br Discussion br Flavonoids constitute common components in
Flavonoids constitute common components in human diet that exhibit many health benefits. Among them, polyhydroxy flavones, such as nor-wogonin and wogonin have been reported to exhibit many pharmacological activities which include anxiolytic, antimi-crobial, and anticancer activities [13–16]. Previously, nor-wogonin showed more potent anti-leukemic effects than wogonin due to potent proapoptotic activity . However, unlike wogonin, the anticancer effects and mechanisms of actions of nor-wogonin were poorly understood. Here we sought to investigate the anticancer activity and mechanisms of action of nor-wogonin in human TNBC cells. Our findings showed that nor-wogonin is both potent and specific anti-cancer agent; it selectively inhibited the proliferation and induced cytotoxicity in four human TNBC cell lines, MDA-MB-
231, BT-549, HCC70, and HCC1806, (IC50: 32.24–56.2 mM), whereas its effects in non-tumorigenic breast cells (MCF-10A and AG11132) were minimal (IC50 > 100 mM). Furthermore, nor-wogonin is a more potent antiproliferative and cytotoxic agent in TNBC cells when compared to structurally related anticancer compounds, such as wogonin (50–200 mM) and wogonoside (50–400 mM) [17,18]. From a structure-activity relationship (SAR) point of view, methoxylated flavones are less active in vitro as antiproliferative compounds, in comparison with the hydroxylated flavones. For example, Chow et al., analyzed the effect of six structurally related compounds, including 5-OH, 7-OH, 5,7-diOH, 5,7-diOCH3, 7,8-diOCH3, and 7-OCH3-8-OH flavones on HL-60 cells . Their results suggest that the hydroxyl group at C5 and C7 is essential for the anticancer activity of flavones . Moreover, Androutsopou-los et al., reported that hydroxylation of eupatorin in MDA-MB-468 human breast cancer cells as a result of CYP1-family enzymes-mediated metabolism can enhance its anti-proliferative effect. Our results are consistent with the above findings with regards to the importance of the hydroxyl groups in nor-wogonin, when compared to the methoxy (in wogonin) or the glycosides (in wogonoside) groups for anti-cancer activities .
The antiproliferative effects of polyhydroxy flavone derivatives have been previously shown to involve 178603-78-6 arrest at the G1 and G2/M phases [20,21]. We found that nor-wogonin inhibited the growth of MDA-MB-231 cells via cell cycle arrest at both the G1 and G2/M phases, although the induction of G2/M arrest was more significant. The mammalian cell cycle is controlled by cyclins and CDKs. Progression through the G1 phase is activated by the binding of cyclin D family (D1, D2, and D3) to CDK4 or its homolog, CDK6 (CDK4/CDK6). Progression through the G2/M phase is activated by the binding of cyclin B to CDK1 [22–24]. The CDK inhibitor p21, also known as p21waf1/cip1, is a well-known inhibitor of cell cycle and can arrest cell cycle progression at the G1/S and G2/M transitions by inhibiting CDK4,6/cyclin-D and CDK1/cyclin-B respectively . In our study, we showed that nor-wogonin upregulated the expres-sion of p21 in a dose- and time-dependent manner. Moreover, nor-wogonin downregulated the expression of the G1 and G2/M regulatory proteins in a dose-dependent manner. While cyclin D1 is typically associated with G1/S phase transition, cyclin D1 levels are also increased during the G2 phase [26,27]. Together our results suggest nor-wogonin-induced cell cycle arrest in MDA-MB-231
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 cell cycle 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.