TNF-α induces expression of urokinase-type plasminogen activator and β-catenin activation through generation of ROS in human breast epithelial cells
Graphical abstract
Introduction
Metastasis, one of the most detrimental causes associated with malignant progression, is a complex cascade of events involving tumor dissemination from the primary site of growth to distant organs. Metastasis is multistep events involving degradation or rearrangement of the extracellular matrix (ECM), local invasion, angiogenesis, intravasation, survival of malignant cells during the circulation, extravasation and the growth at a secondary site [1]. One of the key mediators causally involved in the aforementioned processes is the serine protease, urokinase-type plasminogen activator (uPA) [2]. Members of the plasminogen activator system, including urokinase plasminogen activated receptor (uPAR), have been found to be over-expressed in a large number of tumors, particularly breast cancer, which is associated with a poor prognosis [2], [3]. uPA catalyzes the formation of plasmin from plasminogen to generate the proteolytic cascade that contributes to the breakdown of ECM, a key step in cancer metastasis [4], [5].
During the metastatic cascade, many pathogenic changes occur. These include inflammation mediated by over-secretion of cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-8 [6], [7]. TNF-α is a pleiotropic pro-inflammatory cytokine that has a wide range of biological activities. TNF-α can mediate tumor progression by inducing the cellular transformation, promotion, survival, proliferation, invasion, angiogenesis, and metastasis [8], [9], [10]. Many of TNF-α-induced cellular responses and alterations have been associated with accumulation of reactive oxygen species (ROS) [11], [12]. ROS are relatively harmless, but when produced excessively or in the absence of sufficient antioxidant defense, the balance between oxidants and antioxidants is disturbed. This may lead to the initiation and promotion of cancer [13], [14], [15]. ROS produced by TNF-α activates several intracellular signaling molecules, such as β-catenin and its partner T-cell factor (Tcf)-4 [16], [17]. β-Catenin has been shown to have a dual role as a major structural component of cell–cell adherence junctions and also as a transcription activator. In addition, β-catenin is involved in tumorigenesis by transactivating the lymphoid enhancer factor/T-cell factor (Lef/Tcf) transcription factor. Some of the genes whose expression is up-regulated by β-catenin/Tcf signaling are c-jun, c-myc, fibronectin, cyclin D1, mmp and uPA [18], [19].
In the present study, we found that treatment of immortalized human breast epithelial (MCF-10A) cells with TNF-α resulted in the increased expression of uPA with concurrent enhancement of invasive capacity. Interestingly, TNF-α-induced uPA up-regulation and cell invasion were mediated by ROS that triggered the activation of β-catenin signaling. As TNF-α is a typical pro-inflammatory cytokine, its overactivation of β-catenin signaling may contribute to the inflammation-associated carcinogenesis. In this context, it is noticeable that β-catenin-mediated signaling has recently been recognized as an important molecular target for chemoprevention with anti-inflammatory substances [19]. Artemisia asiatica (Asteracease), widespread in nature, has been frequently used in traditional oriental medicine for the treatment of inflammation, cancer, and microbial infections [20], [21], [22]. Our previous studies have shown that eupatilin (5,7-dihydoxy-3′,4′,6-tri-methoxy-flavone), one of the pharmacologically active ingredients of A. asiatica, possesses anti-inflammatory and anti-tumor promoting activities [21]. This prompted us to examine the ability of this flavonoid to inhibit TNF-α-induced expression of uPA, to activate β-catenin and to cause invasion of MCF-10A cells.
Section snippets
Materials
Euaptilin was supplied from Dong-A Pharmaceutical Co. Ltd. (Yong-In Si, South Korea) and dissolved in DMSO for treatment. TNF-α, sodium dodecylsulfate (SDS), N-acetylcysteine (NAC), cholera toxin, hydrocortisone, insulin and human recombinant epidermal growth factor (h-EGF) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM)/Ham's nutrient mixture F-12 (1:1) and horse serum were obtained from Gibco BRL (Grand Island, NY, USA).
TNF-α induced expression of uPA and invasion in MCF-10A cells
When human breast epithelial (MCF-10A) cells were stimulated with TNF-α (10 ng/ml), mRNA (Fig. 1A) and protein (Fig. 1B) levels of uPA were elevated in a time-dependent manner. In addition, the invasive capacity of MCF-10A cells was significantly increased by TNF-α treatment for 24 h (Fig. 1C), suggesting that the upregulation of uPA expression is essential for development of the invasiveness in TNF-α treated MCF-10A cells.
TNF-α-induced activation of β-catenin in MCF-10A cells
In another experiment, we examined whether TNF-α could induce β-catenin
Discussion
TNF-α is produced by tumors and can act as an endogenous tumor promoter. As a prototypic pro-inflammatory cytokine, TNF-α is considered as a molecular link between inflammation and cancer [9], [24]. It also induces expression/production of other cytokines, angiogenic factors, MMPs, and uPA, contributing to increased growth and survival of tumor cells. Thus, Yin et al. have reported that TNF-α enhances the invasive capacity of human breast cancer (MCF-7) cells through upregulation of adhesion
Acknowledgements
This work was supported by the grant for the Biofood Research Program and also by the ERC/IDRC grant (R11-2007-107-01002-0), Ministry of Education, Science and Technology, Republic of Korea.
