Annals of Surgical Oncology

, Volume 18, Issue 12, pp 3514–3527 | Cite as

Butylidenephthalide Suppresses Human Telomerase Reverse Transcriptase (TERT) in Human Glioblastomas

  • Po-Cheng Lin
  • Shinn-Zong Lin
  • Yi-Lin Chen
  • Jeng-Shou Chang
  • Li-Ing Ho
  • Po-Yen Liu
  • Li-Fu Chang
  • Yeu-Chern Harn
  • Shee-Ping Chen
  • Li-Yi Sun
  • Pi-Chun Huang
  • Jung-Ting Chein
  • Chang-Hai Tsai
  • Chii-Wen Chou
  • Horng-Jyh Harn
  • Tzyy-Wen Chiou
Translational Research and Biomarkers



Telomerase is widely expressed in most human cancers, but is almost undetectable in normal somatic cells and is therefore a potential drug target. Using the human telomerase promoter platform, the naturally occurring compound butylidenephthalide (BP) was selected for subsequent investigation of antitumor activity in vitro and in vivo.


We treated human glioblastoma cells with BP and found a dose-dependent decrease in human telomerase reverse transcriptase (hTERT) mRNA expression and a concomitant increase in p16 and p21 expression. Because c-Myc and Sp1 are involved in transcriptional regulation of hTERT, the effect of BP on c-Myc and Sp1 expression was examined.


Using electrophoretic mobility shift assays and western blotting, we showed that BP represses hTERT transcriptional activity via downregulation of Sp1 expression. Using the telomerase repeat amplification protocol, an association between BP concentration and suppression of telomerase activity, induction of human glioblastoma senescence, and inhibition of cellular proliferation was identified. This was supported by a mouse xenograft model, in which BP repressed telomerase and inhibited tumor proliferation, resulting in tumor senescence. Overexpression of hTERT restored telomerase activity in human glioblastoma cells and overcame replicative senescence.


These findings suggest that BP inhibits proliferation and induces senescence in human glioblastomas by downregulating hTERT expression and consequently telomerase activity. This is the first study to describe regulation of telomerase activity by BP in human glioblastomas.


hTERT Expression hTERT mRNA Expression Brain Cancer Cell Butylidenephthalide hTERT Core Promoter 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Robert A. Weinberg for the pCI neo-hEST2-HA (Addgene plasmid 1782) construct. Grant: This work was supported by grants from the National Science Council of the Republic of China (96-2320-B-039-044-MY3), from the Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan, and from China Medical University and Hospital, Taichung, Taiwan (DMR-99-076). This study is supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH99-TD-B-111-004).

Supplementary material

10434_2011_1644_MOESM1_ESM.eps (1.4 mb)
Electronic Supplementary Material: Supplementary Figures 1 and 2 are available for this article at doi: 10.1245/s10434-011-1644-0 and are accessible for authorized users. (EPS 1430 kb)
10434_2011_1644_MOESM2_ESM.eps (697 kb)
Supplementary material 2 (EPS 697 kb)


