Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins
David S. Ziegler, Renee D. Wright, Santosh Kesari, Madeleine E. Lemieux, Mary A. Tran, Monish Jain, Leigh Zawel, Andrew L. Kung
David S. Ziegler, Renee D. Wright, Santosh Kesari, Madeleine E. Lemieux, Mary A. Tran, Monish Jain, Leigh Zawel, Andrew L. Kung
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Research Article Oncology

Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

Abstract

Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.

Authors

David S. Ziegler, Renee D. Wright, Santosh Kesari, Madeleine E. Lemieux, Mary A. Tran, Monish Jain, Leigh Zawel, Andrew L. Kung

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Figure 5

NOL3 is expressed in gliomas and is modulated by imatinib.

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NOL3 is expressed in gliomas and is modulated by imatinib. (A) Quantitat...
(A) Quantitative RT-PCR evaluation of NOL3 mRNA expression in LN827 cells following treatment with 10 μM imatinib. Data represent mean ± SEM of triplicates. *P < 0.01, 2-tailed Student’s t test. (B) Western blot showing NOL3 protein levels in LN827 cells following treatment with imatinib 10 μM for the indicated times. (C) Comparison of NOL3 mRNA expression levels in normal brain tissue, compared with GBM specimens. P values were calculated using unpaired t test with Welch's correction. Data values were obtained from the GSE4290 dataset (see Methods). (D) Comparison of NOL3 mRNA expression levels in grade III, grade IV, and recurrent high-grade gliomas, with P values calculated using the 1-way ANOVA test. Data values were obtained from the GSE4271 dataset (see Methods). (E) Kaplan-Meier survival curves for patients with high-grade gliomas based on NOL3 expression level. Results were separated above or below the median NOL3 level, and P values were calculated by the log-rank test. Data values were obtained from the GSE4271 dataset. (F) Western blot showing NOL3 protein levels in 293T cells following transfection with NOL3 plasmid. (G) LN827 cells were transfected with CD19 plasmid and either NOL3 plasmid or control vector. Cells were then treated with LBW242 50 μM plus imatinib 10 μM or with vehicle control for 72 hours. Cells were stained with annexin V–PE and CD19-FITC antibodies and analyzed by flow cytometry, with CD19 used as a marker of successful transfection and annexin V–PE as a marker of apoptosis. P values were calculated by 2-tailed Student’s t test. (H) LN827 cells were transfected with 50 nM NOL3 siRNA, control (CT) siRNA, or Lipofectamine 2000 only (Nil). Western blot was performed for NOL3 protein after 72 hours, with all samples on a single gel but not in contiguous wells. (I) LN827 cells were transfected with either control or NOL3 siRNA. LBW242 50 μM was added 5 hours after transfection. Apoptosis was determined by annexin V and PI staining after 72 hours of treatment. (J) LN827 cells were similarly treated with etoposide 50 μg/ml for 48 hours. P values were calculated using the 2-tailed Student’s t test.