Top

Published in:

01-11-2007 | Lab. Investigation-Human/Animal Tissue

Valproic acid induces p21 and topoisomerase-II (α/β) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines

Authors: Chandra M. Das, Dolly Aguilera, Hernan Vasquez, Preethi Prasad, Ming Zhang, Johannes E. Wolff, Vidya Gopalakrishnan

Published in: Journal of Neuro-Oncology | Issue 2/2007

Abstract

Object

Etoposide, a topoisomerase-II inhibitor promotes DNA damage and apoptosis of cancer cells. In this study, we have examined the ability of the histone deacetylase inhibitor, valproic acid (VPA) to modulate gene expression and sensitize glioblastoma cell lines to the cytotoxic effects of etoposide in vitro.

Methods

The effect of VPA and etoposide alone or a combination of the two drugs on the growth of three different glioblastoma cell lines (U87, LN18, and U251) were measured by MTT assays. Drug treated cells were analyzed for their cell cycle profile, gene expression, differentiation status, and induction of apoptosis by flow-cytometry, western blotting, immunofluorescence assays, and caspase activity measurements.

Results

We observed that while VPA and etoposide independently inhibited the growth of U87, U251, and LN18 cells, exposure of tumor cells to both drugs significantly enhanced the cytotoxicity of etoposide in all cell lines. VPA promoted a G₁ accumulation of U87, while an increase in the G₂/M population of U251 and LN18 cells was observed upon exposure to the drug. Treatment with etoposide resulted in a G₂/M arrest of U87, U251, and LN18 cells, whereas, exposure to both drugs increased the fraction of cells with a G2/M and sub-G1 DNA content. Further, VPA and not etoposide, promoted acetylation of histone H4 and induced the expression of the cyclin-dependent kinase inhibitor (CDKI), p21/WAF1. VPA also up-regulated the expression of the α and β isoforms of topoisomerase-II, as well as the glial differentiation marker, glial fibrillary acidic protein. Finally, a significant increase in caspase-3 activity and apoptosis was observed in the presence of both VPA and etoposide compared to either agent alone.

Conclusion

Our study demonstrates that VPA sensitizes U87, U251, and LN18 cells to the cytotoxic effects of etoposide in vitro by inducing differentiation and up-regulating the expression of p21/WAF1 and both isoforms of topoisomerase-II.

Hanada R, Inaba T, Yaginuma A, Hayashi Y, Yamamoto K, Nishimoto H, Tsukiyama T (1989) [Effect of high-dose cyclophosphamide plus high-dose etoposide in malignant brain tumors of children followed by autologous bone marrow rescue]. Gan To Kagaku Ryoho 16:263–268PubMed

Leo E, Schlegel PG, Lindemann A (1997) Chemotherapeutic induction of long-term remission in metastatic medulloblastoma. J Neurooncol 32:149–154PubMedCrossRef

Kageji T, Nagahiro S, Horiguchi H, Watanabe T, Suzuya H, Okamoto Y, Kuroda Y (2003) Successful high-dose chemotherapy for widespread neuroaxis dissemination of an optico-hypothalamic juvenile pilocytic astrocytoma in an infant: a case report. J Neurooncol 62:281–287PubMedCrossRef

Postmus PE, Mulder NH, Sleijfer DT, Meinesz AF, Vriesendorp R, de Vries EG (1984) High-dose etoposide for refractory malignancies: a phase I study. Cancer Treat Rep 68:1471–1474PubMed

Parney IF, Chang SM (2003) Current chemotherapy for glioblastoma. Cancer J 9:149–156PubMedCrossRef

Yin D, Tamaki N, Parent AD, Zhang JH (2005) Insulin-like growth factor-I decreased etoposide-induced apoptosis in glioma cells by increasing bcl-2 expression and decreasing CPP32 activity. Neurol Res 27:27–35PubMedCrossRef

El-Deiry WS (2003) The role of p53 in chemosensitivity and radiosensitivity. Oncogene 22:7486–7495PubMedCrossRef

Wu GS, El-Diery WS (1996) p53 and chemosensitivity. Nat Med 2:255–256PubMedCrossRef

Valkov NI, Sullivan DM (2003) Tumor p53 status and response to topoisomerase II inhibitors. Drug Resist Updat 6:27–39PubMedCrossRef

10.

Kim EH, Kim HS, Kim SU, Noh EJ, Lee JS, Choi KS (2005) Sodium butyrate sensitizes human glioma cells to TRAIL-mediated apoptosis through inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP. Oncogene 24:6877–6889PubMedCrossRef

11.

