Elsevier

Phytomedicine

Volume 23, Issue 13, 1 December 2016, Pages 1680-1690
Phytomedicine

Original article
Inhibitory effect of quercetin on colorectal lung metastasis through inducing apoptosis, and suppression of metastatic ability

https://doi.org/10.1016/j.phymed.2016.09.011 Get rights and content

Abstract

Background

Quercetin is a major dietary flavonoid found in a various fruits, vegetables, and grains. Although the inhibitory effects of quercetin have previously been observed in several types of cancer cells, the anti-metastatic effect of quercetin on colorectal metastasis has not been determined.

Purpose

This study investigated whether quercetin exhibits inhibitory effect on colorectal lung metastasis.

Study design

The effects of quercetin on cell viability, mitogen-activated protein kinases (MAPKs) activation, migration, invasion, epithelial-mesenchymal transition (EMT) and lung metastasis were investigated.

Methods

We investigated the effect of quercetin on metastatic colon cancer cells using WST assay, Annexin V assay, real-time RT-PCR, western blot analysis and gelatin zymography. The anti-metastatic effect of quercetin in vivo was confirmed in a colorectal lung metastasis model.

Results

Quercetin inhibited the cell viability of colon 26 (CT26) and colon 38 (MC38) cells and induced apoptosis through the MAPKs pathway in CT26 cells. Expression of EMT markers, such as E-, N-cadherin, β-catenin, and snail, were regulated by non-toxic concentrations of quercetin. Moreover, the migration and invasion abilities of CT26 cells were inhibited by quercetin through expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) regulation. Quercetin markedly decreased lung metastasis of CT26 cells in an experimental in vivo metastasis model.

Conclusion

In conclusion, this study demonstrates for the first time that quercetin can inhibit the survival and metastatic ability of CT26 cells, and it can subsequently suppress colorectal lung metastasis in the mouse model. These results indicate that quercetin may be a potent therapeutic agent for the treatment of metastatic colorectal cancer.

Introduction

Colorectal cancer (CRC) is the most commonly diagnosed cancer worldwide, and up to 50% of patients suffer from aggravated metastatic disease (Jemal et al., 2011). Over the past 15 years, the incidence and mortality rates of CRC have also increased in Korea (Jung et al., 2013). In particular, colorectal lung metastasis will be seen in 10–20% of patients with CRC, with studies reporting 5-year survival rates of 20–60% in patients who undergo resection (Cidón 2010).

Apoptosis is the process of programmed cell death and apoptotic cells are eliminated by phagocytes, such as macrophages, without eliciting inflammation (Kerr et al., 1972). The intrinsic apoptotic pathway is regulated by the anti-apoptotic and pro-apoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-xL (Fulda and Debatin 2006). After intrinsic changes, activated caspase-9 induces the activation of caspase-3, which in turn cleaves poly (ADP-ribose) polymerase (PARP), which finally leads to apoptosis (Boulares et al., 1999).

Mitogen-activated protein kinases (MAPKs), including c-Jun-N-terminal kinase (JNK), extracellular-regulated protein kinase (ERK), and p38 kinase, are important mediators of cell membrane-to-nucleus signal transduction and are activated by diverse extracellular stimulation. MAPK pathway is linked to the triggering of apoptosis because they are often activated in response to various cellular stress and growth factors (Gomez-Sarosi et al., 2009, Chang and Karin, 2001). Thus, many anticancer agents show their effect through the regulation of MAPKs activation in most cancer cell lines (Wada and Penninger 2004).

Tumor metastasis occurs through a multistep process that includes vessel formation, cell attachment, invasion, and abnormal cell growth (van Zijl et al., 2011). This event initiates cytophysiological changes of cancer cells, such as epithelial-mesenchymal transition (EMT), in which primary tumor cells lose cellular adhesion and there is increased cellular motility. EMT is a cellular process during which epithelial cells become mesenchymal-like cells, resulting in the loss of epithelial polarity and intercellular adhesion (Thiery and Sleeman 2006). After this process, metastatic cancer cells migrate and invade the lymph and blood vessels. Then, they adhere and survive in the target organs (Gupta and Massagué 2006). Matrix metalloproteinases (MMPs) are important extracellular proteases, and at least 20 types of MMPs are known. The activation of MMPs enables the degradation of the extracellular matrix (ECM) and provides the opportunity for cancer cells to access the vasculature, migrate, and ultimately invade target organs (Nabeshima et al., 2002, Itoh and Nagase, 2002). Among the MMPs, MMP-2 and MMP-9 efficiently degrade collagen, fibronectin, and elastin, which are associated with tumor migration and invasion (Khasigov et al., 2003). Therefore, the inhibition of MMP activity is extremely affective in slowing cancer progression, including suppression glio, aof metastasis.

Quercetin, a flavonoid contained in various foods, such as red onion, cranberry, broccoli, and black or green tea, plays a role in improving several diseases, such as psoriasis, neurodegenerative diseases, diabetes, inflammation, and allergic diseases (Chen et al., 2006, Formica and Regelson, 1995, Shen et al., 2012). In particular, quercetin has shown an anti-cancer effect through the regulation of angiogenesis, apoptosis, cell cycle arrest, and inhibition of cell migration and invasion ability in several types of cancer, including breast, prostate, and lung cancer, without any damage to normal cells (Ansó et al., 2010, Lin et al., 2008, Temraz et al., 2013, Hung, 2007). It has been reported that quercetin inhibits CRC by inducing cell cycle arrest and apoptosis as well as enhancing the effects of anti-cancer drugs (Zhang et al., 2012, Kim et al., 2010, Atashpour et al., 2015). However, it has not been reported whether quercetin could suppress colorectal metastasis. In this study, we attempted to confirm the effect of quercetin on colorectal metastasis and related molecular mechanisms.

Section snippets

Materials

Quercetin (Que, purity > 95%) and 5-Fluorouracil (5-FU) were obtained from Sigma (St Louis, MO, USA). Anti-phospho-p38, -ERK, -JNK, PARP, and caspase-3 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Caspase-9 antibody was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Bcl-2 and α-tubulin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Bcl-xL antibody was purchased from Bioworld Technology (Louis Park, MN, USA).

Cell culture

Effect of quercetin on the cell proliferation of metastatic colon cancer cells

In the present study, we examined the effects of quercetin on the growth of CT26 and MC38 metastatic murine colon cancer cells to confirm whether quercetin suppresses cell growth. The chemical structure of this compound is illustrated in Fig. 1A. Cells were treated with various concentrations of quercetin for 24–72 h, and their viability was evaluated by WST assay. As shown in Fig. 1B, morphologic changes in CT26, MC38, and HT29 cells after quercetin treatment for 48 h were detected by microscope

Discussion

The induction of cancer cell apoptosis is considered an effective therapeutic strategy against cancer progression. Many studies have reported that various natural products and compounds have anti-metastatic effects by inducing the apoptosis of cancer cells and decreasing their metastatic abilities (Surh 2003). Thus, the use of dietary bioactive compounds can be a safe and desirable approach to treating cancer. Quercetin, a flavonoid found in many fruits, vegetables, and grains, has been shown

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgments

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2015R1C1A1A02036733).

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