Anti‐cancer effect of entacaponeon esophageal cancer cells via apoptosis induction and cell cycle modulation

Abstract Background Esophageal cancer (EC) is the sixth leading cause of cancer‐related death, despite many advances in treatment, the survival of patients still remains poor. In recent years, the N6‐methyladenosine (m6A) has been introduced as one of the most important modifications at the epitranscriptome level, with an important role in the mRNA regulation in various diseases, such as cancers. The m6A is regulated by different factors, including FTO as a demethylase. The m6A modification, especially through FTO overexpression has an oncogenic role in different cancer types such as EC. Recent studies showed that entacapone, a catechol‐o‐methyl transferase (COMT) inhibitor currently applied for Parkinson's disease, can inhibit FTO enzyme. Aims In this study, we aimed to investigate the effect of entacapone as an FTO inhibitor on the m6A level and also apoptosis and cell cycle response in KYSE‐30 and YM‐1 of esophageal squamous cancer cell (ESCC) lines. Methods Cell toxicity and IC50 of entacapone were evaluated using The MTT assay in YM‐1 and KYSE‐30 cells. Cells were treated into two groups: DMSO (control) and entacapone (mean IC50). Total RNA was extracted, and m6A levels were measured via the ELISA method. Subsequently, the apoptosis and cell cycle dys‐regulation were detected by annexin‐V‐FITC/PI staining and PI staining via flow cytometry. Results Entacapone has the cytotoxicity effect on both esophageal cancer cell lines compared to normal PBMC cells. As well, entacapone treatment (140 μM) can induce apoptosis (KYSE‐30: 50%. YM‐1:22.6%) and has a modulatory effect on cell cycle progression in both YM‐1 and KYSE‐30 cells (p‐value<.05). However, no significant difference in the m6A concentration was observed. Conclusion Our findings suggested that entacapone has the inhibitory effect on ESCC cell lines through induction of the apoptosis and modulation of the cell cycle without toxicity on the normal PBMC.


| INTRODUCTION
Esophageal cancer (EC) is the eighth most common malignancy and sixth leading cause of cancer-related death worldwide. 1 Esophageal cancer is divided into two common histologic subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). 2 In Asia, ESCC is the most frequent histologic subtype of esophageal cancer. [3][4][5] In recent years, despite of the great improvements in treatment of ESCC patients, the prognosis of patients remains poor and overall survival rate is still low. 2,6 Cancer is considered as a complex disease and different environmental, genetic and epigenetic factors can play role in cancer development or progression. More recently, epitranscriptome changes particularly with N6-methyladenine (m6A) modification, has been reported with association to multiple diseases, such as cancer. [7][8][9] The m6A is the common chemical modification of messenger RNA (mRNA), involved in mRNA splicing, stability, transcription, splicing, localization, translation regulation process. 10 The level of m6A modification in mRNAs is regulated by multiple proteins called, "writer," "eraser", and "reader" proteins. 11,12 The writers are consisted of methyl-transferase complex, METTL3, METTL14 and WTAP. 13 In contrast the eraser proteins, namely FTO and ALKBH5 have the role of m6A demethylation. 13 The Reader protein family includes the YTH domain-containing family proteins YTHDF1/2/3, YTHDC1, and YTHDC2 in which the YTH domain is responsible for recognition and binding to the m6A sites. 14,15 The previous studies have suggested that overexpression of the FTO has an oncogenic role in different cancer types 16 such as acute myeloid leukemia, 17 gastric cancer, 18 cervical squamous cell carcinoma, 19 and ESCC. 20 It has been reported that down-regulation of the FTO expression inhibits cell proliferation, migration and invasion abilities of ESCC cells. 20 Recent studies have established, entacapone, a catechol-o-methyl transferase (COMT) inhibitor, can compete for the binding to the cofactor-and substrate binding sites on FTO and thereby can inhibit FTO activity. 21 The entacapone, is the FDA approved drug which is currently in use for treatment of the Parkinson's disease in combination with levodopa. 22 In this study, we aimed to investigate the potential effect of entacapone on m6a concentration and also its anti-cancer effect on the cell viability and apoptosis

| Cell culture
In the present study, the two cell lines of human ESCC, YM-1 and KYSE-30, were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% streptomycin/penicillin. Cells were kept in a humidified incubator containing 5% CO 2 , at 37 C.

| PBMC Isolation
The peripheral blood mononuclear cells isolation was performed using the ficoll reagent. Thus, 10 ml of peripheral blood containing anticoagulants was collected from a healthy person. The blood was diluted with equal volume of PBS and then was added to ficoll, in two separate layers. Then, to separate the PBMC containing layer, solution was centrifuged at 2500 rpm for 30 min. cells were rinsed by PBS. The peripheral blood mononuclear cells (PBMC) then were suspended in 1 ml of RPMI supplemented with 10% FBS and 1% streptomycin/ penicillin and used for next experiments.

| Viability assay
The toxicity of entacapone was evaluated in 48 h at different doses, in both esophageal squamous carcinoma cell lines, YM1 and KYSE 30, using MTT assay. Cells were seeded into 96-well plates at a density of 1 Â 10 4 cells/well after 24 incubations, the culture medium was removed and then then cells were treated with different concentrations (25, 50, 75, 100, 125, and 150 μM) or IC50 (140 μM) of entacapone with a final volume of 200 μl. As vehicle control cells were treated with DMSO (0.3%) with same condition. After 48 h treatment, 10 λ of MTT assay (10 mg/mL) solution, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), was added to each well and incubated at 37 C for 4 h. Then, the supernatant was aspirated off and 100 μl of DMSO was added to solve the precipitates. The absorbance of each well was measured at wavelength 570 nm using a micro-plate reader. Also the viability was checked with dye exclusion assay using Trypan blue dye. Then the viable cell percentage was calculated as (trypan blue negative cell/total cell) Â 100.

