates and incubated at 37°C in a non‐CO2 incubator. After 18 h, the cells were treated with NaB (1.56–12.5 mM), MG115 (0.25–2.0 μM), MG132 (0.25–2 μM), PSI‐1 (0.013–0.1 μM), and the combinations of NaB and MG115 (A), MG132 (B), or PSI‐1 (C) starting 18 h after seeding the cells in culture. Control cells were left untreated or treated with vehicle (DMSO) at a final concentration (0.1%). Cell growth was monitored by MTT assay.

with a usage of NaB alone (mean, 87.0 ± 2.0%). The other results of SW837 cells inhibition with NaB, PSI‐2, epoxomicin, and their combinations are shown in **Figure 3**.

We also investigated the effect of another proteasome PSI‐1 alone and in combination with NaB. A 24 h treatment of SW837 tumor cells resulted in (mean, 12 ± 1.0%; 1.3 ± 0.4% and 3.0 ± 0.4%) for NaB, PSI‐1, and combination of the two agents, respectively (**Figure 2C**). A striking difference (mean, 96.0 ± 4.0%) was observed when SW837 was treated with a combination of NaB and PSI‐1, compared with PSI‐1 alone (mean, 54.0 ± 2.0%) for 72 h. The change in inhibition of SW837 after a combination treatment of NaB/PSI‐1 and a sole treatment with PSI‐1 was statistically significant (*P* ≤ 0.001). The change in inhibition of SW837 cells after a combination treatment of NaB/PSI‐1 and a sole treatment with PSI‐1 was statistically nonsignificant (*P* ≤ 0.32). A prolonged treatment of SW837 cells for 120 h produced comparable results **Figure 2C**. The combination action showed a higher inhibition for SW837 cells, compared

**Figure 2.** Enhancement of the anticancer effect of proteasome inhibitors MG115, MG132, and PSI‐1 with NaB on human

 incubator. After 18 h, the cells were treated with NaB (1.56–12.5 mM), MG115 (0.25–2.0 μM), MG132 (0.25–2 μM), PSI‐1 (0.013–0.1 μM), and the combinations of NaB and MG115 (A), MG132 (B), or PSI‐1 (C) starting 18 h after seeding the cells in culture. Control cells were left untreated or treated with vehicle (DMSO) at a final concentration

cells/well) into 96‐well plates and incubated at 37°C in

colorectal cancer SW837 cells. SW837 cells were plated (27 × 103

(0.1%). Cell growth was monitored by MTT assay.

a non‐CO2

62 Fatty Acids

**Figure 3.** Enhancement of the anticancer effect of proteasome inhibitors PSI‐2 and epoxomicin with NaB on human colorectal cancer SW837 cells. SW837 cells were plated (27 × 103 cells/well) into 96‐well plates and incubated at 37°C in a non‐CO2 incubator. After 18 h, the cells were treated with NaB (1.56–12.5 mM), PSI‐2 (0.375–3.0 μM), epoxomicin (3.0–12 nM), and the combinations of NaB and PSI‐2 (A) or epoxomicin (B) starting 18 h after seeding the cells in culture. Control cells were left untreated or treated with vehicle (DMSO) at a final concentration (0.1%). Cell growth was monitored by MTT assay.

An analysis of our investigations has established that human colorectal cancer cell line, SW837, when treated with 3 mM NaB caused amassing of cells in the G1‐phase (82.7%), and a corresponding reduction in the number of cells in G2/M‐ (2.61%) and S‐ (14.7%) phases (**Figure 4**). In addition, treatment with 1.0 μM MG115, 0.1 μM MG132, 0.1 μM PSI‐1, 1.5 μM PSI‐2, or 12 nM epoxomicin followed a buildup of cells in the S‐phase (55.5, 33.7, 41.5, 42.7, and 32.2%, respectively) and G2‐phase (12.2, 36.1, 44.3, 29.9, and 45.7%, respectively) with a consequent reduction in the total cells in the G1‐phase (29.0, 29.7, 14.1, 27.4 and 22.3%, respectively).

A combination of NaB at 3 mM and MG115 or MG132 at 1.0 μM concentration caused the colorectal cancer cells arrest in the G1‐phase (79.8 or 75.5%, respectively) and the G2‐phase (6.73 or 14.4%, respectively). The upturn in the G1‐phase complemented by an equivalent reduction in the S‐phase of the cells (13.5 or 10%, respectively) (**Figure 4**). A combination treatment with NaB (3 mM) and PSI‐1 (1.0 μM), PSI‐2 (1.5 μM), or epoxomicin (12 nM) resulted in an increase in the number of cells in the G2‐phase (45.2, 92.7, and 88.3%, respectively). This was accompanied by a parallel reduction in the quantity of cells in the G1‐phase (55.4, 7.29, or 12.3%, respectively) and the S‐phase (0.0%) (**Figure 4**).

Next, we turned to analyze the effect of antineoplastic agents on the DNA of treated cells by agarose gel electrophoresis. The DNA was extracted from the untreated and treated human colorectal cancer cells that displayed a discrete ladder pattern, displaying apoptosis. These consequences obviously displayed that the action of NaB, proteasome inhibitors, or their combination triggered the apoptotic trail. The magnitude of apoptosis of cancer cells, treated with a combination of NaB and proteasome inhibitors was prominent compared with the NaB or proteasome inhibitors alone (**Figure 5**).

