Samuel N. Rodman

Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism

The goal of this study was to determine if depletion of glutathione (GSH) and inhibition of thioredoxin (Trx) reductase (TrxR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol-dependent oxidative stress. The following were used to inhibit GSH and Trx metabolism: buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc– cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2-AAPA, a GSH-reductase inhibitor. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24–ESA+ or ALDH1) and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO and radiation also significantly decreased breast cancer cell migration and invasion in a thiol-dependent manner that could be inhibited by NAC. In addition, pretreating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24–ESA+ or ALDH1. Combined administration of Au and BSO, given prior to irradiation, significantly increased the survival of mice with human breast cancer xenografts, and decreased the number of ALDH1+ cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and 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.

Augmentation of intracellular iron using iron sucrose enhances the toxicity of pharmacological ascorbate in colon cancer cells

Pharmacological doses (> 1 mM) of ascorbate (a.k.a., vitamin C) have been shown to selectively kill cancer cells through a mechanism that is dependent on the generation of H2O2 at doses that are safely achievable in humans using intravenous administration. The process by which ascorbate oxidizes to form H2O2 is thought to be mediated catalytically by redox active metal ions such as iron (Fe). Because intravenous iron sucrose is often administered to colon cancer patients to help mitigate anemia, the current study assessed the ability of pharmacological ascorbate to kill colon cancer cells in the presence and absence of iron sucrose. In vitro survival assays showed that 10 mM ascorbate exposure (2 h) clonogenically inactivated 40–80% of exponentially growing colon cancer cell lines (HCT116 and HT29). When the H2O2 scavenging enzyme, catalase, was added to the media, or conditionally over-expressed using a doxycycline inducible vector, the toxicity of pharmacological ascorbate was significantly blunted. When colon cancer cells were treated in the presence or absence of 250 µM iron sucrose, then rinsed, and treated with 10 mM ascorbate, the cells demonstrated increased levels of labile iron that resulted in significantly increased clonogenic cell killing, compared to pharmacological ascorbate alone. Interestingly, when colon cancer cells were treated with iron sucrose for 1 h and then 10 mM ascorbate was added to the media in the continued presence of iron sucrose, there was no enhancement of toxicity despite similar increases in intracellular labile iron. The combination of iron chelators, deferoxamine and diethylenetriaminepentaacetic acid, significantly inhibited the toxicity of either ascorbate alone or ascorbate following iron sucrose. These observations support the hypothesis that increasing intracellular labile iron pools, using iron sucrose, can be used to increase the toxicity of pharmacological ascorbate in human colon cancer cells by a mechanism involving increased generation of H2O2.

D-penicillamine combined with inhibitors of hydroperoxide metabolism enhances lung and breast cancer cell responses to radiation and carboplatin via H2O2-mediated oxidative stress

Abstract D‐penicillamine (DPEN), a copper chelator, has been used in the treatment of Wilsons disease, cystinuria, and rheumatoid arthritis. Recent evidence suggests that DPEN in combination with biologically relevant copper (Cu) concentrations generates H2O2 in cancer cell cultures, but the effects of this on cancer cell responses to ionizing radiation and chemotherapy are unknown. Increased steady‐state levels of H2O2 were detected in MB231 breast and H1299 lung cancer cells following treatment with DPEN (100 &mgr;M) and copper sulfate (15 &mgr;M). Clonogenic survival demonstrated that DPEN‐induced cancer cell toxicity was dependent on Cu and was significantly enhanced by depletion of glutathione [using buthionine sulfoximine (BSO)] as well as inhibition of thioredoxin reductase [using Auranofin (Au)] prior to exposure. Treatment with catalase inhibited DPEN toxicity confirming H2O2 as the toxic species. Furthermore, pretreating cancer cells with iron sucrose enhanced DPEN toxicity while treating with deferoxamine, an Fe chelator that inhibits redox cycling, inhibited DPEN toxicity. Importantly, DPEN also demonstrated selective toxicity in human breast and lung cancer cells, relative to normal untransformed human lung or mammary epithelial cells and enhanced cancer cell killing when combined with ionizing radiation or carboplatin. Consistent with the selective cancer cell toxicity, normal untransformed human lung epithelial cells had significantly lower labile iron pools than lung cancer cells. These results support the hypothesis that DPEN mediates selective cancer cell killing as well as radio‐chemo‐sensitization by a mechanism involving metal ion catalyzed H2O2‐mediated oxidative stress and suggest that DPEN could be repurposed as an adjuvant in conventional cancer therapy. Graphical abstract Figure. No Caption available. HighlightsDPEN+Cu at physiologic concentrations increase H2O2 levels in cancer cells.DPENs clonogenic toxicity is enhanced using auranofin and buthionine sulfoximine.DPEN+Cu treatment is more toxic to cancer cells than to normal epithelial cells.DPEN toxicity in correlated with intracellular labile iron pools.Labile iron pools are higher in cancer cells verses normal lung epithelial cells.