| Apoptosis assay
To perform the apoptosis assay, 4 Â 10 5 cells were seeded in 6-well plates. After 24 h cells were treated with 0.3% DMSO (control) and  was isolated. Then, it was precipitated by adding equal volume of isopropanol followed with incubation for 15 min on ice and centrifugation. The RNA was washed twice by adding 1 ml 75% ethanol, and centrifugation at 7500 rpm at 4 C for 8 min. Finally, the purified RNA samples were dissolved in RNase-free water and stored at À80 C.

| Measurement of m6a by ELISA method
The level of m6A modification in RNA samples, was measured by m6 A Elisa Kit (Zellbio, Germany), following the manufacturer's protocol.
Briefly, a total amount of 5 μg RNA for each group was used to determine the amount of m6A. To remove any RNA secondary structure, RNA samples were heated at 95 C for 5 min, and then was rapidly chilled on ice. The RNA was digested to nucleosides; by incubating the denatured RNA with 5 unit of nuclease P1 for 1 h at 37 C. Subsequently, 5 units of alkaline phosphatase plus sufficient of tris buffer to a final concentration of 100 mM Tris, pH 7.5 was added, and then was incubated for 1 h at 37 C. The supernatant was collected and was used for the ELISA experiment. The absorbance was measured at the wavelength 450 nm using a microplate reader. The standard curve was constructed to calculate the concentration of the samples.

| Cell cycle assay
The cell cycle analysis was performed using flow cytometer with PI staining method. Labeling DNA with PI allows for fluorescence-

| Statistics
In this study the p-value < .05 was considered as significant level. All the experiments were carried out in replicates (N = 3-5) for each group. For data visualization, the Graph Pad Prism v.5.04 was used and the data was reported as mean ± SE. for statistical analysis, SPSS v.19 was used. The normality of data was checked with the Shapirowik test and then in the case of normal distribution one-way Anova test was used. In the case of non-normal distribution the nonparametric test was used for data analysis.  Figure 1A). As a normal cell control, the PBMC, isolated from healthy individual was used. The viability was evaluated at a dose of 140 μM of entacapone after 48 h (mean IC 50 for cancer cells). Our findings illustrated that the mean survival of PBMC cells treated with entacapone (Mean ± SE:100 ± 1.39) was not significantly different (pvalue: .693) from control group(Mean ± SE: 95.70 ± 2.56),indicating the selective toxicity of entacapone in cancer cells ( Figure 1B). The dye exclusion assay using trypan blue also indicated selective toxicity of entacapone (140 μM) on YM-1 (p-value = .004) and KYSE-30 cells (p-value = .008) but no toxicity in PBMC was detected ( Figure 1C).

| Entacapone treatment induces apoptotic cell death in ESCC
The programmed cell death was measured using annexinV/PI staining protocol and flow cytometry technique. As shown in Figure 2A

| Measurement of m6A concentration in ESCC cell lines treated with entacapone
The entacapone previously was introduced as inhibitor of FTO demethylase which removes the methyl group from m6A, then we hypothesized it may increase the m6A level as substrate of FTO. To test this hypothesis, the concentration of m6A in total RNA samples extracted from both cell lines was measured using ELISA in entacapone treated in comparison to the control cells. We found a small increase in m6A level in entacapone treated ESCC; however the change was not statistically significant ( Figure 3).

| DISCUSSION
The esophagus squamous cell carcinoma is one of the most common types of esophageal malignancies with aggressive nature 24 and poor survival rate. 20 There are many factors playing role in ESCC pathogenesis, including genetic and epigenetic, as well as epitranscriptomic changes. 24,25 The role of epitranscriptome changes in cancer progression of different types has been investigated in recent years. 26 The most common type of the epitranscriptome modification is N6-methyladenosine (m6A) which can be modulated by methyltransferases METTL3/14, demethylases FTO and ALKBH5 and m6Abinding proteins called YTHDF1-3. 25,27 Increased expression of FTO as m6A demethylase has an onco- In other hand a recent drug virtual screening introduced entacapone as a potential inhibitor of FTO which can directly bind to it and suppress its activity in vitro. 21 Then here in current study we evaluated the potential anticancer properties of the entacapone as FTO inhibitor in ESCC cells.
We found a significant dose-dependent toxicity of entacapone in  35 In another study it was reported that entacapone or tolcapone in combination with EGCG can reduce expression of cyclin D1 and consequently, resulting to G1 arrest induction in H1299 cells, whereas in CL-13 lung cancer cells they found significant G2/M arrest, which was in parallel to our findings. 32 Similar studies support the role of the FTO in cell cycle progression through regulation of cyclin D1 m6A modification. It has been reported that FTO inhibition can prolong G1 phase by cyclin D1 suppression. 36 Altogether although there are various effects of FTO siRNA, or chemical compounds in cell cycle progression in different cell lines, but all of these share common outcome in general in cell cycle inhibition.

| CONCLUSION
Our findings indicated selective anti-cancer effect of entacapone in esophageal cancer cells in vitro through apoptosis induction and cell cycle regulation. These results suggest potential application of entacapone in ESCC treatment however further investigations to discover the underlying mechanisms are needed.

ACKNOWLEDGMENT
The authors would like to Acknowledge the fanda pharma company for the gift of entacapone powder.

FUNDING INFORMATION
This project was extracted from a master student thesis which was financially supported by Golestan University of Medical Sciences (grant number: 111290).

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Marie Saghaeian Jazi https://orcid.org/0000-0003-0647-9545