Abstract
Background/Aim: Chlorogenic acid (CGA) is a polyphenol compound found in a variety of foods, including coffee, tea, cherries, and apples. It has been found by a number of studies to affect the viability of human cancer cells. No study has investigated its effect on esophageal squamous cell carcinoma (ESCC) metastasis or the molecular mechanism underlying its effect on this disease. Materials and Methods: We first used the Taiwanese ESCC cell line CE81T/VGH to create CE81T-M4 cells. Treatment of higher motility cells with chlorogenic acid for 24 h led to inhibition of cell migration and invasion as shown by scratch migration and transwell assays. Results: Western blotting showed that chlorogenic acid halted the activation of EGFR/p-Akt/Snail pathway and suppressed the expression of MMP-2 and MMP-9. Knockdown of either EGFR or Akt inhibited Snail, MMP2, and MMP9 activity as well as cell migration and invasion. Conclusion: Chlorogenic acid inhibited cancer cell motility via the EGFR/p-Akt/Snail pathway and could potentially be used to develop an antimetastatic agent for ESCC in the future.
Esophageal cancer was the ninth leading cause of death due to cancer in 2018. More than 572,034 new cases and 508,585 deaths from esophageal cancer were expected worldwide that year (1). In Taiwan, according to the Ministry of Health and Welfare, esophageal cancer was the ninth leading cause of cancer death in 2019 with a mortality rate of 8.4 deaths per 100,000 people. The histological subtypes of esophageal cancer are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC makes up approximately 90% of all cases of esophageal cancer worldwide (2). Despite the many advances in endoscopic resection (ER), surgery, chemotherapy, and radiation, the five-year survival rate for ESCC remains 15% to 25% (3). Thus, there is a great need to find a molecular mechanism that could potentially be targeted to inhibit ESCC tumor progression. There are different genetic mutations in ESCC, which have been associated with growth factor receptors, one being epidermal growth factor receptor (EGFR), known to have tyrosine kinase activity (4, 5). When EGFR tyrosine kinase is activated, many intracellular signaling pathways are activated, leading to cancer cell proliferation and processes crucial to the progression of cancer, including angiogenesis, metastatic spread, and inhibition of apoptosis (6). Furthermore, EGFR is over-expressed in a large percentage of ESCC cells, and its over-expression has been correlated with higher stages, metastasis, and poorer survival (7).
EGFR activates lipid kinase phosphatidylinositol 3-kinase, and when this activation occurs, a second messenger phosphatidylinositol 3, 4, 5-trisphosphate is produced, leading to the activation of Akt (8). AKT activation is crucial to ESCC cell growth, migration, invasion, and metastasis (9-11). Two regulatory transcription factors, Slug (SNAI2) and Snail (SNAI1), play roles in cancer cell epithelial-to-mesenchymal transition (EMT) (12, 13). When Snail is over-expressed in epidermoid cancer cells, E-cadherin expression is lost, the cells acquire fibroblastic phenotypes, and the expression of vimentin gene is upregulated, indicating Snail-induced EMT (14). E-cadherin is important for epithelial cell-to-cell adhesion (15). When there is a reduction or loss of E-cadherin, there is loss of cell-to-cell adhesion, making it easier for epithelial cells to move to other organs, a change that facilitates cancer invasion and metastasis (16). Also overexpressed in cancer cells, matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) have been reported to decompose extracellular matrices, including Type IV collagen and play important roles in invasion and metastasis; they have also been associated with poor prognosis (14-17).
