Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP5+, augments glucocorticoid-induced apoptosis in WEHI7.2 murine thymic lymphoma cells, suggesting that it may have potential as a lymphoma therapeutic. However, the mechanism by which MnTE-2-PyP5+ potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H2O2]ss) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP5+ is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H2O2]ss levels. The combined treatment depletes GSH, oxidizes the 2GSH:GSSG ratio, and causes protein glutathionylation to a greater extent than glucocorticoid treatment alone. Removal of the glucocorticoid-generated H2O2 or depletion of glutathione by BSO prevents MnTE-2-PyP5+ from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP5+ glutathionylates p65 NF-κB and inhibits NF-κB activity. Inhibition of NF-κB with SN50, an NF-κB inhibitor, enhances glucocorticoid-induced apoptosis to the same extent as MnTE-2-PyP5+. Taken together, these findings indicate that: 1) H2O2 is important for MnTE-2-PyP5+ activity; 2) Mn-TE-2-PyP5+ cycles with GSH; and 3) MnTE-2-PyP5+ potentiates glucocorticoid-induced apoptosis by glutathionylating and inhibiting critical survival proteins, including NF-κB. In the clinic, over-expression of NF-κB is associated with a poor prognosis in lymphoma. MnTE-2-PyP5+ may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which approximately 40% of the patients respond well to current chemotherapy, but the prognosis for the other 60% is poor. The Leukemia/Lymphoma Molecular Profiling Project (LLMPP) used microarray technology to define a molecular profile for each of 240 patients with DLBCL and develop a molecular outcome predictor score that accurately predicted patient survival. Data from our laboratory and others suggest that alterations in antioxidant defense enzyme levels and redox environment can be oncogenic and affect the response to glucocorticoid treatment, one of the components of combination chemotherapy regimens for lymphoma. The goal of the current study was to reanalyze the LLMPP microarray data to determine whether the levels of antioxidant defense enzymes and redox proteins were correlated with prognosis in DLBCL. We found that patients with DLBCL with the worst prognosis, according to the outcome predictor score, had decreased expression of catalase, glutathione peroxidase, manganese superoxide dismutase, and VDUP1, a protein that inhibits thioredoxin activity. The data suggest that the patients with the worst prognosis combine a decrease in antioxidant defense enzyme expression with an increase in thioredoxin system function (the redox signature score). IntroductionDiffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. 1 It is a heterogeneous disease in which approximately 40% of the patients respond well to current chemotherapy, but the prognosis for the other 60% is poor. 1 To identify the patients who will not respond to current therapies, Staudt and coworkers 2 designed the Leukemia/Lymphoma Molecular Profiling Project (LLMPP). This project used microarray technology to define a molecular profile for each of 240 patients with DLBCL. By using these profiles, the authors identified two gene-expression subsets, germinal center B-cell-like and activated B-cell-like, that corresponded to 2 subpopulations of patients with significantly different survival. 2 A third subpopulation, type III, fit neither gene-expression subset. 3 Subsequent analysis of these data resulted in the development of a molecular outcome predictor score (OPS) that accurately predicted patient survival, which includes the response to therapy. 3 The success in using molecular profiling to predict patient outcome suggests that the same data can be used for a systems biology approach to identify pathways or groups of genes involved in biologic processes that affect prognosis, and to suggest new chemotherapeutic targets. In the current study, we took this approach to examine the role of antioxidant defense enzymes and other proteins that modulate the cellular redox environment in prognosis of DLBCL. A large number of studies suggest the levels of these proteins play a role in diverse tumor types [4][5][6] ; however, the role of these proteins in DLBCL prognosis is unknown.The intracellular redox environment depends on the relative production and r...

Glucocorticoid-induced apoptosis is exploited for the treatment of hematologic malignancies. Innate and acquired resistance limits treatment efficacy; however, resistance mechanisms are not well understood. Previously, using WEHI7.2 murine thymic lymphoma cells, we found that increasing the resistance to hydrogen peroxide (H 2 O 2 ) by catalase transfection or selection for H 2 O 2 resistance caused glucocorticoid resistance. This suggests the possibility that increasing H 2 O 2 sensitivity could sensitize the cells to glucocorticoids. In other cell types, increasing manganese superoxide dismutase (MnSOD) can increase intracellular H 2 O 2 . The current study showed that increased expression of MnSOD sensitized WEHI7.2 cells to glucocorticoid-induced apoptosis and H 2 O 2 . Treatment of WEHI7.2 cells with the catalytic antioxidant Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP 5+ ), a manganoporphyrin, mimicked the effects of increased MnSOD expression. MnTE-2-PyP 5+ also sensitized WEHI7.2 cells to cyclophosphamide and inhibited cell growth; it had no effect on the WEHI7.2 cell response to doxorubicin or vincristine. In primary follicular lymphoma cells, MnTE-2-PyP 5+ increased cell death due to dexamethasone. Treatment of H9c2 cardiomyocytes with MnTE-2-PyP 5+ inhibited doxorubicin cytotoxicity. The profile of MnTE-2-PyP 5+ effects suggests MnTE-2-PyP 5+ has potential for use in hematologic malignancies that are treated with glucocorticoids, cyclophosphamide, and doxorubicin. [Cancer Res 2009;69(13):5450-7]

The interplay between the accumulation of cationic MnPs and their potency as catalysts for oxidation of Asc, protein cysteines, and GSH controls the magnitude of their anticancer therapeutic effects.