References (47)
The biochemistry of metastasis
Adv Clin Chem
(1996)- et al.
Tumor necrosis factor alpha stimulates invasion of Src-activated intestinal cells
Gastroenterology
(2002) - et al.
Tumour necrosis factor alpha: a potential target for the therapy of solid tumours
Lancet Oncol
(2003) - et al.
Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation
J Biol Chem
(2004) - et al.
Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes
Biochem Pharmacol
(2006) - et al.
Free radicals, metals and antioxidants in oxidative stress-induced cancer
Chem Biol Interact
(2006) - et al.
beta-Catenin-mediated signaling: a novel molecular target for chemoprevention with anti-inflammatory substances
Biochim Biophys Acta
(2006) - et al.
Eupatilin, a pharmacologically active flavone derived from Artemisia plants, induces cell cycle arrest in ras-transformed human mammary epithelial cells
Biochem Pharmacol
(2004) - et al.
Selective inhibition of 5-lipoxygenase by natural compounds isolated from Chinese plants, Artemisia rubripes Nakai
FEBS Lett
(1983) - et al.
Gene expression of the invasive phenotype of TNF-alpha-treated MCF-7 cells
Biomed Pharmacother
(2009)
Upregulation of cell-surface-associated plasminogen activation in cultured keratinocytes by interleukin-1 beta and tumor necrosis factor-alpha
Exp Cell Res
Angiogenesis: regulators and clinical applications
Biochem Pharmacol
Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer
Cell Biol Int
Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis
Drug Discov Today
Urokinase receptor and integrin partnership: coordination of signaling for cell adhesion, migration and growth
Curr Opin Cell Biol
Sulindac suppresses beta-catenin expression in human cancer cells
Eur J Pharmacol
Urokinase-type plasminogen activator: a potent marker of metastatic potential in human cancers
Biochem Soc Trans
Urokinase-plasminogen activator, a marker for aggressive breast carcinomas. Preliminary report
Cancer
Biology and biochemistry of proteinases in tumor invasion
Physiol Rev
The role and regulation of urokinase-type plasminogen activator receptor gene expression in cancer invasion and metastasis
Med Res Rev
Rapid induction of cytokine and E-selectin expression in the liver in response to metastatic tumor cells
Cancer Res
Essential roles of tumor necrosis factor receptor p55 in liver metastasis of intrasplenic administration of colon 26 cells
Cancer Res
TNF: a master switch for inflammation to cancer
Front Biosci
Cited by (29)
Eupatilin: a natural pharmacologically active flavone compound with its wide range applications
2020, Journal of Asian Natural Products ResearchFlavone glucosides from Artemisia juncea
2019, Natural Product ResearchEthnopharmacological properties of Artemisia asiatica: A comprehensive review
2018, Journal of EthnopharmacologyCitation Excerpt :The anti-inflammatory activity of DA-9601 (1–5 μg/ear) was also evaluated in a female BALB/c mouse atopic dermatitis (AD) model; DA-9601 significantly reduced AD-like lesions and serum immunoglobulin IgE levels and also downregulated the increase of IL-4, IL-13, IL-31, and TNF-α (Choi et al., 2011a, 2011b). Pre-incubation of eupatilin (0–50 μM) in MCF-10A cells significantly (P < 0.01) inhibited TNF-α-induced intracellular accumulation of ROS, β-catenin accumulation, and β-catenin-Tcf-4-DNA binding activity (Kim et al., 2010). The in vitro anti-inflammatory mechanism and action of DA-9601 was studied in a human mast cell line, HMC-1 cells under stimulation with phorbol 12-mysristate 13-acetate (PMA) and calcium ionophore A23187.
DATS sensitizes glioma cells to TRAIL-mediated apoptosis by up-regulation of death receptor 5 via ROS
2017, Food and Chemical ToxicologyCitation Excerpt :We found that ROS generation occurs during treatment with DATS. ROS, including the superoxide anion, hydrogen peroxide and hydroxyl radical, are known to mediate apoptosis induced by some cancer chemopreventive and therapeutic agents (Chandra-Kuntal et al., 2013; Khan et al., 2012; Kim et al., 2010; Luo et al., 2013). Intracellular ROS may interact with cellular membrane lipids, proteins, and DNA and cause oxidative injury (Mai et al., 2010; Saitoh et al., 2011).
Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen-plasmin system
2016, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :Plasminogen activators convert zymogen plasminogen into plasmin which then digests the fibrin in blood clot to restore blood flow to the occluded vessels and the downstream tissue. Increases in uPA mRNA and protein expressions have also been shown in various human cell lines such as microvascular and umbilical vein endothelial cells and breast epithelial cells exposed to TNF-α [9–11]. Although elevations in intracellular calcium and NADPH oxidase activity can explain the enhanced activities of tPA and uPA during ischaemic injury, the nature of link between plasminogen activators and TNF-α with reference to BBB integrity remains unexplored [12].
- 1
The first two authors equally contributed to this work.