Effect of Conjugated Linoleic Acid Supplementation on Inflammatory Factors and Matrix Metalloproteinase Enzymes in Rectal Cancer Patients Undergoing Chemoradiotherapy

Abstract

Objectives. The aims of this study were to determine the effect of conjugated linoleic acid (CLA) supplementation on inflammatory factors and matrix metalloproteinase (MMP) enzymes in rectal cancer patients undergoing chemoradiothetrapy. Method and Material. In this randomized, double-blind, placebo-controlled pilot study, 34 volunteer patients with rectal cancer undergoing chemoradiotherapy assigned into the CLA group (n = 16), receiving 3 g CLA/d, and placebo group (n = 18) receiving placebo capsules (sunflower oil) for 6 weeks. The supplementation began 1 week before starting RT (loading period) and continued every day during treatment. Before and after intervention, serum tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), IL-6, MMP-2, MMP-9, and high-sensitivity C-reactive protein (hsCRP) were measured by enzyme-linked immunosorbent assay (ELISA) kits and immunoturbidimetric method, respectively. Independent t tests and paired t tests were used to compare parameters between and within groups, respectively. Results. In the CLA group, the mean serum TNF-α, IL-1β, hsCRP, MMP-9, and MMP-2 levels reduced insignificantly. However, significant changes in TNF-α (P = 0.04), hsCRP (P = 0.03), and MMP-9 (P = 0.04) concentrations were observed in the CLA group when compared with the placebo group. The mean serum IL-6 level remained unchanged in the CLA group but increased remarkably in the placebo group. Conclusion. According to our results, CLA supplementation improved inflammatory factors, MMP-2, and MMP-9 as biomarkers of angiogenesis and tumor invasion. It seems that CLA may provide new complementary treatment by reducing tumor invasion and resistance to cancer treatment in patients with rectal cancer.

Keywords

rectal cancer chemoradiotherapy conjugated linoleic acid supplementation inflammatory factors

Introduction

Colorectal cancer is one of the most prevalent cancers in the world, and it is the third most common cancer in both men and women.¹ Surgery and preoperative chemoradiotherapy (CRT) or postoperative CRT are the main treatments for rectal cancer.¹ In most oncology departments, preoperative chemoradiation is now the current standard of care for locally advanced operable rectal cancer.²

It is now known that exposure to clinically relevant doses of ionizing radiation stimulates various biological responses at the cell and tissue levels through the early activation of cytokine cascades.³ Increases in proinflammatory cytokines such as tumor necrosis factor α (TNF-α),interleukin 1 (IL-1), IL-6, and IL-8 as a result of cancer or cancer treatments may be responsible for the incidence of symptoms such as pain, fatigue, distress, and so on.³ Moreover, many of these inflammatory downstream responses to radiation are harmful to normal tissue, and they gave a survival advantage to tumor cells.³ Therefore, new adjuvant anti-inflammatory therapeutic approaches may be a useful tool to downregulate inflammatory signaling pathways that include TNF-α, IL-1, and Cox-2 activities for the prevention of radiotherapy (RT) failure and tumor recurrence and the enhancement of tumor radiosensitivity while reducing tumor vascularization and cell invasion.^4-6