The regulation of gene expression and inhibition of histone deacylases are regulated by NaB [39]. The hyperacetylation of histones and an amelioration of the availability of the transcription factors to nucleosomal DNA are due to inhibition of histone deacylases [40]. Hyperacetylation of nonhistone proteins, modification of DNA methylation, careful inhibition of histone phosphorylation, and alteration of intracellular kinase signaling may be the other cellular targets of NaB [39]. This multistage mechanism of butyrate explains the gene expression regulation and its impact on the crucial regulators of apoptosis and the cell cycle.

The synergistic apoptotic consequences of NaB and proteasome inhibitors may offer new opportunities in research to develop therapeutic strategies to contain human colorectal cancer. The proteasome inhibitors seem to act as apoptotic agents only in the rapidly dividing cells while shielding quiescent cells from apoptosis that may be activated by many diverse compounds [30]. For this specific action, proteasome inhibitors may be used as a substitute in the treatment of some proliferative disorders. Moreover, treatment with a combination of proteasome inhibitors, effective apoptotic agents, such as NaB, and other short‐chain fatty acids may be a valuable therapeutic strategy for the treatment of proliferative diseases such as colorectal cancer. Thus, further research in this area may be very rewarding and offer hope to the suffering patients around the globe.

An analysis of our investigations has established that human colorectal cancer cell line, SW837, when treated with 3 mM NaB caused amassing of cells in the G1‐phase (82.7%), and a corresponding reduction in the number of cells in G2/M‐ (2.61%) and S‐ (14.7%) phases (**Figure 4**). In addition, treatment with 1.0 μM MG115, 0.1 μM MG132, 0.1 μM PSI‐1, 1.5 μM PSI‐2, or 12 nM epoxomicin followed a buildup of cells in the S‐phase (55.5, 33.7, 41.5, 42.7, and 32.2%, respectively) and G2‐phase (12.2, 36.1, 44.3, 29.9, and 45.7%, respectively) with a consequent reduction in the total cells in the G1‐phase (29.0, 29.7, 14.1, 27.4 and 22.3%,

A combination of NaB at 3 mM and MG115 or MG132 at 1.0 μM concentration caused the colorectal cancer cells arrest in the G1‐phase (79.8 or 75.5%, respectively) and the G2‐phase (6.73 or 14.4%, respectively). The upturn in the G1‐phase complemented by an equivalent reduction in the S‐phase of the cells (13.5 or 10%, respectively) (**Figure 4**). A combination treatment with NaB (3 mM) and PSI‐1 (1.0 μM), PSI‐2 (1.5 μM), or epoxomicin (12 nM) resulted in an increase in the number of cells in the G2‐phase (45.2, 92.7, and 88.3%, respectively). This was accompanied by a parallel reduction in the quantity of cells in the G1‐phase (55.4, 7.29, or

Next, we turned to analyze the effect of antineoplastic agents on the DNA of treated cells by agarose gel electrophoresis. The DNA was extracted from the untreated and treated human colorectal cancer cells that displayed a discrete ladder pattern, displaying apoptosis. These consequences obviously displayed that the action of NaB, proteasome inhibitors, or their combination triggered the apoptotic trail. The magnitude of apoptosis of cancer cells, treated with a combination of NaB and proteasome inhibitors was prominent compared with the NaB

The regulation of gene expression and inhibition of histone deacylases are regulated by NaB [39]. The hyperacetylation of histones and an amelioration of the availability of the transcription factors to nucleosomal DNA are due to inhibition of histone deacylases [40]. Hyperacetylation of nonhistone proteins, modification of DNA methylation, careful inhibition of histone phosphorylation, and alteration of intracellular kinase signaling may be the other cellular targets of NaB [39]. This multistage mechanism of butyrate explains the gene expression regulation and its impact on the crucial regulators of apoptosis and the cell cycle.

The synergistic apoptotic consequences of NaB and proteasome inhibitors may offer new opportunities in research to develop therapeutic strategies to contain human colorectal cancer. The proteasome inhibitors seem to act as apoptotic agents only in the rapidly dividing cells while shielding quiescent cells from apoptosis that may be activated by many diverse compounds [30]. For this specific action, proteasome inhibitors may be used as a substitute in the treatment of some proliferative disorders. Moreover, treatment with a combination of proteasome inhibitors, effective apoptotic agents, such as NaB, and other short‐chain fatty acids may be a valuable therapeutic strategy for the treatment of proliferative diseases such as colorectal cancer. Thus, further research in this area may be very rewarding and offer hope

12.3%, respectively) and the S‐phase (0.0%) (**Figure 4**).

or proteasome inhibitors alone (**Figure 5**).

to the suffering patients around the globe.

respectively).

