INTRODUCTION:

Cancer is a condition of disease where abnormal cells continuously proliferate and can invade nearby tissues.[1] Globally, cancer is one of the leading cause of death. In WHO records, 1 of 6 deaths in the world is caused by cancer. [2] In 2015, cancer caused 8.8 million deaths in the world. [3] According Indonesian Ministry of Health’s

Data and Information Center, the biggest cause of cancer deaths every year is lung, stomach, liver, breast, and colorectal cancer. [2] Colorectal cancer itself is the third most common malignancy in women and men throughout the world.

Based on Globocan (IARC) data in 2012, incidence of colorectal cancer in Indonesia reached 12.8 per 100.000 adult population, with mortality rate reaching 9.5% of all cancer cases. [4]

Most of colorectal cancer cases in Indonesia occurs with a sporadic pattern. Based on epidemiological studies in Indonesia, the age average of diagnosed patients is around 45-50 years old. The initial onset of colorectal cancer in Indonesia also appears at a younger age than developed countries, which is at the age of 40 or lower. Localization characteristic of colorectal cancer in developed countries shows proximal localization in ascending colon, with low metastasis and good prognosis. Whereas in Indonesia, the localization occurs in the distal of rectum, with low survival and poor prognosis. With the severe condition of colorectal cancer in Indonesia, comprehensive understanding and management are needed. [5]

A study from BMC Gastroenterology Journal stated that cyclooxygenase-2 (COX-2) plays a role in cancer development and progression. COX-2 overexpression is associated with premalignant and malignant lesion originating from epithelial cell, especially in the gastrointestinal tracts such as colon and rectum. [6] The high expression of COX-2 in colorectal cancer set COX-2 as one of the targets to cancer drugs. Until now, COXIB (COX-2 inhibitor) and NSAID (nonsteroidal anti-inflammatory drugs) are the most promising chemopreventive agents to treat colorectal cancer. However, these two agents aren’t recommended because of the serious cardiovascular side effects. [7] For this reason, it is necessary to find another agent that can inhibitor COX-2, but with minimal side effects.

Previous study states that there is an herbal medicine that could inhibit colorectal cancer growth on mice, named ethanol extract from Phaleria macrocarpa fruit or known as Mahkota Dewa originating from Indonesia. With the potential of Phaleria macrocarpa fruit, it is possible for other parts of the plant to have the same potentials as the fruit, including the stem bark. [8] The stem bark will also be dissolved in an ethanol solvent, which is a solvent that can extract polar phytochemistry contents needed and also it is one of the safest solvents. Based on these possibilities, we are interested to see the effect of ethanol extract of Phaleria macrocarpa stem bark on growth inhibition and COX-2 expression in colorectal cancer cell line HCT116.

MATERIALS AND METHODS:

Plant and Cell Line Materials:

The stem bark of Phaleria macrocarpa was harvested from Cigadung Village, Kuningan (West Java, Indonesia) in June 2018. The stem bark was dried inside a room before making the extract. Colorectal cancer cell line HCT116 was obtained from Pathology Anatomy Department, University of Indonesia. HCT116 cells were used in this study because of its similar characteristic to colorectal cancer growth pattern Indonesia with late onset, distal localization and low survival rate. [9] The cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco) consisting of fetal bovine serum medium 10% (Gibco) and penicillin-streptomycin 1% (Gibco).

Extract Preparation:

Preparation of the extract was done at Medical Chemistry Department, Faculty of Medicine, University of Indonesia.

About 500 gram of dry powder Phaleria macrocarpa stem bark is macerated in 1500 ml of ethanol solvent for 24 hours inside a beaker glass container while stirred for 2-3 times a day. Maceration process is repeated for three times to get as many filtrates as possible. The filtrate is dried using a rotary evaporator to get the crude ethanol extract of Phaleria macrocarpa stem bark.

MTT cytotoxic Assay:

In vitro anticancer cytotoxic activity of ethanol extract of Phaleria macrocarpa stem bark against colorectal cancer cell line HCT116 was tested using MTT assay. The comparison used in the MTT assay as the positive control was Cisplatin.

