Sanjit K. Dhar

Manganese superoxide dismutase regulation and cancer

Mitochondria are the power plants of the eukaryotic cell and the integrators of many metabolic activities and signaling pathways important for the life and death of a cell. Normal aerobic cells use oxidative phosphorylation to generate ATP, which supplies energy for metabolism. To drive ATP production, electrons are passed along the electron transport chain, with some leaking as superoxide during the process. It is estimated that, during normal respiration, intramitochondrial superoxide concentrations can reach 10⁻¹² M. This extremely high level of endogenous superoxide production dictates that mitochondria are equipped with antioxidant systems that prevent consequential oxidative injury to mitochondria and maintain normal mitochondrial functions. The major antioxidant enzyme that scavenges superoxide anion radical in mitochondria is manganese superoxide dismutase (MnSOD). Extensive studies on MnSOD have demonstrated that MnSOD plays a critical role in the development and progression of cancer. Many human cancer cells harbor low levels of MnSOD proteins and enzymatic activity, whereas some cancer cells possess high levels of MnSOD expression and activity. This apparent variation in MnSOD level among cancer cells suggests that differential regulation of MnSOD exists in cancer cells and that this regulation may be linked to the type and stage of cancer development. This review summarizes current knowledge of the relationship between MnSOD levels and cancer with a focus on the mechanisms regulating MnSOD expression.

Specificity Protein 1-dependent p53-mediated Suppression of Human Manganese Superoxide Dismutase Gene Expression

Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme necessary for the survival of aerobic life. Previously, we demonstrated that specificity protein 1 (Sp1) is essential for the basal transcription of the MnSOD gene. We also identified nucleophosmin (NPM), an RNA-binding protein, as an important co-activator of NF-κB in the induction of MnSOD by cytokine and tumor promoter. Here, using chromatin immunoprecipitation (ChIP) analysis, we demonstrate that Sp1 and NPM interact in vivo to enhance NF-κB-mediated MnSOD induction. Interaction between NPM and Sp1 or NF-κB at the promoter and enhancer of the MnSOD gene in vivo were verified by the presence of the PCR products from the promoter and enhancer elements in the ChIP assay. Unexpectedly, we also found p53, another transcription factor, to be a component of the complex detected by ChIP assay. The presence of p53 in this transcription complex was verified by immunoprecipitation of p53 proteins with antibody to Sp1 in nuclear extracts. Using a vector expressing full-length p53 cDNA, we demonstrated that p53 overexpression suppresses MnSOD mRNA and protein levels. Consistent with the negative role of p53 in the expression of the MnSOD gene, expression of small interfering RNA for p53 leads to an increase of MnSOD mRNA and protein levels. Using ChIP assays and immunoprecipitation, we further demonstrated that p53 interacts with Sp1 to suppress both the constitutive and 12-O-tetradecanoylphorbol-13-acetate-stimulated expression of the MnSOD gene. Inhibition of the MnSOD gene by p53 was abolished when Sp1 sites on the MnSOD promoter were mutated or when the Sp1 protein was reduced by siRNA approaches. Because expression of MnSOD protects against cell death, our findings reveal a previously unrecognized mechanism of p53-mediated cell death and demonstrate an intricate relationship between the positive and negative control of MnSOD expression.

Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle

The identification of potential endogenous or synthetic ligands for orphan receptors in the steroid receptor superfamily is important both for discerning endogenous regulatory pathways and for designing receptor inhibitors. The insect nuclear receptor Ultraspiracle (USP), an ortholog of vertebrate RXR, has long been treated as an orphan receptor. We have tested here the fit of terpenoid ligands to the JH III-binding site of monomeric and homo-oligomeric USP from Drosophila melanogaster (dUSP). dUSP specifically bound juvenile hormone III (JH III), but not control farnesol or JH III acid, and also specifically changed in conformation upon binding of JH III in a fluorescence binding assay. Juvenile hormone III binding caused intramolecular changes in receptor conformation, and stabilized the receptors dimeric/oligomeric quaternary structure. In both a radiometric competition assay and the fluorescence binding assay the synthetic JH III agonist methoprene specifically competed with JH III for binding to dUSP, the first demonstration of specific binding of a biologically active JH III analog to an insect nuclear receptor. The recombinant dUSP bound with specificity to a DR12 hormone response element in a gel shift assay. The same DR12 element conferred enhanced transcriptional responsiveness of a transfected juvenile hormone esterase core promoter to treatment of transfected cells with JH III, but not to treatment with retinoic acid or T3. The activity of JH III or JH III-like structures, but not structures without JH III biological activity, to bind specifically to dUSP and activate its conformational change, provide evidence of a terpenoid endogenous ligand for Ultraspiracle, and offer the prospect that synthetic, terpenoid structures may be discovered that can agonize or antagonize USP function in vivo.

