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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 67  |  Page : 57-60  

The effect of Phyllanthus debilis methanolic extract on DNA methylation of TAC1 gene in colorectal cancer cell line


Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Penang, Malaysia

Date of Submission29-May-2019
Date of Decision29-Jul-2019
Date of Web Publication11-Feb-2020

Correspondence Address:
Wan Adnan Wan Omar
Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Pulau Pinang
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_226_19

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   Abstract 


Aim/Background: In colorectal cancer, TAC1 was shown to be highly methylated in early carcinogenesis. Using HT29 cell line as a model of colorectal cancer, we postulated that Phyllanthus debilis methanolic extract could regulate DNA methylation in TAC1 gene promoter region and thus could alter the progression of colorectal cancer. Methodology: Cell culture study was done using HT29 cells, which were treated with 0.117 mg/ml P. debilis methanolic extract or 0.5 μM 5-Aza-2-Deoxycytidine (5-Aza) for 72 h. Cells were harvested at 72 h and were extracted for DNA. The DNA was bisulfite modified before been PCR and later was pyrosequenced. Results: The treatment with P. debilis significantly decreased the DNA methylation of TAC1 gene at site 1 (93.1% ± 2.4% vs. 100% ± 0.1%, P < 0.05), site 2 (86.5% ± 1.3% vs. 91.7% ± 0.2%, P < 0.05), and site 3 (96.7% ± 0.9% vs. 100% ± 0%, P < 0.05); but, no significant changes of DNA methylation were seen at site 4 (96.5% ± 1.9% vs. 93.6% ±0.6%, P > 0.05). The average of all Cytosine nucleotide followed by Guanine nucleotide (CpG) sites methylation was reduced, but not statistically significant when compared to the untreated cells (mean methylation 93.2% ± 2.4% vs. 96.3% ± 2.2%, P > 0.05). For cells treated with 5-Aza, DNA methylation was significantly decreased only at site 2 (88.6% ±0.8% vs. 91.7% ±0.2%, P < 0.05), but there was no significant methylation changes at site 1 (94.8% ± 2.4% vs. 100.0% ±0.06%, P > 0.05), site 3 (98.9% ± 1.1% vs. 100% ± 0%, P > 0.05), site 4 (90.6% ±1.6% vs. 93.6% ±0.6%, P > 0.05), and the average of all CpG sites (mean methylation 93.2% ± 2.3% vs. 96.3% ± 2.2%, P > 0.05). Conclusion: Treatment of methanolic extract of P. debilis reduces the methylation of promoter region of TAC1 gene, with better effect than low dose 5-Aza at 72 h of treatment. The anticancer effect of P. debilis may be partly been regulated through DNA methylation.

Keywords: HT29, methylation, Phyllanthus, pyrosequencing, TAC1


How to cite this article:
Mohd Zain SN, Wan Omar WA. The effect of Phyllanthus debilis methanolic extract on DNA methylation of TAC1 gene in colorectal cancer cell line. Phcog Mag 2020;16:57-60

How to cite this URL:
Mohd Zain SN, Wan Omar WA. The effect of Phyllanthus debilis methanolic extract on DNA methylation of TAC1 gene in colorectal cancer cell line. Phcog Mag [serial online] 2020 [cited 2020 Feb 17];16:57-60. Available from: http://www.phcog.com/text.asp?2020/16/67/57/278001



SUMMARY

  • The methanolic extract of Phyllanthus debilis reduces the methylation of promoter region of TAC1 gene. The anticancer effect of P. debilis may partly been regulated through DNA methylation.




Abbreviations used: °C: Degree celcius; mg/mL: Milligram per milliliter; μL: Microliter; min: Minutes; ng: Nanogram; nM: nanoMolar; mM: MilliMolar; μM: MicroMolar; HT29: Colon cancer cell line; h: Hour; PCR: Polymerase chain reaction.


