Tieli Wang

Manganese Superoxide Dismutase-Mediated Gene Expression in Radiation-Induced Adaptive Responses

ABSTRACT Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (−/−) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-κB transcriptional activity in MCF+FIR cells, by using mutant IκBα, inhibited radioresistance as well as reducing steady-state levels of MnSOD, 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNA. In contrast, mutant IκBα was unable to inhibit radioresistance or reduce 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNAs in MCF+SOD cells, where MnSOD overexpression was independent of NF-κB. These results support the hypothesis that NF-κB is capable of regulating the expression of MnSOD, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.

A Ferrous-Triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase.

Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapine-induced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo. [Mol Cancer Ther 2006;5(3):586–92]

The Role of Peroxiredoxin II in Radiation-Resistant MCF-7 Breast Cancer Cells

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.

Expression of ErbB2 enhances radiation-induced NF-κB activation

Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NF-κB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-κB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2). Stable transfection of dominant-negative mutant IκB (MCF-7/ErbB2/mIκB) or treatment with anti-ErbB2 antibody, Herceptin, inhibited NF-κB activation and radiosensitized MCF-7/ErbB2 cells. Consistent with NF-κB regulation, basal and IR-induced Akt, a kinase downstream of ErbB2, was activated in MCF-7/ErbB2 cells and inhibited by Herceptin. To identify specific genes affected by ErbB2-mediated NF-κB activation, a group of IR-responsive elements Cyclin B1, Cyclin D1, Bcl-2, Bcl/XL, BAD and BAX were evaluated. Basal levels of prosurvival elements Cyclin B1, Cyclin D1, Bcl-2 and Bcl/XL but not apoptotic BAD and BAX were upregulated in MCF-7/ErbB2 cells with striking enhancements in Bcl-2 and Bcl/XL. IR further induced Cyclin B1 and Cyclin D1 expression that was reduced by Herceptin. Bcl-2 kept a high steady level after Herceptin+IR treatment and, in contrast to control MCF-7/Vector cells, Bcl/XL was inhibited in MCF-7/ErbB2 cells by Herceptin+IR treatment. However, all four prosurvival proteins were downregulated by inhibition of NF-κB in MCF-7/ErbB2/mIκB cells. These results thus provide evidence suggesting that overexpression of ErbB2 is able to enhance NF-κB response to IR, and that a specific prosurvival network downstream of NF-κB is triggered by treatments using anti-ErbB2 antibody combined with radiation.

Co-activation of ERK, NF-κB, and GADD45β in Response to Ionizing Radiation

NF-κB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45β is a primary cell cycle element responsive to NF-κB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-κB, ERK and GADD45β are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-κB transactivation and GADD45β expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-κB. Transfection of dominant negative mutant IκB that blocks NF-κB nuclear translocation, inhibited ERK activity and GADD45β expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45β strikingly blocked IR-induced NF-κB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-κB, ERK, and GADD45β are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

Structurally Dependent Redox Property of Ribonucleotide Reductase Subunit p53R2

p53R2 is a newly identified small subunit of ribonucleotide reductase (RR) and plays a key role in supplying precursors for DNA repair in a p53-dependent manner. Currently, we are studying the redox property, structure, and function of p53R2. In cell-free systems, p53R2 did not oxidize a reactive oxygen species (ROS) indicator carboxy-H2DCFDA, but another class I RR small subunit, hRRM2, did. Further studies showed that purified recombinant p53R2 protein has catalase activity, which breaks down H2O2. Overexpression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas overexpression of hRRM2 did not and resulted in a collapse of mitochondrial membrane potential. In a site-directed mutagenesis study, antioxidant activity was abrogated in p53R2 mutants Y331F, Y285F, Y49F, and Y241H, but not Y164F or Y164C. The fluorescence intensity in mutants oxidizing carboxy-H2DCFDA, in order from highest to lowest, was Y331F > Y285F > Y49F > Y241H > wild-type p53R2. This indicates that Y331, Y285, Y49, and Y241 in p53R2 are critical residues involved in scavenging ROS. Of interest, the ability to oxidize carboxy-H2DCFDA indicated by fluorescence intensity was negatively correlated with RR activity from wild-type p53R2, mutants Y331F, Y285F, and Y49F. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its fundamental enzymatic activity.

Synthesis and characterization of a novel aluminophosphate with layer structure

An aluminophosphate with a layer structure has been synthesized hydrothermally. The ratio P/Al = 2 for the framework; its structure consists of Al-centered tetrahedra and P-centered tetrahedra as basic building units to form a two-dimensional layer structure. The protonated ethylenediamine molecules are located in the interlayer space and sustain the framework. The water molecules are located in the space between two layers. Layers are joined together by van der Waals force to form a three-dimensional structure which crystallizes in the space group Pbnb, with a = 8.052(6), b = 8.760(2), c = 17.037(7) {angstrom}, and V = 1201.88 {angstrom}{sup 3}. The crystal structure has been refined to yield values of R = 0.0418, R{sub w} = 0.0447.

The role of peroxiredoxin II in chemoresistance of breast cancer cells.

Peroxiredoxin (Prx)II belongs to a family of redox-active proteins that use redox-sensitive cysteine in the active site to reduce peroxides. PrxII is induced by various oxidative stimuli and plays an important protective role against oxidative radical damage by reactive oxygen species. PrxII expression levels are correlated with resistance to radiation therapy or certain anti-cancer drugs in radioresistant breast cancer cells, glioblastomas, and head and neck cancer cells as well as in tissue isolated from head and neck patients who do not respond to radiation therapy. Small interfering RNA (siRNA) that inhibits the PrxII gene expression has been shown to partially reverse the radioresistant phenotype in radiation resistant breast cancer cells and sensitizes glioma cells to oxidative stress, highlighting the potential clinical importance of PrxII in radiation resistance in cancer. This article focuses on the role that PrxII may play in chemoresistant breast cancer cells.

Enhanced radiation response in radioresistant MCF-7 cells by targeting peroxiredoxin II.

In our previous study, we identified that a protein target, peroxiredoxin II (PrxII), is overexpressed in radioresistant MCF+FIR3 breast-cancer cells and found that its expression and function is associated with breast-cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast-cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by the siRNA that inhibits PrxII gene expression. Our results, presented here, show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells, and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to this technology’s property of targeting to specific cancer-related genes.

Abstract 3953: Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953