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Chemotherapy-induced cognitive impairment (CICI) has been observed in a large fraction of cancer survivors. Although many of the chemotherapeutic drugs do not cross the blood-brain barrier, following treatment, the structure and function of the brain are altered and cognitive dysfunction occurs in a significant number of cancer survivors. The means by which CICI occurs is becoming better understood, but there still remain unsolved questions of the mechanisms involved. The hypotheses to explain CICI are numerous. More than 50% of FDA-approved cancer chemotherapy agents are associated with reactive oxygen species (ROS) that lead to oxidative stress and activate a myriad of pathways as well as inhibit pathways necessary for proper brain function. Oxidative stress triggers the activation of different proteins, one in particular is tumor necrosis factor alpha (TNFα). Following treatment with various chemotherapy agents, this pro-inflammatory cytokine binds to its receptors at the blood-brain barrier and translocates to the parenchyma via receptor-mediated endocytosis. Once in brain, TNFα initiates pathways that may eventually lead to neuronal death and ultimately cognitive impairment. TNFα activation of the c-jun N-terminal kinases (JNK) and Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathways may contribute to both memory decline and loss of higher executive functions reported in patients after chemotherapy treatment. Chemotherapy also affects the brain's antioxidant capacity, allowing for accumulation of ROS. This review expands on these topics to provide insights into the possible mechanisms by which the intersection of oxidative stress and TNFΑ are involved in chemotherapy-induced cognitive impairment.

Aim Extracellular superoxide dismutase (ECSOD) and the cysteine/glutamate transporter (Cys)/(xCT) are tumor microenvironment (TME) redox state homeostasis regulators. Altered expression of ECSOD and xCT can lead to imbalance of the TME redox state and likely have a profound effect on cancer invasion. In the present study, we investigated whether ECSOD and xCT could be therapeutic targets for prostate cancer (PCa) invasion. Results Immunohistochemistry of tumor microarray PCa tissues (N = 165) with high Gleason scores indicated that xCT protein expression is significantly increased while ECSOD protein expression is significantly decreased. Metastatic PCa indicated ECSOD protein expression is significantly decreased in epithelial area whereas xCT protein expression is significantly increased in stromal area. Furthermore, inhibition of extracellular O2•- by overexpression of ECSOD or alteration of the extracellular Cys/CySS ratio by knockdown of xCT protein inhibited PCa cell invasion. Simultaneous overexpression of ECSOD and knockdown xCT inhibited PCa cell invasion more than overexpression of ECSOD or knockdown of xCT alone. In the co-culturing system, simultaneous overexpression of ECSOD and knockdown of xCT in prostate stromal WPMY-1 cells inhibited PCa cell invasiveness more than overexpression of ECSOD alone. The decrease in PCa invasion correlated with increased of extracellular H2O2 levels. Notably, overexpression of catalase in TME reversed the inhibitory effect of ECSOD on cancer cell invasion. Conclusion Impaired ECSOD activity and an upregulated of xCT protein expression may be clinical features of an aggressive PCa, particularly metastatic cancers and/or those with a high Gleason score. Therefore, shifting the extracellular redox state toward an oxidizing status by targeted modulation of ECSOD and xCT, in both cancer and stromal cells, may provide a greater strategy for potential therapeutic interventions of aggressive PCa.

Radiation therapy (RT) is commonly used for the treatment of localized prostate cancer (PCa). However, cancer cells often develop resistance to radiation through unknown mechanisms and pose an intractable challenge. Radiation resistance is highly unpredictable, rendering the treatment less effective in many patients and frequently causing metastasis and cancer recurrence. Understanding the molecular events that cause radioresistance in PCa will enable us to develop adjuvant treatments for enhancing the efficacy of RT. Radioresistant PCa depends on the elevated DNA repair system and the intracellular levels of reactive oxygen species (ROS) to proliferate, self-renew, and scavenge anti-cancer regimens, whereas the elevated heat shock protein 90 (HSP90) and the epithelial-mesenchymal transition (EMT) enable radioresistant PCa cells to metastasize after exposure to radiation. The up-regulation of the DNA repairing system, ROS, HSP90, and EMT effectors has been studied extensively, but not targeted by adjuvant therapy of radioresistant PCa. Here, we emphasize the effects of ionizing radiation and the mechanisms driving the emergence of radioresistant PCa. We also address the markers of radioresistance, the gene signatures for the predictive response to radiotherapy, and novel therapeutic platforms for targeting radioresistant PCa. This review provides significant insights into enhancing the current knowledge and the understanding toward optimization of these markers for the treatment of radioresistant PCa.

