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.