64 Fatty Acids

**Figure 4.** Cell cycle distribution of human colorectal cancer SW837 cell treated with NaB, proteasome inhibitors, and their combinations. SW837 cells were plated (5 × 105 cells/well) into 24‐well plates and incubated at 37°C in a non‐CO2 incubator. After 18 h, the cells were treated individually with NaB (3.0 mM), MG115 (1.0 μM), MG132 (1.0 μM), PSI‐1 (0.1 μM), PSI‐2 (1.5 μM), and epoxomicin (12 nM) or treated with the combinations NaB/MG115 (3 mM/1.0 μM), NaB/MG132 (3.0 mM/1.0 μM), NaB/PSI‐1 (3.0 mM/0.1 μM), NaB/PSI‐2 (3.0 mM/1.5 μM), and Na/ epoxomicin (3.0 mM/12 nM) for 72 h. At least duplicate samples were analyzed and 20,000 events were scored for each sample. The vertical axis represents the relative number of events and the horizontal axis represents the fluorescence intensity. The percentage of cells in different cell cycle phases was calculated using Phoenix statistical software package. A‐E: Single and combined treatments with NaB and MG11S, MG132, PSI‐1, PSI‐2 or Epox, respectively.

**Figure 5.** Assessment of apoptosis in human colorectal SW837 cells treated with NaB, proteasome inhibitors, and their combinations. SW837 cells were plated (5 × 105 cells/well) into 24‐well plates and incubated at 37°C in a non‐CO2 incubator. After 18 h, the cells were treated individually with NaB (3.0 mM), MG115 (1.0 μM), MG132 (1.0 μM), PSI‐1 (0.1 μM), PSI‐2 (1.5 μM), and epoxomicin (12 nM) or treated with the combinations NaB/MG115 (3 mM/1.0 μM), NaB/ MG132 (3.0 mM/1.0 μM), NaB/PSI‐1 (3.0 mM/0.1 μM), NaB/PSI‐2 (3.0 mM/1.5 μM), and Na/epoxomicin (3.0 mM/ 12 nM) for 72 h. DNA fragments were extracted and analyzed on 1.0% agrose gel. A‐E: Single (+) and combined (++) treatments with NaB and MG115, MG132, PSI‐1, PSI‐2 or Epox, respectively.

### **Author details**

Mohammad Salah Abaza1 , Aneela Afzal2 and Mohammad Afzal1 \*

\*Address all correspondence to: afzalq8@gmail.com


### **References**

**Figure 5.** Assessment of apoptosis in human colorectal SW837 cells treated with NaB, proteasome inhibitors, and

incubator. After 18 h, the cells were treated individually with NaB (3.0 mM), MG115 (1.0 μM), MG132 (1.0 μM), PSI‐1 (0.1 μM), PSI‐2 (1.5 μM), and epoxomicin (12 nM) or treated with the combinations NaB/MG115 (3 mM/1.0 μM), NaB/ MG132 (3.0 mM/1.0 μM), NaB/PSI‐1 (3.0 mM/0.1 μM), NaB/PSI‐2 (3.0 mM/1.5 μM), and Na/epoxomicin (3.0 mM/ 12 nM) for 72 h. DNA fragments were extracted and analyzed on 1.0% agrose gel. A‐E: Single (+) and combined (++) treatments

cells/well) into 24‐well plates and incubated at 37°C in a non‐CO2

their combinations. SW837 cells were plated (5 × 105

66 Fatty Acids

with NaB and MG115, MG132, PSI‐1, PSI‐2 or Epox, respectively.


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## **Fatty Acids and Their Analogues as Anticancer Agents**

### Jubie Selvaraj

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68171

#### **Abstract**

Recent research supports the beneficial effects of dietary polyunsaturated fatty acids (PUFAs) on inhibiting tumour development. Long‐chain fatty acids modulate the tumour cell response to chemotherapeutic drugs. Investigators recently claimed high dietary intake of omega‐6 polyunsaturated fatty acids such as linoleic acid especially in associa‐ tion with a low intake of omega‐3 polyunsaturated fatty acids such as docosahexaenoic acid to increase risks for cancers of the breast, colon and possibly prostate. In addition to these facts, a number of investigations have demonstrated that a modified fatty acid ana‐ logues are promising molecules in cancer prevention and have potential in the treatment of cancer. Although billions of dollars have been spent on research and development on anticancer drugs, the disease remains uncontrolled. It is expected that anticancer agents preferentially kill tumour cells without causing adverse effects on normal cells. But this is rarely achieved with the existing cancer therapy. Hence, polyunsaturated fatty acids have come under the category of nutraceuticals/functional foods; their exploration in the treat‐ ment of cancer may be considered as safe. This chapter describes the effects of long‐chain fatty acids and their analogues in cancer chemotherapy.

**Keywords:** fatty acids, cancer, PUFA, fatty acid synthase, omega‐3

### **1. Introduction to fatty acids**

Plants, animals and microbes generally contain even number of carbon atoms in straight chains, with a carboxylic group at one end and double bonds with *cis* configuration on the another end. The chain length of the common fatty acids varies between 14 and 22, but on occasions can span between 2 and 36 or even more in animal tissues. Fatty acids found in ani‐ mal tissues have one to six double bonds, whereas those in algae have up to five bonds. Higher plants rarely have more than three, whereas microbial fatty acids occasionally have more than one. The fatty acids, which are derived from triglycerides or phospholipids, have a chain of

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

4–28 carbons. Fatty acids, which are not attached to other molecules, are known as free fatty acids which on breakdown yield large quantities of ATP. Many cell types use either glucose or fatty acids for this purpose. In particular, heart and skeletal muscle prefer fatty acids [1].

Fatty acids may be monounsaturated, polyunsaturated or saturated (**Figure 1**). They help in moving oxygen through the blood stream to all parts of the body, aid cell membrane develop‐ ment and strengthen the organs and tissue. They also help in healthy skin and prevent early ageing and more importantly help rid the arteries of cholesterol build‐up.

