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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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Changes in cholesterol metabolism induced by DHA. A: Western blot analysis of HMGCR, mSREBP2, and NPC1 protein levels in total protein extracts from SW620 cells treated with DHA for the indicated time periods (h). Controls were harvested at all time points; only 24 h control is shown for mSREBP2 and NPC1. For HMGCR, controls are shown for all time points. β-actin was used as a control for equal protein loading. One blot, representing three independent experiments, is shown. The blots were quantified and protein band intensities normalized relative loading control. The actin adjusted band intensities from the DHA and control membranes were further normalized relative to the 24 h control band, present at all membranes, to adjust for differences in signal intensities between the membranes. The numbers under the blots represent mean fold change (with SD) of DHA samples relative to control at indicted time points for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05). B: Alterations in HMGCR and mSREBP2 protein levels in control (baseline) and DHA treated cells at the indicated time periods. The plots show the mean value of the actin adjusted band intensitites normalized relative to the 24 h control band for DHA treated cells (gray bars) and control cells (black bars). The data represent the mean and SD of three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).
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fig5: Changes in cholesterol metabolism induced by DHA. A: Western blot analysis of HMGCR, mSREBP2, and NPC1 protein levels in total protein extracts from SW620 cells treated with DHA for the indicated time periods (h). Controls were harvested at all time points; only 24 h control is shown for mSREBP2 and NPC1. For HMGCR, controls are shown for all time points. β-actin was used as a control for equal protein loading. One blot, representing three independent experiments, is shown. The blots were quantified and protein band intensities normalized relative loading control. The actin adjusted band intensities from the DHA and control membranes were further normalized relative to the 24 h control band, present at all membranes, to adjust for differences in signal intensities between the membranes. The numbers under the blots represent mean fold change (with SD) of DHA samples relative to control at indicted time points for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05). B: Alterations in HMGCR and mSREBP2 protein levels in control (baseline) and DHA treated cells at the indicated time periods. The plots show the mean value of the actin adjusted band intensitites normalized relative to the 24 h control band for DHA treated cells (gray bars) and control cells (black bars). The data represent the mean and SD of three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).

Mentions: From the gene expression data, it was apparent that several genes encoding proteins involved in cholesterol biosynthesis were down-regulated at the mRNA level in DHA-treated SW620 cells (Table 1). These include 7- and 24-dehydrocholesterol reductase (DHCR7, DHCR24), farnesyl diphosphate synthase (FDPS), phosphomevalonate kinase (PMVK), 3β-hydroxysterol Δ14-reductase (TM7SF2) (12, 24 h), and lanosterol synthase (LSS) (12 h). However, some transcripts involved in cholesterol uptake and intracellular cholesterol transport, such as low and very low density lipoprotein receptor (LDLR, VLDLR), the Niemann-Pick C1 protein (NPC1), NPC2, and the oxysterol binding protein (OSBP), were found to be up-regulated in SW620 cells after DHA-treatment (Table 1). NPC1 protein levels were in addition analyzed by Western blot and were increased in DHA-treated cells compared with control at 24 h (Fig. 5A). Since several of the differentially expressed transcripts listed above are regulated by sterol regulatory element binding protein 2 (SREBP2), the protein levels of mSREBP2 (mature) and pSREBP2 (precursor) were analyzed by Western blot. An increase in mSREPB2 levels was observed in control and DHA-treated cells over the time period assayed, but DHA-treated cells displayed higher levels of mSREBP2 compared with control cells at all time points (Fig. 5A, B). The level of pSREBP2 was unchanged in SW620 control cells at all time points, while a slight decrease was observed after 48 h treatment with DHA (data not shown). We also analyzed the level of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) by Western blot after DHA treatment. The protein levels of HMGCR in DHA-treated and control cells were similar at 3 and 6 h. At 12–48 h, the HMGCR protein levels were reduced in controls, while the protein level in DHA-treated cells increased slightly (Fig. 5A, B).