  1. 1.
    Sathornsumetee S, Rich JN. Designer therapies for glioblastoma multiforme. Ann N Y Acad Sci. 2008;1142:108–32.PubMedCrossRefGoogle Scholar
  2. 2.
    Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352:997–1003.PubMedCrossRefGoogle Scholar
  3. 3.
    Morrison SJ, Prowse KR, Ho P, Weissman IL. Telomerase activity in hematopoietic cells is associated with self-renewal potential. Immunity. 1996;5:207–16.PubMedCrossRefGoogle Scholar
  4. 4.
    Lee HW, Blasco MA, Gottlieb GJ, Horner JW II, Greider CW, DePinho RA. Essential role of mouse telomerase in highly proliferative organs. Nature. 1998;392:569–74.PubMedCrossRefGoogle Scholar
  5. 5.
    Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, et al. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA. 1992;89:10114–8.PubMedCrossRefGoogle Scholar
  6. 6.
    von Zglinicki T, Saretzki G, Docke W, Lotze C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res. 1995;220:186–93.CrossRefGoogle Scholar
  7. 7.
    Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts. Cancer Res. 1998;58:4168–72.PubMedGoogle Scholar
  8. 8.
    Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, et al. The RNA component of human telomerase. Science. 1995;269:1236–41.PubMedCrossRefGoogle Scholar
  9. 9.
    Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 1997;90:785–95.PubMedCrossRefGoogle Scholar
  10. 10.
    Counter CM, Meyerson M, Eaton EN, Ellisen LW, Caddle SD, Haber DA, et al. Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene. 1998;16:1217–22.PubMedCrossRefGoogle Scholar
  11. 11.
    Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet. 1996;18:173–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Bryan TM, Englezou A, Dalla-Pozza L, Dunham MA, Reddel RR. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat Med. 1997;3:1271–4.PubMedCrossRefGoogle Scholar
  13. 13.
    Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer. 1997;33:787–91.PubMedCrossRefGoogle Scholar
  14. 14.
    Le S, Zhu JJ, Anthony DC, Greider CW, Black PM. Telomerase activity in human gliomas. Neurosurgery. 1998;42:1120–4; discussion 1124–5.Google Scholar
  15. 15.
    Ozawa T, Gryaznov SM, Hu LJ, Pongracz K, Santos RA, Bollen AW, et al. Antitumor effects of specific telomerase inhibitor GRN163 in human glioblastoma xenografts. Neuro Oncol. 2004;6:218–26.PubMedCrossRefGoogle Scholar
  16. 16.
    Cheng YL, Chang WL, Lee SC, Liu YG, Chen CJ, Lin SZ, et al. Acetone extract of Bupleurum scorzonerifolium inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing telomerase activity. Life Sci. 2003;73:2383–94.PubMedCrossRefGoogle Scholar
  17. 17.
    Yim TK, Wu WK, Pak WF, Mak DH, Liang SM, Ko KM. Myocardial protection against ischaemia-reperfusion injury by a Polygonum multiflorum extract supplemented ‘Dang-Gui decoction for enriching blood’, a compound formulation, ex vivo. Phytother Res. 2000;14:195–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Ye YN, Koo MW, Li Y, Matsui H, Cho CH. Angelica sinensis modulates migration and proliferation of gastric epithelial cells. Life Sci. 2001;68:961–8.PubMedCrossRefGoogle Scholar
  19. 19.
    Ye YN, Liu ES, Li Y, So HL, Cho CC, Sheng HP, et al. Protective effect of polysaccharides-enriched fraction from Angelica sinensis on hepatic injury. Life Sci. 2001;69:637–46.PubMedCrossRefGoogle Scholar
  20. 20.
    Ye YN, Liu ES, Shin VY, Koo MW, Li Y, Wei EQ, et al. A mechanistic study of proliferation induced by Angelica sinensis in a normal gastric epithelial cell line. Biochem Pharmacol. 2001;61:1439–48.PubMedCrossRefGoogle Scholar
  21. 21.
    Ye YN, So HL, Liu ES, Shin VY, Cho CH. Effect of polysaccharides from Angelica sinensis on gastric ulcer healing. Life Sci. 2003;72:925–32.PubMedCrossRefGoogle Scholar
  22. 22.
    Abebe W. Herbal medication: potential for adverse interactions with analgesic drugs. J Clin Pharm Ther. 2002;27:391–401.PubMedCrossRefGoogle Scholar
  23. 23.
    Tsai NM, Lin SZ, Lee CC, Chen SP, Su HC, Chang WL, et al. The antitumor effects of Angelica sinensis on malignant brain tumors in vitro and in vivo. Clin Cancer Res. 2005;11:3475–84.PubMedCrossRefGoogle Scholar
  24. 24.
    Tsai NM, Chen YL, Lee CC, Lin PC, Cheng YL, Chang WL, et al. The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem. 2006;99:1251–62.PubMedCrossRefGoogle Scholar
  25. 25.
    Chen YL, Jian MH, Lin CC, Kang JC, Chen SP, Lin PC, et al. The induction of orphan nuclear receptor Nur77 expression by n-butylenephthalide as pharmaceuticals on hepatocellular carcinoma (HCC) cells therapy. Mol Pharmacol. 2008;74:1046–58.PubMedCrossRefGoogle Scholar
  26. 26.
    Lin PC, Chen YL, Chiu SC, Yu YL, Chen SP, Chien MH, et al. Orphan nuclear receptor, Nurr-77 was a possible target gene of butylidenephthalide chemotherapy on glioblastoma multiform brain tumor. J Neurochem. 2008;106:1017–26.PubMedCrossRefGoogle Scholar
  27. 27.
    Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994;266:2011–5.PubMedCrossRefGoogle Scholar
  28. 28.
