Young-Mee Park

Human prx1 Gene Is a Target of Nrf2 and Is Up-regulated by Hypoxia/Reoxygenation: Implication to Tumor Biology

Peroxiredoxin 1 (Prx1) has been found to be elevated in several human cancers. The cell survival-enhancing function of Prx1 is traditionally attributed to its reactive oxygen species-removing capacity, although the growth-promoting role of Prx1 independent of this antioxidant activity is increasingly gaining attention. Although much progress has been made in understanding the behavior of Prx1, little information is available on the mechanism responsible for the abnormal elevation of Prx1 level in cancer. We hypothesized that the hypoxic and unstable oxygenation microenvironment of a tumor might be crucial for prx1 up-regulation. In this study, we cloned the human prx1 promoter and identified nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) as a key transcription factor. Hypoxia/reoxygenation, an in vitro condition suited to mimic changes of oxygenation, increased Nrf2 nuclear localization and its binding to the electrophile-responsive elements located at the proximal (-536 to -528) and distal (-1429 to -1421) regions of the prx1 promoter. A significant reduction of both steady-state and hypoxia/reoxygenation-mediated prx1 gene expression was shown in Nrf2 knock-out cells. Our results indicated that decreased Kelch-like ECH-associated protein, Keap1, might be an important mechanism for the increased nuclear translocation and activation of Nrf2 in response to hypoxia/reoxygenation. A constitutive elevation of prx1 mRNA and protein was observed in Keap1 knock-out cells. The above information suggests that the Nrf2-Prx1 axis may be a fruitful target for intervention with respect to inhibiting the malignant progression and/or reducing the treatment resistance of cancer cells.

Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2.

Human peroxiredoxins 1 and 2, also known as Prx1 and Prx2, are more than 90% homologous in their amino acid sequences. Prx1 and Prx2 are elevated in various cancers and are shown to influence diverse cellular processes. Although their growth regulatory role has traditionally been attributed to the peroxidase activity, the physiological significance of this function is unclear because the proteins are highly susceptible to inactivation by H2O2. A chaperone activity appears to emerge when their peroxidase activity is lost. Structural studies suggest that they may form a homodimer or doughnut-shaped homodecamer. However, little information is available whether human Prx1 and Prx2 are duplicative in structure and function. We noted that Prx1 contains a cysteine (Cys83) at the putative dimer-dimer interface, which is absent in Prx2. We studied the role of Cys83 in regulating the peroxidase and chaperone activities of Prx1, because the redox status of Cys83 might influence the oligomeric structure and consequently the functions of Prx1. We show that Prx1 is more efficient as a molecular chaperone, whereas Prx2 is better suited as a peroxidase enzyme. Substituting Cys83 with Ser83 (Prx1C83S) results in dramatic changes in the structural and functional characteristics of Prx1 in a direction similar to those of Prx2. Here we also report the first crystal structure of human Prx1 and the presence of the Cys83–Cys83 bond at the dimer-dimer interface of decameric Prx1. These findings are consistent with the hypothesis that human Prx1 and Prx2 possess unique functions and regulatory mechanisms and that Cys83 bestows a distinctive identity to Prx1.

Prx1 Suppresses Radiation-Induced c-Jun NH2-Terminal Kinase Signaling in Lung Cancer Cells through Interaction with the Glutathione S-Transferase Pi/c-Jun NH2-Terminal Kinase Complex

Radiotherapy is one of the major treatment modalities for lung cancer. Cell killing by ionizing radiation is mediated primarily through the reactive oxygen species (ROS) and ROS-driven oxidative stress. Prx1, a peroxiredoxin family member, was shown to be frequently elevated in lung cancer cells and tissues. Although the antioxidant function of Prx1 is expected to affect the radiotherapy response of lung cancer, the physiologic significance of its peroxidase activity in irradiated cells is unclear because the catalytic Cys52 is easily inactivated by ROS due to its overoxidation to sulfinic or sulfonic acid. In this study, we investigated the role of Prx1 in radiation sensitivity of human lung cancer cells, with special emphasis on the redox status of the catalytic Cys52. We found that overexpression of Prx1 enhances the clonogenic survival of irradiated cells and suppresses ionizing radiation-induced c-Jun NH2-terminal kinase (JNK) activation and apoptosis. The peroxidase activity of Prx1, however, is not essential for inhibiting JNK activation. The latter effect is mediated through its association with the glutathione S-transferase pi (GSTpi)-JNK complex, thereby preventing JNK release from the complex. Reduced JNK activation is observed when the peroxidase activity of Prx1 is compromised by Cys52 overoxidation or in the presence of the Cys52 to Ser52 mutant (Prx1C52S) lacking peroxidase activity. We show that both Prx1 and Prx1C52S interact with the GSTpi-JNK complex and suppress the release of JNK from the complex. Our study provides new insight into the antiapoptotic function of Prx1 in modulating radiosensitivity and provides the impetus to monitor the influence of Prx1 levels in the management of lung cancer.

