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  • br NF kB is a transcription factor that is

    2020-08-02


    NF-kB, is a transcription factor that is present as an inactive complex bound to its endogenous inhibitor, IkB in the cytoplasm [38]. Phosphorylation of IkB induces its degradation and release of NF-kB from the complex, allowing NF-kB to translocate to the nucleus [39]. Nuclear translocation of NF-kB activates genes involved in cell proliferation (cyclins/CDKs), thereby promoting cell growth [40]. Furthermore, there is a positive correlation between the activation of NF-kB and increase in the expression of antiapoptotic Bcl-2 family members, resulting in resistance to apoptosis [41]. Many polyhydroxy flavones have been shown to block NF-kB activation leading to tumor growth inhibition. For example, wogonoside (50–150 mM) inhibited the activation of NF-kB signaling via suppression of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and TRAF4 expression [42]. In addition, wogonin (15–60 mM) blocked the NF-kB pathway via inhibition of the phosphorylation of IkBα and inhibitor-kappa B kinase alpha (IKKα) in B16-F10 cells [43]. In the present study, we observed that nor-wogonin treatment (20–40 mM) significantly downregulated expression of NF-Kb/p65 that can be correlated with a reduction in the level of phospho-IkBα. Since nor-wogonin treatment reduced the expression of Bcl-2 and cyclin/CDK proteins, which are downstream targets of NF-kB, nor-wogonin-induced apoptosis and Amiloride HCL arrest may be partially attributed to the inhibition of the NF-kB pathway.
    STAT3 pathway is a major pathway that plays critical roles in the pathogenesis of many cancers, including breast cancer [44]. Constitutively active STAT3 induces gene expression changes and molecular events that result in dysregulated cell growth,  resistance to apoptosis, and promotion of metastasis [45]. The present study showed that nor-wogonin (10–40 mM) inhibited STAT3 activation in MDA-MB-231 cells via inhibition of STAT3 phosphorylation at Ser727. Interestingly, wogonin showed similar effects in CD4+ T cells, but at a slightly higher concentration (50 mM) [46]. Overall, inhibition of STAT3 and NF-kB activation could be the primary underlying mechanisms for nor-wogonin-induced cell cycle arrest and apoptosis in MDA-MB-231 cells. Furthermore, we investigated the effects of nor-wogonin on an upstream regulator of STAT3 and NF-kB in breast cancer cells, TAK1. TAK1 is a mitogen-activated protein (MAP) kinase that plays important roles in the activation of NF-kB and STAT3. TAK1-mediated NF-kB activation involves direct phosphorylation of IKK complex [47]. Moreover, Ohkawara et al. showed that the TAK1-NLK pathway plays a critical role in the phosphorylation of STAT3 at Ser727 [7]. In the present study, we found that nor-wogonin-induced inhibition of the NF-kB and STAT3 pathways in MDA-MB-231 cells can be correlated with a decrease in TAK1 expression (Fig. 4E and F). Moreover, the inhibitory actions of nor-wogonin on NF-kB, STAT3, and TAK1 pathways were not evident in a non-tumorigenic cell line (Fig. 5A and B), which is consistent with selective antiproliferative and cytotoxic effects on TNBC cells when compared to non-tumorigenic cells.
    In summary, our studies provided critical insights into the mechanistic basis for the antitumor activities of nor-wogonin in TNBC cells. Nor-wogonin inhibited the growth of TNBC cells by inducing cell cycle arrest and apoptosis. Furthermore, nor-wogonin inhibited TAK1 expression, which can be correlated with downregulation of NF-kB and STAT3 pathways. Therefore, nor-wogonin might be an attractive multi-target candidate drug for treatment of TNBC.
    Author’s contribution
    Conflict of interests
    All authors declare that they have no conflicts of interest.
    Financial support
    This research did not receive any funding.
    References
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    [8] Smith SM, Lyu YL, Cai L. NF-kB affects proliferation and invasiveness of breast cancer cells by regulating CD44 expression. PLoS One 2014;9:e106966.
    [9] Zhang L, Fu Z, Li X, Tang H, Luo J, Zhang D, et al. Transforming growth factor β-activated kinase 1 inhibitor suppresses the proliferation in triple-negative breast cancer through TGF-β/TGFR pathway. Chem Biol Drug Des 2017.