Pentaazamacrocyclic Mn (II)-containing (MnPAM) superoxide dismutase mimetics can selectively modulate the effects of radiotherapy in tumors and normal tissues by enhancing oxidative stress in tumors and mitigating it in normal tissues. However, radiosensitizing effects in tumors are only achieved with stereotactic body radiotherapy-type dosing (≥7 Gy per fraction). These effects are limited at lower fractional doses that are clinically relevant for many tumors, including locally advanced rectal cancer. Here, we show that combining the MnPAM dismutase mimetic, rucosopasem manganese (GC4711), with pharmacological ascorbate significantly enhances radiosensitization to fractionated radiation doses relevant in colorectal cancer treatment (2–5 Gy) compared to either agent alone in multiple tumor models in vitro and significantly prolongs tumor growth delay in vivo. Mechanistically, enhanced radiosensitization with combination therapy is mediated by increased oxygen consumption and hydrogen peroxide generation. Despite having no effect on oxygen consumption in tumor cell media on its own, GC4711 significantly increases pharmacological ascorbate-induced oxygen consumption. Inducible catalase expression, or administration of exogenous catalase, attenuates the anti-cancer activity when combined. As predicted, increased peroxide fluxes with combination therapy enhanced postirradiation DNA damage and G 2 /M cell cycle accumulation. Importantly, treatment with pharmacological ascorbate and/or GC4711 had no significant effect on postirradiation clonogenic survival or DNA damage in non-malignant FHs74 enterocytes. These results support the hypothesis that adding pharmacological ascorbate to MnPAM dismutase mimetics selectively induces more peroxide-mediated radiosensitization than either agent alone in tumors without increasing radiosensitivity in cells treated with clinically relevant radiation fraction sizes used for radiotherapy of colorectal cancers.

BACKGROUND:Therapeutic ionizing radiation is a standard treatment modality for thoracic cancers that may affect surrounding normal tissues, including the cardiovascular system. The increasing recognition of mitochondrial health in cellular responses to oxidative stress, particularly in ionizing radiation-induced cardiac implications, highlights mitochondria as a critical target for therapeutic interventions. Optimizing mitochondrial function to attenuate ionizing radiation-induced cardiac pathology necessitates balancing killing cancer cells with sparing nonmalignant tissues. METHODS:Using a cardiac targeted radiation injury (CTI) model of injury, C57BL/6J female mice were exposed to a single 16 Gy dose and treated with an SOD (superoxide dismutase) mimetic, ucosopasem manganese (RUC) starting 1 hour before CTI, continued daily for 1 week post CTI, and once per week thereafter until euthanasia to investigate cardiopulmonary implications over 9 months. RESULTS:CTI cardiovascular toxicities were observed in all irradiated mice. RUC significantly increased overall survival and alleviated CTI-induced changes in cardiac function as assessed by cardiac echocardiography. Persistent changes in mitochondrial oxidative phosphorylation proteins and tricarboxylic acid cycle enzymes were notably attenuated following RUC treatment. Interestingly, RUC reduced both CTI-induced cardiac fibrosis and the activation of the TGF-β/Smad (transforming growth factor-beta/suppressor of mothers against decapentaplegic transcription factor family) pathway. CONCLUSIONS:This study presents a novel role for selective superoxide dismutase mimetics, such as RUC, in protecting against CTI-induced cardiovascular toxicities. As RUC dismutates superoxide, the results suggest superoxide plays a key part in the modulation of mitochondrial oxidative phosphorylation, activation of the canonical TGF-β/Smad pathway, and ionizing radiation cardiovascular side effects. These findings suggest an association between RUC treatment and TGF-β pathway antagonism that requires additional mechanistic validation.

Pharmacological ascorbate (IV delivery, to plasma levels ≈ 15-20 mM) has been shown to be selectively toxic to cancer vs. normal cells as well as inducing radio-chemo-sensitization in non-small cell lung cancer (NSCLC) via increased generation of hydrogen peroxide (H2O2) and increased intracellular redox-active iron (Fe2+). The current study shows that 24 h pretreatment with an FDA-approved iron-oxide nanoparticle, Ferumoxytol (FMX), enhances the toxicity of P-AscH- in human NSCLC cells (H1299T and A549), but not in primary human bronchiolar epithelial cells (HBEpC). In H1299TCat15 cells engineered to overexpress doxycycline inducible catalase, FMX + P-AscH- also induced cell killing and carboplatin-induced radio-chemo-sensitization that was inhibited by exposure to doxycycline, demonstrating the dependence of the biological effects on H2O2. P-AscH- + FMX induced increases in intracellular redox active Fe2+ in H1299TCat15 cells, that was partially inhibited by doxycycline-inducible catalase overexpression, demonstrating that both P-AscH- and H2O2 participate in the intracellular release of redox active Fe2+ from FMX. Finally, H1299TCat15 cells treated with P-AscH- + FMX demonstrated increased single- and double-strand DNA damage, that was not seen in HBEpCs and was inhibited by doxycycline induced expression of catalase. This study represents the first demonstration that FMX combined with P-AscH- selectively sensitize NSCLC cells (relative to normal cells) to ascorbate toxicity and chemo-radio-sensitization through enhancing H2O2-dependent DNA damage, that is accompanied by increased release of intracellular Fe2+. These results support the hypothesis that FMX can be used to selectively enhance therapy responses to P-AscH- in NSCLC.