### **2. Types of fatty acids**

### **2.1. Saturated fatty acids**

 Saturated fatty acids are straight‐chain compounds with 14, 16 and 18 carbon atoms. The most abundant saturated fatty acids found in animal and plant tissues are esterified with odd‐ and even‐numbered homologues with 2–36 carbon atoms. A list of common saturated fatty acids together with their trivial names and shorthand designations is given in **Table 1**.

#### **2.2. Monoenoic fatty acids**

Monoenoic fatty acids are straight‐chain fatty acids containing 10–30 carbon atoms with one *cis*‐double bond. The double bond can be in different positions and this is specified in the systematic nomenclature in relation to the carboxyl group (**Table 2**).

#### **2.3. Polyunsaturated fatty acids**

Polyunsaturated fatty acids (PUFAs) are fatty acids which contain multiple double bonds and are subdivided into families according to their derivation from specific biosynthetic precur‐ sors. In each instance, the families contain between two and six *cis*‐double bonds separated

**Figure 1.** Naturally occurring fatty acids.


**Table 1.** Saturated fatty acids of general formula CH3 (CH2 )*n* COOH.

4–28 carbons. Fatty acids, which are not attached to other molecules, are known as free fatty acids which on breakdown yield large quantities of ATP. Many cell types use either glucose or fatty acids for this purpose. In particular, heart and skeletal muscle prefer fatty acids [1].

Fatty acids may be monounsaturated, polyunsaturated or saturated (**Figure 1**). They help in moving oxygen through the blood stream to all parts of the body, aid cell membrane develop‐ ment and strengthen the organs and tissue. They also help in healthy skin and prevent early

 Saturated fatty acids are straight‐chain compounds with 14, 16 and 18 carbon atoms. The most abundant saturated fatty acids found in animal and plant tissues are esterified with odd‐ and even‐numbered homologues with 2–36 carbon atoms. A list of common saturated fatty acids together with their trivial names and shorthand designations is given in **Table 1**.

Monoenoic fatty acids are straight‐chain fatty acids containing 10–30 carbon atoms with one *cis*‐double bond. The double bond can be in different positions and this is specified in the

Polyunsaturated fatty acids (PUFAs) are fatty acids which contain multiple double bonds and are subdivided into families according to their derivation from specific biosynthetic precur‐ sors. In each instance, the families contain between two and six *cis*‐double bonds separated

ageing and more importantly help rid the arteries of cholesterol build‐up.

systematic nomenclature in relation to the carboxyl group (**Table 2**).

**2. Types of fatty acids**

72 Fatty Acids

**2.1. Saturated fatty acids**

**2.2. Monoenoic fatty acids**

**2.3. Polyunsaturated fatty acids**

**Figure 1.** Naturally occurring fatty acids.

by single methylene\ groups, and have the same terminal structure [2]. A list of some of the important PUFAs is presented in **Table 3**.

#### **2.4. Branched‐chain and cyclopropane fatty acids**

Branched‐chain fatty acids, which occur widely in nature, are present as minor components except in bacteria, where they appear to replace unsaturated fatty acids functionally. The branch consists of a single methyl group, either on the penultimate (*iso*) or on the antepenul‐ timate (*anteiso*) carbon atoms [3, 4].

#### **2.5. Oxygenated and cyclic fatty acids**

A large number of hydroperoxy, hydroxyl and epoxy fatty acids (eicosanoids) are formed enzymatically as intermediates in the biosynthesis of prostanoids. A large number of hydroxy fatty acids occur in seed oils, and the best known of these is ricinoleic acid which is the prin‐ ciple constituent of castor oil. Polyhydroxy fatty acids are present in plant cutins, shellacs and many seed oils.


**Table 2.** Monoenoic fatty acids of general formula CH3 (CH2 )mCH=CH(CH2 )nCOOH.

#### **2.6. Omega‐3 and omega‐6 fatty acids**

The biological fatty acids are of different lengths, the last position is labelled as *omega* (ω). *Omega‐3* fatty acids are long‐chain polyunsaturated fatty acids (18–22 carbon atoms) with the first of many double bonds beginning with the third carbon atom. However, *omega‐6* fatty acids have the first of many double bonds beginning with the sixth carbon atom. Alpha‐linolenic acid (ALA) and linoleic acid (LA) are the parent compounds of the omega‐3 family and omega‐6 family of fatty acids, respectively.


**Table 3.** Polyunsaturated fatty acids of general formula CH3 (CH2 )m(CH=CHCH2 )x(CH2 ) n COOH. Although the International panel of lipid experts says the ideal ratio of *omega‐3* to *omega‐6* essential fatty acids is approximately 1:1, still we follow the ratio 20:1 in our diet [5]. Long‐ chain polyunsaturated fatty acids cannot be formed *de novo* but can be synthesized from the essential fatty acids like linoleic acid and alpha‐linolenic acid. These two essential fatty acids are desaturated and lengthened progressively by microsomal enzyme systems to form highly unsaturated, long‐chained fatty acids such as arachidonic acid and docosahexaenoic acid (DHA). The *omega‐3 and omega‐6* fatty acids are not interconvertible. Dietary fish and fish oil supplements are a direct source of *omega‐3* fatty acids and dietary oils have large quantity of *omega‐6* fatty acids [6].

