Molecular and cellular pharmacologyThe regulatory effects of metformin on the [SNAIL/miR-34]:[ZEB/miR-200] system in the epithelial-mesenchymal transition(EMT) for colorectal cancer(CRC)
Introduction
The epithelial-mesenchymal transition(EMT) is a process in which epithelial cells lose their epithelial characteristics and acquire mesenchymal ones (Peinado et al., 2007). EMT usually occurs in embryogenesis, tissue healing and tumour progression (Armstrong et al., 2011). In pathological practices, EMT has been proven to be associated with the invasion, metastasis and drug resistance of tumour cells (Singh et al., 2010; Christine, 2011). Previous studies found that EMT can be triggered by activating signaling pathways such as Wnt, Notch, TGF, JAK-STAT, etc. (Thiery et al., 2014), and signals from the upstream pathways eventually converge on a core control system which contains two double negative feedback chimaeric regulatory circuits: the upstream circuit SNAIL/miR-34 and the downstream circuit ZEB/miR-200 (Lu et al., 2013). In each circuit, the transcription factor family(ZEB/SNAIL) component and the miRNA family(miR-200/miR34) component act as mutual inhibitors to each other (Burk et al., 2008; Siemens et al., 2011). Based on the interactive mechanism, the SNAIL/miR-34 & ZEB/miR-200 system acts as the master regulator of the EMT process (Berx et al., 2007).
Metformin is a classic hypoglycaemic agent for type 2 diabetes mellitus(DM2) and was proven in the last decade to possess anti-neoplastic activities. In previous clinical studies, metformin was shown to decrease the morbidity (Josie et al., 2005; Libby et al., 2009) and to improve the prognosis (Garrett et al., 2012; Medairos et al., 2016; Gadducci et al., 2016; Lin et al., 2017; Gash et al., 2017; Haukka et al., 2017) for many cancers. In the laboratory, the anti-neoplastic effects were found to be associated with the alterations of several cellular signaling pathways, such as AMPK, mTOR, IGF, etc. (Pollak, 2008; Zakikhani et al., 2008; Vazquez-Martin et al., 2009). In addition, metformin also showed inhibitory effects on the EMT of tumour cells in a few other studies (Cufí et al., 2010; Vazquez-Martin et al., 2010; Zhao et al., 2014; Laskov et al., 2016). However, the exact underlying mechanism of metformin in anti-EMT regulation is still elusive, and the concrete alterations of the [SNAIL/miR-34]:[ZEB/miR-200] core control system are worthwhile to study.
To explore the anti-EMT activities and the mechanisms of metformin, we carried out our study on the human colorectal cancer cell lines SW480 and HCT116. By inducing EMT and by treating with metformin, we tested the EMT extent and detected the alterations of TFs and miRNAs in the [SNAIL/miR-34]:[ZEB/miR-200] system of CRC cells to further discern metformin's pharmacological mechanisms in the EMT process.
Section snippets
Cell culture and reagents
The human colorectal cancer cell lines SW480 and HCT116 were from the Type Culture Collection of the Chinese Academy of Sciences. The cell lines was maintained in RPMI 1640(Corning Inc.) medium containing 10% foetal bovine serum(HyClone, Inc.) at 37 °C in an atmosphere of 5% CO2. Before TGF-β(10 ng/ml, Prepro Tech Inc.) induction, the cells were starved in serum-free RPMI 1640 medium overnight. Metformin was obtained from Solarbio Science﹠Technology Co, stored sealed at −20 °C and dissolved in
Metformin suppresses the proliferation, migration and invasion of human SW480 CRC cells
To testify the inhibitory effect of metformin on CRC cells growth and proliferation, metformin was added by gradient concentration(0, 1, 2, 5, 10 mM) and the viability of the cells detected chronologically(0, 24, 48, 72 h) with a microplate reader using a CCK8 kit. The OD values showed that SW480 cells growth and proliferation were chrono- and dose-dependently inhibited by metformin, consistent with previous studies [Fig. 1B].
In the wound-healing assay, TGF-β(10 ng/ml, 48 h)-induced
Discussion
Colorectal cancer(CRC) is the third most common global cancer. Mainly caused by distant metastasis, CRC is also the fourth highest cause of cancer death worldwide (Ferlay et al., 2015). Previous studies showed decreased miR-200 at the invasive front than at the primary site of CRC, suggesting the role of miR-200 and [SNAIL/miR-34];[ZEB/miR-200] in the EMT, invasion and metastasis of tumours (Hur et al., 2013; Paterson et al., 2013). In our study, metformin exhibited inhibitory effects on the
Funding
This study was funded by the Natural Science Foundation of Shandong Province [Z2005C02] and Science and Technology Development Project of Shandong Province [2014GSF118052].
