Icariin selectively inhibits breast cancer cell proliferation in vitro.
To investigate the anti-proliferation effect of icariin on breast cancer cells, the human triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-453, murine breast cancer cell line 4T1 and human mammary epithelium cell line MCF-10A were assessed via MTT assay. As shown in Fig. 1B, C, D and E, icariin exhibited a concentration-dependent and time-dependent cytotoxicity towards both the MDA-MB-231, MDA-MB-453 and 4T1 cell lines, but had little cytotoxicity to MCF-10A cells. Furthermore, colony formation assays were exploited to confirm the anti-proliferation effect of icariin. As displayed in Fig. 1F and G, the colony formation ability of the cells treated with 10 μM or 20 μM of icariin was significantly inhibited in comparison with the control group (P <0.001). Moreover, the size of the colonies exposed to icariin was smaller than those of the control group. Colony formation of MCF-10A cells were hardly inhibited by treatment with icariin.
Icariin selectively induces apoptosis in breast cancer cells via the mitochondria-mediated apoptotic pathway.
For the purpose of examining whether the anti-proliferation effect of icariin towards breast cancer cells was associated with apoptosis, Annexin V-FITC/PI dual-staining assays utilizing FCM were conducted after treatment with icariin. As shown in Fig. 2A and B, compared with the control group, the apoptotic rate of the MDA-MB-231 cells treated with 10 μM or 20 μM icariin was 26.53±2.3% (P <0.001) and 45.17±2.4% (P <0.001), respectively. Treatment of 10 μM or 20 μM induced 13.1±2.1% (P <0.001) and 18.2±1.6% (P <0.001) apoptosis in MDA-MB-453 cells respectively in comparison with control group. As for the 4T1 cells, cell apoptosis was noticeably triggered following treatment with icariin (20 μM, P <0.001) in comparison with the control group. However, little apoptosis was observed in MCF-10A cells after treatment with icariin. From the results of western blot analysis (Fig. 2C), the expression levels of cleaved caspase3 and Bax were increased in a concentration-dependent manner, whereas that of Bcl-2 was distinctly inhibited. In addition, the ratio of Bax/Bcl-2 was significantly increased after treatment with icariin (Fig. 2D), suggesting that the mitochondrial apoptotic pathway might participate in the observed icariin-induced apoptosis.
To verify the above hypothesis and assess the role of mitochondria in icariin-induced cell apoptosis, we investigated whether icariin could induce a loss of ΔΨm in 4T1 cells. As shown in Fig. 2E, icariin caused a significant loss of ΔΨm in a concentration-dependent manner in comparison with the control group. Because a loss of ΔΨm participates in the generation of ROS,(Chauhan et al., 2003) we measured the ROS levels in the 4T1 cells. As displayed in Fig. 2F, the level of ROS was dramatically increased after treatment with icariin in a concentration-dependent manner; however, pre-treatment with NAC, a ROS inhibitor, significantly inhibited this increased ROS generation (Fig. 2G). Together, these results suggested that icariin triggers breast cancer cell apoptosis via the mitochondrial-mediated apoptotic pathway (Fig. 2H).
Icariin suppresses migration and invasion of breast cancer cells viathe SIRT6/ NF-κB/EMT pathway.
Wound healing assays were used to assess the anti-migration ability of icariin in 4T1 and MDA-MB-231 cells. As shown in Fig. 2A and B, the mobilities of the MDA-MB-231 and 4T1 cells were inhibited following treatment with icariin in a concentration-dependent manner. Furthermore, transwell-migration assays were conducted to investigate the anti-migration effect of icariin. The results of this analysis showed that icariin could significantly suppress the migration of both MDA-MB-231 and 4T1 cells in a concentration-dependent manner in comparison with the control group (P <0.001). Moreover, the results of transwell-invasion analyses indicated that, compared with the control group, icariin exhibited an obvious anti-invasion effect on both MDA-MB-231 and 4T1 cells in a concentration-dependent manner (P <0.001).
Accumulated evidences have shown that activation of the NF-κB pathway plays an important role in cancer cell migration and invasion.(Wu & Zhou, 2009) As such, we next examined whether icariin could impair breast cancer cell migration and invasion via the NF-κB pathway. SIRT6 (sirtuin 6) is a specific histone H3 lysine 9 (H3K9) deacetylase that modulates chromatin structure, and deacetylation of H3K9 has been shown to play an important role in gene suppression.(Kouzarides, 2007; Michishita et al., 2008) Recent studies have indicated that SIRT6 deacetylates H3K9 at the promoters of NF-κB target genes to destabilize NF-κB.(Kawahara et al., 2009) Therefore, we attempted to detect the expression level of SIRT6 and acetylated H3K9 in MDA-MB-231 cells after treatment with icariin. As displayed in Fig. 4A and B, when the cells were exposed to 20 μM of icariin the expression level of SIRT6 was significantly increased, whereas acetylated H3K9 was significantly reduced, indicating that icariin served to upregulate the expression level of SIRT6 and promote the deacetylation of H3K9. To investigate the possible inhibition of the NF-κB pathway by icariin, both phosphorylated IκBα, which binds to the NF-κB transcription complex and suppresses its nuclear translocation, and the NF-κB p65 subunit, which serves as a transcription factor and oncogene, were assessed via western blot analysis.(Hayden & Ghosh, 2008; Kim, Hawke & Baldwin, 2006; Xiao et al., 2016) As shown in Fig. 4C and D, the expression levels of p-IκBα in the cytoplasm and NF-κB p65 in the nucleus were both significantly inhibited following treatment with icariin. Importantly, abnormal activation of NF-κB might function as a contributor to the EMT,(Chua, Bhat-Nakshatri, Clare, Morimiya, Badve & Nakshatri, 2007) and we found that MDA-MB-231 cells exposed to icariin dramatically upregulated the expression level of E-cadherin and downregulated the expression level of N-cadherin and MMP-2 (Fig. 4E and F).
Additionally, we used oss-128167, an inhibitor of SIRT6, to investigate whether icariin targets SIRT6 and subsequently affects the activation of the NF-κB pathway. After pre-treatment with 20 μM oss-128167 for 2 h, icariin demonstrated little effect on the expression levels of acetylated H3K9 or p-IκBα (Fig. 4G and H). In addition, the anti-migration and anti-invasion effects of icariin in MDA-MB-231 cells were both significantly attenuated following pre-treatment with oss-128167 (Fig. 4I, P <0.001). Furthermore, we evaluated the translocation of NF-κB after treatment with icariin in MDA-MB-231 cells. As illustrated by Figure 4J, the expression of NF-κB p65 located in both cytoplasm and nucleus, resulting from constitutive activation of NF-κB in MDA-MB-231 cells. When treated with icariin, the expression of NF-κB p65 in nucleus was significantly reduced. More importantly, icariin could significantly attenuate nuclear translocation of NF-κB p65 protein which was induced by treatment of TNF-α. Taken together, result above suggested that icariin could upregulate the expression level of SIRT6 and deacetylate H3K9, followed by inhibiting the activation of NF-κB and the EMT process. Ultimately, icariin treatment clearly impaired MDA-MB-231 cell migration and invasion in vitro .