Identification of Nucleophosmin as an NF-κB Co-activator for the Induction of the Human SOD2 Gene *

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme essential for the survival of life. We have reported that NF-κB is essential but not sufficient for the synergistic induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. To further identify transcription factors and co-activators that participate in the induction of MnSOD, we used NF-κB affinity chromatography to isolate potential NF-κB interacting proteins. Proteins eluted from the NF-κB affinity column were subjected to proteomic analysis and verified by Western analysis. Nucleophosmin (NPM), a nucleolar phosphoprotein, is the most abundant single protein identified. Co-immunoprecipitation studies suggest a physical interaction between NPM and NF-κB proteins. To verify the role of NPM on MnSOD gene transcription, cells were transfected with constructs expressing NPM in sense or antisense orientation as well as interference RNA. The results indicate that an increase NPM expression leads to increased MnSOD gene transcription in a dose-dependent manner. Consistent with this, expression of small interfering RNA for NPM leads to inhibition of MnSOD gene transcription but does not have any effect on the expression of interleukin-8, suggesting that the effect of NPM is selective. These results identify NPM as a partner of the NF-κB transcription complex in the induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines.

RelB regulates manganese superoxide dismutase gene and resistance to ionizing radiation of prostate cancer cells.

The relationship between NF-κB and resistance to radiation treatment in many tumor cell types has been generally well recognized. However, which members of the NF-κB family contribute to radiation resistance is unclear. In the present study, we demonstrate that RelB plays an important radioprotective role in aggressive prostate cancer cells, in part by the induction of antioxidant and antiapoptotic manganese superoxide dismutase (MnSOD) gene. RelB is both constitutively present and is inducible by radiation in aggressive prostate cancer cells. Using ectopically expressed dominant negative inhibitor, p100 mutant, and the siRNA approach, we demonstrate that selective inhibition of RelB significantly decreases the levels of MnSOD resulting in a significant increase in the sensitivity of prostate cancer cells to radiation treatment. These results demonstrate that RelB plays an important role in redox regulation of the cell and protects aggressive prostate cancer cells against radiation-induced cell death. Thus, inhibition of RelB could be a novel mechanism to radiosensitize prostate cancer.

Manganese superoxide dismutase: beyond life and death

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant enzyme that localizes to the mitochondria. Expression of MnSOD is essential for the survival of aerobic life. Transgenic mice expressing a luciferase reporter gene under the control of the human MnSOD promoter demonstrate that the level of MnSOD is reduced prior to the formation of cancer. Overexpression of MnSOD in transgenic mice reduces the incidences and multiplicity of papillomas in a DMBA/TPA skin carcinogenesis model. However, MnSOD deficiency does not lead to enhanced tumorigenicity of skin tissue similarly treated because MnSOD can modulate both the p53-mediated apoptosis and AP-1-mediated cell proliferation pathways. Apoptosis is associated with an increase in mitochondrial levels of p53 suggesting a link between MnSOD deficiency and mitochondrial-mediated apoptosis. Activation of p53 is preventable by application of a SOD mimetic (MnTE-2-PyP5+). Thus, p53 translocation to mitochondria and subsequent inactivation of MnSOD explain the observed mitochondrial dysfunction that leads to transcription-dependent mechanisms of p53-induced apoptosis. Administration of MnTE-2-PyP5+ following apoptosis but prior to proliferation leads to suppression of protein carbonyls and reduces the activity of AP-1 and the level of the proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Remarkably, the incidence and multiplicity of skin tumors are drastically reduced in mice that receive MnTE-2-PyP5+ prior to cell proliferation. The results demonstrate the role of MnSOD beyond its essential role for survival and suggest a novel strategy for an antioxidant approach to cancer intervention.

Manganese Superoxide Dismutase Is a p53-Regulated Gene That Switches Cancers between Early and Advanced Stages

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

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

Redox proteomic identification of HNE-bound mitochondrial proteins in cardiac tissues reveals a systemic effect on energy metabolism after doxorubicin treatment.

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria after DOX treatment. A redox proteomics method involving two-dimensional electrophoresis followed by mass spectrometry and investigation of protein databases identified several HNE-modified mitochondrial proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle and electron transport chain. The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE-adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate was also significantly reduced after DOX treatment. Treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE-protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury, suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

MANGANESE SUPEROXIDE DISMUTASE VS. P53: REGULATION OF MITOCHONDRIAL ROS

Coordination of mitochondrial and nuclear activities is vital for cellular homeostasis, and many signaling molecules and transcription factors are regulated by mitochondria-derived reactive oxygen species (ROS) to carry out this interorganellar communication. The tumor suppressor p53 regulates myriad cellular functions through transcription-dependent and -independent mechanisms at both the nucleus and mitochondria. p53 affect mitochondrial ROS production, in part, by regulating the expression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Recent evidence suggests mitochondrial regulation of p53 activity through mechanisms that affect ROS production, and a breakdown of communication amongst mitochondria, p53, and the nucleus can have broad implications in disease development.