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.

Clinical radiation resistance continues to be a major problem in the treatment of cancer. The molecular mechanisms underlying tumor-adaptive radioresistance remains to be elucidated. It has been well documented that ionizing radiation produces free radicals in biological tissues. Those radicals are the primary source of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS/RNS, such as superoxide anion, hydrogen peroxide, and peroxynitrite, seem to be transiently produced in response to ionizing radiation, growth factor, and cytokine stimulation (1, 2). They are involved in both the lethal clastogenic and mutagenic effects of ionizing radiation (3). Radioprotective effects by modifications of antioxidant enzyme expression or by addition of free-radical scavengers have been described (36). Accumulating evidence show that ROS and RNS are not only toxic byproducts but have been shown to act as signal transduction agents involved in regulating normal functions, including cell growth and differentiation, immune responses by phagocytic cells, and apoptosis (7). ROS and RNS play a significant but paradoxical role acting as a “double-edged sword” to regulate cellular processes.

In the past few years, nuclear DNA damage–sensing mechanisms activated by ionizing radiation have been identified, including ataxia-telangiectasia mutated (ATM)/ATM and Rad-3 related and the DNA-dependent protein kinase (810). Less is known about sensing mechanisms for cytoplasmic ionization events and how these events influence nuclear processes. Several studies have shown the importance of cytoplasmic signaling pathways in cytoprotection and mutagenesis. For cytoplasmic signaling, radiation-stimulated ROS and RNS are essential activators of the cytoplasmic signaling pathways (11). Although as yet unproven, the nature and extent of the ROS and RNS insult may determine the threshold of the cellular response, manifesting as proliferation, stress response, and damage repair or apoptosis (12, 13). Elevation of ROS in excess of the buffering capacity and enzymatic activities designed to modulate ROS levels results in potentially cytotoxic oxidative stress. Under these pro-oxidant conditions, highly reactive radicals can damage DNA, RNA, protein, and lipid components, which may lead to cell death. Oxidative stress induced by ROS/RNS, such as hydrogen peroxide, has been implicated in the pathogenesis of several neurodegenerative diseases and cancers (1215). To cope with oxidative insult, the cell has developed a number of defense strategies, both at the level of oxidative damage repair and ROS/RNS-scavenging mechanisms (2). Repair mechanisms that confer resistance to oxidative stress include various protein disulfide reductase enzymes as well as multifunctional DNA repair and thiol-reducing proteins. Antioxidant mechanisms comprise small proteins with redox-active sulfhydryl moieties as well as enzymatic ROS-metabolizing systems, including superoxide dismutase, catalase, and peroxidase. We hypothesize that radio adaptation could arise from the activation of damage repair and/or the reactions of the antioxidant defense systems.

To address the above questions, we have established an effective model system to further explore the radiation resistance phenotype. This model is based on the MCF-7 human breast cancer cell line that was exposed to chronic ionizing radiation and became resistant to radiation treatment (16). Because most advanced and recurrent tumors are resistant to chemotherapy, it is desirable to investigate persons or cells that express such abnormalities. Acquired radiation-resistant cancer cells (MCF+FIR30) were obtained by exposure of MCF-7 breast cancer cells to fractionated ionizing radiation (FIR) with a total dose of 60 Gy of γ-irradiation (2 Gy per fraction, five times per week for 6 weeks; ref. 16). Several radiation-resistant clones were independently isolated and characterized. A radioresistant clone (MCF+FIR3) and a radiosensitive clone (MCF+FIS4) isolated from the same MCF+FIR30 populations were cultured for further study. Comparative proteomic analysis was done to search for expression alterations between the two clones. We found that the protein expression level of peroxiredoxin II (PrxII) is significantly up-regulated in radioresistant MCF+FIR3 cells compared with that in radiosensitive MCF+FIS4 cells. Western blot analysis of radioresistant MCF+FIR3 cells and radiosensitive MCF+FIS4 cells confirmed the increased expression of PrxII protein in MCF+FIR3 radioresistant breast cancer cells. We introduced dsRNA into MCF+FIR3 radioresistant breast cancer cells, which leads to gene silencing in a sequence-specific manner. Clonogenic survival assays were done to show that silencing the PrxII gene sensitized radioresistant MCF+FIR3 breast cancer cells. In addition, overexpression of PrxII in radiosensitive MCF+FIS4 cells results in increased radioresistance in breast cancer cells. ROS are known to contribute to the toxicity of ionizing radiation, and the intracellular scavenging of these species is an important modulator of ionizing radiation–induced damage. Our experimental results clearly indicate that radiation-resistant cells contain elevated levels of the radical scavenger PrxII protein, a member of a family of proteins that provide a protective role in redox damage. Induction of PrxII protein in resistant breast cancer cells provides evidence for the involvement of ROS in breast cancer cells resistance to radiation. Our study helps to understand and manipulate resistance in radiation therapy and may elucidate an alternative mechanism by which breast cancer can be sensitized to radiation.

