Acquired temozolomide resistance in human malignant gliomas: a proteomics approach and the role of prolyl 4-hydroxylase, beta polypeptide

Abstract

Temozolomide (TMZ) provides significant benefits in the treatment of malignant glioma, but many patients continue to suffer from progressive or recurrent disease because of the intrinsic or acquired resistance of their tumors to TMZ. In this study, we employed proteomic profiling to study changes in global protein expression in TMZ-resistant malignant glioma cells. We also investigated the potential role of one of the protein candidates, prolyl 4-hydroxylase, beta polypeptide (P4HB), that may be involved in the development of acquired TMZ resistance. Human malignant glioma cell lines (D54 and U87) with acquired TMZ resistance were first developed, and their biological behaviors were characterized before protein profiling analysis using 2-dimensional gel electrophoresis. Potential protein spot candidates were identified by MALDI-TOF/TOF. Transient transfection with small interfering RNAs was used to knock down potential candidates to study their biological roles. Our results revealed 15 protein spots that were highly upregulated or downregulated proteins in TMZ-resistant glioma cells. Among these, the protein P4HB was found to be consistently dysregulated in TMZ-resistant D54 sublines when compared to control sublines. Dysregulated expression of P4HB was also confirmed in U87 cells. Transient knockdown of P4HB expression was found to resensitize TMZ-resistant cells to subsequent treatment with TMZ. P4HB is known to play an important role in hypoxia-induced cell death. To our knowledge, this study is the first to describe the roles of P4HB in the regulation of TMZ-induced apoptosis in malignant glioma cells and in the development of acquired TMZ resistance. P4HB is a potential candidate for future investigations of TMZ-induced cytotoxicity and novel therapeutic strategies for the treatment of TMZ-resistant malignant glioma.