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    2020-08-12

    r> Statistical analyses
    Data are represented as mean SD. Non-repeated ANOVA with post hoc Dunnett’s test was performed to determine the statistical significance compared to a corresponding negative control. Statistical significance was defined as p < 0.05. The IC50 of nor-wogonin was calculated using GraphPad Prism 5 (Version 5.01, GraphPad Software, San Diego, CA, USA) from the results of the trypan blue exclusion assay. Data are representative of three independent experiments.
    Results
    Nor-wogonin has superior antiproliferative and cytotoxic activities in human TNBC cells, compared to its structurally related compounds
    Cell proliferation of four TNBC cell lines (MDA-MB-231, BT-549, HCC70, and HCC1806) and two non-tumorigenic breast cell lines  (MCF-10A and AG11132) were assessed using BrdU incorporation assays after treatment with different polyhydroxy flavones, including nor-wogonin, wogonin, and wogonoside (Fig. 1A). We found that nor-wogonin inhibited the proliferation of TNBC cells in a dose-dependent manner; however, it had little or no impact on the growth of the non-tumorigenic breast cells (Fig. 1B). A head-to-head comparison of anti-proliferative effect against TNBC cell lines showed that nor-wogonin (40 mM) was more potent than wogonin (100 mM) or wogonoside (100 mM) in all lines tested. Unlike nor-wogonin, wogonin and wogonoside significantly reduced the growth of the non-tumorigenic breast cells (Fig. 1B). These findings were further confirmed using an alternate assay to monitor cell viability, as determined using the widely-accepted trypan blue exclusion assay (Fig. 1C). Nor-wogonin (80 mM) reduced the percent viability of MDA-MB-231, BT-549, HCC70, and HCC1806 cells to 32, 31.8, 40.5, and 35.3%, respectively. Nor-wogonin did not significantly reduce the viability of MCF-10A and AG11132 cells. The IC50s of nor-wogonin in MDA-MB-231, BT-549, HCC70, and HCC1806 were 32.24, 56.2, 39.05 and 37.3 mM, respectively, while the IC50s of nor-wogonin for non-tumorigenic breast cells (MCF-10A and AG11132) were more than 100 mM. Therefore, the effects of nor-wogonin on proliferation and viability is more evident in cancer cells when compared to non-tumorigenic cells.
    Nor-wogonin induces ABT888 arrest in TNBC cells that can be correlated with modulation of the expression regulators of cell cycle progression
    To elucidate the mechanism of action underlying the antiproliferative effects of nor-wogonin in TNBC cells, next we tested whether nor-wogonin affected cell cycle progression of MDA-MB-231 cells. As shown in Fig. 2A and B, treatment of MDA-MB-231 cells with nor-wogonin resulted in a dose-dependent increase in the percentage of cells in the G1 phase with a concomitant decrease in the percentage of those in the S phase, and a dose- and time-dependent increase in the G2/M phases. These results suggest that nor-wogonin induces cell cycle arrest at both the G1 and G2/M phases, although the induction of G2/M arrest was more significant. To determine the molecular mechanism by which nor-wogonin induced cell cycle arrest, we examined the effects of nor-wogonin on the expression of a few well-known cell cycle regulatory proteins, e.g., the cyclin dependent kinase inhibitor p21, cyclin dependent kinases (CDK1 and CDK4), and cyclins (cyclin B1 and cyclin D1). We observed that nor-wogonin upregulated p21 protein expression and downregulated cyclin D1, cyclin B1, and CDK1 protein expressions in a dose-and time-dependent manner (Fig. 2C, D, E, and F). Moreover, nor-wogonin downregulated CDK4 in a dose-depen-dent manner only (Fig. 2C and E). These results show that nor-wogonin induced cell cycle arrest in TNBC that can be correlated with upregulation of p21 and downregulation of expression of cyclins and CDKs that regulate cell cycle progression.
    Nor-wogonin induces apoptosis in TNBC cells via a caspase-dependent mitochondrial mechanism
    The cytotoxic effects of nor-wogonin (Fig. 1C), as well as nor-wogonin-induced increase in the percentage of cells in the sub G1 phase (Fig. 2A and B) suggested that nor-wogonin might induce apoptosis. To test this hypothesis, we further characterized the proapoptotic effects of nor-wogonin in MDA-MB-231 cells. Results from flow cytometry experiments indicated that nor-wogonin increased the percentage of both early and late apoptotic cells in a dose-dependent manner (annexin-V positive cells; Fig. 3A). Mitochondrial changes, including loss of mitochondrial membrane potential (DCm), are considered as key events in
    phytochemical agents-induced apoptosis in cancer cells. Thus, the effects of nor-wogonin on the mitochondria, in particular the changes in DCm, were examined using the lipophilic dye JC-1. Results presented in Fig. 3B indicate that nor-wogonin induced decreases in DCm in a dose-dependent manner in MDA-MB-231 cells. To further investigate whether nor-wogonin induced apoptosis by triggering the mitochondrial apoptosis pathway,