Development radioresistance cell line. MCF-7 human breast cancer cells were purchased from the American Type Culture Collection (Manassas, VA). MCF+FIR30 cells were obtained from MCF-7 cells by exposure to FIR with a total dose of 60 Gy of γ-irradiation (2 Gy per fraction, five times per week for 6 weeks). Radiation was delivered at room temperature at a rate of 5 Gy/1.15 minutes with a Cesium-137 (Cs-137) Irradiator (JL Shepherd Mark I, San Fernando, CA). A moderate radioresistance [dose-modifying factor (DMF) = 1.6-2.3 at 10% isosurvival rate] in MCF-7 human breast cancer cells that was exposed to FIR for 20 fractions (MCF+FIR20) or for 30 fractions (MCF+FIR30) was observed (16). These FIR-derived MCF-7 populations showed increased radioresistant phenotype for 7 to 10 passages after FIR treatment. There is no resistance observed in cells after 10 passages without FIR. Several clones isolated from the resistant MCF+FIR30 breast cancer cell population were individually cultured. Clonogenic assays were used to determine the resistance level among those clones. A radiation-resistant clone (MCF+FIR3) and a radiation-sensitive clone (MCF+FIS4) isolated from the MCF+FIR30 clonal populations were used for comparative proteomic analysis.

Clonogenic assay. Experiments were done to compare the radioresistant level between the MCF+FIR3 and MCF+FIS4 cells. Radioresistance was measured by clonogenic survival following exposure to IR. Cells were plated into T-25 flasks and cultured for 6 hours before exposure to graded dose of Cs-137. After irradiation, cells were cultured for 21 days, and colonies having >50 cells were counted. Plating efficiencies were calculated as colonies per 10 cells. Surviving fractions were normalized to the plating efficiencies. DMFs were calculated using the following expression: DMF = dose to reach the specified survival in resistant cells ÷ dose to reach the same survival in the control (1).

Silencing peroxiredoxin II expression by small interfering RNA. Small interfering RNA (siRNA) capable of targeting mRNAs encoding PrxII was custom synthesized by Dharmacon Research (Lafayette, CO). The cells were seeded to achieve 30% to 50% confluence on the day of transfection. Transient transfection of siRNAs was done using a protocol recommended from the manufacturer (Dharmacon Research). In brief, the cells were harvested by exposure to trypsin, diluted with fresh medium without antibiotics, and transferred to T-25 flask. After incubation for 24 hours, the cells were transfected with the siRNAs for 2.5 days with the use of Oligofectamine (Invitrogen, Carlsbad, CA). The Scramble RNA Duplex (City of Hope, Duarte, CA) served as a control for siRNA activity. A mixture of 20 μL of Opti-MEM medium (Invitrogen) and 5 μL of Oligofectamine were incubated for 10 minutes at room temperature and were then combined with 20 μL of 20 μmol/L siRNA diluted with 355 μL of Opti-MEM. The resulting mixture (400 μL) was incubated for 20 minutes at room temperature to allow complex formation and then overlaid onto each flask containing the cells for a final volume of 3.6 mL/well. Approximately 105 MCF+FIS4 cells were transfected using Oligofectamine (Invitrogen) with specific siRNAs. PrxII expression of the transiently transfected cells was analyzed by Western blot at 24, 48, 72, and 144 hours after transfection using antibodies specific for PrxII (LabFrontier, Seoul, South Korea). All the transfectants were maintained in 10% fetal bovine serum (FBS)/DMEM (Mediatech, Inc., Herndon, VA) until collected for analysis.

Sample preparation for proteomics analysis. Radioresistant MCF+FIR3 and radiosensitive MCF+FIS4 cells were cultured for comparative proteomics analysis. Exponentially growing cells were rinsed thrice with ice-cold PBS and lysed in lysis buffer (8 mol/L urea, 4% CHAPS, and 40 mmol/L Tris base) by sonication in a sonic bath three to four times for an interval of 30 seconds. After removal of insoluble materials by centrifugation at 14,000 × g for 30 minutes, cell lysates were mixed with 10 volumes of rehydration buffer (8 mol/L urea, 2% CHAPS, 0.5% immobilized pH gradient buffer, 20 mmol/L DTT, and 0.005% bromphenol blue) and loaded onto immobilized pH gradient strips (pH 3-10, nonlinear and pH 4-7; Amersham Biosciences, Piscataway, NJ). The protein concentration in the supernatant fraction was determined by bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL) using bovine serum albumin as a standard. Carrier ampholytes (0.5% final concentration) were added, and the protein extracts were stored at −20°C.

Gel electrophoresis and analysis. Proteins were separated by two-dimensional electrophoresis, using immobilized pH gradient strip (pH 3-10 and pH 4-7). The samples were loaded on the immobilized pH gradient strip by in-gel sample rehydration, using a urea-thiourea mixture as solubilizing agent. Total extract (240-450 μg) was loaded on the first dimension gel. Isoelectric focusing on an IPGPhor isoelectrofocusing unit (Amersham Biosciences) and preparation (reduction and alkylation) of the immobilized pH gradient strips for the second-dimension SDS-PAGE was carried out according to the procedures recommended by the manufacturer. The isoelectric focusing was done over 24 hours for a total of 55,000 V-h. SDS-PAGE was conducted on 12% gels using an Amersham Biosciences SE600 vertical unit, and the protein spots were visualized by staining with colloidal Coomassie (Bio-Rad, Hercules, CA). Colloidal Coomassie two-dimensional gels were scanned and quantified with the GS 800 Bio-Rad densitometry and PdQuest software. All two-dimensional gels were run a minimum of three independent times under each condition to insure reproducibility.

