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Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy Kranti A

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Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy Kranti A Published OnlineFirst August 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0106 Cancer Therapeutics, Targets, and Chemical Biology Research Mitochondrial Superoxide Increases Age- Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy Kranti A. Mapuskar1, Kyle H. Flippo2, Joshua D. Schoenfeld1, Dennis P. Riley3, Stefan Strack2, Taher Abu Hejleh4, Muhammad Furqan4, Varun Monga4, Frederick E. Domann1, John M. Buatti1, Prabhat C. Goswami1, Douglas R. Spitz1, and Bryan G. Allen1 Abstract Elderly cancer patients treated with ionizing radiation (IR) or adenoviral-mediated overexpression of SOD2 activity (5–7- chemotherapy experience more frequent and greater normal fold), mitochondrial ETC activity and aconitase activity were *À tissue toxicity relative to younger patients. The current study restored, demonstrating a role for mitochondrial O2 in these demonstrates that exponentially growing fibroblasts from effects. Old fibroblasts also demonstrated elevated levels elderly (old) male donor subjects (70, 72, and 78 years) are of endogenous DNA damage that were increased following significantly more sensitive to clonogenic killing mediated by treatment with IR and chemotherapy. Most importantly, treat- platinum-based chemotherapy and IR (70%–80% killing) ment with the small-molecule, superoxide dismutase mimetic relative to young fibroblasts (5 months and 1 year; 10%– (GC4419; 0.25 mmol/L) significantly mitigated the increased 20% killing) and adult fibroblasts (20 years old; 10%–30% sensitivity of old fibroblasts to IR and chemotherapy and killing). Old fibroblasts also displayed significantly increased partially restored mitochondrial function without affecting IR *À (2–4-fold) steady-state levels of O2 ,O2 consumption, and or chemotherapy-induced cancer cell killing. These results *À mitochondrial membrane potential as well as significantly support the hypothesis that age-associated increased O2 and decreased (40%–50%) electron transport chain (ETC) complex resulting DNA damage mediate the increased susceptibility of I, II, IV, V, and aconitase (70%) activities, decreased ATP levels, old fibroblasts to IR and chemotherapy that can be mitigated by and significantly altered mitochondrial structure. Following GC4419. Cancer Res; 77(18); 5054–67. Ó2017 AACR. Introduction Numerous studies examining pathways that connect the pro- cesses governing aging and cancer have focused on cellular oxi- Cancer incidence and survival rates are expected to increase by dative metabolism (4–6). Furthermore, normal tissue injury over 50% during the next 20 years predominantly due to an aging associated with cancer therapy has been hypothesized to involve population and improved therapeutic outcomes (1). By the year alterations to mitochondrial oxidative metabolism, leading to 2020, there will be approximately 18,000,000 cancer survivors in increased steady-state levels of reactive oxygen species (ROS), the United States (1). Acute and chronic normal tissue injuries *À including superoxide (O ) and hydrogen peroxide (H O ; resulting from combined modality radiation and chemotherapies 2 2 2 refs. 7, 8). Recently, several studies have utilized mitochondrially represent highly significant treatment limiting complications that targeted antioxidants to mitigate IR-induced skin damage, pre- are exacerbated in elderly patients (2, 3). Currently, there are venting dermatitis, inflammation, and fibrosis, thereby preserving limited data available on the age-associated mechanisms of the antioxidant capacity of the skin following IR exposure (9, 10). normal tissue injury associated with ionizing radiation (IR) and The mitochondrial ETC complexes located on the inner mito- chemotherapy as well as few treatment options for preventing or chondrial membrane are believed to be a major metabolic site of mitigating this injury. ROS production in mammalian cells with 0.1% to 1% of the electrons that flow through the ETCs predicted to undergo one- *À electron reductions of oxygen forming O2 and H2O2 (11). 