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The Effect of Omega-3 Polyunsaturated Fatty Acids on Human Cancer Cells: – Molecular Mechanisms Involved
Norwegian University of Science and Technology, Faculty of Medicine, Department of Laboratory Medicine, Children's and Women's Health.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fatty fish and cod liver oil have been common ingredients in the Norwegian diet for centuries. In the latter half of the 1800s the apothecary Møller produced pure cod liver oil for medicinal use. In the 1950s and 1960s, the Norwegian Professor Notevarp found that fish oil contained the omega-3 polyunsaturated fatty acid (n-3 PUFA) docosahexaenoic acid (DHA). He was also the first to describe a beneficial effect of n-3 PUFAs on cardiovascular disease. In the 1970s, the doctors Bang and Dyerberg found that Greenland Inuits had low incidence of cardiovascular disease, and related it to increased n-3 PUFA content in the blood. Some epidemiological observational studies have found that n-3 PUFAs may reduce the incidence of some cancer types like colon cancer; however, the results have not been consistent. Yet, some clinical studies, as well as several animal and cell culture experiments have shown that n-3 PUFAs have a growth inhibiting effect on some types of cancers, but the mechanisms behind this effect are unclear. The aim of the studies has been to explore the molecular mechanisms behind the growth reducing effect of n-3 PUFAs on cancer cells in vitro.

Supplementation of DHA to human colon cancer cells (SW620) in culture strongly reduced cell growth in physiological relevant concentrations, and gene expression analysis indicated changed expression of thousands of genes involved in different biological pathways. The DHA treatment resulted in stress in the endoplasmic reticulum (ER) and induction of unfolded protein response (UPR). Some of the main functions of the ER are protein folding, regulation of calcium levels, as well as synthesis of lipids and sterols. Disruption of any of these mechanisms such as accumulation of unfolded/misfolded proteins may result in ER stress and induction of UPR. This stress response is characterized by phosphorylation of the eukaryotic translation initiation factor 2 α (eIF2α-P) which was induced as early as 3 hours after DHA administration. Increased eIF2α-P arrests protein translation, reduces the cell cycle regulator cyclin D and induces cell cycle arrest, allowing the cells to repair the damage/restore ER homeostasis. However, if damage is too severe, the cell death machinery (apoptosis) will be activated.

The gene expression results also indicated changes in calcium and cholesterol homeostasis. Addition of DHA to SW620 cells increased the intracellular calcium level, probably as a result of calcium release from the ER and uptake of exogenous calcium. Cellular cholesterol synthesis is regulated by e.g. sterol regulatory element binding protein 2 (SREBP2). Despite active SREBP2, only some of its target genes were up-regulated by DHA supplementation. New synthesis of cholesterol and incorporation of newly synthesized cholesterol into cholesteryl esters were down-regulated, while total cholesterol levels were not markedly affected. Changes in calcium homeostasis were further investigated using a human leukemia cell line (HL-60) and a mutant HL-60 clone (E2R2) which is resistant towards the calcium homeostasis-disturbing chemical econazole. The HL-60 cells were strongly growth inhibited by addition of eicosapentaenoic acid (EPA), while the growth of E2R2 cells was only moderately affected. Gene expression analysis and induction of eIF2α-P in EPA-treated HL-60 cells indicated changes in calcium homeostasis and induction of UPR in the mother cell line, while there was no evidence of ER stress and UPR in E2R2 cells.

Administration of DHA to SW620 cells also resulted in changed expression of several genes and proteins involved in regulation of cell cycle and cell death. Protein levels of survivin, livin and nuclear factor κ B (NFκB) were reduced, which is interesting since they are induced in different tumors and are known targetproteins of chemotherapy. Whether n-3 PUFAs have a place as supplements to clinical cancer treatment depends, however, on further research within this field.

Place, publisher, year, edition, pages
NTNU, 2012.
Dr.ingeniøravhandling, ISSN 0809-103X ; 2012:172Dissertations at the Faculty of Medicine, ISSN 0805-7680 ; 555
URN: urn:nbn:no:ntnu:diva-17173ISBN: 978-82-471-3636-2 (printed ver.)ISBN: 978-82-471-3637-9 (electronic ver.)OAI: diva2:545447
Public defence
2012-06-14, 00:00
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2012-08-20Bibliographically approved
List of papers
1. DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis.
Open this publication in new window or tab >>DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis.
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2008 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 49, no 10, 2089-100 p.Article in journal (Refereed) Published
Abstract [en]