    Kurz DJ, Decary S, Hong Y, Erusalimsky JD. Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci. 2000;113:3613–22.PubMedGoogle Scholar
  29. 29.
    Janik P, Briand P, Hartmann NR. The effect of estrone-progesterone treatment on cell proliferation kinetics of hormone-dependent GR mouse mammary tumors. Cancer Res. 1975;35:3698–704.PubMedGoogle Scholar
  30. 30.
    Nakano K, Watney E, McDougall JK. Telomerase activity and expression of telomerase RNA component and telomerase catalytic subunit gene in cervical cancer. Am J Pathol. 1998;153:857–64.PubMedCrossRefGoogle Scholar
  31. 31.
    Boldrini L, Faviana P, Gisfredi S, Zucconi Y, Di Quirico D, Donati V, et al. Evaluation of telomerase in the development and progression of colon cancer. Int J Mol Med. 2002;10:589–92.PubMedGoogle Scholar
  32. 32.
    Park TW, Riethdorf S, Riethdorf L, Löning T, Jänicke F. Differential telomerase activity, expression of the telomerase catalytic sub-unit and telomerase-RNA in ovarian tumors. Int J Cancer. 1999;84:426–31.PubMedCrossRefGoogle Scholar
  33. 33.
    Kanaya T, Kyo S, Takakura M, Ito H, Namiki M, Inoue M. hTERT is a critical determinant of telomerase activity in renal-cell carcinoma. Int J Cancer. 1998;78:539–43.PubMedCrossRefGoogle Scholar
  34. 34.
    Kelland LR. Telomerase inhibitors: targeting the vulnerable end of cancer? Anticancer Drugs. 2000;11:503–13.PubMedCrossRefGoogle Scholar
  35. 35.
    Suenaga M, Soda H, Oka M, Yamaguchi A, Nakatomi K, Shiozawa K, et al. Histone deacetylase inhibitors suppress telomerase reverse transcriptase mRNA expression in prostate cancer cells. Int J Cancer. 2002;97:621–5.PubMedCrossRefGoogle Scholar
  36. 36.
    Djojosubroto MW, Chin AC, Go N, Schaetzlein S, Manns MP, Gryaznov S, et al. Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma. Hepatology. 2005;42:1127–36.PubMedCrossRefGoogle Scholar
  37. 37.
    Hochreiter AE, Xiao H, Goldblatt EM, Gryaznov SM, Miller KD, Badve S, et al. Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer. Clin Cancer Res. 2006;12:3184–92.PubMedCrossRefGoogle Scholar
  38. 38.
    Cookson JC, Dai F, Smith V, Heald RA, Laughton CA, Stevens MF, et al. Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in vitro: activity in human tumor cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents. Mol Pharmacol. 2005;68:1551–8.PubMedGoogle Scholar
  39. 39.
    Gowan SM, Harrison JR, Patterson L, Valenti M, Read MA, Neidle S, et al. A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity. Mol Pharmacol. 2002;61:1154–62.PubMedCrossRefGoogle Scholar
  40. 40.
    Flores I, Benetti R, Blasco MA. Telomerase regulation and stem cell behaviour. Curr Opin Cell Biol. 2006;18:254–60.PubMedCrossRefGoogle Scholar
  41. 41.
    Wu KJ, Grandori C, Amacker M, Simon-Vermot N, Polack A, Lingner J, et al. Direct activation of TERT transcription by c-MYC. Nat Genet. 1999;21:220–4.PubMedCrossRefGoogle Scholar
  42. 42.
    Kyo S, Takakura M, Taira T, Kanaya T, Itoh H, Yutsudo M, et al. Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT). Nucleic Acids Res. 2000;28:669–77.PubMedCrossRefGoogle Scholar
  43. 43.
    Counter CM, Hahn WC, Wei W, Caddle SD, Beijersbergen RL, Lansdorp PM, et al. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc Natl Acad Sci U S A. 1998;95:14723–8.PubMedCrossRefGoogle Scholar
  44. 44.
    Jagadeesh S, Kyo S, Banerjee PP. Genistein represses telomerase activity via both transcriptional and posttranslational mechanisms in human prostate cancer cells. Cancer Res. 2006;66:2107–15.PubMedCrossRefGoogle Scholar
  45. 45.
    Schmitt CA. Cellular senescence and cancer treatment. Biochim Biophys Acta. 2007;1775:5–20.PubMedGoogle Scholar
  46. 46.
    Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol Cell. 2004;14:501–13.PubMedCrossRefGoogle Scholar
  47. 47.
    Keith WN, Thomson CM, Howcroft J, Maitland NJ, Shay JW. Seeding drug discovery: integrating telomerase cancer biology and cellular senescence to uncover new therapeutic opportunities in targeting cancer stem cells. Drug Discov Today. 2007;12:611–21.PubMedCrossRefGoogle Scholar
  48. 48.
    Chang BD, Broude EV, Dokmanovic M, Zhu H, Ruth A, Xuan Y, et al. A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res. 1999;59:3761–7.PubMedGoogle Scholar
  49. 49.
    Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92:9363–7.PubMedCrossRefGoogle Scholar
  50. 50.
    Coates SS, Lehnert BE, Sharma S, Kindell SM, Gary RK. Beryllium induces premature senescence in human fibroblasts. J Pharmacol Exp Ther. 2007;322:70–9.PubMedCrossRefGoogle Scholar
  51. 51.
    Liu JP. Telomerase: not just black and white, but shades of gray. Mol Cell Biol Res Commun. 2000;3:129–35.PubMedCrossRefGoogle Scholar
  52. 52.
    Harley CB. Telomerase and cancer therapeutics. Nat Rev Cancer. 2008;8:167–79.PubMedCrossRefGoogle Scholar
  53. 53.
    Keith WN, Bilsland A, Evans TR, Glasspool RM. Telomerase-directed molecular therapeutics. Expert Rev Mol Med. 2002;4:1–25.PubMedCrossRefGoogle Scholar

