Arctigenin is a plant lignan extracted from Arctium lappa that has been shown to have estrogenic properties. In spite of the health benefits of phytoestrogens reducing the risk of osteoporosis, heart disease, and menopausal symptoms, its benefits against the risk of breast cancer have not been fully elucidated. Thus, we investigated the effects of arctigenin on metastasis of breast cancer using both estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cell lines to see if the effects are dependent on the status of ER expression. In ER-positive MCF-7 cells, arctigenin efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration and invasion. The activity of crucial metastatic protease matrix metalloprotease (MMP)-9 in gelatin zymography was also efficiently decreased by arctigenin, as well as its mRNA expression. Notably, arctigenin exhibited similar anti-metastatic effects even in ER-negative MDA-MB-231 cells, suggesting that the anti-metastatic effects of arctigenin were not exerted via the ER. The upstream signaling pathways involved in the regulation of MMP-9 and urokinase plasminogen activator (uPA) were analyzed using western blotting. The activation of Akt, NF-κB and MAPK (ERK 1/2 and JNK 1/2) was found to be inhibited. Taken together, these data suggest that arctigenin confers anti-metastatic effects by inhibiting MMP-9 and uPA via the Akt, NF-κB and MAPK signaling pathways on breast cancer, regardless of ER expression. Therefore, we propose that the intake of arctigenin could be an effective supplement for breast cancer patients.

Arctigenin, a member of the Asteraceae family, is a biologically active lignan that is consumed worldwide due to its several health benefits. However, its use may pose a problem for patients with estrogen receptor (ER)α-positive breast cancer, since studies have shown that arctigenin is a phytoestrogen that exerts a proliferative effect by binding to the ER. Thus, in this study, we examined the effect of arctigenin on ERα-positive MCF-7 human breast cancer cells to determine whether the consumption of arctigenin is safe for patients with breast cancer. First, we found that arctigenin inhibited the viability of the MCF-7 cells, and colony formation assay confirmed that this effect was cytotoxic rather than cytostatic. The cytotoxic effects were not mediated by cell cycle arrest, apoptosis, or necroptosis, despite DNA damage, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage and phosphorylated H2A.X. An increase in lipidated LC3, a marker of autophagosome formation, was observed, indicating that autophagy was induced by arctigenin, which was found to be triggered by the inhibition of the mechanistic target of rapamycin (mTOR) pathway. We then examined the effects of arctigenin on ERα expression and determined whether it affects the sensitivity of the cells to tamoxifen, as tamoxifen is commonly used against hormone-responsive cancers and is known to act via the ERα. We found that treatment with arctigenin effectively downregulated ERα expression, which was found to be a consequence of the inhibition of the mTOR pathway. However, treatment with arctigenin in combination with tamoxifen did not affect the sensitivity of the cells to tamoxifen, but instead, exerted a synergistic effect. On the whole, our data indicate that the phytoestrogen, arctigenin, mainly targeted the mTOR pathway in ERα-positive MCF-7 human breast cancer cells, leading to autophagy-induced cell death and the downregulation of ERα expression. Furthermore, the synergistic effects between arctigenin and tamoxifen suggest that the consumption of arctigenin is not only safe for patients with hormone-sensitive cancers, but may also be an effective co-treatment.

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BackgroundArctigenin is a biologically active plant lignan extracted from Arctium lappa that is consumed worldwide for its several health benefits. However, it might pose a problem for estrogen receptor (ER)-positive human breast cancer patients since several studies show that arctigenin is a phytoestrogen that could exert a proliferative effect by binding to the ER. Therefore, we studied the effect of arctigenin in MCF-7 ER-positive and MDA-MB-231 triple-negative human breast cancer cells. Methods and Results SRB demonstrated that arctigenin inhibited the cell viability of MCF-7 cells, and colony forming assay confirmed that this effect was cytotoxic rather than cytostatic. Western blot analysis indicated that arctigenin induced DNA damage without cell cycle arrest, apoptosis, or necroptosis. The increase in lipidated LC3 indicated that arctigenin caused autophagyinduced cell death, which was triggered by an inhibition of mTOR pathway and activation of AMPK. Arctigenin downregulated ERa and its downstream signaling, which was a consequence of the inhibition of mTOR pathway. SRB assay showed that arctigenin did not interfere with the sensitivity of the cells to tamoxifen, anti-cancer drug commonly used in ER-positive breast cancer treatment, but instead exerted a synergistic effect. Furthermore, wound-healing assay and invasion assay demonstrated the antimetastatic effects of arctigenin. RT-PCR, zymography and western blotting revealed that arctigenin exerted its effects by inhibiting MMP9 via the Akt/NF-kB and MAPK/AP-1 signaling pathways. The antimetastatic effects were observed in both ERa positive MCF-7 and triple-negative MDA-MB-231 human breast cancer cells. ConclusionArctigenin was cytotoxic to ER-positive human breast cancer cells, via inhibition of mTOR pathway resulting in autophagy-induced cell death and DNA damage, as well as down regulation of ERa and its downstream signaling. Arctigenin also inhibited cell migration and invasion of human breast cancer cells, irrespective of ERa expression, by inhibiting crucial tumorigenic signaling pathways. Hence, arctigenin is not only safe for consumption by ER-positive breast cancer patients it may also be a natural anti-breast cancer supplement.