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Phyllanthus debilis Methanolic Extract Reduces the Viability of Human Colorectal Adenocarcinoma (HT-29) Cells and Increases LINE-1 and Alu DNA Methylation

Siti Nur Dalila Mohd Zain and Wan Adnan Wan Omar

Pertanika Journal of Tropical Agricultural Science, Volume 45, Issue 1, February 2022


Keywords: 5-aza-2-deoxycytidine, Alu, global methylation, LINE-1, Phyllanthus debilis

Published on: 10 Febuary 2022

Phyllanthus debilis was shown to have a strong anti-proliferative effect on cancer cells with less effect in normal cells. However, its mechanism on the epigenetic mechanism at repeat sequences is unknown. This study was carried out to determine the effect of P. debilis extract on long interspersed nuclear element-1 (LINE-1) and Alu DNA methylation. The anti-proliferative effect of P. debilis methanolic extract on human colorectal adenocarcinoma (HT-29) at 24 hours was done using trypan blue assay. LINE-1 and Alu methylation measurement on the HT-29 cell line was done after 72 hours of treatment using Pyrosequencing. The effect of P. debilis methanolic extract at 24 hours on the viability of HT-29 cells was dose-dependent with the half-maximal inhibitory concentration (IC50) concentration of 0.1 mg/mL. Treatment with P. debilis methanolic extract showed significantly higher Alu DNA methylation when compared with the untreated HT-29 cells (37.0 ± 2.5% vs 32.3 ± 4.3%, p<0.05). Similarly, treatment with 5-aza-2-deoxycytidine also significantly increased the Alu DNA methylation compared with the untreated HT-29 cells (46.0 ± 2.3% vs 37.0 ± 2.5%, p<0.05). For LINE-1, there was a significant increase of LINE-1 methylation when treated with P. debilis extract (80.3 ± 1.3% vs 76.3 ± 2.1%, p<0.05) and with 5-aza-2-deoxycytidine (81.8 ± 4.3% vs 76.3 ± 2.1%, p<0.05) when compared with untreated cells. In conclusion, treatment of P. debilis methanolic extract on HT-29 cell line reduces the viability of HT-29 cells and increases the methylation of Alu and LINE 1. Similar changes in methylation were also seen in the 5-aza treatment. These epigenetic changes by P. debilis methanolic extract may contribute to its anti-cancer properties.

  • Ahmed, B., Khan, S., Verma, A., & Habibullah. (2009). Antihepatotoxic activity of debelalactone, a new oxirano-furanocoumarin from Phyllanthus debilis. Journal of Asian Natural Products Research, 11(8), 687–692.

  • Bae, J. M., Shin, S., Kwon, H., Park, S., Kook, M. C., Kim, Y., Cho, N., Kim, N., Kim, T., Kim, D., & Kang, G. H. (2012). ALU and LINE-1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications. International Journal of Cancer, 131(6), 1323-1331.

  • Chalitchagorn, K., Shuangshoti, S., Hourpai, N., Kongruttanachok, N., Tangkijvanich, P., Thong-ngam, D., Voravud, N., Sriuranpong, V., & Mutirangura, A. (2004). Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene, 23(54), 8841-8846.

  • Chandrashekar, K. S., Joshi, A. B., Satyanarayana, D., & Pai, P. (2005). Analgesic and anti-inflammatory activities of Phyllanthus debilis. Whole plant. Pharmaceutical Biology, 43(7), 586-588.

  • Das, P. M., & Singal, R. (2004). DNA methylation and cancer. Journal of Clinical Oncology, 22(22), 4632-4642.

  • Ehrlich, M. (2002). DNA methylation in cancer: Too much, but also too little. Oncogene, 21(35), 5400-5413.

  • Feinberg, A. P., & Tycko, B. (2004). The history of cancer epigenetics. Nature Reviews Cancer, 4(2), 143-153.

  • Fraga, M. F., Herranz, M., Espada, J., Ballestar, E., Paz, M. F., Ropero, S., Erkek, E., Bozdogan, O., Peinado, H., Niveleau, A., Mao, J., Balmain, A., Cano, A., & Esteller, M. (2004). A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors. Cancer Research, 64(16), 5527-5534.

  • Ishiguro, M., Iida, S., Uetake, H., Morita, S., Makino, H., Kato, K., Takagi, Y., Enomoto, M., & Sugihara, K. (2007). Effect of combined therapy with low-dose 5-aza-2′-deoxycytidine and irinotecan on colon cancer cell line HCT-15. Annals of Surgical Oncology, 14(5), 1752-1762.