Endoplasmic Reticulum Stress Signal Mediators Are Targets of Selenium Action

A monomethylated selenium metabolite, called methylseleninic acid (MSA), has recently been shown to cause global thiol redox modification of proteins. These changes represent a form of cellular stress due to protein misfolding or unfolding. An accumulation of aberrantly folded proteins in the endoplasmic reticulum (ER) triggers a defined set of transducers to correct the defects or commit the cells to apoptosis if the rescue effort is exhausted. Treatment of PC-3 human prostate cancer cells with MSA was found to induce a number of signature ER stress markers: (a) the survival/rescue molecules such as phosphorylated protein kinase-like ER-resident kinase (phospho-PERK), phosphorylated eukaryotic initiation factor-2alpha (phospho-eIF2alpha), glucose-regulated protein (GRP)-78, and GRP94; and (b) the apoptotic molecules such as caspase-12, caspase-7, and CAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153). Additional evidence suggested that CHOP/GADD153 might be an important transcription factor in apoptosis induction by MSA. In general, a higher concentration of MSA was required to elicit the apoptotic markers compared with the rescue markers. The apoptotic markers increased proportionally with the dose of MSA, whereas the rescue markers failed to keep pace with the increasing challenge from MSA. GRP78 is the rheostat of the ER stress transducers. In GRP78-overexpressing cells, the ability of MSA to up-regulate phospho-PERK, phospho-eIF2alpha, GRP94, caspase-12, caspase-7, and CHOP/GADD153 was significantly muted. A generous supply of GRP78 would allow cells to cope better with ER stress, thereby improving the odds for survival and negating the commitment to apoptotic death. The present study thus provides strong evidence to support an important role of ER stress response in mediating the anticancer effect of selenium.

Peroxiredoxin 1 Interacts with Androgen Receptor and Enhances Its Transactivation

Although hypoxia is accepted as an important microenvironmental factor influencing tumor progression and treatment response, it is usually regarded as a static global phenomenon. Consequently, less attention is given to the impact of dynamic changes in tumor oxygenation in regulating the behavior of cancer cells. Androgen receptor (AR) signaling plays a critical role in prostate cancer. We previously reported that hypoxia/reoxygenation, an in vitro condition used to mimic an unstable oxygenation climate in a tumor, stimulates AR activation. In the present study, we showed that peroxiredoxin 1 (Prx1), a member of the peroxiredoxin protein family, acts as a key mediator in this process. We found that the aggressive LN3, C4-2, and C4-2B prostate cancer cell lines derived from LNCaP possess constitutively elevated Prx1 compared with parental cells, and display greater AR activation in response to hypoxia/reoxygenation. Although the cell survival-enhancing property of Prx1 has traditionally been attributed to its antioxidant activity, the reactive oxygen species-scavenging activity of Prx1 was not essential for AR stimulation because Prx1 itself was oxidized and inactivated by hypoxia/reoxygenation. Increased AR transactivation was observed when wild-type Prx1 or mutant Prx1 (C52S) lacking antioxidant activity was introduced into LNCaP cells. Reciprocal immunoprecipitation, chromatin immunoprecipitation, and in vitro pull-down assays corroborated that Prx1 interacts with AR and enhances its transactivation. We also show that Prx1 is capable of sensitizing a ligand-stimulated AR. Based on the above information, we suggest that disrupting the interaction between Prx1 and AR may serve as a fruitful new target in the management of prostate cancer.

Phase II Study of Weekly Intravenous Oxaliplatin Combined With Oral Daily Capecitabine and Radiotherapy With Biologic Correlates in Neoadjuvant Treatment of Rectal Adenocarcinoma

