References
Amin AR, Kucuk O, Khuri FR, & Shin DM (2009). Perspectives for cancer
prevention with natural compounds. J Clin Oncol 27: 2712-2725.
Brady G, Haas DA, Farrell PJ, Pichlmair A, & Bowie AG (2017). Molluscum
Contagiosum Virus Protein MC005 Inhibits NF-kappaB Activation by
Targeting NEMO-Regulated IkappaB Kinase Activation. J Virol 91.
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, & Jemal A
(2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence
and mortality worldwide for 36 cancers in 185 countries. CA Cancer J
Clin 68: 394-424.
Capece D, Verzella D, Di Francesco B, Alesse E, Franzoso G, & Zazzeroni
F (2019). NF-kappaB and mitochondria cross paths in cancer:
mitochondrial metabolism and beyond. Semin Cell Dev Biol.
Chambers AF, Groom AC, & MacDonald IC (2002). Dissemination and growth
of cancer cells in metastatic sites. Nat Rev Cancer 2: 563-572.
Chauhan D, Li G, Sattler M, Podar K, Mitsiades C, Mitsiades N, et
al. (2003). Superoxide-dependent and -independent mitochondrial
signaling during apoptosis in multiple myeloma cells. Oncogene
22: 6296-6300.
Chen L, Li S, Guo X, Xie P, & Chen J (2016). The role of GSH in
microcystin-induced apoptosis in rat liver: Involvement of oxidative
stress and NF-kappaB. Environ Toxicol 31: 552-560.
Chen M, Wu J, Luo Q, Mo S, Lyu Y, Wei Y, et al. (2016). The
Anticancer Properties of Herba Epimedii and Its Main Bioactive
Componentsicariin and Icariside II. Nutrients 8.
Chen S, Han Q, Wang X, Yang M, Zhang Z, Li P, et al. (2013).
IBP-mediated suppression of autophagy promotes growth and metastasis of
breast cancer cells via activating mTORC2/Akt/FOXO3a signaling pathway.
Cell Death Dis 4: e842.
Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, & Nakshatri H
(2007). NF-kappaB represses E-cadherin expression and enhances
epithelial to mesenchymal transition of mammary epithelial cells:
potential involvement of ZEB-1 and ZEB-2. Oncogene 26: 711-724.
Fidler IJ, & Poste G (2008). The ”seed and soil” hypothesis revisited.
Lancet Oncol 9: 808.
Fusella F, Secli L, Busso E, Krepelova A, Moiso E, Rocca S, et
al. (2017). The IKK/NF-kappaB signaling pathway requires Morgana to
drive breast cancer metastasis. Nat Commun 8: 1636.
Geng YD, Yang L, Zhang C, & Kong LY (2014). Blockade of epidermal
growth factor receptor/mammalian target of rapamycin pathway by
Icariside II results in reduced cell proliferation of osteosarcoma
cells. Food Chem Toxicol 73: 7-16.
Gu ZF, Zhang ZT, Wang JY, & Xu BB (2017). Icariin exerts inhibitory
effects on the growth and metastasis of KYSE70 human esophageal
carcinoma cells via PI3K/AKT and STAT3 pathways. Environ Toxicol
Pharmacol 54: 7-13.
Hayden MS, & Ghosh S (2008). Shared principles in NF-kappaB signaling.
Cell 132: 344-362.
He W, Sun H, Yang B, Zhang D, & Kabelitz D (1995). Immunoregulatory
effects of the herba Epimediia glycoside icariin. Arzneimittelforschung
45: 910-913.
Huang S, Pettaway CA, Uehara H, Bucana CD, & Fidler IJ (2001). Blockade
of NF-kappaB activity in human prostate cancer cells is associated with
suppression of angiogenesis, invasion, and metastasis. Oncogene
20: 4188-4197.
Inigo-Marco I, & Alonso MM (2019). Destress and do not suppress:
targeting adrenergic signaling in tumor immunosuppression. J Clin Invest
129: 5086-5088.
Jezek J, Cooper KF, & Strich R (2018). Reactive Oxygen Species and
Mitochondrial Dynamics: The Yin and Yang of Mitochondrial Dysfunction
and Cancer Progression. Antioxidants (Basel) 7.
Karagiannis GS, Pastoriza JM, Wang Y, Harney AS, Entenberg D, Pignatelli
J, et al. (2017). Neoadjuvant chemotherapy induces breast cancer
metastasis through a TMEM-mediated mechanism. Sci Transl Med 9.
