Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate.

AIMS: We aim here to demonstrate that radiation (RT) enhances tumor sensitization by only those Mn complexes that are redox active and cycle with ascorbate (Asc), thereby producing H2O2 and utilizing it subsequently in protein S-glutathionylation in a glutathione peroxidase (GPx)-like manner. In turn, such compounds affect cellular redox environment, described by glutathione disulfide (GSSG)/glutathione (GSH) ratio, and tumor growth. To achieve our goal, we tested several Mn complexes of different chemical and physical properties in cellular and animal flank models of 4T1 breast cancer cell. Four other cancer cell lines were used to substantiate key findings. RESULTS: Joint administration of cationic Mn porphyrin (MnP)-based redox active compounds, MnTE-2-PyP5+ or MnTnBuOE-2-PyP5+ with RT and Asc contributes to high H2O2 production in cancer cells and tumor, which along with high MnP accumulation in cancer cells and tumor induces the largest suppression of cell viability and tumor growth, while increasing GSSG/GSH ratio and levels of total S-glutathionylated proteins. Redox-inert MnP, MnTBAP3- and two other different types of redox-active Mn complexes (EUK-8 and M40403) were neither efficacious in the cellular nor in the animal model. Such outcome is in accordance with their inability to catalyze Asc oxidation and mimic GPx. INNOVATION: We provided here the first evidence how structure-activity relationship between the catalytic potency and the redox properties of Mn complexes controls their ability to impact cellular redox environment and thus enhance the radiation and ascorbate-mediated tumor suppression. CONCLUSIONS: 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.

Manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin acts as a pro-oxidant to inhibit electron transport chain proteins, modulate bioenergetics, and enhance the response to chemotherapy in lymphoma cells.

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.

Induction of autophagy contributes to cisplatin resistance in human ovarian cancer cells.

Cisplatin resistance is a major challenge in the clinical treatment of ovarian cancer, of which the underlying mechanisms remain unknown. The aim of the present study was to explore the role of autophagy in cisplatin resistance in ovarian cancer cells. A2780cp cisplatin-resistant ovarian carcinoma cells and the A2780 parental cell line, were used as a model throughout the present study. The cell viability was determined using a water soluble tetrazolium salt-8 assay, and western blot analysis was performed to determine the protein expression levels of microtubule-associated protein 1 light chain 3 (LC3 I and LC3 II), and Beclin 1. Beclin 1 small interfering (si)RNA and 3-methyladenine (3-MA) were used to determine whether inhibition of autophagy may re-sensitize cisplatin-resistant cells to cisplatin. The ultrastructural analysis of autophagosomes was performed using transmission electron microscopy, and apoptosis was measured by flow cytometry. In both A2780cp and A2780 cells, cisplatin induced the formation of autophagosomes and upregulated the expression levels of autophagy protein markers, LC3 II and Beclin 1. However, the levels of autophagy were significantly higher in A2780cp cells, as compared with the A2780 cells. The combined treatment of cisplatin with 3-MA, the autophagy pharmacological inhibitor, increased the cell death rate, but had no effects on apoptosis, as compared with cisplatin treatment alone in A2780cp cells. However, inhibition of autophagy by siRNA knockdown of Beclin 1 expression enhanced cisplatin-induced cell death and apoptosis. The findings of the present study suggest that autophagy has a protective role in human ovarian cancer cells, and that targeting autophagy may promote chemotherapeutic sensitivity.

Mitochondria and redox homoeostasis as chemotherapeutic targets.

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.

Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma.

UNLABELLED: Cisplatin resistance is a major problem affecting ovarian carcinoma treatment. NF-E2-related factor 2 (Nrf2), a nuclear transcription factor, plays an important role in chemotherapy resistance. However, the underlying mechanism by which Nrf2 mediates cisplatin chemoresistance is unclear. METHODS: The human ovarian carcinoma cell line, A2780, and its cisplatin-resistant variant, A2780cp were cultivated. Cell viability was determined with WST-8 assay. Western blot was applied to detect the expression of Nrf2, Nrf2 target genes, and autophagy-related proteins. RNA interference was used to knock down target genes. Annexin V and propidium iodide (PI) staining was utilized to quantify apoptosis. The ultrastructural analysis of autophagosomes was performed by transmission electron microscopy (TEM). RESULTS: Nrf2 and its targeting genes, NQO1 and HO-1, are overexpressed in A2780cp cells compared with A2780 cells. Knocking down Nrf2 sensitized A2780cp cells to cisplatin treatment and decreased autophagy-related genes, Atg3, Atg6, Atg12 and p62 in both mRNA and protein levels. Furthermore, we demonstrated that in both cell lines cisplatin could induce the formation of autophagosomes and upregulate the expression of autophagy-related genes Atg3, Atg6 and Atg12. Treatment with an autophagy inhibitor, 3-Methyladenine (3-MA), or beclin 1 siRNA enhanced cisplatin-induced cell death in A2780cp cells, suggesting that inhibition of autophagy renders resistant cells to be more sensitive to cisplatin. Taken together, Nrf2 signaling may regulate cisplatin resistance by activating autophagy. CONCLUSIONS: Nrf2-activated autophagy may function as a novel mechanism causing cisplatin-resistance.