PURPOSE To evaluate the efficacy of a combination of capecitabine, oxaliplatin, and radiotherapy (RT) in the neoadjuvant treatment of Stage II and III rectal cancers. METHODS Capecitabine was given at 725 mg/m(2) orally twice daily Monday through Friday concurrently with RT. Oxaliplatin was given intravenously at 50 mg/m(2) once weekly five times starting the first day of RT. The radiation dose was 50.4 Gy in 28 fractions (1.8 Gy/fraction), five fractions weekly. Endorectal tumor biopsies were obtained before treatment and on the third day of treatment to explore the effects of treatment on thymidine phosphorylase, thymidylate synthase, excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1), and apoptosis. RESULTS A total of 25 patients were enrolled in this study; 6 patients (24%) had a complete pathologic response. T-downstaging occurred in 52% of patients, and N-downstaging occurred in 53%. Grade 3 diarrhea was the most common Grade 3-4 toxicity, occurring in 20% of patients. Only 2 patients experienced disease recurrence, with a median of 20 months of follow-up. Thymidylate synthase, thymidine phosphorylase, ERCC1, and apoptosis did not vary significantly between the pretreatment and Day 3 tumor biopsies, nor did they predict for T-downstaging or a complete pathologic response. CONCLUSION Capecitabine at 725 mg/m(2) orally twice daily, oxaliplatin 50 mg/m(2)/wk, and RT at 50.4 Gy is an effective neoadjuvant combination for Stage II and III rectal cancer and results in a greater rate of complete pathologic responses than historically shown in fluoropyrimidine plus RT controls.

Measurement of glutathione oxidation and 8-hydroxy-2′-deoxyguanosine accumulation in the gerbil hippocampus following global ischemia

Involvement of oxidative stress in ischemia/reperfusion-induced brain damage has been suggested. However, experimental support of this suggestion was limited partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. We have established biochemical assay systems to assess oxidative brain damage following ischemia. Mongolian gerbil brains were subjected to global ischemia/reperfusion, and the hippocampi were analyzed for oxidative damage by measuring temporal changes in glutathione and 8-ohdG following ischemia. Under oxidative stress, glutathione is known to be oxidized and subsequently depleted from cells. Therefore, glutathione content and its redox status can serve as sensitive indicators of oxidative damage. The accumulation of 8-ohdG has also been recognized as an excellent marker for oxidative DNA damage. The reduced and oxidized glutathione were measured by HPLC method following derivatization with 2,4-dinitrofluorobenzene. The 8-ohdG in DNA hydrolyzate was measured by HPLC with electrochemical detection. While total glutathione content decreased, glutathione oxidation ratio and 8-ohdG accumulation increased over a period of 30 min of reperfusion following ischemia. The results demonstrated that glutathione content, its oxidation ratio, and the accumulated 8-ohdG could be utilized as sensitive indices for the assessment of oxidative brain damage.

Regulation of rat heat shock factor 2 expression during the early organogenic phase of embryogenesis.

A central step in the transcriptional regulation of heat shock protein (hsp) genes is the binding of the heat shock factor (HSF) to the upstream heat shock elements (HSEs). In vertebrates, HSF2 has been suggested to mediate the transcriptional regulation of hsp gene expression during development and differentiation. The expression levels of HSF2 were shown to vary widely among fully developed mouse organs. However, there exists limited information on the regulation of HSF2 expression during the inductive stage of organ formation in mammalian development. In this study, we have cloned the rat HSF2 cDNA and examined embryos for HSF2 expression from days 9.5 (E9.5) to 15.5 (E15.5) of gestation that correspond to the period when the major organ primordia are being actively established. We show that rat HSF2 has 94.6 and 96.3% identity to mouse HSF2 in nucleotide and amino acid sequences, respectively. By establishing a competitive RT-PCR, we show that about 503.6 pg of HSF2 mRNA were present per microgram of embryonic RNA in the primitive streak stage E9.5 embryos. The amounts of HSF2 mRNA then gradually decreased, resulting in an approximately 300-fold reduction in E15.5 embryos. The amounts of HSF2 mRNA in the embryos were found to be closely correlated with those of HSF2 protein and their HSE-binding activities. To our knowledge, this is the first detailed report on the structure and regulation of the rat HSF2 during the early organogenic period of mammalian embryogenesis.

Differential expression of Bcl-2, Bcl-XL and p53 in colorectal cancer

Aims:  The balance between proliferation and apoptosis is often disturbed in cancer. The aim of this study was to investigate the possible role of Bcl‐2 gene family members and p53 as prognostic factors in a series of colorectal cancer.

Analysis of Protein Redox Modification by Hypoxia

Abstract We examined hypoxia‐induced changes in global thiol proteome profile in human prostate cancer cells using a BIAM‐based display method. We analyzed the kinetics of protein thiol modification by using a pattern recognition algorithm, self‐organizing maps (SOM) clustering, and identified the BIAM‐labeled proteins by MALDI‐TOF and ESI‐tandem mass spectrometry. We found 99 out of 215 of total BIAM‐labeled proteins were affected by hypoxia treatment and, yet, with diverse patterns and kinetics of redox modification. Our study proved that proteomics analysis employing the BIAM‐labeling method can provide valuable information pertaining to global changes in the redox status of proteins in response to hypoxia.