Cancer cells typically experience higher oxidative stress than normal cells, such that elevating pro-oxidant levels can trigger cancer cell death. Although pre-exposure to mild oxidative agents will sensitize cancer cells to radiation, this pre-exposure may also activate the adaptive stress defense system in normal cells. Ascorbic acid is a prototype redox modulator that when infused intravenously appears to kill cancers without injury to normal tissues; however, the mechanisms involved remain elusive. In this study, we show how ascorbic acid kills cancer cells and sensitizes prostate cancer to radiation therapy while also conferring protection upon normal prostate epithelial cells against radiation-induced injury. We found that the NF-κB transcription factor RelB is a pivotal determinant in the differential radiosensitization effects of ascorbic acid in prostate cancer cells and normal prostate epithelial cells. Mechanistically, high reactive oxygen species concentrations suppress RelB in cancer cells. RelB suppression decreases expression of the sirtuin SIRT3 and the powerful antioxidant MnSOD, which in turn increases oxidative and metabolic stresses in prostate cancer cells. In contrast, ascorbic acid enhances RelB expression in normal cells, improving antioxidant and metabolic defenses against radiation injury. In addition to showing how RelB mediates the differential effects of ascorbic acid on cancer and normal tissue radiosensitivities, our work also provides a proof of concept for the existence of redox modulators that can improve the efficacy of radiotherapy while protecting against normal tissue injury in cancer settings. Cancer Res; 77(6); 1345–56. ©2017 AACR.

Cancer treatments are developing fast and the number of cancer survivors could arise to 20 million in United State by 2025. However, a large fraction of cancer survivors demonstrate cognitive dysfunction and associated decreased quality of life both shortly, and often long-term, after chemotherapy treatment. The etiologies of chemotherapy induced cognitive impairment (CICI) are complicated, made more so by the fact that many anti-cancer drugs cannot cross the blood-brain barrier (BBB). Multiple related factors and confounders lead to difficulties in determining the underlying mechanisms. Chemotherapy induced, oxidative stress-mediated tumor necrosis factor-alpha (TNF-α) elevation was considered as one of the main candidate mechanisms underlying CICI. Doxorubicin (Dox) is a prototypical reactive oxygen species (ROS)-generating chemotherapeutic agent used to treat solid tumors and lymphomas as part of multi-drug chemotherapeutic regimens. We previously reported that peripheral Dox-administration leads to plasma protein damage and elevation of TNF-α in plasma and brain of mice. In the present study, we used TNF-α null (TNFKO) mice to investigate the role of TNF-α in Dox-induced, oxidative stress-mediated alterations in brain. We report that Dox-induced oxidative stress in brain is ameliorated and brain mitochondrial function assessed by the Seahorse-determined oxygen consumption rate (OCR) is preserved in brains of TNFKO mice. Further, we show that Dox-decreased the level of hippocampal choline-containing compounds and brain phospholipases activity are partially protected in TNFKO group in MRS study. Our results provide strong evidence that Dox-targeted mitochondrial damage and levels of brain choline-containing metabolites, as well as phospholipases changes decreased in the CNS are associated with oxidative stress mediated by TNF-α. These results are consistent with the notion that oxidative stress and elevated TNF-α in brain underlie the damage to mitochondria and other pathological changes that lead to CICI. The results are discussed with reference to our identifying a potential therapeutic target to protect against cognitive problems after chemotherapy.

Significance: Cancer cells that are resistant to radiation and chemotherapy are a major problem limiting the success of cancer therapy. Aggressive cancer cells depend on elevated intracellular levels of reactive oxygen species (ROS) to proliferate, self-renew, and metastasize. As a result, these aggressive cancers maintain high basal levels of ROS compared with normal cells. The prominence of the redox state in cancer cells led us to consider whether increasing the redox state to the condition of oxidative stress could be used as a successful adjuvant therapy for aggressive cancers. Recent Advances: Past attempts using antioxidant compounds to inhibit ROS levels in cancers as redox-based therapy have met with very limited success. However, recent clinical trials using pro-oxidant compounds reveal noteworthy results, which could have a significant impact on the development of strategies for redox-based therapies. Critical Issues: The major objective of this review is to discuss the role of the redox state in aggressive cancers and how to utilize the shift in redox state to improve cancer therapy. We also discuss the paradox of redox state parameters; that is, hydrogen peroxide (H(2)O(2)) as the driver molecule for cancer progression as well as a target for cancer treatment. Future Directions: Based on the biological significance of the redox state, we postulate that this system could potentially be used to create a new avenue for targeted therapy, including the potential to incorporate personalized redox therapy for cancer treatment.

