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Cancer trends over a two-decade period show a greater reduction in cancer mortality rates for non-Hispanic Whites than for Native Americans. The Partnership for Native American Cancer Prevention (NACP) was established to address cancer health disparities that impact Native Americans. The partners are Northern Arizona University, the University of Arizona Cancer Center, Arizona's tribal communities and the National Cancer Institute. The activities include outreach, research and cancer education. Overall, NACP seeks to expand capacity for culturally-sensitive and community-relevant research on cancer, and to continue developing respectful collaborations that will empower sovereign Native American communities to define, implement, and achieve their goals for cancer health equity., Competing Interests: Conflict of Interest The authors declare that no competing or conflict of interests exists. The funders had no role in study design, writing of the manuscript, or decision to publish.

This concept paper addresses communication issues arising between physicians and their patients. To facilitate the communication of essential diagnostic pathology information to patients, and address their questions and concerns, we propose that "Pathology Explanation Clinics" be created. The Pathology Explanation Clinics would provide a channel for direct communications between pathologists and patients. Pathologists would receive special training as "Certified Pathologist Navigators" in preparation for this role. The goal of Pathology Explanation Clinics would be to help fill gaps in communication of information contained in laboratory reports to patients, further explain its relevance, and improve patient understanding of the meaning of such information and its impact on their health and health-care choices. Effort would be made to ensure that Certified Pathologist Navigators work within the overall coordination of care by the health-care team., Competing Interests: Declaration of Conflicting Interests: The author(s) received no financial support for the research, authorship, and/or publication of this article.

Starting in 1910, the "Flexner Revolution" in medical education catalyzed the transformation of the US medical education enterprise from a proprietary medical school dominated system into a university-based medical school system. In the 21st century, what we refer to as the "Second Flexner Century" shifts focus from the education of medical students to the education of the general population in the "4 health literacies." Compared with the remarkable success of the first Flexner Revolution, retrofitting medical science education into the US general population today, starting with K-12 students, is a more daunting task. The stakes are high. The emergence of the patient-centered medical home as a health-care delivery model and the revelation that medical errors are the third leading cause of adult deaths in the United States are drivers of population education reform. In this century, patients will be expected to assume far greater responsibility for their own health care as full members of health-care teams. For us, this process began in the run-up to the "Second Flexner Century" with the creation and testing of a general pathology course, repurposed as a series of "gateway" courses on mechanisms of diseases, suitable for introduction at multiple insertion points in the US education continuum. In this article, we describe nomenclature for these gateway courses and a "top-down" strategy for creating pathology coursework for nonmedical students. Finally, we list opportunities for academic pathology departments to engage in a national "Democratization of Medical Knowledge" initiative., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The aim of the study is to better understand where uranium deposits in mice kidneys. The spatial distribution of uranium was examined in the kidneys of C57BL/6 mice using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Mice were exposed to varying levels of uranyl nitrate in their drinking water. Calibration standards were developed to allow for semi-quantitative measurement of uranium in the cortical and medullary regions of mice kidney by LA-ICP-MS. Scanning electron microscopy was used to image the ablation patterns on the kidney. Uranium levels were observed to increase in kidney tissue as uranyl nitrate treatment exposure levels increased. A trend towards a higher uranium concentration in the medullary versus cortical region of the kidneys was observed. These results show the usefulness of LA-ICP-MS in toxicity studies by providing a quantitative, spatial assessment of uranium deposition in a target organ., Competing Interests: Competing interest: The authors have declared that no competing interest exist.

Faculty members from the Department of Pathology at The University of Arizona College of Medicine-Tucson have offered a 4-credit course on enhanced general pathology for graduate students since 1996. The course is titled, "Mechanisms of Human Disease." Between 1997 and 2016, 270 graduate students completed Mechanisms of Human Disease. The students came from 21 programs of study. Analysis of Variance, using course grade as the dependent and degree, program, gender, and year (1997-2016) as independent variables, indicated that there was no significant difference in final grade (F = 0.112; P = .8856) as a function of degree (doctorate: mean = 89.60, standard deviation = 5.75; master's: mean = 89.34, standard deviation = 6.00; certificate program: mean = 88.64, standard deviation = 8.25), specific type of degree program (F = 2.066, P = .1316; life sciences: mean = 89.95, standard deviation = 6.40; pharmaceutical sciences: mean = 90.71, standard deviation = 4.57; physical sciences: mean = 87.79, standard deviation = 5.17), or as a function of gender (F = 2.96, P = .0865; males: mean = 88.09, standard deviation = 8.36; females: mean = 89.58, standard deviation = 5.82). Students in the physical and life sciences performed equally well. Mechanisms of Human Disease is a popular course that provides students enrolled in a variety of graduate programs with a medical school-based course on mechanisms of diseases. The addition of 2 new medically oriented Master of Science degree programs has nearly tripled enrollment. This graduate level course also potentially expands the interdisciplinary diversity of participants in our interprofessional education and collaborative practice exercises., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In the absence of oxygen human life is measured in minutes. In the presence of oxygen, normal metabolism generates reactive species (ROS) that have the potential to cause cell injury contributing to human aging and disease. Between these extremes, organisms have developed means for sensing oxygen and ROS and regulating their cellular processes in response. Redox signaling contributes to the control of cell proliferation and death. Aberrant redox signaling underlies many human diseases. The attributes acquired by altered redox homeostasis in cancer cells illustrate this particularly well. This teaching review and the accompanying illustrations provide an introduction to redox biology and signaling aimed at instructors of graduate and medical students., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The manganese porphyrin, manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin (MnTE-2-PyP(5+)), 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-PyP(5+) and that we could exploit this effect to enhance the chemotherapeutic response in lymphoma. In this study, we demonstrate that MnTE-2-PyP(5+) modulates the mitochondrial redox environment and sensitizes lymphoma cells to antilymphoma chemotherapeutics. MnTE-2-PyP(5+) 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-PyP(5+) also augmented the effects of 2-deoxy-D-glucose (2DG), an antiglycolytic agent. In combination with 2DG, MnTE-2-PyP(5+) increased protein glutathionylation, decreased ATP levels more than 2DG treatment alone, and enhanced 2DG-induced cell death in primary B-ALL cells. MnTE-2-PyP(5+) did not enhance dexamethasone- or 2DG-induced cell death in normal cells. Our findings suggest that MnTE-2-PyP(5+) has potential as an adjuvant for the treatment of hematologic malignancies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Lymphoma cells are subject to higher levels of oxidative stress compared with their normal counterparts and may be vulnerable to manipulations of the cellular redox balance. We therefore designed a phase 2 study of imexon (Amplimexon/NSC-714597), a prooxidant molecule, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Imexon was administered at 1000 mg/m(2) IV daily for 5 days in 21-day cycles. Gene expression analysis performed on pretreatment tumor specimens included 13 transcripts used to generate a redox signature score, previously demonstrated to correlate with lymphoma prognosis. Twenty-two patients were enrolled having follicular (n = 9), diffuse large B-cell (DLBCL) (n = 5), mantle cell (n = 3), transformed follicular (n = 2), small lymphocytic (n = 2), and Burkitt (n = 1) lymphoma. The most common grade 3/4 adverse events were anemia (14%) and neutropenia (9%). The overall response rate was 30%, including responses in follicular lymphoma (4 of 9) and DLBCL (2 of 5). Gene expression analyses revealed CD68 and the redox-related genes, GPX1 and SOD2, as well as a higher redox score to correlate with clinical responses. Therefore, pretreatment markers of oxidative stress may identify patients likely to respond to this therapeutic approach. This trial was registered at www.clinicaltrials.gov as #NCT01314014., (© 2014 by The American Society of Hematology.)