Indolent lymphoma: follicular lymphoma and the microenvironment-insights from gene expression profiling.

As shown with gene expression profiling (GEP), the development and progression of follicular lymphoma (FL) involves complex interactions between neoplastic B cells and the surrounding microenvironment. GEP further reveals that the tumor microenvironment may predict survival in patients with FL and influence the response to therapy and the risk of transformation. Here, we briefly review GEP technology and summarize the role of the tumor microenvironment in FL diagnosis, prognosis, and transformation. Genes expressed by infiltrating T cells and macrophages appear to be the most important predictors of survival, clinical behavior, and outcome. These findings provide a basis for future studies into the pathogenesis and pathophysiology of FL and may ultimately provide guidance in the choice of therapy and the identification of potential therapeutic targets.

Poly(ADP-ribose) polymerase-1 modulates Nrf2-dependent transcription.

The basic leucine zipper transcription factor Nrf2 has emerged as a master regulator of intracellular redox homeostasis by controlling the expression of a battery of redox-balancing antioxidants and phase II detoxification enzymes. Under oxidative stress conditions, Nrf2 is induced at the protein level through redox-sensitive modifications on critical cysteine residues in Keap1, a component of an E3 ubiquitin ligase complex that targets Nrf2 for proteasomal degradation. Poly(ADP-ribose) polymerase-1 (PARP-1) is historically known to function in DNA damage detection and repair; however, recently PARP-1 has been shown to play an important role in other biochemical activities, such as DNA methylation and imprinting, insulator activity, chromosome organization, and transcriptional regulation. The exact role of PARP-1 in transcription modulation and the underlying mechanisms remain poorly defined. In this study, we report that PARP-1 forms complexes with the antioxidant response element (ARE) within the promoter region of Nrf2 target genes and upregulates the transcriptional activity of Nrf2. Interestingly, PARP-1 neither physically interacts with Nrf2 nor promotes the expression of Nrf2. In addition, PARP-1 does not target Nrf2 for poly(ADP-ribosyl)ation. Instead, PARP-1 interacts directly with small Maf proteins and the ARE of Nrf2 target genes, which augments ARE-specific DNA-binding of Nrf2 and enhances the transcription of Nrf2 target genes. Collectively, these results suggest that PARP-1 serves as a transcriptional coactivator, upregulating the transcriptional activity of Nrf2 by enhancing the interaction among Nrf2, MafG, and the ARE.

The emerging role of the Nrf2-Keap1 signaling pathway in cancer.

The Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2])-Keap1 (Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1) signaling pathway is one of the most important cell defense and survival pathways. Nrf2 can protect cells and tissues from a variety of toxicants and carcinogens by increasing the expression of a number of cytoprotective genes. As a result, several Nrf2 activators are currently being tested as chemopreventive compounds in clinical trials. Just as Nrf2 protects normal cells, studies have shown that Nrf2 may also protect cancer cells from chemotherapeutic agents and facilitate cancer progression. Nrf2 is aberrantly accumulated in many types of cancer, and its expression is associated with a poor prognosis in patients. In addition, Nrf2 expression is induced during the course of drug resistance. Collectively, these studies suggest that Nrf2 contributes to both intrinsic and acquired chemoresistance. This discovery has opened up a broad spectrum of research geared toward a better understanding of the role of Nrf2 in cancer. This review provides an overview of (1) the Nrf2-Keap1 signaling pathway, (2) the dual role of Nrf2 in cancer, (3) the molecular basis of Nrf2 activation in cancer cells, and (4) the challenges in the development of Nrf2-based drugs for chemoprevention and chemotherapy.

Tanshinone I activates the Nrf2-dependent antioxidant response and protects against As(III)-induced lung inflammation in vitro and in vivo.