Significant advances in the efficacy of cancer therapy have been accompanied by an escalation in side effects that result from therapy-induced injury to normal tissues. Cardiac injury is a major cause of death in cancer survivors, but biomarkers for it are detectable only after tissue injury has occurred. Extracellular vesicles (EVs) sort out oxidized proteins from tissues into the circulation as a compensatory mechanism for preventing cellular proteotoxicity. Thus, the protein contents of EVs released during the pre-degeneration stage reflects oxidative stress phenomenon that is occurring in the early damaged tissue. Here, we evaluate the potential of using circulating EVs as early diagnostic markers for long-term cardiac injury. Using a mouse model of doxorubicin (DOX)-induced cardiac injury, we found a significant increase in circulating EVs (DOX_EVs) compared to saline-treated controls. DOX_EVs exhibited a higher level of 4-hydroxynonenal adducted proteins, a lipid peroxidation product linked to DOX-induced cardiotoxicity. Proteomic profiling of DOX_EVs revealed the distinctive presence of brain/heart, muscle, and liver isoforms of glycogen phosphorylase (GP), and their origins were verified to be heart, skeletal muscle, and liver, respectively. The presence of brain/heart GP (PYGB) in DOX_EVs correlated with a reduction of PYGB in heart, but not brain tissues. Manganese superoxide dismutase (MnSOD) overexpression as well as pretreatment with FDA- approved cardioprotective agents and MnSOD mimetics resulted in a reduction of EV-associated PYGB in mice treated with DOX. Kinetic studies indicated that EVs containing PYGB are released prior to the rise of cardiac troponin (the current standard-of-care marker of cardiac injury) in the blood after DOX treatment, suggesting that PYGB is an earlier indicator of cardiac injury than conventional serum biomarkers. Thus, EVs containing PYGB serve as a liquid biopsy that is clinically attractive diagnostic biomarker for therapeutic interventions.

Prostate cancer (PCa), the second leading cause of male cancer deaths in the United States, is most frequently treated with radiation therapy (RT). We screened 786 FDA-approved compounds to identify drugs that can radiosensitize PCa cells while radioprotecting non-cancer prostate epithelial cells and identified Betamethasone (BET) as a lead compound with these properties. BET increases ROS levels in PCa cells (high oxidized redox state) but not in normal prostate epithelial cells (low oxidized redox state). Remarkably, BET has an opposite effect on RelB, a non-canonical redox-sensitive NF-κB pathway. BET suppresses the expression of RelB and RelB target genes including MnSOD and survivin in PCa cells, while it induces expression of RelB and MnSOD in non-cancer cells. The differential effects of BET are, in part, attributable to the intrinsic differences in the cellular redox states of normal versus PCa cells. Intriguingly, RNA seq and ChIP assays identify B-cell linker protein (BLNK) as a novel RelB transcription regulator that is differentially regulated by BET in normal and cancer cells. Proximity-ligation assays and knockdown of BLNK confirm that RelB-BLNK possibly interact in the nuclear compartment. BET/RT treatments that target disruptions of RelB and BLNK expressions reverse the survival of PCa cells but not of normal prostate epithelial cells. Our study is the first to establish that the RelB-BLNK mediated NF-κB non-canonical pathway is a novel mechanism to regulate the differential responses to radiation therapy of normal prostate epithelial and prostate cancer cells. The results of this study fill in a critical gap in scientific knowledge of how the differences in redox conditions of normal and tumor cells trigger differential responses to pro-oxidants. They will facilitate the translation of therapeutic approaches that use prooxidants to push cancer cells into oxidative stress overload but stimulate adaptive responses in normal cells to selectively enhance RT efficacy.

Antioxidant compounds that can mimic the activity of endogenous enzymes via redox cycling offer a novel method of targeted cancer treatment. Here, we investigated the therapeutic effect of a mitochondrial- targeted, MnSOD mimetic, Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin, (MnTnBuOE) within sensitive and resistant myeloid lineage hematopoietic malignancies and utilize the hematopoietic disorder myelodysplastic syndrome (MDS) for the presentation of mechanistic insights to MnTnBuOE efficacy. MnTnBuOE treatment results in an increase in both CD34+CD38‒ and CD34+CD38+ normal stem/progenitor cell viability. Similar treatment shows significant cytotoxicity within AML and MDS models. Furthermore, manipulation of the cellular redox state via inhibition of glutathione synthesis, glutathione reductase activity, and treatment with glutathione disulfide sensitizes drug resistant CD34+ CML cells to MnTnBuOE treatment induced cytotoxicity. These results demonstrate glutathione metabolism and redox balance as a potential determinant of MnTnBuOE efficacy. Transcriptional analysis indicates that MnTnBuOE treatment initiates activator protein 1 (AP-1) activity in MDSL cells. DNA binding and immunoprecipitation identifies JunB as a transcriptional mediator of MnTnBuOE induced cytotoxicity. This is supported by siRNA knock down of JunB, resulting in protection against MnTnBuOE induced cytotoxicity. JunB precipitation via reaction with biotin-1,3-cyclopentadione demonstrates MnTnBuOE mediated oxidation of the JunB DNA binding region via hydrogen peroxide. This, in concert with co-immunoprecipitation of JunB, offers an alternative mechanism of JunB transcriptional activation through the identification of Ref-1; a protein specifically involved in the redox-modification of JunB. The finding that MnTnBuOE can target varying cellular redox states and exert selective cytotoxicity in malignant cells via differential activation of molecular pathways controlling lifespan and cell fate will significantly aid further development of safe, effective redox-active therapeutics for clinical applications.

The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox) balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC) and intra- and extracellular glutathione (GSH)/glutathione disulfide (GSSG) ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF) than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens.

