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.

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.

A whole-food approach to the in vitro assessment of the antitumor activity of gazpacho.

Gazpacho is a traditional cold soup of the Mediterranean diet consisting of a main base of fresh pureed tomato and other vegetables. Tomato and tomato products have demonstrated chemopreventive activity against several types of cancer through in vitro studies, and in animal and clinical research. Here we have applied a whole-food approach for the preclinical assessment of the antitumor potential of gazpacho. Colon cancer cells (HT-29) were exposed to growing concentrations of gazpacho previously digested in vitro to simulate the delivery of bioactive molecules to colon cells after food consumption. The cytotoxicity of gazpacho ingredients was also tested in independent experiments. Programmed cell death by apoptosis was detected by using a multiparametric analysis that combines image-based bright-field and fluorescence cytometry, intracellular ATP level determination and enzymatic activity of caspase-3/7. Modulation of gene expression of key regulatory genes (p53, Bcl-2, BAX, and cyclin D1) was also investigated. Our cytotoxicity data showed that in vitro digestion of samples allowed the delivery of bioactive levels of antitumor phytochemicals to cultured cells. Controlled experiments showed significant repetitive dose and time-response cytotoxicity of gazpacho. Gazpacho digestates caused net cell death of cultures suggesting synergic activity among phytochemicals from its vegetable ingredients. Multiparametric and genetic analyses showed that gazpacho digestates can trigger colon cancer cells death by apoptosis through the activation of caspase cascade. Our results show that coupled in vitro methodology employed can be applied to investigate the antitumor potential of complex food matrixes or combinations of foods in the diet.

Proteomics Study Reveals That Docosahexaenoic and Arachidonic Acids Exert Different In Vitro Anticancer Activities in Colorectal Cancer Cells.

Two polyunsaturated fatty acids, docosahexaenoic acid (DHA) and arachidonic acid (ARA), as well as derivatives, such as eicosanoids, regulate different activities, affecting transcription factors and, therefore, DNA transcription, being a critical step for the functioning of fatty-acid-derived signaling. This work has attempted to determine the in vitro anticancer activities of these molecules linked to the gene transcription regulation of HT-29 colorectal cancer cells. We applied the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test along with lactate dehydrogenase and caspase-3 assays; proteome changes were assessed by "sequential windowed acquisition of all theoretical mass spectra" quantitative proteomics, followed by pathway analysis, to determine the affected molecular mechanisms. In all assays, DHA inhibited cell proliferation of HT-29 cells to a higher extent than ARA and acted primarily by downregulating proteasome particles, while ARA presented a dramatic effect on all six DNA replication helicase particles. The results indicated that both DHA and ARA are potential chemopreventive agent candidates.

Phytochemical composition and in vitro anti-tumour activities of selected tomato varieties.

BACKGROUND: Previous studies indicated that tomato is a rich source of phytochemicals that act on different tumours. In this research, the phytochemical composition of selected tomato varieties was assessed by GLC and UHPLC/HPLC-MS, as well as their anti-tumour activities on HT-29 colorectal cancer cells. RESULTS: Significant differences were found among tomato varieties; lycopene was high in Racimo, phenolics in Pera, sterols in Cherry, and linoleic acid predominated in all varieties. The MTT and LDH assays showed significant time- and concentration-dependent inhibitory/cytotoxic effects of all tomato varieties on HT-29 cells. Furthermore, the joint addition of tomato carotenoids and olive oil to HT-29 cell cultures induced inhibitory effects significantly higher than those obtained from each of them acting separately, while no actions were exercised in CCD-18 normal cells. CONCLUSION: Tomato fruits constitute a healthy source of phytochemicals, although differences exist among varieties. In vitro, all of them inhibit colorectal cancer cell proliferation with Racimo variety at the top, and exercising a selective action on cancer cells by considering the lack of effects on CCD-18 cells. Furthermore, synergy was observed between olive oil and tomato carotenoids in inhibiting HT-29 cancer cell proliferation; conversely, phenolics showed no significant effects and hindered carotenoids actions. © 2016 Society of Chemical Industry.

Various Acylglycerols from Common Oils Exert Different Antitumor Activities on Colorectal Cancer Cells.

Colorectal cancer is one of the leading causes of death in Western countries; therefore, the implementation of healthy dietary habits in order to prevent its occurrence is a desirable action. We show here that both free fatty acids (FFAs) and some acylglycerols induce antitumoral actions in the colorectal cancer cell line HT-29. We tested several C18 polyunsaturated fatty acid-enriched oils (e.g., sunflower and Echium) as well as other oils, such as arachidonic acid-enriched (Arasco®) and docosahexaenoic acid-enriched (Marinol® and cod liver oil), in addition to coconut and olive oils. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test indicated inhibitory effects on HT-29 cells viability for FFAs, and monoacylglycerol and diacylglycerol (DAG) species, while the lactate dehydrogenase test proved that FFAs were the more effective species to induce membrane injury. Conversely, all species did not exhibit actions on CCD-18 normal human colon cells viability. Furthermore, transmission electron microscopy showed the presence of necrosis and apoptosis, while the monoacylglycerol lipase (MAGL) inhibition test demonstrated high activity for 2-monoacylglycerols derived from Arasco and sunflower oils. However, different monoacylglycerols and DAGs have also the potential for MAGL inhibition. Therefore, checking for activity on colon cancer cells of specifically designed acylglycerol-derivative species would be a suitable way to design functional foods destined to avoid colorectal cancer initiation.