Cytotoxicity, Cell Cycle Arrest, and Apoptosis Induction Activity of Ethyl-p-methoxycinnamate in Cholangiocarcinoma Cell

Objective: To investigate cytotoxic activity of ethyl-p-methoxycinnamate (EPMC) including its effect on p-glycoprotein (multidrug resistance-1: mdr-1 gene) in human cholangiocarcinoma cell. Methods: Cytotoxic activity of EPMC against human cholangiocarcinoma (CL-6), fibroblast (OUMS-36T-1F), and colon cancer (Caco-2) cell lines were assessed using MTT assay. Selectivity index (SI) was determined as the ratio of IC50 (concentration that inhibits cell growth by 50%) of EPMC in OUMS-36T-1F and that in CL-6 cell. Cell cycle arrest and apoptosis in CL-6 cells were investigated by flow cytometry and fluorescent microscopy. Effect of EPMC on mdr-1 gene expression in CL-6 and Caco-2 was determined by real-time PCR. Results: The median (95% CI) IC50 values of EPMC in CL-6, OUMS-36T-1F, and Caco-2 were 245.5 (243.1-266.7), 899.60 (855.8-966.3) and 347.0 (340.3-356.9) µg/ml, respectively. The SI value of the compound for the CL-6 cell was 3.70. EPMC at IC50 inhibited CL-6 cell division and induced apoptosis compared to untreated control. EPMC exposure did not induce mdr-1 gene expression in both CL-6 and Caco-2 cells. Conclusion: The results suggest the potential role of EPMC in cholangiocarcinoma with a low possibility of drug resistance induction.


Introduction
Cholangiocarcinoma is the bile duct malignancy, which the highest incidence has been reported in the population living in the North-Eastern region of Thailand (Kirstein andVogel, 2016, Xia et al., 2015). The major risk factors are ingestion of Opisthorchis viverrini metacercaria contaminated raw cyprinoid fish, family history of cancer, and liquor consumption (Hughes et al., 2017;Kamsa-ard et al., 2018). Most cholangiocarcinoma cases are diagnosed when the disease progresses to late stage and must receive treatment by chemotherapeutic drugs. The 5-Fluorouracil (5-FU) is the first-line drug for cholangiocarcinoma but drug resistance results in reduced treatment efficacy (Konstantinidis et al., 2016).
Treatment failure in cholangiocarcinoma patients promotes the requirement of novel anticancer agents to replace the commonly used anticancer drugs. Therefore, the study aimed to investigate cytotoxic activity of EPMC in in various human cell lines and its effect on drug resistance.

Wound healing assay
Wound healing assay was performed according to the method of Pinto and Ramenzoni with modification (Pinto BI et al., 2018, Ramenzoni et al., 2017. The CL-6 cells (1.0×10 4 cell/well) were seeded onto a 96-well plate and incubated overnight at 37 o C under 5% CO 2 atmosphere. The cell monolayers were scratched using a 10 µl pipette tip, and cell debris was removed by rinsing with 1xPBS. The cells were exposed to EPMC at IC 25 and IC 50 concentrations for 24 and 48 hours; control cells were exposed to complete RPMI 1640 medium. The images of the scratched areas were examined under the inverted microscope (40x magnification) before EPMC exposure, and at 24 and 48 hours after exposure.

Cell cycle arrest investigation
The CL-6 cell line was grown overnight in a 6-well plate (5×10 5 cells/well) before exposing to EPMC at the IC 25 and IC 50 concentrations for 12, 24, and 48 hours. Cells were harvested by trypsinization, and DNA content was stained by fluorescence dye according to the manufacturer's protocol (BD Cycletest TM Plus DNA Reagent Kit, BD Biosciences, CA, USA). Briefly, cell pellets were collected through centrifugation at 250 x g for 5 minutes and incubated with the provided reagent before staining with propidium iodide (PI). Cell number and copy number of DNA were determined using FACTverse flow cytometer (BD Biosciences, CA, USA) at 278 energy voltage. The experiment was performed in triplicate, and cell development to G0/G1, S and G2/M phases in tested samples were compared to control.