Abstract 3219: Sensitizing hypoxic small cell lung cancer cells to radiation and hydrogen peroxide-producing agents using CuATSM

BACKGROUND: Cancer cells have increased steady state levels of reactive oxygen species (ROS; O2•and H2O2) compared to normal cells. It has been proposed that using redox active agents that further increase ROS levels will result is selective cancer cell death. This metabolic frailty can be targeted using drugs deemed safe for human use, ascorbate (ASC) and disulfiram (DSF), via a mechanism of H2O2 production. CuATSM is a drug being used in clinical trials to treat ALS disease. Imaging studies have shown that CuATSM preferentially concentrates in hypoxic tissues, releasing its Cu after entering cells. Copper (Cu) can participate in oxidation reactions that result in highly toxic hydroxyl radicals. Tumors often have areas of hypoxic tissue that exhibit resistance to ionizing radiation (IR). Our hypothesis is that CuATSM can be used to sensitize hypoxic regions of tumors to IR, ASC and/or DSF.

Abstract 1177: Ketogenic diet sensitizes FaDu human head and neck cancer xenografts to cisplatin as well as ionizing radiation combined with cetuximab

Purpose: Ketogenic diets (KDs) used in the treatment of epilepsy are high fat, low carbohydrate diets that force cells to utilize fatty acid oxidation for mitochondrial energy metabolism. KDs have been suggested to sensitize tumors to standard radio-chemotherapy because cancer cells, relative to normal cells, produce more reactive oxygen species from mitochondrial metabolism and increased cancer cell glucose metabolism is thought to provide reducing equivalents to compensate for the metabolic production of hydroperoxides (PMID: 23743570). The current study was designed to determine if the improved KetoCal® formula that includes more polyunsaturated fatty acids was efficacious in sensitizing human head and neck cancer xenografts to standard radio-chemotherapies. Experimental Design: Mice bearing FaDu human head and neck cancer xenografts were assigned to either standard mouse chow or new KetoCal® KD and treated with cetuximab (6 mg/kg i.p. 2 times per week for 2 weeks), cisplatin (2 mg/kg 2 i.p. times per week for 2 weeks), and/or IR (6 Gy x 2 dose followed by 1.8 Gy x 2 doses). In similar experiments mice were fed either standard chow or the new KetoCal® formula and treated with IR (6 Gy 2 times a week for 2 weeks, and then 1.8 Gy 3 times a week indefinitely) and cetuximab (6 mg/kg cetuximab 2 times a week indefinitely). In parallel, tumors were harvested from treated animals and blood samples were collected from head and neck cancer patients treated with standard of care radio-chemotherapy and the new KetoCal® KD. Blood samples were used to confirm ketosis as well as using tumors and blood to measure markers of redox homeostasis (protein carbonyls, GSH/GSSG, and 4HNE-modified proteins). Results: Mice tolerated all treatments as indicated by no change in general health status as assessed by body condition scoring. Mice treated with new KetoCal® + cisplatin demonstrated increased survival relative to cisplatin alone. Furthermore, mice treated with new KetoCal® + IR + cetuximab showed a significant decrease in tumor growth rate and survival, relative to any other group. Western blot analysis of tumor homogenates demonstrated increased phosphorylated epidermal growth factor in mice fed a KD which was suppressed with the addition of cetuximab as well as increased 4HNE modified proteins. Preliminary results from human studies indicated ketosis was achieved without serious adverse event. Furthermore, patients on KD demonstrate an increase in carbonyl content and a decrease in both GSH and GSSG. Conclusions: KD remains an attractive adjunct therapy for cancer patients, especially for head and neck cancer patients due to their feeding status. The mice xenograft studies support the hypothesis that the new KetoCal® diet combined with standard of care treatments for head and neck cancer has the potential to improve clinical outcomes. Note: This abstract was not presented at the meeting. Citation Format: Daniel C. Ma, Samuel N. Rodman, John M. Buatti, Carryn Anderson, Bryan G. Allen, Douglas R. Spitz, Melissa A. Fath. Ketogenic diet sensitizes FaDu human head and neck cancer xenografts to cisplatin as well as ionizing radiation combined with cetuximab. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1177. doi:10.1158/1538-7445.AM2015-1177

