br Article history br Keywords br Breast cancer br
Cell cycle arrest
Background: Nor-wogonin, a polyhydroxy flavone, has been shown to possess antitumor activity. However, the mechanisms responsible for its antitumor activity are poorly studied. Herein, we investigated the mechanisms of nor-wogonin actions in triple-negative breast cancer (TNBC) cells.
Methods: Effects of nor-wogonin on cell proliferation and viability of four TNBC cell lines (MDA-MB-231, BT-549, HCC70, and HCC1806) and two non-tumorigenic breast cell lines (MCF-10A and AG11132) were assessed by BrdU incorporation assays and trypan blue dye exclusion tests. Cell cycle and apoptosis analyses were carried out by flow cytometry. Protein expression was analyzed by immunoblotting.
Results: Nor-wogonin significantly inhibited the growth and decreased the viability of TNBC cells; however, it exhibited no or minimal effects in non-tumorigenic breast cells. Nor-wogonin (40 mM) was a more potent anti-proliferative and cytotoxic agent than wogonin (100 mM) and wogonoside (100 mM), which are structurally related to nor-wogonin. The antitumor effects of nor-wogonin can be attributed to GSK 3 arrest via reduction of the expression of cyclin D1, cyclin B1, and CDK1. Furthermore, nor-wogonin induced mitochondrial apoptosis, (as evidenced by the increase in % of cells that are apoptotic), decreases in the mitochondrial membrane potential (DCm), increases in Bax/Bcl-2 ratio, and caspase-3 cleavage. Moreover, nor-wogonin attenuated the expression of the nuclear factor kappa-B and activation of signal transducer and activator of transcription 3 pathways, which can be correlated with suppression of transforming growth factor-β-activated kinase 1 in TNBC cells.
Conclusion: These results showed that nor-wogonin might be a potential multi-target agent for TNBC treatment.
© 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.
* Corresponding author.
E-mail address: [email protected] (A.A. Abd El-Hafeez).
1734-1140/© 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.
Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks estrogen receptors, progesterone receptors, and human epidermal growth factor 2 overexpression . It comprises 15–20% of all breast cancer cases and is considered a clinical challenge because this cancer does not respond to available targeted agents, mandating the search for new agents for treatment of TNBC .
Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a serine/threonine kinase, which is frequently involved in human cancer development . Activation of TAK1 in cancer cells results in the activation of nuclear factor kappa-B (NF-kB) and activator protein-1 (AP-1), which are key transcription factors that regulate the expression of many genes involved in cancer progression [3,4]. Moreover, signal transducer and activator of transcription 3 (STAT3) plays pivotal roles in tumor cell proliferation, survival, and invasion . STAT3 must be activated by its phosphorylation in
Fig. 1. Proliferation and viability of TNBC cells and non-tumorigenic breast cells after treatment with nor-wogonin and structurally related compounds. A. Chemical structures of nor-wogonin, wogonin, and wogonoside. B. Effects of nor-wogonin (5–80 mM), wogonin (100 mM), and wogonoside (100 mM) on the proliferation of TNBC cells (MDA-MB-231, BT-549, HCC70, and HCC1806) and non-tumorigenic breast cells (MCF-10A and AG11132) were determined by BrdU incorporation assays. Dimethyl sulfoxide (DMSO vehicle) was used as a negative control. C. The cytotoxic effects of nor-wogonin (5–80 mM), wogonin (100 mM), and wogonoside (100 mM) on TNBC cells (MDA-MB-231, BT-549, HCC70, and HCC1806) and non-tumorigenic breast cells (MCF-10A and AG11132) were determined by trypan blue exclusion assays. DMSO was used as a vehicle control. Data are expressed as the means SD based on three independent experiments.
different sites . Ohkawara et al. showed that the TAK1/Nemo-like kinase (NLK) pathway is responsible for the phosphorylation of STAT3 at Ser727 . Collectively, since TAK1, NF-kB, and STAT3 were found to be activated in TNBC and play critical roles in TNBC progression and chemoresistance, new chemotherapeutic agents that can inhibit TAK1, NF-kB, and STAT3 signaling have the potential to improve the outcome of patients with TNBC [8–10].
In this context, several natural products have been shown to possess potent antitumor activities [11,12]. Wogonin (5,7-dihy-droxy-8-methoxyflavone) and nor-wogonin (5,7,8-trihydroxyfla-vone) are bioactive polyhydroxy flavones isolated from Scutellaria baicalensis Georgi [13,14]. The antitumor activity and mechanisms of action of wogonin have been studied in several cancers, including breast, leukemia, and colorectal cancers . Nor-wogonin is a flavone that is structurally related to wogonin; they differ in the presence of OH group at the C-8 position in nor-wogonin instead of methoxy (OMe) group in wogonin (Fig.1A). The anticancer activity and mechanisms of action of nor-wogonin are poorly studied. Chow et al., reported that nor-wogonin was more effective than wogonin in inducing apoptosis in HL-60 leukemia cells . However, the molecular mechanisms under-lying the antitumor effects of nor-wogonin have been poorly investigated. The structural similarity between wogonin and nor-wogonin prompted us to elucidate the mechanisms of nor-wogonin actions in TNBC cells.