Copyright information

© Society of Surgical Oncology 2011

Authors and Affiliations

  • Po-Cheng Lin
    • 1
  • Shinn-Zong Lin
    • 2
    • 3
    • 4
  • Yi-Lin Chen
    • 5
  • Jeng-Shou Chang
    • 6
  • Li-Ing Ho
    • 7
  • Po-Yen Liu
    • 8
  • Li-Fu Chang
    • 1
  • Yeu-Chern Harn
    • 9
    • 10
  • Shee-Ping Chen
    • 11
  • Li-Yi Sun
    • 12
  • Pi-Chun Huang
    • 13
  • Jung-Ting Chein
    • 14
  • Chang-Hai Tsai
    • 15
    • 16
  • Chii-Wen Chou
    • 2
  • Horng-Jyh Harn
    • 17
    • 18
  • Tzyy-Wen Chiou
    • 1
  1. 1.Department of Life Science and Graduate Institute of BiotechnologyNational Dong Hwa UniversityHualienTaiwan, Republic of China
  2. 2.Center for Neuropsychiatry and Department of NeurosurgeryChina Medical University HospitalTaichungTaiwan, Republic of China
  3. 3.Graduate Institute of ImmunologyChina Medical UniversityTaichungTaiwan, Republic of China
  4. 4.Department of NeurosurgeryChina Medical University Beigan HospitalYunlinTaiwan, Republic of China
  5. 5.Graduate Institute of BiotechnologyNational Ilan UniversityIlanTaiwan, Republic of China
  6. 6.Graduate Institute of Basic Medical ScienceChina Medical UniversityTaichungTaiwan, Republic of China
  7. 7.Department of Respiratory CareVeterans General Hospital-TaipeiTaipeiTaiwan, Republic of China
  8. 8.Graduate Institute of Chinese MedicineChina Medical UniversityTaichungTaiwan, Republic of China
  9. 9.Graduate Institute of Networking and MultimediaNational Taiwain UniversityTaipeiTaiwan, Republic of China
  10. 10.The Metabolomics GroupNational Taiwan UniversityTaipeiTaiwan, Republic of China
  11. 11.Buddhist Tzu Chi Stem Cells CenterBuddhist Tzu Chi Medical CenterHualienTaiwan, Republic of China
  12. 12.Department of Biological Science and TechnologyNational Chiao Tung UniversityHsinchuTaiwan, Republic of China
  13. 13.Department of Stem Cell Applied TechnologyGwo Xi Stem Cell Applied TechnologyHsinchuTaiwan, Republic of China
  14. 14.Departmeant of Medical TechnologyChina Medical UniversityTaichungTaiwan, Republic of China
  15. 15.Department of PediatricsChina Medical University HospitalTaichungTaiwan, Republic of China
  16. 16.Department of Healthcare AdministrationAsia UniversityTaichungTaiwan, Republic of China
  17. 17.Department of PathologyChina Medical University HospitalTaichungTaiwan, Republic of China
  18. 18.Department of MedicineChina Medical UniversityTaichungTaiwan, Republic of China

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