|Year : 2019 | Volume
| Issue : 1 | Page : 157-163
Myricetin ameliorates cytokine-induced migration and invasion of cholangiocarcinoma cells via suppression of STAT3 pathway
Pattarapon Tuponchai1, Veerapol Kukongviriyapan2, Auemduan Prawan2, Sarinya Kongpetch2, Laddawan Senggunprai2
1 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand
|Date of Web Publication||13-Mar-2019|
Dr. Laddawan Senggunprai
Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002
Source of Support: None, Conflict of Interest: None
Aim of Study: Cholangiocarcinoma (CCA) is an aggressive cancer with considerable metastatic potential. Various cytokines secreted by tumor cells or cells in the tumor environment can promote the metastasis of CCA. The aim of the present study was to investigate the effect of myricetin on the inhibition of cytokine-induced migration and invasion and the associated cellular mechanisms in human CCA cells.
Materials and Methods: CCA KKU-100 cells were treated with a pro-inflammatory cytokine mixture consisting of interleukin-6, interferon-γ, and tumor necrosis factor-α. The migratory and invasive ability of KKU-100 cells were determined using a wound-healing assay and transwell invasion assay. The effect of myricetin on cytokine-induced STAT3 activation in CCA cells was determined using Western blot analysis. The real-time polymerase chain reaction was performed to determine messenger RNA expression.
Results: Myricetin significantly inhibited cytokine-induced migration and invasion of KKU-100 cells. Detailed molecular analyses revealed that myricetin suppressed the activation of the STAT3 pathway, evidently by a decrease of the active phospho-STAT3 protein expression after myricetin treatment. The cytokine-mediated upregulation of metastasis- and inflammatory-associated genes, which are downstream genes of STAT3 including the intercellular adhesion molecule-1, matrix metalloproteinase-9, inducible nitric oxide synthase, and cyclo-oxygenase 2 (COX-2), were also significantly abolished by myricetin treatment. Moreover, the anti-migratory and anti-invasive activities of a widely prescribed COX inhibitor, indomethacin, were also revealed.
Conclusion: This finding reveals the anti-metastatic effect of myricetin against CCA cells which is mediated partly through suppression of the STAT3 pathway. This compound could be potentially useful as a therapeutic agent against CCA.
Keywords: Cholangiocarcinoma, inflammation, metastasis, myricetin, STAT3
|How to cite this article:|
Tuponchai P, Kukongviriyapan V, Prawan A, Kongpetch S, Senggunprai L. Myricetin ameliorates cytokine-induced migration and invasion of cholangiocarcinoma cells via suppression of STAT3 pathway. J Can Res Ther 2019;15:157-63
|How to cite this URL:|
Tuponchai P, Kukongviriyapan V, Prawan A, Kongpetch S, Senggunprai L. Myricetin ameliorates cytokine-induced migration and invasion of cholangiocarcinoma cells via suppression of STAT3 pathway. J Can Res Ther [serial online] 2019 [cited 2019 Oct 17];15:157-63. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/157/237383
| > Introduction|| |
Cholangiocarcinoma (CCA) is an extremely aggressive malignancy arising from biliary epithelial cells lining extrahepatic or intrahepatic bile ducts. It is the most common form of liver cancer in the Southeast Asia region. Although surgery is the only curative treatment for CCA, most of the patients are inappropriate for the surgical treatment. Conventional cytotoxic therapy exhibits low efficacy for prolonged overall survival in patients with unresected CCA and disease recurrence is common.
Chronic infection with the liver flukes, Opisthorchis viverrini and Clonorchis sinensis, is a predominant risk factor for the development of CCA in Asia. The infection causes persistent inflammation that can create a local environment enriched with cytokines and growth factors. These inflammatory mediators contribute to alteration of intracellular signaling pathways regulating cell proliferation, survival, apoptosis, and motility. JAK/STAT3 is one of the most important signaling cascades linking inflammation and carcinogenesis. An aberrant activation of this pathway has been reported in various cancers including CCA.,
Cancer metastasis is one of the factors affecting cancer treatment and is the primary cause of morbidity and mortality in cancer patients. This process involves multiple sequential steps including migration and invasion. Various signaling transduction cascades including JAK/STAT3 have been implicated in several features of the metastatic process of CCA., STAT3 acts as a transcription factor that regulates the expression of metastasis-associated genes such as vascular endothelial growth factor, intercellular adhesion molecule-1 (ICAM-1), and matrix metalloproteinase-9 (MMP-9). Indeed, overexpression of phosphorylated STAT3 was associated with a high level of metastasis in CCA patients. Given the apparent role of STAT3 in the metastatic process of CCA, compounds aimed at suppressing its activity would likely be therapeutically useful.