### **3. Polyunsaturated fatty acids as anticancer agents**

**2.6. Omega‐3 and omega‐6 fatty acids**

74 Fatty Acids

**Table 2.** Monoenoic fatty acids of general formula CH3

family of fatty acids, respectively.

\*The double bond configuration in each instance is *cis*.

**Table 3.** Polyunsaturated fatty acids of general formula CH3 (CH2

The biological fatty acids are of different lengths, the last position is labelled as *omega* (ω). *Omega‐3* fatty acids are long‐chain polyunsaturated fatty acids (18–22 carbon atoms) with the first of many double bonds beginning with the third carbon atom. However, *omega‐6* fatty acids have the first of many double bonds beginning with the sixth carbon atom. Alpha‐linolenic acid (ALA) and linoleic acid (LA) are the parent compounds of the omega‐3 family and omega‐6

)mCH=CH(CH2

)nCOOH.

(CH2

*cis*‐6‐Octadecenoic 18:1(n‐12) Petraselenic

**S. no. Systematic name Shorthand designation Trivial name** 1. 9,12‐Octadecadienoic\* 18:2(n‐6) Linoleic 2. 6,9,12‐Octadecatrienoic 18:3(n‐6) γ‐Linolenic 3. 8,11,14‐Eicosatrienoic 18:3(n‐6) Homo‐γ‐linolenic 4. 5,8,11,14‐Eicosatetraenoic 20:4(n‐6) Arachidonic

**S. no. Systematic name Shorthand designation Trivial name** 1. *cis*‐9‐Tetradecenoic 14:1(n‐5) Myristoleic 2. *cis*‐9‐Hexadecenoic 16:1(n‐7) Palmitoleic

3. *trans*‐3‐Hexadecenoic **– –**

4. *cis*‐9‐Octadecenoic 18:1(n‐9) Oleic 5. *cis*‐11‐Octadecenoic 18:1(n‐7) *cis*‐Vaccenic 6. *trans*‐11‐Octadecenoic **–** Elaidic 7. *cis*‐9‐Eicosenoic 20:1(n‐11) Gadoleic 8. *cis*‐11‐Octadecenoic 18:1(n‐9) Gondic 9. *cis*‐13‐Docosenoic 22:1(n‐9) Erucic 10. *cis*‐15‐Tetracosenoic 24:1(n‐9) Nervonic

5. 4,7,10,13,16‐Eicosapentaenoic 20:5(n‐6) –

7. 5,8,11,14,17‐Eicosapentaenoic 20:5(n‐3) EPA 8. 7,10,13,16,19‐Docosapentaenoic 22:5(n‐3) – 9. 4,7,10,13,16,19‐Docosahexaenoic 22:5(n‐3) DHA 10. 5,8,11‐Eicosatrienoic 20:3(n‐9) Mead's acid

6. 9,12,15‐Octadecatrienoic 18:3(n‐6) α‐Linolenic

)m(CH=CHCH2

)x(CH2

) n COOH.

Yonesawa and co‐workers carried out the inhibitory effect of conjugated eicosapentaenoic acid (cEPA) on mammalian DNA polymerase and topoisomerase activities and human cell proliferation. They found that the inhibitory effect of cEPA was stronger than that of the non‐ conjugated EPA and suggested the therapeutic potential of cEPA as a leading anticancer com‐ pound that poisons mammalian DNA polymerase (POLS) [7]. The work carried by *Unduri* revealed the tumouricidal and antiangiogenic actions of gamma‐linolenic acid (GLA) and its derivatives. It was found that GLA being an endogenous naturally occurring molecule had no significant side effects [8]. Paul *et al.* reported that the long‐chain eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) have been consistently shown to inhibit the proliferation of breast and prostate cancer cell lines *in vitro* and to reduce the risk and progression of these tumours in animal experiments. Many investigations revealed that the above‐said fatty acids inhibit cyclooxygenase‐2 and the oxidative metabolism of arachidonic acid (AA) to PGE2 . EPA and DHA also have been shown to inhibit lipoxygenase which metabolizes AA to hydroxyl eicosatetraenoic acids and leucotrienes which suppress apoptosis, stimulate angiogenesis and stimulate tumour cell division (**Figure 2**). Further, they explained that the n‐3 PUFAs potentially affect carcinogenesis by specific mechanisms [9]. These mechanisms are as fol‐ lows: (1) alteration of the response of immune system to cancer cells through the suppression of arachidonic acid (AA, 20:4n‐6)‐derived eicosanoid biosynthesis; (2) alteration of metabo‐ lism, cell growth and differentiation; (3) alteration of oestrogen metabolism, which leads to reduced oestrogen‐stimulated cell growth; (4) alteration of free radicals and productivity; and (5) alteration of the mechanisms involving insulin sensitivity and membrane fluidity. Interest in the use of supplementary omega‐3‐fatty acids to reduce the risk of cancer and other chronic‐debilitating conditions, including cardiovascular disease and cognitive impairment, stems from several long‐standing avenues of registration [9, 10]. Furthermore, the anticancer activity of fatty acids is well evidenced by Helmut *et al*. in experimental and human studies, which summarize that a high intake of omega‐3 PUFAs and monounsaturated fatty acids is protective in breast, colon and prostate cancers [11].