Karin M, & Greten FR (2005). NF-kappaB: linking inflammation and
immunity to cancer development and progression. Nat Rev Immunol
5: 749-759.
Kawahara TL, Michishita E, Adler AS, Damian M, Berber E, Lin M, et
al. (2009). SIRT6 links histone H3 lysine 9 deacetylation to
NF-kappaB-dependent gene expression and organismal life span. Cell
136: 62-74.
Kim B, & Song YS (2016). Mitochondrial dynamics altered by oxidative
stress in cancer. Free Radic Res 50: 1065-1070.
Kim HJ, Hawke N, & Baldwin AS (2006). NF-kappaB and IKK as therapeutic
targets in cancer. Cell Death Differ 13: 738-747.
Kouzarides T (2007). Chromatin modifications and their function. Cell
128: 693-705.
Lee KS, Lee HJ, Ahn KS, Kim SH, Nam D, Kim DK, et al. (2009).
Cyclooxygenase-2/prostaglandin E2 pathway mediates icariside II induced
apoptosis in human PC-3 prostate cancer cells. Cancer Lett 280:93-100.
Li S, Dong P, Wang J, Zhang J, Gu J, Wu X, et al. (2010).
Icariin, a natural flavonol glycoside, induces apoptosis in human
hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway.
Cancer Lett 298: 222-230.
Li Y, Li X, Cole A, McLaughlin S, & Du W (2018). Icariin improves
Fanconi anemia hematopoietic stem cell function through SIRT6-mediated
NF-kappa B inhibition. Cell Cycle 17: 367-376.
Maeda T, Hiraki M, Jin C, Rajabi H, Tagde A, Alam M, et al.(2018). MUC1-C Induces PD-L1 and Immune Evasion in Triple-Negative
Breast Cancer. Cancer Res 78: 205-215.
Menezes SV, Fouani L, Huang MLH, Geleta B, Maleki S, Richardson A,
et al. (2018). The Metastasis Suppressor, NDRG1, Attenuates Oncogenic
TGF-beta and NF-kB Signaling to Enhance Membrane E-Cadherin Expression
in Pancreatic Cancer Cells. Carcinogenesis.
Michishita E, McCord RA, Berber E, Kioi M, Padilla-Nash H, Damian
M, et al. (2008). SIRT6 is a histone H3 lysine 9 deacetylase that
modulates telomeric chromatin. Nature 452: 492-496.
Pan Y, Kong LD, Li YC, Xia X, Kung HF, & Jiang FX (2007). Icariin from
Epimedium brevicornum attenuates chronic mild stress-induced behavioral
and neuroendocrinological alterations in male Wistar rats. Pharmacol
Biochem Behav 87: 130-140.
Peart O (2017). Metastatic Breast Cancer. Radiol Technol 88:519M-539M.
Rogers C, Erkes DA, Nardone A, Aplin AE, Fernandes-Alnemri T, & Alnemri
ES (2019). Gasdermin pores permeabilize mitochondria to augment
caspase-3 activation during apoptosis and inflammasome activation. Nat
Commun 10: 1689.
Sen R, & Baltimore D (1986). Multiple nuclear factors interact with the
immunoglobulin enhancer sequences. Cell 46: 705-716.
Sharpe R, Pearson A, Herrera-Abreu MT, Johnson D, Mackay A, Welti
JC, et al. (2011). FGFR signaling promotes the growth of
triple-negative and basal-like breast cancer cell lines both in vitro
and in vivo. Clin Cancer Res 17: 5275-5286.
Shinde P, Banerjee P, & Mandhare A (2019). Marine natural products as
source of new drugs: a patent review (2015-2018). Expert Opin Ther Pat
29: 283-309.
Siegel RL, Miller KD, & Jemal A (2016). Cancer statistics, 2016. CA
Cancer J Clin 66: 7-30.
Staudt LM (2010). Oncogenic activation of NF-kappaB. Cold Spring Harb
Perspect Biol 2: a000109.
Steeg PS (2016). Targeting metastasis. Nat Rev Cancer 16:201-218.
Su B, Ye H, You X, Ni H, Chen X, & Li L (2018). Icariin alleviates
murine lupus nephritis via inhibiting NF-kappaB activation pathway and
NLRP3 inflammasome. Life Sci 208: 26-32.