Conjugated linoleic acids (CLAs), which represent a heterogeneous group of positional and geometric isomers of linoleic acid are found mainly in foods derived from ruminant animals (such as dairy products and meats).⁷ CLA consists of a mixture of geometric and positional isomers (cis or trans double-bond positioning at ([7,9], [8,10], [9,11], [10,12], or [11,13]). The most abundant isomer in dietary sources is cis-9,trans-11-CLA (more than 75%-90% of total CLA).⁷ The trans-10,cis-12 is the other main isomer, which represents 1% to 10% of total CLA from food sources.⁸ According to findings from animal and human studies, CLA has beneficial effects on cancer, inflammation, body composition, diabetes and cardiovascular disease.⁹ Nowadays, most research is focused on CLA because of its anti-inflammatory and anticancer properties. The findings of in vitro and in vivo studies indicate that CLA has anti-inflammatory effects, for example, reducing colonic inflammation¹⁰ and modulating the production of cytokines and prostaglandins.^11,12 In humans, CLA has modest anti-inflammatory effects and improves immune function.¹³ The results of another clinical trial showed that 6 g of CLA mixture per day in patients with Crohn’s disease suppressed the production of proinflammatory cytokines.¹⁴ Furthermore, results from animal studies showed that CLA supplementation inhibited colon cancer incidence, progression, and metastasis.^15-17 Also, several in vitro studies have demonstrated that CLA supplementation inhibited metastasis and immigration of human colon cancer cell line Colo320¹⁸ and SW480 cells¹⁷ by different mechanisms. Recently, Grądzka et al¹⁹ reported that C9,t11-CLA increased sensitivity of HT-29 cells to X radiation. Also, Cho et al²⁰ found that t10,c12-CLA isomer inhibited HT-29 cells growth via the induction of G1 cell cycle arrest. The results of another study indicated that the t10,c12 isomer has more inhibitory effects than the c9,t11 isomer on colorectal cancer proliferation.²¹

Anti-inflammatory properties of CLA have been investigated less in human studies, and to the best of our knowledge, there is no published article about the effect of mixed CLA supplementation in cancer patients. Therefore, we evaluated the effect of CLA supplementation on serum inflammatory factors and matrix metalloproteinase (MMP) enzymes as biomarkers of angiogenesis and tumor invasion, and serum liver enzyme levels in rectal cancer patients undergoing CRT.

Methods and Materials

This randomized, double-blind, placebo-controlled pilot study was approved by the Ethics Committee of Tabriz University of Medical Sciences and registered as an RCT study (IRCT:201012041197N9). In all, 34 volunteer patients with rectal cancer who were referred to the RT center of Imama Hospital in Tabriz were recruited. Written informed consent was obtained from all patients.

Those included were ambulatory rectal cancer patients in stage II or III (based on TNM [tumor-node–metastasis] staging), who were slated to receive standard preoperative CRT treatment. RT treatment was administered in 25 fractions of 1.8-Gy (total dose, 45 Gy) concomitant chemotherapy with 5-fluorouracil and leucovorin (intravenous bolus infusion for 5 consecutive days in weeks 1 and 5). Exclusion criteria were history of any other cancer, RT and chemotherapy treatments, underweight (BMI < 18.5 kg/m²), vitamin and mineral supplementation within the past month, diabetes, and liver, renal, or endocrine dysfunction.

Patients and those involved in doing the assessment and chemical analyses were blinded to group assignments. Patients were assigned to the CLA group (n = 16), receiving four 1000-mg capsules (providing 3 g CLA) 3 times/d (1 capsule at breakfast and dinner and 2 capsules at lunch) or the placebo group (n = 18) receiving 4 placebo capsules for 6 weeks. Placebo capsules were made up with sunflower oil and were carefully matched in appearance with that of the CLA capsules. The CLA capsules (Tonalin, Natural factor, Canada) contained 2 active isomers 18:2 c9, t11 and 18:2 t10,c12 in a 50/50 ratio. The dose of 3 g/d of CLA was chosen on the basis of previous studies carried out in healthy individuals or in those with other diseases.^13,22 The supplementation began 1 week before starting RT (loading period) and continued every day during RT.

Patients were monitored weekly for any side effects of supplementation. Compliance was assessed by a capsule count every 2 weeks. Patients who consumed less than 90% of the planned number of capsules were excluded from the study. At the onset of the study, height was measured using a mounted tape, with the participants’ arms hanging freely by their sides, and recorded to the nearest 0.5 cm. After ensuring that they were barefoot and wore light clothing, their weight was recorded to the nearest 0.1 kg with a Seca scale. BMI was calculated by dividing weight (in kilograms) by the square of height (in meters).