Antioxidant compounds that can mimic the activity of endogenous enzymes via redox cycling offer a novel method of targeted cancer treatment. We investigated the therapeutic effect of a mitochondrial- targeted, MnSOD mimetic, Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin, (MnTnBuOE) within myeloid lineage hematopoietic malignancies and utilize the hematopoietic disorder myelodysplastic syndrome (MDS) for the presentation of mechanistic insights to MnTnBuOE efficacy. Consistent with our previous report, MnTnBuOE treatment offers protection of normal HSCs demonstrated by an increase in both CD34+CD38- and CD34+CD38+ stem/progenitor cell viability. However, similar treatment shows significant cytotoxicity mediated by MnTnBuOE induced hydrogen peroxide within acute myeloid leukemia (AML) and MDS models. Transcriptional array analysis indicates that MnTnBuOE treatment initiates activator protein 1 (AP-1) activity. DNA mediated pull down and immunoprecipitation identifies JunB as a transcriptional mediator of MnTnBuOE induced cytotoxicity. Stable knock down of JunB provides protection against MnTnBuOE induced cytotoxicity confirming JunB’s critical role in MnTnBuOE induced hematopoietic malignant cell death. Precipitation with biotin-1,3-cyclopentadione combined with LC-MS/MS analysis of JunB identified Cys285 as the critical cysteine within the DNA binding region of JunB that is susceptible to oxidation by hydrogen peroxide. This, in concert with co-immunoprecipitation of JunB placed Ref-1 as a protein specifically involved in the redox-modification of JunB. Lentiviral mediated knock down of Ref-1 indicates that the interaction between Ref-1 and JunB is critical for MnTnBuOE induced cytotoxicity. The AP-1 ability to bind consensus promoter sequence regardless of methylation status, indicates MnTnBuOE mediated activation of JunB as a potential alternative therapy for aggressive or chemoresistant malignancies in a p53 independent manner. The finding that MnTnBuOE can exert selective cytotoxicity in malignant cells via redox-dependent differential activation of molecular pathways controlling cell fate will significantly aid further development of safe, effective redox-active therapeutics to prevent normal tissue injury and enhance chemotherapeutic efficacy.

Previous studies in our laboratory demonstrated that 7, 12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) treatment induced apoptosis and mitochondrial translocation of the tumor suppressor p53 in a mouse skin carcinogenesis model, suggesting that oncogenic versus cell death signaling involve a common mediator. Mutational activation of oncogenic Ras is an early event and has been demonstrated to play a critical role in skin carcinogenesis. A malignant skin keratinocyte cell line (308), which carries a H-ras mutation at codon 61, showed elevated p53 levels, increased caspase 3 activity and enhanced apoptosis after TPA treatment. In contrast, the non-malignant counterpart (C50) showed undetectable levels of p53 and less apoptosis than 308 cells similarly treated. Inhibition of NADPH-oxidase (NOX) by diphenyleneiodonium suppressed p53 activation and apoptosis in 308 cells, linking Ras mutation to NOX-induced p53 activation, which was further supported by the finding that siRNA to Rac1 inhibited p53 activation after TPA treatment. Application of DPI to DMBA-initiated skin tissue significantly blocked TPA-mediated increased p53 levels and reduced apoptosis in skin epidermal tissues. Taken together, our results suggest that NOX bridges oncogenic activation and p53 mitochondrial translocation to apoptosis in the multistage chemical-induced skin carcinogenesis model.

In the present study, we used genetically engineered B6C3 mice [mice overexpressing manganese superoxide dismutase (TgM+/+), mice in which inducible nitric oxide synthase had been inactivated (iNOSKO−/−), and crosses of these two genotypes] to study the role of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in the development of acute Adriamycin-induced cardiotoxicity. Both nontransgenic and genetically engineered mice were treated with 20 mg/kg Adriamycin and cardiac left ventricular tissues studied at 0, 3, 6, and 24 hours. Ultrastructural damage and levels of 4-hydroxy-2-nonenal (4HNE) protein adducts and 3-nitrotyrosine (3NT) were determined in cardiomyocytes using immunogold ultrastructural techniques. Our previous results showed that Adriamycin caused mitochondrial injury without significant nuclear or cytoplasmic damage at early time points. Interestingly, overexpression of MnSOD protected against acute mitochondrial injury, whereas deficiency in iNOS potentiated mitochondrial injury in comparison with levels of injury present in cardiomyocyte mitochondria of nontransgenic mice. In TgM+/+ mice, there was a significant inverse correlation between mitochondrial injury and 4HNE/3NT levels at all time points analyzed, suggesting that reactive oxygen species/reactive nitrogen species damage products directly regulated acute Adriamycin-induced mitochondrial injury in these mice. The present studies are the first to directly quantify the effects of MnSOD and iNOS on mitochondrial injury during acute Adriamycin-induced cardiotoxicity and show extensive and specific patterns of posttranslational modifications of mitochondrial proteins following Adriamycin treatment.