Apoptosis investigation
The CL-6 cells (5×10 5 cells) were grown overnight in a 25 cm 2 filter flask. Cells were harvested by trypsinization after exposure to EPMC at the IC 25 and IC 50 concentrations for 24 and 48 hours. The apoptotic protein marker phosphatidylserine and intracellular DNA content were stained by fluorescence dye according to the manufacturer's protocol (BD Pharmingen TM FITC Annexin V Apoptosis Detection Kit, BD Biosciences, CA, USA). The numbers of both living and apoptotic (early and late) cells were determined by FACTverse flow cytometer (BD Biosciences, CA, USA) at 281 and 278 energy voltages for FITC and PI, respectively. The experiment was performed in triplicate.
The apoptotic effect of EPMC on CL-6 cell line was also investigated based on caspase 3/7 expression (Panrit

Cytotoxic activity of EPMC against CL-6, OUMS-36T-1F, and Caco-2 cells
Growth inhibitory effects of EPMC and/or 5-FU on CL-6, OUMS-36T-1F, and Caco-2 cell lines expressed as IC 25 , IC 50 , and IC 90 are summarized in Table 1. The potency of activity of EPMC varied with concentrations. The potency of activity based on IC 50 was about 50% of 5-FU. The SI value of EPMC at the three concentrations ranged from 3.1 to 4.1. The cytotoxic activity of EPMC was also investigated in the Caco-2 cell line to identify optimal concentration to be used in the mdr-1 gene induction experiment. The concentration that produced 100% cell viability was used as the maximum concentration for investigation of mdr-1 gene expression in CL-6 and Caco-2 cell lines.

Effect of EPMC on cell migration
Wound healing assay was performed to determine the effect of EPMC on CL-6 cell migration. CL-6 cell et al., 2018). The cells (3.5×10 4 cells/well) were seeded in Corning™ Falcon™ Chambered Cell Culture Slides (Corning, NY, USA) and incubated overnight at 37 o C under 5% CO 2 atmosphere before exposing to EPMC at IC 25 and IC 50 concentrations for 24 and 48 hours. The apoptotic proteins caspase 3/7 were stained with fluorescence CellEvent™ Caspase-3/7 Green detection assay (Thermo Fisher Scientific, NY, USA) and cells with caspase-3/7 expression were examined under ZOE fluorescent microscope (Bio-rad, CA, USA) using 40x objective lens. The green stained apoptotic cells were observed in 10 fields and the ratio of cell apoptosis was estimated as follow: Caspase 3/7 expression (%) = (Mean fluorescencestained cells/Mean of total cell number) × 100

Mdr-1 gene expression
The CL-6 cells (5.0 × 10 5 cells) were grown in a 6-well plate for 24 hours before exposing to EPMC (0.08, 0.8, and 8 µg/ml). The Caco-2 cells were seeded onto a 6-well plate at the same number for 48 hours before exposing to EPMC (0.2, 2, and 20 µg/ml). Following 24 and 48 hours incubation, RNA contents of both cells were collected using Trizol TM reagent (Invitrogen, NY, USA). The RNA was then converted to cDNA according to the manufacturer's protocol (SuperScript™ III Reverse Transcriptase kit, Invitrogen, NY, USA). Gene expression was determined by SYBR green real-time PCR using MDR1-F: GTCTTTGGTGCCATGGCCGT, MDR1-R: ATGTCCGGTCGGGTGGGATA for mdr-1 gene (target gene) and GAPDH-F: CAACAGCCTCAAGATCATC AGC, GAPDH-R: TTCTAGACGGCAGGTCAGGTC for gapdh gene (reference gene or housekeeping gene). The cDNA template was amplified according to the following conditions: pre-denaturation at 95 o C for 5 min, denaturation at 95 o C for 15 sec, and annealing at 60 o C for 1 min with fluorescence intensity detection. The melting curve was plotted every 0.5 o C until reaching 95 o C (Chaijaroenkul et al., 2011). Mdr-1 gene expression level was calculated relative to gapdh gene expression and normalized with H0 sample according to delta-delta C t method (Sumsakul and Na-Bangchang, 2015) as follows: ΔΔC t = δC t (tested or control) -δC t (H0) Relative expression = 2 -δδCt

Data analysis
Each experiment was performed in triplicate. Cytotoxic activity of EPMC against CL-6, OUMS-36T-1F and Caco-2 cells was expressed as median (95% CI) values of IC 25 , IC 50 , and IC 90 . Quantitative data are presented as median (95% CI) values. Difference between the two independent quantitative groups was determined using Mann Whitney U test at a statistical significance level of α = 0.05.

