Kranti A. Mapuskar

Ketogenic diets as an adjuvant cancer therapy: History and potential mechanism

Cancer cells, relative to normal cells, demonstrate significant alterations in metabolism that are proposed to result in increased steady-state levels of mitochondrial-derived reactive oxygen species (ROS) such as O2•−and H2O2. It has also been proposed that cancer cells increase glucose and hydroperoxide metabolism to compensate for increased levels of ROS. Given this theoretical construct, it is reasonable to propose that forcing cancer cells to use mitochondrial oxidative metabolism by feeding ketogenic diets that are high in fats and low in glucose and other carbohydrates, would selectively cause metabolic oxidative stress in cancer versus normal cells. Increased metabolic oxidative stress in cancer cells would in turn be predicted to selectively sensitize cancer cells to conventional radiation and chemotherapies. This review summarizes the evidence supporting the hypothesis that ketogenic diets may be safely used as an adjuvant therapy to conventional radiation and chemotherapies and discusses the proposed mechanisms by which ketogenic diets may enhance cancer cell therapeutic responses.

Simultaneous inhibition of glutathione- and thioredoxin-dependent metabolism is necessary to potentiate 17AAG-induced cancer cell killing via oxidative stress.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is an experimental chemotherapeutic agent believed to form free radicals in vivo, and cancer cell resistance to 17AAG is believed to be a thiol-dependent process. Inhibitors of thiol-dependent hydroperoxide metabolism [L-buthionine-S,R-sulfoximine (BSO) and auranofin] were combined with the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) to determine if 17AAG-mediated cancer cell killing could be enhanced. When 2DG (20mM, 24h), BSO (1mM, 24h), and auranofin (500nM, 3h) were combined with 17AAG, cell killing was significantly enhanced in three human cancer cell lines (PC-3, SUM159, MDA-MB-231). Furthermore, the toxicity of this drug combination was significantly greater in SUM159 human breast cancer cells, relative to HMEC normal human breast epithelial cells. Increases in toxicity seen with this drug combination also correlated with increased glutathione (GSH) and thioredoxin (Trx) oxidation and depletion. Furthermore, treatment with the thiol antioxidant NAC (15mM, 24h) was able to significantly protect from drug-induced toxicity and ameliorate GSH oxidation, Trx oxidation, and Trx depletion. These data strongly support the hypothesis that simultaneous inhibition of GSH- and Trx-dependent metabolism is necessary to sensitize human breast and prostate cancer cells to 2DG+17AAG-mediated killing via enhancement of thiol-dependent oxidative stress. These results suggest that simultaneous targeting of both GSH and Trx metabolism could represent an effective strategy for chemosensitization in human cancer cells.

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Superoxide Mediates Acute Liver Injury in Irradiated Mice Lacking Sirtuin 3

AIMS This study determined whether acute radiation-induced liver injury seen in Sirtuin3(-/-) mice after exposure to Cs-137 γ-rays was mediated by superoxide anion (O2(•-)). RESULTS Male wild-type (WT) and SIRT3(-/-) mice were given 2×2 Gy whole-body radiation doses separated by 24 h and livers were harvested 20 h after the second dose. Ex vivo measurements in fresh frozen liver sections demonstrated 50% increases in dihydroethidium oxidation from SIRT3(-/-) animals, relative to WT animals, before irradiation, but this increase was not detected 20 h after radiation exposure. In addition, irradiated livers from SIRT3(-/-) animals showed significant hydropic degeneration, loss of MitoTracker Green FM staining, increased immunohistochemical staining for 3-nitrotyrosine, loss of Ki67 staining, and increased mitochondrial localization of p53. These parameters of radiation-induced injury were significantly attenuated by an intraperitoneal injection of 2 mg/kg of the highly specific superoxide dismutase mimic, GC4401, 30 min before each fraction. INNOVATION Sirtuin 3 (SIRT3) is believed to regulate mitochondrial oxidative metabolism and antioxidant defenses in response to acute radiation-induced liver injury. This work provides strong evidence for the causal role of O2(•-) in the liver injury process initiated by whole-body irradiation in SIRT3(-/-) mice. CONCLUSION These results support the hypothesis that O2(•-) mediates acute liver injury in SIRT3(-/-) animals exposed to whole-body γ-radiation and suggest that GC4401 could be used as a radio-protective compound in vivo.

Consuming a Ketogenic Diet while Receiving Radiation and Chemotherapy for Locally Advanced Lung Cancer and Pancreatic Cancer: The University of Iowa Experience of Two Phase 1 Clinical Trials