5-O-caffeoylquinic acid (5-CQA), a common chlorogenic acid, is an ester of caffeic and quinic acid widely found in the leaves and fruits of various plants, and in particularly high concentrations in some vegetables, fruits, and medicinal herbs (18, 19). Chlorogenic acid has recently be found in vitro and in vivo to inhibit tumor cell growth, migration, and invasion (20-22). It modulates mTORC2-associated signaling pathways by decreasing p-protein kinase C alpha (PKCα) phosphorylation and Akt and decreasing the expression of Rictor and F-actin, both found to activate cell growth and organize the actin cytoskeleton (23). Chlorogenic acid has been found to hinder tumor cell growth via EGFR/PI3K/mTOR, HIF, VEGF, and MAPK/ERK pathways (24). It has also been found to improve Regorafenib’s ability to induce apoptosis via its activation of pro-apoptotic Annexin V, Bax, and Caspase 3/7 and inhibition of anti-apoptotic Bcl-2 and Bcl-xL. The combined use of chlorogenic acid and Regorafenib has been found to increase the ability MAPK (mitogen-activated protein kinase) and PI3K (phosphatidylinositol-3-kinase)/Akt/mTORC ability to inhibit tumor progression (25). Metabolites of chlorogenic acid have been found to reduce the proliferation of human colon cancer Caco-2 cell line by arresting cells in the S phase and inducing apoptosis (26). Chlorogenic acid has additionally been reported to hinder hepatoma cell line HepG2 proliferation in vitro and in vivo using nude mice xenografts by inhibiting the activation of Erk1/2 and reducing MMP-2 and MMP-9 expression in HepG2 (27).
To date, no study has investigated the effect of chlorogenic acid on ESCC tumor growth, its metastasis, or the molecular mechanism underlying its potential effect on this disease. Therefore, we created CE81T-M4 cells using a Taiwanese ESCC cell line CE81T/VGH, tested their motility, and treated them with chlorogenic acid for 24 h. We then used migration and transwell assays to assess their invasiveness and metastasis ability and used western blot analysis to study the possible mechanisms through which chlorogenic acid exerts its effects in knockdown models.
Materials and Methods
Cell lines and culture conditions. Taiwanese ESCC cell line CE81T/VGH were purchased from Food Industry Research and Development Institute (Hsinchu City, Taiwan, ROC). The esophageal cancer cell lines CE81T/VGH (BCRC 60166) and CE146T/VGH (BCRC 60167) were obtained from a 57-year-old and a 50-year-old Taiwanese male, respectively. Using a membrane invasion culture system (MICS), we chose CE81T-M1, CE81T-M2, CE81T-M3, CE81T-M4 for this study (28). Cells were cultured in a Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Gibco BRL). It was maintained at a temperature of 37°C in an atmosphere of 5% CO2.
Cytotoxicity of chlorogenic acid treatment assessed by MTT assay. We used a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium (MTT, Sigma Chemical Co., St. Louis, MO, USA) assay to assess cellular chemosensitivity in vitro. To do this, we placed cells mixed in a 100 μl culture medium into 96-well microplates and incubated them at 37°C for 24 h. We titrated the cell numbers to make sure the cells grew exponentially throughout the incubation period. Cells were then placed into 96-well plates (3,000 cells per well) and incubated for 24 h in the presence of different concentrations of chlorogenic acid (0, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 μM). After 24 h, we added 50 μl of MTT (2 mg/ml) to each well and let them incubate at 37°C for 4 h. The plates were then centrifuged, and the supernatant was disposed of. DMSO solution (150 μl) was then added to each well. An ELISA reader, MRX (DYNEXCO) (540 nm) was used to determine OD values. Proliferation of treated CE81T-M4 cells was recorded and expressed as mean±standard deviation (SD).
In vitro scratch migration assay. The cancer cells were placed in 24-well plates and cultured in a medium containing 10% FBS until cell monolayers neared confluence. For each well, we used plastic pipette tips to carefully produce a linear scratch in the cell monolayer. The scratches were exposed to 0, 700, or 900 μM of chlorogenic acid (C3878, Sigma-Aldrich). The number of cells that had migrated into the cell-free zone was recorded. Cultures were photographed before incubation and then 24 h afterwards at 37°C. Closure of the scratch was observed under a microscope. Scratch closure was expressed as the difference between wound width at 0 h and 24 h based on digital images of cells captured by a Leica microsystem (DMi8, Leica, Wetzlar, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to measure the scratch area. All assessments were performed three times. At least two researchers counted the migrating cells in a double-blind fashion.
