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  • br we measured the expression levels

    2020-08-02


    we measured the expression levels of the antiapoptotic protein (Bcl-2) and proapoptotic proteins (Bax and caspase-3), which are involved in the mitochondrial apoptosis pathway. Western blot analysis results showed that nor-wogonin downregulated the expression of Bcl-2 and upregulated the expression of Bax, resulting in an increase in Bax/Bcl-2 ratio (Fig. 3C and D). Moreover, nor-wogonin induced caspase-3 activation (Fig. 3C and
    Fig. 3. Proapoptotic actions of nor-wogonin in MDA-MB-231 cells. A. Quantification of annexin V-positive apoptotic Nocodazole after treatment with nor-wogonin B. The average JC-1 red/green fluorescence ratio in MDA-MB-231 cells C. The expression of Bcl-2, Bax, and cleaved caspase-3 were determined by western blot analyses. GAPDH served as a loading control. D. Bax/Bcl-2 ratio was quantified by ImageJ software. E. The protein expression levels of cleaved caspase-3 relative to GAPDH, were quantified by ImageJ software. F. The expression levels of cleaved caspase-3 in vehicle or nor-wogonin (40 mM)-treated cells in the absence or presence of the pan-caspase inhibitor, Z-VAD-FMK (30 mM) were determined by western blot analysis. DMSO and GAPDH were used as negative and loading controls, respectively. G. Proportion of annexin-V positive cells after 24-h incubation with vehicle or nor-wogonin (40 mM)-treated cells in the absence or presence of the pan-caspase inhibitor, Z-VAD-FMK (30 mM). Values represent the means SD of three independent experiments. *p < 0.05 indicate significant differences, compared to the control or nor-wogonin-treated cells.
    E). To test whether caspase-3 pathway was essential for nor-wogonin-induced apoptosis, the pan-caspase inhibitor, Z-VAD-FMK was used. We found that Z-VAD-FMK reduced nor-wogonin-induced caspase-3 cleavage and percentage of apoptotic cells (Fig. 3F and G). These results indicated that nor-wogonin might induce apoptosis via a caspase-3 dependent mitochondrial pathway.
    Nor-wogonin suppressed the activation of NF-kB and STAT3 pathways that can be correlated with suppression of TAK1 expression in TNBC cells
    NF-kB and STAT3 are transcription factors involved in cancer progression. Activation of both NF-kB and STAT3 can contribute to TNBC cell proliferation and resistance to apoptosis [8,10]. To further understand the molecular mechanisms by which nor-
    Fig. 4. Effects of nor-wogonin on expression of NF-kB, STAT3, and TAK1 in MDA-MB-231 cells. A. The expression levels of NF-kB (p65), phospho-IKBα, and IKBα were determined by western blot analyses. B. The protein expression level of NF-kB (p65; nuclear fraction) was quantified relative to Histone H3, while the protein expression levels of NF-kB (p65; whole cell lysate), phospho-IKBα, and IkBα were quantified relative to GAPDH. C. The expression of p-STAT3 (Ser727) and STAT3 were determined by western blot analyses. D. The protein expression levels of p-STAT3 relative to STAT3 were quantified. E. The expression of TAK1 was measured by western blot analyses. F. The protein expression levels of TAK1 were quantified relative to GAPDH. Values represent the means SD of three independent experiments. *p < 0.05 indicate significant differences, compared to the negative control.
    wogonin induced cell cycle arrest and apoptosis, we next investigated whether nor-wogonin affected the NF-kB and STAT3 pathways in MDA-MB-231 cells using Western blot analysis. As shown in Fig. 4A and B, nor-wogonin treatment resulted in a dose-dependent decrease in NF-kB (p65), in the nuclear fraction as well as the whole cell lysate (WCL), and phospho-IkBα protein levels and increase in that of IkBα. Nor-wogonin inhibited the phosphor-ylation of STAT3 at Ser727 in a dose-dependent manner, resulting in a decrease in pSTAT3/STAT3 ratio (Fig. 4C and D). Both NF-kB and STAT3 are downstream targets for TAK1. Therefore, to understand of the molecular basis for the inhibitory effects of nor-wogonin on NF-kB and STAT3 expression, the effects of nor-wogonin on the expression of TAK1 was explored. We found that nor-wogonin reduced the expression of TAK1 in MDA-MB-231 cells, compared to that in the control (Fig. 4E and F). To test whether or not nor-wogonin-inhibited NF-kB, STAT3, and TAK1 pathways is selective for TNBC cells when compared to non-tumorigenic cells, we tested the effect of nor-wogonin on the expression of NF-kB (p65), p-STAT3, STAT3, and TAK1in MCF-10A non-tumorigenic breast cells. Results in Fig. 5A and B showed that nor-wogonin (40 mM) had no effect on the expression of NF-kB (p65), p-STAT3, STAT3, and TAK1in MCF-10A non-tumorigenic breast cells. These results showed that nor-wogonin inhibited the activation of the NF-kB and STAT3 pathways in TNBC cells that can be correlated with suppression of TAK1 expression in TNBC cells.