For several decades, anticancer drug discovery has focused toward natural compounds., Myricetin is a naturally occurring flavonoid abundant in fruits, vegetables, and tea. Previous studies have revealed several biological activities of myricetin such as antioxidant, anti-inflammatory, and anticancer effects. The aim of the present study was to evaluate the anti-metastatic potential of myricetin against CCA cells and to gain insight into the cellular mechanism mediating its effect.
| > Materials and Methods|| |
Myricetin and indomethacin were purchased from Sigma Chemical (St. Louis, MO). IL-6, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were obtained from Prospec-Bio (Israel). RIPA Lysis Buffer was obtained from Amresco (Solon, OH, USA). The primary antibody against phospho-STAT3 (p-STAT3) (Tyr705) was purchased from Cell Signaling Technology (Dancer, MA, USA). The primary antibody against β-actin and the secondary horseradish peroxidase (HRP)-linked antibodies were obtained from Santa Cruz Biotechnology, Inc.(California, USA). Reagents for cell culture were from Gibco-BRL Life Technologies (Grand Island, New York).
A human CCA cell line, KKU-100, was grown in Ham's F12 medium supplemented with sodium bicarbonate, 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.3), 100 U/mL penicillin, 100 μg/mL gentamycin sulfate, and 10% fetal calf serum and maintained under 5% CO2 in air at 37°C. The cells were subcultured every 2 days using 0.25% trypsin– ethylenediaminetetraacetic acid.
Cell viability assay
Sulforhodamine B (SRB) was used to determine the effect of myricetin on the viability of CCA cells. KKU-100 cells were treated with various concentrations of myricetin for 24 h. Cultured cells were fixed with ice-cold 10% trichloroacetic acid and stained with 0.4% SRB in 1% acetic acid. Excess dye was removed with 1% acetic acid, and protein-bound dye was solubilized with 10 mM Tris for determination of absorbance with a microplate reader with a filter wavelength of 570 nm.
KKU-100 cells were grown in a 24-well plate, and a scratch wound was made using a sterile 200 μL pipette tip. After washing with PBS to remove any detached cells, the wound was photographed as a baseline. The cells were then treated with the cytokine mixture (IL-6 100 ng/mL + IFN-γ 100 ng/mL + TNF-α 20 ng/mL) with or without various concentrations of myricetin or indomethacin. A series of images of the scratched wound were taken at 24 h after treatment. The closing of the scratched wound, representing the migration process, was determined by the capture of the denuded area along the scratch using Image-Pro Plus software (Media Cybernetics, LP, USA). The wound distance was calculated by dividing the area by the length of scratch.
The invasive behavior of CCA cells was analyzed using transwell chambers (Corning, Lowell, MA). The polycarbonate membrane of an 8 μm pore size of transwell inserts were coated with 0.3 mg/mL Matrigel (BD Biosciences, Bedford, MA) overnight. The KKU-100 cells suspended in serum-free medium containing vehicle, cytokine mixture, or cytokine with myricetin or indomethacin were then added to the upper chamber. The lower part of the chamber was filled with medium containing 10% fetal calf serum. After incubation for 24 h, the cells on the upper surface were gently removed by scraping with a cotton swab. The invaded cells were fixed with methanol, stained with crystal violet, and then photographed.
Protein extraction and Western blot analysis
After treatments at designated time points, whole cell lysates were prepared using RIPA cell lysis buffer according to the manufacturer's instructions. The 20 μg of proteins were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were electrophoretically transferred to a polyvinylidene fluoride membrane, blocked with 5% bovine serum albumin, and incubated with primary antibodies against p-STAT3 (1:1000) and β-actin (1:5000) overnight at 4°C. After washing, the membranes were exposed to HRP-conjugated secondary antibody for 2 h. The specific protein bands were subsequently examined by Luminata™ Forte Western HRP Substrate (Merck Millipore, Watford, UK).