   Introduction Top


Colorectal cancer is one of the most common cancers in Malaysia. It is the second most common cancer affecting males after lung cancer and the third most common cancer affecting females, after breast and uterine cancer.[1] Most of the patients were diagnosed at the late stage, and their 5 years' survival rate was lower than other Asian developed countries.[1] There is no formal/structured national colorectal cancer screening program in Malaysia at the moment. In the developed countries such as in Japan, South Korea, and Singapore, colorectal cancer is one of the most common cancer found in their population.[1] However, the incidence and mortality rates have been stable and are even declining in these countries. This trend may be associated to colorectal cancer screening programs, reduced prevalence of risk factors and/or improved treatments in these countries.[1] Colorectal cancer screening program was shown to reduce colorectal cancer mortality up to 53% in developed countries.[2] Colorectal screening program however was difficult to implement in other parts of the world due to lack of optimal strategy and public acceptance.[2] The most common methods of detection of colorectal cancer are through fecal occult blood test (FOBT) and sigmoidoscopy. However, these two methods had been shown to have poor sensitivity and specificity with the latter at risk of getting gut perforation.[2]

For the last 10 years, DNA methylation-based detection was becoming more common with the availability of sequencing machine. The measurement of SEPT9 gene methylation in peripheral blood had been approved by the United States Food and Drug Administration (FDA)as one of screening methods to detect early colorectal cancer.[3] The monitoring of the changes in the methylation of this gene had shown that it is more accurate and sensitive compared with the current detection of colorectal cancer such as FOBT, carcinoembryonic antigen, and Ca-199.[3],[4] Apart from SEPT9 gene, other gene that is candidate for colorectal cancer biomarker is TAC1. DNA hypermethylation of TAC1 gene was common observation in early colorectal cancer.[5] In this study, we postulated that Phyllanthus debilis methanolic extract could regulate the methylation of the TAC1 gene, in which it was commonly methylated in HT29 cells.

Phyllanthus sp. is one of the herbals that has been used traditionally for treatment of many illnesses such as hepatitis, renal stone, and cancer. Phyllanthus sp. has been shown to possess hepatoprotective, anticarcinogenic, antibacterial, antiviral, and anti-inflammatory activity. We had shown in our previous studies that P. debilis contained strong antioxidant activity with higher anticancer activity and less toxicity than other Phyllanthus species such as Phyllanthus urinaria and Phyllanthus niruri.[6],[7] Based on this result, we had chosen P. debilis as our candidate herb to study its mechanism on DNA methylation. Our study is aimed to look at the effect of methanolic extract of P. debilis on the methylation of HT29, a colorectal cancer cell line. We compared the effect of P. debilis with 5-Aza, a chemotherapeutic drug which the main mechanism as anticancer drug was through regulation of DNA methylation.


   Methodology Top


Herbal specimen

P. debilis was collected from local collection at Tasek Gelugor, Penang, Malaysia. It was identified and deposited at the Universiti Sains Malaysia herbarium (voucher specimen: 11623).

Methanol extraction

Briefly, whole plant of P. debilis was dried in the oven at 50°C for 3 days. Once dried, it was grounded and prepared in the powder form. Five gram of sample was then used to be extracted with 100 mL of methanol (Fisher Chemical) in ultrasonic bath (Power Sonic 405) for 20 min and then filtered. The process was repeated twice with the remaining residual extract. The extracts were dried using rotary evaporator (Buchi Rotary Evaporator RII). Dried extract was kept at −20°C for further use.

Cell culture

HT29 cells were cultured in 24 well plate in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). Cells were maintained at 37°C in a 5% CO2 atmosphere.

When the cells reach to 80% confluency, they were treated with 0.117 mg/mL P. debilis extract or 0.5 μM of the demethylating agent 5-Aza-2-Deoxycytidine (Acros). Treatments were continued for 3 days (72 h), while replacing RPMI-1640 culture medium (Gibco) plus P. debilis or 5-Aza daily. Cells were harvested at 72 h and were pooled for further used.

