Conjugated Linolenic acids: Implication in Cancer

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Conjugated Linolenic acids: Implication in Cancer Kaushik K Dhar Dubey, Girish Sharma, and Aruna Kumar J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01379 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Journal of Agricultural and Food Chemistry

Conjugated Linolenic acids: Implication in Cancer Kaushik K Dhar Dubey a, Girish Sharma a, b*, Aruna Kumar a* a

Molecular Genetics Lab, Amity Institute of Biotechnology, Amity University Uttar Pradesh,

Noida-201303, India. b

Amity Center for Cancer Epidemiology & Cancer Research, Amity University Uttar

Pradesh, Noida-201303, India. *Corresponding Author Girish Sharma and Aruna Kumar Email address: [email protected] (Aruna Kumar)

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ABSTRACT

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Conjugated fatty acids (CFAs) including both conjugated linoleic acids (CLAs) and

3

conjugated linolenic acids (CLNAs) have various health promoting effects. These beneficial

4

effects are comprised by their anti-oxidant, anti-atherogenecity, anti-carcinogenic activities,

5

etc. Several reports indicate that CLNAs such as eleostearic acid, punicic acid, jacaric acid

6

and calendic acid possess anti-cancer properties. These CLNAs are produced and

7

accumulated in seeds of certain commonly available plants. This review discusses their role

8

in chemoprevention of cancer. Using in vitro as well as in vivo models of cancer, bioactivities

9

of these CLNAs have been explored in detail. CLNAs have been shown to have potent anti-

10

cancer activity as compared to the CLAs. Although the molecular basis of these effects has

11

been summarized here, more detailed studies are needed to explore the underlying

12

mechanisms. Further clinical trials are obligatory for assessing the safety and efficacy of

13

CLNAs as an anti-cancer agent.

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KEYWORDS: Conjugated fatty acids (CFAs), conjugated-linolenic acids (CLNAs), anti-

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cancer agent.

16 17 18 19 20 21 22 2 ACS Paragon Plus Environment

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INTRODUCTION

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From ancient times, plant products have been used as preventive and therapeutic agents. With

25

advances in biomedical research, naturally occurring compounds in leaves, roots, stem or

26

seeds of plants have been identified which are bioactive against many diseases such as

27

cardiovascular, liver, kidney or blood disorders and cancer etc. Fatty acids are a class of

28

compounds which are varied in composition. Plants and animals are the source of fatty acids

29

in the human diet. They not only serve as a source of energy but are also important structural

30

and functional component of the cell, and hence their consumption also influences human

31

health. Depending on the presence or absence of double bonds, fatty acids may be classified

32

into two major classes: saturated and unsaturated. Saturated fatty acids contain only single

33

bonds, whereas unsaturated fatty acids contain double or triple bonds. Unsaturated fatty acids

34

that contain two or more double bonds are referred to as polyunsaturated fatty acids (PUFAs).

35

PUFAs containing double bonds are generally of two types; one includes fatty acids in which

36

double bonds are separated by a methylene (-CH2-) group and are in cis-configuration.

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Examples include linoleic (C18:2 Δ9 cis, 12 cis) and α-linolenic (C18:3 Δ9 cis, 12 cis, 15 cis)

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acids, commonly found in seed oil of plants. The other types include non-methylene

39

interrupted fatty acids in cis- or trans-configuration and are known as conjugated fatty acids

40

(CFAs). These are fatty acid in which at least one pair of double bonds is separated by a

41

single bond. The CFAs occur as diene (2 C=C), triene (3 C=C), and tetraene (4 C=C) fatty

42

acids. In addition to the number of double bonds, there are many cis- and trans- geometric

43

isomers of CFAs. Some CFAs also have specialized moieties or groups such as a hydroxyl

44

group (-OH) or keto (-C=O) group. For example, licanic acid has a keto group, while

45

dimorphecolic has a hydroxyl group etc.

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CFAs which have 18 carbons and two C=C double bonds are known as conjugated linoleic

47

acids (CLAs), while those having 18 carbons and three double bonds are referred to as 3 ACS Paragon Plus Environment


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conjugated linolenic acids (CLNAs). CFAs are now being reported to have several beneficial

49

effects on human health. There are several examples of CLAs possessing anti-oxidant , anti-

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obesity, anti-carcinogenic, anti-atherogenicity and anti-diabetic activities1;2;3;4. Some CLAs

51

also influence fat composition or have immunomodulatory effects 5;6;7.

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CLNAs which have conjugated trienes (3 C=C), have now been reported to have beneficial

53

effects that include anti-oxidant, anti-cancer, anti-inflammatory, anti-obesity, anti-diabetic,

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anti-atherosclerosis activities, etc. 4;8;9;10;11. This review focuses on role of different types of

55

CLNAs in chemoprevention of cancer.

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Structure and Sources of CFAs:

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Different cis- and trans- geometrical configurations exist for CLNAs as shown in Table 1.

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CFAs may be derived from plants or dairy or microbial sources. CFAs that are synthesized

59

and stored in seed oils of various plant species are mostly CLNAs. Interestingly, many of

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these distinctive plant species are not domesticated. For example, α-eleostearic acid is

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obtained from Aleuritis fordii

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mahaleb 16 while β-eleostearic acid is derived from Aleuritis fordii and Momordica charantia

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17.