Real-time polymerase chain reaction
Total RNA was extracted from the cells using trizol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed to single-stranded cDNA using iScript reverse transcriptase (Bio-Rad, Hercules, CA). The primers for polymerase chain reaction (PCR) were as follows: MMP-9 forward 5'-GAAGATGCTGCTGTTCAGCG-3' and MMP-9 reverse 5'-ACTTGGTCCACCTGGTTCAA-3'; ICAM-1 forward 5'-CAAGGCCTCAGTCAGTGTGA-3' and ICAM-1 reverse 5'-CCTCTGGCTTCGTCAGAATC-3'; inducible nitric oxide synthase (iNOS) forward 5'-GTTCTCACGGCACAGGTCTC-3' and iNOS reverse 5'-GCAGGTCACTTATGTCACTTATC-3'; cyclo-oxygenase-2 (COX-2) forward 5'-TGAGCATCTACGGTTTGCTG-3' and COX-2 reverse 5'-TGCTTGTCTGGAACAACTGC-3'; and β-actin forward 5'-TGCCATCCTAAAAGCCAC-3' and β-actin reverse 5'-TCAACTGGTCTCAAGTCAGTG-3'. The quantitative real-time-PCR (RT-PCR) was carried out using QPCR Green Master Mix (Biotechrabbit GmbH, Germany), and thermal cycling was performed using Light Cycler 480II/384 (Roche Applied Science) under the following conditions: denaturation at 95°C for 3 min and amplification by cycling 40 times at 95°C for 15 s and 60°C for 31 s.
Data were analyzed using the Prism 5 program (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard deviation. Data were statistically analyzed using one-way analysis of variance. Post hoc analyses were carried out using the Student–Newman–Keuls multiple comparison test. Results were considered to be statistically significant at a value of P < 0.05.
| > Results|| |
The effect of myricetin on cholangiocarcinoma cell viability
The effect of myricetin on the viability of CCA cells was first determined by the SRB assay. The KKU-100 cells were treated with different concentrations of myricetin (1, 3, 5, 7, 10, and 25 μM) for 24 h. The results showed that under these conditions, myricetin did not significantly affect the viability of CCA cells [Figure 1].
|Figure 1: Effect of myricetin on the viability of cholangiocarcinoma cells. KKU-100 cells were treated with various concentrations of myricetin for 24 h. After treatment, the cell viability was determined by using the sulforhodamine B assay. Each bar represents the mean ± standard deviation of three independent experiments|
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The effect of myricetin on cholangiocarcinoma cell migration and invasion
Migration and invasion are two critical events in the metastasis process of cancer cells. In this study, the effect of myricetin on cytokine-stimulated migration and invasion of CCA cells was investigated using the wound-healing and transwell invasion assays. Results showed that the migratory ability induced by cytokine of CCA cells treated with myricetin was markedly abolished in a dose-dependent manner [Figure 2]a and [Figure 2]b. Similarly, myricetin could significantly suppress cytokine-induced CCA cell invasion [Figure 2]c and [Figure 2]d.
|Figure 2: Effect of myricetin on cytokine-induced cholangiocarcinoma cell migration and invasion. (a) Scratch wounds of monolayer KKU-100 cells were treated with vehicle, cytokine mixture (IL-6 100 ng/mL + interferon-γ 100 ng/mL + tumor necrosis factor-α 20 ng/mL) or a combination of cytokine and myricetin (5, 10, and 25 μM) for 24 h. Representative images of wound healing were obtained at the time of the scratch and 24 h later. (b) The graph shows the level of cell migration into the wound scratch quantified as the percentage of wound closure at 24 h. Data represent the mean ± standard deviation of three independent experiments. (c) The invasion ability was assessed using the transwell invasion assay. KKU-100 cells were treated with vehicle, a cytokine mixture or a combination of cytokine and myricetin for 24 h. Invaded cells were fixed, stained with crystal violet, and photographed. Results shown are representative of three independent experiments.(d) The graph shows the quantitative analysis of invaded cells. Data represent the mean ± standard deviation. CT = Control; CK = Cytokine mixture; MR = Myricetin. *P < 0.05 versus control. #P < 0.05 versus cytokine mixture alone|
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The effect of myricetin on the STAT3 signaling pathway of cholangiocarcinoma cells
Previous studies suggested the involvement of JAK/STAT3 cascades in the metastasis of CCA. In the present study, the effect of myricetin on inflammatory cytokine-induced STAT3 pathway activation of CCA cells was investigated using Western blot analysis. The results showed that the cytokine mixture (IL-6+ IFN-γ+ TNF-α) induced STAT3 pathway activation, evidently by an increased phosphorylation of STAT3. Treatment of cells with myricetin suppressed STAT3 phosphorylation in a dose-dependent manner [Figure 3].