  • Khamas, A., Ishikawa, T., Mogushi, K., Iida, S., Ishiguro, M., Tanaka, H., Uetake, H., & Sugihara, K. (2012). Genome-wide screening for methylation-silenced genes in colorectal cancer. International Journal of Oncology, 41(2), 490-496.

  • Kumaran, A., & Joel Karunakaran, R. (2007). In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWT - Food Science and Technology, 40(2), 344-352.

  • Kwon, H., Kim, J. H., Bae, J. M., Cho, N., Kim, T., & Kang, G. H. (2010). DNA methylation changes in ex-adenoma carcinoma of the large intestine. Virchows Archiv, 457(4), 433-441.

  • Lander, E. S. (2011). Initial impact of the sequencing of the human genome. Nature, 470(7333), 187-197.

  • Lee, H. S., Kim, B., Cho, N., Yoo, E. J., Choi, M., Shin, S., Jang, J., Suh, K., Kim, Y. S., & Kang, G. H. (2009). Prognostic implications of and relationship between CPG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma. Clinical Cancer Research, 15(3), 812-820.

  • Lisanti, S., Omar, W. A., Tomaszewski, B., De Prins, S., Jacobs, G., Koppen, G., Mathers, J. C., & Langie, S. A. (2013). Comparison of methods for quantification of global DNA methylation in human cells and tissues. PLOS One, 8(11), e79044.

  • Momparler, R. L. (2013). Epigenetic therapy of non-small cell lung cancer using decitabine (5-aza-2′-deoxycytidine). Frontiers in Oncology, 3, 188.

  • Natsume, A., Wakabayashi, T., Tsujimura, K., Shimato, S., Ito, M., Kuzushima, K., Kondo, Y., Sekido, Y., Kawatsura, H., Narita, Y., & Yoshida, J. (2008). The DNA demethylating agent 5-aza-2′-deoxycytidine activates NY-ESO-1 antigenicity in orthotopic human glioma. International Journal of Cancer, 122(11), 2542-2553.

  • Omar, W. A. W., & Zain, S. N. D. M. (2018). Therapeutic index of methanolic extracts of three Malaysian Phyllanthus species on MCF-7 and MCF-10A cell lines. Pharmacognosy Journal, 10(6s), s30-s32.

  • Rodić, N., & Burns, K. H. (2013). Long interspersed element–1 (LINE-1): Passenger or driver in human neoplasms?. PLOS Genetics, 9(3), e1003402.

  • Saito, K., Kawakami, K., Matsumoto, I., Oda, M., Watanabe, G., & Minamoto, T. (2010). Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non–small cell lung cancer. Clinical Cancer Research, 16(8), 2418-2426.

  • Sarin, B., Verma, N., Martín, J. P., & Mohanty, A. (2014). An overview of important ethnomedicinal herbs of Phyllanthus species: Present status and future prospects. The Scientific World Journal, 2014, 839172.

  • Sellis, D., Provata, A., & Almirantis, Y. (2007). Alu and LINE-1 distributions in the human chromosomes: Evidence of global genomic organization expressed in the form of power laws. Molecular Biology and Evolution, 24(11), 2385-2399.

  • Sharma, G., Mirza, S., Parshad, R., Srivastava, A., Gupta, S. D., Pandya, P., & Ralhan, R. (2010). Clinical significance of promoter hypermethylation of DNA repair genes in tumor and serum DNA in invasive ductal breast carcinoma patients. Life Sciences, 87(3-4), 83-91.

  • So, F. V., Guthrie, N., Chambers, A. F., Moussa, M., & Carroll, K. K. (1996). Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices. Nutrition and Cancer, 26(2), 167-181.

  • Sugimura, T., & Ushijima, T. (2000). Genetic and epigenetic alterations in carcinogenesis. Mutation Research/Reviews in Mutation Research, 462(2-3), 235-246.

  • Wanniarachchi, K. K., Peiris, L. D., & Ratnasooriya, W. (2009). Antihyperglycemic and hypoglycemic activities of Phyllanthus debilis aqueous plant extract in mice. Pharmaceutical Biology, 47(3), 260-265.

  • Whitelaw, E., & Martin, D. I. (2001). Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nature Genetics, 27(4), 361-365.

  • Yoder, J. A., Walsh, C. P., & Bestor, T. H. (1997). Cytosine methylation and the ecology of intragenomic parasites. Trends in Genetics, 13(8), 335-340.

  • Zain, S. N. D. M., & Omar, W. A. W. (2020). The effect of Phyllanthus debilis methanolic extract on DNA methylation of TAC1 gene in colorectal cancer cell line. Pharmacognosy Magazine, 16(67), 57.

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