Sains Malaysiana 48(9)(2019): 1833–1840

http://dx.doi.org/10.17576/jsm-2019-4809-04

Investigation of Boldine as a Potential Telomerase Inhibitor by Downregulation of hTERT/hTERC in HCT 116 Human Colon Carcinoma Cells

(Kajian Boldina sebagai Perencat Telomerase Berpotensi oleh Pengawalaturan Rendah hTERT/hTERC dalam HCT 116 Karsinoma Sel Kolon Manusia)

NADHIRAH AHMAD, CHING JOO JIE & NAZIA ABDUL MAJID*

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

Received: 25 September 2018/Accepted: 24 June 2019

ABSTRACT

Telomerase, a ribonucleoprotein (RNP) complex, is a type of RNA-dependent DNA polymerase that synthesises telomeric DNA repeats (TTAGGG) at the 3’ end of chromosomes. Most of the cancer cells express high level of telomerase which results in cellular immortality. The high telomerase activity in cancer cells can be detected via expression of human TERT (hTERT), the catalytic protein subunit of telomerase, and expression of human TERC (hTERC), the RNA component in telomerase. Boldine, a natural alkaloid compound, was shown to have anticancer properties on various types of cancer cells, but the anti-telomerase property was poorly understood. This study was carried out to investigate the ability of boldine in targeting telomerase on the human colon cancer cell line, HCT 116, by analyzing the expression of hTERT and hTERC. Boldine was shown to have a time- and dose-dependent cytotoxic effect on HCT 116 cell line in SRB assay. The protein expression of hTERT was assessed through Western blot where it was observed to be down-regulated upon boldine treatment compared to control. The cells treated with boldine also exhibited a down-regulation of mRNA expression for both hTERT and hTERC in Real Time PCR (qRT-PCR). The down-regulation of hTERT protein expression correlated with the reduced hTERC mRNA expression in qRT-PCR. The observations on the down-regulation of protein and mRNA expressions of telomerase related genes, hTERT and hTERC, in this study suggested that boldine might become a significant candidate for telomerase-targeted anti-cancer therapy.

Keywords: Boldine; cancer; hTERC; hTERT; telomerase

ABSTRAK

Telomerase, iaitu satu kompleks ribonukleoprotein (RNP) merupakan sejenis DNA polimerase bersandar RNA yang mensintesis telomerik DNA berulangan (TTAGGG) pada hujung kromosom. Kebanyakan sel kanser mengekspres telomerase pada tahap yang tinggi dan akan mengakibatkan sel mencapai keabadian. Aktiviti telomerase yang tinggi dalam sel kanser dapat dikesan melalui pengekspresan TERT manusia (hTERT) iaitu subunit protein katalitik telomerase dan pengekspresan TERC manusia (hTERC) iaitu komponen RNA dalam telomerase. Boldina merupakan sebatian alkaloid semula jadi yang mempunyai sifat antikanser terhadap pelbagai jenis sel kanser, namun begitu sifat anti-telomerase masih kurang difahami. Kajian ini dijalankan untuk mengkaji keupayaan boldina untuk mensasarkan telomerase dalam titisan sel kanser kolon manusia, HCT 116 dengan menganalisis pengekspresan hTERT dan hTERC. Boldina dikatakan mempunyai kesan sitotoksik yang bersandar kepada masa and dos terhadap titisan sel HCT 116 dalam asai SRB. Pengekspresan protein hTERT telah dinilai melalui Western blot dengan pengawalaturannya menurun selepas rawatan dengan boldina berbanding kawalan. Sel yang dirawat dengan boldina juga menunjukkan pengawalaturan yang menurun bagi pengekspresan mRNA untuk kedua-dua hTERT dan hTERC melalui PCR masa sebenar (qRT-PCR). Pengawalaturan yang menurun bagi pengekspresan protein hTERT didapati berkorelasi dengan pengurangan pengekspresan mRNA hTERC melalui qRT-PCR. Pemerhatian terhadap pengawalaturan yang menurun bagi pengekspresan protein dan mRNA gen berkait telomerase, hTERT dan hTERC dalam kajian ini mencadangkan bahawa boldina berkemungkinan menjadi calon penting untuk terapi antikanser yang mensasarkan telomerase.

