Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells

PLOS ONE, Dec 2019

The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin’s reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in VM-M3 GBM cells. Metformin potentiated DCA-induced superoxide production, which was required for enhanced cytotoxicity towards VM-M3 cells observed with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells.

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Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells

June Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells Nathan P. Ward 0 1 Angela M. Poff 0 1 Andrew P. Koutnik 0 1 Dominic P. D'Agostino 0 1 0 Department of Molecular Pharmacology & Physiology, University of South Florida , Tampa, FL , United States of America 1 Editor: Jianhua Zhang, University of Alabama at Birmingham , UNITED STATES The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin's reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in VM-M3 GBM cells. Metformin potentiated DCA-induced superoxide production, which was required for enhanced cytotoxicity towards VM-M3 cells observed with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells. - Funding: This study was funded through a charitable donation from Scivation, Inc. to DPD; however, they had no role in the design of the study, collection, analysis and interpretation of the data, or writing of this manuscript. Competing interests: This study was funded through a charitable donation from Scivation, Inc. Introduction A consequence of the hallmark metabolic alterations associated with neoplastic growth is elevated oxidative stress [ 1 ]. Hypoxia, mitochondrial abnormalities, and organellar inputs, such as ER stress, not only direct cancer metabolism but also greatly influence the generation of reactive oxygen species (ROS) and oxidative stress [ 2, 3 ]. Concurrently, these energetic and redox stresses dictate a compensatory increase in antioxidant capacity that permits cancer cell resilience and proliferation [4]. ROS modulate cellular function and integrity through oxidation of macromolecular structures. Moderate oxidative stress can therefore contribute to the genomic instability that is characteristic of cancer as well as enhance oncogenic signaling through oxidation of constituents of mitogenic pathways [ 5 ]. However, excessive ROS can promote membrane dysfunction and the loss of mitochondrial integrity, ultimately leading to cell death [ 6 ]. Ionizing radiation as well as many traditional chemotherapies such as 5-fluorouracil and doxorubicin elicit cytotoxicity towards cancer cells in part through induction of ROS and overwhelming cellular redox balance [ 7 ]. Yet there is accumulating evidence that robust antioxidant capacity contributes to chemo- and radiotherapy resistance and the eventual failure of these therapies in patients [8±10]. Therefore, it is vital to identify adjuvant agents that further enhance oxidative stress to overwhelm the antioxidant system and overcome this mechanism of resistance. The small-molecule pyruvate mimetic dichloroacetate (DCA) is being evaluated as an adjuvant to chemotherapy because of its propensity to enhance oxidative stress [11±16]. DCA, an inhibitor of pyruvate dehydrogenase kinase (PDK), promotes oxidative metabolism through activation of the pyruvate dehydrogenase complex (PDH) and subsequent flux of glucose carbon through the citric acid cycle (TCA) [ 17 ]. PDK is upregulated in a number of cancers and DCA is shown to reverse the glycolytic phenotype resultant from its enhanced activity [ 18 ]. A consequence of DCA-induced oxidative metabolism is ROS production, and this enhanced oxidative stress is shown to promote cancer cell death [19±21]. DCA potentiates the cytotoxicity of several chemotherapies and reverses HIF-mediated resistance to bevacizumab in a model of glioblastoma [11±16]. Moreover, DCA promoted stable disease in patients with malignant brain tumors in a Phase I trial [ 22 ]. However, following a separate Phase I doseescalation study, Siu-Chung Chu et al concluded that DCA will be minimally effective as a single-agent and would be best used in combination with therapies that would benefit from enhanced oxidative metabolism [ 23 ]. There is recent evidence to suggest that DCA efficacy is enhanced by the anti-diabetic drug metformin [ 24, 25 ]. Metformin, a cationic biguanide, readil (...truncated)


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Nathan P. Ward, Angela M. Poff, Andrew P. Koutnik, Dominic P. D’Agostino. Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells, PLOS ONE, 2017, Volume 12, Issue 6, DOI: 10.1371/journal.pone.0180061