|Ahead of print publication
Plasma fatty acids composition and estimated delta desaturases activity in women with breast cancer
A Preethika1, Suchetha N Kumari1, Jayarama Shetty2, Vijith Shetty2
1 Department of Biochemistry, K S Hegde Medical Academy, Mangalore, Karnataka, India
2 Department of Oncology, K S Hegde Medical Academy, Mangalore, Karnataka, India
|Date of Submission||25-Apr-2019|
|Date of Decision||13-Oct-2019|
|Date of Acceptance||01-Dec-2019|
|Date of Web Publication||13-Oct-2020|
K S Hegde Medical Academy, Mangalore - 575 018, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Fatty acids (FAs) are the vital constituents of membrane structures. De novo synthesis of FAs includes an enzymatic complex of FA synthase and delta desaturases. These enzymes are overexpressed in tumors, and inhibition of these enzymes is gaining interest. Our aim was to determine if delta desaturase activities are altered in breast cancer (BC) cases and if altered whether delta desaturase activities differ among BC genotypes.
Materials and Methods: In this observational comparative study, 50 women with BC and 30 control women were recruited for the study. Gas chromatography-flame ionization detector was used to measure the plasma FA levels. Desaturase activities were assessed as product-to-precursor FA ratios. The Wilcoxon signed-rank test was used to compare between two groups, and P ≤ 0.05 was considered as statistically significant.
Results: The FA analysis revealed higher levels of monounsaturated FAs (MUFAs) and linolenic acid metabolites (C18:3n-6, C20:4n-6) in BC patients, whereas C20:5n-3 was higher in controls. The Delta 9 desaturase (D9D) and D6D were higher in BC cases suggesting greater conversion saturated FA to MUFA and linoleic acid to its metabolites. D9D-16 activity was statistically significant (P = 0.03) in BC women, particularly in estrogen-receptor-positive patients.
There is limited evidence to substantiate the link between diet and cancer. The current study showed there is an altered lipid desaturase activity. Nutritional intervention and drugs that target the FA pathway may provide a new approach to prevent and treat BC.
Keywords: Breast cancer, delta desaturases, estrogen receptors, fatty acids
|How to cite this URL:|
Preethika A, Kumari SN, Shetty J, Shetty V. Plasma fatty acids composition and estimated delta desaturases activity in women with breast cancer. J Can Res Ther [Epub ahead of print] [cited 2020 Oct 28]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=298073
| > Introduction|| |
Breast cancer (BC) is one of the most common cancers among women leading to mortality worldwide. BC has two genotypes with discrete possibility and outcome; estrogen-receptor-positive (ER + ve) and estrogen-receptor negative (ER − ve) BCs. The pathogenesis of BC is influenced by the estrogen, where the estrogen signaling pathway is influenced by genetic factors and diet. Various studies show the role of dietary fatty acids (FAs) in BC risk., Saturated FAs (SFAs), monounsaturated FAs (MUFAs), and polyunsaturated FAs (PUFAs) are found to be associated with BC risk.,
The formation of SFAs and MUFAs is the basic step in FA synthesis. Since these are fundamental for cellular activities, the balance between SFA and MUFA has to be maintained. Delta 9 desaturase (D9D) or Stearoyl CoA desaturases (SCD) is the key regulator catalyzing the introduction of the first double bond in the cis-delta-9 position.
Omega-3 (n-3) FAs include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). Linoleic acid (LA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), and arachidonic acid (AA) are the omega 6 (n-6) FAs. Both n-3 and n-6 FAs are important for development and biological functions in humans. Specific PUFAs have anticancer effects. Omega-3 PUFAs have shown suppressive activity in cancer cells, whereas n-6 PUFAs inhibit apoptosis in cancer cells.,
Omega FAs are absorbed directly from the food or synthesized by the essential FAs LA and ALA, which involves a cascade of desaturation and elongation. SCD, delta 6-desaturase (D6D), and delta 5-desaturase (D5D) catalyze the endogenous synthesis of PUFAs. D6D is the rate-limiting step in the conversion of LA to arachidonic acid and ALA to EPA. D5D enzyme metabolizes DGLA to arachidonic acid., N-3 and n-6 FAs strive for the same enzymes and produce eicosanoids with contrary effects; it has been hypothesized that the balance of the n-3 and n-6 FAs may be imperative in the BC pathogenesis.
FA ratios are used as alternate measures of desaturase activities that might be at least as important for metabolic changes as are individual FAs for each and have been related to cancer. Our objective was to assess FA desaturases activity among the BC cases and controls and to find if these variables differ based on the estrogen receptor (ER) types in BC.
| > Materials and Methods|| |
The use of human subjects for the study was approved by the institutional review board. From January 2018 to December 2018, 50 histopathologically proven BC patients and 30 control women who attended OPD for general health check-up between the age group of 25–60 years were recruited for the study. Each participant was recruited after obtaining informed consent. Demographic data such as age, body mass index, food habits, alcohol and smoking status, family history, and obstetric history were noted. Molecular subtype ER status was noted in BC patients.
