Abstract
Human telomerase reverse transcriptase (hTERT) plays a key role in conferring immortality to human malignant neuroblastomas. We first determined differential expression of hTERT in four human malignant neuroblastoma SH-SY5Y, SK-N-DZ, SK-N-BE2, and IMR-32 cell lines. We then used SK-N-DZ and SK-N-BE2 cell lines, which showed the highest expression of hTERT, to investigate the therapeutic effects of sequential hTERT knockdown and apigenin (APG) treatment. We performed cell invasion assay and studied alterations in expression of matrix metalloproteinases and cell cycle regulatory molecules after this combination therapy. We also investigated induction of apoptosis by using in situ Wright staining, Annexin V staining, and Western blotting. Sequential hTERT knockdown and APG treatment significantly downregulated expression of hTERT so as to cause over 90 % inhibition of cell invasion and 70 % induction of apoptosis in both SK-N-DZ and SK-N-BE2 cell lines. Western blotting demonstrated downregulation of the molecules involved in cell invasion and proliferation, but upregulation of the cell cycle inhibitor and apoptosis-inducing molecules. In conclusion, our current results clearly showed that sequential hTERT knockdown and APG treatment could be a promising therapeutic strategy for effective inhibition of invasion and proliferation and induction of apoptosis in hTERT overexpressing malignant neuroblastoma cells.
Similar content being viewed by others
References
Cabuy E, de Ridder L (2001) Telomerase activity and expression of telomerase reverse transcriptase correlated with cell proliferation in meningiomas and malignant brain tumors in vivo. Virchows Arch 439:176–184
Carpentier C, Lejeune J, Gros F, Everhard S, Marie Y, Kaloshi G, Laigle-Donadey F, Hoang-Xuan K, Delattre JY, Sanson M (2007) Association of telomerase gene hTERT polymorphism and malignant gliomas. J Neurooncol 84:249–253
Chakrabarti M, Khandkar M, Banik NL, Ray SK (2012) Alterations in expression of specific microRNAs by combination of 4-HPR and EGCG inhibited growth of human malignant neuroblastoma cells. Brain Res 1454:1–13
Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR (2007) Protein composition of catalytically active human telomerase from immortal cells. Science 315:1850–1853
Ganeshan VR, Schor NF (2011) Pharmacologic management of high-risk neuroblastoma in children. Paediatr Drugs 13:245–255
Ge L, Shao W, Zhang Y, Qiu Y, Cui D, Huang D, Deng Z (2011) RNAi targeting of hTERT gene expression induces apoptosis and inhibits the proliferation of lung cancer cells. Oncol Lett 2:1121–1129
George J, Tsutsumi M (2007) siRNA-mediated knockdown of connective tissue growth factor prevents N-nitrosodimethylamine-induced hepatic fibrosis in rats. Gene Ther 14:790–803
George J, Banik NL, Ray SK (2009a) Combination of hTERT knockdown and IFN-γ treatment inhibited angiogenesis and tumor progression in glioblastoma. Clin Cancer Res 15:7186–7195
George J, Banik NL, Ray SK (2009b) Combination of taxol and Bcl-2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis and tumor growth. J Cell Mol Med 13:4205–4218
George J, Banik NL, Ray SK (2010) Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL-2 knockdown in human malignant neuroblastoma SK-N-DZ cells. J Neurosci Res 88:877–886
Hiraga S, Ohnishi T, Izumoto S, Miyahara E, Kanemura Y, Matsumura H, Arita N (1998) Telomerase activity and alterations in telomere length in human brain tumors. Cancer Res 58:2117–2125
Jayasooriya RG, Kang SH, Kang CH, Choi YH, Moon DO, Hyun JW, Chang WY, Kim GY (2012) Apigenin decreases cell viability and telomerase activity in human leukemia cell lines. Food Chem Toxicol 50:2605–2611
Karmakar S, Davis KA, Choudhury SR, Deeconda A, Banik NL, Ray SK (2009) Bcl-2 inhibitor and apigenin worked synergistically in human malignant neuroblastoma cell lines and increased apoptosis with activation of extrinsic and intrinsic pathways. Biochem Biophys Res Commun 388:705–710
Kirkpatrick KL, Mokbel K (2001) The significance of human telomerase reverse transcriptase (hTERT) in cancer. Eur J Surg Oncol 27:754–760
Lee SH, Kim JW, Oh SH, Kim YJ, Rho SB, Park K, Park KL, Lee JH (2005) IFN-γ/IRF-1-induced p27kip1 down-regulates telomerase activity and human telomerase reverse transcriptase expression in human cervical cancer. FEBS Lett 579:1027–1033
Maher SG, Romero-Weaver AL, Scarzello AJ, Gamero AM (2007) Interferon: cellular executioner or white knight? Curr Med Chem 14:1279–1289
Masutomi K, Hahn WC (2003) Telomerase and tumorigenesis. Cancer Lett 194:163–172
Molenaar JJ, Ebus ME, Geerts D, Koster J, Lamers F, Valentijn LJ, Westerhout EM, Versteeg R, Caron HN (2009) Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells. Proc Natl Acad Sci U S A 106:12968–12973
Qian X, Cheng J, Chen A, Wang Y, Tao Y, Cao J, Feng Z (2008) Long-term effects of short hairpin RNA-targeted human telomerase reverse transcriptase on suppression of SGC-7901 cell proliferation by inhibition of telomerase activity. Oncol Rep 19:575–581
Sato N, Maehara N, Mizumoto K, Nagai E, Yasoshima T, Hirata K, Tanaka M (2001) Telomerase activity of cultured human pancreatic carcinoma cell lines correlates with their potential for migration and invasion. Cancer 91:496–504
Shay JW, Wright WE (1998) Role of telomeres and telomerase in cancer. Semin Cancer Biol 21:349–353
Shay JW, Zou Y, Hiyama E, Wright WE (2001) Telomerase and cancer. Hum Mol Genet 10:677–685
Tchirkov A, Rolhion C, Kémény JL, Irthum B, Puget S, Khalil T, Chinot O, Kwiatkowski F, Périssel B, Vago P, Verrelle P (2003) Clinical implications of quantitative real-time RT-PCR analysis of hTERT gene expression in human gliomas. Br J Cancer 88:516–520
Wang L, Wei Q, Wang LE, Aldape KD, Cao Y, Okcu MF, Hess KR, El-Zein R, Gilbert MR, Woo SY, Prabhu SS, Fuller GN, Bondy ML (2006) Survival prediction in patients with glioblastoma multiforme by human telomerase genetic variation. J Clin Oncol 24:1627–1632
Xi L, Chen G, Zhou J, Xu G, Wang S, Wu P, Zhu T, Zhang A, Yang W, Xu Q, Lu Y, Ma D (2006) Inhibition of telomerase enhances apoptosis induced by sodium butyrate via mitochondrial pathway. Apoptosis 11:789–798
Acknowledgments
This work was supported in part by a grant (R01 NS65456) from the National Institutes of Health (Bethesda, MD, USA) and another grant (SCIRF-11-002) from the South Carolina Spinal Cord Injury Research Foundation (Columbia, SC, USA).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chakrabarti, M., Banik, N.L. & Ray, S.K. Sequential hTERT Knockdown and Apigenin Treatment Inhibited Invasion and Proliferation and Induced Apoptosis in Human Malignant Neuroblastoma SK-N-DZ and SK-N-BE2 Cells. J Mol Neurosci 51, 187–198 (2013). https://doi.org/10.1007/s12031-013-9975-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12031-013-9975-x