Kata kunci: Boldina; hTERC; hTERT; kanser; telomerase

REFERENCES

Akıncılar, S.C., Low, K.C., Liu, C.Y., Yan, T.D., Oji, A., Ikawa, M., Li, S. & Tergaonkar, V. 2015. Quantitative assessment of telomerase components in cancer cell lines. FEBS Letters 589(9): 974-984.

Aljarbou, F., Almousa, N., Bazzi, M., Aldaihan, S., Alanazi, M., Alharbi, O., Almadi, M., Aljebreen, A.M., Azzam, N.A., Arafa, M., Aldbass, A., Shaik, J., Alasirri, S., Warsy, A., Alamri, A., Parine, N.R. & Alamro, G. 2018. The expression of telomere-related proteins and DNA damage response and their association with telomere length in colorectal cancer in Saudi patients. PloS ONE 13(6): e0197154.

Avilion, A.A., Piatyszek, M.A., Gupta, J., Shay, J.W., Bacchetti, S. & Greider, C.W. 1996. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Research 56(3): 645-650.

Ayiomamitis, G.D., Notas, G., Zaravinos, A., Zizi-Sermpetzoglou, A., Georgiadou, M., Sfakianaki, O. & Kouroumallis, E. 2014. Differences in telomerase activity between colon and rectal cancer. Canadian Journal of Surgery 57(3): 199-208.

Baena-del Valle, J.A., Zheng, Q., Esopi, D.M., Rubenstein, M., Hubbard, G.K., Moncaliano, M.C., Hruszkewycz, A., Vaghasia, A., Yegnasubramanian, S., Wheelan, S.J., Meeker, A.K., Heaphy, C.M., Graham, M.K. & De Marzo, A.M. 2018. MYC drives overexpression of telomerase RNA (hTR/TERC) in prostate cancer. The Journal of Pathology 244(1): 11-24.

Beattie, T.L., Zhou, W., Robinson, M.O. & Harrington, L. 1998. Reconstitution of human telomerase activity in vitro. Current Biology 8(3): 177-180.

Bertorelle, R., Rampazzo, E., Pucciarelli, S., Nitti, D. & De Rossi, A. 2014. Telomeres, telomerase and colorectal cancer. World Journal of Gastroenterology 20(8): 1940-1950.

Boscolo-Rizzo, P., Da Mosto, M.C., Rampazzo, E., Giunco, S., Del Mistro, A., Menegaldo, A., Baboci, L., Mantovani, M., Tirelli, G. & De Rossi, A. 2016. Telomeres and telomerase in head and neck squamous cell carcinoma: From pathogenesis to clinical implications. Cancer and Metastasis Reviews 35(3): 457-474.

Cayuela, M.L., Flores, J.M. & Blasco, M.A. 2005. The telomerase RNA component Terc is required for the tumour‐promoting effects of Tert overexpression. EMBO Reports 6(3): 268-274.

Celeghin, A., Giunco, S., Freguja, R., Zangrossi, M., Nalio, S., Dolcetti, R. & De Rossi, A. 2016. Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells. Cell Death & Disease 7(12): e2562.

Chadeneau, C., Hay, K., Hirte, H.W., Gallinger, S. & Bacchetti, S. 1995. Telomerase activity associated with acquisition of malignancy in human colorectal cancer. Cancer Research 55(12): 2533-2536.

Chen, R.J., Wu, P.H., Ho, C.T., Way, T.D., Pan, M.H., Chen, H.M., Ho, Y.S. & Wang, Y.J. 2017. P53-dependent downregulation of hTERT protein expression and telomerase activity induces senescence in lung cancer cells as a result of pterostilbene treatment. Cell Death & Disease 8(8): e2985.

Cong, Y.S., Wright, W.E. & Shay, J.W. 2002. Human telomerase and its regulation. Microbiology and Molecular Biology Reviews 66(3): 407-425.

Cristofari, G. & Lingner, J. 2006. Telomere length homeostasis requires that telomerase levels are limiting. The EMBO Journal 25(3): 565-574.

Feng, J., Funk, W.D., Wang, S.S., Weinrich, S.L., Avilion, A.A., Chiu, C.P., Adams, R.R., Chang, E., Allsopp, R.C., Yu, J., Le, S., West, M.D., Harley, C.B., Andrews, W.H., Greider, C.W. & Villeponteau, B. 1995. The RNA component of human telomerase. Science 269(5228): 1236-1241.