Purpose: Cisplatin contributes to acute kidney injury (AKI) and chronic kidney disease (CKD) that occurs with greater frequency and severity in older patients. Age-associated cisplatin sensitivity in human fibroblasts involves increased mitochondrial superoxide produced by older donor cells. Experimental design: Young and old C57BL/6 J murine models of cisplatin-induced AKI and CKD were treated with the SOD mimetic avasopasem manganese to investigate the potential antioxidant and anti-inflammatory effects. Adverse event reporting from a phase 2 and a phase 3 randomized clinical trial (NCT02508389 and NCT03689712) conducted in patients treated with cisplatin and AVA was determined to have established the incidence and severity of AKI. Results: Cisplatin-induced AKI and CKD occurred in all mice, however, was more pronounced in older mice. AVA reduced cisplatin-induced mortality, AKI, and CKD, in older animals. AVA also alleviated cisplatin-induced alterations in mitochondrial electron transport chain (ETC) complex activities and NADPH Oxidase 4 (NOX4) and inhibited the increased levels of the inflammation markers, TNF alpha, IL1, ICAM-1, and VCAM-1. Analysis of agestratified subjects treated with cisplatin from clinical trials (NCT02508389, NCT03689712) also supported that the incidence of AKI increased with age and AVA reduced age-associated therapy-induced adverse events (AE), including hypomagnesemia, increased creatinine, and AKI. Conclusions: Older mice and humans are more susceptible to cisplatin-induced kidney injury, and treatment with AVA mitigates age-associated damage. Mitochondrial ETC and NOX4 activities represent sources of superoxide production contributing to cisplatin-induced kidney injury, and pro-inflammatory cytokine production and endothelial dysfunction may also be increased by superoxide formation.

Abstract High dose Vitamin C (pharmacological ascorbate; P-AscH‒ > 20 mM plasma levels) given intravenously (I.V.), has made a remarkable re-emergence as an adjuvant to combined modality approaches in cancer therapy. P-AscH‒ can be readily oxidized in the presence of redox active metals (i.e., P-AscH‒ serves as an iron reducing agent), which can result in increased fluxes of H2O2. This chemical phenomenon allows for the tumor-selective enhancement of chemo-radiation in NSCLC. We hypothesized that enhancing labile Fe2+ in cancer cells using iron-oxide nanoparticles (ferumoxytol, FMX) can increase the effectiveness of P-AscH‒ in enhancing cancer cell sensitivity to chemo-radiation in NSCLC by increasing H2O2 production. To test this hypothesis, we performed clonogenic cell survival assays in-vitro. To test the role of H2O2 in the combined effect of P-AscH‒ and FMX, we developed lentiviral-based doxycycline-inducible overexpression models for catalase, and to determine the changes in Fe, we used fluorescent probes that specifically detect changes in labile Fe2+. Results from these experiments showed FMX enhanced clonogenic cell killing in NSCLC. Furthermore, catalase overexpression inhibited this effect, suggesting that enhanced H2O2 production plays a central role in the additive effect of P-AscH- and FMX. Finally, staining the cells with the fluorescent iron probes showed that FMX enhanced labile Fe2+ in cells treated with P-AscH‒. These studies continue to support the hypothesis that H2O2 plays a significant role in P-AscH‒ toxicity in cancer cells that can be exacerbated using iron oxide nanoparticles. Future studies will test these findings in-vivo using preclinical xenograft models. (supported by P01 CA217797, P30 CA086862, T32 CA078586, and P01 CA244091). Citation Format: Mekhla Singhania, Miachael S. Petronek, Casey F. Pulliam, Kranti A. Mapuskar, Amira Zaher, Douglas R. Spitz. Enhancing therapeutic responses in NSCLC using iron-oxide nanoparticles combined with pharmacological ascorbate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 484.

Thioredoxin Reductase (TrxR) functions to recycle thioredoxin (Trx) during hydroperoxide metabolism mediated by peroxiredoxins and is currently being targeted using the FDA-approved anti-rheumatic drug, auranofin (AF), to selectively sensitize cancer cells to therapy. AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the H727 atypical lung carcinoid cell line. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, an FDA-approved multi-kinase inhibitor that depleted intracellular glutathione (GSH). The pharmacokinetic, pharmacodynamic, and safety profile of AF was examined in nude mice with DMS273 xenografts administered AF intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1−5 d. Plasma levels of AF were 10–20 μM (determined by mass spectrometry of gold), and the optimal inhibition of TrxR activity was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. This AF treatment extended for 14 d, inhibited TrxR (>75%), and resulted in a significant prolongation of median overall survival from 19 to 23 d (p = .04, N = 30 controls, 28 AF). In this experiment, there were no observed changes in animal bodyweight, complete blood counts (CBCs), bone marrow toxicity, blood urea nitrogen, or creatinine. These results support the hypothesis that AF effectively inhibits TrxR both in vitro and in vivo in SCLC, sensitizes NETs and SCLC to sorafenib, and could be repurposed as an adjuvant therapy with targeted agents that induce disruptions in thiol metabolism.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T2* MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T2* relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T2* mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

List of RT-RTPCR primers used in the manuscript.