64

balsamina

65

found in seed oil of Jacaranda mimosifiola 22. Likewise, source of α- and β-calendic acid is

66

Calendula officinalis

67

bignonioides

68

acid is present in from bovine milk

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such as (C18:3 Δ 9 cis, 11 trans, 15 cis and C18:3 Δ 9 cis, 13 trans, 15 cis) are produced in

70

ruminants through bioconversion process by several bacteria for example Bifidobacterium

12,

Momordica charantia

Sources of punicic acid are Punica granatum 19,Trichosanthes

24.

23.

anguina

20

18,

13;14,

Parinarium spp

Fevillea trilobata

and Trichosanthes kirilowii

21

15

13;12,

and Prunus

Momordica

while Jacaric acid is

Catalpic acid is obtained from Catalpa ovata

12

and Catalpa

Some CLNAs are also present in dairy sources, for example- α-rumelenic 25;26,

bovine

27;28

as well as goat meat


29.

Some CLNAs

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9.

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breve, Lactobacillus plantarum, Propionibacterium freudenreichii etc

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concentrations of major CLNAs have also been reviewed in detail 9;30.

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CLNAs and Cancer:

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Cancer is one of the deadliest diseases associated with the uncontrolled growth of malignant

75

cells. By the process of angiogenesis, invasion and metastasis, cancer spreads to various parts

76

of the body thereby causing a heavy toll of life globally. Prevention, early detection and

77

timely treatment are the keys to curb this fatal disease

78

most commonly occurring cancers worldwide, causing huge mortality amongst men and

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women, respectively. Although, in more developed countries prostate cancer in men and lung

80

cancer in women are the leading causes of cancer-related death

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various approaches are being applied worldwide. One such approach could be the use of

82

CLNAs as an alternative strategy to either prevent or treat cancer. The anti-cancer effects of

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CLNAs have been studied in both in vitro as well as in vivo models, as detailed in Table 2

84

and Table 3.

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In vitro Anti-cancer Effects of CLNAs:

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Role of eleostearic acid (ESA) on various cancer cells:

87

ESA is found in the tung oil which is obtained from seeds of tung tree (A. fordii) that belongs

88

to family Euphorbiaceae. It has several industrial applications such as in wood finish, paints

89

and varnishes, due to its drying property on exposure to air. ESA has three double bonds at

90

9, 11, 13 carbon and may exist as two isomers (α-ESA and β-ESA).

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Alpha-eleostearic acid (α-ESA): α-ESA is a type of CFA with three conjugated double

92

bonds (18:3 Δ9 cis, 11 trans, 13 trans). α-ESA is produced and stored in seed oil of plants

93

such as A. fordii, M. charantia, P. spp and P. mahaleb. A. fordii, also known as tung tree

31.


Sources and

Lung and breast cancers are the

32.

For treatment of cancer,


94

accumulates nearly 70% ESA in its seed. Besides tung tree, other plant that produces ESA is

95

M. charantia or bitter melon that accumulates up to 50% ESA in its seed.

96

Besides having industrial applications, ESA has been documented to have anti-cancer

97

properties (Table 2). Igarashi et al. studied cytotoxic effect of CLNA on five tumor cell lines

98

namely, A549 (lung), MCF-7 (breast), DLD1 (colorectal), MKN-7 (stomach), HepG2

99

(hepatoma), in comparison to CLA, and found CLNA and tung oil fatty acids to be more

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potent in its anti-cancer effect. They further suggested the underlying mechanism as lipid

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peroxidation

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(SV-T2) and human monocytic leukemia cells (U-937). Other CLNAs from pomegranate,

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catalpa and pot marigold were also used in their study. They showed that tung oil inhibited

104

the growth of these tumors and the cytotoxicity involved lipid peroxidation pathway. Except

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pot marigold (C18:3 Δ 8,10,12 CLNA), all others (C18:3 Δ 9,11,13 CLNAs) showed

106

comparable cytotoxic effect on cancer cells 34.

107

Tsuzuki et al. studied the anti-cancer activity of tung oil and karela seed oil containing 60-

108

80% ESA, with that of CLA isomers (C18.2 Δ 9 cis, 11 trans and C18:2 Δ 10 trans 12 cis -

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CLA) on four types of human cancer cells namely, A549 (lung), DLD-1 (colorectal), HL-60

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(acute promyelocytic leukemia) and HepG2 (hepatoma). α-ESA was found to be more

111

effective as an anti-cancer agent in comparison to CLA isomers in a dose-dependent

112

manner.Even at 5 µg/mL, the cytotoxic effects were more in DLD-1 and HL-60 cells. An

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increase in mRNA expression as well as caspase activity (Casp3, Casp8 and Casp9) was

114

observed in DLD-I cells following α-ESA treatment. α-ESA induced apoptosis in cancer cells

115

with DNA fragmentation involving lipid peroxidation 17. Similar effect of α-ESA from bitter

116

gourd seed oil was found on human colon cancer cells (Caco2). The cell viability of colon

117

cancer Caco-2 cells was remarkably reduced when treated with free fatty acids and purified

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C18:3 Δ 9 cis, 11 trans, 13 trans-CLNA, at a concentration of 25 μmol/L and 50 μmol/L in a

33.