|Figure 3: Effect of myricetin on cytokine-induced STAT3 activation. KKU-100 cells were pretreated with indicated concentrations of myricetin for 2 h and then were treated with the cytokine mixture (IL-6 100 ng/mL + interferon-γ 100 ng/mL + tumor necrosis factor-α 20 ng/mL) for 30 min. After that, whole cell lysates were prepared and analyzed by Western blotting. Results shown are representative of two reproducible independent experiments. CK = Cytokine mixture; MR = Myricetin|
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The effect of myricetin on the expression of genes involving in cholangiocarcinoma metastasis
STAT3 has been shown to regulate the expression of several genes involving in metastasis process of cancer cells. In the present study, the effects of myricetin on the expression of metastasis-associated genes, MMP-9, and ICAM-1, were further evaluated. The results showed that cytokine treatment prominently upregulated the expression of MMP-9 and ICAM-1. Treatment with myricetin significantly suppressed cytokine-mediated upregulation of these two genes [Figure 4]. In addition, myricetin also abolished cytokine-induced expression of iNOS and COX-2, which are the critical molecules involved in inflammatory and carcinogenesis processes [Figure 4].
|Figure 4: Effects of myricetin on the cytokine-induced expression of matrix metalloproteinase-9, intercellular adhesion molecule-1, inducible nitric oxide synthase, and cyclo-oxygenase-2. KKU-100 cells were treated with vehicle, cytokine mixture (IL-6 100 ng/mL + interferon-γ 100 ng/mL + tumor necrosis factor-α 20 ng/mL), or a combination of cytokine and myricetin (10 or 25 μM) for 6 h. At the end of treatments, total RNA of cells were collected, and real-time-polymerase chain reaction was carried out for indicated gene expression. The data represent the mean ± standard deviation of three independent experiments as follows: CT = Control; CK = Cytokine mixture; and MR = Myricetin. *P < 0.05 vs control #P < 0.05 versus cytokine mixture alone|
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The effect of indomethacin on cholangiocarcinoma cell migration and invasion
Since the inhibition of COX-2 expression could attribute in part to the mechanism for anti-metastasis action, the effect of indomethacin, a commonly used non-steroidal anti-inflammatory COX inhibitor drug, on the migration and invasion properties of CCA cells were also investigated in this study. Results showed that 25 μM indomethacin significantly suppressed cytokine-induced CCA cell migration [Figure 5]a and [Figure 5]b. Similarly, exposure to indomethacin also impaired the invasiveness of CCA cells induced by cytokine [Figure 5]c and [Figure 5]d. Notably, under these conditions, the cell viability was not affected (data not shown).