Ketogenic diets are low in carbohydrates and high in fat, which forces cells to rely more heavily upon mitochondrial oxidation of fatty acids for energy. Relative to normal cells, cancer cells are believed to exist under a condition of chronic mitochondrial oxidative stress that is compensated for by increases in glucose metabolism to generate reducing equivalents. In this study we tested the hypothesis that a ketogenic diet concurrent with radiation and chemotherapy would be clinically tolerable in locally advanced non-small cell lung cancer (NSCLC) and pancreatic cancer and could potentially exploit cancer cell oxidative metabolism to improve therapeutic outcomes. Mice bearing MIA PaCa-2 pancreatic cancer xenografts were fed either a ketogenic diet or standard rodent chow, treated with conventionally fractionated radiation (2 Gy/fraction), and tumor growth rates were assessed daily. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modfied proteins as a marker of oxidative stress. Based on this and another previously published preclinical study, phase 1 clinical trials in locally advanced NSCLC and pancreatic cancer were initiated, combining standard radiation and chemotherapy with a ketogenic diet for six weeks (NSCLC) or five weeks (pancreatic cancer). The xenograft experiments demonstrated prolonged survival and increased 4HNE-modfied proteins in animals consuming a ketogenic diet combined with radiation compared to radiation alone. In the phase 1 clinical trial, over a period of three years, seven NSCLC patients enrolled in the study. Of these, four were unable to comply with the diet and withdrew, two completed the study and one was withdrawn due to a dose-limiting toxicity. Over the same time period, two pancreatic cancer patients enrolled in the trial. Of these, one completed the study and the other was withdrawn due to a dose-limiting toxicity. The preclinical experiments demonstrate that a ketogenic diet increases radiation sensitivity in a pancreatic cancer xenograft model. However, patients with locally advanced NSCLC and pancreatic cancer receiving concurrent radiotherapy and chemotherapy had suboptimal compliance to the oral ketogenic diet and thus, poor tolerance.

Size-dependent cytotoxicity of copper oxide nanoparticles in lung epithelial cells

The increasing use of copper oxide (CuO) nanoparticles (NPs) in medicine and industry demands an understanding of their potential toxicities. In this study, we compared the in vitro cytotoxicity of CuO NPs of two distinct sizes (4 and 24 nm) using the A549 human lung cell line. Despite possessing similar surface and core oxide compositions, 24 nm CuO NPs were significantly more cytotoxic than 4 nm CuO NPs. The difference in size may have affected the rate of entry of NPs into the cell, potentially influencing the amount of intracellular dissolution of Cu2+ and causing a differential impact on cytotoxicity.

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.

Erratum: O2 ⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate (Cancer Cell (2017) 32 (2)(268) (S1535610817300624) (10.1016/j.ccell.2017.02.018))

Joshua D. Schoenfeld1, Zita A. Sibenaller1, Kranti A. Mapuskar1, Brett A. Wagner1, Kimberly L. Cramer-Morales1, Muhammad Furqan2, Sonia Sandhu2, Thomas L. Carlisle2, Mark C. Smith1, Taher Abu Hejleh2, Daniel J. Berg2, Jun Zhang2, John Keech3, Kalpaj R. Parekh3, Sudershan Bhatia1, Varun Monga2, Kellie L. Bodeker1, Logan Ahmann1, Sandy Vollstedt1, Heather Brown1, Erin P. Shanahan Kauffman2, Mary E. Schall2, Ray J. Hohl2, Gerald H. Clamon2, Jeremy D. Greenlee4, Matthew A. Howard4, Michael K. Shultz5, Brian J. Smith6, Dennis P. Riley7, Frederick E. Domann1, Joseph J. Cullen3, Garry R. Buettner1, John M. Buatti1, Douglas R. Spitz1,*,#, and Bryan G. Allen1,* 1Free Radical and Radiation Biology Program, Department of Radiation Oncology, Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA, 52242, USA

The absence of CpG in plasmid DNA-chitosan polyplexes enhances transfection efficiencies and reduces inflammatory responses in murine lungs.

Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200–400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(−)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(−)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(−)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.

Mitochondrial Superoxide Increases Age-Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy

Elderly cancer patients treated with ionizing radiation (IR) or chemotherapy experience more frequent and greater normal tissue toxicity relative to younger patients. The current study demonstrates that exponentially growing fibroblasts from elderly (old) male donor subjects (70, 72, and 78 years) are significantly more sensitive to clonogenic killing mediated by platinum-based chemotherapy and IR (∼70%-80% killing) relative to young fibroblasts (5 months and 1 year; ∼10%-20% killing) and adult fibroblasts (20 years old; ∼10%-30% killing). Old fibroblasts also displayed significantly increased (2-4-fold) steady-state levels of O2•-, O2 consumption, and mitochondrial membrane potential as well as significantly decreased (40%-50%) electron transport chain (ETC) complex I, II, IV, V, and aconitase (70%) activities, decreased ATP levels, and significantly altered mitochondrial structure. Following adenoviral-mediated overexpression of SOD2 activity (5-7-fold), mitochondrial ETC activity and aconitase activity were restored, demonstrating a role for mitochondrial O2•- in these effects. Old fibroblasts also demonstrated elevated levels of endogenous DNA damage that were increased following treatment with IR and chemotherapy. Most importantly, treatment with the small-molecule, superoxide dismutase mimetic (GC4419; 0.25 μmol/L) significantly mitigated the increased sensitivity of old fibroblasts to IR and chemotherapy and partially restored mitochondrial function without affecting IR or chemotherapy-induced cancer cell killing. These results support the hypothesis that age-associated increased O2•- and resulting DNA damage mediate the increased susceptibility of old fibroblasts to IR and chemotherapy that can be mitigated by GC4419. Cancer Res; 77(18); 5054-67. ©2017 AACR.