Generation of reactive oxygen species (ROS) has been implicated in the development of cancer. Groundwork establishing mitochondria as a critical source of ROS generation and the role of manganese superoxide dismutase (MnSOD) in preventing mitochondria-mediated cell death have been well established. In a seemingly contradictory role, it also is well documented that increased MnSOD expression suppresses the carcinogenesis effect of ROS. Our recent studies demonstrated that overexpression of MnSOD reduced tumor incidence in the two-stage 7,12-dimethylbenz(a)-anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) skin carcinogenesis model. However, reduction of MnSOD by heterozygous knockout of the MnSOD gene (Sod 2+/-) did not lead to an increase in tumor incidence. Thus, how modulation of mitochondrial ROS levels alter the outcome of developing cancer is unclear. This review will provide background information on the sequence of ROS-mediated events in the mitochondria and evidence that suggests that the antioxidant and tumor suppressor functions of MnSOD are indeed inter-related. It also will offer insights into the mechanisms by which MnSOD modulates the outcome of early stage skin carcinogenesis.

The tumor suppressor gene p53 is activated by reactive oxygen species–generating agents. After activation, p53 migrates to mitochondria and nucleus, a response that eventually leads to apoptosis, but how the two events are related is unknown. Herein, we show that p53 translocation to mitochondria precedes its translocation to nucleus in JB6 skin epidermal cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Translocation of p53 to mitochondria occurs within 10 minutes after TPA application. In the mitochondria, p53 interacts with the primary antioxidant enzyme, manganese superoxide dismutase (MnSOD), consistent with the reduction of its superoxide scavenging activity, and a subsequent decrease of mitochondrial membrane potential. In contrast to the immediate action on mitochondria, p53 transcriptional activity in the nucleus increases at 1 hour following TPA application, accompanied by an increase in the levels of its target gene bax at 15 hours following TPA treatment. Activation of p53 transcriptional activity is preventable by application of a SOD mimetic (MnTE-2-PyP5+). Thus, p53 translocation to mitochondria and subsequent inactivation of MnSOD explains the observed mitochondrial dysfunction, which leads to transcription-dependent mechanisms of p53-induced apoptosis.

Studies in our laboratories showed that overexpression of manganese superoxide dismutase (MnSOD) reduced tumor incidence in a multistage skin carcinogenesis mouse model. However, reduction of MnSOD by heterozygous knockout of the MnSOD gene (MnSOD KO) did not lead to an increase in tumor incidence, because a reduction of MnSOD enhanced both cell proliferation and apoptosis. The present study extends our previous studies in the MnSOD KO mice and shows that apoptosis in mouse epidermis occurred prior to cell proliferation (6 versus 24 hours) when treated with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the possibility that a timed administration of SOD following apoptosis but before proliferation may lead to suppression of tumor incidence, we applied a SOD mimetic (MnTE-2-PyP5+) 12 hours after each TPA treatment. Biochemical studies showed that MnTE-2-PyP5+ suppressed the level of protein carbonyls and reduced the activity of activator protein-1 and the level of proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Histologic examination confirmed that MnTE-2-PyP5+ suppressed mitosis without interfering with apoptosis. Remarkably, the incidence and multiplicity of skin tumors were reduced in mice that received MnTE-2-PyP5+ before cell proliferation. These results show a novel strategy for an antioxidant approach to cancer intervention.

Resistance to platinum-based drugs (i.e., carboplatin) is a major clinical challenge in the current treatment of serous ovarian cancer. The molecular mechanisms underlying resistance are not yet fully clarified. In this study, we investigated whether the differences in redox state (levels of antioxidants and ROS) is correlated with carboplatin resistant phenotype of ovarian cancer cells (OV90C). We found that OV90C cells exhibited an oxidized redox state with a high level of H2O2 but a low level of GPxs and reduced form of thioredoxin 1. Furthermore, OV90C cells displayed an increase of O2 consumption and oxidative phosphorylation activity when compared to their parental cancer cells (OV90) and immortalized ovarian cells (hTER7). Next, we used MnTnBuOE-2-PyP5+(MnP), a redox- active MnSOD mimetic to investigate the dependence of ovarian cancer cell growth on the redox state. We found that OV90C cells preferentially uptake MnP more than OV90 cells, resulting in a greater inhibition of cell growth in OV90C cells versus OV90 cells. Combination treatment of MnP and carboplatin enhanced the killing effect of carboplatin in both OV90C cells and OV90 cells when compared to either MnP or carboplatin treatment alone. Moreover, MnP protected hTER7 cells from carboplatin-induced cell death. Mechanistically, the carboplatin enhanced redox cycle ability of MnP; which led to the induction of H2O2 levels and reduction of GSH/GSSG ratio, in ovarian cancer cells but not in normal cells. Thus, increasing of oxidized redox state sensitized ovarian cancer cells to carboplatin, especially carboplatin resistant cells. In conclusion, our data provide evidence that the oxidized redox state plays an important role in carboplatin resistant ovarian cancer cells survival and pushing ROS levels beyond their antioxidant capacity might serve as a novel treatment strategy for ovarian cancer patients with resistance toward platinum-based drugs.

