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Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide.

ABSTRACT: We previously found that BRCA1-BRCA2-containing complex subunit 3 (BRCC3) was highly expressed in tumorigenic rat glioma cells. However, the functional role of BRCC3 in human glioma cells remains to be characterized. This study indicated that the upregulation of BRCC3 expression was induced in two human malignant glioblastoma U251 and A172 cell lines following exposure to the alkylating agent, temozolomide (TMZ). Homologous recombination (HR)-dependent DNA repair-associated genes (i.e. BRCA1, BRCA2, RAD51 and FANCD2) were also increased in U251 and A172 cells after treatment with TMZ. BRCC3 gene knockdown through lentivirus-mediated gene knockdown approach not only significantly reduced the clonogenic and migratory abilities of U251 and A172 cells, but also enhanced their sensitization to TMZ. The increase in phosphorylated H2AX foci (?H2AX) formation, an indicator of DNA damage, persisted in TMZ-treated glioma cells with stable knockdown BRCC3 expression, suggesting that BRCC3 gene deficiency is associated with DNA repair impairment. In summary, we demonstrate that by inducing DNA repair, BRCC3 renders glioma cells resistant to TMZ. The findings point to BRCC3 as a potential target for treatment of alkylating drug-resistant glioma.

SUBMITTER: Chai KM

PROVIDER: S-EPMC4279418 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide.

Chai Kit Man KM Wang Chih-Yen CY Liaw Hung-Jiun HJ Fang Kuan-Min KM Yang Chung-Shi CS Tzeng Shun-Fen SF

Oncotarget 20141101 21

We previously found that BRCA1-BRCA2-containing complex subunit 3 (BRCC3) was highly expressed in tumorigenic rat glioma cells. However, the functional role of BRCC3 in human glioma cells remains to be characterized. This study indicated that the upregulation of BRCC3 expression was induced in two human malignant glioblastoma U251 and A172 cell lines following exposure to the alkylating agent, temozolomide (TMZ). Homologous recombination (HR)-dependent DNA repair-associated genes (i.e. BRCA1, BR ...[more]

PMID: 25337721

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Project description:ObjectiveRho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells.MethodsGlycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration.ResultsOur results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone.ConclusionsIn conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.

Project description:Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3-7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.

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Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide.

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Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide.

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