Suzuki et al. studied the cytotoxic effect of tung oil on mouse tumor cells


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dose-dependent manner. CLA C18:2 Δ 9 cis, 11 trans did not affect the cell viability even at

120

higher dose (150 μmol/L). Mechanism of cytotoxicity involved reduced expression of anti-

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apoptotic Bcl-2 protein whereas up-regulation of GADD45 (Growth arrest and DNA

122

damage), p53, and PPARγ (Peroxisome proliferator-activated receptor γ) was observed. p53

123

and GADD45 plays important role in pathways involving induction of apoptosis. PPARγ is a

124

ligand-activated transcription factor that regulate expression of genes and acts as a modulator

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of carcinogenesis 35. Anti-cancer and apoptosis inducing activity of α-ESA on different colon

126

cancer cell lines DLD-1, HT-29 and Caco-2 were tested by Yasui et al. The level of PPARγ

127

protein was found to be higher in HT-29 and DLD-1 cells as compared to Caco-2 cells. This

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study also involved use of troglitazone (a synthetic ligand of PPARγ) that induce apoptosis in

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many cancer cells. It was found that the anti-proliferative effect of α-ESA and troglitazone

130

were more pronounced in HT-29 cells as compared to Caco-2 cells

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growth and induction of apoptosis were reported in HL60 leukemia and HT-29 colon cancer

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cells by HPLC-fraction of bitter gourd extract which contained α-ESA 37. In another study by

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Tsuzuki et al., α-ESA was found to have suppressive effect on cancer cell growth by

134

decreasing tumor angiogenesis, both in vitro as well as in vivo. In vitro studies done using

135

human umbilical vein endothelial (HUVEC) cells

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angiogenesis by suppressing expression of vascular endothelial growth factor receptors 1 and

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-2 (VEGFR-1,-2), and activation of PPARγ, whose role in tumorigenesis is now well

138

documented, at a concentration one-tenth of CLA 38.

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α-ESA was also found to induce apoptosis in HeLa cells. The underlying mechanism

140

involved autophagy-dependent cell death. α-ESA treatment resulted in concomitant

141

production of reactive oxygen species and the signaling pathway involved activation of

142

pERK1/2, as well as decreased phosphorylation of AKT and P70S6K, in a dose- and time-

143

dependent manner 39. α-ESA also checks the proliferation of breast cancer which is the most

36.

An inhibition of

demonstrated that α-ESA inhibited



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devastating cancer that affects women globally. Studies on MCF-7, a human breast cancer

145

cell line, have shown anti-cancer activity of α-ESA in addition to cellular and molecular

146

mechanism behind the activity. In a study by Moon et al., treatment with α-ESA activated

147

PPARγ and prevented phosphorylation of ERK1/2 MAPK. There was up-regulation of tumor

148

suppressor gene p53, and cdk inhibitor p21, and pro-apoptotic Bax whereas anti-apoptotic

149

Bcl-2 and procaspase-9 was down-regulated 40. Similar results were reported by Zhang et al.,

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using MCF-7 cell line. They confirmed that α-ESA from tung oil has potent anti-cancer

151

activity in both time- and dose- dependent manner. α-ESA significantly induced apoptosis in

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breast cancer cells whereas normal cells (liver) were not affected. The anti-cancer activity

153

was in part due to inhibition of DNA synthesis and cell proliferation. There was induction of

154

apoptosis and cells were arrested in the G2/M phase. At molecular level, there was also

155

upregulation in the expression levels of p53, Bax, PPARγ, p21 and caspase-3 mRNA

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Grossmann et al. studied the effects of α-ESA on human breast cancer cells with or without

157

estrogen receptor (ER). α-ESA inhibited cell proliferation and induced apoptosis in both ER–

158

negative MDA-MB-231 (MDA-wt) and ER-positive MDA-ERα7 cells. Mitochondrial

159

membrane potential was also disrupted. At molecular level, the apoptosis-inducing factor and

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endonuclease G was translocated from mitochondria to the nucleus. Study indicated that the

161

mechanism involved in growth inhibition may be oxidation- dependent and possibly a

162

caspase-independent mechanism

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was found to actively suppress proliferation of adult T-cell leukemia (ATL) cell lines (ED

164

and Su9To1) and moderately inhibited phytohemagglutinin-activated human peripheral blood

165

mononuclear cells 43.

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Beta-eleostearic acid (β-ESA): β-ESA (C18:3 Δ9 cis, 11 trans, 13 trans), is an isomer of α-

167

ESA. Interestingly, β-ESA was reported to have stronger anti-proliferative effect than α-

168

ESA. Yasui et al. compared anti-cancer effect of α-ESA, β-ESA, α-calendic acid (α-CDA)

42.

41.

Bitter gourd seed extract which contains ~50% α-ESA


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and β-calendic acid (β-CDA) on human colon cancer cells (Caco-2). All trans CLNAs: β-

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ESA and β-CDA showed effective anti-cancerous activity and apoptosis induction in Caco-2

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cells in comparison to their cis-isomer. At molecular level, there was decrease in bcl-2 and

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increase in bax mRNA expression along with DNA fragmentation. This study also indicated

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that β-ESA and β-CDA exert their effect through a different signaling pathway than their cis-

174

isomers 44. Similarly, β-ESA affected viability of human bladder cancer cells (T24) in a dose-

175

and time-dependent manner. There was induction of apoptosis involving reactive oxygen

176

species (ROS) accumulation. Specific mechanism involved activation of PPARγ andcaspase-

177

3 while Bcl-2 expression was down-regulated 45.