Mass spectrometry analysis. Excised, chopped, destained, and dehydrated protein spots from gels were reduced in reducing buffer (10 mmol/L DTT in 100 mmol/L NH4HCO3). The samples were then alkylated in 100 mmol/L iodoacetamide solution in the dark. Gel pieces were rehydrated on ice for 45 minutes in 1 mmol/L HCl/100 mmol/L ammonium bicarbonate containing 50 ng/μL sequencing grade pig trypsin (Promega, Madison, WI). Digestion was carried out at 30°C for 15 hours. The resulting peptides were extracted by sequential extraction for 20 minutes in 20 mmol/L ammonium bicarbonate followed by 0.1% trifluoroacetic acid in 60% acetonitrile. Combined extracts were concentrated in a Speed Vac to 20 μL. Samples were loaded from a stainless steel chamber pressurized to 1,000 p.s.i. onto fused silica capillary (150 μm, inner diameter) slurry packed to 75 mm with Everest C18 beads (Michrom, Auburn, CA). The column was developed with a linear gradient of 5% to 50% acetonitrile in 0.4% acetic acid and 0.005% hepta-fluorobutyric acid over 25 minutes at 300 to 400 nL/min. Electrospray ionization was conducted by applying 1.25 kV to a Valco stainless steel union. Capillary columns terminated inside the Valco union, and a fused silica capillary (75 mm × 150 μm × 360 μm) tapered to ∼5-μm inner diameter served as an emitter. The peptides eluting from the column were analyzed directly on a TFinnigan LCQ Classic liquid chromatography tandem mass spectrometry (LC/MS/MS) spectrometer equipped with an in-house built microspray device at Core Facility of City of Hope. Data-dependent MS/MS spectra were acquired automatically by an Instrument Control Language procedure. Acquired MS/MS spectra were searched with SEQUEST against the National Center for Biotechnology Information (NCBI) protein database.

Western blot analysis. For immunoblot analyses of Prx enzymes, proteins on one-dimensional gels were transferred electrophoretically to a nitrocellulose membrane, and the membrane was incubated with rabbit antibodies against PrxII. Immunocomplexes were detected with horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Biosciences or Pierce Biotechnology). All immunoblot and SDS-PAGE gels were scanned using a EPSON expression 800 flatbed with Adobe Photoshop software for quantification. Scanned images were quantified by Kodak 1D image software. Excel software was used for data analysis.

Overexpression of the PrxII gene in breast cancer cells. The coding region of the PrxII gene was amplified by PCR with full-length human cDNA as a template and subcloned into a mammalian expression vector pcDNA3.1. The forward primer sequence is 5′-CCTTTGCCCACGAAGCTTTCAGTCA, where the underlined A base pair was mutated from C to create the HindIII enzyme digestion site. The reverse primer is 5′-CTCACTATCCGGGATCCAGCCTAATT, where the underlined A and C base pairs were mutated from A residues to create the BamHI enzyme digestion site. The PCR product was digested with HindIII and BamHI enzymes and ligated into pcDNA3.1 vector digested by the same enzyme. The vector contains the neomycin phosphotransferase gene, a selectable marker for mammalian cells. PrxII transfectants were obtained using published method with LipofectAMINE reagent (Life Technologies, Inc., Gaithersburg, MD). The transfected MCF+FIS4 cells were selected with the antibiotic G418. A pair of cell lines was established by transfecting the recombinant plasmid of PrxII and the empty vector control. Clonogenic assay was done to compare the resistant level between PrxII-overexpressing cells and MCF+FIS4 vector control cells.

Heterogeneity of radioresistant breast cancer cells. Radioresistance developing during consecutive treatments with ionizing radiation is an extremely complex and poorly understood phenomenon within the cells that develop radioresistance. There is tremendous heterogeneity as shown by their wide range of responses to different radiation doses. Tumor heterogeneity has also been well documented in the response to a variety of anticancer reagents. Previous experiments have shown that NSAIDs induce heterogeneous changes in human tumor cells, including growth arrest, apoptosis, and necrosis (17). To understand the mechanism underlying the resistant phenotype, we acquired a radiation-resistant breast cancer cell population (MCF+FIR30) and used it as a model system to study the adaptive survival to chronic FIR (16). Clonogenic survival assays have revealed that the heterogeneity exists in the MCF+FIR30 population. Figure 1 shows the results from clonogenic survival assays for the radioresistant MCF+FIR3 and a reverted radiosensitive MCF+FIR4 cloned cell line. This unique pair of cells were used for proteomics analysis.

Figure 1.