Before and after intervention, blood samples were collected after an overnight fast of 12 hours. The serums of patients were kept at −80°C until biochemical analyses. Serum TNF-α, IL-1β, IL-6, and high-sensitivity C-reactive protein (hsCRP) were measured by platinum enzyme-linked immunosorbent assay (ELISA) kits (Bender MedSystem eBioscience, Vienna, Austria) and immunoturbidimetric method. Serum MMP-2 and MMP-9 were assessed by ELISA kits (Boster, China, and Bender MedSystem eBioscience, Vienna, Austria). Serum liver enzyme levels (alkaline phosphatase [ALP], alanine transaminase [ALT], and aspartate transaminase [AST]) were determined by the photometric method.

Statistical Analysis

The data were analyzed using Statistical Package for the Social Sciences (SPSS, version 11.5, Chicago, IL).We used the independent t test on quantitative parameters and the χ² test on qualitative variables (gender and stage of disease) to determine whether baseline characteristics differed between the CLA and placebo groups. Because all quantitative parameters had normal distributions according to the Kolmogorov-Smirnov test, data were presented as mean ± standard deviation. The paired t test was used to compare serum biochemical factors at the end of the study with baseline values. An independent t test was performed to compare changes (from preintervention to the end of study) of biochemical factors between the 2 groups. An analysis of covariance test was used to adjust the effect of confounding factors (baseline values of biochemical parameters). A P value of less than 0.05 was considered statistically significant.

Results

In this study, 34 patients were recruited. In the CLA group, 1 patient consumed less than 90% of the planned number of capsules because of forgetting to take CLA capsules regularly and was excluded from the study. In the placebo group, one patient was hospitalized and excluded from the study. Finally, statistical analysis was performed on 32 patients (CLA group, n = 15; placebo group, n = 17). There were no significant differences in baseline characteristics between the 2 groups, as presented in Table 1. The mean biochemical factors and comparison of changes between the study groups before and after intervention are indicated in Tables 2 and 3, respectively. Only the baseline values of MMP-9 were significantly (P = 0.01) different from those of the control group (Table 2).

Table 1.

Baseline Characteristics of Study Groups.

	Supplemented Group (n = 15)	Placebo Group (n = 17)	P
Age (year)	62.4 ± 15.6	58.05 ± 16.4	0.44^a
Gender, n (%)			0.75^c
Male	8 (53.38%)	10 (58.8%)
Female	7 (46.7%)	7 (41.2%)
Stage rectal cancer, n (%)^b			0.78^c
II	6 (40%)	6 (35.3%)
III	9 (60%)	11 (64.7%)
Height (cm)	161.7 ± 8.7	156.5 ± 22.9	0.42^a
Weight (kg)	66.1 ± 11.2	64.8 ± 10.1	0.73^a
BMI (kg/m²)	25.3 ± 4.2	24.6 ± 3.7	0.61^a

Independent t test.

Based on TNM staging.

χ² test.

Table 2.

The Mean Serum Biochemical Factors Before and After Intervention in Both Supplemented and Placebo Groups.^a.

	CLA Group (n = 15)			Placebo Group (n = 17)
	Before	After	P ^b	Before	After	P ^b
TNF-α (pg/mL)	55.6 ± 8.67	54.5 ± 3.06	0.68	55.8 ± 7.2	57.7 ± 4.39	0.30
IL-1β (pg/mL)	45.4 ± 5.18	45.1 ± 4.84	0.81	44.7 ± 5.3	45.7 ± 10.84	0.35
IL-6 (pg/mL)	6.1 ± 3.9	6.5 ± 4.5	0.89	8.03 ± 5.4	12.3 ± 11.8	0.19
hsCRP (mg/L)	2.5 ± 2.1	1.7 ± 1.5	0.45	2.4 ± 2.26	3.8 ± 3.2	0.06
MMP-9 (ng/mL)	578.01 ± 237.14^c	499.8 ± 297.3	0.31	839.1 ± 302.43	815.7 ± 291.72	0.80
MMP-2 (ng/mL)	21.28 ± 16.17	21.19 ± 4.69	0.98	22.26 ± 18.58	25.11 ± 20.74	0.64
ALT (U/L)	15.1 ± 11.4	15.3 ± 6.0	0.90	14.8 ± 9.04	13.6 ± 9.7	0.57
AST (U/L)	15.6 ± 3.2	15.1 ± 5.34	0.31	17.93 ± 4.25	16.2 ± 6.01	0.28
ALP (U/L)	218.7 ± 55.16	190.4 ± 36.95	0.02	221.2 ± 53.60	220.3 ± 56.29	0.94