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Role of punicic acid (PA) on leukemia, prostate and breast cancer cells:

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Other CLNAs closely related to ESA is PA (C 18: 3 Δ 9 cis, 11 trans, 13 cis). This fatty acid

180

is derived from the fruit of P. granatum (commonly known as pomegranate) and from a

181

lesser known source T. kirilowii. Pomegranate is widely grown for its fruit juice. Its seeds

182

are used as spice or in culinary preparations. PA is another example of a potent CFA reported

183

to have several health benefits such as anti-cancer activity, prevention of obesity and insulin

184

resistance in mice 46;47;48.

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The pomegranate seed oil (PSO) contains approximately 80% PA. As mentioned earlier, PA

186

derived from pomegranate was cytotoxic to human monocytic leukemia cells at concentration

187

greater than 5 μmol/L and the mechanism involved was lipid peroxidation

188

was also observed on human prostate cancer cell line, which is an androgen-dependent and

189

androgen receptor (AR)-positive cell line (LNCaP). Growth of LNCaP cells was found to be

190

inhibited on treatment with PA. At molecular level, there was activation of caspase-9 with

191

accumulation of cleaved PARP. There was significant reduction in the expression levels of

192

AR proteins in a dose-dependent manner besides reduction in PSA and AR-dependent

193

SRD5A1 (steroid 5 reductase type 1) 49. PA also induced apoptosis in breast cancer cells. At 9 ACS Paragon Plus Environment

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Effect of PA


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40 μmol/L of PA, proliferation of MDA-MB-231, an estrogen insensitive breast cancer cell

195

line, was inhibited by 92%. Similarly, a 96% decrease was observed for MDA-ER-7, an

196

estrogen sensitive cell line. Inhibitor of protein kinase C, bisindolylmaleimide (BIM),

197

partially blocked proliferation by PA which showed PKC signaling pathway was involved in

198

cytotoxicity. As antioxidant tocotrienol addition also reversed the effect of PA which

199

indicated involvement of lipid peroxidation 46. PA alone and in combination with luteolin and

200

ellaigic acid also inhibited invasion of prostate cancer cells (PC-3) in artificial membranes 50.

201

PA also induced apoptosis and mitochondrial dysfunction in two prostate cancer cell lines,

202

LNCaP and PC-3, which could be blocked by anti-oxidant α-tocotrienol. This indicated that

203

the mechanism of inhibition was lipid peroxidation-dependent. Additionally, the PKC

204

pathway was also partially involved 51. Different studies using PA showed debatable results,

205

i.e. anti-oxidant as well as pro-oxidant activity of CLNAs. The crucial point has been the

206

dose of CLNAs used. Pre-clinical studies have shown that PA exhibits both pro-oxidant (at

207

1.2 g kg-1) and anti-oxidant (at 0.6 g kg-1) that warrants more studies in this direction 11;20;48.

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Effect of jacaric acid (JA) on leukemia and prostate cancer cells:

209

JA (C18:3 Δ 8 cis, l0 trans, 12 cis) is a natural fatty acid present in the seed oil of J.

210

mimosifolia, a subtropical tree of South America. There are now increasing evidences that JA

211

is a powerful and selective inducer of programmed cell death (PCD) or apoptosis in cancer

212

cells. JA exhibited anti-cancer activity in prostate cancer cell lines by activating apoptosis in

213

both androgen-dependent (LNCaP) and -independent (PC-3) prostate cancer cell lines. There

214

was an increase in lipid peroxidation in DLD-1 human colorectal adenocarcinoma cells

215

(Table 2). At the same concentration, JA also exerted a stronger effect in inducing apoptosis

216

when compared to α-ESA, PA, catalpic acid (CPA) and the trans-isomers ESA and CDA 52.

217

Gasmi et al. studied the effect of seven fatty acids viz PA, JA, α-CDA, β-CDA, CPA, trans


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and cis- vaccenic acid on the same cancer cell lines. They also reported similar induction of

219

apoptosis in these cancer cell lines which was both dose and time-dependent. Mechanism

220

involved triggering of both intrinsic as well as extrinsic signaling pathways of apoptosis,

221

modulation of Bcl-2 proteins and activation of cysteine proteases that play an important role

222

in apoptosis such as caspase-3, -8, -9 and PARP-1

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induced apoptotic cell death in human leukemia HL-60 cells, while linoleic acid and CLA

224

C18:2 Δ 10 trans 12 cis had no effect. The mechanism of inhibition involved increase in

225

DNA fragmentation and oxidative stress

226

culture of human eosinophilic leukemia (EoL-1) cells. JA had potent anti-cancer effect on

227

EoL-1 cells when compared to any other CLN isomers and showed growth inhibitory activity

228

by arresting cell cycle in G0/G1 phase, inducing apoptosis and eosinophilic differentiation of

229

leukemia cells. It induced apoptosis in temporal and concentration-dependent manner and

230

therefore showed potential in treatment of myeloid leukemia

231

effect on proliferation of murine macrophage-like leukemia PU5-1.8 cells in a time- and dose

232

-dependent manner with minimal effect on normal murine cell. JA had most potent effect

233

(IC50 6 µmol/L) on these cells, when compared to α-CDA, β-CDA, α-ESA, β-ESA, and PA.

234

Similarly, JA also inhibited two other murine macrophage-like leukemia J774 A.1 cells and

235

P388D1 cells. It induced fragmentation of DNA, mitochondrial dysfunction and apoptosis.