Elevated oxidative stress is observed more frequently in cancer cells than in normal cells. It is therefore expected that additional exposure to a low level of reactive oxygen species (ROS) will push cancer cells toward death, whereas normal cells might maintain redox homeostasis through adaptive antioxidant responses. We previously showed that parthenolide enhances ROS production in prostate cancer cells through activation of NADPH oxidase. The present study identifies KEAP1 as the downstream redox target that contributes to parthenolide's radiosensitization effect in prostate cancer cells. In vivo, parthenolide increases radiosensitivity of mouse xenograft tumors but protects normal prostate and bladder tissues against radiation-induced injury. Mechanistically, parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX) in prostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1, which in turn leads to KEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In contrast, parthenolide increases oxidation of KEAP1 in normal prostate epithelial cells, leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of antioxidant enzymes. These results reveal a novel redox-mediated modification of KEAP1 in controlling the differential effect of parthenolide on tumor and normal cell radiosensitivity. Furthermore, they show it is possible to develop a tumor-specific radiosensitizing agent with radioprotective properties in normal cells. Cancer Res; 73(14); 4406–17. ©2013 AACR.

Integrins, a family of heterodimeric receptors for extracellular matrix, are promising therapeutic targets for ovarian cancer, particularly high-grade serous-type (HGSOC), as they drive tumor cell attachment, migration, proliferation and survival by activating focal adhesion kinase (FAK)-dependent signaling. Owing to the potential off-target effects of FAK inhibitors, disruption of the integrin signaling axis remains to be a challenge. Here, we tackled this barrier by screening for inhibitors being functionally cooperative with small-molecule VS-6063, a phase II FAK inhibitor. From this screening, JQ1, a potent inhibitor of Myc oncogenic network, emerged as the most robust collaborator. Treatment with a combination of VS-6063 and JQ1 synergistically caused an arrest of tumor cells at the G2/M phase and a decrease in the XIAP-linked cell survival. Our subsequent mechanistic analyses indicate that this functional cooperation was strongly associated with the concomitant disruption of activation or expression of FAK and c-Myc as well as their downstream signaling through the PI3K/Akt pathway. In line with these observations, we detected a strong co-amplification or upregulation at genomic or protein level for FAK and c-Myc in a large portion of primary tumors in the TCGA or a local HGSOC patient cohort. Taken together, our results suggest that the integrin–FAK signaling axis and c-Myc synergistically drive cell proliferation, survival and oncogenic potential in HGSOC. As such, our study provides key genetic, functional and signaling bases for the small-molecule-based co-targeting of these two distinct oncogenic drivers as a new line of targeted therapy against human ovarian cancer.

Extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies are released from most cell types; especially under cellular stress conditions, including oxidative stress. The high abundance of EVs in biological fluids and their unique stability make them a potential biomarker for many diseases. It is well recognized that in addition to ionizing radiation, nearly 50% of current FDA approved chemotherapeutic drugs cause reactive oxygen species (ROS) in target tissues. However, the molecular cargo contained within EVs that are released from chemotherapy-treated cells, as well as their contribution to tissue injury are unknown. Here we report that EV contents mirror pathological conditions of the tissues affected by chemotherapy. In mice, systemic injection of doxorubicin (DOX), a potent and known ROS generator chemotherapeutic agent used for aggressive cancer, caused approximately two-fold increase in circulating EVs (DOX_EVs) compared to the saline treated controls (SAL_EVs). In mouse (n=4) and human serum (n=24), EVs exhibits a significantly higher level of 4-hydroxynonenal adducted proteins post chemotherapy (P

Cancer cells exhibit a plasticity of redox state so that the conversion between redox status in tumor microenvironment (TME) can be accomplished rapidly, thus, extracellular redox-related proteins may be a new potential therapeutic targets for metastasis. Cysteine(CyS)/glutamate transporter(xCT) and extracellular superoxide dismutase (ECSOD) are mainly TME-redox state hemeostasis regulator, one control redox coupling ratio CyS/Cystine(CySS), the latter control prooxidants level. Herein, we demonstrated xCT protein expression is significantly increased and correlated with high Gleason score PCa tissues. In contrast, ECSOD protein expression is significantly decreased in high Gleason score PCa tissues. A decrease in ECSOD may result in elevation of extracellular prooxidant levels due to the increase in O2●– and OONO●, while overexpression of xCT may result in imbalance of extracellular thiol couples. These changes in the extracellular redox state are likely to have a profound effect on activation of invasion-related proteins. Inhibition of extracellular O2●– by overexpression of ECSOD using adenovirus SOD3 transduction or altering extracellular Cys/CySS levels by knock-downed xCT protein expression using siRNA xCT resulted in inhibition of PCa cell invasion and decreased activity of MMP2. Simultaneously overexpressed ECSOD and knock-downed xCT inhibited PCa cell invasion greater than overexpressed ECSOD or knock-downed xCT alone (~70%, 50%, and 30%, respectively). In the co-culturing system, overexpressed ECSOD and knock-downed xCT in both PC3 and stromal cells (WPMY-1) decreased PCa cell invasion greater than overexpressed ECSOD and knock-downed xCT in only PC3 cells or stromal cells. In conclusion, our data indicated that synergic modulation of ECSOD and xCT expression would elevate the success of site- specific redox based therapies at TME. Since, paracrine signaling from stromal cells induce PCa cells to grow and metastatic, targeting ECSOD and xCT in both cancer and stromal cells would provide a greater inhibition of PCa metastasis.