Effect of EPMC on cell cycle arrest
The effect of EPMC on CL-6 cell cycle arrest (G0/G1, S, and G2/M) is shown in Figure 2. Following 12 hours of EPMC exposure, CL-6 cell division was obstructed at the G0/G1 phase. Percentage of cell population at this phase was significantly higher than control cells after exposing to EPMC at both IC 25

Effect of EPMC on mdr-1 gene expression
The mdr-1 gene was not significantly upregulated in CL-6 and Caco-2 cells following exposure to EPMC (0.08, 0.8, and 8 µg/ml and 0.2, 2, and 20 µg/ml, respectively) for 24 and 48 hours, compared to control sample (p>0.05) ( Figure 5). Decreasing of mdr-1 gene expression was observed in all compound exposed CL-6 cells and conferred to mdr-1 expression inhibition activity of EPMC.
(a) (b) Figure 3. Apoptosis Investigation of CL-6 Cell Following Exposure to EPMC at IC 25 (172 µg/ml) and IC 50 (245 µg/ml) Concentrations for 24 and 48 Hours. (a) Scatter plots of apoptosis in the untreated control and EPMC-exposed (at IC 50 ) CL-6 cell for 48 hours and (b) apoptosis degree of CL-6 cell population following EPMC exposure at IC 25 and IC 50 for 24 and 48 hours. Data are presented as median (95% CI) and (*) showed a significant increase in cell population after EPMC exposure compared to untreated control. EPMC, ethyl p-methoxycinnamate; H24, 24 hours; H48, 48 hours