Purpose: Green tea consumption has been associated with decreased risk of certain types of cancers in humans. Induction of detoxification enzymes has been suggested as one of the biochemical mechanisms responsible for the cancer-preventive effect of green tea. We conducted this clinical study to determine the effect of repeated green tea polyphenol administration on a major group of detoxification enzymes, glutathione S-transferases (GST). Methods: A total of 42 healthy volunteers underwent a 4-week washout period by refraining from tea or tea-related products. At the end of the washout period, a fasting blood sample was collected, and plasma and lymphocytes were isolated for assessment of GST activity and level. Following the baseline evaluation, study participants underwent 4 weeks of green tea polyphenol intervention in the form of a standardized Polyphenon E preparation at a dose that contains 800 mg epigallocatechin gallate (EGCG) once a day. Polyphenon E was taken on an empty stomach to optimize the oral bioavailability of EGCG. Upon completion of the intervention, samples were collected for postintervention GST assessment. Results: Four weeks of Polyphenon E intervention enhanced the GST activity in blood lymphocytes from 30.7 F 12.2 to 35.1 F 14.3 nmol/min/mg protein, P = 0.058. Analysis based on baseline activity showed that a statistically significant increase (80%, P = 0.004) in GST activity was observed in individuals with baseline activity in the lowest tertile, whereas a statistically significant decrease (20%, P = 0.02) in GST activity was observed in the highest tertile. In addition, Polyphenon E intervention significantly increased the GST-P level in blood lymphocytes from 2,252.9 F 734.2 to 2,634.4 F 1,138.3 ng/mg protein, P = 0.035. Analysis based on baseline level showed that this increase was only significant (P = 0.003) in individuals with baseline level in the lowest tertile, with a mean increase of 80%. Repeated Polyphenon E administration had minimal effects on lymphocyte GST-M and plasma GST-A levels. There was a small but statistically significant decrease (8%, P = 0.003) in plasma GST-A levels in the highest tertile. Conclusions: We conclude that 4 weeks of Polyphenon E administration resulted in differential effects on GST activity and level based on baseline enzyme activity/level, with GST activity and GST-P level increased significantly in individuals with low baseline enzyme activity/level. This suggests that green tea polyphenol intervention may enhance the detoxification of carcinogens in individuals with low baseline detoxification capacity. (Cancer Epidemiol Biomarkers Prev 2007;16(8):1662 -6)

The manganese porphyrin, manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin (MnTE-2-PyP5+), acts as a pro-oxidant in the presence of intracellular H2O2. Mitochondria are the most prominent source of intracellular ROS and important regulators of the intrinsic apoptotic pathway. Due to the increased oxidants near and within the mitochondria, we hypothesized that the mitochondria are a target of the pro-oxidative activity of MnTE-2-PyP5+ and that we could exploit this effect to enhance the chemotherapeutic response in lymphoma. In this study, we demonstrate that MnTE-2-PyP5+ modulates the mitochondrial redox environment and sensitizes lymphoma cells to anti-lymphoma chemotherapeutics. MnTE-2-PyP5+ increased dexamethasone-induced mitochondrial ROS and oxidation of the mitochondrial glutathione pool in lymphoma cells. The combination treatment induced glutathionylation of Complexes I, III and IV in the electron transport chain, and decreased the activity of Complexes I and III, but not the activity of Complex IV. Treatment with the porphyrin and dexamethasone also decreased cellular ATP levels. Rho(0) malignant T-cells with impaired mitochondrial electron transport chain function were less sensitive to the combination treatment than wild-type cells. These findings suggest that mitochondria are important for the porphyrin’s ability to enhance cell death. MnTE-2-PyP5+ also augmented the effects of 2-deoxy-D-glucose (2DG), an antiglycolytic agent. In combination with 2DG, MnTE-2-PyP5+ increased protein glutathionylation, decreased ATP levels more than 2DG treatment alone and enhanced 2DG-induced cell death in primary B-ALL cells. MnTE-2-PyP5+ did not enhance dexamethasone- or 2DG-induced cell death in normal cells. Our findings suggest that MnTE-2-PyP5+ has potential as an adjuvant for the treatment of hematologic malignancies.

Superoxide dismutases play an important role in human health and disease. Three decades of effort have gone into synthesizing SOD mimics for clinical use. The result is the Mn porphyrins which have SOD-like activity. Several clinical trials are underway to test the efficacy of these compounds in patients, particularly as radioprotectors of normal tissue during cancer treatment. However, aqueous chemistry data indicate that the Mn porphyrins react equally well with multiple redox active species in cells including H2O2, O2•-, ONOO-, thiols, and ascorbate among others. The redox potential of the Mn porphyrins is midway between the potentials for the oxidation and reduction of O2•-. This positions them to react equally well as oxidants and reductants in cells. The result of this unique chemistry is that: 1) the species the Mn porphyrins react with in vivo will depend on the relative concentrations of the reactive species and Mn porphyrins in the cell of interest, and 2) the Mn porphyrins will act as catalytic (redox cycling) agents in vivo. The ability of the Mn porphyrins to catalyze protein S-glutathionylation means that Mn porphyrins have the potential to globally modulate cellular redox regulatory signaling networks. The purpose of this review is to summarize the data that indicate the Mn porphyrins have diverse reactions in vivo that are the basis of the observed biological effects. The ability to catalyze multiple reactions in vivo expands the potential therapeutic use of the Mn porphyrins to disease models that are not SOD based.

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