Manganese superoxide dismutase (MnSOD) plays a critical role in the survival of aerobic life, and its aberrant expression has been implicated in carcinogenesis and tumor resistance to therapy. However, despite extensive studies in MnSOD regulation and its role in cancer, when and how the alteration of MnSOD expression occurs during the process of tumor development in vivo are unknown. Here, we generated transgenic mice expressing a luciferase reporter gene under the control of human MnSOD promoter-enhancer elements and investigated the changes of MnSOD transcription using the 7,12-dimethylbenz(α)anthracene (DMBA)/12-O-tetradecanoylphorbol-l3-acetate (TPA) multistage skin carcinogenesis model. The results show that MnSOD expression was suppressed at a very early stage but increased at late stages of skin carcinogenesis. The suppression and subsequent restoration of MnSOD expression were mediated by two transcription-factors, Sp1 and p53. Exposure to DMBA and TPA activated p53 and decreased MnSOD expression via p53-mediated suppression of Sp1 binding to the MnSOD promoter in normal-appearing skin and benign papillomas. In squamous cell carcinomas, Sp1 binding increased because of the loss of functional p53. We used chromatin immunoprecipitation, electrophoretic mobility shift assay, and both knockdown and overexpression of Sp1 and p53 to verify their roles in the expression of MnSOD at each stage of cancer development. The results identify MnSOD as a p53-regulated gene that switches between early and advanced stages of cancer. These findings also provide strong support for the development of means to reactivate p53 for the prevention of tumor progression. Cancer Res; 71(21); 6684–95. ©2011 AACR.

Protein kinase C epsilon (PKCepsilon) overexpressing transgenic (PKCepsilon Tg) mice develop papilloma-independent squamous cell carcinomas (SCC) elicited by 7,12-dimethylbenz[a]anthracene (DMBA) tumor initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) tumor promotion. We examined whether epidermal cell turnover kinetics was altered during the development of SCC in PKCepsilon Tg mice. Dorsal skin samples were fixed for histological examination. A single application of TPA resulted in extensive infiltration of polymorphonuclear neutrophils (PMNs) into the epidermis at 24 h after TPA treatment in PKCepsilon Tg mice while wild-type (WT) mouse skin showed focal infiltration by PMNs. Complete epidermal necrosis was observed at 48 h in PKCepsilon Tg mice only; at 72 h, epidermal cell regeneration beginning from hair follicles was observed in PKCepsilon Tg mice. Since the first TPA treatment to DMBA-initiated PKCepsilon Tg mouse skin led to epidermal destruction analogous to skin abrasion, we propose the papilloma-independent phenotype may be explained by death of initiated interfollicular cells originally destined to become papillomas. Epidermal destruction did not occur after multiple doses of TPA, presumably reflecting adaptation of epidermis to chronic TPA treatment. Prolonged hyperplasia in the hair follicle may result in the early neoplastic lesions originally described by Jansen et al. (2001) by expanding initiated cells in the hair follicles resulting in the subsequent development of SCC.

Previous studies in our laboratories demonstrated that overexpression of manganese superoxide dismutase (MnSOD) suppressed both the incidence and multiplicity of papillomas in a DMBA/TPA multi-stage skin carcinogenesis model. The activity of activator protein-1 (AP-1), which is associated with tumor promotion, was reduced in MnSOD transgenic mice overexpressing MnSOD in the skin, suggesting that MnSOD may reduce tumor incidence by suppressing AP-1 activation. In the present study, we report that reduction of MnSOD by heterozygous knockout of the MnSOD gene (Sod2 -/+, MnSOD KO) increased the levels of oxidative damage proteins and the activity of AP-1 following TPA treatment. RNA levels of ornithine decarboxylase (ODC) were also increased, suggesting an increase in cell proliferation in the KO mice. Histological examination confirmed that the number of proliferating cells in DMBA/TPA-treated mouse skin were higher in the KO mice. Interestingly, histological examination also demonstrated greater numbers of apoptotic cells in the KO mice after DMBA/TPA treatment. Evidence of apoptosis, including DNA fragmentation, cytochrome c release from mitochondria, and caspase 3 activation were also observed by biochemical assays of the skin tissues. Apoptosis was associated with an increase in nuclear levels of p53 as determined by Western analysis. Quantitative immunogold ultrastructural analysis confirmed that p53 immunoreactive protein levels were increased to a greater level in the nuclei of epidermal cells from MnSOD KO mice compared to epidermal nuclei from wild type mice similarly treated. Moreover, p53 levels further increased in the mitochondria of DMBA/TPA treated mice, and this increase was much greater in the MnSOD KO than in the wild type mice, suggesting a link between MnSOD deficiency and mitochondrial-mediated apoptosis. Pathological examination reveals no difference in the incidence and frequency of papillomas comparing the KO mice and their wild type littermates. Taken together, these results suggest that: (1) MnSOD deficiency enhanced TPA-induced oxidative stress and AP-1 and p53 levels, consistent with the increase in both proliferation and apoptosis events in the MnSOD KO mice, and (2) increased apoptosis may negate increased proliferation in the MnSOD deficient mice during an early stage of tumor development.

