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

Accepted Manuscript

Inhibitory effect of quercetin on colorectal lung metastasis through inducing apoptosis, cell cycle arrest and suppression of metastatic ability Ji-Ye Kee , Yo-Han Han , Dae-Seung Kim , Jeong-Geon Mun , Jinbong Park , Mi-Young Jeong , Jae-Young Um , Seung-Heon Hong PII: DOI: Reference:

S0944-7113(16)30175-1 10.1016/j.phymed.2016.09.011 PHYMED 52083

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

7 June 2016 26 September 2016 29 September 2016

Please cite this article as: Ji-Ye Kee , Yo-Han Han , Dae-Seung Kim , Jeong-Geon Mun , Jinbong Park , Mi-Young Jeong , Jae-Young Um , Seung-Heon Hong , Inhibitory effect of quercetin on colorectal lung metastasis through inducing apoptosis, cell cycle arrest and suppression of metastatic ability, Phytomedicine (2016), doi: 10.1016/j.phymed.2016.09.011

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Inhibitory effect of quercetin on colorectal lung metastasis through inducing apoptosis, cell cycle arrest and suppression of metastatic ability

Ji-Ye Keea, Yo-Han Hana, Dae-Seung Kima, Jeong-Geon Muna, Jinbong Parkb, Mi-Young

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Jeong,a,b, Jae-Young Umb,*, Seung-Heon Honga,**

Department of Oriental Pharmacy, College of Pharmacy, Wonkwang-Oriental Medicines

Research Institute, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 54538, Republic

b

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of Korea

Department of Pharmacology, College of Korean Medicine, Kyung Hee University, 26,

Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea * Corresponding author

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Jae-Young Um, Department of Pharmacology, College of Korean Medicine, Kyung Hee

Tel.: +82 2 961 9262

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University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea

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E-mail address: [email protected] (J.Y. Um). ** Corresponding author

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Seung-Heon Hong, Department of Oriental Pharmacy, College of Pharmacy, Wonkwang-

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Oriental Medicines Research Institute, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 54538, Republic of Korea Tel.: +82 63 850 6805; fax: +82 63 843 3421 E-mail address: [email protected] (S.H. Hong). *,** These two authors equally contributed as corresponding authors. 1

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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

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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

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(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.

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The anti-metastatic effect of quercetin in vivo was confirmed in a colorectal lung metastasis

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model.

Results: Quercetin inhibited the cell viability of colon 26 (CT26) and colon 38 (MC38) cells

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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

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concentrations of quercetin. Moreover, the migration and invasion abilities of CT26 cells

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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 2

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lung metastasis in the mouse model. These results indicate that quercetin may be a potent therapeutic agent for the treatment of metastatic colorectal cancer.

Keywords: Quercetin, Colorectal cancer, Lung metastasis, Apoptosis, Mitogen-activated

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protein kinases

Abbreviations: CRC, colorectal cancer; ECM, extracellular matrix; EMT, epithelialmesenchymal transition; ERK, extracellular-regulated protein kinase; JNK, c-Jun-N-terminal

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kinase; MAPK, mitogen-activated protein kinases; MMP, matrix metalloproteinases; PARP,

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poly (ADP-ribose) polymerase; TIMP, tissue inhibitor of metalloproteinases

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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.,

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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

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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

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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).

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Mitogen-activated protein kinases (MAPKs), including c-Jun-N-terminal kinase (JNK),

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extracellular-regulated protein kinase (ERK), and p38 kinase, are important mediators of cell membrane-to-nucleus signal transduction and are activated by diverse extracellular

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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;

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Chang et al., 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), 4

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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

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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,

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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

suppression glio,aof metastasis.

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inhibition of MMP activity is extremely affective in slowing cancer progression, including

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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,

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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-

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cancer effect through the regulation of angiogenesis, apoptosis, cell cycle arrest, and

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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 at al., 2012; Kim et al., 2010; Atashpour et al., 2015). However, it has not been 5

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reported whether quercetin could suppress colorectal metastasis. In this study, we attempted

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to confirm the effect of quercetin on colorectal metastasis and related molecular mechanisms.

