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DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis.

Jakobsen CH, Størvold GL, Bremseth H, Follestad T, Sand K, Mack M, Olsen KS, Lundemo AG, Iversen JG, Krokan HE, Schønberg SA - J. Lipid Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology (NTNU), Erling Skjalgssons gate 1, N-7006 Trondheim, Norway.

ABSTRACT
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.

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ER stress signaling and UPR in response to n-3 polyunsaturated fatty acids (PUFAs) and oleic acid (OA). Western blot analysis of proteins involved in ER stress signaling and UPR (ATF4: nuclear extracts; cyclin D1, eIF2α: cytoplasmic extracts) in SW620 cells treated with complete growth medium supplemented with either OA (70 μM), DHA (35 μM), eicosapentaenoic acid (EPA, 70μM), or ethanol (control media, C) for 6 and 24 h. β-actin (cytoplasmic extracts), lamin C (nuclear extracts), or total eIF2α was used as a control for equal protein loading. One representative blot is shown. The blots were quantified and intensities normalized relative to loading control. The numbers under the blots represent mean fold change (SD) relative to control for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).
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fig3: ER stress signaling and UPR in response to n-3 polyunsaturated fatty acids (PUFAs) and oleic acid (OA). Western blot analysis of proteins involved in ER stress signaling and UPR (ATF4: nuclear extracts; cyclin D1, eIF2α: cytoplasmic extracts) in SW620 cells treated with complete growth medium supplemented with either OA (70 μM), DHA (35 μM), eicosapentaenoic acid (EPA, 70μM), or ethanol (control media, C) for 6 and 24 h. β-actin (cytoplasmic extracts), lamin C (nuclear extracts), or total eIF2α was used as a control for equal protein loading. One representative blot is shown. The blots were quantified and intensities normalized relative to loading control. The numbers under the blots represent mean fold change (SD) relative to control for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).

Mentions: We have previously shown that EPA has an antiproliferative effect on SW620 cells, although to a lesser extent than DHA, while OA has no effect (5). Key mediators of ER stress and UPR were also induced at protein level by EPA, but not OA, at equal molar concentrations (Fig. 3). DHA-treatment (70 μM) of SW620 cells induced phosphorylation of eIF2α already after 3 h (Fig. 2A), while a weaker response is observed after treatment with EPA (70 μM) or half molar concentrations of DHA (35 μM) first at 24 h (Fig. 3). A similar time- and concentration-dependent response is also observed for the induction of ATF4 (Fig. 3). Also, EPA (70 μM) and DHA (35 and 70 μM) reduced the level of cyclin D1 after 24 h (Fig. 2B, C and Fig. 3). After 6 h, only DHA (70 μM) reduced the level of cyclin D1 significantly, whereas DHA (70 μM) and EPA (70 μM) both reduced cyclin D1 substantially and with comparable effects after 24 h. OA did not affect the proliferation of SW620 cells. However, OA reduced the level of cyclin D1 after 24 h, although to a much lesser extent (Fig. 3). This reflects the differences in the antiproliferative effect between the PUFAs observed earlier.


DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis.

Jakobsen CH, Størvold GL, Bremseth H, Follestad T, Sand K, Mack M, Olsen KS, Lundemo AG, Iversen JG, Krokan HE, Schønberg SA - J. Lipid Res. (2008)

ER stress signaling and UPR in response to n-3 polyunsaturated fatty acids (PUFAs) and oleic acid (OA). Western blot analysis of proteins involved in ER stress signaling and UPR (ATF4: nuclear extracts; cyclin D1, eIF2α: cytoplasmic extracts) in SW620 cells treated with complete growth medium supplemented with either OA (70 μM), DHA (35 μM), eicosapentaenoic acid (EPA, 70μM), or ethanol (control media, C) for 6 and 24 h. β-actin (cytoplasmic extracts), lamin C (nuclear extracts), or total eIF2α was used as a control for equal protein loading. One representative blot is shown. The blots were quantified and intensities normalized relative to loading control. The numbers under the blots represent mean fold change (SD) relative to control for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533412&req=5

fig3: ER stress signaling and UPR in response to n-3 polyunsaturated fatty acids (PUFAs) and oleic acid (OA). Western blot analysis of proteins involved in ER stress signaling and UPR (ATF4: nuclear extracts; cyclin D1, eIF2α: cytoplasmic extracts) in SW620 cells treated with complete growth medium supplemented with either OA (70 μM), DHA (35 μM), eicosapentaenoic acid (EPA, 70μM), or ethanol (control media, C) for 6 and 24 h. β-actin (cytoplasmic extracts), lamin C (nuclear extracts), or total eIF2α was used as a control for equal protein loading. One representative blot is shown. The blots were quantified and intensities normalized relative to loading control. The numbers under the blots represent mean fold change (SD) relative to control for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).
Mentions: We have previously shown that EPA has an antiproliferative effect on SW620 cells, although to a lesser extent than DHA, while OA has no effect (5). Key mediators of ER stress and UPR were also induced at protein level by EPA, but not OA, at equal molar concentrations (Fig. 3). DHA-treatment (70 μM) of SW620 cells induced phosphorylation of eIF2α already after 3 h (Fig. 2A), while a weaker response is observed after treatment with EPA (70 μM) or half molar concentrations of DHA (35 μM) first at 24 h (Fig. 3). A similar time- and concentration-dependent response is also observed for the induction of ATF4 (Fig. 3). Also, EPA (70 μM) and DHA (35 and 70 μM) reduced the level of cyclin D1 after 24 h (Fig. 2B, C and Fig. 3). After 6 h, only DHA (70 μM) reduced the level of cyclin D1 significantly, whereas DHA (70 μM) and EPA (70 μM) both reduced cyclin D1 substantially and with comparable effects after 24 h. OA did not affect the proliferation of SW620 cells. However, OA reduced the level of cyclin D1 after 24 h, although to a much lesser extent (Fig. 3). This reflects the differences in the antiproliferative effect between the PUFAs observed earlier.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology (NTNU), Erling Skjalgssons gate 1, N-7006 Trondheim, Norway.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus