Free Radic Biol Med.2016 Jan;90:158-172
DHA-induced stress response in human colon cancer cells – Focus on oxidative stress and autophagy
Kristine Pettersen a,b,d,1 , Vivi Talstad Monsen a,1 , Caroline Hild Hakvåg Pettersen a , Hilde Bremseth Overland a,c , Grete Pettersen a , Helle Samdal a , Almaz Nigatu Tesfahun a , Anne Gøril Lundemo a , Geir Bjørkøy b,d,2 , Svanhild A. Schønberg a,*,2
a Department of Laboratory Medicine, Children’s and Women’s Health, Faculty of Medicine, Norwegian University of Science and Technology, NTNU, 7006 Trondheim, Norway
b Department of Technology, Erling Skjalgssons gt. 1, University College of Sør-Trøndelag, 7006 Trondheim, Norway
c Central Norway Regional Health Authority, 7055 Stjørdal, Norway
d Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway
1 These authors have contributed equally to this work
2 Shared senior authorship
* Corresponding author
Polyunsaturated fatty acids (PUFAs) are important constituents of the diet and health benefits of omega- 3/n3 PUFAs, especially eicosapentaenoic acid (EPA, 20:5 n3) and docosahexaenoic acid (DHA, 22:6 n3) have been well documented in relation to several diseases. Increasing evidence suggests that n3 PUFAs may have anticancer activity and improve the effect of conventional cancer therapy. The mechanisms behind these effects are still unclear and need to be elucidated. We have examined the DHA-induced stress response in two human colon cancer cell lines, SW620 and Caco-2. SW620 cells are growth-inhibited at early time points by DHA, while the growth of Caco-2 cells almost remains unaffected by the same treatment. Gene expression analysis of SW620 cells treated with DHA revealed changes at early time points; transcripts involved in oxidative stress and autophagy were among the first to be differentially expressed. We find that oxidative stress is induced in both cell lines, although at different time points and to different extent. DHA induced nuclear translocation of the oxidative stress sensor NFE2L2 in both cell lines, indicating an induction of an anti-oxidative response. However, vitamin E did not counteract ROS-production or the translocation of NFE2L2 to the nucleus. Neither vitamin E nor the antioxidants butylated hydoxyanisole (BHA) and butylated hydoxytoluene (BHT) did affect the growth inhibition in SW620 cells after DHA-treatment. Also, siRNA-mediated downregulation of NFE2L2 did not sensitize SW620 and Caco-2 cells to DHA. These results indicate that oxidative stress response is not the cause of DHA-induced cytotoxicity in SW620 cells. Using biochemical and imaging based functional assays, we found a low basal level of autophagy and no increase in autophagic flux after adding DHA to the SW620 cells. However, Caco-2 cells displayed a higher level of autophagy, both in the absence and presence of DHA. Inhibition of autophagy by siRNA mediated knock down of ATG5 and ATG7 sensitized both SW620 and Caco-2 cells to DHA. Stimulation of autophagy by rapamycin in SW620 and Caco-2 cells resulted in decreased DHA-sensitivity and inhibition of autophagy in Caco-2 cells by chloroquine resulted in increased DHA-sensitivity. These results suggest that autophagy is important for the DHA sensitivity of colon cancer cells and imply possible therapeutic effects of this fatty acid against cancer cells with low autophagy.
The mechanisms behind the potential anti-cancer properties of omega-3 polyunsaturated fatty acids (PUFAs) have been studied for several years. New research has focused on the ability of n-3 PUFAs like docosahexaenoic acid (DHA) to induce autophagy in human cancer cells. Autophagy is the cell’s renovation system where damaged organelles or proteins are degraded to yield new building blocks that the cell may reuse.
In this study we used two human colon cancer cell lines that responded very differently to supplementation with DHA; the SW620 cells were strongly growth inhibited, while the Caco-2 cells were not. In DHA-treated SW620 cells, early response genes were linked to oxidative stress and autophagy. However, when measuring the level of autophagy after DHA supplementation we found no increase in autophagic flux. We found both oxidative stress and lipid peroxidation to be induced upon DHA-treatment, but neither antioxidants nor siRNA knock down of nuclear factor erythroid 2-like 2 (NFE2L2/Nrf-2) or heme oxygenase 1 (HMOX 1) was able to affect the cells’ sensitivity towards DHA.
Importantly, we found that the level of basal autophagy was important for the sensitivity towards DHA in SW620 and Caco-2 cells. The Caco-2 cells had a much higher level of basal autophagy compared to the SW620 cells. Also, we were able to affect the cells’ sensitivity towards DHA by interfering with the autophagic process. Inhibition of autophagy by chloroquine increased the sensitivity of Caco-2 cells towards DHA, while stimulation of autophagy by rapamycin left the SW620 cells less sensitive to DHA supplementation. In addition, knock down of the autophagy related genes 5 and 7 (Atg5/7) increased the DHA sensitivity of both SW620 and Caco-2 cells.
Altogether, this emphasizes the importance of the autophagic process when it comes to handling cellular stress in cancer cells. Monitoring autophagy levels may potentially help us predict whether growth of tumor cells may be affected by supplementation with DHA.
Figure 1. The colon cancer cell line SW620 has a low level of basal autophagy and is strongly growth inhibited by suppplementation with DHA, while the Caco-2 cells have a high level of basal autophagy and is much less affected by DHA-supplementation.
Caroline H Pettersen, Associated Professor
Dept of Laboratory Medicine, Children’s and Women’s Health Norwegian University of Science and Technology Caroline.email@example.com
Svanhild A Schønberg, Professor
Dept of Laboratory Medicine, Children’s and Women’s Health Norwegian University of Science and Technology Svanhild.firstname.lastname@example.org