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Materials and methods Materials Quercetin (Que, purity > 95%) and 5-Fluorouracil (5-FU) were obtained from Sigma (St

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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-

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xL antibody was purchased from Bioworld Technology (Louis Park, MN, USA).

Cell culture

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The mouse colon carcinoma cell line colon 26 (CT26), colon 38 (MC38), and CCD18Co cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL,

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Grand Island, NY), and the human colon adenocarcinoma cell line HT29 was maintained in RPMI 1640 (Gibco BRL, Grand Island, NY). These mediums were supplemented with 10%

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fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in humidified

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Mice

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air containing 5% CO2 in a 37 °C incubator.

BALB/c female mice (5 weeks, 19-20 g) were purchased from Da-Mool Science

(Daejeon, Korea). All mice were housed six per cage in a laminar air-flow room maintained at a temperature of 22 ± 1 °C and a reactive humidity of 55 ± 1% throughout the study. The research was carried out in accordance with the internationally accepted principles for 7

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laboratory animal use and care as found in the Wonkwang University guidelines (WKU1416).

WST assay

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Cells were seeded in 96-well microplates (3 × 103 cells/well) and a quercetin-containing medium was added to the wells. After 24-72 h incubation, WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt)

absorbance was measured at 450 nm.

Annexin V assay

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solution (Enzo Life Sciences, Farmingdale, NY, USA) was added with new medium, and

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Annexin V assay was performed using an FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA). Harvested cells were washed twice with cold

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phosphate-buffered saline (PBS), and the cells were resuspended in 1X Annexin V binding

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buffer (1 × 106 cells/ml). Then, 100 μl of the solution (1 × 105 cells) was transferred to a 5 ml tube and added with 5 µl titrated FITC Annexin V and Propidium Iodide (PI) Staining

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Solution. Cells were incubated for 15 min at room temperature in the dark. The volume was made up to 700 μl and analyzed with the FACS Calibur system (BD Biosciences, San Diego,

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CA, USA).

Western blot analysis CT26 cells (1 × 106 cells/well) were incubated with various concentrations of quercetin. 8

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Stimulated cells were rinsed with PBS and then lysed in lysis buffer (iNtRon Biotech, Seoul, Korea) for 1 h. Cell lysates were centrifuged for 10 min, and the quantity of protein in the supernatant was evaluated by using a bicinchoninic acid (BCA) protein assay. The supernatant was mixed with 2X sample buffer for sulfate-polyacrylamide gel electrophoresis

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and transferred to a membrane. The membranes were blocked with 5% skim milk for 1 h 30 min and incubated over 3 h with primary antibodies. The antibodies were detected using horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and immunoglobulin G (Dako,

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Glostrup, Denmark), and blots were detected using the ECL system (Santa Cruz, CA, USA).

Wound healing assay

CT26 cells were seeded in a 6-well plate (5 × 105 cells/well) to form a monolayer

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overnight. Using a 200 μl pipette tip, a scratch of ~ 1 mm width was made in triplicate. Detached cells were removed, and the scratches were monitored at regular intervals over the

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course of 0-24 h under serum-free conditions containing quercetin at the indicated concentrations. Photographs were taken under phase contrast microscopy (Leica, Wetzlar,

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Germany). The cells that migrated across the lines were counted in five random fields.

Invasion assay

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CT26 cells (5 × 104 cells) were suspended in a serum-free medium with quercetin and

added to the upper part of a BD BioCoat GFR matrigel invasion chamber. The lower part of the transwell chamber was filled with 10% FBS in DMEM as a chemoattractant and incubated for 24 h. The membrane inserts were washed twice with PBS and fixed in 3.7% paraformaldehyde in PBS for 5 min. The fixed cells were added with 100% methanol for 20 9

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min and stained with Giemsa stain solution for 15 min. After twice washing with PBS, the inside of the chamber was wiped out with a cotton swab to eliminate non-attached cells. The membrane inserts were dried and observed under a microscope. Cell numbers in randomly selected fields were counted under a light microscope at ×400 magnification (EVOS, Thermo

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Fisher Scientific, USA). Cell numbers in the randomly selected fields were counted.