Different sensitivities to radiation between cloned MCF+FIR30 cell lines. The cloned cells (MCF+FIR3 and MCF+FIS4) were expanded in T-25 flasks for two to four passages to get enough cells for the measurement of clonogenic survival. Cloned cell lines were trypsinized, and clonogenic radiosensitivity was measured following irradiation with a range of single doses. Points, mean (n = 3); bars, SE.

Figure 1.

Different sensitivities to radiation between cloned MCF+FIR30 cell lines. The cloned cells (MCF+FIR3 and MCF+FIS4) were expanded in T-25 flasks for two to four passages to get enough cells for the measurement of clonogenic survival. Cloned cell lines were trypsinized, and clonogenic radiosensitivity was measured following irradiation with a range of single doses. Points, mean (n = 3); bars, SE.

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Changes in MCF+FIR3 proteomic profile. Radioresistant MCF+FIR3 clone and radiosensitive MCF+FIS4 clones were cultured for four passages. MCF+FIR3 cells still retained the resistance phenotype, whereas MCF+FIS4 cells were relatively sensitive to ionizing radiation. Comparative proteomic analysis was done to search for expression alterations between the two clones MCF+FIR3 and MCF+FIS4. Figure 2 shows the typical two-dimensional gels done on total cellular extracts for both MCF+FIR3 and MCF+FIS4 cells. Figure 2A is the two-dimensional gel for MCF+FIS4 radiosensitive breast cancer cells, whereas Fig. 2B is for radioresistant MCF+FIR3 breast cancer cells. After colloidal Coomassie staining, 100 different spots focalized in the pH range 3 to 10 used for the analysis of each sample. A pair of spots (Fig. 2A and B, No. 1) was visible on the gels; one spot intensified in radioresistance MCF+FIR3 cells and diminished in intensity in radiosensitive MCF+FIS4 cells. PdQuest software analysis showed about 4-fold increase in the area of the spot from the radioresistant MCF+FIR3 breast cancer cells compared with the one from radiosensitive MCF+FIS4 breast cancer cells (Fig. 2C). To get a clearer picture of them, proteins from both cells were further analyzed in two-dimensional gels by narrowing down the pH range from 3 to 10 to 4 to 7 (Fig. 2D and E). Figure 2D is the two-dimensional gel image for radiosensitive MCF+FIR4 breast cancer cells, whereas Fig. 2E is the image for radioresistant MCF+FIR3 breast cancer cells. PdQuest software analysis showed about the same 4-fold increase in the area of the spot from the radioresistant MCF+FIR3 breast cancer cells versus MCF+FIS4 breast cancer cells for pH range between 4 and 7 as for pH range 3 to 10 (Fig. 2F). The spots 1 from Fig. 2B and E were excised from the gel, digested with trypsin, and analyzed by LC/MS/MS. Acquired MS/MS spectra were searched with SEQUEST software against the NCBI protein database. Peptide mass fingerprint analysis and nonredundant sequence database matching allowed the unambiguous identification of the analyzed species. The result showed that the spots from MCF+FIR3 two-dimensional gels (pH 3-10 and pH 4-7) are PrxII proteins. Table 1 reports the nature of the identified spot, the measured two-dimensional coordinates, and relative sequence coverage. The nature of the mass signals occurring in the spectra excluded the simultaneous presence of different protein species in the analyzed spot. Figure 2G shows an electrospray LC/MS/MS of the single and double charged tryptic fragment 5 of PrxII. Unlike H2O2-treated cells, PrxII protein does not occur as different isoforms with various isoelectric point values in the radioresistant MCF+FIR3 breast cancer cells compared with that in radiosensitive MCF+FIS4 breast cancer cells.

Figure 2.

Two-dimensional gel images. A, proteins extracted from MCF+FIR3 radioresistant breast cancer cells were separated on an immobilized pH 3 to 10 gradient strip followed by a 12% polyacrylamide gel, as stated in Materials and Methods. The gels were stained with Coomassie blue. B, proteins extracted from the MCF+FIS4 radiosensitive breast cancer cells were run in the same condition as in (A). The two-dimensional images of MCF+FIR3 and MCF+FIS4 cells were analyzed by PdQuest software. C, fold increases for No.1 spot in radioresistant MCF+FIR3 cells compared with that in radiosensitive MCF+FIS4 cells. D, proteins extracted from MCF+FIR3 cells were separated on an immobilized pH 4 to 7 gradient strip followed by a 12% polyacrylamide gel, as stated in Materials and Methods. The gels were stained with Coomassie blue. E, proteins extracted from the MCF+FIS4 cells were run in the same condition as in (A). The two-dimensional images of MCF+FIR3 and MCF+FIS4 cells were analyzed by PdQuest software. F, fold increases for No.1 spot in MCF+FIR3 cells compared with that in MCF+FIS4 cells. G, partial two-dimensional gel images; protein corresponding to PrxII (arrow). An electrospray LC/MS/MS of the single- and double-charged tryptic fragment 5 of PrxII.

Figure 2.

