br Altered expression of cell cycle regulatory proteins and
2.3. Altered expression of AICAR regulatory proteins and activation of the p38 MAP kinase pathway in breast cancer cells after treatment with CTU analogues
In view of findings from flow cytometry we evaluated the effect of CTU analogues on the expression of important cell cycle regulatory proteins in MDA-MB-231 cells. Several of the most active analogues, especially 2–4 and CTU (10 μM, 24 h; P < 0.001), and to a lesser ex-tent 5, 6, and 7 decreased the expression of cyclin D1 immunoreactive protein (P < 0.01; Fig. 3A). Expression of CDK4, which is a major catalytic partner for cyclin D1, was also strongly decreased by 3 and 4, while lesser decreases were effected by 2, 6, 7 and CTU. Expression of cyclin D1 and CDK4 was essentially unchanged by the analogues that did not markedly alter ATP production or cell cycle distribution.
We also assessed the effects of the analogues on the expression of other cell cycle regulatory proteins in MDA-MB-231 cells. The most active CTU analogues also decreased the expression of other regulatory cyclins (Fig. 3A). Cyclin E1 immunoreactive protein in MDA-MB-231 cells was decreased after treatment with the highly active agents 4 and 7, as well as 2, 3, 5–7 and CTU. Similarly, cyclin B1 expression was also decreased by most analogues, except 5 and 8. CDK6, which is an al-ternate catalytic partner for cyclin D1, was also significantly down-regulated by most of the more effective agents (2, 4, 7 and CTU; Fig. 3A).
We found previously that ω-3-17,18-epoxy-EPA downregulated the cyclin D1/CDK4 holoenzyme complex by activating the p38 MAP ki-nase pathway (Cui et al., 2011). Accordingly we tested the capacity of the present CTU analogues to activate this pathway in MDA-MB-231 cells. As shown in Fig. 3B, active analogues increased the expression of the activated (phosphorylated) form of the p38 MAP kinase; as antici-pated, the expression of total p38 MAP kinase protein was unchanged from control. Consistent with these findings, phospho-p38 MAP kinase expression was not markedly increased by the inactive analogues. Taken together, it emerged from these studies that the CTU analogues that effectively decreased the viability of MDA-MB-231 cells also de-creased the expression of multiple cell cycle regulatory cyclins and CDKs, and activated the p38 MAP kinase signalling pathway.
2.4. SAR of ring-substituted aryl-ureas
From our earlier study it appeared that strong electron-withdrawing groups were required to impart activity to the aryl-urea scaffold (Rawling et al., 2017). In accord with this finding, it emerged from the present study that the most active CTU analogues contained strongly electron-withdrawing substituents in the meta- and para-positions of
inactive analogues 1, 8, 9, and 10 did not attain this value (σtotal 0.43 to −0.17; Table 1). The σtotal value for 5 was 0.77 but this analogue ex-hibited a low capacity to modulate ATP formation and cell cycle dis- tribution. However, it is noteworthy that this was the only ortho-sub-stituted agent in the test set, suggesting that ortho-substitution is not tolerated.
To further test this SAR we prepared two additional analogues: 11 which contains the strongly electron-withdrawing CF3 and SF5 sub-
stituents in the meta-positions on the aryl ring (σtotal 0.98), and 12, which had a calculated value for σtotal of 1.00 but, like 5, contained ortho-substituents in the aromatic ring. In agreement with findings from the initial set of analogues, we found that 11 (10 μM, 24 h) strongly decreased ATP production (to 42 ± 9% of control,
P < 0.001; Fig. 4B). In accord with these findings the expression of cyclins D1, E1 and B1, and CDKs 4 and 6, were all decreased and phospho-p38 MAP kinase expression was strongly activated by 11 (Fig. 4C). In contrast, the ortho-disubstituted analogue 12 was inactive and did not impair ATP production, or cell cycle distribution (Fig. 4A, B). Furthermore, 12 did not alter the expression of cell cycle regulatory
proteins or phospho-p38 MAP kinase even though the aryl-substituents were strongly electron-withdrawing (Fig. 4C).
Finally, we directly tested the most active analogues that emerged from these studies for the capacity to modulate proliferation and apoptosis in MDA-MB-231 cells. We used the BrdU assay, which mea-sures 5-bromo-2′-deoxyuridine incorporation into DNA, as an index of proliferation. As shown in Fig. 5A, the active analogues CTU, 3 and 4 decreased BrdU incorporation most effectively to 61–69% of control, while less pronounced decreases to 75–83% of control were effected by three other analogues that also decreased ATP production in cells (2, 6 and 11). Three other analogues that did not alter ATP production, disrupt cell cycle regulation or modulate phospho-p38 MAP kinase expression (1, 9 and 12) were found not to decrease BrdU incorporation (Fig. 5A).