Several cancer chemotherapeutic drugs and radiation mediate their effects, at least in part, by generation of reactive oxygen/nitrogen species. Therefore, the levels of antioxidants and status of cellular redox state have tremendous influence on the outcome of these therapies. We analyzed antioxidant proteins in normal prostate epithelial cells and tissues as well as prostate cancer cell lines and tissues. Highly aggressive androgen-independent prostate cancer PC3 and LNCAP-C42B cells demonstrated significantly higher levels of superoxide dismutase (SOD) 2, peroxyredoxin (Prx) 1, 2, 3, and 4, thioredoxin (Trx) 1 and 2, and thioredoxin reductase (TR) 1 and 2, relative to normal prostate epithelial PrEC cells and immortalized RWPE1 human prostate epithelial cells. Similarity, the highly aggressive human prostate cancer tissues (Gleason scores 4+4 and 4+5) demonstrated significantly higher levels of SOD1 and 2, Prx 3 and 4, Trx 1 and 2, and TR 1 and 2 relative to adjacent normal prostate tissues. In contrast, the expression levels of these antioxidant proteins were lower or were not altered in less aggressive human prostate cancer tissues (Gleason score 3+3) relative to adjacent normal prostate tissues. Utilizing Trx1 redox western as a marker of cellular redox states, we found more oxidized form of Trx1 in PC3 and LNCAP-C42B cells and human prostate cancer tissues (Gleason scores 4+4 and 4+5) despite their higher levels of Trx1 protein expression. Our data suggest that alteration in antioxidant proteins and cellular redox state may differentiate cancers from non-cancer tissues. Additionally, knowledge of redox state profiles can help to predict the response of each cancer cell type to chemotherapeutic drugs and radiation. 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 2072. doi:1538-7445.AM2012-2072

Cancer cells have aberrant manganese superoxide dismutase (MnSOD) activity and MnSOD deficient mice exhibit abnormal mitochondrial pathology. Recent data from human and animal studies indicate that the activity of MnSOD and the tumor suppressor gene, p53, are important for the maintenance of cellular energy metabolism. In addition, these critical proteins regulate oxidative stress to control the switch from oxidative phosphorylation to the aerobic glycolysis that is observed in cancer cells (Warburg's effect). It has been well-documented that MnSOD level is reduced in transformed cells and benign cancer tissues but increased in some aggressive cancers. However, there is little information concerning the regulation of MnSOD during carcinogenesis in vivo. Furthermore, when and how the alteration of MnSOD transcription occurs during the process of tumor development is unclear. To address these questions, we employed three complementary approaches in vitro and in vivo. First, we generated transgenic mice expressing luciferase reporter gene under the control of human MnSOD promoter/enhancer elements for investigating changes in MnSOD expression in animals by a non-invasive in vivo imaging. Second we used the 7-12-dimethylbenz(a)anthracene (DMBA)/TPA treated multistage skin carcinogenesis model to generate tumors and identify transcription factors associated with the expression of MnSOD in pathologically normal-appearing skin tissue, papillomas and squamous cell carcinoma. Third, we used Chromatin-immunoprecipation, EMSA, RNAi mediated knock-down and over-expression of candidates transcription factors in cultured cells to verify their roles in regulation of MnSOD at each stage of cancer development. The results demonstrate that reduction of MnSOD occurred very early in the carcinogenesis process and preceded the formation of any detectable tumor. The reduction of MnSOD transcription continued until papillomas were detected. However, as the benign tumor progressed into carcinoma, MnSOD expression was drastically increased. Contributing factors leading to the suppression and de-repression of MnSOD expression included Sp1, members of the NF-κB family, and p53. Among these important transcription factors, p53 appeared to play a major role in the switch from suppressed expression of MnSOD in the early carcinogenesis process to overexpression of MnSOD at late stages of cancer development. These results have identified MnSOD as a p53 regulated gene during cancer development and progression and highlighted the importance of mitochondrial oxidative stress in carcinogenesis at the early stages and chemo- and radio-resistance at the late stages of cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2011-1606