Gelatin zymography

Cells were seeded in 6-well plates (3 × 105 cells/well) and treated with quercetin for 24 h.

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Conditioned medium was collected from the cells and mixed with 5× sample buffer and subjected to electrophoresis on an 8% SDS-PAGE gel containing 1% gelatin. The gels were washed two times in a renaturing buffer (pH 7.5, 2.5% Triton X-100 in D.W) for 30 min and

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incubated in a developing buffer (50 mM Tris-HCl pH 7.5, 10 mM CaCl2, and 150 mM NaCl) at 37°C for 24 h to allow digestion of the gelatin. The gelatinolytic activity of MMPs

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was visualized by staining the gels with Coomassie blue R-250 solution for 20 min and the

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samples were destained with destaining buffer (40% methanol, and 10% acetic acid in D.W).

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Real-time RT-PCR

Total RNA was extracted from cells by using an RNA-spinTM Total RNA Extraction Kit

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(iNtRon Biotech, Seoul, Korea) according to the manufacturer’s instruction. First-strand cDNA was synthesized using a Power cDNA Synthesis Kit (iNtRon Biotech, Seoul, Korea). Reverse transcription was performed at 42 °C for 50 min and then at 70 °C for 15 min. Realtime RT-PCR was carried out using Power SYBR® Green PCR Master Mix and step-one plusTM real-time pcr systems (Applied Biosystems, Foster City, CA, USA). The sequences of 10

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the

primers

used

for

murine

genes

were

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following:

E-cadherin,

5’-

AATGGCGGCAATGCAATCCCAAGA-3’ and 5’-TGCCACAGACCGATTGTGGAGATA3’;

5’-TGGAGAACCCCATTGACATT-3’

N-cadherin,

and

5’-

TGATCCCTCAGGAACTGTCC-3’; β-catenin, 5’-ACTGCTGGGACTCTG-3’ and 5’-

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TGATGGCGTAGAACAG-3’; Snail, 5’-TCCAAACCCACTCGGATGTGAAGA-3’ and 5’TTGGTGCTTGTGGAGCAAGGACAT-3’; MMP-2, 5’-CCCCATGAAGCCTTGTTTACC3’

5’-TTGTAGGAGGTGCCCTGGAA-3’;

and

AGACCAAGGGTACAGCCTGTTC-3’

and

MMP-9,

5’-

5’-GGCACGCTGGAATGATCTAAG-3’;

3’;

TIMP-2,

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TIMP-1, 5’-GCCTACACCCCAGTCATGGA-3’ and 5’-GGCCCGTGATGAGAAACTCTT5’-AGGAGATGTAGCAAGGGATCA-3’

and

5’-

GAGCCTGAACCACAGGTACCA-3’ ; GAPDH, 5’-GACATGCCGCCTGGAGAAAC-3’ and 5’-AGCCCAGGATGCCCTTTAGT-3’. All data were normalized to GAPDH mRNA

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level.

Experimental lung metastasis model

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For experimental lung metastasis, CT26 cells were harvested and resuspended in PBS.

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Mice were inoculated with cells (3 × 104 cells/200 μl of PBS) via the lateral tail vein (i.v.). Quercetin and DMSO were administrated by intraperitoneal (i.p) injection 2 h prior to the

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injection of cancer cells, and the mice were treated once every 2 days. The mice were sacrificed 14 days later, and the lungs were excised and fixed in Bouin’s solution (Sigma, St Louis, MO, USA). The lung weights were measured, and the tumor colonies were counted to evaluate the inhibition of metastasis.

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Statistical analysis All experiments were performed at least three times, and the data were analyzed for statistical significance using the Student’s t-test and one-way ANOVA followed by post-hoc Tukey’s multiple comparisons tests. P values < 0.05 were considered to indicate statistically

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significant differences. The mean ± SD values were calculated for all variables.