Colorectal cancer cell line HCT116 were cultured in DMEM and supplemented by fetal bovine serum 10% and penicillin-streptomycin 1%. The supplemented cultured cells were incubated in 37oC and 5% of CO2 in humid conditions for 24 hours. The crude ethanol extract was prepared to be dissolved with the medium until the concentration reached 100; 50; 25; 12,5; and 6 µg/ml, while cisplatin was prepared to be dissolved until the concentration reached 64; 32; 16; 4; and 0,5 µg/ml. The dissolved samples of each concentration were added to HCT116 cells in the well plate (Costar) in triplicates and were incubated for 24 hours. A total of 100 µl of 5 mg/ml MTT solution was also added to the well plate (Costar). The mixture in the well plate was incubated for 4 hours, and centrifuged with a separate medium. 100 µl of dimethyl sulfoxide was added until blue-purple deposits was formed. The absorbance was counted in 590 nm wavelength with micro plate reader (Model 550, Bio-Rad, USA). Inhibition percentage was counted with this equation:

Inhibition percentage (%):

Absorbance of Treatment Group

= 1 - ^{------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------}x 100%

Absorbance of Control Group

The IC50 score was counted with Microsoft Excel 2013, by using simple connecting log concentration of extract in the x-axis with inhibition percentage in the y-axis using simple linear regression equation of y = bx + a. To count the IC50, 50 was added to the “y” in the simple linear regression equation. The “x” then will be calculated, and IC50 was attained by calculating the antilog of “x”.

Immunochemistry Assay:

Extracts in 50; 100; and 200 ppm are administered to colorectal cancer cell lines that have been added with COX-2 antibodies. The comparison used as the negative control is the cell culture in DMEM without given ethanol extract of Phaleria macrocarpa. Then, COX-2 expression on the colorectal cell line culture is assessed by counting the H-score from the immunochemistry staining.

A total of 50 mg of ethanol extract of Phaleria macrocarpa stem bark was dissolved with 5000 µl of dimethyl sulfoxide. The dissolved ethanol extract of Phaleria macrocarpa stem bark was divided into three treated concentration of 50; 100; and 200 ppm. The solution used as a negative control in the experiment was a cell culture preparations in DMEM medium without treated with ethanol extract of Phaleria macrocarpa stem bark.

HCT116 cells were placed into petri dish that already has sterile object glass and incubated under 5% CO2 incubator at 37oC for 24 hours. The extract at 50; 100; and 200 ppm were added to the cells and was incubated for another 24 hours. The solution used as the negative control in the experiment was a cell culture preparations in DMEM medium without treated with ethanol extract of Phaleria macrocarpa stem bark. After incubation, the cells were washed with PBS solutions. The cells were fixed in the sterile object glass by dropping absolute methanol on the object glass surface and left it for 10 minutes. Hydrogen peroxidase (H2O2) 3% was dropped to the surface to eliminate endogen peroxidase. The cells were then dipped in 0.01 M citrate buffer (pH = 6) and dropped with normal serum. The cells were incubated overnight with anti-COX-2 antibody inside buffer phosphate solution. The cells were incubated again with secondary antibody for 10 minutes, and HRP-conjugated streptavidin for 3-8 minutes. The cells were washed by running distilled water and added with hematoxylin-harris solution. The expression of COX-2 was observed under light microscope with 400x magnification by counting H-score based on cell color intensity. The intensity ranged from 0-2 which represents negative to strongly colored cells. Strongly colored cells appear as dark brown colored dots in more than 50% of the cells, weakly colored cells appear as dark brown colored dots in less than 50% of the cells, and negative apprears as light brown colored dots. The judgement of cells color intensity was done using ImageJ and TMARKER application. H-score was counted with this equation:

H-score = (% of cells with color intensity 0 x 1) + (% of cells with color intensity 1 x 2) + (% of cells with color intensity 2 x 3)

The result of H-score ranges from 0 to 300, where the value 300 means there are 100% strongly colored cells (+2). High expression of COX-2 is indicated by H-score > 200, while H-score < 200 indicates low expression of COX-2.

