Abstract
Temozolomide (TMZ) is a novel cytotoxic alkylating agent for chemotherapy of malignant gliomas. However, intrinsic or acquired resistance to TMZ often defines poor efficacy of chemotherapy in malignant gliomas. A growing number of studies indicate that expression of O 6-methylguanine-DNA methyltransferase (MGMT) is one of the principal mechanisms responsible for this chemoresistance. In the present study, we evaluated the relationship between expression of MGMT and resistance to TMZ. We generated a TMZ-resistant cell line, U251/TR, by stepwise (8 months) exposure of parental U251 cells to TMZ. The resistance to TMZ was quantified using SRB assay. MGMT expression was evaluated at mRNA (RT-PCR) and protein (Western blot) levels. U251/TR cells showed increased (~ sevenfold) resistance to TMZ. The MGMT expression (both mRNA and protein) was significantly (P < 0.01) increased in U251/TR cells compared with parental U251 cells. Further, MGMT expression fluctuated during exposure of U251/TR cells to TMZ. The resistance of U251/TR cells to TMZ could be overcome by application of elevated doses of TMZ when MGMT expression was at the lowest level. In conclusion, our results demonstrate that the primary mechanism responsible for resistance of U251/TR cells to TMZ is associated with increased expression of MGMT. Resistance of malignant gliomas to TMZ can be overcome by synchronizing metronomic TMZ regimen with MGMT expression.
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Ohgaki, H., & Kleihues, P. (2005). Epidemiology and etiology of gliomas. Acta Neuropathologica, 109, 93–108. doi:10.1007/s00401-005-0991-y.
Palanichamy, K., Erkkinen, M., & Chakravarti, A. (2006). Predictive and prognostic markers in human glioblastomas. Current Treatment Options in Oncology, 7, 490–504.
Grauer, O. M., Wesseling, P., & Adema, G. J. (2009). Immunotherapy of diffuse gliomas: Biological background, current status and future developments. Brain Pathology, 19, 674–693. doi:10.1111/j.1750-3639.2009.00315.x.
Gao, S., Yang, X. J., Zhang, W. G., et al. (2009). Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro. Chinese Medical Journal (English Edition), 122, 1260–1266.
Ma, J., Murphy, M., O’Dwyer, P. J., et al. (2002). Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance. Biochemical Pharmacology, 63, 1219–1228.
Middleton, M. R., Grob, J. J., Aaronson, N., et al. (2000). Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. Journal of Clinical Oncology, 18, 158–166.
Hammond, L. A., Eckardt, J. R., Baker, S. D., et al. (1999). Phase I and pharmacokinetic study of temozolomide on a daily-for-5-days schedule in patients with advanced solid malignancies. Journal of Clinical Oncology, 17, 2604–2613.
Hegi, M. E., Diserens, A. C., Godard, S., et al. (2004). Clinical trial substantiates the predictive value of O-6-methylguanine-DNA methyltransferase promoter methylation in glioblastoma patients treated with temozolomide. Clinical Cancer Research, 10, 1871–1874.
Lee, S. M., Lee, E. J., Ko, Y. H., et al. (2009). Prognostic significance of O6-methylguanine DNA methyltransferase and p57 methylation in patients with diffuse large B-cell lymphomas. APMIS, 117, 87–94. doi:10.1111/j.1600-0463.2008.00017.x.
Glas, M., Happold, C., Rieger, J., et al. (2009). Long-term survival of patients with glioblastoma treated with radiotherapy and lomustine plus temozolomide. Journal of Clinical Oncology, 27, 1257–1261. doi:10.1200/JCO.2008.19.2195.
Criniere, E., Kaloshi, G., Laigle-Donadey, F., et al. (2007). MGMT prognostic impact on glioblastoma is dependent on therapeutic modalities. Journal of Neuro-oncology, 83, 173–179. doi:10.1007/s11060-006-9320-0.
Hegi, M. E., Diserens, A. C., Gorlia, T., et al. (2005). MGMT gene silencing and benefit from temozolomide in glioblastoma. New England Journal of Medicine, 352, 997–1003. doi:10.1056/NEJMoa043331.