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Results 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

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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

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quercetin treatment for 48 h were detected by microscope observation. They showed apoptotic phenotypes such as cell shrinkage, volume reduction, and irregularities in shape. It was confirmed that quercetin inhibited the proliferation of CT26, MC38, and HT29 cells in a dose- and time-dependent manner (Fig. 1C-E). Moreover, CCD-18Co cells did not show

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cytotoxicity up to 100 μM quercetin treatment for 72 h (Fig. 1F). These results suggest that quercetin selectively decreased the cell viability of CRC cells. Since CT26 is a highly

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metastatic CRC cell line, it is commonly used for studies regarding experimental metastasis

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models. In particular, quercetin showed much stronger growth inhibitory effect in CT26 cells

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than in MC38 cells. Therefore, we selected CT26 cells for subsequent experiments.

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Effect of quercetin on apoptotic cell death in CT26 and HT29 cells To confirm whether quercetin-induced cell death is mediated by apoptosis, we evaluated

apoptotic cell death by DAPI nuclear staining and flow cytometric analysis. As expected, quercetin (50, 100 μM) treatment for 24 h led to morphological changes in cell apoptosis, including chromatin condensation (Fig. 2A). In addition, flow cytometric analysis of 13

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quercetin-treated CT26 and HT29 cells showed a dose-dependent increase in the percentage of Annexin V-positive cells, indicative of apoptosis (Fig. 2B-D). These results indicate that quercetin induced apoptosis in CT26 and HT29 cells.

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Effect of quercetin on apoptotic pathway in CT26 cells Because quercetin induced the apoptosis of CT26 cells, the molecular mechanism was investigated by western blot analysis. Exposure of CT26 cells to quercetin caused the cleavage of caspases-3, -9 and PARP, whereas expressions of Bcl-2 and Bcl-xL decreased in a

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dose- and time-dependent manner (Fig. 3A and B). In addition, quercetin increased the phosphorylation of ERK, JNK and p38 within early phase (Fig. 3C). To investigate the relation between MAPK activation and quercetin-induced apoptosis, we applied a

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pretreatment with specific MAPK inhibitors, SB203580, U0126, and SP600125, to quercetintreated CT26 cells. Pretreatment with MAPK inhibitors effectively prevented quercetin-

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induced apoptosis (Fig. 3D). Expressions of apoptosis-related proteins including caspase-3, 9, PARP, Bcl-2, and Bcl-xL were also regulated by MAPK inhibitors (Fig. 3E and F). These

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results suggest that quercetin induces apoptosis through regulation of Bcl-2 family protein

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expression and cleavage of caspases and PARP as well as activation of MAPK-mediated

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signaling in CT26 cells.

Effect of quercetin on the migration and invasion ability of CT26 cells Migration and invasion is an important step in the metastasis process. Therefore, we evaluated the inhibitory effects of quercetin on migration and invasion. As shown in Fig. 4A, quercetin (0-10 μM) induced significant decreases in the migration of CT26 cells. Invasion 14

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assay was carried out to determine the effect of quercetin on invasion ability using a matrigelcoated transwell chamber. Quercetin inhibited the invasion of cells in comparison with the control group in a dose-dependent manner (Fig. 4B). MMPs are a family of ECM-degrading enzymes that play a critical role in the migration and invasion of cancer cells (Nabeshima et

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al., 2002). Among 20 types of MMPs, MMP-2 and MMP-9 are known to degrade the ECM in a process associated with metastasis. The activity of MMP-2 and MMP-9 is strongly related to that of their specific inhibitors, TIMP-2 and TIMP-1 (Itoh and Nagase 2002). Quercetin inhibited the activity of MMP-2 and MMP-9 in a dose-dependent manner (Fig. 4C). The

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activity of MMP-2 and MMP-9 was analyzed by gelatin zymography. As shown in Fig. 4D, quercetin decreases the expression of MMP-2 and MMP-9 in CT26 cells. TIMP-2 and TIMP1 mRNA expression was significantly increased by quercetin in CT26 cells (Fig. 4E). These results suggest that quercetin decreases migration and invasion abilities through the inhibition

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of MMP-2 and MMP-9 expression and activity in CT26 cells.

Effect of quercetin on the expressions of EMT-related genes in CT26 cells.