Fatty acid analysis
The plasma FA composition was performed in the 2 mL of blood samples collected in an ethylenediaminetetraacetic acid vacutainer. Samples were stored at −20°C until further analysis. Lipid transesterification of stored plasma to FA methyl esters (FAMEs) was performed according to the modified protocol of Metcalfe et al., and described briefly below. Plasma samples were thawed, and one microgram in one microliter (1 mg/ml) of methyl heptadecanoate (C17:0 methyl ester; Sigma-Aldrich) was added to the plasma samples as an internal standard. Extraction of the total plasma FAs by the hydrolysis of esters and then derivatization of esters under alkaline conditions in 14% boron trifluoride-methanol for 5 min at 100°C to form FAMEs.
FAs were measured in gas chromatography (GC). FAMEs extracted were analyzed on a 7820A Agilent GC-flame ionization detector (FID system) equipped with JandW DB-23 high-quality column. Individual FAs were identified by the elution times compared with the relative FA standards. FAs were calculated by their comparative abundance to the internal standard added. The amount of individual FA was calculated as the percentage of the total FA concentration within each sample.
The FA profile considered 12 FAs, representative of three families: SFAs, C16:0 palmitic acid; C18:0 stearic acid; MUFAs, C16:1 palmitoleic acid; C18:1n-9c oleic acid; omega 3 FAs, C18:3 ALA; C20:5 EPA; C22:5 DPA; C22:6 DHA; omega 6 FAs, C18:2 LA; C18:3 GLA; C20:3 DGLA; C20:4 arachidonic acid.
Microsoft ® Office Excel 2010 was used for the compilation of the data. Data were analyzed in SPSS software SPSS version 16.0 (IBM Corp, USA). Variables are expressed in the mean percentage. The Wilcoxon signed-rank test was used to compare two groups. P ≤ 0.05 was considered as statistically significant.
Considering the FAs, different desaturase activities were calculated:
- D5D activity = C20:4 (arachidonic acid)/C20:3 (DGLA)
- D6D activity = C18:3 (GLA)/C18:2 (LA)
- D9D-16 activity = C16:1 (Palmitoleic acid)/C16:0 (Palmitic acid)
- D9D-18 activity = C18:1 (oleic acid)/C18:0 (Stearic acid).
| > Results|| |
[Table 1] shows the baseline characteristics of control and BC cases. [Figure 1] shows the distribution of FAs in 80 samples. SFA levels were high in BC cases compared to the control group. Plasma concentration of MUFAs, oleic acid levels were higher, and stearic acid levels were lesser in the control group than in the case. Among the omega-6 FAs, cancer patients had a higher percentage of LA, which differed significantly (P = 0.01) compared to controls (18% vs. 13%). A statistically significant difference between the mean percentage EPA levels of cancer cases (1.2%) and control subjects (2.6%) was observed in our study (P = 0.049). No other significant differences in omega-3 and omega-6 FAs were observed between cases and controls.
|Figure 1: Fatty acid profile in breast cancer cases and controls. Figure represents the mean percentage of individual fatty acids, saturated fatty acids, C1 palmitic acid; C18:0 stearic acid; monounsaturated fatty acids, C16:1 palmitoleic acid; C18:19 oleic acid; Omega 3 FAs, C18:3 alpha-linolenic acid; C20:5 eicosapentaenoic acid; C22:5 docosapentaenoic acid; C22:6 docosahexaenoic acid; omega 6 FAs, C18:2 linoleic acid; C18:3 Gamma-linolenic acid; C20:3 dihomo-gamma linolenic acid; C20:4 arachidonic acid. **P < 0.03, *P < 0.05|
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BC cases had higher ratios of D5D (GLA: LA), D9D-16 (POA: PA), and D9D-18 (OA: SA) and lower D6D (AA: DGLA) activity. As shown in [Figure 2], the D9D-16 activity was statistically significant in BC cases compared with the control group (1.45: 0.3; P = 0.03).
|Figure 2: Delta desaturase activity in breast cancer cases and control groups. Delta desaturase activity is expressed as the ratio of the sum of individual fatty acids (**P ≤ 0.03). D5D activity = C20:4 (arachidonic acid)/C20:3 (Dihomo-gamma linolenic acid). D6D activity = C18:3 (Gamma linolenic acid)/C18:2 (Linoleic acid). Delta 9 desaturase-16 activity = C16:1 (Palmitoleic acid)/C16:0 (Palmitic acid). Delta 9 desaturase-18 activity = C18:1 (oleic acid)/C18:0 (Stearic acid)|
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We also considered the differences in delta desaturase activity between the molecular subtypes among the BC cases. [Figure 3] shows the desaturase activity ratio in ER +ve and ER −ve patients. D5D activity was high in ER −ve BC patients. The ratio D6D activity was very similar in both molecular subtypes (ER +ve: ER −ve = 0.04: 0.03). ER +ve BC cases had higher D9D activities. D9D-16 was significantly increased in ER +ve patients versus ER −ve patients (2.2: 0.2, P = 0.03).