Discussion
The potential anticancer activity of K. galanga L. crude extract has previously been reported in the CL-6 cell (Amuamuta et al., 2017;Mahavorasirikul et al., 2010). However, the study about the anticancer activity of the isolated compound, EPMC, in cholangiocarcinoma cell still limited. Investigation of EPMC as a promising anticancer in CL-6 cell line revealed its biological activities i.e.; cytotoxicity, wound healing inhibition, apoptosis induction, cell cycle arrest without mdr-1 gene expression induction.
The potency of activity of EPMC in CL-6 cell varied with concentrations as its IC 25 , IC 50 , and IC 90 was 172.3, 245.5, and 498.3 µg/ml, respectively (Table 1). The weak cytotoxic activity of EPMC against CL-6 cells (about 50% of 5-FU) is explained by the ester functional group containing chemical structure which results in weak interaction with other cellular components (Sakagami et al., 2017;Soult, 2018). The selectivity to CL-6 cell was 3.7 times compared to a normal cell, OUMS-36T-1F (Table 1). It is noted for the variable activity, and selectivity of EPMC reported in this study compared with the previous study against the same cell lines (Amuamuta, et al., 2017). In the previous study, the IC 50 (median [95% CI]) and selectivity index of EPMC were shown to be 49.19 (48.16-52.29) μg/ml and 2.09, respectively. The potency of activity of this active compound was about 1.3 times of the crude ethanolic extract (median IC 50 64.2 µg/ml) (Amuamuta, et al., 2017). The anticancer activity of EPMC could be improved when combined with other compounds as the components of K. galanga L. such as kaempferol or kaempferide. This remains to be confirmed.
EPMC at IC 25 and IC 50 was found to inhibit wound healing in CL-6 cell line after 24 and 48 hours of exposure. This suggests the potential role of the compound to interrupt cell migration and metastasis. Results of the previous toxicity study showed that K. galangal L. extract exhibited promising anti-cholangiocarcinoma activity in CL6-xenografted nude mice as determined Hours. Data are presented as median (95% CI). EPMC, ethyl p-methoxycinnamate; H24, 24 hours; H48, 48 hours by significant inhibitory activity on tumor growth and lung metastasis, as well as prolongation of survival time (Amuamuta, et al., 2017). Several drugs or compounds produce cytotoxic effect through the initiation of cell death mechanisms such as loss of function of essential organelles, apoptosis activation, and cell cycle arrest (Iorga et al., 2017;Sharifi-Rad et al., 2017). Results of the current study suggest that induction of cell cycle arrest and apoptosis are essential anti-cholangiocarcinoma action of EPMC. To determine action in cell cycle arrest, the phases of cell growth in normal condition has been compared with the EPMC exposed sample. CL-6 cell required 24 hours to complete cell division cycle and remained at G0/G1 phase as well as 1 copy or 2n chromosome has been detected. Cell division processes or G2/M phase are active at about mid of cell cycle or 12 hours incubation period, and 2 copies or 4n chromosome has been observed. The study found that most of the cell population stopped at G0/G1 phase after EPMC treatment for 12 hours while the untreated control sample developed to G2/M phase. Besides, 24 and 48 hours incubation duration allowed CL-6 cells to complete 1 and 2 cell division cycles by most cells presented in G0/ G1 phase whereas most of EPMC exposed sample found at the G2/M phase. Cell cycle arrest occurs in response to DNA damage (Wenzel and Singh, 2018). Mechanism of cell cycle arrest at G0/G1 is p53-dependent, resulting in inhibition of the synthesis of DNA, cyclin-dependent kinase 4 (CDK4) and cyclin D (García-Reyes et al., 2018, Liu et al., 2003. P53-dependent arrested the transition of cells from G2 to M phase and -independent pathways through inhibition of cyclin B and Cdc2 proteins which are involved in mitosis consequences such as inhibition of mitotic spindle, centromere, or cytokinesis (Stark and Taylor, 2006;Wenzel and Singh, 2018).
EPMC exposure at concentrations of IC 25 and IC 50 for 24 and 48 hours induced apoptosis in CL-6 cell line according to the nuclear envelope permeability and cell membrane alteration with the appearance of phosphatidylserine (PS) and expression of caspase 3/7. Apoptosis is a final effect of caspases 3 and 7 which are cysteine proteases activated by the initiator caspase 8 via death receptor in the extrinsic pathway or caspase 9 by DNA damage in the intrinsic pathway (McIlwain et al., 2013). Caspases 3 and 7 exert almost the same action during apoptosis, but caspase 3 is clearly related to plasma membrane blebbing, whereas caspase 7 is also involved in inflammation process (Tomita, 2017;Zhang et al., 2018). Both cell cycle arrest and apoptosis are downstream consequences of p53 protein (Chen, 2016). EPMC exposure may induce DNA damage leading to cell cycle arrest and activate caspase 3/7 expression results in cell apoptosis.
Several anticancer agents derived from medicinal plants fail for treatment because it is the substrate of P-glycoprotein and the situation becomes worse if the compound can induce mdr-1 gene expression (Barthomeuf et al., 2005;Lopes-Rodrigues et al., 2016). Results of the study showed that even CL-6 and Caco-2 have different EPMC sensitivity, but it does not effect on mdr-1 expression in both cell lines. This suggests low potential of cholangiocarcinoma cells to develop resistance to EPMC. Furthermore, the compound may has mdr-1 expression inhibition activity in CL-6 cell as decreasing of mdr-1 gene expression was observed in all compound exposed samples ( Figure 5). Co-administration of p-glycoprotein inhibitor together with medicinal plant anticancer is an interesting option to improve cancer treatment efficacy. Also, investigation of EPMC on mdr-1 expression mechanisms will expand knowledge for treatment innovation (Santos and Paulo, 2013).
In summary, EPMC exerted low to moderate cytotoxic activity against cholangiocarcinoma through cell cycle arrest and cell apoptosis without inducing of mdr-1 expression. Therefore, the compound has suitable biological activities for further development to be used as an adjunct therapy.