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Temozolomide sensitizes stem-like cells of glioma spheres to TRAIL-induced apoptosis via upregulation of casitas B-lineage lymphoma (c-Cbl) protein

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Tumor Biology

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has potent antitumor effects in glioma cell lines but has shown little clinical benefit for patients. We investigated whether the widely used chemotherapeutic agent temozolomide (TMZ) can sensitize glioma stem-like cells (GSCs) from human glioblastoma multiforme (GBM) to TRAIL-induced apoptosis. GSCs were isolated from GBM, and stem cell properties were confirmed by immunocytochemistry and in vivo tumorigenicity. Primary GSCs (PGCs) were produced by serum treatment of GBM-derived cells. Changes in expression levels of various TRAIL-related signaling factors before and after TRAIL or TRAIL + TMZ treatment were measured by Western blotting. Overexpression vectors and siRNAs were used to investigate mechanism of TRAIL sensitivity. GSCs showed greater resistance to TRAIL-induced apoptosis than PGCs and had lower basal caspase activity. Caspase knockdown in PGCs reduced TRAIL sensitivity. Expression levels of c-Fas-associated death domain-like interleukin 1-converting enzyme-like inhibitory protein long and short isoforms (c-FLIPL and c-FLIPS) were significantly higher in GSCs than PGCs, and siRNA-mediated c-FLIP knockdown in GSCs enhanced TRAIL-induced apoptosis. TMZ enhanced TRAIL-induced apoptosis in GSCs and downregulated c-FLIP expression. Add of TMZ also upregulated the expression of the E3 ubiquitin ligase casitas B-lineage lymphoma (c-Cbl). Moreover, overexpression of c-Cbl alone reduced c-FLIP expression, and c-Cbl knockdown both enhanced c-FLIP expression and reduced the potentiating effect of TMZ on TRAIL-induced apoptosis. The result indicated that TMZ may overcome TRAIL resistance in GSCs by suppressing c-FLIP expression through c-Cbl-mediated ubiquitination and degradation.

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Acknowledgments

This work was supported by the Chinese National Natural Science Foundation (nos. 81101917 and 81172409) (http://www.nsfc.gov.cn/) SL and by the Liaoning Province Natural Science Foundation (no. 2013021045) (http://www.lninfo.gov.cn/).

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Authors and Affiliations

  1. Department of Neurosurgery, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China

    Jing Zhitao, Li Long, Liu Jia, Ban Yunchao & Wu Anhua

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  1. Jing Zhitao
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Correspondence to Wu Anhua.

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S1 Fig

Role of c-Fas-associated death domain-like interleukin 1-converting enzyme-like inhibitory protein long isoform (c-FLIPL) in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in primary glioma cells (PGC-No.1 and PGC-No.2). (PDF 94 kb)

S2 Fig

Immunoblot analysis c-FLIPL and c-FLIPS expression in GSC-No.1 and GSC-No.2 transfected with control or c-Cbl cDNA. (PDF 26 kb)

S3 Fig

A total of 295 samples from the Chinese Glioma Genome Atlas (CGGA) database were analyzed; data included whole genome mRNA expression profiles from microarray analyses and corresponding clinical information. The relationship between c-FLIP mRNA expression and glioma grade (II–IV) is shown. (PDF 30 kb)

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Zhitao, J., Long, L., Jia, L. et al. Temozolomide sensitizes stem-like cells of glioma spheres to TRAIL-induced apoptosis via upregulation of casitas B-lineage lymphoma (c-Cbl) protein. Tumor Biol. 36, 9621–9630 (2015). https://doi.org/10.1007/s13277-015-3720-8

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  • DOI: https://doi.org/10.1007/s13277-015-3720-8

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