Development of Novel Omega-3 Fatty Acid Epoxide Analogues as Anti-Proliferative and Anti-Migration Agents in Triple Negative Human Breast Cancer Cells

Abstract

Metastasis is a multi-stage process in which primary tumour cells proliferate, invade the surround tissue and migrate through blood or lymph stream to distant tissues and form secondary tumours. Recently, a novel aryl-urea fatty acid named CTU was developed based on the epoxides of ω-3 fatty acids that impaired the viability of the highly metastatic MDA-MB-231 triple negative breast cancer (TNBC) cells in vitro and in mouse xenograft models in vivo. In this project, new CTU analogues were synthesized and examined for the anti-proliferative activity against MDA-MB-231 breast cancer cells. The structural requirements for their activities were also identified. CTU and selected analogues were also tested for their ability to inhibit the invasion/migration of the same cell line. Furthermore, the mechanism of the anti-invasion/migration was characterized. In the first part of this project, many of the new CTU analogues exhibited an improved anti-proliferative activity over CTU. Compared with control, 3-Cl, 5-CF3-substituted 4 was the most effective analogue in decreasing ATP production (26±2% of control; 10 μM, 24 hours; P<0.001) in MDA-MB-231 breast cancer cells followed by 3-NO2 ,5-CF3-substituted 3 (55±5% of control; 10 μM, 24 hours; P<0.001) and CTU (60±3% of control; 10 μM, 24 hours; P<0.001), and to a lesser extent 4-CF3, 5-Cl-substituted 2 and 4-Br, 5-CF3-substituted 6. The 3, 5-CF3 -substituted 7 was also active and strongly decreased ATP production when tested at 20 µM (18±3% of contro; 24 hours; P<0.001), while other analogues produced minimal decreases in ATP production, even at higher concentrations. To corroborate the effects on ATP production, the impact of CTU and the new analogues on cell cycle kinetics was investigated by flow cytometry. After 24 hours of treatment analogue 2, 4, 6, 7 and CTU increased the proportion of cells in sub-G1 phase (~7.5-14-fold of control). Moreover, the cell population in S-phase was significantly increased by 4 and 7 (17.7±0.5% to 16.9±1.1% of cells, respectively, compared with 13.2±0.3% in DMSO-treated control, P<0.01) and, to a lesser extent 2 and 3. Consistent with these findings, the expression of the cell cycle regulatory proteins cyclins D1, E1 and B1, and the cyclin-dependent kinases (CDKs) 4 and 6 was also down-regulated by the effective agents (2-4, 6, 7 and CTU), and unchanged by the analogues that did not markedly alter ATP production or cell cycle distribution. Additionally, the active analogues stimulated the phosphorylation and activation of the P38 mitogen-activated protein kinase (MAPK) signalling pathway which inhibits the expression of the cell cycle regulatory proteins. As a preliminary evaluation of the structure-activity relationship of these analogues, it was found that most active analogues have strong electron-withdrawing groups with the sum of the Hammett constants (σtotal) values ranging between 0.66-1.14, while the inactive analogues did not attain this value (σtotal= -0.17 to 0.43). Furthermore, analogues that contained strong electron-withdrawing groups at the meta- and para-positions of the aryl ring (analogue 4, 7 and 11) were very active in comparison to analogues with strong electron-withdrawing groups; substituents of the ortho-positions (analogue 5 and 12) were inactive. This suggests that strong electron withdrawing substituents (σtotal≥ 0.66) in the meta- and para-positions were required for anti-proliferative activity, and that ortho-substitutions were not tolerated.