Kim TY, Zhong S, Fields CR, Kim JH, Robertson KD (2006) Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma. Cancer Res 66:7490–7501PubMedCrossRef

12.

Yin D, Ong JM, Hu J, Desmond JC, Kawamata N, Konda BM, Black KL, Koeffler HP (2007) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor: effects on gene expression and growth of glioma cells in vitro and in vivo. Clin Cancer Res 13:1045–1052PubMedCrossRef

13.

Minucci S, Pelicci PG (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6:38–51PubMedCrossRef

14.

Eyupoglu IY, Hahnen E, Trankle C, Savaskan NE, Siebzehnrubl FA, Buslei R, Lemke D, Wick W, Fahlbusch R, Blumcke I (2006) Experimental therapy of malignant gliomas using the inhibitor of histone deacetylase MS-275. Mol Cancer Ther 5:1248–1255PubMedCrossRef

15.

Wetzel M, Premkumar DR, Arnold B, Pollack IF (2005) Effect of trichostatin A, a histone deacetylase inhibitor, on glioma proliferation in vitro by inducing cell cycle arrest and apoptosis. J Neurosurg 103:549–556PubMed

16.

Entin-Meer M, Rephaeli A, Yang X, Nudelman A, VandenBerg SR, Haas-Kogan DA (2005) Butyric acid prodrugs are histone deacetylase inhibitors that show antineoplastic activity and radiosensitizing capacity in the treatment of malignant gliomas. Mol Cancer Ther 4:1952–1961PubMedCrossRef

17.

Chapman A, Keane PE, Meldrum BS, Simiand J, Vernieres JC (1982) Mechanism of anticonvulsant action of valproate. Prog Neurobiol 19:315–359PubMedCrossRef

18.

Davis R, Peters DH, McTavish D (1994) Valproic acid. A reappraisal of its pharmacological properties and clinical efficacy in epilepsy. Drugs 47:332–372PubMed

19.

Driever PH, Knupfer MM, Cinatl J, Wolff JE (1999) Valproic acid for the treatment of pediatric malignant glioma. Klin Padiatr 211:323–328PubMedCrossRef

20.

Witt O, Schweigerer L, Driever PH, Wolff J, Pekrun A (2004) Valproic acid treatment of glioblastoma multiforme in a child. Pediatr Blood Cancer 43:181PubMedCrossRef

21.

Hanson JA, Bentley DP, Bean EA, Nute SR, Moore JL (1991) In vitro chemosensitivity testing in chronic lymphocytic leukaemia patients. Leuk Res 15:565–569PubMedCrossRef

22.

Su X, Gopalakrishnan V, Stearns D, Aldape K, Lang FF, Fuller G, Snyder E, Eberhart CG, Majumder S (2006) Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation. Mol Cell Biol 26:1666–1678PubMedCrossRef

23.

Kagan VE, Gleiss B, Tyurina YY, Tyurin VA, Elenstrom-Magnusson C, Liu SX, Serinkan FB, Arroyo A, Chandra J, Orrenius S, Fadeel B (2002) A role for oxidative stress in apoptosis: oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fas-mediated apoptosis. J Immunol 169:487–499PubMed

24.

Yang H, Hoshino K, Sanchez-Gonzalez B, Kantarjian H, Garcia-Manero G (2005) Antileukemia activity of the combination of 5-aza-2′-deoxycytidine with valproic acid. Leuk Res 29:739–748PubMedCrossRef

25.

Stockhausen MT, Sjolund J, Manetopoulos C, Axelson H (2005) Effects of the histone deacetylase inhibitor valproic acid on Notch signalling in human neuroblastoma cells. Br J Cancer 92:751–759PubMedCrossRef

26.

Li XN, Shu Q, Su JM, Perlaky L, Blaney SM, Lau CC (2005) Valproic acid induces growth arrest, apoptosis, and senescence in medulloblastomas by increasing histone hyperacetylation and regulating expression of p21Cip1, CDK4, and CMYC. Mol Cancer Ther 4:1912–1922PubMedCrossRef

27.

Yoo EJ, Chung JJ, Choe SS, Kim KH, Kim JB (2006) Down-regulation of histone deacetylases stimulates adipocyte differentiation. J Biol Chem 281:6608–6615PubMedCrossRef

28.

Wolff JE, Finlay JL (2004) High-dose chemotherapy in childhood brain tumors. Onkologie 27:239–245PubMedCrossRef

29.