Two-dimensional gel images. A, proteins extracted from MCF+FIR3 radioresistant breast cancer cells were separated on an immobilized pH 3 to 10 gradient strip followed by a 12% polyacrylamide gel, as stated in Materials and Methods. The gels were stained with Coomassie blue. B, proteins extracted from the MCF+FIS4 radiosensitive breast cancer cells were run in the same condition as in (A). The two-dimensional images of MCF+FIR3 and MCF+FIS4 cells were analyzed by PdQuest software. C, fold increases for No.1 spot in radioresistant MCF+FIR3 cells compared with that in radiosensitive MCF+FIS4 cells. D, proteins extracted from MCF+FIR3 cells were separated on an immobilized pH 4 to 7 gradient strip followed by a 12% polyacrylamide gel, as stated in Materials and Methods. The gels were stained with Coomassie blue. E, proteins extracted from the MCF+FIS4 cells were run in the same condition as in (A). The two-dimensional images of MCF+FIR3 and MCF+FIS4 cells were analyzed by PdQuest software. F, fold increases for No.1 spot in MCF+FIR3 cells compared with that in MCF+FIS4 cells. G, partial two-dimensional gel images; protein corresponding to PrxII (arrow). An electrospray LC/MS/MS of the single- and double-charged tryptic fragment 5 of PrxII.

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Table 1.

Spot no.2D gel pH rangeProteinQ scoreSequencing coverage (%)plMr (kDa)
3-10 Prx II human 57.74 55 5.41 21.891 
4-7 Prx II human 53.66 68 5.41 21.891 
Spot no.2D gel pH rangeProteinQ scoreSequencing coverage (%)plMr (kDa)
3-10 Prx II human 57.74 55 5.41 21.891 
4-7 Prx II human 53.66 68 5.41 21.891 

Abbreviation: 2d, two-dimensional.

Peroxiredoxin II isoform is selectively up-regulated in radioresistant breast cancer cells. The Western blot for all Prxs cytosol isoforms showed that PrxII, PrxIII, PrxV, and PrxVI are constitutively expressed higher (Fig. 3), whereas PrxIV is low. No PrxIV isoform can be detected in MCF+FIR3 and MCF+FIS4 breast cancer cells, whereas PrxIV can be detected from HK18 cell lines (data not shown). Enhanced PrxII expression was seen in the radioresistant MCF+FIR3 cells compared with MCF+FIS4 cells. A slightly increased expression of PrxI isozyme was also observed in radioresistant MCF+FIR3 cells. PrxI protein can be detected, but the signal is not as strong as that of PrxII.

Figure 3.

A, Western blot analysis of Prx proteins in MCF+FIR3 radioresistant breast cancer cells and MCF+FIS4 radiosensitive breast cancer cells. β-Actins were used as control for the experiments. Kodak 1D image analyses software was used to quantify the gel bands. □, MCF+FIR3 breast cancer cells. ▪, MCF+FIS4 radioresistant breast cancer cells.

Figure 3.

A, Western blot analysis of Prx proteins in MCF+FIR3 radioresistant breast cancer cells and MCF+FIS4 radiosensitive breast cancer cells. β-Actins were used as control for the experiments. Kodak 1D image analyses software was used to quantify the gel bands. □, MCF+FIR3 breast cancer cells. ▪, MCF+FIS4 radioresistant breast cancer cells.

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PrxII protein is in the reduced form in both radioresistant MCF+FIR3 cells and in radiosensitive MCF+FIS4 breast cancer cells. Anti-SO3-PrxII antibody that recognizes both oxidized forms of cysteine residues, Cys-SO2H and Cys-SO3H, was used to detect overoxidized PrxII enzyme. Our Western blot analysis did not show any oxidized PrxII protein in MCF+FIR3 and MCF+FIS4 cells (data not shown). PrxII isozyme does not form large, insoluble, noncovalent aggregates when PrxII is present in high concentration from the result of the Western blot. Our experimental results show selectivity for Prx isozymes overexpression in breast cancer–resistant cells. PrxII has been shown able to protect both DNA from nicking and protein from inactivation mediated by oxidative damage in mixed-function oxidation system (18, 19). The overexpression of PrxII in an endothelial line protects the cells from oxidative stress caused by the heavy metal mercury, inorganic hydrogen peroxides, and organic t-butyl-hydroperoxide (2022). It has been shown to protect RBC membranes from peroxidation (23, 24). PrxII has a wide variety of antioxidant functions throughout the cell.