The author and her research group isolated methyl gamma linolenate (**GLA‐ME**) (**1**) from *Spirulina platensis* and the compound showed strong cytotoxicity against A‐549 cells [13] when compared with the standard drug Rutin. Rutin is a bioflavanol which is a well‐established

**Figure 2.** Overview of the metabolism of n‐6 and n‐3 polyunsaturated fatty acids (PUFAs) into eicosanoids involved in inflammation and carcinogenesis [12].

promising anticancer agent, and its mechanism may be due to the induction of apoptosis [14]. The comparative results are given in **Figure 3** and **Table 4**, respectively. The probable mechanism may be due to the induction of apoptosis of tumour cells by augmenting free radical generation. It is evidenced by the research work carried out by Unduri *et al.* [8]. They also reported that the induction of apoptosis of tumour cells by GLA is due to its action at the gene/oncogene level and by altering BCl‐2 expression. Hence, it may be concluded that the cytotoxicity shown by GLA‐ME may be due to the induction of apoptosis effect. However, a detailed study of this mechanism is in progress.

**Figure 3.** *In vitro* cytotoxic studies □: GLA‐ME, Δ: standard rutin.


**Table 4.** Determination of cytotoxicity by SRB method.

promising anticancer agent, and its mechanism may be due to the induction of apoptosis [14]. The comparative results are given in **Figure 3** and **Table 4**, respectively. The probable mechanism may be due to the induction of apoptosis of tumour cells by augmenting free radical generation. It is evidenced by the research work carried out by Unduri *et al.* [8]. They also reported that the induction of apoptosis of tumour cells by GLA is due to its action at the gene/oncogene level and by altering BCl‐2 expression. Hence, it may be concluded that the cytotoxicity shown by GLA‐ME may be due to the induction of apoptosis effect. However, a

**1**

**3**

**5**

**8**

**10 9**

**1**

**11**

12

14

**16**

**13**

**15**

**17**

**2**

**4**

**6**

**7**

O

**Figure 2.** Overview of the metabolism of n‐6 and n‐3 polyunsaturated fatty acids (PUFAs) into eicosanoids involved in

detailed study of this mechanism is in progress.

19

O

**18**

inflammation and carcinogenesis [12].

76 Fatty Acids

### **4. Polyunsaturated fatty acids as adjunct to chemotherapeutic agents**

Kong and co‐workers found out that gamma linolenic acid modulates the response of multi‐ drug‐resistant K562 leukaemic cells to anticancer drugs. The study also revealed that GLA could modulate the response to anticancer drugs in P‐gp overexpressing multidrug‐resistant cells, which could be due to decrease P‐gp expression [15]. In another study, Julie and co‐ workers reported that alpha linolenic acid and docosahexaenoic acid alone combined with trastuzumab reduced HER2 overexpressing breast cancer cell growth but differentially regu‐ lated HER2‐signalling pathways. Their finding is different in classic mechanisms whereby n‐3 PUFAs exert their effect in breast cancer. The results strongly suggest that DHA reduces growth factor receptor signalling as indicated by reductions in the phosphorylation of AKT and MAPK while the opposite effect is seen for the plant‐based n‐3 PUFA ALA [16]. Effenberger and co‐workers synthesized novel N‐acylhydrazones of doxorubicin which were derived from saturated, unsaturated and methyl or bornyl terminated fatty acids. The mode of cyto‐ toxic action of the hydrazones was largely apoptotic. They led to a distinct long‐term decrease of bcl‐2 MRNA expression, the precise apoptotic mechanism and the involvement of caspases varied for the individual cell lines and test compounds. The apoptosis of 518A2 melanoma cells treated with some compounds was characterized by an early onset of initiator caspase‐9 activity. By contrast, apoptosis elicited in 518A2 or in HL‐60 cells by remaining compounds was accompanied by high‐initiator caspase‐8 activity. The genuine slump of the bcl‐2 mRNA expression may be the reason for the observed quick and steep hike of the ratio of bax mRNA to bcl‐2 mRNA in 518A2 cells. Apoptosis induced by doxorubicin **(2)** and its derivatives (**3)** and (**4)** in HL‐60 and 518A2 cells also proceeds with a swift and distinct loss of mitochondrial membrane potential regardless of the divergent caspase kinetics. This was a proof that fatty acid analogues are more than just lipophilic shuttle groups [17].

Piyali *et al.* studied the antiproliferative activity of somatostatin analogue with N‐terminal acylation with long‐chain fatty acids in human breast adenocarcinoma cell lines. The antiprolif‐ erative activity of the somatostatin analogue RC‐160 (D‐Phe‐Cys‐Tyr‐D‐Trp‐Lys‐Val‐Cys‐Trp‐ NH2 ) is limited by its short serum half‐life. To circumvent this limitation, fatty acids of chain lengths ranging from 4 to 18 were individually conjugated to the N‐terminal residue of RC‐160. Although the affinity of palmitoyl –RC‐160 towards somatostatin receptors remains unaltered when compared to the –RC‐160, it exhibited significantly higher antiproliferative activity on MCF‐7 cells. On further increase in the lipopeptide chain, the bioactivity of lipophilized –RC‐160 was reduced. Increasing the peptide hydrophobicity beyond this range reduced the bioactivity of lipophilized –RC‐160. Accordingly, stearoyl –RC‐160 manifested lower antineoplastic activity and receptor‐binding affinity relative to palmitoyl –RC‐160 and RC‐160 itself. It was observed that an increase in bioactivity was manifested within an optimum range of the lipopeptide. The probable mechanisms may be alterations of the signalling pathways. Lipophilization of RC‐160 with long‐chain fatty acids like palmitic acid improves its stability and antiproliferative activity, thereby improving the scope of enhancing its therapeutic index [18].