PCR and Pyrosequencing

HT29 cells were assayed for gene-specific methylation at the promoter of TAC1 gene. Each of the samples was technically replicates for four times. The promoter of TAC1 gene (GX_94242 [TAC1/human]) was identified using Genomatix software (Intrexon Bioinformatics Germany).

DNA was extracted from HT29 cells using DNA extraction kit (Promega, USA). The DNA was then bisulfite converted using EZ DNA Methylation Gold kit (Zymo Research) according to the manufacturer's protocol. Briefly, 500 ng of DNA was incubated with CT conversion reagent at the following temperatures: 98°C for 10 min, 64°C for 2.5 h, held at 4°C. Once completed, the DNA was transferred to a spin column, washed, and desulphonated. It was further purified using wash buffer before being eluted in 20 μL deionized water.

The bisulfite modified DNA (3 μL) was used in a polymerase chain reaction (PCR) reaction containing 12.5 μL GoTaq Green mastermix (Promega), 400 nM forward primer, and 400 nM biotin-labeled reverse primer in a total volume of 25 μL. PCR amplification was carried out using the following protocol: 95°C 15 min, then 50 cycles of 95°C 15 s, annealing temperature 50°C for 30 s, 72°C for 30 s, followed by 72°C for 5 min. The primer sequences and sequence to analyze were summarized in [Table 1].
Table 1: The primers sequences used in the polymerase chain reaction and pyrosequencing and the sequence to analyze for TAC1 assay

Click here to view


The PCR product which was biotin-labeled was captured with Streptavidin Sepharose beads (GE Healthcare), and using a Pyrosequencing Vacuum Prep Tool (Qiagen), the PCR product was made into single stranded DNA. The sequencing primer was annealed to this strand by heating to 80°C, followed by slow cooling to the room temperature. Pyrosequencing was then carried out on a Pyromark ID (Qiagen). The methylation at CpG sites was quantified using PyroMark Q96 2.5.8 software. Primer sequences used in the PCR reaction and pyrosequencing and sequence to analyze for TAC1 assay were shown in [Table 1].

Statistical analysis

The differences of mean in treated and untreated cells were analyzed using student t-test (GraphPad Prism Version 6.01). All the values are presented as mean ± standard deviation and mean ± standard error of mean.


   Results Top


We measured the methylation changes of TAC1 DNA methylation at 4 CpG sites, with the treatment of the HT29 cells with P. debilis and 5-Aza for 72 h. We compared the methylation changes of the untreated cells with treated cells at each CpG site and overall CpG sites methylation.

On treatment with P. debilis methanolic extract at 72 h, DNA methylation showed a significant reduction of DNA methylation at site 1 (P < 0.05), site 2 (P < 0.05), and site 3 (P < 0.05), but not significantly reduced at site 4 (P > 0.05) when compared with untreated cells.

When HT29 cells were treated with 0.5 μM 5-Aza, there was a significant decrease of DNA methylation at only 1 CpG site, which was site 2 (P < 0.05) when compared to untreated cells. Whereas, no significant DNA methylation changes was observed at site 1, site 3, and site 4 (P > 0.05).

For both treatments, the average reduction of DNA methylation at all CpG sites (reduction of 3% when compared with untreated cells) did not show any significant decrease of methylation when compared to the untreated cells (P > 0.05). Results of DNA methylation at each CpG site and overall CpG sites for both treatments were shown in [Table 2].
Table 2: Percentage of TAC1 DNA methylation at 4 CpG sites and its mean of overall sites

Click here to view



   Discussion Top


Methylation at CpG islands, which happened commonly at the promoter region of tumor suppressor genes, can induce gene silences and promote the early changes to cancer.[8]TAC1 gene encodes 4 products of the tachykinin peptide hormone family, which are mainly substance P and neurokinin A and also neuropeptide K and neuropeptide gamma. They are functioning as neurotransmitter, which interact with nerve receptor and smooth muscle cells and as antimicrobial peptide (substance P), vasodilators, and secretagogues.[9]