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

Background Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes. Methods Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 x 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements. Results The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels. Conclusions Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

The disposition and toxicity of lower chlorinated PCBs (LC-PCBs) with less than five chlorine substituents have received little attention. This study characterizes the distribution and metabolomic effects of PCB 52, an LC-PCB found in indoor and outdoor air, three weeks after intraperitoneal exposure of female Sprague Dawley rats to 0, 1, 10, or 100 mg/kg BW. PCB 52 exposure did not affect overall body weight. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis identified PCB 52 in all tissues investigated. Hydroxylated, sulfated, and methylated PCB metabolites, identified using GC-MS/MS and nontarget liquid chromatography-high resolution mass spectrometry (Nt-LCMS), were primarily found in the serum and liver of rats exposed to 100 mg/kg BW. Metabolomic analysis revealed minor effects on L-cysteine, glycine, cytosine, sphingosine, thymine, linoleic acid, orotic acid, L-histidine, and erythrose serum levels. Thus, the metabolism of PCB 52 and its effects on the metabolome must be considered in toxicity studies.

Cisplatin, a potent chemotherapeutic agent, is marred by severe nephrotoxicity that is governed by mechanisms involving oxidative stress, inflammation, and apoptosis pathways. The transcription factor Nrf2, pivotal in cellular defense against oxidative stress and inflammation, is the master regulator of the antioxidant response, upregulating antioxidants and cytoprotective genes under oxidative stress. This review discusses the mechanisms underlying chemotherapy-induced kidney injury, focusing on the role of Nrf2 in cancer therapy and its redox regulation in cisplatin-induced kidney injury. We also explore Nrf2's signaling pathways, post-translational modifications, and its involvement in autophagy, as well as examine redox-based strategies for modulating Nrf2 in cisplatin-induced kidney injury while considering the limitations and potential off-target effects of Nrf2 modulation. Understanding the redox regulation of Nrf2 in cisplatin-induced kidney injury holds significant promise for developing novel therapeutic interventions. This knowledge could provide valuable insights into potential strategies for mitigating the nephrotoxicity associated with cisplatin, ultimately enhancing the safety and efficacy of cancer treatment.

PURPOSE:Platinum-based chemotherapy with or without immunotherapy is the mainstay of treatment for advanced stage non-small cell lung cancer (NSCLC) lacking a molecular driver alteration. Pre-clinical studies have reported that pharmacological ascorbate (P-AscH-) enhances NSCLC response to platinum-based therapy. We conducted a phase II clinical trial combining P-AscH- with carboplatin-paclitaxel chemotherapy.EXPERIMENTAL DESIGN:Chemotherapy naïve advanced stage NSCLC patients received 75 g ascorbate twice per week intravenously with carboplatin and paclitaxel every three weeks for four cycles. The primary endpoint was to improve tumor response per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 compared to the historical control of 20%. The trial was conducted as an optimal Simon's two-stage design. Blood samples were collected for exploratory analyses.RESULTS:The study enrolled 38 patients and met its primary endpoint with an objective response rate of 34.2% (p = 0.03). All were confirmed partial responses (cPR). The disease control rate was 84.2% (stable disease + cPR). Median progression-free and overall survival were 5.7 months and 12.8 months, respectively. Treatment-related adverse events (TRAE) included one grade 5 (neutropenic fever) and five grade 4 events (cytopenias). Cytokine and chemokine data suggest that the combination elicits an immune response. Immunophenotyping of peripheral blood mononuclear cells demonstrated an increase in effector CD8 T-cells in patients with a progression-free survival (PFS) ≥ 6 months.CONCLUSIONS:The addition of P-AscH- to platinum-based chemotherapy improved tumor response in advanced stage NSCLC. P-AscH- appears to alter the host immune response and needs further investigation as a potential adjuvant to immunotherapy.

Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2●− and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.

Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in vivo study is needed to further confirm the findings from the in vitro study.

Lipoprotein lipase (LPL) is a gatekeeper for the delivery of fat to tissues in the body. Dietary and endogenous fat circulated as triglycerides in triglyceride-rich lipoproteins. LPL hydrolyses these circulating triglycerides, releasing fatty acids that can be taken up by tissues. The activity of LPL is regulated at many stages and by many different proteins in a tissue specific manner, allowing for the distribution of fatty acid fuel among tissues to be altered according to metabolic conditions. The central role of LPL in lipid delivery also makes it relevant to the pathogenesis of metabolic disease and a potential therapeutic target.