236

Other changes involved were increase in levels of pro-apoptotic Bax protein besides decrease

237

in Bcl-2 and Bcl-xL proteins 56. Another study by Gafar et al. showed that JA (similar to PA)

238

induced apoptosis and mitochondrial dysfunction in LNCaP and PC-3 at 30 µmol/L and 100

239

µmol/L doses (Table 2). The mechanism of inhibition was lipid peroxidation-dependent and

240

partially through the PKC signaling pathway 51.

241

Cytotoxic effect of calendic acid (CDA) on colon cancer cells:

54.

53.

Yamasaki et al. also found that JA

Jacaric acid also had inhibitory effect on cell


55.

JA has growth inhibitory


242

CDA (C 18: 3 Δ 8 trans, 10 trans, 12 cis), is a major constituent of seed oil of pot marigold

243

(C. officinalis) which finds its use in cosmetic products. It has great medicinal value as

244

ointments for reducing inflammation, skin rashes, anti-fungal/bacterial and wound healing

245

properties etc. There are two isomers of CDA namely, α-calendic acid (α-CDA) and β-

246

calendic acid (β-CDA). Both α-CDA and β-CDA possess potent anti-cancer properties. As

247

mentioned earlier, Yasui et al. showed that both α-CDA and β-CDA induced apoptosis in

248

Caco-2 cells. Trans isomer (β-CDA) was more potent than cis-isomer (α–CDA) in terms of

249

inhibition of growth of Caco-2 cells and DNA fragmentation. The cytotoxicity mechanism

250

involved lipid peroxidation. As anti-oxidant α-tocopherol completely diminished the

251

cytotoxic effect of α-CDA but not that of β-CDA, author hypothesized that a different

252

pathway was involved which needs to be explored 44.

253

Similarly, both α-CDA and β-CDA were shown to induce apoptosis and cytotoxicity in

254

human choriocarcinoma JEG-3 cells. With β-CDA, there was 2.13-fold increase in

255

cytotoxicity than α-CDA (1.85 fold). Levels of apoptotic markers Caspase-3, Caspase-9 and

256

Bax were increased, while Bcl-2 was decreased. These CLNAs also partially inhibited the

257

invasion of JEG-3. Cytotoxic effects involved lipid peroxidation and activation of p38

258

MAPK. Analysis revealed increase in oxidative stress which activated the phosphorylation of

259

p38 MAPK. SB203580, a selective inhibitor of p38 MAPK, blocked the apoptosis induced by

260

α- and β-CDA by inhibiting p38 MAPK activation, reversing expression of Bcl-2 and Bax

261

and that of Caspase-3 and Caspase-9 57.

262

Catalpic acid (CPA):

263

CPA is another CLNA that accumulates naturally in the seed oil of C. ovata. Anti-cancer

264

activity of CPA was reported in mouse tumor cells and human monocytic leukemia cells 34.

265

Not much work has been conducted for studying the effect of CPA on cancer. 12 ACS Paragon Plus Environment

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In vivo Anti-cancer Effects of CLNAs:

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Besides effect of CLNAs on in vitro models, studies demonstrated their role in in vivo models

268

as well. Both α-ESA and PA when fed to mice and rat inhibited the growth of tumor.

269

Similarly, JA and CPA have shown anti-tumor effect in mice and rats, respectively. However,

270

more studies are warranted to support their role.

271

Kohno et al. studied the effect of bitter melon seed oil (BMSO) on proliferation of colonic

272

aberrant cryptic foci (ACF) induced in male F344 rats by azoxymethane. BMSO inhibited the

273

growth of ACF and induced apoptosis 58. In another study by Kohno et al., BMSO inhibited

274

proliferation of azoxymethane induced ACF

275

cancer cell line (DLD-1) into nude mice which caused formation of tumors. Mice were fed

276

with tung oil containing α-ESA. Growth of the tumor was strongly suppressed in mice fed

277

with tung oil as compared to control and other CLAs. The DNA fragmentation rate was

278

higher in mice fed with α-ESA, suggesting induction of apoptosis similar to the in vitro

279

results

280

orally fed mice, at concentrations of 50 and 100 mg/kg/day in a dose-dependent manner,

281

which was significantly lower than that of CLA (500 mg/kg/day). The effect of ESA on

282

cancer cell growth was presented by decreasing tumor angiogenesis 38 (Table 3).

283

Besides ESA, several in vivo studies also implicated anti-cancer effect of PA. Hora et al.

284

investigated chemopreventive efficacy of PSO in chemically induced skin tumor

285

development in SCID mice. Results highlighted significant decrease in tumor incidence and

286

its progression. Dietary feeding of PSO to 6-week old male rats prior to azoxymethane

287

treatment, also suppressed progression of azoxymethane-induced adenocarcinoma of colon in

288

vivo and therefore showed as a promising chemopreventive agent 60. Kohno et al. investigated

289

the efficacy of dietary PSO and BMSO in comparison to CLA (C18:2 Δ 9 cis, 11 trans ;

17.

59.

Tsuzuki et al. transplanted human colon

In another study by Tsuzuki et al., ESA clearly inhibited formation of vessels in



290

C18:2 Δ 10 trans 12 cis), on inhibition of azoxymethane-induced malignancy of colon in rats

291

61.