Hake SB, Xiao A, Allis CD (2004) Linking the epigenetic ‘language’ of covalent histone modifications to cancer. Br J Cancer 90:761–769PubMedCrossRef

30.

Johnstone RW (2002) Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 1:287–299PubMedCrossRef

31.

Dokmanovic M, Marks PA (2005) Prospects: histone deacetylase inhibitors. J Cell Biochem 96:293–304PubMedCrossRef

32.

Takai N, Kawamata N, Gui D, Said JW, Miyakawa I, Koeffler HP (2004) Human ovarian carcinoma cells: histone deacetylase inhibitors exhibit antiproliferative activity and potently induce apoptosis. Cancer 101:2760–2770PubMedCrossRef

33.

Peart MJ, Smyth GK, van Laar RK, Bowtell DD, Richon VM, Marks PA, Holloway AJ, Johnstone RW (2005) Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors. Proc Natl Acad Sci USA 102:3697–3702PubMedCrossRef

34.

Tsai SC, Valkov N, Yang WM, Gump J, Sullivan D, Seto E (2000) Histone deacetylase interacts directly with DNA topoisomerase II. Nat Genet 26:349–353PubMedCrossRef

35.

Kurz EU, Wilson SE, Leader KB, Sampey BP, Allan WP, Yalowich JC, Kroll DJ (2001) The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines. Mol Cancer Ther 1:121–131PubMed

36.

Marchion DC, Bicaku E, Daud AI, Richon V, Sullivan DM, Munster PN (2004) Sequence-specific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. J Cell Biochem 92:223–237PubMedCrossRef

37.

Marin-Husstege M, Muggironi M, Liu A, Casaccia-Bonnefil P (2002) Histone deacetylase activity is necessary for oligodendrocyte lineage progression. J Neurosci 22:10333–10345PubMed

38.

Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH (2004) Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci USA 101:16659–16664PubMedCrossRef

39.

Asklund T, Appelskog IB, Ammerpohl O, Ekstrom TJ, Almqvist PM (2004) Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer 40:1073–1081PubMedCrossRef

40.

Marchion DC, Bicaku E, Turner JG, Daud AI, Sullivan DM, Munster PN (2005) Synergistic interaction between histone deacetylase and topoisomerase II inhibitors is mediated through topoisomerase IIbeta. Clin Cancer Res 11:8467–8475PubMedCrossRef

41.

Tews DS, Fleissner C, Tiziani B, Gaumann AK (2001) Intrinsic expression of drug resistance-associated factors in meningiomas. Appl Immunohistochem Mol Morphol 9:242–249PubMedCrossRef

42.

Takano H, Kohno K, Matsuo K, Matsuda T, Kuwano M (1992) DNA topoisomerase-targeting antitumor agents and drug resistance. Anticancer Drugs 3:323–330PubMedCrossRef

43.

Wang Q, Zambetti GP, Suttle DP (1997) Inhibition of DNA topoisomerase II alpha gene expression by the p53 tumor suppressor. Mol Cell Biol 17:389–397PubMed

44.

Clifford B, Beljin M, Stark GR, Taylor WR (2003) G2 arrest in response to topoisomerase II inhibitors: the role of p53. Cancer Res 63:4074–4081PubMed

45.

Kim MS, Blake M, Baek JH, Kohlhagen G, Pommier Y, Carrier F (2003) Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res 63:7291–7300PubMed

Title: Valproic acid induces p21 and topoisomerase-II (α/β) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines
Authors: Chandra M. Das
Dolly Aguilera
Hernan Vasquez
Preethi Prasad
Ming Zhang
Johannes E. Wolff
Vidya Gopalakrishnan
Publication date: 01-11-2007
Publisher: Springer US
Published in: Journal of Neuro-Oncology / Issue 2/2007
Print ISSN: 0167-594X
Electronic ISSN: 1573-7373
DOI: https://doi.org/10.1007/s11060-007-9402-7

At a glance: The STEP trials

Springer Medicine

Valproic acid induces p21 and topoisomerase-II (α/β) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines

Abstract

Object

Methods

Results

Conclusion

At a glance: The STEP trials

Springer Medicine

Abstract

Object

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 2/2007

Hyperoxia retards growth and induces apoptosis, changes in vascular density and gene expression in transplanted gliomas in nude rats

Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression

A cerebral teratoma in an unusual location

Prolonged and severe myelosuppression in two patients after low-dose temozolomide treatment- case study and review of literature

Myelosuppression in patients benefiting from imatinib with hydroxyurea for recurrent malignant gliomas

Capecitabine Therapy of Central Nervous System Metastases from Breast Cancer