|Figure 5: Effect of indomethacin on cytokine-induced cholangiocarcinoma cell migration and invasion. (a) Scratch wounds of monolayer KKU-100 cells were treated with vehicle, cytokine mixture (IL-6 100 ng/mL + interferon-γ 100 ng/mL + tumor necrosis factor-α 20 ng/mL), or a combination of cytokine and indomethacin (5, 10, and 25 μM) for 24 h. Representative images of wound healing were obtained at the time of the scratch and 24 h later. (b) The graph shows the level of cell migration into the wound scratch quantified as the percentage of wound closure at 24 h. Data represent the mean ± standard deviation averaged from quadruplets of one experiment. (c) The invasion ability was assessed using the transwell invasion assay. KKU-100 cells were treated with vehicle, a cytokine mixture, or a combination of cytokine and indomethacin for 24 h. Invaded cells were fixed, stained with crystal violet, and photographed. (d) The graph shows the quantitative analysis of invaded cells. Data represent the mean ± standard deviation of three independent experiments as follows: CT = Control; CK = Cytokine mixture; and IDC = Indomethacin. *P < 0.05 versus control. #P < 0.05 versus cytokine mixture alone|
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| > Discussion|| |
CCA is a malignant cancer with significant metastatic potential. The development of a novel effective agent targeting metastasis could be valuable to improve current treatments in CCA patients. The JAK/STAT3 signaling pathway, which modulates several molecules involved in metastasis process, is usually dysregulated in many cancers., In the present study, it was revealed that myricetin inhibits cytokine-induced migration and invasion of CCA cells. Suppression of the metastatic potential of CCA cells is associated with reduced active STAT3 after myricetin treatment. In addition, myricetin downregulated the expression of metastasis- and inflammatory-associated genes. Thus, the compound may be a promising agent for treating CCA by targeting metastasis-associated events.
The metastasis process of cancers is complicated including cell migration and invasion. Previous studies have revealed the anti-migratory activity of myricetin in several cancer cells., Consistent with previous reports, in this study, it was found that myricetin abolished cytokine-stimulated CCA cell migration and invasion. The anti-metastatic effect was observed on myricetin treatment at low concentrations which negligibly affected cell growth. Cumulative evidence suggests that blocking of the STAT3 pathway leads to suppression of the metastatic potential of cancer cells., This pathway gets activated by various pro-inflammatory cytokines. It has been earlier reported that myricetin inhibited epidermal growth factor-mediated phosphorylation of STAT3 which resulted in the blocking of cell transformation of mouse epidermal JB6 cells. To gain further insight into the anti-metastasis effect of myricetin, the effect of this compound on the STAT3 signaling transduction cascade was then examined. As expected, myricetin showed a suppressive effect on cytokine-mediated STAT3 pathway activation in CCA cells.
The metastasis process requires degradation of extracellular matrix by proteolytic enzyme activity of MMPs. Moreover, a group of cell surface proteins such as cell adhesion molecules is also involved in this process. In this study, cytokine treatment significantly induced the expression of MMP-9 and ICAM-1 genes in CCA cells. Exposure of the cells to myricetin could downregulate the expression of these two genes. Because inflammation is closely related to cancer metastasis, the effect of myricetin on inflammation-related genes, iNOS and COX-2, was evaluated in the present study. The results revealed that myricetin also inhibited the mRNA expression of iNOS and COX-2 induced by cytokines. Indeed, previous studies have indicated that STAT3 is involved in the regulation of these gene expressions., Since the activation of STAT3 was inhibited by myricetin, it is, therefore, conceivable that STAT3 may, in part, account for the anti-metastatic activity of myricetin against CCA.
COX-2 serves a role in tumor progression including metastatic spread. In the present study, it has been found that inhibition of COX-2 could be a partly mechanism of myricetin to suppress metastasis of CCA cells. The anti-migratory and anti-invasive activities of a widely prescribed COX inhibitor, indomethacin, were then investigated. The results showed that the drug was capable of inhibiting cytokine-induced metastasis properties of CCA cells. Consistent with previous reports, the anti-metastasis effect of indomethacin was found in several cancers such as breast cancer and colorectal cancer. Indeed, previous studies have revealed the inhibitory effect of indomethacin on STAT3 pathway., Further study is required to elucidate the action of the drug on signaling pathways involved in CCA metastasis.
| > Conclusion|| |
This finding revealed that myricetin elicits an anti-metastasis effect against CCA cells. Blocking the activation of STAT3 pathway contributes in part to its effect. Thus, this compound may be a potential candidate for treating metastasis CCA. Further study is required to obtain more pharmacological data and to verify the effects in vivo.
This work was supported by the grant of the Faculty of Medicine, Khon Kaen University (Grant No. IN60218), and the grant from Khon Kaen University, Thailand (Grant No. 581401). The authors would like to thank Prof. James A. Will, University of Wisconsin-Madison, for editing the manuscript and the English Editing Publication Clinic, Faculty of Medicine, Khon Kaen University, Thailand.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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