TAC1 gene was frequently used as a panel for detection of early cancer, in which the detection of TAC1 methylation was shown to increase the sensitivity of detection in early lung cancer to up to 93%[8] whereas in colorectal cancer, TAC1 combined with SEPT9 methylation can detect early colorectal cancer with 73.1% sensitivity and 92.3% specificity,[5] much higher than current detection methods using FOBT and sigmoidoscopy. Furthermore, TAC1 methylation can predict the recurrent of colorectal cancer postsurgery.[10]

Our study showed that treatment with P. debilis induced small changes in DNA methylation of TAC1 genes. Although the changes were very small, they did give a statistical significance at 3 out of 4 CpG sites, which were CpG site 1, site 2, and site 3. When compared with 5-Aza-treated cells, the methylation changes were seen at only 1 CpG sites (site 2). For both treatments (P. debilis methanolic extract and 5-Aza), the overall changes of methylation were almost similar, even though it was insignificant (both treatments showed 3% reduction of DNA methylation in overall CpG sites). We believed that the effect of P. debilis exerted in the DNA methylation at TAC1 promoter region may be site specific. To our surprise, we did not see the marked changes in methylation in majority of the CpG sites in TAC1 genes when treated with the lower concentration of 5-Aza (0.5 μM). The dose that we used was probably too low to induce site-specific demethylating changes at TAC1-targeted promoter region. The concentration of 5-Aza at 0.5 μM used in this study, however, was shown previously to demethylate genes at genome-wide scale.[11],[12]

Result from our study showed that P. debilis was able to reduce DNA methylation of TAC1 gene, a possible mechanism that P. debelis exerts as anticancer properties. Previously, we had shown that P. debilis methanolic extract had a strong anticancer activity in breast cancer cell line (MCF-7) with less toxicity in the normal breast cells (MCF-10A).[6]P. debilis contained high total phenolic and total flavonoid compounds with high antioxidant activity.[7] Analysis of P. debilis by high-performance liquid chromatography showed that the extract contains some beneficial compounds such as caffeic acid, p-coumaric acid, myricetin, and kaempferol (data not shown). Natural compounds such as caffeic acid and dietary catechol were shown previously to regulate DNA methylation by demethylating the RARβ gene through inhibition of DNA methyltransferase 1 (DNMT1) enzyme.[13] The process was through an increased formation of S-adenosyl-L-homocysteine, a by-product of catalyzed methylation process, which acts as a feedback inhibitor to S-adenosyl-methionine (a methyl donor of methylation reaction)-dependent methylation processes.[13] [Figure 1] showed the methylation process, which was catalyzed by the DNMT enzyme family. This process requires S-adenosylmethionine as a cofactor.[14],[15]
Figure 1: Methylation process catalyzed by the DNA methyltransferases enzyme family. This process requires S-adenosylmethionine as a methyl donor. S-adenosyl-L-homocysteine, a by-product of catalyzed methylation process of SAM. 'M' indicates methyl group

Click here to view


In cancer, hypermethylation of genes promoter region of tumour suppressor genes were commonly occurred, which silenced its expressions. Reswitching the expression of these genes will exert protective effect to cancer development and will have the possibilities of changing back the cancer phenotype to normal phenotype. We believe that the re-expression of TAC1 in colorectal cancer cells may have beneficial effect in the long-term by improving the abnormal TAC1 function (deranged inflammatory and immune) back to normal.


   Conclusion Top


Methanolic extract of P. debilis reduced the methylation of TAC1 gene in HT29 cancer cell line. This reduction of methylation may have an effect on the gene functionality. The P. debilis methanolic extract may exert its anticancer through regulation of DNA methylation.

Acknowledgements

We would like to acknowledge the generosity of Professor John Mathers of Human Nutrition Research Centre, Newcastle University in providing and helping us to do pyrosequencing at his laboratory.

