Quercetin restrains TGF-β1-induced epithelial–mesenchymal transition by inhibiting Twist1 and regulating E-cadherin expression

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Abstract

Emerging evidence has indicated that transforming growth factor-beta 1 (TGF-β1) induces the epithelial–mesenchymal transition (EMT) in cancer cells, thus promoting their motility and invasiveness. Quercetin, a member of the polyphenolic flavonoid family, has been reported to display anticancer activity against a broad range of cancer cell types. Indeed, numerous studies have shown the cancer preventive effects and molecular mechanisms of quercetin in vitro using diverse cell model systems. However, the potential effect of quercetin on EMT remains unclear. In this study, we identified a unique function of quercetin in inhibiting the EMT process induced by TGF-β1. In particular, quercetin rescued the morphological changes and EMT-like phenotypes in TGF-β1-activated SW480 cells, and this inhibition of TGF-β1-induced EMT was mediated via the suppression of Twist1 expression. In addition, quercetin strongly suppressed TGF-β1-induced invasion of SW480 cells. Thus, quercetin may be considered a novel therapeutic agent for the treatment of patients with refractory cancer and for the prevention of the metastatic cascade initiated by EMT.

Introduction

The epithelial–mesenchymal transition (EMT) is regarded as a crucial process during tumor initiation and progression, and this process is also recognized as a key step toward cancer metastasis [1]. Loss of E-cadherin is considered a hallmark event of EMT that initiates a series of signal transduction pathways and major cytoskeletal reorganization processes [2]. In response, a cohort of EMT-inducing transcription factors, including Twist, Snail, Slug, and ZEB1, has been reported to activate EMT programs in epithelial cells, and their elevated expression has been well documented in invasive tumors [[3], [4], [5]].

Transforming growth factor-beta 1 (TGF-β1), a potent regulator of EMT, regulates a wide range of cellular functions, including tumor initiation, progression, and extracellular matrix remodeling [6]. Upon TGF-β1 treatment, epithelial cells switch from a cuboidal to an elongated spindle shape and demonstrate enhanced expression of Snail1 and Twist1 and subsequently decreased expression of E-cadherin [7]. As a transcription factor, Twist1 plays a vital role in inducing metastasis in tumor cells through the EMT process [8]. Indeed, ectopic expression of Twist1 decreases E-cadherin, occludin and claudin-7 expression and increases Vimentin and N-cadherin expression [3], although its role in the occurrence of TGF-β1-induced EMT has not been fully elucidated.

Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a member of the polyphenolic flavonoid family, is universally distributed across edible plant species and is one of the most potent antioxidants of plant origin [9]. Quercetin is also the most abundant dietary flavonoid in fruit and vegetables [10]. Numerous studies have indicated that quercetin exhibits a range of biological properties, including antioxidant, anti-inflammatory, antiproliferative, and anticancer activities [[11], [12], [13]]. However, little information exists concerning the role of quercetin in tumorigenesis and tumor progression in TGF-β1-activated CRC cells.

In this study, we investigated novel effects of quercetin on TGF-β1-induced EMT in CRC cells and elucidated the underlying downstream signaling mechanism. Our results show that quercetin inhibits E-cadherin expression by decreasing Twist1 expression, leading to the suppression of TGF-β1-induced EMT and migration in CRC.

Section snippets

Reagents

Quercetin, dimethyl sulfoxide (DMSO), propidium iodide (PI), fetal bovine serum (FBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Purity of quercetin (>98%) was confirmed by high-performance liquid chromatography (HPLC). Stock solutions (200 mM) were prepared in DMSO. Triton X-100 and EDTA were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Recombinant human TGF-β1 (AF-100-21C) was purchased

TGF-β1 triggers EMT in SW480 cells

To assess whether TGF-β1 affects EMT markers in epithelial cells, SW480 cells were treated with TGF-β1 at various concentrations for various durations to induce an EMT-like process. We found that SW480 cells cultured with TGF-β1 displayed an elongated fibroblast-like morphology with a scattered distribution, whereas cells cultured without TGF-β1 presented a cobblestone-like morphology with tight cell-cell contact in culture (Fig. 1A). Consistent with this observation, Western blot analyses of

Discussion

Quercetin is a naturally occurring chemopreventive agent whose biological activities include anticancer, antioxidant, anti-inflammatory, anti-angiogenic, growth-suppressing, pro-apoptotic, and anti-aging properties [10]. As a cancer-preventing agent, quercetin can also decrease cellular migration, which is a prerequisite for cellular invasion and an often fatal tumorigenic property [14]. Although some reports have indicated an effect of quercetin on tumor metastasis [15,16], its role in

Competing interests

The authors declare no competing financial interests.

Acknowledgments

This research was supported by the Program of National Natural Science Foundation of China (NO.81560407), The Key Project on Social Development by Department of Science and Technology of Guizhou Province (No. 2015SY3046), Natural Science Foundation of Jiangsu Province (BK20160174), the Joint Foundation of Guizhou Province (No. 2015LH7483 and 2015LH7505).

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1

These authors contributed equally to this work.

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