SWATH Differential Abundance Proteomics and Cellular Assays Show In Vitro Anticancer Activity of Arachidonic Acid- and Docosahexaenoic Acid-Based Monoacylglycerols in HT-29 Colorectal Cancer Cells

Abstract

Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on CRC cells demonstrated for two very long-chain PUFA. These were docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6) in the free fatty acid (FFA) form. In this work, similar design and technology have been used to investigate the actions of both DHA and ARA as monoacylglycerol (MAG) molecules, and results have been compared with those obtained using the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. Sequential window acquisition of all theoretical mass spectra (SWATH) - mass spectrometry massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while the ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted as inhibited by DHA-MAG. Results clearly demonstrated the ability of both ARA- and DHA-MAG to induce cell death in colon cancer cells, which suggests a direct relationship between chemical structure and antitumoral actions.

Keywords: HT-29 cells; SWATH; arachidonic acid; colorectal cancer; docosahexaenoic acid; monoacylglycerols; proteomics.

Figure 1

Plots showing results of cell assays. (a) Dose-dependent viability of HT-29 cells after exposure to docosahexaenoic acid (DHA)- and arachidonic acid (ARA)-monoacylglycerol (MAG). (b) Dose-dependent lactate dehydrogenase (LDH) release from HT-29 colon cancer cells after exposure to DHA- and ARA-MAG. (c) Dose-dependent caspase-3 activity from HT-29 colon cancer cells in comparison with untreated cells (control). Data represent the mean of three complete independent experiments ± SD (error bars). Data were analyzed using generalized linear models (GZLMs). There are no significant differences (p < 0.05) among series sharing the same letter.

Figure 2

Quantitative proteomics result overview. Volcano plot showing proteins with differential expression as a result of the addition of (a) DHA-MAG; and (b) ARA-MAG. Red dotted lines show p-value < 0.01 and two-fold change cut-offs; proteins above these thresholds are shown in red. (c) Heat map including all 1882 quantified proteins; samples treated with DHA-MAG and ARA-MAG are separated from each other and from the control group.

Figure 3

Pathway impact and gene ontology (GO) analysis for DHA-MAG and ARA-MAG. (a) Significantly impacted pathways and their associated adjusted p-values. (b,c) GO analysis showing significant (adjusted p-values < 0.05) (b) biological processes or (c) cellular components.

Figure 4

Effect of DHA-MAG extract on HT-29 cells’ proteasome pathway. (a) Proteasome pathway diagram highlighting regulated proteins. (b) Gene perturbation bar plot for affected proteins of the proteasome pathway.

Figure 5

Effect of ARA-MAG extract on HT-29 cells’ DNA replication pathway. (a) DNA replication pathway diagram highlighting regulated proteins. (b) Gene perturbation bar plot for affected proteins of the DNA replication pathway.

Figure 6

Differential expression of nine proteins from DHA-MAG- and ARA-MAG-treated HT-29 colorectal cancer cells by PRM quantification (* adjusted p-value <0.05, ** adjusted p-value <0.01; group comparison to control).