Fernández-Marcelo, T., Gómez, A., Pascua, I., de Juan, C., Head, J., Hernando, F., Jarabo, J.R., Calatayud, J., Torres- García, A.J. & Iniesta, P. 2015. Telomere length and telomerase activity in non-small cell lung cancer prognosis: Clinical usefulness of a specific telomere status. Journal of Experimental & Clinical Cancer Research 34(1): 78.

Günes, Ç., Lichtsteiner, S., Vasserot, A.P. & Englert, C. 2000. Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1. Cancer Research 60(8): 2116-2121.

Herbert, B.S., Pitts, A.E., Baker, S.I., Hamilton, S.E., Wright, W.E., Shay, J.W. & Corey, D.R. 1999. Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proceedings of The National Academy of Sciences 96(25): 14276-14281.

Houghton, P., Fang, R., Techatanawat, I., Steventon, G., Hylands, P.J. & Lee, C.C. 2007. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 42(4): 377-387.

Ishikawa, F. 1997. Regulation mechanisms of mammalian telomerase. A review. Biochemistry-New York-English Translation of Biokhimiya 62(11): 1332-1337.

Jafri, M.A., Ansari, S.A., Alqahtani, M.H. & Shay, J.W. 2016. Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Medicine 8(1): 69.

Leão, R., Apolónio, J.D., Lee, D., Figueiredo, A., Tabori, U. & Castelo-Branco, P. 2018. Mechanisms of human telomerase reverse transcriptase (h TERT) regulation: Clinical impacts in cancer. Journal of Biomedical Science 25(1): 22.

Makki, J. 2015. Telomerase activity in breast cancer, promising marker of disease progression. Telomere and Telomerase 2: e681

Nakajima, A., Tauchi, T., Sashida, G., Sumi, M., Abe, K., Yamamoto, K., Ohyashiki, J.H. & Ohyashiki, K. 2003. Telomerase inhibition enhances apoptosis in human acute leukemia cells: Possibility of antitelomerase therapy. Leukemia 17(3): 560-567.

Nakamura, R.M. & Kasahara, Y. 2010. Molecular diagnostics in the evaluation of cancer: Modern concepts and overview. In Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory. Massachusetts: Academic Press. pp. 215-223

Natarajan, S., Chen, Z., Wancewicz, E.V., Monia, B.P. & Corey, D.R. 2004. Telomerase reverse transcriptase (hTERT) mRNA and telomerase RNA (hTR) as targets for downregulation of telomerase activity. Oligonucleotides 14(4): 263-273.

Noureini, S.K. & Wink, M. 2015a. Dose-dependent cytotoxic effects of boldine in HepG-2 cells-Telomerase inhibition and apoptosis induction. Molecules 20(3): 3730-3743.

Noureini, S.K. & Tanavar, F. 2015b. Boldine, a natural aporphine alkaloid, inhibits telomerase at non-toxic concentrations. Chemico-Biological Interactions 231: 27-34.

Noureini, S.K., Kheirabadi, M., Masoumi, F., Khosrogerdi, F., Zarei, Y., Suárez-Rozas, C., Salas-Norambuena, J. & Kennedy Cassels, B. 2018. Telomerase inhibition by a new synthetic derivative of the aporphine alkaloid boldine. International Journal of Molecular Sciences 19(4): 1239.

Shimojima, M., Komine, F., Hisatomi, H., Shimizu, T., Moriyama, M. & Arakawa, Y. 2004. Detection of telomerase activity, telomerase RNA component, and telomerase reverse transcriptase in human hepatocellular carcinoma. Hepatology Research 29(1): 31-38.

Weinrich, S.L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V.M., Holt, S.E., Bodnar, A.G., Lichtsteiner, S., Kim, N.W., Trager, J.B., Taylor, R.D., Carlos, R., Andrews, W.H., Wright, W.E., Shay, J.W., Harley, C.B. & Morin, G.B. 1997. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nature Genetics 17(4): 498-502.

Wong, M.S., Wright, W.E. & Shay, J.W. 2014. Alternative splicing regulation of telomerase: A new paradigm? Trends in Genetics 30(10): 430-438.

Xi, L. & Cech, T.R. 2014. Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT. Nucleic Acids Research 42(13): 8565-8577.

Xu, Y. & Goldkorn, A. 2016. Telomere and telomerase therapeutics in cancer. Genes 7(6): 22.

*Corresponding author; email: nazia@um.edu.my