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The process of EMT is an essential event for cancer cell migration and invasion. Loss of

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the epithelial phenotypic marker expression and increase of mesenchymal phenotypic marker expression are necessary in the EMT process (Thiery and Sleeman 2006). Thus, as shown in

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Fig. 5, the mRNA and protein expression of epithelial and mesenchymal markers was investigated in quercetin-treated CT26 cells. The expression of E-cadherin was increased, whereas expression of N-cadherin, β-catenin, and snail was decreased by quercetin. These results suggest that the inhibitory effects of quercetin on the migration and invasion of CT26 cells are mediated by regulation of the EMT process. 15

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Effect of quercetin on the lung metastasis of CT26 cells. Previous results have shown that quercetin significantly induced apoptosis and suppressed the migration and invasion ability of CT26 cells. Therefore, we evaluated the anti-

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metastatic effect of quercetin in in vivo using an experimental lung metastasis model. To assess the toxicity of quercetin, we measured body weight. Quercetin-administered groups (10 or 50 mg/kg) did not shown changes in body weight compared with the control group (Fig. 6A). The number of tumor nodules and lung weights were significantly decreased by

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quercetin (Fig. 6B-D). These results support the anti-metastatic effect of quercetin on

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colorectal lung metastasis.

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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

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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 to have an anti-tumor effect on colon cancer cells through inducing apoptosis (Kim et al., 2010; Zhang et al., 2015). Our data

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shows that quercetin inhibits the proliferation of metastatic colon cancer cells in vitro via the induction of apoptosis (Fig. 1, Fig. 2A and B).

Apoptosis occurs through extrinsic and intrinsic pathways. The intrinsic pathway is

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activated by several signals, including heat shock, loss of cell-survival or growth factors, and damage to cellular DNA. This pathway is mainly maintained by the balance between pro- and

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anti-apoptotic proteins of the Bcl-2 family. Among the Bcl-2 family proteins, anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, initiate caspase cascade (Kerr et al., 1972; Fulda and

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Debatin 2006). In the present study, we determined the molecular mechanisms of quercetin-

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induced apoptosis of CT26 cells. The expression of Bcl-2 and Bcl-xL was decreased after treatment with quercetin. Caspase-9 was subsequently activated by quercetin treatment, and it

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led to the cleavage of caspase-3 and PARP (Fig. 3A and B). Other studies have shown that caspase cascade in apoptosis is related to the regulation of MAPK activity in colon cancer cells (Ryu and Chung 2015). Quercetin induces apoptosis via the sestrin 2-AMPK-p38 MAPK signaling pathway in HT29 cells (Kim et al., 2010). In addition, p38 MAPK, JNK and ERK play a critical role in quercetin-induced apoptosis in AGS cells (Kim et al., 2014). 17

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Therefore, we assumed that quercetin-induced apoptosis might be related to the MAPK pathway in CT26 cells. In the present study, quercetin increased phosphorylation of ERK, JNK, and p38 (Fig. 3C). These results suggest that quercetin induces apoptosis through the modulation of the intrinsic apoptotic pathway via MAPK pathway in CT26 cells.

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The EMT process contributes to a more stromal cellular adhesion profile because deregulated cell-cell or cell-matrix adhesion and increased migration and invasion are required for the progression of metastatic cancers (Gupta and Massagué 2006). The migration and invasion abilities of cancer cells are increased by decreased E-cadherin expression, which

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is a cell-cell adhesion molecule whereas N-cadherin expression is decreased, which is inversely associated with E-cadherin (Thiery and Sleeman 2006). Quercetin regulates the expression of EMT markers, such as E-cadherin, vimentin, β-catenin and twist in head and neck cancer-derived sphere cells and breast cancer line MDA-MB-231 and MDA-MB-468

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cells (Chang et al., 2013; Srinivasan et al., 2016). In addition, quercetin prevented the EGF-

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induced expression of N-cadherin and vimentin and increased the expression of E-cadherin in prostate cell line PC-3 cells (Bhat et al., 2014). Based on these reports, it is assumed that

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quercetin treatment may modulate the expression of EMT-related markers. Quercetin, in this study, actually suppressed EMT through increasing the expression of E-cadherin and

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decreasing the expression of N-cadherin, β-catenin, and snail (Fig. 5). Therefore, quercetin

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inhibits EMT through modulating the expression of EMT-related genes. Since quercetin is a lipophilic compound, normally it is dissolved in DMSO for

experimental usage. It has been reported that trichostatin A (TSA) combination with quercetin administered by intraperitoneal (i.p.) injection shows greater decrease in the tumor size of A549 in xenograft mice when compared to oral administration (Chan et al., 2014). A recent 18

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study also demonstrated that i.p. injection of quercetin (50 mg/kg/day, for 9 days) reduces cisplatin nephrotoxicity in rats (Sanchez-Gonzalez et al., 2011). Therefore, we used quercetin intraperitoneally injected at maximum dose of 50 mg/kg for once every other day in this study.

