ER stress, reactive oxygen species and autophagy in colon cancer cells treated with polyunsaturated marine fatty acids

Abstract

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are abundantly found in fatty fish, canola oil, flaxseed and walnuts. Alternatively, they may be obtained through intake of fish oil or fish oil capsules. They have been associated with numerous health promoting effects, including prevention of some cancer types as demonstrated in a few epidemiological studies. These studies, however, have not been entirely conclusive. Nevertheless, several studies using cell culture and animal models have shown that n-3 PUFAs may have a potential in cancer treatment, since they have been shown to inhibit growth of and / or induce cell death (apoptosis) in cancer cells at concentrations where normal cells are not harmed.

The molecular mechanisms mediating the anticancer effects of n-3 PUFAs are complex and still not elucidated in detail. Lipid peroxidation has been proposed as one mechanism explaining how these fatty acids inhibit growth of cancer cells. Increased levels of the lipid peroxidation product malondialdehyde (MDA) were found in two cancer cell lines from colon (SW480 and SW620) after administration of the n-3 fatty acid docosahexaenoic acid (DHA), but either lipid peroxidation or defects in the antioxidant defence appeared to be related to the observed growth inhibition. Both cell lines accumulated lipid droplets, mainly in the form of cholesteryl esters in SW620 and triglycerides in SW480. This indicates that the cells have different mechanisms for processing potentially harmful molecules like DHA. In addition, the active form of sterol regulatory element-binding protein 1 (SREBP1) was found downregulated both at mRNA and protein level in both cell lines. Gene expression analysis showed activation of a cellular stress response called unfolded protein response (UPR) in SW620 cells after DHA treatment. UPR is induced by accumulation of unfolded and misfolded proteins in endoplasmic reticulum (ER), also called ER stress. ER stress was further demonstrated by increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Transcripts encoding proteins associated with ER stress / UPR, oxidative stress / antioxidant defence and autophagy were shown upregulated already after 3 hours of DHA treatment. In addition, the gene expression results indicated disturbances in cholesterol and calcium homeostasis in SW620 cells after DHA treatment. DHA was shown to activate SREBP2, which is involved in the regulation of cholesterol synthesis,but this did not lead to increased de novo synthesis of cholesterol. DHA treatment also resulted in an increased level of calcium in the cytosol, which was likely a result of the induced ER stress in SW620 cells. Autophagy is a prosurvival mechanism that may contribute to restore cellular homeostasis after ER stress, and which can be induced by ER stress, oxidative stress and disturbances in calcium homeostasis.

Autophagy is also shown to be involved in the regulation of lipid metabolism. Co-localization of the autophagic markers LC3B and p62 is a method to identify autophagic structures. Despite the induction of autophagy at the transcriptional level in SW620 cells after DHA addition, co-localization of LC3B and p62 was not found. Such co-localization was however identified in the DHAresistant cancer cell line from colon, Caco-2. Furthermore, increased levels of reactive oxygen species (ROS) were found after treatment with DHA in SW620, but not in Caco-2. Nrf2, a transcription factor regulating gene expression of antioxidant enzymes, was however shown to accumulate in the nucleus after 6 hours of DHA treatment in both cell lines. Knockdown of Nrf2 in Caco-2 cells by siRNA made these cells more sensitive to DHA. Taken together, these findings suggest ER stress and autophagy as important cellular responses to DHA in cancer cells. Furthermore, the results indicate that the DHA-sensitivity of different cancer cells may be affected by their ability to induce autophagy and counteract oxidative stress via activation of Nrf2.