### **5. Fatty acid analogues as anticancer agents**

of bcl‐2 MRNA expression, the precise apoptotic mechanism and the involvement of caspases varied for the individual cell lines and test compounds. The apoptosis of 518A2 melanoma cells treated with some compounds was characterized by an early onset of initiator caspase‐9 activity. By contrast, apoptosis elicited in 518A2 or in HL‐60 cells by remaining compounds was accompanied by high‐initiator caspase‐8 activity. The genuine slump of the bcl‐2 mRNA expression may be the reason for the observed quick and steep hike of the ratio of bax mRNA to bcl‐2 mRNA in 518A2 cells. Apoptosis induced by doxorubicin **(2)** and its derivatives (**3)** and (**4)** in HL‐60 and 518A2 cells also proceeds with a swift and distinct loss of mitochondrial membrane potential regardless of the divergent caspase kinetics. This was a proof that fatty

acid analogues are more than just lipophilic shuttle groups [17].

78 Fatty Acids

O

OH

OH

OCH3 O

2

O

O

OH

OH

O O

O

OH

OH

NH2

OH

O

OH NH2

OH

OH NH2

OH

**3**

OH

N

H N O

N

H N O

OH

OH

OH

OCH3 O

N N NH2

O O R O

OH

R O O

O

**4**

OCH3 O

O

O

O

A number of investigations have demonstrated that a variety of modified fatty acid analogues are promising molecules in cancer prevention and have potential in the treatment of cancer. Bhupender *et al.*synthesized fatty acyl amide derivatives of doxorubicin (**5**) and evaluated their *in vitro* anticancer activities. The results indicated that the designed molecule with com‐ parable antileukaemia activity to cytarabine with sustained release effect is possible by struc‐ ture modification [19].

They also synthesized fatty acyl ester derivatives (**6**) of cytarabine and evaluated them for anti‐ leukaemia activity. Some of 2',5'‐dimyristoyl derivatives of cytarabine were found to inhibit the growth of CCRF‐CEM cells [20]. Liu *et al.* reported the synthesis and antitumour evaluation of N<sup>4</sup> fatty acyl amino derivatives of cytarabine. The bioavailability of cytarabine is low due to its low lipophilicity. In order to improve the lipophilicity and bioavailability of cytarabine, a series of fatty acyl amino acid cytarabine analogues (**7**) were synthesized. It was found that the deriv‐ atives synthesized were more lipophilic than cytarabine. The antitumour activity determined in HL‐600 and HeLa cells showed that the derivatives were more active in HeLa cells than cytarabine while most of them demonstrated similar activity to cytarbine in HL‐60 cells. The length of fatty acids in the derivatives seemed to have an impact on the activity observed [21].

AA=Amino acids R -sugar

Zhang Chun‐hong and co‐workers synthesized new panaxadiol fatty acid esters (**8**) and eval‐ uated them for their antitumour activity. Tumour cell used was Vero cell line. Positive con‐ trol was 5‐FU, blank was an RPMI1640 culture medium, negative control was an RPMI1640 culture medium and the solvent for drugs to be tested. The compounds show the strongest antitumour activity [22].

Earlier, the author of the present chapter has reported some novel fatty acid heterocyclic conjugates and their anticancer evaluation on human lung carcinoma cell lines [23, 24]. The compounds have shown comparable cytotoxicity towards human lung carcinoma cell lines. The compound (**9**), fatty acid chain substituted 1,3,4‐oxadiazole showed maximum cytotoxic activity. It was observed that the presence of toxophoric –N=C‐O‐ linkage in 1,3,4 oxadiazole nucleus may be responsible for the antitumour activity. Further, 1,3,4 oxadiazole is a good bioisostere of amide and ester functionalities with substantial improvement in biological activity in hydrogen‐bonding interactions with different targets responsible for the tumour development. The 1,2,4‐triazole substituted fatty acid analogues (**10**) displayed promising cytotoxicity towards human lung carcinoma cell lines. It was also observed that the length of the fatty acids plays a vital role in antitumour activity.

### **6. Fatty acid synthase as a potential target in cancer**

They also synthesized fatty acyl ester derivatives (**6**) of cytarabine and evaluated them for anti‐ leukaemia activity. Some of 2',5'‐dimyristoyl derivatives of cytarabine were found to inhibit the growth of CCRF‐CEM cells [20]. Liu *et al.* reported the synthesis and antitumour evaluation of

 fatty acyl amino derivatives of cytarabine. The bioavailability of cytarabine is low due to its low lipophilicity. In order to improve the lipophilicity and bioavailability of cytarabine, a series of fatty acyl amino acid cytarabine analogues (**7**) were synthesized. It was found that the deriv‐ atives synthesized were more lipophilic than cytarabine. The antitumour activity determined in HL‐600 and HeLa cells showed that the derivatives were more active in HeLa cells than cytarabine while most of them demonstrated similar activity to cytarbine in HL‐60 cells. The length of fatty acids in the derivatives seemed to have an impact on the activity observed [21].