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)

Changes in cholesterol metabolism induced by DHA. A: Western blot analysis of HMGCR, mSREBP2, and NPC1 protein levels in total protein extracts from SW620 cells treated with DHA for the indicated time periods (h). Controls were harvested at all time points; only 24 h control is shown for mSREBP2 and NPC1. For HMGCR, controls are shown for all time points. β-actin was used as a control for equal protein loading. One blot, representing three independent experiments, is shown. The blots were quantified and protein band intensities normalized relative loading control. The actin adjusted band intensities from the DHA and control membranes were further normalized relative to the 24 h control band, present at all membranes, to adjust for differences in signal intensities between the membranes. The numbers under the blots represent mean fold change (with SD) of DHA samples relative to control at indicted time points for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05). B: Alterations in HMGCR and mSREBP2 protein levels in control (baseline) and DHA treated cells at the indicated time periods. The plots show the mean value of the actin adjusted band intensitites normalized relative to the 24 h control band for DHA treated cells (gray bars) and control cells (black bars). The data represent the mean and SD of 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

fig5: Changes in cholesterol metabolism induced by DHA. A: Western blot analysis of HMGCR, mSREBP2, and NPC1 protein levels in total protein extracts from SW620 cells treated with DHA for the indicated time periods (h). Controls were harvested at all time points; only 24 h control is shown for mSREBP2 and NPC1. For HMGCR, controls are shown for all time points. β-actin was used as a control for equal protein loading. One blot, representing three independent experiments, is shown. The blots were quantified and protein band intensities normalized relative loading control. The actin adjusted band intensities from the DHA and control membranes were further normalized relative to the 24 h control band, present at all membranes, to adjust for differences in signal intensities between the membranes. The numbers under the blots represent mean fold change (with SD) of DHA samples relative to control at indicted time points for three independent experiments. * Significantly different from control (Student's t-test, P < 0.05). B: Alterations in HMGCR and mSREBP2 protein levels in control (baseline) and DHA treated cells at the indicated time periods. The plots show the mean value of the actin adjusted band intensitites normalized relative to the 24 h control band for DHA treated cells (gray bars) and control cells (black bars). The data represent the mean and SD of three independent experiments. * Significantly different from control (Student's t-test, P < 0.05).
Mentions: From the gene expression data, it was apparent that several genes encoding proteins involved in cholesterol biosynthesis were down-regulated at the mRNA level in DHA-treated SW620 cells (Table 1). These include 7- and 24-dehydrocholesterol reductase (DHCR7, DHCR24), farnesyl diphosphate synthase (FDPS), phosphomevalonate kinase (PMVK), 3β-hydroxysterol Δ14-reductase (TM7SF2) (12, 24 h), and lanosterol synthase (LSS) (12 h). However, some transcripts involved in cholesterol uptake and intracellular cholesterol transport, such as low and very low density lipoprotein receptor (LDLR, VLDLR), the Niemann-Pick C1 protein (NPC1), NPC2, and the oxysterol binding protein (OSBP), were found to be up-regulated in SW620 cells after DHA-treatment (Table 1). NPC1 protein levels were in addition analyzed by Western blot and were increased in DHA-treated cells compared with control at 24 h (Fig. 5A). Since several of the differentially expressed transcripts listed above are regulated by sterol regulatory element binding protein 2 (SREBP2), the protein levels of mSREBP2 (mature) and pSREBP2 (precursor) were analyzed by Western blot. An increase in mSREPB2 levels was observed in control and DHA-treated cells over the time period assayed, but DHA-treated cells displayed higher levels of mSREBP2 compared with control cells at all time points (Fig. 5A, B). The level of pSREBP2 was unchanged in SW620 control cells at all time points, while a slight decrease was observed after 48 h treatment with DHA (data not shown). We also analyzed the level of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) by Western blot after DHA treatment. The protein levels of HMGCR in DHA-treated and control cells were similar at 3 and 6 h. At 12–48 h, the HMGCR protein levels were reduced in controls, while the protein level in DHA-treated cells increased slightly (Fig. 5A, B).

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