In vitro transwell migration and invasion assays. Assays were performed using Millicell Hanging Cell Culture Insert (MCEP24H48. Merck KGaA, Darmstadt, Germany). Invasion assays were performed using membranes coated uniformly with reconstituted basement gel (Matrigel; 354262, Corning, AZ, USA), and migration assays were performed without the gel. In each well, 2×105 cells were suspended in DMEM medium without serum and then seeded into the upper wells of the Boyden chamber. After 24 h incubation with chlorogenic acid (0 μM, 900 μM) at 37°C, cells that had migrated or invaded the membrane were fixed with 3.7% paraformaldehyde (Merck KGaA, Darmstadt, Germany), stained with 0.3% crystal violet (ACROS, NJ, USA), and visualized by excitation at 540 nm. Images were captured using a Leica microsystem (DMi8, Leica, Wetzlar, Germany). Experiments were repeated in triplicate.
Western blot analysis. Western blot analysis was performed using EGFR (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-EGFR (Cell Signaling, Danvers, MA, USA), phospho-AKT (Thr308; Cell Signaling), phospho-AKT (Ser473; Cell Signaling), AKT1/2 (Santa Cruz Biotechnology), phospho-ERK (Cell Signaling), ERK1/2 (Santa Cruz Biotechnology,), Snail (Cell Signaling), Slug (Cell Signaling), E-cadherin (Cell Signaling), Vimentin (Cell Signaling), MMP1 (Santa Cruz Biotechnology), MMP2 (GeneTex, Alton Pkwy Irvine, CA, USA), and MMP9 (GeneTex) antibody. An internal control protein, GAPDH or Lamin-b1, was re-probed with an anti-GAPDH monoclonal (GeneTex) or anti-Lamin-b1 monoclonal (Abcam, Burlingame, CA, USA) antibody. We scraped cells (2×105) from a 100-mm petri dish and resuspended them in 50 μl RIPA lysis buffer consisting of 0.5% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl (pH 7.5), 1 mM sodium vanadate, and 0.1% sodium dodecyl sulfate (SDS). The resuspended cells were placed on ice for 30 min. Afterwards, we centrifuged the lysate at 13,000 × g for 30 min at 4°C. We collected the supernatant and measured the total amount of protein using a BCA Protein Assay Reagent (Pierce Life Science Co., Rockford, IL, USA). For our western blot analyses, we separated protein extracts (50 μg) on 10% SDS-polyacrylamide gels and transferred them to microporous polyvinylidene difluoride membranes. TBST buffer (20 mM Tris- HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween-20) plus 5% skim milk was added, and membranes were cultured with different primary antibodies at 4°C for 12 h. They were then washed for 4 min three times with TBST buffer and incubated with secondary antibody for 1 h at room temperature. Afterwards, the membranes were washed again with TBST buffer three times followed by incubation with western blotting luminol reagent (Millpore Co., Bedford, MA, USA) for protein detection.
Gelatin zymography. 2×105 cells were cultured in DMEM with 10% FBS until they reached 70-80% subconfluence. The cells were treated with chlorogenic acid (0 and 900 μM) in DMEM without serum for 24 h. The media were then collected, concentrated with Amicon Ultra (Millipore, Carrigtwohill, Co. Cork, Ireland) by repeated centrifugation at 14,000 × g for 30 min, followed by mixing with Laemmli SDS sample buffer (without β-mercaptoethanol), which consisted of 25 mM Tris/HCl (pH 6.8), 10% glycerin, 1% SDS, and 0.1% bromophenol blue for electrophoresis performed on 8% polyacrylamide gels that contained 0.1% SDS and gelatin at a final concentration of 0.1% (w/v). Afterwards, to remove the SDS, we washed the polyacrylamide gels three times in a buffer containing 50 mM Tris/HCl (pH 7.6), 10 mM CaCl2, 1 μM ZnCl2, and 2.5% TritonX 100 for 30 min. They were then incubated for 24 h in a reaction buffer [50 mM Tris/HCl (pH 7.6), 10 mM CaCl2, 1 μM ZnCl2, 0.1% NaN3, and 1% TritonX 100], and stained with Coomassie blue for 1 h. Destaining was performed in a 45% (vol/vol) methanol/10% (vol/vol) acetic acid solution until the appearance of transparent bands on the blue background. Images were captured using UVP bioimaging system.