Data Analysis:

Cell cytotoxicity was analyzed by using simple linear regressions in Microsoft Excel 2013 to calculate IC50 of ethanol extract of Phaleria macrocarpa stem bark and cisplatin from triplicates treatment. H-score from immunocytochemistry staining was analyzed by one-way ANOVA using IBM SPSS Statistics Version 20 program.

RESULT AND DISCUSSION:

This study evaluates anticancer activity of ethanol extract of Phaleria macrocarpa stem bark on colorectal cancer cell line HCT116, and also to prove whether the anticancer activity is mediated COX-2 inhibition. COX-2 plays a big part on colorectal cancer progression and one of the target site of anticancer medicine, such as COXIB and NSAID. [7]

MTT Assay:

From this study, Phaleria macrocarpa stem bark is proven to perform anticancer effect on colorectal cancer HCT116. Based from U.S NCI plant screening program, crude extract with IC50 value of < 20 µg/ml is considered to have cytotoxic activity. [10] The IC50 value of ethanol extract of Phaleria macrocarpa stem bark is 1.327 µg/ml (R2 = 1,00). Compared to cisplatin (IC50 = 0,0139 µg/ml; R2 = 0,9083) as one of the most effective anticancer agent on the treatment of solid tumor such as colorectal cancer, the IC50 value of ethanol extract of Phaleria macrocarpa stem bark is close to cisplatin. With the IC50 value < 20 µg/ml, both ethanol extract of Phaleria macrocarpa stem bark and cisplatin are proven to have cytotoxic activity on colorectal cancer cell line HCT116. This study also shows very high correlation between log concentration of both extract and cisplatin and its growth inhibition, shown by the R value of ethanol ranging from 0,9 – 1. [11]

The result of Cisplatin anticancer effect is congruent with previous study from Gurbuz V et al, stating that there is antiapoptosis effect of cisplatin on colorectal cancer cell line HT29, shown in the concentration of 50 µM. [12] Son DJ et al, using HCT116 cells as the target cell, states that cisplatin inhibits HCT116 cells growth with IC50 value of 4,2 µg/ml. [13]

Table 1: H-score from the observation on COX-2 expression of HCT116 cells

Variable	Take	H-score	Average H-score
Negative control	1	219.64
	2	217.16	214,00
	3	204,91
Extract 50 ppm	1	212,30
	2	198,70	206,00
	3	206,79
Extract 100 ppm	1	209,36
	2	198,70	206,00
	3	204,18
Extract 200 ppm	1	173,78
	2	157,88	173,3
	3	187,50

The anticancer effect of ethanol extract of Phaleria macrocarpa stem bark in the result are in accordance with previous study from Rahmawati E et al. stating that ethanol extract of Phaleria macrocarpa fruit pulp shows anticancer effect on C3H mouse mammary tumor induced by transplantation. [14] Shwter AN et al. also done an in-vivo study stating that Phaleria macrocarpa fruit crude extract is proven to perform antioxidant activity on colorectal cancer aberrant crypt foci. [15]

Figure 1: Effect of various concentration of ethanol extract of Phaleria macrocarpa on HCT116 cells inhibition

Table 2: One-way ANOVA test result

Treatment	Mean + SD	Sig
Negative control	214,00 + 7,937	0,003
Extract 50 ppm	206,00 + 6,557
Extract 100 ppm	207,7 + 3,215
Extract 200 ppm	173,3, + 15,011

Immunochemistry Assay:

Low IC50 value of ethanol extract of Phaleria macrocarpa stem bark shows the ability of the extract in inhibiting colorectal cancer cell growth. Colorectal cancer growth is caused by several mechanisms, either by mutation of Wnt signaling pathway or disturbance in other signaling pathways such as cyclooxygenase-2 (COX-2), EGF receptor (EGFR) and RAS pathway that plays a role in increasing cells’ activity. [16,17] A study by Sostres et al. states that overexpression of COX-2 was found in 50% of colorectal adenoma and 80-90% of colorectal cancer tumor, and most of it were related to worse survival in colorectal cancer patients. [18] The characteristic of colorectal cancer in Indonesia is in coherence with worse prognosis as the onset starts at a younger age, with distal localization in rectum, and worse survival. [5] One of the mechanism underlying colorectal cancer growth is assumed to be caused by overexpression of COX-2. Thus, it could be assumed that by inhibiting the COX-2 expression, the colorectal cancer growth will be inhibited.

