Resveratrol inhibits TGF-β1-induced epithelial-to-mesenchymal transition and suppresses lung cancer invasion and metastasis

Abstract

Epithelial-to-mesenchymal transition (EMT) is a cellular process during which epithelial polarized cells become motile mesenchymal-appearing cells, which in turn promotes carcinoma invasion and metastasis. Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural polyphenolic compound found in grapes, red wine and several other plants. Numerous reports in the literature indicate that resveratrol can suppress cancer invasion and metastasis. However, the underlying mechanisms of inhibiting metastasis by resveratrol are complex, not fully elucidated and the subject of intense scientific debate. Despite evidence indicating that EMT can be a target for resveratrol, little is known about the effect of resveratrol on lung cancer cells. Our previous studies demonstrated that TGF-β1 induces EMT to promote lung adenocarcinoma invasion and metastasis. To understand the repressive role of resveratrol in lung cancer invasion and metastasis, we sought to investigate the potential use of resveratrol as an inhibitor of TGF-β1-induced EMT development in A549 lung cancer cells in vitro. Here we show that when A549 cells are treated with TGF-β1 and resveratrol, the latter inhibits the initiation of TGF-β1-induced EMT. Our results show that 20 μM resveratrol increases expression of the epithelial phenotype marker E-cadherin and represses the expression of the mesenchymal phenotype markers, Fibronectin and Vimentin during the initiation of TGF-β1-induced EMT. Resveratrol also inhibits expression of EMT-inducing transcription factors Snail1 and Slug, although the expression of the Twist1 transcription factor remained unchanged. Resveratrol inhibits the TGF-β1-induced increase in cell adhesion, migration and invasion of A549 lung cancer cells. Taken together, our findings provide new evidence that resveratrol suppresses lung cancer invasion and metastasis in vitro through inhibiting TGF-β1-induced EMT.

Introduction

Lung cancer is one of the most common cancers, and is the leading cause of death among cancer patients. Metastasis causes 90% of deaths from lung cancer, so it is the overwhelming cause of mortality in patients with lung cancer. Metastasis is a complex process whereby cancer cells spread from a primary site and form tumors at distant sites. It occurs through a multistep process by which primary tumor cells lose cellular adhesion, increase cellular motility, invade adjacent tissue, and enter and survive in the systemic circulation (intravasate). They then translocate through the vasculature, arrest in distant capillaries, extravasate into the surrounding tissue parenchyma, and finally proliferate from microscopic growths (micrometastases) into macroscopic secondary tumors at distant sites (Ma et al., 2007). There is evidence to suggest that epithelial-to-mesenchymal transition (EMT) contributes to cancer progression, invasion and metastasis of various cancers (Chang et al., 2011, Thiery et al., 2009).

Recent evidence implicates EMT induction in cancer progression (Chang et al., 2011, Thiery et al., 2009). EMT is a cellular process during which epithelial polarized cells become motile mesenchymal-appearing cells (Chang et al., 2011, Thiery et al., 2009, Voulgari and Pintzas, 2009). During EMT, three major changes occur: (i) epithelial cells lose cell–cell contacts, cell polarity and epithelial markers, especially E-cadherin; (ii) acquire mesenchymal markers (Vimentin and Fibronectin); and (iii) undergo major changes in their cytoskeleton that enables them to acquire mesenchymal phenotypes with increased motility and invasiveness (Xie et al., 2010). EMT is closely related to the loss of E-cadherin expression and acquisition of Vimentin and Fibronectin expression, and is activated by transcriptional regulators, including Twist, Zeb2 (SIP1), Snail1 and Slug (Snail2) (Moreno-Bueno et al., 2009, Thiery et al., 2009). EMT regulatory transcription factors are expressed in many malignant cancers. EMT transcription factors change the epithelial gene expression profile, decrease the expression of genes encoding epithelial junctional complexes (e.g., E-cadherin) and cytokeratins, and induce the expression of mesenchymal Vimentin, Fibronectin and N-cadherin (Gjerdrum et al., 2010, Voulgari and Pintzas, 2009). The growth factors, cytokines, extracellular matrix (ECM) and hypoxia all play an important role in EMT induction (Thiery et al., 2009). Studies have found that growth factors, including TGF-β, epidermal growth factor (EGF), VEGF, PDGF and HGF induce morphological cellular changes that are consistent with the acquisition of the EMT phenotype as characterized by the loss of epithelial marker expression and the gain or increased expression of mesenchymal markers (Cai et al., 2010, Gonzalez-Moreno et al., 2010, Kong et al., 2008, Thiery et al., 2009). The growth factors control expression of Twist1, Zeb2 (SIP1), Snail1 and Slug (Snail2) during EMT induction by activating cell signaling pathways (TGF-β/Smad, PI3K/AKT, MEK/ERK, and WNT/β-catenin) (Chang et al., 2011, Moreno-Bueno et al., 2009).

