br NF kB is a transcription factor that is
NF-kB, is a transcription factor that is present as an inactive complex bound to its endogenous inhibitor, IkB in the cytoplasm . Phosphorylation of IkB induces its degradation and release of NF-kB from the complex, allowing NF-kB to translocate to the nucleus . Nuclear translocation of NF-kB activates genes involved in cell proliferation (cyclins/CDKs), thereby promoting cell growth . 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 . 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 . 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 . 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 . Constitutively active STAT3 induces gene expression changes and molecular events that result in dysregulated cell growth, resistance to apoptosis, and promotion of metastasis . 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) . 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 . Moreover, Ohkawara et al. showed that the TAK1-NLK pathway plays a critical role in the phosphorylation of STAT3 at Ser727 . 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.
Conflict of interests
All authors declare that they have no conflicts of interest.
This research did not receive any funding.
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