Vaisitti T, Gaudino F, Ouk S, Moscvin M, Vitale N, Serra S, et
al. (2017). Targeting metabolism and survival in chronic lymphocytic
leukemia and Richter syndrome cells by a novel NF-kappaB inhibitor.
Haematologica 102: 1878-1889.
van Loo G, Saelens X, van Gurp M, MacFarlane M, Martin SJ, &
Vandenabeele P (2002). The role of mitochondrial factors in apoptosis: a
Russian roulette with more than one bullet. Cell Death Differ
9: 1031-1042.
Vanamee ES, & Faustman DL (2017). TNFR2: A Novel Target for Cancer
Immunotherapy. Trends Mol Med 23: 1037-1046.
Wang Y, Dong H, Zhu M, Ou Y, Zhang J, Luo H, et al. (2010).
Icariin exterts negative effects on human gastric cancer cell invasion
and migration by vasodilator-stimulated phosphoprotein via Rac1 pathway.
Eur J Pharmacol 635: 40-48.
Wu C, Tan X, Hu X, Zhou M, Yan J, & Ding C (2019). Tumor
Microenvironment following Gemcitabine Treatment Favors Differentiation
of Immunosuppressive Ly6C(high) Myeloid Cells. J Immunol.
Wu Y, & Zhou BP (2009). Inflammation: a driving force speeds cancer
metastasis. Cell Cycle 8: 3267-3273.
Xiao X, Yang G, Bai P, Gui S, Nyuyen TM, Mercado-Uribe I, et al.(2016). Inhibition of nuclear factor-kappa B enhances the tumor growth
of ovarian cancer cell line derived from a low-grade papillary serous
carcinoma in p53-independent pathway. BMC Cancer 16: 582.
Xu HB, & Huang ZQ (2007). Icariin enhances endothelial nitric-oxide
synthase expression on human endothelial cells in vitro. Vascul
Pharmacol 47: 18-24.
Yang L, Tian Y, Leong WS, Song H, Yang W, Wang M, et al. (2018).
Efficient and tumor-specific knockdown of MTDH gene attenuates
paclitaxel resistance of breast cancer cells both in vivo and in vitro.
Breast Cancer Res 20: 113.
Zhang Q, Lenardo MJ, & Baltimore D (2017). 30 Years of NF-kappaB: A
Blossoming of Relevance to Human Pathobiology. Cell 168: 37-57.
Zhang X, Chen LX, Ouyang L, Cheng Y, & Liu B (2012). Plant natural
compounds: targeting pathways of autophagy as anti-cancer therapeutic
agents. Cell Prolif 45: 466-476.
Zhao Z, Lu P, Zhang H, Xu H, Gao N, Li M, et al. (2014). Nestin
positively regulates the Wnt/beta-catenin pathway and the proliferation,
survival and invasiveness of breast cancer stem cells. Breast Cancer Res
16: 408.
Figure
legends
Fig.
1. Icariin selectively inhibited breast cancer cells proliferationin vitro . (A) Natural resources and chemical structure of
icariin. (B, C, D and E) Cell viability analysis of MDA-MB-231,
MDA-MB-453, 4T1 and MCF-10A after treatment with indicated
concentrations of icariin for 24, 48 and 72 h by MTT assay. (F and G)
Colony formation of MDA-MB-231, MDA-MB-453, 4T1 and MCF-10A after
treatment with different concentrations of icariin. Bars represent means
± SD of at least three independent experiments; *, P <
0.01, **, P < 0.005 and ***, P < 0.001
in comparison with control group.
Fig.2. Effects of icariin on the intrinsic apoptosis mechanism in
breast cancer cells. (A and B) MDA-MB-231, MDA-MB-453, 4T1 and MCF-10A
cells were exposed to indicated concentrations of icariin and then
analyzed apoptosis by FCM using Annexin V/PI dual-staining assay. (C)
Western-blot analysis of apoptotic proteins in MDA-MB-231 cells after
treatment with indicated concentrations of icariin. (D) The protein
expression ratio of Bax/Bcl-2 in icariin treated MDA-MB-231 cells. (E)
Changes of mitochondrial membrane potential (ΔΨm) in MDA-MB-231 cells
after treatment of icariin. (F and G) The level of ROS was measured in
MDA-MB-231 cell after exposed to icariin with or without pretreatment
with NAC (10 μM). (H) Overview of
mitochondrial apoptosis triggered by icariin in breast cancer cells.
Bars represent means ± SD of at least three independent experiments; *,P < 0.01, **, P < 0.005 and ***,P < 0.001 in comparison with control group.