Idbaih, A., Omuro, A., Ducray, F., et al. (2007). Molecular genetic markers as predictors of response to chemotherapy in gliomas. Current Opinion in Oncology, 19, 606–611. doi:10.1097/CCO.0b013e3282f075f3.
Ishii, D., Natsume, A., Wakabayashi, T., et al. (2007). Efficacy of temozolomide is correlated with 1p loss and methylation of the deoxyribonucleic acid repair gene MGMT in malignant gliomas. Neurologia Medico-Chirurgica (Tokyo), 47, 341–349.
Martinez, R., Schackert, G., Yaya-Tur, R., et al. (2007). Frequent hypermethylation of the DNA repair gene MGMT in long-term survivors of glioblastoma multiforme. Journal of Neuro-oncology, 83, 91–93. doi:10.1007/s11060-006-9292-0.
Paz, M. F., Yaya-Tur, R., Rojas-Marcos, I., et al. (2004). CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas. Clinical Cancer Research, 10, 4933–4938. doi:10.1158/1078-0432.CCR-04-0392.
Chinot, O. L., Barrie, M., Fuentes, S., et al. (2007). Correlation between O6-methylguanine-DNA methyltransferase and survival in inoperable newly diagnosed glioblastoma patients treated with neoadjuvant temozolomide. Journal of Clinical Oncology, 25, 1470–1475. doi:10.1200/JCO.2006.07.4807.
Capper, D., Mittelbronn, M., Meyermann, R., et al. (2008). Pitfalls in the assessment of MGMT expression and in its correlation with survival in diffuse astrocytomas: Proposal of a feasible immunohistochemical approach. Acta Neuropathologica, 115, 249–259. doi:10.1007/s00401-007-0310-x.
Bobola, M. S., Silber, J. R., Ellenbogen, R. G., et al. (2005). O6-methylguanine-DNA methyltransferase, O6-benzylguanine, and resistance to clinical alkylators in pediatric primary brain tumor cell lines. Clinical Cancer Research, 11, 2747–2755. doi:10.1158/1078-0432.CCR-04-2045.
Hermisson, M., Klumpp, A., Wick, W., et al. (2006). O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells. Journal of Neurochemistry, 96, 766–776. doi:10.1111/j.1471-4159.2005.03583.x.
Kanzawa, T., Germano, I. M., Komata, T., et al. (2004). Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death and Differentiation, 11, 448–457. doi:10.1038/sj.cdd.4401359.
Skehan, P., Storeng, R., Scudiero, D., et al. (1990). New colorimetric cytotoxicity assay for anticancer-drug screening. Journal of the National Cancer Institute, 82, 1107–1112.
Rodriguez, F. J., Thibodeau, S. N., Jenkins, R. B., et al. (2008). MGMT immunohistochemical expression and promoter methylation in human glioblastoma. Applied Immunohistochemistry & Molecular Morphology, 16, 59–65. doi:10.1097/PAI.0b013e31802fac2f.
Hampson, R., Humbert, O., Macpherson, P., et al. (1997). Mismatch repair defects and O6-methylguanine-DNA methyltransferase expression in acquired resistance to methylating agents in human cells. Journal of Biological Chemistry, 272, 28596–28606.
Balana, C., Carrato, J., Ramirez, J., et al. (2008). Concordance and clinical value of the MGMT promoter methylation pattern in tissue with paired serum and MGMT protein expression in a series of glioblastoma (GB) patients. Journal of Clinical Oncology, 26, 2037.
Paus, C., Murat, A., Stupp, R., et al. (2007). Role of MGMT and clinical applications in brain tumours. Bull Cancer, 94, 769–773.
Acknowledgments
The authors gratefully acknowledge the financial support by the National Natural Science Foundation of China (grants no. 30571904 and 30772228).
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Qiang Pan and Xue-jun Yang are contributed equally.
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Pan, Q., Yang, Xj., Wang, Hm. et al. Chemoresistance to Temozolomide in Human Glioma Cell Line U251 is Associated with Increased Activity of O 6-methylguanine-DNA Methyltransferase and Can be Overcome by Metronomic Temozolomide Regimen. Cell Biochem Biophys 62, 185–191 (2012). https://doi.org/10.1007/s12013-011-9280-7
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DOI: https://doi.org/10.1007/s12013-011-9280-7