DHA-induced stress response in human colon cancer cells – Focus on oxidative stress and autophagy

ABSTRACTPolyunsaturated fatty acids (PUFAs) are important constituents of the diet and health benefits of omega-3/n−3 PUFAs, especially eicosapentaenoic acid (EPA, 20:5 n−3) and docosahexaenoic acid (DHA, 22:6 n−3) have been well documented in relation to several diseases. Increasing evidence suggests that n−3 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 down-regulation 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

Tetradecylthioacetic acid inhibits proliferation of human SW620 colon cancer cells - gene expression profiling implies endoplasmic reticulum stress

Background: Previous reports have shown an antiproliferative effect of the synthetic, 3-thia fatty acid tetradecylthioacetic acid (TTA) on different cancer cells in vitro and in vivo. The mechanisms behind the observed effects are poorly understood. We therefore wanted to explore the molecular mechanisms involved in TTA-induced growth inhibition of the human colon cancer cell line SW620 by gene expression profiling. Methods: An antiproliferative effect of TTA on SW620 cells in vitro was displayed in real time using the xCELLigence System (Roche). Affymetrix gene expression profiling was performed to elucidate the molecular mechanisms behind the antiproliferative effect of TTA. Changes in gene expression were verified at protein level by western blotting. Results: TTA reduced SW620 cell growth, measured as baseline cell index, by 35% and 55% after 48 h and 72 h, respectively. We show for the first time that TTA induces an endoplasmic reticulum (ER) stress response in cancer cells. Gene expression analysis revealed changes related to ER stress and unfolded protein response (UPR). This was verified at protein level by phosphorylation of eukaryote translation initiation factor 2 alpha (eIF2a) and downstream upregulation of activating transcription factor 4 (ATF4). Transcripts for positive and negative cell cycle regulators were down- and up-regulated, respectively. This, together with a down-regulation of Cyclin D1 at protein level, indicates inhibition of cell cycle progression. TTA also affected transcripts involved in calcium homeostasis. Moreover, mRNA and protein level of the ER stress inducible C/EBP-homologous protein (CHOP), Tribbles homolog 3 (Drosophila) (TRIB3) and CCAAT/enhancer binding protein beta (C/EBPb) were enhanced, and the C/EBPb LIP/LAP ratio was significantly increased. These results indicate prolonged ER stress and a possible link to induction of cell death. Conclusion: We find that TTA-induced growth inhibition of SW620 cells seems to be mediated through induction of ER stress and activation of the UPR pathway

Tetradecylthioacetic acid inhibits proliferation of human SW620 colon cancer cells - gene expression profiling implies endoplasmic reticulum stress

Abstract Background Previous reports have shown an antiproliferative effect of the synthetic, 3-thia fatty acid tetradecylthioacetic acid (TTA) on different cancer cells in vitro and in vivo. The mechanisms behind the observed effects are poorly understood. We therefore wanted to explore the molecular mechanisms involved in TTA-induced growth inhibition of the human colon cancer cell line SW620 by gene expression profiling. Methods An antiproliferative effect of TTA on SW620 cells in vitro was displayed in real time using the xCELLigence System (Roche). Affymetrix gene expression profiling was performed to elucidate the molecular mechanisms behind the antiproliferative effect of TTA. Changes in gene expression were verified at protein level by western blotting. Results TTA reduced SW620 cell growth, measured as baseline cell index, by 35% and 55% after 48 h and 72 h, respectively. We show for the first time that TTA induces an endoplasmic reticulum (ER) stress response in cancer cells. Gene expression analysis revealed changes related to ER stress and unfolded protein response (UPR). This was verified at protein level by phosphorylation of eukaryote translation initiation factor 2 alpha (eIF2α) and downstream up-regulation of activating transcription factor 4 (ATF4). Transcripts for positive and negative cell cycle regulators were down- and up-regulated, respectively. This, together with a down-regulation of Cyclin D1 at protein level, indicates inhibition of cell cycle progression. TTA also affected transcripts involved in calcium homeostasis. Moreover, mRNA and protein level of the ER stress inducible C/EBP-homologous protein (CHOP), Tribbles homolog 3 (Drosophila) (TRIB3) and CCAAT/enhancer binding protein beta (C/EBPβ) were enhanced, and the C/EBPβ LIP/LAP ratio was significantly increased. These results indicate prolonged ER stress and a possible link to induction of cell death. Conclusion We find that TTA-induced growth inhibition of SW620 cells seems to be mediated through induction of ER stress and activation of the UPR pathway.</p