Characteristics of cancer cells include a more oxidized redox environment, metabolic reprogramming and apoptosis resistance. Our studies with a lymphoma model have explored connections between the cellular redox environment and cancer cell phenotypes. Alterations seen in lymphoma cells made resistant to oxidative stress include: a more oxidized redox environment despite increased expression of antioxidant enzymes, enhanced net tumour growth, metabolic changes involving the mitochondria and resistance to the mitochondrial pathway to apoptosis. Of particular importance, the cells show cross-resistance to multiple chemotherapeutic agents used to treat aggressive lymphomas. Analyses of clinical and tumour data reveal the worst prognosis when patients' lymphomas have gene expression patterns consistent with the most oxidized redox environment. Lymphomas from patients with the worst survival outcomes express increased levels of proteins involved in oxidative phosphorylation, including cytochrome c. This is consistent with these cells functioning as metabolic opportunists. Using lymphoma cell models and primary lymphoma cultures, we observed enhanced killing using genetic and drug approaches which further oxidize the cellular redox environment. These approaches include increased expression of SOD2 (superoxide dismutase 2), treatment with a manganoporphyrin that oxidizes the glutathione redox couple, or treatment with a copper chelator that inhibits SOD1 and leads to peroxynitrite-dependent cell death. The latter approach effectively kills lymphoma cells that overexpress the anti-apoptotic protein Bcl-2. Given the central role of mitochondria in redox homoeostasis, metabolism and the intrinsic pathway to apoptosis, our studies support the development of new anti-cancer drugs to target this organelle.

Bcl-2 and other anti-apoptotic proteins are associated with defective caspase-dependent apoptotic pathways, resulting in chemoresistance. We have previously shown that ATN-224, a copper chelator drug, induces cell death in murine thymic lymphoma cells transfected with Bcl-2. In the current study, we tested whether ATN-224 was effective in diffuse large B cell lymphoma (DLBCL) cells, which have increased anti‑apoptotic proteins through translocation or amplification. We found that nanomolar concentrations of ATN-224 induced cell death in DLBCL cells independent of Bcl-2, Bcl-xL or Mcl-1 status. ATN-224 treatment resulted in mitochondrial dysfunction, release of apoptosis-inducing factor (AIF) and induction of caspase-independent cell death. In addition, ATN-224 degraded Mcl-1 and enhanced the effect of the BH3 mimetic ABT-263. These findings indicate that ATN-224 has potential as a therapeutic for the treatment of DLBCL. Induction of caspase‑independent cell death in apoptosis‑resistant DLBCL would provide a therapeutic alternative for the treatment of refractory disease.

Variable survival outcomes are seen following treatment for aggressive non-Hodgkin lymphoma (NHL). This study examined whether outcomes for aggressive B-cell NHL are associated with single nucleotide polymorphisms (SNPs) in oxidative stress-related genes, which can alter drug metabolism and immune responses. Genotypes for 53 SNPs in 29 genes were determined for 337 patients given anthracycline-based therapies. Their associations with progression-free survival (PFS) and overall survival (OS) were estimated by Cox proportional hazard regression; associations with hematologic toxicity were estimated by logistic regression. To validate the findings, the top three SNPs were tested in an independent cohort of 572 DLBCL patients. The top SNPs associated with PFS in the discovery cohort were the rare homozygotes for MPO rs2243828 (hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.14-3.06, P = 0.013), AKR1C3 rs10508293 (HR = 2.09, 95% CI = 1.28-3.41, P = 0.0032) and NCF4 rs1883112 (HR = 0.66, 95% CI = 0.43-1.02, P = 0.06). The association of the NCF4 SNP with PFS was replicated in the validation dataset (HR = 0.66, 95% CI = 0.44-1.01, P = 0.05) and the meta-analysis was significant (HR = 0.66, 95% CI = 0.49-0.89, P < 0.01). The association of the MPO SNP was attenuated in the validation dataset, while the meta-analysis remained significant (HR = 1.64, 95% CI = 1.12-2.41). These two SNPs showed similar trends with OS in the meta-analysis (for NCF4, HR = 0.72, 95% CI = 0.51-1.02, P = 0.07 and for MPO, HR = 2.06, 95% CI = 1.36-3.12, P < 0.01). In addition, patients with the rare homozygote of the NCF4 SNP had an increased risk of hematologic toxicity. We concluded that genetic variations in NCF4 may contribute to treatment outcomes for patients with aggressive NHL., (© 2014 Wiley Periodicals, Inc.)

Chemoresistance due to oxidative stress resistance or upregulation of Bcl-2 contributes to poor outcome in the treatment of hematological malignancies. In this study, we utilize the copper-chelator drug ATN-224 (choline tetrathiomolybdate) to induce cell death in oxidative stress-resistant cells and cells overexpressing Bcl-2 by modulating the cellular redox environment and causing mitochondrial dysfunction. ATN-224 treatment decreases superoxide dismutase 1 (SOD1) activity, increases intracellular oxidants, and induces peroxynitrite-dependent cell death. ATN-224 also targets the mitochondria, decreasing both cytochrome c oxidase (CcOX) activity and mitochondrial membrane potential. The concentration of ATN-224 required to induce cell death is proportional to SOD1 levels, but independent of Bcl-2 status. In combination with doxorubicin, ATN-224 enhances cell death. In primary B-cell acute lymphoblastic leukemia patient samples, ATN-224 decreases the viable cell number. Our findings suggest that ATN-224's dual targeting of SOD1 and CcOX is a promising approach for treatment of hematological malignancies either as an adjuvant or as a single agent., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chronic inflammation increases lymphoma risk. Chronic inflammation exposes cells to increased reactive oxygen species (ROS). Constant exposure to ROS selects for oxidative stress-resistant cells with upregulated anti-oxidant defense enzymes. The impact of oxidative stress resistance on the redox biology and chemotherapy response in lymphoma has not been rigorously tested. To measure the effect of antioxidant defense enzyme upregulation in lymphoid cells, we created oxidative stress-resistant WEHI7.2 thymic lymphoma cell variants. We selected a population of WEHI7.2 cells for resistance to hydrogen peroxide and constructed catalase-overexpressing WEHI7.2 transfectants. The WEHI7.2 variants had: i) increased catalase and total superoxide dismutase activities; ii) an altered GSSG/2GSH redox potential; iii) a more oxidized NADP(+)/NADPH pool; and iv) increased phase 2 enzymes, NAD(P)H:quinone oxidoreductase and glutathione S-transferases μ and π. Regression analysis showed a correlation between the GSSG/2GSH redox potential and the increased phase 2 enzyme activities. As predicted from the anti-oxidant defense enzyme profile, the variants were more resistant to the oxidants hydrogen peroxide and paraquat. The variants exhibited resistance to the common lymphoma chemotherapeutics, cyclophosphamide, doxorubicin, vincristine and glucocorticoids. These data indicate that chronic ROS exposure results in lymphoid cells with multiple changes in their redox biology and a chemoresistance phenotype. These data further suggest that lymphomas that arise at the site of chronic inflammation develop chemoresistance due to a combination of drug detoxification and removal of ROS.

Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP(5+), 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-PyP(5+) potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H(2)O(2)](ss)) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP(5+) is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H(2)O(2)](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 H(2)O(2) or depletion of glutathione by BSO prevents MnTE-2-PyP(5+) from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP(5+) 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-PyP(5+). Taken together, these findings indicate that: 1) H(2)O(2) is important for MnTE-2-PyP(5+) activity; 2) Mn-TE-2-PyP(5+) cycles with GSH; and 3) MnTE-2-PyP(5+) 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-PyP(5+) may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glucocorticoids are a class of steroid hormones commonly used for the treatment of hematological malignancies due to their ability to induce apoptosis in lymphoid cells. An understanding of the critical steps in glucocorticoid-induced apoptosis is required to identify sources of drug resistance. Previously, we found that an increase in hydrogen peroxide is a necessary signal for glucocorticoid-induced apoptosis. In the current study, we found that mitochondria are the source of the signal. Glucocorticoid treatment inhibited Complex I and Complex III of the electron transport chain (ETC). Mitochondrial matrix reactive oxygen species (ROS) increased concomitantly with the oxidation of the mitochondrial glutathione pool. Treatment with Tiron, a superoxide scavenger, inhibited the signal. This suggests that the hydrogen peroxide signal originates as superoxide from the mitochondria and is metabolized to hydrogen peroxide. An inability to generate mitochondrial oxidants in response to glucocorticoids could cause drug resistance.

Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis.

Previously, arsenic trioxide showed impressive regression rates of acute promyelocytic leukemia. Here, we investigated molecular determinants of sensitivity and resistance of cell lines of different tumor types towards arsenic trioxide. Arsenic trioxide was the most cytotoxic compound among 8 arsenicals investigated in the NCI cell line panel. We correlated transcriptome-wide microarray-based mRNA expression to the IC(50) values for arsenic trioxide by bioinformatic approaches (COMPARE and hierarchical cluster analyses, Ingenuity signaling pathway analysis). Among the identified pathways were signaling routes for p53, integrin-linked kinase, and actin cytoskeleton. Genes from these pathways significantly predicted cellular response to arsenic trioxide. Then, we analyzed whether classical drug resistance factors may also play a role for arsenic trioxide. Cell lines transfected with cDNAs for catalase, thioredoxin, or the anti-apoptotic bcl-2 gene were more resistant to arsenic trioxide than mock vector transfected cells. Multidrug-resistant cells overexpressing the MDR1, MRP1 or BCRP genes were not cross-resistant to arsenic trioxide. Our approach revealed that response of tumor cells towards arsenic trioxide is multi-factorial.

Glucocorticoid-induced apoptosis is exploited clinically for the treatment of hematologic malignancies. Determining the required molecular events for glucocorticoid-induced apoptosis will identify resistance mechanisms and suggest strategies for overcoming resistance. In this study, we found that glucocorticoid treatment of WEHI7.2 murine thymic lymphoma cells increased the steady-state [H(2)O(2)] and oxidized the intracellular redox environment before cytochrome c release. Removal of glucocorticoids after the H(2)O(2) increase resulted in a 30% clonogenicity; treatment with PEG-CAT increased clonogenicity to 65%. Human leukemia cell lines also showed increased H(2)O(2) in response to glucocorticoids and attenuated apoptosis after PEG-CAT treatment. WEHI7.2 cells that overexpress catalase (CAT2, CAT38) or were selected for resistance to H(2)O(2) (200R) removed enough of the H(2)O(2) generated by glucocorticoids to prevent oxidation of the intracellular redox environment. CAT2, CAT38, and 200R cells showed a 90-100% clonogenicity. The resistant cells maintained pERK survival signaling in response to glucocorticoids, whereas the sensitive cells did not. Treating the resistant cells with a MEK inhibitor sensitized them to glucocorticoids. These data indicate that: (1) an increase in H(2)O(2) is necessary for glucocorticoid-induced apoptosis in lymphoid cells, (2) increased H(2)O(2) removal causes glucocorticoid resistance, and (3) MEK inhibition can sensitize oxidative stress-resistant cells to glucocorticoids., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mitochondria are central to a variety of cellular processes, from metabolism to cell death. In this study, we demonstrated that an increase in the critical mitochondrial protein, cytochrome c, correlated with drug resistance in a cell culture model of aggressive lymphoma. Increased cytochrome c expression was also correlated with decreased survival in the aggressive diffuse large B-cell and mantle cell lymphomas, but not in the indolent follicular lymphoma. This suggests that an increased reliance on the mitochondria for energy allows tumor cells to be metabolic opportunists and contribute to tumor development and drug resistance.

Background: There has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols., Methodology/principal Findings: To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Using flow cytometry, and western blotting to label free thiols with Alexa Fluor 633 C(5) maleimide dye and N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), respectively, we show that parthenolide decreases the level of free exofacial thiols on Granta mantle lymphoma cells. In addition, we used immuno-precipitation techniques to identify the central redox regulator thioredoxin, as one of the surface protein thiol targets modified by parthenolide. To examine the functional role of parthenolide induced surface protein thiol modification, we pretreated Granta cells with cell impermeable glutathione (GSH), prior to exposure to parthenolide, and showed that GSH pretreatment; (a) inhibited the interaction of parthenolide with exofacial thiols; (b) inhibited parthenolide mediated activation of JNK and inhibition of NFkappaB, two well established mechanisms of parthenolide activity and; (c) blocked the cytotoxic activity of parthenolide. That GSH had no effect on the parthenolide induced generation of intracellular reactive oxygen species supports the fact that GSH had no effect on intracellular redox. Together these data support the likelihood that GSH inhibits the effect of parthenolide on JNK, NFkappaB and cell death through its direct inhibition of parthenolide's modulation of exofacial thiols., Conclusions/significance: Based on these data, we postulate that one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Further, we propose that cancer cell exofacial thiols may be important and novel targets for therapy.

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.

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 +/- 12.2 to 35.1 +/- 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-pi level in blood lymphocytes from 2,252.9 +/- 734.2 to 2,634.4 +/- 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-mu and plasma GST-alpha levels. There was a small but statistically significant decrease (8%, P = 0.003) in plasma GST-alpha 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-pi 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.

Glucocorticoids are one component of combined treatment regimens for many types of lymphoma due to their ability to induce apoptosis in lymphoid cells. In WEHI7.2 murine thymic lymphoma cells, altering catalase and glutathione peroxidase activity by transfection or the use of chemical agents modulates the ability of glucocorticoids to induce apoptosis. This suggests that the oxidative stress response is important in determining the glucocorticoid sensitivity of the cells. For glutathione peroxidase and catalase to detoxify reactive oxygen species (ROS), reducing equivalents in the form of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) are ultimately required. The major source of NADPH in the cell is glucose 6-phosphate dehydrogenase (G6PDH). Therefore, we created G6PDH-overexpressing WEHI7.2 variants to test whether G6PDH activity is a key determinant of glucocorticoid sensitivity in WEHI7.2 cells. G6PDH-overexpressing WEHI7.2 cells were more sensitive to oxidative stress and glucocorticoids. The G6PDH-overexpressing WEHI7.2 variants appeared similar to cells undergoing glucose deprivation with decreased adenosine triphosphate (ATP) synthesis by the mitochondria and increased basal levels of ROS. Overexpression of G6PDH also sensitized the cells to other standard lymphoma chemotherapeutics including cyclophosphamide, doxorubicin, and vincristine. The decreased ATP and elevated ROS due to G6PDH overexpression may be key factors in increasing the sensitivity of the WEHI7.2 cells to lymphoma chemotherapeutics.

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).

Cancer chemotherapy is often limited by patient's toxicity and tumor drug resistance indicating that new drug development and modification of existing drugs is critical for improving the therapeutic response. Traditional Chinese medicine is a rich source of potential anticancer agents. In particular, cantharidin (CAN), the active principle ingredient from the blister beetle, Mylabris, has anti-tumor activity, but the cytotoxic mechanism is unknown. In leukemia cells, cantharidin induces apoptosis by a p53-dependent mechanism. Cantharidin causes both DNA single- and double-strand breaks. Colony-forming assays with knockout and transfectant cells lines showed that DNA polymerase beta, but not ERCC1, conferred increased cell survival after cantharidin treatment, indicating that base excision repair (BER), rather than nucleotide excision repair (NER), is important for CAN-induced DNA lesions. Oxidative stress-resistant thymic lymphoma-derived WEHI7.2 variants are also more resistant to cantharidin. These data suggest that cantharidin treatment causes oxidative stress that provokes DNA damage and p53-dependent apoptosis.

Chronic inflammation is often associated with increased cancer frequency. Continuous exposure to reactive oxygen species, as at the site of chronic inflammation, can result in cells with increased antioxidant defense enzymes. In WEHI7.2 cells, overexpression of catalase or thioredoxin by transfection or selection of a cell population resistant to hydrogen peroxide has resulted in WEHI7.2 variants with altered glucose and energy metabolism. This metabolic change would favor survival in a tumor environment. We conclude that metabolic alterations, due to increased antioxidant enzyme expression, may underlie the increased tumorigenicity seen previously in the variants and contribute to the increased tumor risk associated with chronic inflammation.

Glucocorticoids induce apoptosis in lymphocytes by causing the release of cytochrome c into the cytosol; however, the events in the signaling phase between translocation of the steroid-receptor complex to the nucleus and the release of cytochrome c have not been elucidated. Previously, we found that, in response to steroid treatment, WEHI7.2 mouse thymic lymphoma cells overexpressing catalase (CAT38) show delayed apoptosis (delayed cytochrome c release) compared to the parental cells, while Bcl-2 overexpressing cells (Hb12) are protected from steroid-induced apoptosis. In lymphocytes, glucocorticoid treatment decreases glucose uptake. Both glucose deprivation and the attendant ATP drop are known inducers of apoptosis. Therefore, we used (31)P and (1)H NMR spectroscopy to compare metabolic profiles of WEHI7.2, CAT38 and Hb12 cells in the presence and absence of dexamethasone to determine: (1) whether glucocorticoid effects on glucose metabolism contribute to the mechanism of steroid-induced apoptosis; and (2) whether catalase or Bcl-2 overexpression altered metabolism thereby providing a mechanism of steroid resistance. Loss of mitochondrial hexokinase activity was correlated to the induction of apoptosis in WEHI7.2 and CAT38 cells. CAT38 and Hb12 cells have an altered basal metabolism which includes increases in hexokinase activity, lactate production when subcultured into new medium, use of mitochondria for ATP production and potentially increased glutaminolysis. These data suggest that: (1) glucocorticoid effects on glucose metabolism may contribute to the mechanism of steroid-induced lymphocyte apoptosis; and (2) the altered metabolism seen in catalase and Bcl-2 overexpressing cells may contribute to both the steroid resistance and increased tumorigenicity of these variants.

Peroxisome proliferator activated receptors are nuclear hormone receptors that regulate the expression of genes containing a peroxisome proliferator activated receptor response element. We report here that the human bcl-2 gene contains a functional peroxisome proliferator activated receptor response element in the 3' untranslated region. Peroxisome proliferator activated receptor gamma bound the human bcl-2 peroxisome proliferator activated receptor response element in gel shift assays and co-transfection of this receptor led to increased luciferase activity from a reporter plasmid containing the human bcl-2 peroxisome proliferator activated receptor response element. Examination of peroxisome proliferator activated receptor gamma-transfected cells demonstrated an increased amount of bcl-2 message compared to empty vector-transfected cells. Confocal analyses confirmed that more Bcl-2 protein was present in peroxisome proliferator activated receptor gamma-transfected cells compared to control-transfected cells. The functionality of the increased Bcl-2 protein was examined using resistance to bile salt-induced apoptosis as the endpoint. Peroxisome proliferator activated receptor gamma-transfected cells were almost twice as resistant as control-transfected cells. These data show that PPARgamma can mediate transcription of bcl-2, resulting in an increase in Bcl-2 protein and protection from apoptosis. We discuss these findings with regards to their potential implications for colon carcinogenesis.

Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2.

The antimalaria drug, artesunate (ART), is very cytotoxic in tumor cell lines. The active moiety of ART is an endoperoxide bridge that generates carbon-centered free radicals and oxidative stress upon cleavage. Oxidative stress appears to be necessary for the antimalarial activity of ART. To test whether antioxidant gene expression affects the ART response in tumor cell lines we compared the baseline antioxidant mRNA gene expression in the 55 human tumor cell line panel from the National Cancer Institute Developmental Therapeutics Program to the ART IC50. Thioredoxin reductase expression showed a significant positive correlation to the ART IC50 and catalase expression was inversely correlated with the ART IC50 (p<0.05). WEHI7.2 mouse thymoma cells selected for resistance to hydrogen peroxide or transfected with thioredoxin, manganese superoxide dismutase, catalase or bcl-2 showed resistance to ART compared to the parental cell line. Taken together these data support a role for oxidative stress in the mechanism of ART action in tumor cells and suggest that antioxidant defenses act in combination to affect the cellular response to ART.

Resistance to apoptosis may be a critical phenotype that cells must acquire during skin carcinogenesis. The Akt kinase is a known upstream regulator of apoptosis in many cell types and has been shown to be activated by increased reactive oxygen species (ROS). We have previously demonstrated that two malignant variants (6M90 and 6R90) of the mouse keratinocyte 308 cell line have elevated ROS because of loss of catalase activity, and that this elevated ROS confers a growth advantage. We report here that in addition to a growth advantage, chronically increased ROS in the variants resulted in an increase in resistance to ultraviolet (UV) B-induced apoptosis. This resistance was due to basal increases of Akt phosphorylation in the malignant variants compared to the 308 cells. Modulation of ROS in 6M90 and 6R90 cells by catalase overexpression or antioxidant treatment resulted in decreased levels of Akt phosphorylation and subsequent loss of resistance to UVB-induced apoptosis. Conversely, treatment of 308 cells with hydrogen peroxide caused increases in Akt phosphorylation and increased apoptosis resistance. These results indicate that the chronically elevated ROS often observed in tumors may contribute to a malignant phenotype by keeping Akt in a phosphorylated state, resulting in increased apoptosis resistance., (Copyright 2003 Wiley-Liss, Inc.)

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Treatment of the mouse thymoma-derived WEHI7.2 cell line with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous studies have shown that WEHI7.2 cell variants with an increased antioxidant defense exhibit increased resistance to dexamethasone-induced apoptosis, suggesting that oxidative stress may play a role in glucocorticoid-induced apoptosis. In this work we compared metabolic profiles of WEHI7.2 parental cells with those of WEHI7.2 variants with an increased antioxidant defense or overexpressing bcl-2, to determine whether bolstering the antioxidant defense results in altered metabolic parameters that could translate into increased resistance to dexamethasone-induced apoptosis. WEHI7.2 parental cells and cells overexpressing catalase, thioredoxin or bcl-2, or selected for resistance to 200 micro M H(2)O(2) were cultured in low-glucose DMEM medium supplemented with 10% calf serum, and extracted using chloroform-methanol-water (1:1:1). Metabolites contained in the aqueous and organic phases of the extracts were processed separately and subjected to high-resolution (31)P NMR spectroscopy. In most of the steroid-resistant variants, ATP levels and energetic status were decreased compared with the steroid-sensitive parental cell line, while the concentrations of hexose and triose phosphates were increased. Furthermore, the ratio of choline-containing phospholipids to ethanolamine-containing phospholipids was generally reduced in steroid-resistant cells. Phosphatidylethanolamine and its derivatives contain a higher amount of polyunsaturated fatty acids (PUFA) than the choline-containing analogs, and PUFA are readily oxidized by reactive oxygen species. Therefore, an increased initial amount of phosphatidylethanolamine may increase the 'buffering capacity' of this antioxidant and may thus contribute to the steroid resistance of WEHI7.2 variants., (Copyright 2002 John Wiley & Sons, Ltd.)

Imexon is an aziridine-containing iminopyrrolidone with selective growth-inhibitory potency for multiple myeloma. Our previous research indicates that imexon induces mitochondrial alterations, oxidative stress, and apoptosis. This drug represents an interesting model drug with a nonmyelosuppressive profile to study the basic mechanisms leading to antitumor activity and resistance. The major purpose of this study was to characterize an imexon-resistant RPMI8226/I cell line that was developed from RPMI8226 cells by continuous exposure to imexon. No significant differences were observed in the sensitivity to several cytotoxic drugs, including mitoxantrone, mitomycin C, melphalan, methotrexate, cytarabine, cisplatin, vincristine, and paclitaxel, in the imexon-resistant cells. However, RPMI8226/I cells were cross-resistant to arsenic trioxide, doxorubicin, fluorouracil, etoposide, irinotecan, and especially IFN-alpha. The data from DNA microarray and Western blot analyses indicated that the levels of antiapoptotic proteins Bcl-2 and thioredoxin-2, which reside mainly in the mitochondria, are increased in RPMI8226/I cells. In addition, increased levels of lung resistance protein were detected in imexon-resistant cells. Expression of P-glycoprotein was not detected in RPMI8226/I cells. No loss of mitochondrial membrane potential or increase in the levels of reactive oxygen species was observed in RPMI8226/I cells after exposure to imexon; however, the levels of glutathione are increased in the RPMI8226/I cells. Transmission electron microscopy revealed significant changes in the mitochondrial morphology of RPMI8226/I cells, whereas no ultrastructural changes were observed in other cellular compartments. Imexon-resistant RPMI8226/I myeloma cells appear to have a unique mechanism of resistance that is associated with morphological alterations of mitochondria, increased protection against oxidative stress, elevated levels of glutathione, and enhanced expression of antiapoptotic mitochondrial proteins.

We have developed an in vitro model to study the molecular mechanisms of tumor progression. Using repeated treatments with ionizing radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we caused malignant progression of a papilloma producing mouse keratinocyte cell line, 308 cells. In a previous study we have shown that the malignant variants of 308 cells have elevated reactive oxygen species (ROS) levels, and have established a functional role for the pro-oxidant state in the progressed phenotype (Carcinogenesis 20 (1999) 2063). In this study, we have evaluated the status of intracellular defense mechanisms for ROS scavenging in the progressed phenotype to identify sources that contribute to their pro-oxidant state. Our results demonstrate that a reduction in several anti-oxidant defense mechanisms, including catalase and glutathione S-transferase mu, correlates with the emergence of the malignant phenotype. We provide evidence that attenuation of catalase activity may play a functional role in the malignant progression of mouse keratinocytes.

Treatment of WEHI7.2 cells, a mouse thymoma-derived cell line, with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous work has shown that treatment of WEHI7.2 cells with dexamethasone results in a downregulation of antioxidant defense enzymes, suggesting that increased oxidative stress may play a role in glucocorticoid-induced apoptosis. To test whether resistance to oxidative stress causes resistance to dexamethasone-induced apoptosis, WEHI7.2 cell variants selected for resistance to 50, 100 and 200 microM H(2)O(2) were developed. Resistance to H(2)O(2) is accompanied by increased antioxidant enzyme activity, resistance to other oxidants and a delayed loss of viable cells after dexamethasone treatment. In the 200 microM H(2)O(2)-resistant cell variant the delay in cell loss is correlated with delayed release of cytochrome c from the mitochondria into the cytosol. This suggests that reactive oxygen species play a role in a signaling event during steroid-mediated apoptosis in lymphocytes.

Imexon is a cyanoaziridine derivative that has antitumor activity in multiple myeloma. Previous studies have shown that imexon induces oxidative stress and apoptosis in the RPMI 8226 myeloma cell line. This study reports that imexon has cytotoxic activity in other malignant cell lines including NCI-H929 myeloma cells and NB-4 acute promyelocytic leukemia cells, whereas normal lymphocytes and U266 myeloma cells are substantially less sensitive. Flow cytometric experiments have shown that imexon treatment is associated with the formation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Deltapsi(m)) in imexon-sensitive myeloma cell lines and NB-4 cells. In contrast, reduction of Deltapsi(m) and increased levels of ROS were not observed in imexon-resistant U266 cells. Treatment of imexon-sensitive RPMI 8226 cells with the antioxidant N-acetyl-L-cysteine (NAC) protects cells against these effects of imexon. Mitochondrial swelling was observed by electron microscopy in RPMI 8226 myeloma cells treated with 180 microM imexon as early as 4 hours. Damage to mitochondrial DNA was detected by a semiquantitative polymerase chain reaction assay in imexon-treated RPMI 8226 cells; however, nuclear DNA was not affected. Finally, partial protection of RPMI 8226 cells against the imexon effects was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex II. These changes are consistent with mitochondrial oxidation and apoptotic signaling as mediators of the growth inhibitory effects of imexon. Interestingly, oxidative damage and decrease of Deltapsi(m) induced by imexon highly correlates with sensitivity to imexon in several myeloma cell lines and an acute promyelocytic leukemia cell line. (Blood. 2001;97:3544-3551)

Glucocorticoids are used for the treatment of lymphoid neoplasms, taking advantage of the well-known ability of these compounds to cause apoptosis in lymphoid tissues. Previously, we have shown that dexamethasone, a synthetic glucocorticoid, causes a down-regulation of several antioxidant defense enzymes and proteins, including catalase and thioredoxin, concomitant with the induction of apoptosis in WEHI7.2 mouse thymoma cells. To test whether this down-regulation plays a critical role in the mechanism of steroid-induced apoptosis, WEHI7.2 cells were transfected with rat catalase. Two clones, expressing 1.4-fold and 2.0-fold higher catalase specific activity, respectively, when compared with vectoronly transfectants were selected for further study. An increase to 1.4-fold parental cell catalase activity delayed cell loss after dexamethasone treatment, whereas a 2.0-fold parental catalase activity prevented dexamethasone-induced cell loss for 48 h after treatment. Dexamethasone treatment of the WEHI7.2 cells stimulated a release of cytochrome c into the cytosol. Catalase-overexpressing cells showed a delay or lack of cytochrome c release from the mitochondria, which correlated temporally with the delay or prevention of cell loss in the culture after dexamethasone treatment. A decreased amount of cell death from WEHI7.2 cells overexpressing catalase was also seen in tumor xenografts in severe combined immunodeficient mice when compared with tumors from vector-only transfected cells. Similarly, thioredoxin-overexpressing WEHI7.2 cells, shown previously to be apoptosis resistant, showed decreased cell death in tumor xenografts. This resulted in larger tumors from cells overexpressing these proteins. Cell death in control transfectant tumor xenografts was primarily attributable to apoptosis. In contrast, the cell death we observed in tumors from thioredoxin- or catalase-overexpressing cells had a higher frequency of a nonapoptotic, nonnecrotic type of cell death termed para-apoptosis. These data suggest that: (a) oxidative stress plays a critical role in steroid-induced apoptosis prior to the commitment of the cells to undergo apoptosis; and (b) resistance to oxidative stress can contribute to tumor growth.

Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule-dependent, and long exposures (> or = 48 hr) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also demonstrated by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione. Oxidative stress in 8226 cells exposed to imexon was detected by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG), followed by confocal microscopy. These images showed increased levels of 8-OHdG in the cytoplasm of cells treated with different concentrations of imexon at 8, 16, and 48 hr. Interestingly, 8-OHdG staining was not observed in the nuclei of imexon-treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis upon electron microscopy, and increased levels of phosphatidylserine exposure, detected as Annexin-V binding, on the cell surface. To prevent depletion of thiols, 8226 myeloma cells exposed to imexon were treated with N-acetylcysteine (NAC). Simultaneous, as well as sequential, treatment with NAC before imexon exposure resulted in protection of myeloma cells against imexon-induced cytotoxicity. Conversely, the glutathione synthesis inhibitor buthionine sulfoximine increased imexon cytotoxicity. These data suggest that imexon perturbs cellular thiols and induces oxidative stress leading to apoptosis in human myeloma cells.

Evidence from a number of studies suggests that the mechanism by which tumor necrosis factor (TNF) kills transformed cells involves oxidative stress. NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme with particular relevance to cancer. The MCF-7 breast cancer cell line was stably transfected with rat NQO1 cDNA to determine whether increased NQO1 activity alters sensitivity to TNF-induced apoptosis. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the MCF-7 line, and two control transfectants were examined. Northern blot hybridization analyses and reverse transcription-PCR demonstrated that the increase in NQO1 activity in the transfectants was attributable to expression from the transfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indoloquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants were significantly more sensitive to TNF than the control transfectants or MCF-7 parent. This increased sensitivity could not be explained by changes in superoxide dismutase or catalase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using dichlorodihydrofluorescein diacetate as a probe, we found that the NQO1 transfectants had no difference in baseline level of oxidative stress compared to the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated pathway to apoptosis.

Oxidative stress has been implicated in the mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis, raising a question about the status of antioxidant defenses in TNF-sensitive cells. Antioxidant defenses were examined in MCF-7 cells after treatment with TNF. Cell morphology and DNA fragmentation assays were used to confirm increased apoptosis as a result of TNF treatment. The expression and activity of antioxidant defenses were assessed using Northern blot hybridization analyses and biochemical assays, respectively. Five- and ten-fold increases in manganese superoxide dismutase (MnSOD) mRNA were measured after one and five days of TNF treatment, respectively. The expression of copper,zinc superoxide dismutase, catalase or thioredoxin was not altered. An approximate five-fold increase in MnSOD activity followed the change in gene expression, but no difference in the activity of catalase or glutathione peroxidase was seen. Thus, increased MnSOD activity was not accompanied by an increase in other antioxidant defenses and in particular, H2O2-scavenging enzymes. MnSOD has previously been shown to afford protection against TNF-mediated cytotoxicity. The observed lack of increased peroxidase activity is consistent with mitochondrially-generated superoxide anion radical contributing to the mechanism of TNF-induced apoptosis.

The redox protein thioredoxin plays an important role in controlling cancer cell growth through regulation of DNA synthesis and transcription factor activity. Thioredoxin is overexpressed by a number of human primary cancers and its expression is decreased during dexamethasone-induced apoptosis of mouse WEHI7.2 thymoma cells. We examined the ability of WEHI7.2 cells stably transfected with human thioredoxin cDNA showing increased levels of cytoplasmic thioredoxin to undergo apoptosis in vitro and in vivo. The cells were protected from apoptosis induced by dexamethasone, staurosporine, etoposide, and thapsigargin, but not by N-acetyl-sphingosine. When inoculated into severe combined immunodeficient mice, the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as bcl-2-transfected, WEHI7.2 cells. The trx- and bcl-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type cells. Unlike tumors formed by the wild-type and bcl-2-transfected WEHI7.2 cells, trx-transfected cell tumors did not show growth inhibition upon treatment with dexamethasone. This study suggests that increased thioredoxin expression in human cancers may result in an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of the tumor to drug-induced apoptosis.

Understanding the fundamental mechanism of apoptosis is crucial to developing therapeutic strategies for controlling apoptosis in diseased tissues. We are using model systems with relevance to cancer treatment to investigate the mechanism of apoptosis. Subtraction hybridization cloning was used to identify transcripts present at higher levels in regressing vs. normal prostate; these may be important for apoptosis. One of the genes cloned from regressing prostate is also upregulated in the murine W7.2 lymphocyte cell line induced to undergo apoptosis by treatment with the synthetic glucocorticoid, dexamethasone. This gene encodes a mu class glutathione S-transferase (EC 2.5.1.18), a protein that can protect the cell against oxidative stress by repairing oxidized lipids, proteins, and DNA. Glutathione S-transferase expression does not increase with dexamethasone treatment of lymphocyte cell lines expressing nonfunctional glucocorticoid receptors or a mutation in the apoptotic pathway. Other antioxidant defenses, including catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1), decline following dexamethasone treatment of W7.2 cells. Overexpression of the bcl-2 oncogene protects these cells against dexamethasone-mediated apoptosis and prevents the decrease in antioxidant enzyme activity. These findings support the hypothesis that control of the cellular redox state is important to the mechanism of glucocorticoid-mediated lymphocyte apoptosis. Another model system we are using is tumor necrosis factor-alpha treatment of MCF-7 human breast cancer cells. Our preliminary results suggest that, in this system, activation of nuclear factor-kappa B and increased expression of manganese superoxide dismutase may afford protection from apoptosis.

Thioredoxin, a redox protein with growth factor activity that modulates the activity of several proteins important for cell growth, has been reported to be overexpressed in a number of human primary cancers. In the present study, the effects of stably transfecting mouse NIH 3T3 cells and MCF-7 human breast cancer cells with cDNA for wild-type human thioredoxin or a redox-inactive mutant thioredoxin, Cys32-->Ser32/Cys35-->Ser35 (C32S/C35S), on cell proliferation and transformed phenotype have been investigated. NIH 3T3 cells transfected with thioredoxin achieved increased saturation densities compared with vector alone-transfected cells, but were not transformed as assessed by tumor formation in immunodeficient mice. Thioredoxin-transfected MCF-7 cells showed unaltered monolayer growth on plastic surfaces compared with vector alone-transfected cells, but exhibited severalfold increased colony formation in soft agarose. Stable transfection of NIH 3T3 and MCF-7 cells with C32S/C35S resulted in inhibition of monolayer growth on plastic surfaces, and up to 73% inhibition of colony formation by MCF-7 cells in soft agarose. When inoculated into immunodeficient mice, thioredoxin-transfected MCF-7 cells formed tumors, although with a 38-57% growth rate compared with vector alone-transfected cells, whereas tumor formation by C32S/C35S-transfected MCF-7 cells was almost completely inhibited. The results of the study suggest that thioredoxin plays an important role in the growth and transformed phenotype of some human cancers. The inhibition of tumor cell growth by the dominant-negative redox-inactive mutant thioredoxin suggests that thioredoxin could be a novel target for the development of drugs to treat human cancer.

When the WEHI7.2 mouse lymphoid cell line was treated with dexamethasone to induce apoptosis the activities and transcript levels of the antioxidant defence enzymes catalase, superoxide dismutase (SOD) and DT-diaphorase exhibited a progressive decrease over 48 hours. Catalase activity was maintained and total SOD and DT-diaphorase activity showed smaller decreases following dexamethasone treatment of WEHI7.2 cells transfected with the bcl-2 oncogene, which protects the cells against apoptosis. Treatment of wild-type WEHI7.2 and bcl-2 transfected cells with a catalase inhibitor, aminotriazole, was not sufficient to induce apoptosis. Antioxidants, including bovine liver catalase, bovine erythrocyte CuZn-SOD, sodium selenite and Trolox, a water soluble vitamin E analogue, as well as hypoxia, inhibited dexamethasone-induced apoptosis. These results suggest that oxidant stress due to the decreased activity of antioxidant defence enzymes may play a role in dexamethasone-mediated lymphoid cell apoptosis and that bcl-2 may prevent apoptosis by maintaining the level of critical antioxidant defence mechanisms, which include catalase.

This review focuses on evidence that oxidative stress during apoptosis is controlled, at least in part, by modulating cellular antioxidant defences. Evidence is presented from studies of apoptosis induced by glucocorticoids, HIV-1 infection and tumour necrosis factor-alpha. Glucocorticoid treatment of murine lymphocyte cell lines leads to the down-regulation of primary antioxidant defence enzymes, including catalase, superoxide dismutases, thioredoxin and DT-diaphorase. Following HIV-1 infection, disturbances in glutathione metabolism are seen, and decreased antioxidant enzyme activities have been reported for HIV-1-infected cell lines. The viral protein Tat may mediate these effects. Cellular resistance to apoptosis induced by tumour necrosis factor-alpha is modulated by the expression of manganese superoxide dismutase or Bcl-2. The loss of antioxidant defences is predicted to lead to oxidative stress, which could contribute to the mechanism of apoptosis through an effect on redox-sensitive transcription factors, calcium homeostasis or cysteine proteases.

Recent studies implicate oxidative stress in the mechanism of apoptosis. We have examined the expression of genes, whose products counteract oxidative stress, during glucocorticoid-mediated apoptosis of a murine thymoma-derived cell line. Using Northern blot hybridisation analyses, we observed a progressive decline over a 24 h period in the transcript levels for catalase, manganese superoxide dismutase, copper, zinc-superoxide dismutase, DT-diaphorase and thioredoxin. The changes were first seen within 2-8 h of the addition of the hormone which is well in advance of appreciable apoptosis. Using Western blot hybridisation analyses we found that a dexamethasone-mediated increase in glutathione S-transferase message level was followed closely by an increase in glutathione S-transferase mu class protein and a 20% decrease in reduced glutathione levels. Our findings suggest that the downregulation of cellular oxidant defense enzymes with a consequent increase in oxidant damage could contribute to the molecular mechanism of apoptosis.

Based on the finding that glutathione S-transferase Yb1 (GST) gene expression is elevated in the regressing prostate of androgen-ablated rats, we analyzed GST transcript levels during steroid-induced lymphocyte cell death. It was found that GST gene expression was induced in steroid-sensitive cells within 4 hr of dexamethasone treatment, required functional glucocorticoid receptor, and was dose-dependent with regard to hormone. GST expression was not induced in an apoptosis-defective variant that contained normal levels of functional receptor, indicating that GST up-regulation was the result of secondary events that occur during steroid-mediated apoptosis. Using the calcium ionophore A23817 to induce lymphocyte cell death, GST RNA levels were increased in both steroid-sensitive and steroid-resistant cell lines, supporting the conclusion that elevated GST expression was the result of cellular processes associated with apoptosis, rather than a direct consequence of steroid-mediated transcriptional control. The cells were also treated with dibutyryl cAMP to cause cell death; however, this mode of killing did not result in GST up-regulation. Taken together, these results suggest that GST induction in dexamethasone-treated T-lymphocytes occurs early in the steroid-regulated apoptotic pathway and that this may be a marker of calcium-stimulated cell death. Based on the known function of GST as an antioxidant defense enzyme and its transcriptional regulation by reactive oxygen intermediates, we propose that the gene product of a primary GR target gene(s) directly or indirectly effects the redox state of the cell. Thus activation of GST gene expression in apoptotic lymphocytes is likely a indicator of oxidative stress, rather than a required step in the pathway.