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Over the past decade, the use of natural products and their compounds comprising dietary supplements and therapeutic agents for various diseases has steadily been on the rise. In the case of quercetin, this compound enhances 5-FU-induced apoptosis in microsatellite instability (MSI) CRC cells through p53 modulation and potentiates doxorubicin mediated

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anti-tumor effects against liver cancer through regulation of p53/Bcl-xL (Xavier et al., 2011; Wang et al., 2012). However, combination of quercetin and several drugs may cause altered pharmacokinetics due to various mechanisms of interaction. Although quercetin increased the area under the curve (AUC) of oral doxorubicin, it did not affect the intravenous

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pharmacokinetics of doxorubicin as well as Tmax and T1/2 values after oral doxorubicin.

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Moreover, quercetin decreased the Cmax and AUC of cyclosporine, whereas the Cmax and AUC values of oral etoposide were increased by quercetin co-administration (Srinivas, 2015).

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Thus, further research is required for elucidation of herb-drug interactions to prevent unexpected side effects.

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This study demonstrated the inhibitory effect of quercetin on colorectal metastasis in in

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vitro and in vivo models. Recent studies have reported that various natural compounds are effective in colorectal metastasis and their therapeutic mechanisms. Berberine inhibits CRC cells migration via AMP-activated protein kinase-mediated downregulation of integrin β1 signaling. Triptolide from Tripterygium wilfordii can reduce proliferation and migration of CRC cells by inhibition of cell cycle regulators and cytokine receptors. In addition, clinical 19

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studies showed that herbal therapy also can improve colorectal metastasis. The co-treatment of Yi-Qi-Zhu-Yu Decoction and FOLFOX4 regimen significantly decrease the side effects and prolong the overall survival time. Additionally, various decoctions such as Jian-Pi-XiaoLiu Decoction, Fu-Zheng Capsule, and Qu-Xie Capsule prevent or decrease colorectal

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metastasis (Deng et al., 2012). Therefore, it is expected that future studies with quercetin will clarify the various molecular mechanisms and combination of anti-cancer drugs for colorectal metastasis.

In conclusion, this study provides new evidence to support the anti-metastatic effect of

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quercetin against metastatic colorectal cancer cells. Our data shows that quercetin induces the apoptosis of CT26 cells by modulating the intrinsic apoptotic pathway and activating the ERK, JNK, and p38 MAPK signaling pathways. Moreover, quercetin inhibited the migration and invasion abilities of CT26 cells in vitro by decreasing MMP-2 and MMP-9 activity and

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regulating the expression of EMT-related genes, including E-cadherin, N-cadherin, β-catenin,

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and snail. This compound inhibited the lung metastasis of colon cancer cells in an experimental in vivo metastasis model by the induction of apoptosis and suppression of

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metastatic abilities. Based on these results, we conclude that quercetin may serve as an

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effective therapeutic agent for colorectal lung metastasis treatment.

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Conflict of interest The authors have declared no conflict of interest.

Acknowledgments

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This research was supported by the National Research Foundation of Korea (NRF) grant

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funded by the Korean government (MSIP) (NRF-2015R1C1A1A02036733).

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Figure legends

Fig. 1. Quercetin decreases cell viability of metastatic colon carcinoma cells. (A) Chemical structure of quercetin. (B) Morphology of quercetin-treated cells with a scale bar of 200 μm.

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After 48 h incubation with quercetin, images were acquired by microscopy. Photographs are representative of three independent experiments. (C-E) Cell viability of quercetin-treated CT26 (C), MC38 (D), and HT29 (E) cells. After 24-72 h treatment with various concentrations of quercetin, cell viability was determined by WST assay. 5-FU was used as a

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positive control. (F) Cell viability of CCD-18Co cells after quercetin treatment for 72 h.

