Acquisition of temozolomide chemoresistance in gliomas leads to remodeling of mitochondrial electron transport chain

Abstract

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.

FIGURE 1.

TMZ treatment affects mtDNA integrity. A, mtDNA copy number was determined by comparing the ratio of mtDNA to nDNA amplified using quantitative PCR on genomic DNA from U251 and UTMZ cells. B, agarose gel electrophoresis shows purified mtDNA (lanes 1 and 2) and full-length mtDNA PCR amplification (lanes 3 and 4) of mtDNA from U251 and UTMZ cells.

FIGURE 2.

TMZ treatment induces changes in mitochondrial function. A, relative activities of ETC complexes normalized to citrate synthase (CS) activity from U251 (TMZ-sensitive) and UTMZ (TMZ-resistant) glioma cells are shown. Columns represent the average from triplicate determinations from at least three independent experiments. B, shown is analysis of the expression of CcO subunits by Western blot. The membranes were also probed with anti-voltage-dependent anion channel (VDAC) protein antibody as a loading control.

FIGURE 3.

Measurement of bioenergetic parameters of U251 and UTMZ cells using extracellular flux technology. A, a schematic diagram demonstrating the use of specific inhibitors to determine the sites of cellular oxygen consumption is shown. After three base-line OCR measurements, oligomycin (Oligo), FCCP, and antimycin A (Ant A) were injected sequentially with OCR measurements recorded after each injection. ATP-linked oxygen consumption (ATP) and the OCR due to proton leak (Proton) can be calculated using the basal and the oligomycin-sensitive rate. Injection of FCCP is used to determine the maximal respiratory capacity. Injection of antimycin A allows for the measurement of oxygen consumption independent of Complex IV (Other). The reserve respiratory capacity (Reserve) is calculated by subtracting the basal from the maximal rate of oxygen consumption. B, bioenergetic profiles were measured using sequential injection of oligomycin (0.3 μg/ml), FCCP (0.3 μm for U251 and 1 μm for UTMZ), and antimycin A (10 μm). Basal OCR (C), reserve respiratory capacity (D), and basal ECAR (E) are shown. Seahorse XF24 Analyzer protocol included 2 min of mixing, 2 min of waiting, and 3 min of measurement times for each measurement. Results represent the means ± S.E., n = 8–10. **, p < 0.01 compared with U251, Student's t test.

FIGURE 4.

NMP preferentially blocks complexes II-III and CcO activities and abrogates TMZ resistance of GBM. A, shown are mitochondrial complex activities normalized to citrate synthase (CS) activity in control UTMZ cells and after 3 days of culture in the presence of 5 μm NMP. B, shown is apoptosis activation in UTMZ cells after treatment with different doses of TMZ in presence or absence of 5 μm NMP. Columns represent average from triplicate determinations of the percentage of apoptotic cells. C, cleaved PARP was determined by Western blot analysis. Levels of uncleaved PARP protein served as the loading control.

FIGURE 5.

shRNA reduces COX-IV-1 expression and CcO activity and abrogates TMZ resistance in UTMZ cells. A, shown is Western blot analysis of COX-IV-1 expression in COX-IV-1-shRNA or empty vector-transfected UTMZ cells. B, densitometric quantification of COX-IV-1 expression is shown. C, shown is CcO activity of COX-IV-1-shRNA transfected versus control, empty vector-transfected UTMZ cells. D, shown is apoptosis activation in shRNA-UTMZ cells after treatment with different doses of TMZ. Columns represent the average from triplicate determinations of the percentage of apoptotic cells. E, cleaved PARP was determined by Western blot analysis. Levels of uncleaved PARP protein served as loading control.

FIGURE 6.

Altered activities of mitochondrial complexes of human GBM specimens. A, scatter plots show the activities of mitochondrial complexes of pair tumor biopsies from the same patients who have relapsed after initial treatment. B, activities of mitochondrial complexes from TMZ-sensitive xenograft lines (GBM12, GBM22) and TMZ-resistant xenograft lines (GBM12-TMZ, GBM22-TMZ) are shown. Activities were normalized to citrate synthase (CS) activity. Columns represent the average from triplicate determinations.