COX-2 is an immediate-early response gene that appears when induced by pro-inflammatory cytokines such as IFN-g, IL1A/B, and TNF-a. The cytokines were produced by inflammatory cells and tumor promotors like TPA and RAS. [19]

The immunocytochemistry assay shows that H-score from negative control, the treatment of 50 ppm, 100 ppm are above 200, and H-score from treatment of 200 ppm is below 200. Only HCT116 cells treated with 200 ppm of Phaleria macrocarpa stem bark ethanol extract has low expression of COX-2, while the other groups have high expression of COX-2. From post hoc test, only the treatment with 200 ppm of the extract shows significant difference with other groups (Sig. < 0,05). The significance of 200 ppm groups shows that treatment with 200 ppm of ethanol extract of Phaleria macrocarpa stem bark could inhibit HCT116 cell growth by inhibiting COX-2 expression. This result is in concordance to study conducted by Kusmardi et al. that proves the Phaleria macrocarpa pericarp extract could inhibit COX-2 expression on mice colon induced by dextran sodium sulfate. [20]

Figure 2: The observation of COX-2 expression on HCT116 cells on immunocytochemistry method under light microscope using 400x magnification

The phytochemistry screening of Phaleria macrocarpa shows the presence of secondary metabolites such as flavonoids, phenolic compounds, saponin glycosides, glycosides, tannins, steroids, and terpenoids. [21] Kaempferol-3-O-b-d-glucoside has been isolated from the fruit and was proven to protect H4IIE rat hepatoma cells from oxidative stress [22], gallic acid from the fruit is also proven to have growth inhibitory effect on OV-CAR3 and A2780/CP70 ovarian cell lines [23], tannin from fruits and leaves is correlated to induction of apoptosis in leukemia cells [24], and benzophenone glycoside has been isolated from the bark and shows inhibitory activity to leukemia mouse L1210 with IC50 of 5,1 µg/ml. [25]

Previous study by Lee et al. demonstrated that kaempferol inhibits UVB-induced COX-2 expression in in vitro and in vivo studies by directly targeting the Src protein kinase. [26] García-Mediacilla et al. reported that a significant concentration-dependent effect of COX-2 was produced by kaempferol. This may contribute to anti-inflammatory effect which also inhibits colorectal cancer growth. [27] Docking analysis done by Amaravani M et al. shows that the 2-[(2E,4E)-hexa-2,4-dienyl]-3,4,5-trihydroxybenzoic acid which is one of top ten ranked gallic acid structural has more affinity at active site of COX-2 than other gallic acid. [28] Shen YC et al. demonstrated that tannin from the heated tomatoes shows suppression on COX-2 expression. [29] Research on benzophenone glycoside effect on COX-2 expression hasn't been done before.

CONCLUSION:

Anticancer activity of Phaleria macrocarpa stem bark ethanol extract on colorectal cancer cell line HCT116 was mediated by COX-2 inhibition. Ethanol extract of Phaleria macrocarpa stem bark offers a new potential as promising chemopreventive agent on colorectal cancer, replacing COXIB and NSAID that have dangerous cardiovascular side effect on human. However, significant effect of Phaleria macrocarpa stem bark ethanol extract on HCT116 cells only shows when given at high dose (200 ppm). Phytochemical properties which is expected to play a role on inhibiting COX-2 expression are kaempferol, gallic acid, and tannin. The molecular mechanism of Phaleria macrocarpa stem bark ethanol extract wasn’t assessed in this study. Therefore, further research on the molecular mechanism needs to be done in the future.

ACKNOWLEDGMENT:

Authors are very grateful to Faculty of medicine Universitas Indonesia, for PITTA grand and for providing facilities to full fill the work.

CONFLICT OF INTREST:

The Authors declare no conflict of interest.

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Received on 04.01.2019 Modified on 28.02.2019

Research J. Pharm. and Tech. 2019; 12(6):2902-2906.

DOI: 10.5958/0974-360X.2019.00489.X