Financial support and sponsorship

The authors gratefully acknowledge the financial support from the Research University Grant, Universiti Sains Malaysia (Grant No:1001/ CIPPT/8012317).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Veettil SK, Lim KG, Chaiyakunapruk N, Ching SM, Abu Hassan MR. Colorectal cancer in Malaysia: Its burden and implications for a multiethnic country. Asian J Surg 2017;40:481-9.  Back to cited text no. 1
    
2.
Ng SC, Wong SH. Colorectal cancer screening in Asia. Br Med Bull 2013;105:29-42.  Back to cited text no. 2
    
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Wang Y, Chen PM, Liu RB. Advance in plasma SEPT9 gene methylation assay for colorectal cancer early detection. World J Gastrointest Oncol 2018;10:15-22.  Back to cited text no. 3
    
4.
Xie L, Jiang X, Li Q, Sun Z, Quan W, Duan Y, et al. Diagnostic value of methylated Septin9 for colorectal cancer detection. Front Oncol 2018;8:247.  Back to cited text no. 4
    
5.
Liu Y, Tham CK, Ong SY, Ho KS, Lim JF, Chew MH, et al. Serum methylation levels of TAC1. SEPT9 and EYA4 as diagnostic markers for early colorectal cancers: A pilot study. Biomarkers 2013;18:399-405.  Back to cited text no. 5
    
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Omar WA, Zain SN. Therapeutic index of methanolic extracts of three Malaysian Phyllanthus species on MCF-7 and MCF-10A cell lines. Pharmacogn J 2018;10.  Back to cited text no. 6
    
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Zain SN, Omar WA. Antioxidant activity, total phenolic content and total flavonoid content of water and methanol extracts of Phyllanthus species from Malaysia. Pharmacogn J 2018;10.  Back to cited text no. 7
    
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Powrózek T, Małecka-Massalska T. DNA hypermethylation of tumor suppressor genes as an early lung cancer biomarker. Transl Cancer Res 2016;5:S1531-8.  Back to cited text no. 8
    
9.
NCBI. TAC1 Tachykinin Precursor 1 [Homo Sapiens (Human)]. USA: NCBI; 2019. Available from: https://www.ncbi.nlm.nih.gov/gene/6863. [Last accessed on 2019, Apr 02].  Back to cited text no. 9
    
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Tham C, Chew M, Soong R, Lim J, Ang M, Tang C, et al. Postoperative serum methylation levels of TAC1 and SEPT9 are independent predictors of recurrence and survival of patients with colorectal cancer. Cancer 2014;120:3131-41.  Back to cited text no. 10
    
11.
Ishiguro M, Iida S, Uetake H, Morita S, Makino H, Kato K, et al. Effect of combined therapy with low-dose 5-aza-2'-deoxycytidine and irinotecan on colon cancer cell line HCT-15. Ann Surg Oncol 2007;14:1752-62.  Back to cited text no. 11
    
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Khamas A, Ishikawa T, Shimokawa K, Mogushi K, Iida S, Ishiguro M, et al. Screening for epigenetically masked genes in colorectal cancer using 5-Aza-2'-deoxycytidine, microarray and gene expression profile. Cancer Genomics Proteomics 2012;9:67-75.  Back to cited text no. 12
    
13.
Lee WJ, Zhu BT. Inhibition of DNA methylation by caffeic acid and chlorogenic acid, two common catechol-containing coffee polyphenols. Carcinogenesis 2006;27:269-77.  Back to cited text no. 13
    
14.
Martin EM, Fry RC. Environmental influences on the epigenome: Exposure- associated DNA methylation in human populations. Annu Rev Public Health 2018;39:309-33.  Back to cited text no. 14
    
15.
Wang Y, Sun Z, Szyf M. S-adenosyl-methionine (SAM) alters the transcriptome and methylome and specifically blocks growth and invasiveness of liver cancer cells. Oncotarget 2017;8:111866-81.  Back to cited text no. 15
    


    Figures

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  [Table 1], [Table 2]



 

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