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    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.
    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 Concanamycin A 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,