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 .