292

colonic adenocarcinomas. Only slight inhibition was seen with CLA. PSO administration

293

resulted in increased expression of PPARγ and levels of CLA in the liver / colon and colon

294

mucosa of rats, respectively (Table 3). An in vivo combinatorial study involving PA along

295

with luteolin, and ellaigic acid inhibited the growth of luciferase-expressing human PC-3M

296

cells which were introduced subcutaneously into prostate of SCID mice. There was inhibition

297

of angiogenesis and PCa metastasis. At molecular level, a decrease in IL-8 and VEGF levels

298

besides inhibition of CXCL12/CXCR4 and AKT signaling was observed 62. JA also exhibited

299

anti-tumor effect in nude mice which were transplanted with DLD-1 human colorectal

300

adenocarcinoma cells

301

studied in rats 63. Dietary feeding of catalpa seed oil to azoxymethane pre-treated five-week-

302

old rats, prevented occurrence of colonic foci significantly when compared to azoxymethane

303

treated controls. Thus, above mentioned studies pointed towards potential chemopreventive

304

role of CLNAs in cancer. However, further research is required to substantiate this

305

information.

306

Conversion of CLNA into CLA:

307

CLAs have various physiological effects on animal and human health. There are several

308

geometric and positional isomers of linoleic acid, of which particularly C18:2 Δ 9 cis, 11

309

trans (rumenic acid) and C18:2 Δ 10 trans 12 cis CLA are of primary focus. There have been

310

well documented evidences that implicated CLAs as having health promoting activities such

311

as anti-inflammatory, anti-oxidant, anti-tumor, anti-atherosclerosis, anti-diabetic and anti-

312

obesity 1;2;3;4. Besides, some of these also influenced bone formation, fat composition or have

313

immunomodulatory effect etc

314

nutraceutical compounds. These fatty acids have several beneficial effects on human health

When compared to CLA feeding, PA clearly inhibited and reduced the formation of

52.

Anti-tumor effects of CPA obtained from catalpa seed oil were

5;6;7.

Hence, they are being recognized as important


Page 14 of 38

Page 15 of 38


315

and therefore are also being projected as chemopreventive or therapeutic agents against

316

diseases.

317

There is a structural similarity in the structure of CLA and CLNA. In mammals, CLNA acts

318

as source of CLA. There are several studies that show that CLNA are metabolized into CLA.

319

Indeed, Tsuzuki et al. reported that ESA is actively metabolized into CLA (C18:2 Δ 9 cis, 11

320

trans) in rats

321

CLA in rats 65;66. ESA when compared to PA, was more effectively metabolized into C18:2 Δ

322

9 cis, 11 trans CLA in mice

323

rats, both ESA and JA got absorbed and converted to CLA. The percentage conversion of JA

324

was less than that of ESA

325

varied in Caco-2 cells which were incubated with α-ESA, β-ESA, CPA or PA

326

conversion rate of CLNA into CLA varies depending on the type of fatty acid. It has been

327

reported that incorporation of PUFA, such as α-linolenic acid found in sunflower-seed and

328

flaxseed in feed, also improved CLA and CLNA levels in animals 27;28;70. Interestingly, CLAs

329

which have been reported to produce several health benefits are also provided by sources

330

such as milk fat, meat, and cheese. However, their levels are very low. The predominant CLA

331

(>90% of total) in milk is C18:2 Δ 9 cis, 11 trans followed by C18:2 Δ 10 trans, 12 cis

332

isomers. Different pathways may be involved in production of CLA from PUFA / CLNA in

333

gut microbiome. The level of different CLA isomers was found to be influenced by the diet

334

such as type of forage or silage that includes rapeseed, sunflower or linseed in cows, goats

335

and ewe 71.

336

CLNA on the other hand are produced and accumulated in the seeds of certain plants. These

337

CLNAs are accumulated to a very high levels; for example PSO produce up to ~83% PA,

338

tung oil has ~68% ESA, calendula oil has ~ 38.9 to 58.4% CDA

339

CLNAs have higher potency as a cytotoxic agent and at a lower dose as compared to CLA in

64;65.

Similarly, PA from P. granatum and T. kirilowii gets metabolized into

67.

68.

When jacaranda seed oil and tung oil were administered to

The efficiency of uptake and conversion of CLNA into CLA


10;12;34;72;73.

69.

Thus, the

In addition,


17;33;38.

340

both in vitro and in vivo studies

CLNAs may be a better alternative because of their

341

greater bioactivities, easy bioavailability and conversion into CLA. They also have a

342

significant potential as chemopreventive and therapeutic agent for cancer. There are only a

343

few reports on the use of CLAs which are in cancer clinical trials 74, but in this aspect CLNAs

344

are still unexplored which warrants further research to assess their mechanism of action and

345

potential role. There are emerging evidences in various model organisms indicating that

346

CLNAs induces apoptosis, decrease tumor incidence and multiplicity, prevent angiogenesis,

347

invasion and metastasis. However, further clinical studies are urgently warranted to

348

understand the role of CLNAs as anti-cancer agent for humans. Till date anti-cancer effect of

349

these CLNAs has been studied taking individual CLNA. Combinatorial studies involving

350

more than one CLNA are also warranted that could show their synergistic effect and different

351

mechanism of action thereby overcoming the multi-drug resistance etc. Additionally,

352

reasonable clinical trials are also obligatory to validate the safety concerns of CLNAs on

353

human health, especially its efficacy as a cancer chemopreventive agent.