Small interfering RNA transfection. EGFR siRNA sequences were VHS41680 5-UUGCAUCAUAGUUAGAUAAGACUGC-3 and 5-GCAGUCUUAUCUAACUAUGAUGCAA-3. Akt siRNA sequences were SG00121093 5’-GAGACUGACACCAGGUAUU[dT][dT]-3’ and SG00121094 5’-AAUACCUGGUGUCAGUCUC[dT][dT]-3. We transfected the cells (1×105) with 80 nM EGFR siRNA and nonspecific control siRNA in Lipofectamine 2000 reagent (Thermo Fisher Scientific, MA, USA) for 6 h. Likewise, we transfected the cells (1×105) with 50 nM Akt siRNA or non-specific control siRNA in Lipofectamine 2000 reagent for 6 h. After 6 h, for growth factor treatment, siRNA-containing medium was replaced with RPMI + 10% FBS, and after 48 h, the cells were harvested and analyzed by transwell assays, and their proteins were extracted for western blot.
Xenograft tumor growth. Eight-week-old male BALB/c Foxlnn mice were purchased from Taiwan’s National Laboratory Animal Center (Taipei, Taiwan, ROC), maintained under strict pathogen-free conditions, with free access to sterile food and water. A total of 1×106 cells of the RFP-luciferase (GL)-labeled CE/81T-M4 cells (Kaohsiung Medical University Gene Recombinant/Infectious Biological Materials Experiment Applications No. 108075) were mixed with 100 μl extracellular matrix (BD Biosciences, NJ, USA) and administered by subcutaneous injection into the dorsal region of nude mice. After one week, the mice were randomly assigned to two groups (n=6 for each group), one treated with DI water and the other with chlorogenic acid. DI water was administered orally and chlorogenic acid (900 μM) by gavage. We used a sliding caliper to measure the subcutaneous tumors weekly. The size was calculated as tumor 3 volume (mm3)=length × width × height. We used an Intensive Vigilance and Intervention System (IVIS) to monitor tumor growth. The protocol of this study was in accordance with the guidelines set forth by the Kaohsiung Medical University Institutional Animal Care and Utilization Committee (approved by IACUC No. 108204). We measured tumor mass once a week over seven weeks. At the end of the 7-week observation period, the mice were killed by asphyxiation with CO2. The lungs, liver, brain, and esophagus were removed and fixed in 4% formalin fixative. To assess metastasis, we counted the number of esophageal tumor colonies in these organs under a dissecting microscope. Representative tumors were then removed, fixed, and embedded in paraffin followed by sectioning into 4-μm layers and staining with hematoxylin and eosin (H&E) for immunohistochemical analysis.
Immunohistochemistry (IHC). Paraffin sections were stained and analyzed using a Polymer Detection System (Thermo Fisher Scientific). We removed the paraffin from sections with xylene, rehydrated the sections with various grades of alcohol, and boiled them for 10 min in a 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by adding 0.3% hydrogen peroxide. The sections were incubated in Ultra-V block solution (Thermo Fisher Scientific) and Klear mouse blocking solution (GBI labs, WA, USA) to stop any non-specific binding followed by overnight incubation with anti-RFP antibody (Santa Cruz Biotechnology) (1:500 dilution) at 4°C. Antigen retrieval was performed by using immunoDNA Digestor (Thermo Fisher Scientific) for 40 min at 37°C and blocking was performed by using Immunodetector (Thermo Fisher Scientific) for 5 min at 37°C. The sections were incubated with a Mouse/Rabbit Poly-Detector secondary antibody (Thermo Fisher Scientific) at room temperature for 30 min. After washing, the antigen-antibody complex was applied to the sections, which were then stained with DAB Chromogen (Thermo Fisher Scientific) as a substrate. Hematoxylin (Merck, Darmstadt, Germany) was then used for light counterstaining. RFP was indicated in the membrane by its brown color.
Statistical analysis. Data are presented as means±SD based on three or more independent experiments. A p<0.05 (two-tailed unpaired Student t-test) was considered significant. All statistical operations were performed using IBM SPSS Statistics 20 software (IBM, Armonk, NY, USA).
Results
Effect of chlorogenic acid on CE81T-M4 cell proliferation. The antitumor effect of chlorogenic acid on ESCC cell lines was evaluated. CE81T-M4 cells were treated with increasing concentrations of chlorogenic acid (0, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1,000 μM) for 24 h and then cell proliferation, was assessed by MTT assay. As shown in Figure 1, treatment with different concentrations of chlorogenic acid resulted in different CE81T-M4 cell proliferation rates. At all concentrations, chlorogenic acid maintained a good viability of esophageal cancer cells.