1Department of Radiation Oncology, The University of Iowa, Iowa City, Iowa. Furthermore, increased steady-state levels of ROS are reported to 2Department of Pharmacology, The University of Iowa, Iowa City, Iowa. 3Galera progressively increase with aging (5, 7). Mutations in mitochon- 4 Therapeutics, Inc., Malvern, Pennsylvania. Department of Internal Medicine, The drial DNA and nuclear genes encoding mitochondrial proteins are University of Iowa, Iowa City, Iowa. known to accumulate during the aging process (12) and are again Note: Supplementary data for this article are available at Cancer Research *À associated with increased levels of O2 ,H2O2, and genomic Online (http://cancerres.aacrjournals.org/). instability (13, 14). In addition, the age-associated vulnerability Corresponding Author: Bryan G. Allen, University of Iowa, B180 Med Labs, Iowa of mammalian cells to accumulate DNA damage is also suggested City, IA 52240. Phone: 319-335-8025; Fax: 319-356-1530; E-mail: to derive from a progressive decline in DNA repair processes (15). [email protected] *À Thus, increased production of O2 and H2O2 as well as the doi: 10.1158/0008-5472.CAN-17-0106 accumulation of DNA damage along with changes in mitochon- Ó2017 American Association for Cancer Research. drial function (6, 7, 15) appear to be fundamental to both cancer 5054 Cancer Res; 77(18) September 15, 2017 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0106 *À Mitochondrial O2 and Age-Associated Normal Tissue Toxicity *À and aging processes, but the relative contribution of O2 to the old) male donors. Data were analyzed by taking the pooled mean increased sensitivity of normal human cells from elderly patients of the data from the different cell sources in each age group. to radiation and chemotherapy is unknown. Exponentially growing cultures of fibroblasts were used from The current study demonstrates that exponentially growing passages 3 to 7 (passage 1 was defined as when the cells were early-passage human fibroblasts, from elderly (old) male donor received from Coriell) for all experiments. subjects (70, 72, 78 years), are significantly more sensitive to clonogenic killing mediated by platinum-based chemotherapy Radiation, chemotherapy, and mimetic treatment and IR, relative to young fibroblasts from male donor subjects (5 Irradiation was performed on cells plated in 60- or 100-mm months and 1 year) and/or adult fibroblasts from a male donor dishes with complete media. Typically, cells were allowed to subject (20-year-old). Furthermore, old fibroblasts also showed attach for at least 24 to 48 hours at 4% O2 and 37 C to obtain significant alterations in mitochondrial ETC and aconitase activ- exponentially growing cultures at around 50%–70% confluence ities. Mitochondrial ETC activity and aconitase activity were for all experiments unless specified otherwise. Cells were irradi- restored by SOD2 overexpression, clearly demonstrating a role ated from 2 to 4 Gy X-rays in the Radiation and Free Radical *À for mitochondrial O2 in these effects. Donor age-associated Research Core laboratory at the University of Iowa. Radiation was alterations in mitochondrial metabolism were found to be given at 1.29 Gy/minute and was delivered using a PANTAK HF- accompanied by downregulation of mitochondrial ETC gene 320 ortho volt X-ray unit. All in vitro irradiations used 200 kVp X- expressioninoldfibroblasts. Furthermore, old fibroblasts also rays, 15 mA with a filter composed of 0.35 mm copper (Cu) þ 1.5 demonstrated elevated levels of endogenous DNA damage that mm aluminum (Al). After irradiation, the cells were plated for were increased following treatment with IR and chemotherapy. clonogenic cell survival within an hour. For the experiments in Most importantly, treatment with a small-molecule, superox- vivo, animals were arranged in lead coffins with only their left legs ide-specific SOD mimetic (GC4419) mitigated the increased exposed to the X-ray source. All in vivo irradiations used 200 kVp X- sensitivity of old fibroblasts to IR and chemotherapy as well as rays, 15 mA with a filter composed of 0.35 mm copper (Cu). partially restored mitochondrial function without affecting IR Radiation was given at 3 Gy/minute with the head height at 30 cm or chemotherapy-induced cancer cell killing. These results show delivered using a PANTAK HF-320 orthovolt X-ray unit. Cisplatin *À age-associated increases in O2 , DNA-damage, and compro- (1 mg/mL; molecular weight 300) was purchased from Hospira, mised mitochondrial function result in increased sensitivity of Inc. and carboplatin (10 mg/mL; molecular weight 371) was old fibroblasts to IR and chemotherapy that can be inhibited by purchased from APP Pharmaceuticals Inc. (Fresenius Kabi USA) an SOD mimetic, GC4419, which is currently being studied and were further diluted in H2O prior to use to achieve the desired clinically as a mitigator for oral mucositis in head and neck concentrations. GC4419 (Galera Therapeutics, molecular weight cancer subjects (NCT02508389). These results support the 483) was prepared at a stock concentration of 10 mmol/L in H2O hypothesis that the use of SOD mimetics in elderly cancer with 10 mmol/L sodium bicarbonate (pH 7.1–7.4) that was patients undergoing radiation and chemotherapy may have diluted in order to achieve a final concentration of 0.25 mmol/L significant benefit in enhancing the tolerability of cancer ther- and was added
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