Furthermore, prolonged exposure to pathogenic environmental factors such as air pollutants or cigarette smoke may result in increased hemoglobin-oxygen affinity leading to chronic hypoxia, endogenous CO₂ increases, and greater generation of peroxynitrite (ONOO⁻) which, by fostering oxidation and the suicidal death of erythrocytes (eryptosis), may promote the release of pro-inflammatory cytokines throughout circulation, enhanced expression of zinc-finger transcriptional factors, stress response activation, and endothelial dysfunctions (Zappulla, 2008). In addition, cigarette smoke extract stimulates EMT (Zhang et al., 2012). Hypoxia also induces EMT development in cancer cells, which affects tumorigenesis and tumor progression (Cheng et al., 2011, Copple, 2010, Du et al., 2012, Thiery et al., 2009). IL-6 inflammatory cytokine promotes tumor metastasis by inducing EMT (Yadav et al., 2011). Therefore, hypoxia, cytokines such as IL-6 inflammatory factor and TGF-β, and especially environmental factor such as cigarette smoke induce EMT development. However, the precise molecular mechanisms of hypoxia, inflammatory cytokines and cigarette smoke factors inducing EMT have not been fully elucidated.

TGF-β plays important roles in EMT (Chen et al., 2012, Ikushima and Miyazono, 2010, Thiery et al., 2009, Zhang et al., 2011). TGF-β signaling regulates expression of snail1, SOX2, SOX4 and ID1 (inhibitor of differentiation/DNA binding-1) (Ikushima and Miyazono, 2010, Thiery et al., 2009). Snail1 and ID1 regulate EMT. Therefore, TGF-β growth factor plays a complex and crucial role in EMT induction via TGF-β signaling pathways (Chen et al., 2012, Thiery et al., 2009, Zhang et al., 2011).

Resveratrol (trans-3,4′,5-trihydroxystilbene, RES) belongs to the stilbene class of naturally polyphenolic compounds produced by plants in response to infection by pathogens (Jang et al., 1997, Kundu and Surh, 2008). Resveratrol exhibits beneficial health effects including anti-oxidant, anti-inflammatory, cardio-protective and anti-tumor properties (Baur and Sinclair, 2006, Qadri et al., 2009), inhibits renal fibrosis induced by TGF-β1 treatment (Li et al., 2010), and affects tumorigenesis and tumor progression (Kundu and Surh, 2008). The findings indicate that resveratrol suppresses angiogenesis and metastasis (Busquets et al., 2007, Tseng et al., 2004). Resveratrol could influence several processes (Kundu and Surh, 2008), and plays an important role in the regulation of various molecular targets, including NF-κB and PPARγ transcription factors (Floyd et al., 2008, Sun et al., 2006), VEGF growth factors (Zhang et al., 2005), TNF-α and IL-6 inflammatory cytokines (Knobloch et al., 2011, Li et al., 2007), p38MAPK and Akt protein kinases (Vergara et al., 2012, Zhong et al., 2012) and other enzymes (COX-2, MMP-9 and PDE4) (Kundu and Surh, 2008, Park et al., 2012). Resveratrol inhibits lung cancer cell invasion and metastasis by suppressing the MAPK and NF-κB pathway and subsequently downregulating expression of MMP-2 and MMP-9 matrix metalloproteinases (Liu et al., 2010, Yang et al., 2009). However, the precise molecular mechanisms by which resveratrol inhibits human lung cancer cell invasion and metastasis have not been fully elucidated.

In particular, our previous studies demonstrated that TGF-β1 induces EMT, which promotes lung adenocarcinoma invasion and metastasis, and that PI3K/Akt and MAPK/Erk1/2 signaling pathways regulate TGF-β1-induced EMT in lung cancer (Chen et al., 2012, Zhang et al., 2011). To understand the repressive role of resveratrol in lung cancer invasion and metastasis, the objective of our study was to investigate the potential use of resveratrol as an inhibitor of TGF-β1-triggered EMT in A549 lung cancer cells. Our findings suggest that resveratrol inhibits TGF-β1-induced EMT development to suppress EMT-aided lung cancer cell invasion and metastasis.