Zhang Chun‐hong and co‐workers synthesized new panaxadiol fatty acid esters (**8**) and eval‐ uated them for their antitumour activity. Tumour cell used was Vero cell line. Positive con‐ trol was 5‐FU, blank was an RPMI1640 culture medium, negative control was an RPMI1640 culture medium and the solvent for drugs to be tested. The compounds show the strongest

> O OH

O C O (CH2)nCH3

**8**

**(7)**

AA=Amino acids R -sugar

N

ROC-AA-HN

O N

N<sup>4</sup>

80 Fatty Acids

antitumour activity [22].

Human fatty acid synthase (HFAS) is a multifunctional enzyme that is essential for the endog‐ enous synthesis of long‐chain fatty acid from its precursor acetyl Co‐A and malonyl Co‐A (**Figure 4**). Blocking HFAS activity causes cytotoxicity [25]. The unique carboxyl terminal thioesterase (TE) domain of fatty acid chain plays a critical role in regulating the chain length of fatty acid releases. Also, the up‐regulation of HFAS in a variety of cancer makes the thio‐ esterase domain a candidate target for therapeutic treatment [26]. It was evident from the literature that the long alkyl/alkenes tail of the fatty acids can bind into the long groove tunnel site of thio‐esterase domain of FAS which may be one of the factors of anticancer activities of fatty acids [27].

Employing these strategies, the author and her research group carried out the *in silico* stud‐ ies on fatty acid analogues. The group designed new derivatives of stearic acid and palmitic acid and studied their *in silico‐*binding affinities towards key enzyme human fatty acid syn‐ thase‐thio‐esterase domain (PDB code 2PX6). The literature clearly says that an identifica‐ tion of oncogenic antigen‐519 (OA‐519) from human breast carcinoma cells as FAS has made it an important diagnostic and prognostic marker for breast cancer patients [28, 29]. By superposing the scaffold structure of all our designed analogues, it is seen that these ana‐ logues bind in the same orientation and similar position in terms of the common structure, that is, long aliphatic chain (**Figure 5**). It complies with the fact that the substrate‐binding site of HFAS is made up of hydrophobic groove. The docking studies revealed that there are two hydrogen‐bonding interactions between the OH group of triazolo thiadiazole of

**Figure 4.** Human fatty acid synthase (PDB id: 2PX6).

synthesized analogues and HIS‐2481 and SER‐2308 residues (**Figure 6**). These interactions revealed the important binding mode, since these two residues are present in the *"cata‐ lytic triad*" of FAS‐TE domain [30]. Further, the long alkyl/alkenyl chain of our synthesized analogues fits into the hydrophobic groove of the substrate‐binding site. The docking pose and hydrogen‐bonding interactions of one of the representative compounds are shown in **Figures 5** and **6**, respectively.

**Figure 5.** Docking pose.

#### **Figure 6.** Hydrogen‐bonding interactions.

Babak Oskouian and co‐workers reported the overexpression of fatty acid synthase in SKBR3 breast cancer cell line and. The objective of this study was to use a breast cancer‐derived cell line, SKBR3 , as a model to define the underlying mechanism for overexpression of FAS in cancer cells [31]. Silva *et al.* reported a clinic pathological study of ErbB2 and Ki‐67 in head and neck squamous cell carcinoma (SCC) and the overexpression of fatty acid synthase enzyme. They showed FAS expression in HNSCC and pointed out ki‐67 as a useful prognostic marker for these tumours [32]. Michelle Agostini *et al.* reported the proliferation of human oral squa‐ mous carcinoma cells and fatty acid synthase. FAS is overexpressed in several human cancers, such as prostate, breast, bladder, liver, lung, melanoma and oral squamous cell carcinoma [33].

### **7. Concluding remarks**

**Figure 4.** Human fatty acid synthase (PDB id: 2PX6).

**Figures 5** and **6**, respectively.

82 Fatty Acids

**Figure 5.** Docking pose.

synthesized analogues and HIS‐2481 and SER‐2308 residues (**Figure 6**). These interactions revealed the important binding mode, since these two residues are present in the *"cata‐ lytic triad*" of FAS‐TE domain [30]. Further, the long alkyl/alkenyl chain of our synthesized analogues fits into the hydrophobic groove of the substrate‐binding site. The docking pose and hydrogen‐bonding interactions of one of the representative compounds are shown in

> As part of a conclusion to our discussion**,** the various studies have shown that fatty acids not only augment the tumouricidal action of anticancer drugs but also enhance the uptake of anti‐ cancer drugs leading to an increase in the intracellular concentration of the anticancer drugs. The omega‐3 fatty acids have become adjutants to chemotherapeutic agents. Although the production of the above‐said fatty acids is a big challenge, a possibility would be gradually implementing the production of these fatty acids in clinical use. Such novel uses of fatty acids in cancer therapy would provide the lipid field with a new avenue to impact public health.

### **Author details**

Jubie Selvaraj

Address all correspondence to: jubiejawahar@gmail.com

Department of Pharmaceutical Chemistry, JSS College of Pharmacy (A Constituent Institution of JSS University‐Mysuru), Rock Lands, Udhagamandalam, Tamil Nadu, India

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