Polyunsaturated fatty acids (PUFAs) are normal constituents of the diet, but have properties different from other fatty acids (e.g., through generation of signaling molecules). N-3 PUFAs reduce cancer cell growth, but no unified mechanism has been identified. We show that docosahexaenoic acid (DHA; 22:6 n-3) causes extensive changes in gene expression patterns at mRNA level in the colon cancer cell line SW620. Early changes include unfolded protein response (UPR) and increased levels of phosphorylated eIF2alpha as verified at protein level. The latter is considered a hallmark of endoplasmic reticulum (ER) stress and is abundantly present already after 3 h. It may coordinate many of the downstream changes observed, including signaling pathways for cell cycle arrest/apoptosis, calcium homeostasis, cholesterol metabolism, ubiquitination, and proteasomal degradation. Also, eicosapentaenoic acid (EPA), but not oleic acid (OA), induced key mediators of ER stress and UPR at protein level. Accumulation of esterified cholesterol was not compensated for by increased total levels of cholesterol, and mRNAs for cholesterol biosynthesis as well as de novo synthesis of cholesterol were reduced. These results suggest that cytotoxic effects of DHA are associated with signaling pathways involving lipid metabolism and ER stress.

urn:nbn:no:ntnu:diva-17171 (URN)10.1194/jlr.M700389-JLR200 (DOI)18566476 (PubMedID)
Free via Creative Commons: CCAvailable from: 2012-08-20 Created: 2012-08-20 Last updated: 2012-08-20Bibliographically approved
2. The antiproliferative effect of EPA in HL60 cells is mediated by alterations in calcium homeostasis.
Open this publication in new window or tab >>The antiproliferative effect of EPA in HL60 cells is mediated by alterations in calcium homeostasis.
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2009 (English)In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 44, no 2, 103-13 p.Article in journal (Refereed) Published
Abstract [en]

Studies show that n-3 polyunsaturated fatty acids (PUFA) inhibit proliferation and induce apoptosis in cancer cells. Recent reports indicate that this effect is due to activation of the unfolded protein response (UPR). However, what causes this activation has been unclear. We examined the effects of eicosapentaenoic acid (EPA) on the human leukemia cell line HL60 and the econazole (Ec) resistant HL60 clone E2R2. Ec depletes Ca(2+) from the ER and blocks Ca(2+) influx in mammalian cells, leading to activation of the UPR and apoptosis. EPA inhibited growth of HL60 cells strongly, while E2R2 cells were much less affected. Gene expression analysis of HL60 cells revealed extensive changes in transcripts related to the ER homeostasis, Ca(2+)-homeostasis and cell cycle/apoptosis. Protein levels of phosphorylated eIF2alpha, a selective translation inhibitor and UPR hallmark, activating transcription factor 4 (ATF4) and sequestosome-1 were moderately increased, whereas the cell cycle/progression protein cyclin D1 was decreased in HL60. In contrast, EPA concentrations that strongly inhibited and caused activation of the UPR in HL60 cells had no effect on the expression level of these UPR markers in E2R2 cells. Given that the only known difference between these cells is Ec-resistance, our results strongly suggest that the inhibitory effect of EPA on HL60 cells is initially meditated through alterations of the Ca(2+)-homeostasis followed by activation of the UPR.

urn:nbn:no:ntnu:diva-5533 (URN)10.1007/s11745-008-3263-5 (DOI)19020915 (PubMedID)
Available from: 2009-06-03 Created: 2009-06-03 Last updated: 2013-07-12Bibliographically approved
3. DHA alters expression of target proteins of cancer therapy in chemotherapy resistant SW620 colon cancer cells.
Open this publication in new window or tab >>DHA alters expression of target proteins of cancer therapy in chemotherapy resistant SW620 colon cancer cells.
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2010 (English)In: Nutrition and Cancer, ISSN 0163-5581, E-ISSN 1532-7914, Vol. 62, no 5, 611-21 p.Article in journal (Refereed) Published
Abstract [en]

Diets rich in n-3 polyunsaturated fatty acids (PUFAs) have been associated with a reduced risk of several types of cancer. Recent reports have suggested that these PUFAs enhance the cytotoxic effect of cancer chemoradiotherapy. The effect of docosahexaenoic acid (DHA) on key cell cycle regulators and target proteins of cancer therapy was investigated in the human malign colon cancer cell line SW620. Cell cycle check point proteins such as p21 and stratifin (14-3-3 sigma) increased at mRNA and protein level, whereas cell cycle progression proteins such as cell division cycle 25 homolog and cyclin-dependent kinase 1 decreased after DHA treatment. Protein levels of inhibitors of apoptosis family members associated with chemotherapy resistance and cancer malignancy, survivin and livin, decreased after the same treatment: likewise the expression of NF-kappaB. Levels of the proapoptotic proteins phosphorylated p38 MAPK and growth arrest-inducible and DNA damage-inducible gene 153/C/EBP-homologous protein (CHOP) increased. The results indicate that DHA treatment causes simultaneous cell cycle arrest in both the G1 and G2 phase. In conclusion, DHA affects several target proteins of chemotherapy in a favorable way. This may explain the observed enhanced chemosensitivity in cancer cells supplemented with n-3 PUFAs and encourage further studies investigating the role of n-3 PUFAs as adjuvant to chemotherapy and radiotherapy in vivo.

urn:nbn:no:ntnu:diva-17172 (URN)10.1080/01635580903532366 (DOI)20574922 (PubMedID)
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2012-08-20Bibliographically approved

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