Chlorogenic acid reduced cell migration of CE81T-M4 cells in a scratch test. We treated CE81T-M4 cells with different concentrations of chlorogenic acid for 24 h and performed a scratch migration assay. Chlorogenic acid 900 μM significantly decreased the migration ratio compared with the control (0 μM) (0.32±0.10 vs. 1±0, respectively, p<0.001) (Figure 2). The experiments were repeated three times, independently. Based on these results, we chose to use chlorogenic acid 900 μM for our treatment group and 0 μM for our control group in all experiments.
Chlorogenic acid inhibited CE81T-M4 cells migration and invasion. To evaluate the impact of chlorogenic acid on cell migration and invasion, we treated CE81T-M4 cells with 900 μM chlorogenic acid for 24 h. We found that chlorogenic acid significantly decreased the migration of CE81T-M4 cells compared with the control (0.72±0.01 vs. 1±0, respectively, both p<0.001) (Figure 3). Consistent with the migration assay findings, 900 μM of chlorogenic acid reduced the invasion ability of CE81-M4 cells, compared with the control (0 μM) (0.44±0.02 vs. 1±0, respectively, p<0.01) (Figure 3). These results suggested that chlorogenic acid played an important role in reducing the migration and invasion of ESCC cells.
Protein expression in CE81T parental, CE81T-M1, CE81T-M2, CE81T-M3, and CE81T-M4 cells. We measured the expression levels of EGFR, phosphorylated EGFR (p-EGFR), Akt, and phosphorylated Akt (Thr308) in different ESCC cells (Figure 4). The expression level of EGFR and Akt in CE81T-M1, CE81T-M2, CE81T-M3, CE81T-M4 cells were higher than those in CE81T parental controls. There was no difference in EGFR expression in other cell types compared to CE81T-M4 cells. However, we found CE81T-M4 cells to have higher expression levels of Akt and phosphorylated Akt (Thr308). Thus, we chose the CE81T-M4 cell line to explore the effect of chlorogenic acid on ESCC.
Chlorogenic acid inhibited EGFR/p-Akt/Snail signaling pathway. To further explore the molecular mechanism underlying the chlorogenic acid-induced inhibition of ESCC cell migration, we treated CE81T-M4 cells with 900 μM of chlorogenic acid for 24 h. Western blot results showed that chlorogenic acid decreased the expression of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt Thr308 and p-Akt Ser473), MMP2, MMP9, phosphorylated Erk (p-Erk), and Snail in CE81T-M4 cells but increased the expression of E-cadherin (Figure 5). These findings suggest that chlorogenic acid may reduce the migration of ESCC by inhibiting the EGFR/p-Akt/Snail signaling pathway.
Chlorogenic acid down-regulated MMP2 and MMP9. CET81T-M4 cells were treated with chlorogenic acid to assess its effect on the enzymatic activity of MMP2 and MMP9. Western blot results showed that 900 μM of chlorogenic acid reduced the enzymatic activity of both, compared to controls (Figure 6).
Knockdown of EGFR expression inhibited the migration and invasion of CE81T-M4 cells. EGFR has been found to be a prognostic indicator of ESCC (7, 29). Migration and invasion have been found to be induced via the EGFR-mediated signaling pathway in some cancer cells (30, 31). In order to assess the effect of EGFR on CE81-M4 cell migration and invasion we transfected ESCC cells with EGFR siRNA for 6 h. Chlorogenic acid (900 μM) was then added to the culture. Twenty-four h later, transwell assay showed the migration ratios to be 1±0 and 0.88±0.02 in the negative control (NC) and EGFR siRNA cells not treated with chlorogenic acid, respectively. However, the migration ratios were 0.46±0.01 and 0.39±0.002 in NCs treated with chlorogenic acid and EGFR siRNA treated with chlorogenic acid, respectively, a significant decrease in the chlorogenic acid-treated group compared to negative control. Results were similar in the invasion assays. Invasion ratios in the untreated negative control and EGFR siRNA transfected cells were 1±0 and 0.80±0.04, respectively. In those that were treated, the ratios were 0.60±0.03 and 0.44±0.03, respectively, indicating that chlorogenic acid significantly reduced CE81-M4 cells in cultures pre-treated with EGFR siRNA compared to negative control (Figure 7).