Abbreviations: CLA, conjugated linoleic acid; TNF-α, tumor necrosis factor α; IL, interleukin 1β; hsCRP, high-sensitivity C-reactive protein; MMP, matrix metalloproteinase; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase.

Mean value ± standard deviation.

P, comparison within group by paired t test.

Baseline values were significantly different from that in the placebo group.

Table 3.

Comparison of Changes in Biochemical Factors Between the Study Groups.^a.

	CLA Group (n = 15)	Placebo Group (n = 17)	P ^b
TNF-α (pg/mL)	−1.07 (−6.64, 4.48)	1.88 (−2.66, 6.42)	0.04
IL-1β (pg/mL)	−0.32 (−3.26, 2.60)	1.00 (−3.6, 5.69)	0.60
IL-6 (pg/mL)	0.38 (−3.06, 3.83)	4.3 (−2.57, 11.20)	0.26
hsCRP (mg/L)	−0.84 (−2.95, 1.26)	1.41 (−0.069, 2.89)	0.03
MMP-9 (ng/mL)	−78.1 (−235.2, 78.88)	−23.4 (−224.3, 177.5)	0.04
MMP-2 (ng/mL)	−0.093 (−9.29, 9.10)	2.85 (−10.07, 15.78)	0.69
ALT (IU/L)	0.21 (−4.3, 4.8)	−1.13 (−5.3, 3.12)	0.60
AST (U/L)	−0.46 (−3.71, 2.78)	−1.73 (−5.09, 1.63)	0.56
ALP (U/L)	−28.3 (−52.31, −4.2)	−0.85 (−27.7, 26.05)	0.05

ANCOVA was used to adjust baseline values of biochemical parameters; mean value (95% confidence interval).

P, comparison between groups by independent t test.

After intervention, the mean serum TNF-α and IL-1β levels did not change significantly within each group (Table 2). However, significant changes (P = 0.04) of TNF-α levels (−1.07 pg/mL) were observed in the CLA group when compared with the placebo group (1.8 pg/mL; Table 3).

The mean serum IL-6 levels remained unchanged in the supplemented group but increased in the placebo group. At the end of study, the mean serum hsCRP levels reduced in the CLA group and increased in the placebo group when compared with baseline (Table 2). The reduction of hsCRP levels in the supplemented group was significant (P = 0.03) in comparison with that in the placebo group (Table 3).

After intervention, the mean serum MMP-9 reduced insignificantly in both groups. However, changes in MMP-9 levels were −78.1 ng/mL for the CLA group and −23.4 ng/mL for the placebo group, which indicated a significant difference from the placebo group (P = 0.04; Table3). This finding was also significant after adjusting for baseline values of MMP-9.

CLA supplementation reduced serum MMP-2 levels (−0.093 ng/mL) insignificantly, whereas they increased in the placebo group (2.85 ng/mL; Table 3) but not significantly.

No significant alterations were observed in ALT and AST levels within each group during the intervention (Table 2). The mean serum ALP concentration decreased significantly (P = 0.02) in the supplemented group, whereas it did not change in the placebo group (Table 2).