Peroxiredoxin II sequence alignment. We searched for sequence homologues, using human PrxII sequence as a query for the Blast program in the NCBI (http://www3.ncbi.nlm.nih.gov/cgi-bin/BLAST/) databases. We identified four new homologous sequences (E = 10−73 to 10−108; see Fig. 4). This result supports the existence of the PrxII family. The PrxII family shows a broad taxonomic distribution. Members of the family are present in Escherichia coli; Saccharomyces cerevisiae; yeast; Rattus norvegicus; Mus musculus, mouse; Cricetulus griseus; human, bovine; Bos taurus; Brugia malayi; Litomosoides sigmodontis; Onchocerca ochengi; Onchocerca volvulus; Echinococcus granulosus; and Schistosoma mansoni (Fig. 4). Note that in most cases, copies of members of the two-cysteine PrxI, PrxIII, and PrxIV are also present. Members of the PrxII family have distinct structural features. They are slightly smaller than the PrxIII and PrxIV proteins: PrxII proteins possess 198 residues; PrxIII proteins possess 238 residues; and PrxIV proteins possess 274 residues. Members of each family display a high degree of sequence conservation (Fig. 4): amino acid residues conserved in aligned sequences (*); identical or closely similar residues (:). The two cysteines of the PrxII family proteins that form a transient disulfide bridge are shown by Cp and CR. Isoleucine, leucine, methionine, and valine (those hydrophobic residues) were colored green. Amino acid residues glycine, proline, serine, and threonine were colored orange. Histine, lysine, and arginine were colored red; phenylalanine, tryptophan, and tyrosine were colored blue. The previous report showed that bacterial two-cysteine Prxs are much less sensitive to oxidative inactivation than are eukaryotic two-cysteine Prxs due to a three-residue insertion associated with a conserved Gly-Gly-Leu-Gly sequence (the GGLG motif) and a COOH-terminal extension associated with a conserved Tyr-Phe sequence (the YF motif). All yeast, parasite, and mammalian PrxII proteins have a three-residue insertion associated with a conserved sequence GGLG and a conserved Tyr-Phe sequence (the YF motif) identified by comparing the crystal structure of a bacterial two-cysteine Prx with other Prx structures (25). They are all possibly sensitive to oxidation. Prokaryotic Prx enzymes, which do not contain the COOH-terminal GGLG motif and the YF motif of their eukaryotic counterparts, are insensitive to oxidation inactivation (26).

Figure 4.

Multiple sequence alignment of PrxII proteins. The alignment was constructed with the use of the ClustalX program. *, positions that have a single, fully conserved residue; :, one of the following “strong” groups is fully conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW; ., one of the following “weaker” groups is fully conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY. Secondary structure elements are depicted as helices (cylinders) or strands (arrows), with structural differences as disconnected elements. Positions of the aligned regions in the respective protein sequences (right-hand sites). Protein sequences are colored by residue as follows: orange, GPST; red, HKR; blue, FWY; green, ILMV. S. cere.,Saccharomyces cerevisiae; Rattus,Rattus norvegicus; Mus,Mus musculus; Crice.,Cricetulus griseus; Bos,Bos taurus; Bruma,Brugia malayi; Lito.,Litomosoides sigmodontis; Ochengi,Onchocerca ochengi; Volvulus,Onchocerca volvulus; ECHGR,Echinococcus granulosus; Schist.,Schistosoma mansoni. Peroxidatic (Cp) and resolving (CR) cysteines.

Figure 4.

Multiple sequence alignment of PrxII proteins. The alignment was constructed with the use of the ClustalX program. *, positions that have a single, fully conserved residue; :, one of the following “strong” groups is fully conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW; ., one of the following “weaker” groups is fully conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY. Secondary structure elements are depicted as helices (cylinders) or strands (arrows), with structural differences as disconnected elements. Positions of the aligned regions in the respective protein sequences (right-hand sites). Protein sequences are colored by residue as follows: orange, GPST; red, HKR; blue, FWY; green, ILMV. S. cere.,Saccharomyces cerevisiae; Rattus,Rattus norvegicus; Mus,Mus musculus; Crice.,Cricetulus griseus; Bos,Bos taurus; Bruma,Brugia malayi; Lito.,Litomosoides sigmodontis; Ochengi,Onchocerca ochengi; Volvulus,Onchocerca volvulus; ECHGR,Echinococcus granulosus; Schist.,Schistosoma mansoni. Peroxidatic (Cp) and resolving (CR) cysteines.

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RNA interference leads to radiosensitization of radioresistant MCF+FIR3 cells. To inhibit protein expression in the highest fraction of cells, the siRNA transfection was repeated 72 hours later. Transfection efficiency was monitored by Western blot analyses using antibodies specific for PrxII (LabFrontier). All the transfectants were maintained in 10% FBS/DMEM. PrxII gene expression in PrxII/siRNA-treated MCF+FIR3 radioresistant breast cancer cells was silenced after 48 hours following the transfection (Fig. 5A), whereas control oligos with scramble sequence was not able to inhibit the PrxII gene expression (Fig. 5B). We wanted to examine whether the down-regulation of PrxII increased cell sensitivity towards ionizing radiation. Figure 5 showed the clonogenic survival assays for MCF+FIS4 radiosensitive cells, MCF+FIR3 radioresistant cells, and PrxII/siRNA-treated MCF+FIR3 cells. Control oligos were not able to sensitize MCF+FIR3 cells (data not shown). The DMFs at 10% isosurvival were found to be 1.91 and 1.57 for MCF+FIR3 and PrxII/siRNA-treated MCF+FIR3 cells, respectively. Clonogenic survival assay in Fig. 5 showed that down-regulation of PrxII could sensitize radioresistant breast cancer cells to ionizing radiation. However, the inhibition of PrxII expression did not completely reverse the radioresistance phenotype of MCF+FIR3 cells. Therefore, it is reasonable to assume that there are other factors that contribute to radiation resistance other than the PrxII protein. These results conclude that the siRNA constructs of PrxII could down-regulate the expression of PrxII and partially reverse the resistant phenotype of breast cancer cells, suggesting that PrxII might play an important role in radiation-resistant cancer cells.

