br Fig Effects of nor wogonin on expression of
Fig. 5. Effects of nor-wogonin on expression of NF-kB, STAT3, and TAK1 in MCF-10A cells. A. The expression levels of NF-kB (p65), p-STAT3 (Ser727), STAT3, and TAK1 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), p-STAT3 (Ser727), STAT3, and TAK1 were quantified relative to GAPDH. Values represent the means SD of three independent experiments.
cells, which may be attributed to direct upregulation of p21 and downregulation of other regulators of the Diphenylterazine including cyclin D1, cyclin B1, and CDK1. Apoptosis is another possible mechanism that can contribute to the cytotoxicity of polyhydroxy flavones in cancer cells . For example, approximately 50% of TNBC cells underwent apoptosis after treatment with 100 mM wogonin  while around 60% of osteosarcoma cells underwent apoptosis after treatment with 75 mM of wogonoside . Herein, approximately 60% of TNBC cells underwent apoptosis after treatment with 40 mM of nor-wogonin which indicates that nor-wogonin is a more potent pro-apoptotic agent than its structurally related compounds, wogonin and wogonoside. Mechanistically, apoptosis can be induced through two different pathways that are known as the death receptor (extrinsic) and mitochondrial (intrinsic) pathways . Changes in the mitochondrial membrane potential (DCm) have been considered to be early events in the mitochondrial pathway [30,31]. Along this line, we observed that nor-wogonin induced loss of DCm. which may support the ability of these phytochem-icals to induce apoptosis via an intrinsic mitochondrial pathway. Some pro-apoptotic members of Bcl-2 family such as Bax and Bak can form membrane channels, resulting in the loss of Dcm . In contrast, anti-apoptotic molecules such as Bcl-2 can prevent the conformational change and oligomerization of Bax and Bak [33,34]. Furthermore, induction of apoptosis is correlated with an increase in the ratio of proapoptotic/antiapoptotic Bax/Bcl-2 [35,36]. Our results showed that nor-wogonin treatment down-regulated Bcl-2 protein expression and upregulated Bax protein expression, resulting in an increase in Bax/Bcl-2 ratio, which could be a possible mechanism by which nor-wogonin induced a loss in the Dcm. An increase in the mitochondrial membrane permeabil-ity results in the release of some apoptotic proteins and eventually caspase-3 activation . Our data showed that nor-wogonin activated caspase-3 in TNBC cells. In addition, nor-wogonin-induced increase in the percent of annexin V positive cells was significantly attenuated in the presence of the pan caspase inhibitor, Z-VAD-FMK, suggesting that nor-wogonin-induced apoptosis through a caspase-dependent mitochondrial pathway.
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 cell cycle arrest may be partially attributed to the inhibition of the NF-kB pathway.