Discussion

A variety of cytokines (TNF-α, Il-6, Il-1β) and other factors such as NF-κB and COX2 are involved in the incidence of inflammation resulting from cancer and cancer treatments.^3,17 It has been reported that inflammation plays an important role in progression, invasion, metastasis, chemoresistance, and radioresistance of colorectal cancer.^23,24 Therefore, today, many studies are focused on the development of new anti-inflammatory therapeutic approaches for blocking their synthesis or action,²⁵ especially by dietary supplementation²⁶ such as CLA. The findings from several animal studies have documented anti-inflammatory properties of CLA in gut inflammation^27,28 and suppressing colon carcinogenesis and progression.^15,29 In pigs with bacterial-induced colitis, CLA supplementation decreased inflammatory colonic lesion developments and upregulated colonic peroxisome proliferator–activated receptor (PPAR) γ expression.²⁷ Also, Evans and colleagues²⁹ reported that CLA supplementation not only attenuated inflammation–induced colorectal cancer in mice but also decreased disease activity and suppressed colitis-related adenoma and tumor formation via activation of PPARγ. Furthermore, findings of other researchers showed that a diet containing 1% t10,c12 or 9t,t11 in rats reduced the incidence and growth of colon cancer by inducing apoptosis in colonic mucosa of rats treated with 1,2-dimethylhydrazine.^30,31 In a clinical trial, Bassaganya-Riera et al¹⁴ found that CLA supplementation (6 g/d for 12 weeks) in patients with Crohn’s disease suppressed the ability of peripheral blood T cells to produce proinflammatory cytokines and decreased disease activity. They concluded that CLA supplementation represents a new complementary treatment for gut inflammation. Taking into account the important role of inflammation in tumor formation and progression of colorectal cancer and the anti-inflammatory and anti-carcinogenic properties of CLA, we assumed that dietary CLA ameliorates inflammatory conditions and, therefore, it can prevent disease progression. In support of this hypothesis, the result of our study showed that 3 g/d CLA supplementation for 6 weeks in rectal cancer patients undergoing CRT reduced TNF-α levels significantly as compared with the placebo group. To the best of our knowledge, there is no published article about the effect of CLA supplementation on inflammatory factors and MMP enzymes in rectal cancer patients undergoing CRT. Therefore, we compared our results with in vitro and animal studies or human studies, which were conducted in healthy individuals or patients with other diseases.

The results of the study by Yang and Cook indicated that the plasma TNF-α level after lipopolysaccharide injection was suppressed in CLA-fed mice.³² Also, Rahman et al³³ demonstrated that CLA mixture supplementation for 10 weeks reduced TNF-α levels in mice. In healthy individuals, 3 CLA mixtures per day (50:50 c9,t11 CLA and t10,c12 CLA for 12 weeks) decreased TNF-α concentration.¹³ In another clinical trial, CLA (cis-9,trans-11 isomer) supplementation in those with birch pollen allergy decreased the in vitro production of TNF-α.³⁴ The finding of the present study is in agreement with results of the above-mentioned studies.^13,32-34

Another result of this study was that IL-1β decreased slightly in the CLA group while increasing in the placebo group. The findings of other studies showed that CLA supplementation had no effect on serum IL-1β concentration in healthy individuals.^35,36 Previously, Song et al¹³ reported that CLA mixture supplementation (3 g/d for 12 weeks) in healthy young individuals reduced serum IL-1β levels significantly.

In this study, CLA supplementation did not change IL-6 levels in cancer patients. Similarly, the findings of previous studies showed that CLA mixture supplementation had no effect on serum IL-6 concentration in healthy individuals^36,37 and patients with diabetes.²²

In contrast to the supplemented group, IL-6 levels increased remarkably in the placebo group, which is compatible with the study by Wang et al, who noted that serum IL-6 concentration increased in non-small-cell lung cancer during CRT.³⁸ According to our results, CLA supplementation may prevent the enhancement of IL-1β and IL-6 concentrations in patients with cancer during CRT.