|Figure 3: Delta desaturase activity in estrogen receptor positive (ER +ve) and estrogen receptor negative (ER −ve) molecular subtypes.(n = 30 for ER +ve samples and n = 20 for ER −ve samples) **P = 0.03. D5D activity = C20:4 (arachidonic acid)/C20:3 (Dihomo-gamma linolenic acid). D6D activity = C18:3 (Gamma linolenic acid)/C18:2 (Linoleic acid). Delta 9 desaturase-16 activity = C16:1 (Palmitoleic acid)/C16:0 (Palmitic acid). Delta 9 desaturase-18 activity = C18:1 (oleic acid)/C18:0 (Stearic acid)|
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| > Discussion|| |
The complex changes in the metabolism of carbohydrates, proteins, and lipids are essential for tumor cell growth and proliferation. Cell proliferation, apoptosis, and anoikis in healthy and cancer cells depend on the metabolism, regulation, and remodeling of the lipids. The relevance of SFA and MUFAs in lipid de novo synthesis and overexpression of the activity of delta desaturases are of interest. We found higher levels of SFA and MUFA in cancer cases compared to the control group except for oleic acid. Since oleic acid is the common FA available from food, it is eliminated from having the food influence. SCD1 or D9D activity was more in the cancer group, which is well recognized in cancer.,
The high activity of SCD1 stimulates the conversion of SFA to MUFA. It plays a key role in augmenting the mitogenic and tumorigenic capacity of tumor cells by activating lipogenesis and AKT (protein kinase B) pathway thereby inhibiting apoptosis and enhancing cell growth. Our study was consistent with the studies of Amezaga et al., Pala V et al., and Mikirova et al. The increase in the D9D activity appears to be metabolic distresses found in metabolic disorders such as diabetes, obesity, where D9D may be associated with BC which can be a common link between these disorders.
The estimation of specific desaturase activity helps to estimate the pathway activity also. Lower activity of D5D observed in cancer patients compared to controls relates to the significant increase of the DGLA, pro-inflammatory omega-6 FA. We also observed the increase in D6D activity, which corresponds to the significant increase of linoleic metabolite GLA. Increased delta 6 and delta 9 activities were also observed in ER +ve patients, whereas delta 5 was activity was more in ER −ve patients. Increased D6D activity in ER +ve was in contrast to the study by Pender-Cudlip et al. However, the sample size of ER –ve was small. Further study with a large population is essential to substantiate the results.
PUFAs are linked with pro-inflammatory and anti-inflammatory signaling. Both omega-3 and omega-6 FAs play an important role in cellular signaling, cellular interaction, and membrane fluidity. In our study, the case group with high amounts of omega-6 LA and low amounts of omega-3 EPA was observed when matched with a control group, which persisted significantly. LA and ALAs are essential FAs. Metabolites produced from the precursor linoleic mainly dependent on the food intake, and therefore, it can be considered as the metabolic indicator in BC patients.
Omega-3 FAs are anti-inflammatory and induce apoptosis. Omega-6 FAs produce prostaglandins E2, which is important for cancer growth. It inhibits apoptosis, increases invasiveness  and angiogenesis  in the tumor through different pathways such as NF-kB,, MAP kinase, and induces COX2 mRNA. This pro-inflammatory hoop can be interjected with omega-3 PUFAs which compete with eicosanoids synthesis enzymes and give rise to metabolites which resolves the inflammation.
Interestingly, both omega FAs compete for the same delta desaturase enzymes, and ALA is predominantly preferred than for LA.,,, Increased LA clearly showcases the increased intake of omega-6 FAs in our diet. On increasing the intake of omega-6 FAs increase the risk of cancer as there is a competition for the enzymes. D6D activities are elated in omega-6 pathway and inhibitors of the D6D desaturases may be therapeutically potential. Our study can be beneficial in explaining the importance of diet in cancer management and prevention.
| > Conclusion|| |
Lipogenic pathways, along with molecular signaling pathways, sense their status in cancer and receiving better consideration. In this study, we have shown that D5D activity is lowered, and D6D and D9D activities were elevated in BC cases which are possibly significant biomarkers to screen, predominantly in women who are at risk of developing cancer. Desaturase activity among BC genotypes also varied among the cases. Therapeutical intervention to address the desaturase activity, aiming at anti-inflammatory FAs rich food for BC prevention should be focused and recommendations could also be made on the genotype basis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]