354

ACKNOWLEDGEMENT

355

Authors acknowledge Amity University Uttar Pradesh, Noida for providing infrastructure and

356

support.

357

FUNDING INFORMATION

358

The authors are grateful to DBT-BioCARe for providing funding to Aruna Kumar and

359

Kaushik K Dhar Dubey.

360 361

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(74)

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593

Perspectives. Nutrients 2019, 11 (2), 370-399.

594 595 596 597 598 599 600 601 602 603



Table1. Conjugated Fatty Acids and Their Isomeric Structure. Nomenclature

Abbreviated formulae

Structure

Alphaeleostearic acid

C18.3 Δ 9 cis, 11 trans, 13 trans

Beta-eleostearic acid

C18.3 Δ 9 trans, 11 trans, 13 trans

Catalpic acid

C18.3 Δ 9 trans, 11 trans, 13 cis

Alpha-calendic acid

C18.3 Δ 8 trans, 10 trans, 12 cis

Beta-calendic acid

C18.3 Δ 8 trans, 10 trans, 12 trans

Jacaric acid

C18.3 Δ 8 cis, l0 trans, 12 cis

Punicic acid

C18.3 Δ 9 cis, 11 trans, 13 cis C18.3 Δ 9 cis, 11 trans, 15 cis

Rumelenic acid C18.3 Δ 9 cis, 11 trans, 15 trans C18.2 Δ 9 cis, 11 trans Conjugated Linoleic acid

C18.2 Δ 10 trans, 12 cis


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Page 29 of 38


Table 2. Conjugated Linolenic Acids and Their Mechanism of Action in Cancer In Vitro.

Fatty Acid


Source

Cancer Type

Cell line

Dose

Mechanisms

References

CLNA (alkali treatment)

Colorectal

DLD1

↑ Lipid peroxidation

33

Hepatoma

HepG2

5- 100 μmol/L

Lung

A549

Breast

MCF-7

Stomach

MKN-7

Mouse tumor cells

SV-T2

↑ Lipid peroxidation

34

Human monocytic leukemia cells

U-937

0-200 μmol/L

Colorectal adenocarcinoma,

DLD-1,

0-25 μg/ mL

↑ DNA fragmentation

17

Tung oil

Tung oil

Karela seed oil

Hepatoma

HepG2,

Lung adenocarcinoma Acute promyelocytic leukemia

A549 HL-60


↑ Caspase activity and mRNA expression ↑ Lipid peroxidation observed in DLD-1 cells


Bitter gourd seed oil-Free Fatty acids

Human colon cancer cell line

Caco-2

0-100 μmol/L

Page 30 of 38

↓ Bcl-2 protein,

35

↑ GADD45, p53, and PPARγ ↑ Apoptosis

Com. α-ESA

Com.α-ESA

Human colon cancer cell line

Caco-2

Colon cancer cell lines

HT-29 DLD-1

2.5-50 μmol/L

↑ Bax mRNA

0-50 μmol/L

↓ Cell viability

Caco-2 Bitter gourd ethanol extract and column and HPLC purified

Human promyelocytic leukemia cells

HL60

Tung oil

--

HUVEC

44

↓ Bcl-2 36

↑ Apoptosis ↑ DNA fragmentation

5 μmol/L

↑ Apoptosis

37

↑ Growth inhibition 5-20 μmol/L

↓VEGFR1, VEGFR2

38

↑ PPAR γ ↑ DNA fragmentation and apoptosis Purified α-ESA

Human breast cancer cells

MDAMB-231 (MDAwt)

0-80 μmol/L

MDAERα7 30 ACS Paragon Plus Environment

Cell proliferation Ψ Loss ↑ Apoptosis involving lipid peroxidation

42

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Com.α-ESA

Human breast cancer cells

MCF-7

0-45 μmol/L

↑ PPARγ ↓ pERK1 ⁄ 2 ↑ p53, p21, and Bax

40

↓ Bcl-2 and procaspase-9 Com.α-ESA

Human cervix carcinoma cells

HeLa

0- 100 μmol/L

↑ Apoptosis and autophagy dependent cell death

39

↓ pAKT and pP70S6K activities ↑ pERK1/2 signal, ↑ ROS α-ESA purified from tung oil