Figure 5.

A, Western analysis of PrxII in PrxII/siRNA-transfected MCF+FIR3 radioresistant cells. B, Western analysis of PrxII in scrambled siRNA-transfected MCF+FIR3 cells. C, clonogenic survival assays for MCF+FIS4, MCF+FIR3 and PrxII/siRNA-treated MCF+FIR3 breast cancer cells. ▪, MCF+FIS4 breast cancer cells. ⧫, radiation-resistant MCF+FIR3 cells. •, PrxII/siRNA-treated MCF+FIR3 resistant breast cancer cells. Clonogenic survival was calculated relative to the corresponding MCF+FIS4 control cells. The DMFs were calculated from the radiation doses corresponding to 10% survival.

Figure 5.

A, Western analysis of PrxII in PrxII/siRNA-transfected MCF+FIR3 radioresistant cells. B, Western analysis of PrxII in scrambled siRNA-transfected MCF+FIR3 cells. C, clonogenic survival assays for MCF+FIS4, MCF+FIR3 and PrxII/siRNA-treated MCF+FIR3 breast cancer cells. ▪, MCF+FIS4 breast cancer cells. ⧫, radiation-resistant MCF+FIR3 cells. •, PrxII/siRNA-treated MCF+FIR3 resistant breast cancer cells. Clonogenic survival was calculated relative to the corresponding MCF+FIS4 control cells. The DMFs were calculated from the radiation doses corresponding to 10% survival.

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Peroxiredoxin II overexpression results in increased radioresistance to ionizing radiation. In this study, we addressed the question whether overexpression of the antioxidant enzyme PrxII may modulate or interfere with ionizing radiation–induced ROS generation leading to increased radiation resistance of MCF+FIS4 breast cancer cells. For this purpose, we have generated transient transfected MCF+FIS4 breast cancer cell lines overexpressing PrxII. To study the effect of PrxII overexpression in radiosensitive MCF+FIS4 breast cancer cells exposed to ionizing radiation, the PrxII-overexpressing MCF+FIS4 breast cancer cells were exposed to ionizing radiation at a dose of 2, 4, 8, and 10 Gy. Clonogenic assays were used to compare the radioresistance level between vector-transfected MCF+FIS4 cells and PrxII-overexpressing MCF+FIS4 cells (Fig. 6). The DMF at 10% isosurvival was found to be 1.2 for PrxII-overexpressing MCF+FIR4 cells. We found that ionizing radiation led to an increase resistant level in PrxII-overexpressing MCF+FIS4 cells compared with vector-transfected control MCF+FIS4 cells. The result shows that overexpression of the PrxII gene confers a significant protection against ionizing radiation–induced cell death.

Figure 6.

Survival of MCF+FIS4/PrxII PrxII-overexpressing cells and MCF+FIS4/vector control cells as measured by the clonogenic assay after exposure to various doses of radiation. ▴, MCF+FIS4/PrxII PrxII-overexpressing cells. ▪, MCF+FIS4/vector control cells. Clonogenic survival was calculated relative to the corresponding MCF+FIS4/vector control. The DMF was calculated from the radiation doses corresponding to 10% survival.

Figure 6.

Survival of MCF+FIS4/PrxII PrxII-overexpressing cells and MCF+FIS4/vector control cells as measured by the clonogenic assay after exposure to various doses of radiation. ▴, MCF+FIS4/PrxII PrxII-overexpressing cells. ▪, MCF+FIS4/vector control cells. Clonogenic survival was calculated relative to the corresponding MCF+FIS4/vector control. The DMF was calculated from the radiation doses corresponding to 10% survival.

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These results also imply indirectly that PrxII overexpression interferes with ionizing radiation–induced ROS generation, leading to the increased radioresistance. It is highly possible that overexpression of PrxII reduce the intracellular ROS to a noncytotoxic level, resulting in increased resistance of breast cancer cells to ionizing radiation.

Anticancer therapy is generally more effective in the early stages, whereas advanced tumors are usually resistant to the same treatments. The molecular basis for this observation is not entirely understood. Therefore, studies involving strategies to overcome this resistance to chemotherapy and radiation or to increase cellular sensitivity to chemotherapy and radiation therapy are crucial to the development of more effective treatments. The hypothesis that key proteins functioning in signal transduction pathways are associated with tumor cellular radiation sensitivity or resistance unifies radiation biology research projects. By identifying such proteins, determining their mechanisms of action, and targeting their modulation by chemical or biological means, new strategies may be developed for improving radiation therapy therapeutic ratios. Other investigations related to DNA damage and repair and carcinogenesis provide a broader base for investigations and additional scientific ideas and goals.