Fig. 3. Icariin suppressed migration and invasion in breast
cancer cells. (A) After treatment with icariin, MDA-MB-231 cell was
measured by wound healing, transwell migration and transwell invasion.
(B) After treatment with icariin, 4T1 cell was measured by wound
healing, transwell migration and transwell invasion. Bars represent
means ± SD of at least three independent experiments; *, P< 0.01, **, P < 0.005 and ***, P< 0.001 in comparison with control group.
Fig. 4. Icariin suppressed
breast cancer cells migration and invasion via
SIRT6/NF-κB/EMT signaling pathway.
(A and B) The levels of SIRT6 and acylated H3K9 of icariin treated cells
determined by western-blot analysis. (C and D)
Expression
levels of
NF-κB
associated proteins of icariin treated cells determined by western-blot
analysis. (E and F) Expression levels of EMT associated proteins of
icariin treated cells determined by western-blot analysis. (G and H) The
levels of acylated H3K9 and p-IκBα of icariin treated cells which was
pre-treated with oss-128167 (20 μM) determined by western-blot analysis.
(I) Immunofluorescent analysis of nuclear transportation of NF-κB p65
protein in MDA-MB-231 cell. (J) Wound healing, transwell migration and
transwell invasion assessment of icariin in MDA-MB-231 cells with or
without pre-treatment with oss-128167. Bars represent means ± SD of at
least three independent experiments; *, P < 0.01, **,P < 0.005 and ***, P < 0.001 in
comparison with control group.
Fig. 5. Transcriptomic analysis of icariin in MDA-MB-231 and
MCF-10A cells. (A) Heatmap depiction of differentially expressed genes
between different treated groups in MDA-MB-231 and MCF-10A cells. (B and
C) KEGG analysis of representative signaling pathways enrichment between
different treated groups in MDA-MB-231 and MCF-10A cells respectively.
(D) Heatmap depiction of differentially expressed genes of NF-κB and TNF
signaling pathways between different treated groups in MDA-MB-231 and
MCF-10A cells.
Fig.
6. Icariin inhibited tumor growth and regulated tumor immunosuppressive
microenvironment. (A) Representative image of MDA-MB-231 tumors of
different groups
at
the
termination
of experiment. (B) The MDA-MB-231 tumor growth curves of different
groups within the treatment process. (C) MDA-MB-231 tumor weight of
different treated groups at the termination of experiment. (D) Variation
of mice body weight of different groups within treatment progress. (E)
Immunohistochemistry analysis of Ki-67 and cleaved caspase3 of
MDA-MB-231 tumor sections after different treatments. (F) Expression
level of NF-κB associated proteins of different treated MDA-MB-231 tumor
tissues were detected by western-bot. (G) Representative image of 4T1
tumors of different groups at the termination of experiment. (H) The 4T1
tumor growth curves of different groups within the treatment process.
(I) 4T1 tumor weight of different treated groups at the termination of
experiment. (J) Variation of mice body weight of different groups within
treatment progress. (K) Immunohistochemistry analysis of Ki-67 and
cleaved caspase3 of 4T1 tumor sections after different treatments. (L)
Expression levels of NF-κB associated proteins of different treated 4T1
tumor tissues were detected by western-bot. (M) Changes of proportion of
CD4+ and CD8+ T cells in 4T1 tumors
after different treatments. (N) Changes of proportion of MDSCs in 4T1
tumors after different treatments. Bars represent means ± SD of at least
three independent experiments; *, P < 0.01, **,P < 0.005 and ***, P < 0.001 in
comparison with control group.
Fig. 7. Icariin inhibited metastasis in the pulmonary
metastatic tumor mouse model of 4T1. (A) Bioluminescence images of mice
bearing pulmonary metastasis model of 4T1-luciferase cells after
different treatments at determined time points (7, 10, 13, 16 days). (B)
Quantitation of bioluminescence signal in mice after different
treatments at determined time points. (C) Number of nodules of lungs
from different treated groups. (D) Images of in vitro lungs from
different treated groups. (E) H&E staining analysis of lung tissues
from different treated groups. (F) Overview of pathways for
icariin-mediated apoptosis induction and anti-metastasis in breast
cancer cells. Icariin induced apoptosis, inhibited metastasis and
regulated immunosuppressive microenvironment by SIRT6/NF-κB signaling
pathway in triple-negative breast cancer cells. oss-127167, the
inhibitor of SIRT6.