Conflict of interests
The authors declare that they have no conflict of interests.
Disclosure statement
The authors declare that they have no conflict of interests.
References (66)
- et al.
Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I
J. Biol. Chem.
(2000) - et al.
Metformin: from mechanisms of action to therapies
Cell Metab.
(2014) - et al.
Metformin use and gynecological cancers: a novel treatment option emerging from drug repositioning
Crit. Rev. Oncol./Hematol.
(2016) - et al.
Metabolism, hypoxia and the diabetic heart
J. Mol. Cell. Cardiol.
(2011) - et al.
Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells
Metab.-Clin. Exp.
(2005) - et al.
Down-regulation of the miRNA-200 family at the invasive front of colorectal cancers with degraded basement membrane indicates EMT is involved in cancer progression
Neoplasia
(2013) - et al.
Coupled reversible and irreversible bistable switches underlying TGFβ-induced epithelial to mesenchymal transition
Biophys. J.
(2013) - et al.
Zeb1 regulates E-cadherin and Epcam (epithelial cell adhesion molecule) expression to control cell behavior in early zebrafish development
J. Biol. Chem.
(2013) - et al.
Metformin and resveratrol ameliorate muscle insulin resistance through preventing lipolysis and inflammation in hypoxic adipose tissue
Cell. Signal.
(2016) - et al.
Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers
Mol. Cancer Res.
(2011)
Molecular interplay between microRNA-34a and sirtuin1 in hyperglycaemia-mediated impaired angiogenesis in endothelial cells: effects of metformin
J. Pharmacol. Exp. Ther.
Pre-EMTing metastasis? Recapitulation of morphogenetic processes in cancer
Clin. Exp. Metastas-.
The ZEB/miR-200 feedback loop—a motor of cellular plasticity in development and cancer?
EMBO Rep.
A reciprocal repression between ZEB1 and members of the miR‐200 family promotes EMT and invasion in cancer cells
Embo Rep.
A perspective on cancer cell metastasis
Science
Metformin inhibits TGF-β1-induced epithelial-to-mesenchymal transition via PKM2 relative-mTOR/p70s6k signaling pathway in cervical carcinoma cells
Int. J. Mol. Sci.
Monotherapy with metformin: does it improve hypoxia in type 2 diabetic patients?
Clin. Chem. Lab. Med. Cclm
Metformin lowers the threshold for stress-induced senescence: a role for the microRNA-200 family and miR-205
Cell Cycle
Metformin against TGFβ-induced epithelial-to-mesenchymal transition (EMT): from cancer stem cells to aging-associated fibrosis
Cell Cycle
Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process
Diabetes
Metformin induces microRNA-34a to downregulate the Sirt1/Pgc-1α/Nrf2 pathway, leading to increased susceptibility of wild-type p53 cancer cells to oxidative stress and therapeutic agents
Free Radic. Biol. Med.
Metformin and reduced risk of cancer in diabetic patients
BMJ (Clinical Res. ed.)
Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012
Int. J. Cancer
Metformin as an adjuvant drug against pediatric sarcomas: hypoxia limits therapeutic effects of the drug
Plos One
Survival advantage observed with the use of metformin in patients with type II diabetes and colorectal cancer
Br. J. Cancer
Potentiating the effects of radiotherapy in rectal cancer: the role of aspirin, statins and metformin as adjuncts to therapy
Br. J. Cancer
The role of oxidative stress and hypoxia in pancreatic beta-cell dysfunction in diabetes mellitus
Antioxid. Redox Signal.
Diabetes and cancer: a consensus report
Ca Cancer J. Clin.
SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial–mesenchymal transition
Embo J.
Metformin is a novel suppressor for transforming growth factor (TGF)-β1
Sci. Rep.
Risk of cause-specific death in individuals with cancer - modifying role diabetes, statins, and metformin
Int. J. Cancer
Metformin protects H9C2 cardiomyocytes from high-glucose and hypoxia/reoxygenation injury via inhibition of reactive oxygen species generation and inflammatory responses: role of AMPK and JNK
J. Diabetes Res.
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