The studies presented here show that the PrxII protein is up-regulated in radiation-resistant breast cancer cells compared with radiation-sensitive breast cancer cells and plays an important protective role from oxidative radical damage. Increased expression of PrxII was observed in tissues isolated from the head and neck cancer patients who did not respond to radiation therapy, whereas PrxII expression was weak in tissues from the patients with regressed tumors. Enhanced expression of PrxII in UMSCC-11A cells was also observed after treatment with γ-radiation (27). This increased expression conferred radiation resistance to cancer cells because overexpression of PrxII protected cancer cells from radiation-induced cell death, suggesting that blocking PrxII expression could enhance radiation sensitivity. Our results showed that silencing PrxII gene expression by PrxII/siRNA treatment of MCF+FIR3 radioresistant breast cancer cells leads to partial radiosensitization of MCF+FIR3 cells. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing cells when compared with vector control cells. However, we found that inhibition of PrxII protein expression did not completely reverse the radioresistant phenotype of MCF+FIR3 cells, suggesting that silencing PrxII gene expression did not completely abrogate radiation-induced DNA or protein damage and cell death. There are other factors that have been shown to contribute to radiation resistance other than PrxII. A number of researchers have shown that markers of apoptosis, reduced proliferation, and signal transduction proteins correlate with breast cancer response to chemotherapy. Several signaling pathways have also been suggested to be involved in cellular radiation sensitivity and response to ionizing radiation (2832).

PrxII has been previously known as a natural killer–enhancing factor B (19). It is induced by various oxidative stimuli and plays an important protective role from oxidative radical damage by ROS/RNS (3335). PrxII-overexpressing cells are more resistant to the oxidative damage caused by H2O2, t-butyl-hydroperxide, and methyl mercury (22). Its expression is correlated with resistance to apoptosis induced by radiation therapy or the anticancer drug cisplatin (27, 36), highlighting the potential clinical importance of PrxII in radiation resistance in cancer.

The up-regulation of PrxII in radioresistant breast cancer cells suggested that human Prx plays an important role in eliminating the generation of reactive oxygen species (37, 38). Prxs represent a major group of detoxification enzymes that use redox-sensitive cysteine, the active site peroxidatic cysteine, to reduce peroxides (3941). In addition, Prxs catalyze decomposition of RNS (26, 42, 43). The importance of thiol-mediated detoxification of oxidative stress that produces ROS/RNS has been addressed over the past few years. There are six mammalian Prx enzymes. All Prxs contain at least one essential cysteine per 21- to 25-kDa subunit. They do not contain metal cofactors or other noncysteine redox centers and share the same basic catalytic mechanism, in which an active site cysteine is oxidized to a sulfenic acid by the peroxide substrate. The catalytic efficiency of mammalian Prxs indicated by the Kcat/Km is significantly lower than that of catalase or glutathione peroxidase. However, catalase is mainly in peroxisomes and glutathione peroxidase, which is mainly in cytosol, exists in low amounts in most tissues. In contrast, five of the six mammalian peroxiredoxins are abundant (0.2-0.4% of total soluble protein) in cytosol, whereas PrxIII is exclusively localized in mitochondria. In addition to its role as a peroxidase, a body of evidence has accumulated to suggest that individual members also serve divergent functions, which are associated with various biological processes, such as the detoxification of oxidants, cell proliferation, differentiation, and gene expression. It seems likely that the divergence is due to unique molecular characteristics intrinsic to each member (37, 39).

It seems probable that PrxII is regulated in vivo by ROS, because not only are some of the most potent inducers capable of generating free radicals by redox cycling, but induction of PrxII by ROS would seem to represent an adaptive response as these enzymes detoxify some of the toxic peroxide- and epoxide-containing metabolites produced within the cell by oxidative stress. PrxII isoenzyme is overexpressed in radiation-resistant breast cancer cells, and increased level of this enzyme probably contributes to the radiation-resistant phenotype. It is similarly known that large amounts of ROS are produced as byproducts of ionizing radiation exposure. Therefore, it is tempting to speculate that the cellular pathway leading to PrxII up-regulation could be triggered by ionizing radiation and ROS exposure in a similar and/or common way. In fact, we found that PrxII was up-regulated after chronic exposure to ionizing radiation, pointing to a role for this protein in stress protection in cancer cells. From these results, we suggest that stress-induced overexpression of PrxII increased radiation resistance via protection of cancer cells from radiation-induced oxidative damage. Induction of the ROS-scavenging proteins could protect cells from oxidative cytolysis and reduce cellular damage by regulating cellular redox status (44, 45). PrxII is not the sole factor responsible for the resistant phenotype, because inhibition of PrxII gene expression did not completely reversed the resistant phenotype. However, PrxII is a potential key player resulting in increased resistance of breast cancer cells to ionizing radiation. This mechanistic study may provide a basis for new targets of therapy based on this contribution to radiation sensitivity and lead to the identification of biomarkers to predict radiation sensitivity.

Grant support: Beckman Foundation Research Fellowship (T. Wang) and Office of Science, U.S. Department of Energy grant DE-FG02-05ER63945 (J.J. Li).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Carlotta Glackin, Binghui Shen, Detlef Schuman, Terry Lee, Mary Young, Roger Moore, Helen Ge, and Jeffery Wong (City of Hope National Medical Center) for insightful discussion; Mian Zhou (Dr. Binghui Shen's lab) for the PrxII plasmid construct; and Kevin Clark for his critical reading of the article.

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