Establishment of a Patient-Derived Xenograft Model of Colorectal Cancer in CIEA NOG Mice and Exploring Smartfish Liquid Diet as a Source of Omega-3 Fatty Acids

Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC (n = 5) were engrafted in four mice per tumor (n = 20). Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks

Power comparisons and clinical meaning of outcome measures in assessing treatment effect in cancer cachexia: secondary analysis from a randomised pilot multimodal intervention trial

Background: New clinical trials in cancer cachexia are essential and outcome measures with high responsiveness to detect meaningful changes are crucial. This secondary analysis from a multimodal intervention trial estimates sensitivity to change and between treatment effect sizes (ESs) of outcome measures associated with body composition, physical function, metabolism and trial intervention. Methods: The study was a multicenter, open label, randomised pilot study investigating the feasibility of a six-week multimodal intervention (exercise, non-steroidal anti-inflammatory drugs and oral nutritional supplements containing polyunsaturated fatty acids (n-3 PUFAs) versus standard cancer care in non-operable non-small cell lung cancer and advanced pancreatic cancer. Body composition measures from computerized tomography scans and circulating biomarkers were analyzed. Results: Forty-six patients were randomised, and the analysis included 22 and 18 patients in the treatment and control groups, respectively. The between group ESs were high for bodyweight (ES=1.2, p0.8, p<0.05 for all) and moderate for 25-hydroxyvitamin D (ES=0.49, p=0.03). In the control group, a moderate sensitivity to change for bodyweight (ES=-0.84, p=0.002) and muscle mass (ES=-0.67, p=0.016), and a high sensitivity to change for plasma levels of 25-hydroxyvitamin D (ES=-0.88, p=0.002) were found. Conclusion: Demonstrating high sensitivity to change and between treatment ES compared to body composition measures, bodyweight still stands out as a clinical and relevant outcome measure in cancer cachexia. Body composition and physical function measures clearly are important to address but demand large sample sizes to detect treatment group differences. Trial registration: ClinicalTrials.gov identifier: NCT01419145

Power comparisons and clinical meaning of outcome measures in assessing treatment effect in cancer cachexia: secondary analysis from a randomised pilot multimodal intervention trial

Background: New clinical trials in cancer cachexia are essential, and outcome measures with high responsiveness to detect meaningful changes are crucial. This secondary analysis from a multimodal intervention trial estimates sensitivity to change and between treatment effect sizes (ESs) of outcome measures associated with body composition, physical function, metabolism, and trial intervention. Methods: The study was a multicenter, open-label, randomized pilot study investigating the feasibility of a 6-week multimodal intervention [exercise, non-steroidal anti-inflammatory drugs, and oral nutritional supplements containing polyunsaturated fatty acids (n−3 PUFAs)] vs. standard cancer care in non-operable non-small-cell lung cancer and advanced pancreatic cancer. Body composition measures from computerized tomography scans and circulating biomarkers were analyzed. Results: Forty-six patients were randomized, and the analysis included 22 and 18 patients in the treatment and control groups, respectively. The between-group ESs were high for body weight (ES = 1.2, p 0.8, p < 0.05 for all) and moderate for 25-OH vitamin D (ES = 0.49, p = 0.03). In the control group, a moderate sensitivity to change for body weight (ES = −0.84, p = 0.002) and muscle mass (ES = −0.67, p = 0.016) and a high sensitivity to change for plasma levels of 25-OH vitamin D (ES = −0.88, p = 0.002) were found. Conclusion: Demonstrating high sensitivity to change and between treatment ES and body composition measures, body weight still stands out as a clinical and relevant outcome measure in cancer cachexia. Body composition and physical function measures clearly are important to address but demand large sample sizes to detect treatment group differences