Melba C. Jaramillo

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.

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.

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.

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.

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.

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.

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 porphyrins 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.

Tumor protein translationally controlled 1 is a p53 target gene that promotes cell survival

Tumor suppressor p53 maintains genome stability by differentially activating target genes that control diverse cellular responses, such as the antioxidant response, cell cycle arrest and apoptosis. Despite the fact that many p53 downstream genes have been well characterized, novel p53 target genes are continuously being identified. Here, we report that Tpt1 is a direct target gene of p53. We found that p53 upregulates the transcription of Tpt1 and identified a p53-responsive element in the promoter of the mouse Tpt1 gene. Furthermore, p53-dependent induction of Tpt1 was able to reduce oxidative stress, minimize apoptosis, and promote cell survival in response to H2O2 challenge. In addition, a positive correlation between the expression of p53 and Tpt1 only existed in normal lung tissues, not in lung tumors. Such positive correlation was also found in lung cell lines that contain wild-type p53, but not mutated p53. Based on the important role of Tpt1 in cancer development, chemoresistance, and cancer reversion, identification of Tpt1 as a direct target gene of p53 not only adds to the complexity of the p53 network, but may also open up a new avenue for cancer prevention and intervention.

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.

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.