Knockdown of EGFR expression suppressed the activation of Akt pathway. We assessed the effect of knocking down EGFR on the activation of the Akt pathway. As shown in Figure 8, transfection of cells with EGFR siRNA led to a reduction in the protein levels of Akt, Snail, MMP2, and MMP9. These findings suggest that EGFR promoted the growth and metastasis of ESCC by activating the Akt signaling pathway.
Knockdown of Akt expression inhibited the migration of CE81T-M4 cells. We also examined the effect of Akt knockdown on ESCC cell migration and invasion by using transwell assay. After 48 h of Akt siRNA transfection, migration of CE81T-M4 cells was significantly decreased compared with the negative control (0±1 vs. 0.85±0.04, respectively, p<0.05) (Figure 9A). Consistent with this finding, invasion of CE81-M4 cells was also reduced in cultures treated with Akt siRNA compared with the negative control (0±1 vs. 0.97±0.01, respectively, p<0.05) (Figure 9B).
Knockdown of Akt downregulated the downstream targets. We knocked down Akt in CE81T-M4 cells with Akt siRNA, and after 48 h its effect on its downstream targets was assessed by using western blotting. Akt knockdown inhibited the expression of transcriptional factor Snail and decreased the matrix metalloproteinases MMP2 and MMP9 (Figure 10).
Nude mice gavage fed with chlorogenic acid had reduced tumor growth rate and lung metastasis. To study the effect of chlorogenic acid in vivo, we subcutaneously injected nude BALB/c Foxlnn mice with RFP-luciferase (GL)-labeled CE/81T-M4 cells (1×106) to induce ESCC. Seven days later, after the tumor was stable, we gavage-fed them with either drinking water or chlorogenic acid to assess tumor growth and lung metastasis. There was no significant difference in body weight between the two groups during the experiment. Seven weeks later, on day 49, the control group was found to have a significantly larger average tumor size than that of mice gavage-fed chlorogenic acid (1.215±0.0944 vs. 0.529±0.0601 cm3; mean± SEM). Mice in the control group were found to have lung metastases and those gavage-fed chlorogenic acid were not (Figure 11).
Discussion
Chlorogenic acid is one of the main polyphenols found in human diets. Nutrition research has found it to be a nutraceutical useful for the prevention and treatment of major chronic diseases (32). It has also been shown to have antioxidant (33), anti-inflammatory (34), and anti-microbial effects (35). Chlorogenic acid has been found in vitro to inhibit cell proliferation or induce apoptosis in several cancers, including melanoma, leukemia, glioblastoma, and breast and liver cancer (36-40). To the best of our knowledge, this study is the first to focus on the effect of chlorogenic acid on CE81T-M4 cells, a Taiwanese ESCC cell line, and the mechanism through which it achieves its effects. We found that chlorogenic acid inhibited CE81T-M4 cell migration and invasion by inhibiting the EGFR/p-Akt/Snail pathway.
Recent studies have reported over-expression of EGFR in 50 to 90 percent of patients with ESCC (41-46) and some of have found its over-expression to be associated with poor overall survival and poor disease-free survival (46, 47). Previously, although studies have found chlorogenic acid to have many anti-disease benefits, no study, to best of our knowledge, has investigated its influence on EGFR activation. Our results showed that chlorogenic acid inhibited EGFR activation by reducing the expression of Akt and snail, and the phosphorylation of Akt (Thr308 and Ser473), while increasing the expression of E-cadherin. Knockdown experiments using EGFR siRNA showed similar results. These findings clearly suggest that chlorogenic acid inhibited ESCC cell growth, migration, and invasion through its effect on the EGFR/p-Akt/Snail pathway.
This study found no difference in the expression of EGFR in various ESCC cell lines, but did find p-EGFR expression to be lower in CE81T-M4 than in CE81T parental cells, possibly because the phosphorylation and activity of EGFR are regulated by several factors, including the presence or absence of EGFR-specific ligands or dimerization partners and the interruption of EGFR-mediated signaling by proteintyrosine phosphatase activity or internalization (48). Additionally, kinases such as Src (49) or co-factors such as integrin can influence the expression EGFR (49, 50); low EGFR phosphorylation may not always indicate that EGFR activity is negligible.