At the end of the study, hsCRP levels were elevated in the placebo group. In line with the present result, Cengiz et al³⁹ observed significant elevation of CRP levels in cancer patients undergoing pelvic RT. As compared with the placebo group, CLA supplementation resulted in significant reduction of hsCRP concentration in the supplemented group. In contrast to our result, Smedman et al⁴⁰ observed significant increases in hsCRP levels in healthy individuals supplemented with 4.2 g of CLA mixture per day. On the other hand, the findings of other studies showed that CLA mixture supplementation (3 g/d and 3.2 g/d) had no effect on CRP levels among overweight³⁷ and obese individuals.⁴¹

It can be assumed that the observed difference between our results and those of previous studies regarding the effect of CLA on CRP may be a result of different doses of CLA supplementation used or the health condition of the individuals. It has been suggested that the anti-inflammatory properties of CLA are more profound in inflammatory conditions.³⁴

In this study, the observed modulatory effects of CLA supplementation on proinflammatory cytokines and hsCRP in rectal cancer patients can be ascribed to differences in the mechanisms which that are proposed by in vitro and in vivo studies. CLA isomers are reported to be a ligand for PPARγ,^42,43 which has a crucial role in the regulation of inflammation, especially inflammatory bowel disease and the incidence and metastasis of colorectal cancer.^44,45 The results of several animal studies have shown that CLA attenuated gut inflammation via activation of PPARγ.^10,27,29 Also, various CLA isomers activate PPARγ in RAW 264.7 cells and reduce the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6.⁴⁵ Therefore, CLA can improve different inflammatory conditions through activation of PPARγ. In addition, CLA reduces proinflammatory eicosanoid (PGE2, LTB4) production^46,47and attenuates the NF-κB pathway,⁴⁸ which is involved in cytokine gene expression, cell cycle activation, apoptosis, and carcinogenesis.²⁶ It seems that CLA may have a beneficial effect on inflammation, which influences initiation, progression, and metastasis of colorectal tumors⁴⁹and also tumor resistance to cancer treatments and side effects of these treatments.

In comparison with the placebo group, noticeable changes in MMP-9 and MMP-2 levels were observed in the CLA group. As far as we know, this is the first study evaluating the effect of CLA supplementation on MMP-2 and MMP-9 levels in rectal cancer patients. The present findings in rectal cancer patients are compatible with those of previous studies.^17,50 Soel et al¹⁷ noted that MMP-9 activity in SW480 human colon cancer cells was significantly suppressed by c9,t11 CLA. The results of another study showed that MMP-2 and MMP-9 synthesis and activity were inhibited by CLA in rats.⁵⁰ MMP-2 and MMP-9 are 2 important members of the MMP family that play vital roles in tumor invasion and angiogenesis; therefore, inhibition of MMP synthesis or activity in different ways can suppress tumor invasion and metastasis.⁵¹ However, more studies are needed to clarify the inhibitory mechanism of CLA on the synthesis and activity of MMP enzymes.

Taking into account that this is the first study to examine the effect of CLA supplementation in cancer patients, we determined serum liver enzymes to assess the safety of CLA in cancer patients. No significant alterations were observed in ALT and AST levels in the CLA group during the intervention. Moreover, the mean serum ALP concentrations decreased significantly in the CLA group. The mean serum liver enzyme levels were in the normal range in both the groups.

In conclusion, the results of the present study indicate that CLA supplementation (50:50 cis-9,trans-11 and trans-10,cis-12 CLA) improved TNF-α and hsCRP levels as indicators of inflammation and MMP-2 and MMP-9 enzymes as biomarkers of angiogenesis and tumor invasion. Moreover, CLA supplementation prevented the elevation of IL-1β and IL-6 concentrations as biomarkers of inflammation when compared with the placebo group in rectal cancer patients undergoing CRT.

Based on the results of previous animal studies and the present study, it seems that CLA may provide a new complementary treatment by reducing tumor invasion and resistance to cancer treatment in patients with rectal cancer. However, further studies with larger sample sizes are needed to achieve more precise results. Moreover, it would be worthwhile to study the effect of different doses of oral CLA supplementation on the production of inflammatory factors and the immune function in cancer patients.

Footnotes

Acknowledgements

The authors are grateful for the financial support of the Nutrition Research Center, Tabriz University of Medical Sciences. The authors also are deeply indebted to all patients who participated in this study.

Authors’ Note

This article was written based on a data set for the PhD thesis (Elnaz Faramarzi) registered in Tabriz University of Medical Sciences.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors are grateful for the financial support of the Nutrition Research Center, Tabriz University of Medical Sciences.

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