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Results are expressed as the mean ± SD of three independent experiments. *p < 0.05.

Fig. 2. Quercetin induces apoptosis of CT26 and HT29 cells. (A) DAPI nuclear staining of 27

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quercetin-treated cells with a scale bar of 100 μm. (B) Flow cytometric analysis of quercetintreated cells. CT26 and HT29 cells were incubated with the indicated concentrations of quercetin for 24 h and stained with Annexin V and 7-AAD. The figure is representative of three independent experiments (n = 3). (C and D) Quantification of apoptotic cells.

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Quantification of the data presented as percentages of apoptotic cells. 5-FU was used as a positive control. Results are expressed as the mean ± SD of three independent experiments.

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*p < 0.05.

Fig. 3. Quercetin induces apoptosis by intrinsic apoptotic pathway via MAPK signaling. (A and B) Expression of apoptosis-related proteins in quercetin-treated CT26 cells. Western blot analysis was performed to determine the expression of caspase-3, -9, PARP, Bcl-2 and Bcl-xL in CT26 cells. α-tubulin was used as a loading control. (C) Regulation of MAPKs in 28

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quercetin-treated CT26 cells. Equal amounts of cell lysates were electrophoresed and ERK, JNK, and p38 and their phosphorylated expression form were detected by Western blotting analysis. (D) Role of MAPK signaling on the quercetin-induced apoptosis of CT26 cells. The cells were stimulated with quercetin after pre-treatment with U0126, SP600125 and

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SB203580, respectively. After 24 h incubation, cell viability was determined by WST assay. (E) Expression of apoptosis-related proteins in MAPK inhibitors and quercetin-treated CT26 cells. (F) The band intensities were measured using an image analyzer and presented as the relative ratio. Results are expressed as the mean ± SD of three independent experiments. #p <

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0.05 versus blank and *p < 0.05 versus quercetin alone.

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Fig. 4. Quercetin reduces migration and invasion ability of CT26 cells through inhibition of MMP-2 and MMP-9 activity. (A) Wound healing assay. Images were photographed using a microscope after 24 h incubation (100× magnification). Mean number of cells at 24 h in the denuded zone and represents the average of three independent experiments. (B) Invasion

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assay using matrigel-coated transwell chamber (scale bar, 200 µm). Photographs are representative of three independent experiments. (C) Activity of MMP-2 and -9 in quercetintreated CT26 cells was determined by gelatin zymography. (D and E) The mRNA expression levels of MMP-2,-9 (D) and TIMP-1, -2 (E) were measured by real-time RT-PCR after

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quercetin treatment for 24 h. Results are expressed as the mean ± SD of three independent

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experiments. *p < 0.05.

Fig. 5. Quercetin regulates the expression of EMT-related genes in CT26 cells. The mRNA expression levels of EMT markers E-cadherin (A), N-cadherin (B), β-catenin (C), and snail 30

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(D) were analyzed by real-time RT-PCR after quercetin treatment for 24 h. CT26 cells (5 × 105 cells/well) were treated with quercetin for 24 h. Results are expressed as the mean ± SD of three independent experiments. (E) The protein levels of EMT markers. (F) The band intensities were measured using an image analyzer and presented as the relative ratio. α-

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tubulin was used as a loading control. * p < 0.05 and ** p < 0.01.

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Fig. 6. Quercetin inhibits the colorectal lung metastasis. CT26 cells (1 × 105 cells) were

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intravenously transplanted into BALB/c mice. Mice were divided into four groups (n = 6) and subjected to i.p injection of quercetin (10 or 50 mg/kg mouse) once every 2 days until sacrifice. Control group mice were administered the same volume of DMSO. (A) Body weight of mice. (B) Lungs were stained with Bouin’s solution to count the tumor nodules. (C and D) The average number of tumor nodules (C) and lung weight (D) are expressed as the mean ± SD. Data are representative of three independent experiments. * p < 0.05, ** p < 0.01. 31

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Graphical abstract:

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