We found a higher expression of Akt in CE81T-M4 cells than in CE81T parental cells. Similarly, other studies have reported Akt to be over-activated in many human solid tumors and hematological malignancies (51, 52) and have associated its activation with poor prognosis (53, 54). We found that in cells treated with chlorogenic acid, there was a decrease in the expression of p-Akt (Thr308 and Ser473). According to a study, it is possible that chlorogenic acid binds to Akt’s pleckstrin homology (PH) domain, where it might induce AKT phosphorylation on Ser-473, initiating the phosphorylation of glycogen synthase kinase 3β (GSK3β) and forkhead box O1 (FOXO1) downstream, which might result in enhanced glucose metabolism (55). These findings led us to hypothesize that chlorogenic acid might also work through the EGFR/p-Akt pathway (Thr308) and bind to the PH domain, directly activating p-AKT (Ser473) and inhibiting ESCC cell migration and invasion. We silenced Akt to explore its role in ESCC and found that its reduced expression led to a down-regulation of the levels of the transcription factor Snail as well as decreased expression of MMP2 and MMP9. Our working model showing the molecular mechanism underlying chlorogenic acid’s ability to decrease ESCC metastasis can be viewed in Figure 12. We concluded that Akt might play an important role in migration and invasion of ESCC cells. Further research is needed to explore this avenue of investigation.
During metastasis, cancer cells detach from the primary tumor, disrupt the basement membrane, invade the surrounding stroma, and from that point on, distal proliferation and angiogenesis occur (56). One barrier preventing tumor growth and cancer cell invasion is the extracellular matrix (ECM) (57, 58). The degradation of ECM marks the beginning of tumor cell invasion (54, 55). Several proteases have been implicated in ECM degradation. Matrix metalloproteinases play a crucial role in invasion, migration, metastasis, and tumorigenesis (59). We evaluated the effect of chlorogenic acid on the gelatinases (MMP2 and MMP9) in one ESCC cell line, CE81T-M4, and found that it significantly decreased MMP2 and MMP9 activity, suggesting that it could potentially be used as an antimetastatic agent.
One previous study has shown that by combining 5-FU and chlorogenic acid together, ERK1/2 could be inactivated (60). That study found that chlorogenic acid enhanced 5-fluorouracil inhibition of human hepatocellular carcinoma cell proliferation by increasing the production of reactive oxygen species, which inactivated ERK. Similarly, we found that chlorogenic acid inhibited the expression of ERK and p-ERK. The downstream targets of ERK in ESCC should be studied further.
This investigation is limited in that we only studied one ESCC cell line (CE81T-M4) from Taiwanese patients. Future studies on the effect of chlorogenic acid on ESCC should focus on a wider diversity of ESCC cell lines. Another limitation is that metastasis and invasion have been related to many factors and signalling pathways. In this study, we focused on the EGFR/p-Akt/Snail pathway. The specific molecular mechanism underlying the chlorogenic acid-induced inhibition of cancer cells requires further investigation.
In summary, this study demonstrated that chlorogenic acid could inhibit Taiwanese ESCC cell growth and metastasis. Based on these findings, chlorogenic acid could potentially be used in the development of a metastasis-prevention agent against ESCC.
Acknowledgements
This research was supported by the grants (MOST 108-2637-B-276-001, MH-110-DFN-001).
Footnotes
Authors’ Contributions
Yu-Kuei Chen, Investigation, Writing- Reviewing and Editing; Ngo Tran My Ngoc, Data curation, Writing- Original draft preparation; Hsi-Wen Chang, Methodology; Ying-Fang Su, Methodology; Chung-Hwan Chen, Supervision, Validation; Yih-Gang Goan, Supervision, Validation; Jeff Yi-Fu Chen, Supervision, Validation; Chun-Wei Tung, Software; Tzyh-Chyuan Hour, Supervision.
Conflicts of Interest
All Authors declare that no support, financial or otherwise, has been received from any organization that may have an interest in the submitted work; and there are no other relationships or activities that could appear to have influenced the submitted work.
- Received April 22, 2022.
- Revision received May 20, 2022.
- Accepted May 26, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.