Original Article
Biomedical
Polyunsaturated Fatty Acids Differentially Modulate Cell Proliferation and Endocannabinoid System in Two Human Cancer Lines

https://doi.org/10.1016/j.arcmed.2017.01.009Get rights and content

Background and Aims

Evidence suggests that quantity and quality of dietary polyunsaturated fatty acids (PUFAs) play a role in the development of cancer. However, the mechanisms involved in this interaction(s) are not clear. Endocannabinoids are lipid metabolites known to have growth modulatory actions. We studied the effect of supplementation with PUFAs ω-6 and ω-3 (essential fatty acids, EFAs), saturated and monounsaturated fatty acids (non-EFAs) on the growth of tumor cells and modifications in their endocannabinoid content.

Methods

Cell cultures of human glioblastoma (T98G) and breast cancer (MCF7) were supplemented with 50 or 100 mmol EFAs and non-EFAs for 72 h. Cell proliferation was then determined by MTT, anandamide (AEA) levels by HPLC, total fatty acids profiles by GLC, CB1 receptor expression by WB and FAAH activity by spectrophotometric method.

Results

Fatty acids profile reflected the incorporation of the lipids supplemented in each assay. Arachidonic acid (EFA ω-6) supplementation increased AEA levels and inhibited the growth of T98G, whereas palmitic acid (non-EFA) enhanced their proliferation. In breast cancer (MCF7) cells, eicosapentaenoic acid (EFA ω-3) reduced and oleic acid (non-EFA) enhanced their proliferation. CB1 expression was higher in T98G and no differences were observed in FAAH activity.

Conclusions

The growth of tumor cells can be differentially modulated by fatty acids and, at least in part, can be attributed to their ability to act on the components of the endocannabinoid system.

Introduction

Clinical and experimental trials indicated some correlation between quantity and quality of dietary fat intake and the incidence and growth of cancer 1, 2. Epidemiologic studies reported a positive correlation with saturated dietary fat intake with an increase in cancer risk for breast, colon, bladder and prostate 1, 2, 3, 4, 5. On the other hand, increased consumption of ω-3 fatty acids (FA) was found to be related to a low incidence of cancer (1) and the same was observed for high dietary intake of essential fatty acids (EFA) 3, 4. Data from studies performed in animal tumor models and experimental treatments of humans show that ω-6 PUFAs can inhibit cell growth and/or promote cell death in central nervous system (CNS) tumors 5, 6, 7, 8. The inhibitory effects of PUFAs may be linked to their ability to serve as precursors to several bioactive lipids that have anti-tumor activities. Some of these bioactive lipids derived mainly from ω-6 PUFAs are eicosanoids and endocannabinoids (EC) (9).

The relative proportions and quality of PUFAs in cell membranes as well as cell type are the primary factors that determine the type and quantity of eicosanoids and EC that will be formed in a given situation. Intriguingly, the hydrolytic release of ω-3 and ω-6 PUFAs from phospholipids appears to occur indiscriminately (6). Since the main PUFA substrate for EC in cell membrane is AA (20:4 ω6), most of the ligands are endogenous AA-derived lipids. These ECs are released in the brain and many other tissues and have been implicated in a wide array of physiological and pathological processes including cancer 10, 11, 12, 13, 14, 15. The two major endogenous cannabinoid ligands are arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG).

The biological effects of EC are due to their binding and activation of the cannabinoid receptors (CBR), called CB1 and CB2, a family of transmembrane G-protein-coupled receptors, densely distributed throughout the autonomic and central nervous system (CNS), immune system and other human tissue (11). Binding of CB1 or CB2 receptors to endogenous ligands can cause excitation or inhibition of certain cellular activities, depending on the enzyme cascades linked to the receptors 12, 13. It is likely that EC arising from other PUFAs (other than AA) or even monounsaturated FAs may have different activities, although this has not yet been fully explored.

EC are not stored as in the case of conventional water-soluble neurotransmitters, but are rather rapidly synthesized from PUFAs of the cell membrane (9). Once extracellularly released by a putative EC transporter, they are quickly degraded by fatty acid amide hydrolase (FAAH), which cleaves AEA and 2-AG into AA and ethanolamide and into AA and glycerol, respectively (16).Thus, EC are akin to eicosanoids and are very short-lived substances, a property very useful for regulatory mechanisms in almost all body tissues 9, 12.

In vitro and in vivo stimulation of CBR by EC ligand influence intracellular events that play a significant role in the proliferation and apoptosis of a wide variety of cancer cells, thereby leading to antitumor effects 10, 17, 18, 19. Studies in mammals (rat, mouse and pigs) showed that dietary PUFAs influence tissue and cellular concentration of EC 20, 21, 22, 23. These data indicate that dietary PUFAs can alter the levels and types of EC to a significant extent mainly due to the availability and release of the precursor AA or other PUFAs from the membrane phospholipids (12). Evidence shows that high availability of different kinds of FA could have pro- or anti-tumor effects and these effects could at least be partially mediated by EC.

Human tumor cell lines with different origins have been reported that express CBR (19) as glioblastoma T98G, an astrocytic-derived line and human breast cancer MCF7, an epidermal ductal strain. The level of expression of receptors, agonists and related enzymes in these different cell lines may lead to diverse physiological responses after activation of the ECS.

Our previous epidemiological and experimental studies revealed the modulatory effects of FA, particularly PUFAs, on cancer process 2, 3, 24, 25, 26, 27. In an extension of these studies, we evaluated supplementation of EFAs (precursors of EC metabolites) and non-EFAs to two human tumors cell lines with different origins, physiology, and metabolic responses. Data obtained confirm that the kind of FA used differentially affects cell growth and viability in vitro. This effect could be due, at least in part, to ECS activation in T98G cells, whereas in the MCF7 line the observed response was independent of endocannabinoids.

Section snippets

Materials and Experimental Procedures

The cell lines were purchased from American Type Culture Collections (ATCC, Rockville, MD). Free and methyl esters of fatty acids were obtained from Nu Check (USA). Cell culture medium was obtained from Gibco (USA). Decanoyl p-nitroaniline, URB597, Rimonabant (SR141716), MTT kit and Anandamide standards were purchased from Cayman Chemicals (USA). Antibodies and Western blot molecular weight standards were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma-Aldrich (St. Louis, MO).

Determination of Profiles of FA in Tumoral Cell Lines

The FA profile of analyzed cells reflected the incorporation of the FA added to the culture medium or its derivatives, and the different treatments showed quantitative and qualitative modifications of these profiles in both cell lines. The total percentages of FAs grouped as saturated, monounsaturated and PUFAs of the ω6 and ω3 families were determined. Using these data, the ratios saturated/PUFAs and ω6/ω3 were calculated, in which the difference in the cell profiles under different treatments

Discussion

Effect of FAs on cancer cell proliferation depends of the type of FA supplemented and the strain/type of cell lines and/or tumor variety. Depending on the FAs that are being tested, they may either enhance or inhibit tumor growth. In general, it is reported that high-fat diets, especially saturated FA, have a cancer-promoting action 1, 3, 4. Epidemiological and experimental studies revealed that optimum amounts of ω-6 and ω-3 PUFAs could prevent or inhibit tumor growth. It is also known that an

Acknowledgments

This work was supported by the funds provided by CONICET PIP 112-200801-03014, SECYT-UNC 05/H297, SECYT-UNLAR 063-2011 (Argentina). We are grateful to Dr. Fulvio Magni for providing CBR antibodies. We are indebted to Mrs. Ester Sticcotti for English technical revision. U.N. Das received a Ramalingaswami Fellowship of the Department of Biotechnology, India during the period of this study.

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