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Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma.

ABSTRACT: The standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemo/radiotherapy. A major limitation on patient improvement is the high resistance of tumors to drug treatment, likely responsible for their subsequent recurrence and rapid progression. Therefore, alternatives to the standard therapy are necessary. The aim of the present study was to evaluate whether mifepristone, an antihormonal agent, has a synergistic effect with temozolomide (used in standard therapy for gliomas). Whereas the mechanism of temozolomide involves damage to tumor DNA leading to apoptosis, tumor resistance is associated with DNA damage repair through the O?-methylguanine-DNA-methyltransferase (MGMT) enzyme. Temozolomide/mifepristone treatment, herein examined in Wistar rats after orthotopically implanting C6 glioma cells, markedly reduced proliferation. This was evidenced by a decreased level of the following parameters: a proliferation marker (Ki-67), a tumor growth marker (18F-fluorothymidine uptake, determined by PET/CT images), and the MGMT enzyme. Increased apoptosis was detected by the relative expression of related proteins, (e.g. Bcl-2 (B-cell lymphoma 2), Bax (bcl-2-like protein 4) and caspase-3). Thus, greater apoptosis of tumor cells caused by their diminished capacity to repair DNA probably contributed significantly to the enhanced activity of temozolomide. The results suggest that mifepristone could possibly act as a chemo-sensitizing agent for temozolomide during chemotherapy for GBM.

SUBMITTER: Llaguno-Munive M

PROVIDER: S-EPMC6356343 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma.

Llaguno-Munive Monserrat M Romero-Piña Mario M Serrano-Bello Janeth J Medina Luis A LA Uribe-Uribe Norma N Salazar Ana Maria AM Rodríguez-Dorantes Mauricio M Garcia-Lopez Patricia P

Cancers 20181222 1

The standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemo/radiotherapy. A major limitation on patient improvement is the high resistance of tumors to drug treatment, likely responsible for their subsequent recurrence and rapid progression. Therefore, alternatives to the standard therapy are necessary. The aim of the present study was to evaluate whether mifepristone, an antihormonal agent, has a synergistic effect with temozolomide (used in standard therapy for glioma ...[more]

PMID: 30583528

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Project description:Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults and is universally fatal. The DNA alkylating agent temozolomide is part of the standard-of-care for GBM. However, these tumors eventually develop therapy-driven resistance and inevitably recur. While loss of mismatch repair (MMR) and re-expression of MGMT have been shown to underlie chemoresistance in a fraction of GBMs, resistance mechanisms operating in the remaining GBMs are not well understood. To better understand the molecular basis for therapy-driven temozolomide resistance, mice bearing orthotopic GBM xenografts were subjected to protracted temozolomide treatment, and cell lines were generated from the primary (untreated) and recurrent (temozolomide-treated) tumors. As expected, the cells derived from primary tumors were sensitive to temozolomide, whereas the cells from the recurrent tumors were significantly resistant to the drug. Importantly, the acquired resistance to temozolomide in the recurrent lines was not driven by re-expression of MGMT or loss of MMR but was due to accelerated repair of temozolomide-induced DNA double-strand breaks (DSB). Temozolomide induces DNA replication-associated DSBs that are primarily repaired by the homologous recombination (HR) pathway. Augmented HR appears to underpin temozolomide resistance in the recurrent lines, as these cells were cross-resistant to other agents that induced replication-associated DSBs, exhibited faster resolution of damage-induced Rad51 foci, and displayed higher levels of sister chromatid exchanges (SCE). Furthermore, in light of recent studies demonstrating that CDK1 and CDK2 promote HR, it was found that CDK1/2 inhibitors countered the heightened HR in recurrent tumors and sensitized these therapy-resistant tumor cells to temozolomide.ImplicationsAugmented HR repair is a novel mechanism underlying acquired temozolomide resistance in GBM, and this raises the possibility of improving the therapeutic response to temozolomide by targeting HR with small-molecule inhibitors of CDK1/2. Mol Cancer Res; 14(10); 928-40. ©2016 AACR.

Project description:BackgroundGlioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance.MethodsMass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ ± calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and ɣH2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation.ResultsCalcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-κB signaling. TP53 showed increased protein abundance, but no transcriptional regulation.ConclusionTMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via "priming" GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.

Dataset Information

Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma.

Publications

Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma.

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