AIMS: The NF-E2 p45-related factor 2 (Nrf2) signaling pathway regulates the cellular antioxidant response and activation of Nrf2 has recently been shown to limit tissue damage from exposure to environmental toxicants, including As(III). In an attempt to identify improved molecular agents for systemic protection against environmental insults, we have focused on the identification of novel medicinal plant-derived Nrf2 activators. RESULTS: Tanshinones [tanshinone I (T-I), tanshinone IIA, dihydrotanshinone, cryptotanshinone], phenanthrenequinone-based redox therapeutics derived from the medicinal herb Salvia miltiorrhiza, have been tested as experimental therapeutics for Nrf2-dependent cytoprotection. Using a dual luciferase reporter assay overexpressing wild-type or mutant Kelch-like ECH-associated protein-1 (Keap1), we demonstrate that T-I is a potent Keap1-C151-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. In human bronchial epithelial cells exposed to As(III), T-I displays pronounced cytoprotective activity with upregulation of Nrf2-orchestrated gene expression. In Nrf2 wild-type mice, systemic administration of T-I attenuates As(III) induced inflammatory lung damage, a protective effect not observed in Nrf2 knockout mice. INNOVATION: Tanshinones have been identified as a novel class of Nrf2-inducers for antioxidant tissue protection in an in vivo As(III) inhalation model, that is relevant to low doses of environmental exposure. CONCLUSION: T-I represents a prototype Nrf2-activator that displays cytoprotective activity upon systemic administration targeting lung damage originating from environmental insults. T-I based Nrf2-directed systemic intervention may provide therapeutic benefit in protecting other organs against environmental insults.

Arsenic-mediated activation of the Nrf2-Keap1 antioxidant pathway.

Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2-Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert-butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2-Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic-induced diseases. The National Institute of Environmental Health Science (NIEHS) Outstanding New Environmental Scientist (ONES) award has provided the opportunity to review the progress both in the fields of arsenic toxicology and Nrf2 biology. Much of the funding has led to (1) the novel discovery that arsenic activates the Nrf2 pathway by a mechanism different to that of other Nrf2 activators, such as sulforaphane and tert-butylhydroquinone, (2) activation of Nrf2 by chemopreventive compounds protects against arsenic toxicity and carcinogenicity both in vitro and in vivo, (3) constitutive activation of Nrf2 by disrupting Keap1-mediated negative regulation contributes to cancer and chemoresistance, (4) p62-mediated sequestration of Keap1 activates the Nrf2 pathway, and (5) arsenic-mediated Nrf2 activation may be through a p62-dependent mechanism. All of these findings have been published and are discussed in this review. This award has laid the foundation for my laboratory to further investigate the molecular mechanism(s) that regulate the Nrf2 pathway and how it may play an integral role in arsenic toxicity. Moreover, understanding the biology behind arsenic toxicity and carcinogenicity will help in the discovery of potential strategies to prevent or control arsenic-mediated adverse effects.

Mitochondria are the primary source of the H(2)O(2) signal for glucocorticoid-induced apoptosis of lymphoma cells.

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.

Manganese porphyrin, MnTE-2-PyP5+, Acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells.

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.

Hydrogen peroxide signaling is required for glucocorticoid-induced apoptosis in lymphoma cells.

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.

Increased manganese superoxide dismutase expression or treatment with manganese porphyrin potentiates dexamethasone-induced apoptosis in lymphoma cells.

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.

Heat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor.

PURPOSE: In cytokine immunotherapy of cancer it is critical to deliver sufficiently high local cytokine concentrations in order to reach the therapeutic threshold needed for clinical efficacy. Simultaneously, for optimal clinical safety adverse effects caused by high systemic cytokine levels must be minimized. One of the most promising anti-cancer therapeutic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), has elicited anti-tumour immune responses in animal studies and clinical trials. However, the clinical efficacy has been limited, with local GM-CSF levels being therapeutically insufficient and systemic toxicity being a limiting factor. METHODS: To address these problems we have developed a novel GM-CSF expression vector, pAD-HotAmp-GM-CSF, which can provide high levels of GM-CSF expression, and induction of cytokine expression to limited tissue areas. This expression system combines inducible and amplifying elements in a single multi-genic construct. The first transcriptional unit contains the inducible element, the heat shock protein 70B (HSP70B) promoter that regulates expression of the transcription-activating factor tat. RESULTS: Upon the binding of tat to the second promoter, the HIV2 long terminal repeat amplifies downstream gene expression of the therapeutic cytokine GM-CSF. Moderate hyperthermia at 42 degrees C for 30 min induced GM-CSF expression in pAD-HotAmp-GM-CSF that was over 2.5- and 2.8-fold higher than levels reached with HSP70B promoter alone and the prototypical human cytomegalovirus promoter. CONCLUSIONS: Thus, the inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system for regulated and enhanced GM-CSF expression, which enables both greater efficacy and safety in cytokine immunotherapy of cancer.