Human breast cancer

MCF-7

5-80 μmol/L

↑ PPARγ, p21, Bax, p53, and caspase-3 mRNA

41

↓ Cell proliferation ↑ Apoptosis ↑ G2/M phase cell population

Betaeleostearic acid

Bitter gourd seed extract

Adult T-cell leukemia

Com. β-ESA

Human colon cancer cells

ED Su9T01

Caco-2

0.5-500 μmol/L

2.5 -50 μmol/L

43

Cell proliferation and differentiation

↓ bcl-2 mRNA ↑ bax mRNA ↑ DNA fragmentation ↑ Apoptosis


44


Com. β-ESA

Human bladder cancer cells

T24

10–80 μmol/L

Page 32 of 38

↑ Apoptosis, ↑ ROS

45

↑ Caspase-3 activity ↓ GSH, ↓ Bcl-2 ↑ PPARγ

Pomegranate seed oil

Mouse tumor cells and

SV-T2 U-937

0-200 μmol/L

Lipid peroxidation possibly involved

34

Human monocytic leukemia cells Punicic acid

Pure PA

Human prostate

PC-3

4 μg/mL

Invasion

50

Com. PA

Prostate

LNCaP

0-100 μmol/L

↑ Antiandrogenic

49

↑ Pro-apoptotic mechanisms ↓ AR protein, PSA and ARdependent SRD5A1

Pomegranate seed oil

Breast

MDAMB-231

0-40 μmol/L

MDAER·7 Com.PA

Prostate

LNCaP PC-3

↑ Apoptosis

46

↑ Lipid peroxidation ↓ PKC pathway

10-100 μmol/L 10-100 μmol/L


↑ Apoptosis ↑ Lipid peroxidation ↓ PKC pathway

51

Page 33 of 38


Com. JA Jacaranda seed oil Com. JA

Colorectal adenocarcinoma

DLD-1

Com. JA

↑ Apoptosis

52

↑ Lipid peroxidation ↓ PKC pathway

Prostate

LNCaP PC-3

Jacaric acid

0-10 μmol/L

Leukemia

HL-60

Jacaranda seed oil

1 – 100 μmol/L

↑ Intrinsic and extrinsic apoptotic pathway

1 – 100 μmol/L

↑ Intrinsic apoptotic pathway

0.625-10 μmol/L

↑ Apoptotic cell death

53

54

↑ Inter-nucleosomal DNA fragmentation ↑ Sub-G1 population

Com. JA

Human eosinophilic leukemia

EoL-1

5-25 μmol/L

Arrest cells in G0/G1 phase ↑ Apoptosis

55

Com. JA

Leukemia

PU5-1.8

4-12 μmol/L

↑ Apoptosis ↑ DNA fragmentation, ↑ Bax

56

Arrest cells in G0/G1 phase ↓ CDK2 and cyclin E ↓ Bcl2 and Bcl-XL Com. JA

Prostate

LNCaP PC-3

1-100 μmol/L 1-100


↑ Apoptotic cell death ↑ Lipid peroxidation ↓ PKC pathway

51


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μmol/L Alpha- and Beta-calendic acid

Com. α- and βCDA

Choriocarcinoma

Betacalendic acid

Com. β-CDA

Colon

Catalpic acid

Catalpa seed oil

JEG-3

Caco-2

Mouse tumor cells and Human monocytic leukemia cells

SV-T2

20-40 μmol/L

↑ Oxidative stress pathways

2.5-50 μmol/L

↑ Bax mRNA

0-350 μmol/L

Lipid peroxidation possibly involved

57

↑ p38 MAPK 44

↓ Bcl-2 34

U-937

Inhibition; Ψ mitochondrial membrane potential; ↑ Increase; ↓ Decrease; ROS Reactive oxygen species, Com. Commercial


Page 35 of 38


Table 3: Conjugated Linolenic Acids and Their Mechanism of Action in Cancer In Vivo.

Fatty Acid


Plant Source

Cancer Type

Cell type

Animal model

Bitter gourd seeds

Colon

Azoxymethane (AOM)induced colonic aberrant crypt foci (ACF)

Male F344 rats treated with Azoxymethane

Dose

Route of

Mechanisms

References

↓ PCNA index

58

Administ ration 0.01%, 0.1% or 1% CLN

Diet

↑ Apoptosis in ACF

0.01%, 0.1% or 1% BMO Diet

↑ PPARγ

59

Tung oil

Colorectal adenocarci noma

DLD-1

DLD-1 Xenograft nude mice: BALB/cAJcl-nu nu/nu

50 mg/animal

Oral

↑ DNA fragmentation and lipid peroxidation

17

Tung oil

Colorectal adenocarci noma

DLD-1

DLD-1 Xenograft s.c ICR mice

50 and 100 mg/kg/day

Oral

↓ Tumor angiogenesis.

38

Pomegra nate seed oil (PSO)

Skin

CD1 mice

Topical

↓ Tumor incidence and multiplicity

60

Female CD1 100 μL of 5% mice initiated PSO with DMBA and promoted by TPA


↓ TPA-stimulated ODC activity


Pomegra nate seed oil

Colon

Punicic acid Prostate


PC-3M-luc2 cells or Pten−/−; KrasG12D

Punicic acid, Luteolin, and Ellagic acid Jacaric acid

Catalpic acid


0.01%, 0.1%, or 1% PGO or 1% CLA

Page 36 of 38

Diet

Adenocarcinoma and multiplicity

61

↑ PPARγ protein in the colon ↑ CLA protein in colonic mucosa and liver s.c. injection of 64 luciferaseμg/componen expressing t/day human PCa cells into prostrate of SCID mice

s.c.

Tumor growth, chemotaxis, angiogenesis and PCa metastasis

62

CXCL12/CXCR4 and AKT signaling ↓ IL-8 and VEGF Jacarand Colorectal a seed oil adenocarci noma

Catalpa seed oil (CPO)

Colon

DLD-1


Male athymic nude mice (BALB/cAJclnu nu/nu

1 mg/day


0.01%, 0.1%, or 1% CPO

Diet

Anti - tumor effect due to Intracellular incorporation of JA

52

↑ Apoptosis via lipid peroxidation Diet

63

Cell proliferation ↓ Serum triglycerides level ↓ COX-2 mRNA


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Inhibition; ↑ Increase; ↓ Decrease; s.c. Subcutaneous



Graphic for Manuscript:


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Conjugated Linolenic acids: Implication in Cancer

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