Discussion
Breast cancer is the most common type of diagnosed cancer among women worldwide and demonstrates considerable metastatic potential, multi-drug resistance, and high mortality.(Peart, 2017) With advancements in medical technology, early diagnosis of breast cancer and treatment with chemoradiation therapy on its own or in combination with surgery can effectively retard the cancer’s progress. However, the treatment of breast cancer remains a significant challenge, and the need for more effective drug candidates is crucial. It has been reported that both abnormal activation and overexpression of NF-κB are both closely associated with the progression and metastasis of breast cancer.(Fusella et al., 2017; Yang et al., 2018) As such, we hypothesized that inhibition of NF-κB might be a promising therapeutic target for the treatment of this cancer.
In this study, we reported that icariin, a natural prenylated flavonol glycoside, exhibited a well-behaved anti-cancer effect towards breast cancer. Our results of MTT and colony formation assays indicated that icariin could selectively inhibit the viability and proliferation of MDA-MB-231 and 4T1 cells in a concentration- and time-dependent manner. Moreover, icariin significantly induced apoptosis in MDA-MB-231 and 4T1 cells. However, understanding the detailed mechanisms underlying how icariin kills breast cancer cells still requires further investigation.
Mitochondria are the major source of ROS generation, which plays an important role in cell apoptosis by altering the mitochondrial membrane potential (ΔΨm).(van Loo, Saelens, van Gurp, MacFarlane, Martin & Vandenabeele, 2002) The anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax clearly participate in the mitochondria-mediated apoptotic pathway.(Rogers, Erkes, Nardone, Aplin, Fernandes-Alnemri & Alnemri, 2019) In this study, activation of cleaved caspase3 and an increase in Bax/Bcl-2 were observed in 4T1 cells after treatment with icariin, as assessed via western blot analysis, implying that icariin could induce cell apoptosis by way of the mitochondria-mediated apoptotic pathway. Moreover, 4T1 cells treated with icariin induced a significant loss of ΔΨm and demonstrated increased ROS production. It was also observed that production of ROS in 4T1 cells induced by icariin could be attenuated by pre-treatment with the antioxidant NAC, suggesting that the balance of ROS in the mitochondria was involved in the induction of cell apoptosis by icariin. Furthermore, icariin showed significant inhibition of tumor growth in a tumor mouse model of 4T1 cells. Similarity, Li et al. demonstrated that icariin could induce apoptosis in human hepatoma cells via a ROS/JNK-dependent mitochondrial pathway,(Li et al., 2010) and Gu et al. reported that icariin could exert an inhibitory effect on the growth of human esophageal carcinoma cells via ROS-mediated alterations in ΔΨm.(Gu, Zhang, Wang & Xu, 2017) Therefore, these results indicated that treatment with icariin likely triggered apoptosis in breast cancer cells via a ROS-mediated mitochondrial apoptotic pathway.
One-eighth of all breast cancers have already initiated cell migration and invasion at the time of diagnosis, having metastasized to the lymph nodes or distant organs.(Chambers, Groom & MacDonald, 2002) Therefore, the anti-metastasis effect of icariin was evaluated in MDA-MB-231 and 4T1 cells via transwell migration and invasion assays. The wound healing and transwell assays showed that icariin dramatically inhibited breast cancer cell migration and invasion, and our in vivo study showed that icariin distinctly inhibited tumor lung metastasis in a tumor mouse pulmonary metastatic model of 4T1 cells. Subsequently, we further investigated the mechanism as to how icariin could effectively inhibit the migration and invasion of breast cancer cells. Aberrant NF-κB pathway activation could function as an important contributor to the EMT process by directly or indirectly affecting SNAIL, SLUG, and ZEB1 expression levels.(Menezes et al., 2018) Additionally, IκBα, which inhibits activation of NF-κB, could be phosphorylated by an activated Iκκ complex (NEMO, Iκκα, and Iκκβ) to induce self-degradation.(Brady, Haas, Farrell, Pichlmair & Bowie, 2017) Subsequently, the NF-κB transcriptional complex is released into the nucleus where it binds the NRE and promotes the transcription of pro- metastatic genes.(Hayden & Ghosh, 2008; Kim, Hawke & Baldwin, 2006) Previous studies have shown that icariin can alleviate murine lupus nephritis and improve anemia hematopoietic stem cell function by inhibiting activation of NF-κB.(Li, Li, Cole, McLaughlin & Du, 2018; Su, Ye, You, Ni, Chen & Li, 2018) In this study, we found that the expression level of p-IκBα in the cytoplasm and NF-κB p65 in the nucleus were both significantly inhibited following treatment with icariin in 4T1 cells. Moreover, 4T1 cells exposed to icariin dramatically upregulated the expression level of E-cadherin and downregulated the expression levels of N-cadherin and MMP-2, suggesting that icariin impaired the migration and invasion of breast cancer cells by inhibiting the NF-κB/EMT pathway.
SIRT6 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 on promoters of NF-κB target genes to destabilize NF-κB.(Kawahara et al., 2009) The western blot results indicated that icariin significantly upregulated the expression level of SIRT6 and inhibited acylation of H3K9 in 4T1 cells. When pre-treated with 20 μM of the SIRT6 inhibitor, oss-128167, icariin had almost no effect on H3K9 acylation or IκBα phosphorylation. Moreover, pre-treatment with oss128167 clearly impaired the anti-migration and anti-invasion effects of icariin in 4T1 cells. Transcriptomic analysis verified that impairment of NF-κB leads to the selective function of icariin in breast cancer cells. Together, the above results indicated that icariin could inhibit breast cancer cell migration and invasion by upregulating expression of SIRT6, which was followed by impaired activation of the NF-κB/EMT pathway (Fig. 7F).
However, what is the relationship between NF-κB and ROS level? It is well established that ROS production is closely associated with mitochondrial structure.(Jezek, Cooper & Strich, 2018; Kim & Song, 2016) NF-κB acts as an oxidative stress response transcription factor, and NF-κB pathway can affect mitochondria dynamics.(Chen, Li, Guo, Xie & Chen, 2016) Vaisitti and colleagues reported that IT-901, a new small-molecule able to inhibit the NF-κB subunit c-Rel, exhibited a dose-dependent mitochondrial ROS production and a remarkable decrease of ATP production in chronic lymphocytic leukemia (CLL) cell lines, resulting from decreased expression levels of NF-κB-regulated gene (ATP5A1), involved in tricarboxylic acid cycle or scavenging processes.(Capece, Verzella, Di Francesco, Alesse, Franzoso & Zazzeroni, 2019; Vaisitti et al., 2017) Therefore, icariin might suppress tricarboxylic acid cycle to produce ROS and then trigger mitochondria dependent apoptosis in breast cancer cells by impairing NF-κB pathway (Fig. 6). More importantly, icariin could significantly ameliorate the tumor immunosuppressive microenvironment by enhancing the proportion of infiltrating CD4+/CD8+T cells and reduced the abundance of MDSCs in tumors. In conclusion, our study showed that icariin could effectively trigger redox-induced apoptosis, inhibit metastasis and regulate immunosuppressive microenvironment of TNBC via the SIRT6/NF-κB signaling pathway, indicating that icariin might serve as a potential candidate drug for the treatment of TNBC.