The composition of each fatty acid was calculated as a percentage of the total amount of fatty acids in the mixture. Statistical N-Methylcytisine analysis All the data were analysed using SPSS 22 software. water-soluble tetrazolium-1 (WST-1) assay kit at 24, 48 and 72?h. Effects of FFA extract of krill oil and EPA on apoptosis and mitochondrial membrane potential were determined using commercial kits after 48?h of treatment. Results Krill oil extract inhibited cell proliferation of all three cell lines in the similar manner as fish oil extract. A significant cell apoptosis and increase N-Methylcytisine in mitochondrial membrane potential were observed after the treatment with krill oil extract. EPA at the concentration of 200?M reduced significantly the proliferation of HCT-15 and SW-480 at 24, 48 and 72?h. In addition, EPA treatment (100 and 200?M) resulted in significant cell apoptosis in all three cell lines. No significant changes were observed after treatment with DHA and AA. Conclusions Our results indicate that the FFA extract of krill oil maybe an effective chemotherapeutic agent to suppress proliferation and induce apoptosis in CRC cells N-Methylcytisine through its bioactive constitute EPA. Although the exact mechanism of the pro-apoptotic properties of krill oil extract is unclear, mitochondrial pathway seems to be implicated. into the cytosol, which activates caspases that in turn, induce apoptosis . In the present study, we investigated the effects of free fatty acid (FFA) extract from krill oil in comparison with that from fish oil on three human CRC cell lines HCT-15, SW-480 and Caco-2. In addition, effects of EPA and DHA on these cells are also assessed. To the best of our knowledge, this is the first study assessing the impacts of krill oil on cell apoptosis and investigating whether the apoptotic process is mediated by changes in MMP. Methods Cell lines and culture conditions The human colon adenocarcinoma cell lines of HCT-15, SW-480 and Caco-2 were obtained from the American Tissue Culture Collection (ATCC) Manassas, VA, USA. HCT-15 and SW-480 cell lines were maintained in RPMI 1640 medium (SAFCO) (Sigma Aldrich, Castle Hill, NSW) supplemented with foetal calf serum (FCS, 10?%) (Hyclone Quantum Scientific, Clayton South, VIC), glutamine (10?mM), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES 10?mM) and penicillin (100U/ml)/streptomycin (100?g/ml) (Sigma Aldrich, Castle Hill, NSW). The Caco-2 cell line was maintained N-Methylcytisine in Dulbeccos Modified Eagles Medium (DMEM) (Sigma Aldrich, Castle Hill, NSW) supplemented with 20%FCS and penicillin (100U/ml)/streptomycin (100?g/ml), 2?mM/L glutamine, 0.1?mM non-essential amino acids. Cells were grown at 37?C in 5?% CO2 humidified atmosphere. Extraction of free fatty acids from oils and fatty acid solution preparation Krill oil and fish oil (Swisse Wellness Pty Ltd., Victoria, Australia) were purchased from the local Chemist. Free fatty acids were extracted from krill oil and fish oil following the hydrolysis (saponification) method of Salimon et al. . The extracts were dissolved in the Dimethyl Sulfoxide (DMSO) and stored at ?20?C. The final treatment solutions contained 1?% DMSO as solvent. Individual PUFA including EPA, DHA and Arachnoid Acid (AA) were purchased from Nu-Chek-Prep, Elysian, USA. Fatty acid solutions were made up by dissolving the individual fatty acid in ethanol. The final treatment solutions contained?0.1?% ethanol as solvent. Cell proliferation assays A water-soluble tetrazolium-1 (WST-1) assay kit (Roche Diagnostics GmbH, Germany) was used E.coli monoclonal to V5 Tag.Posi Tag is a 45 kDa recombinant protein expressed in E.coli. It contains five different Tags as shown in the figure. It is bacterial lysate supplied in reducing SDS-PAGE loading buffer. It is intended for use as a positive control in western blot experiments to determine the proliferative potential of cancer cells. Cells were seeded and cultured at 1??104 cells per well in 96-well plates for 24?h, and then treated with individual PUFA solutions for 24, 48 or 72?h; or free fatty acid extract solutions of oils (krill oil or fish oil) for 48?h. All treatments were performed in quadruplicates. For PUFA treatments, three concentrations of each fatty acid were used including 50?M, 100?M and 200?M. 0.1?% ethanol was used as a vehicle control. Additional assays were performed to observe the effects of various EPA concentrations (100?M, 120?M, 140?M, 160?M, 180?M and 200?M) on cell proliferation of HCT-15 cells after 48?h of treatment. The treatment concentrations of free fatty acid extract of oils (krill oil or fish oil) are 0.03, 0.06, 0.12 or 0.24?L/100?L well. 1?% DMSO was used as a vehicle.