Abstract 1470: Enhancing radiation therapy by simultaneous inhibition of thioredoxin- and glutathione-dependent metabolism

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cancer cells have been shown to be in a constant state of metabolic oxidative stress, concurrent with an elevated level of reactive oxygen species (ROS). In order to facilitate survival in the face of increased steady-sate levels of ROS, cancer cells rely on glutathione (GSH) and thioredoxin (Trx) dependent metabolism. We have recently demonstrated that the simultaneous inhibition of GSH using Buthionine sulphoximine [(BSO), a GHS synthesis inhibitor] and thioredoxin reductase using auronofin [(Au), a thioredoxin reductase inhibitor] effectively chemo-radio-sensitized lung cancer cells via a mechanism involving oxidative stress. In this work we extend this observation to demonstrate that Au 2 mg/kg and BSO 450 mg/kg, given prior to ionizing radiation (6 Gy x 2 doses) can significantly delay tumor growth rate for mice treated with radiation alone in H292 lung cancer xenografts. In addition, we demonstrate through clonogenic survival assays that the combination of Au (250-500 nM) and BSO (100 μM) radio-sensitizes two breast cancer cell lines in vitro, and more importantly the combination of Au and BSO given by intraperitoneal injection prior to radiation decreases the percent of breast cancer stem/progenitor cells in vivo. The rate limiting substrate for GSH synthesis is cysteine, whose level is controlled by the xc- cystine/glutamate antiporter. Sulfasazine (SSZ) has been shown to be a potent inhibitor of the xc- cystine/glutamate antiporter. We demonstrate that SSZ (0.1-0.5 mM) decreases total GSH (up to 90%) levels in a dose dependent manner in both Mia PaCa-2 and PANC-1 human pancreatic cell lines. The combination of Au (250 nM for 3 hours) with SSZ (0.2 mM for 24 hours) demonstrates an 80% and 30% decrease in clonogenic cell survival in MiaPaCa-2 and PANC-1 pancreatic cells that is inhibited by treatment with the thiol antioxidant, 15 mM N-acetylcysteine. These results indicate that inhibition of hydroperoxide metabolism using pharmacologically relevant agents to simultaneously disrupt GSH and Trx system, can sensitize multiple cancer cell lines to radiation. These results support the hypothesis that standard of care radiotherapy protocols could be enhanced using SSZ, Au, and/or BSO. Supported by The Iowa Center for Research by Undergraduates, R21CA139182, R01CA133114 and a Breast Cancer Research Group Seed Grand from the Holden Comprehensive Cancer Center. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1470. doi:1538-7445.AM2012-1470