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  • The finding that the proportion

    2020-08-04

    The finding that the proportion of cells in sub-G1 phase was in-creased by several analogues suggested that apoptosis was activated. To further evaluate this finding the activity of the major apoptotic ex-ecutioner caspases-3/7 was measured in MDA-MB-231 cells that had been treated with a number of aryl-ureas (10 μM, 24 h). As shown in Fig. 5B, several of the analogues that produced marked decreases in ATP production and that dysregulated the PKF118-310 (CTU, 2, 4, 6, 7 and 11) also increased caspase-3/7 activity. In contrast, analogues 1, 9 and 12, that had little effect on ATP production or cell cycle distribution did not increase caspase-3/7 activity (Fig. 5B). Taken together, the aryl-ureas that produced marked decreases in MDA-MB-231 cell viability
    Fig. 3. (A) cyclin and CDK immunoreactive proteins in MDA-MB-231 cells after treatment with aryl-ureas (10 μM; 24 h). All data are mean ± SEM from three separate experiments. Representative Western blot analyses are shown. Different from DMSO-treated control: *P < 0.05, **P < 0.01, ***P < 0.001. (B) Expression of phosphorylated (phospho-p38 MAP kinase) and total p38 MAP kinase immunoreactive proteins in MDA-MB-231 cells after treatment with aryl-ureas (10 μM; 24 h); a representative densitometric analysis from three separate experiments is shown.
    also decreased proliferation and activated apoptosis to varying extents; analogues that minimally altered cell viability did not alter BrdU in-corporation or caspase-3/7 activity.
    3. Discussion
    We recently identified the aryl-urea CTU as the first in a new class of potential anti-cancer agents that had activity against MDA-MB-231 and other tumour cells (Rawling et al., 2017). The 3-trifluoromethyl, 4-chloro-disubstituted aromatic system in CTU produced significant anti-cancer activity in vitro and in vivo, while analogues bearing electron donating or weakly electron withdrawing ring substituents were found to be inactive. The present study evaluated this preliminary SAR and found that the capacities of the analogues 4, 7 and 11, which all con-tained two strongly electron-withdrawing aryl substituents, to decrease
    cell viability were improved over CTU; analogues with σtotal ≤ 0.43 exhibited low activity. Loss of MDA-MB-231 cell viability appeared to be related to decreased proliferation, as reflected by a decrease in BrdU incorporation into DNA, and increased apoptosis, as reflected by an increase in the proportion of cells in sub-G1 phase and by activation of caspase-3/7. However, aryl substituents that were in the ortho-position also produced analogues with low activity and it may be that the steric nature of this configuration detracts from activity.
    Vinca alkaloids and taxanes are established anticancer drugs that impair mitosis by binding to β-tubulin and disrupting microtubule dy-namics. Mitosis and cell proliferation are regulated by the cell cycle, which is controlled by cyclins and CDKs that are expressed at particular 
    stages during cell cycle progression (Walker and Assoian, 2005). Sev-eral newer agents have been developed that selectively target one or more CDKs, rather than disrupting the cytoskeleton, to inhibit tumour cell proliferation and some have entered clinical trials (Law et al., 2015). Thus, the CDK4/6 inhibitor palbociclib is an effective anti-pro-liferative agent in estrogen receptor-positive breast cancer and ovarian cancer cell lines (Finn et al., 2009) and produced a clinical response in some hormone receptor positive patients when used as a single agent (Rocca et al., 2017).
    There is evidence that amplification of cell cycle regulatory proteins diminishes the clinical efficacy of CDK inhibitors. Cyclins D1 and E1 are commonly overexpressed in primary breast cancer and are associated with more aggressive disease and adverse patient outcomes. This may be because overexpression of these genes confers resistance to standard therapies (Butt et al., 2008). Cyclins may be useful targets for anti-cancer drug design. Although palbociclib is a promising agent in pre-clinical and clinical settings, it appears to be less effective in cell lines that over-express cyclin E1, which occurs commonly in tumours (Kanska et al., 2016). Accordingly, instead of selective targeting of CDKs, agents that target multiple cell cycle regulatory proteins may have advantages over CDK inhibitors.
    Although there are no established therapeutic agents that selectively target cyclin proteins that are dysregulated in cancer a number of natural product chemicals reportedly impair the expression of one or more cell cycle regulatory proteins, including cyclins. The fungal pro-duct bassiatin selectively targeted cyclin D1 to induce cell cycle arrest in MCF-7 breast cancer cells (Meng et al., 2011), and the flavonoid