Eun Mi Park

Preferential elevation of Prx I and Trx expression in lung cancer cells following hypoxia and in human lung cancer tissues.

Transient/chronic microenvironmental hypoxia that exists within a majority of solid tumors has been suggested to have a profound influence on tumor growth and therapeutic outcome. Since the functions of novel antioxidant proteins, peroxiredoxin I (Prx I) and II, have been implicated in regulating cell proliferation, differentiation, and apoptosis, it was of our special interest to probe a possible role of Prx I and II in the context of hypoxic tumor microenvironment. Since both Prx I and II use thioredoxin (Trx) as an electron donor and Trx is a substrate for thioredoxin reductase (TrxR), we investigated the regulation of Trx and TrxR as well as Prx expression following hypoxia. Here we show a dynamic change of glutathione homeostasis in lung cancer A549 cells and an up-regulation of Prx I and Trx following hypoxia. Western blot analysis of 10 human lung cancer and paired normal lung tissues also revealed an elevated expression of Prx I and Trx proteins in lung cancer tissues. Immunohistochemical analysis of the lung cancer tissues confirmed an augmented Prx I and Trx expression in cancer cells with respect to the parenchymal cells in adjacent normal lung tissue. Based on these results, we suggest that the redox changes in lung tumor microenvironment could have acted as a trigger for the up-regulation of Prx I and Trx in lung cancer cells. Although the clinical significance of our finding awaits more rigorous future study, preferential augmentation of the Prx I and Trx in lung cancer cells may well represent an attempt of cancer cells to manipulate a dynamic redox change in tumor microenvironment in a manner that is beneficial for their proliferation and malignant progression.

Role of protein kinase Cδ in transmitting hypoxia signal to HSF and HIF-1

An hypoxic microenvironment is an important modulator of gene expression in many pathophysiological conditions. In this study, we show a coordinate activation of heat shock transcription factor (HSF) and hypoxia‐inducible factor‐1 (HIF‐1) in RIF tumor cells by hypoxia. Since heat shock protein (hsp) and angiogenic factor genes that are regulated by HSF and HIF‐1 are thought to contribute to the malignant progression of hypoxic tumor cells, it was of our major interest to identify the components that are responsible for the activation of both HSF and HIF‐1. Our finding that a bioflavonoid quercetin (QCT), a well known inhibitor of hsp gene expression, significantly inhibited the transcriptional activation of HSF and HIF‐1 strongly suggests that QCT‐sensitive molecule(s) is involved in the transcriptional activation of HSF and HIF‐1 by hypoxia. Our results revealed that PCKα, δ and ϵ isoforms are expressed in RIF cells, but only PKCδ was specifically translocated to the membrane by hypoxia. Our results also revealed that the translocation of PKCδ was completely abrogated by QCT. Moreover, inhibiting the PKCδ activation, either pharmacologically with phorbol 12‐myristate 13‐acetate or with bisindolymaleimide II or genetically by transient transfection of a dominant negative PKCδ, significantly inhibited the transcriptional activation of HSF and HIF‐1 by hypoxia. These results strongly substantiate a view that the PKCδ isozyme is the QCT‐sensitive molecule that plays an important role in transmitting hypoxia signals to both HSF and HIF‐1. Here we show that the membrane translocation of PKCδ is dependent on the activation of phosphoinositol 3‐kinase (PI3K). Treatment with PI3K inhibitor, wortmannin or LY294002, abrogated not only PKCδ translocation but the subsequent transcriptional activation of HSF and HIF‐1 by hypoxia. Together, our study shows that the PKCδ isozyme acts as a shared component in transmitting hypoxia‐induced signals to both HSF and HIF‐1, and that the upstream regulator of PKCδ is PI3K.

Effect of ionizing radiation on rat tissue: Proteomic and biochemical analysis

Abstract Reactive oxygen species (ROS), generated by ionizing radiation, has been implicated in its effect on living tissues. We confirmed the changes in the oxidative stress markers upon irradiation. We characterized the changes in the proteome profile in rat liver after administering irradiation, and the affected proteins were identified by MALDI‐TOF‐MS and ESI‐MS/MS. The identified proteins represent diverse sets of proteins participating in the cellular metabolism. Our results demonstrated that proteomics analysis is a useful method for characterization of a global proteome change caused by ionizing radiation to unravel the molecular mechanisms involved in the cellular responses to ionizing radiation. *Equal contribution

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.

An Increased Monocyte Count Predicts Coronary Artery Spasm in Patients with Resting Chest Pain and Insignificant Coronary Artery Stenosis

Background Coronary atherosclerosis with inflammation gives rise to coronary vasospasm in the patients with coronary vasospastic angina. We have postulated that the peripheral leukocyte count and the differential count are associated with vasospastic angina. Methods 144 patients who underwent intracoronary ergonovine provocation testing between January 2002 and December 2004 were divided into two groups: Group I (72 patients with provoked spasm, mean age: 54.8±10.7 years, males: 75%) and Group II (72 without spasm, mean age: 55.3±10.2 years, males: 35%). Blood sampling was done to measure the lipid profiles and inflammatory markers, including the high sensitive C-reactive protein (hsCRP) levels and the monocyte counts. We compared the angiographic findings and laboratory data between the two groups. Results There were no significant differences in the levels of serum lipid and hsCRP between the two groups. The white blood cell count and the monocyte count were higher in Group I than with Group II (7496.4±2622.28 vs. 6703.2±1768.37/mm3, respectively, p=0.035; 627.5±270.70 vs. 426.9±205.76/mm3, respectively, p<0.001). Gensinis score was higher in Group I than in Group II (2.2±2.88 vs. 0.5±1.03, respectively, p<0.001). Multivariate analysis showed that the monocyte count and Gensinis score were independent factors affecting coronary spasm (p=0.047 and p=0.018, respectively). According to a receiver operating characteristics curve analysis, the area under the curve of the monocyte count was 0.738, that of the neutrophil count was 0.577 and that of the WBC count was 0.572. The cut-off value of the monocyte count was 530/mm3; the sensitivity and specificity of this cut-off value were 64% and 76%, respectively. Conclusions The peripheral monocyte count is an independent marker for predicting vasospastic angina in the patients with resting chest pain and insignificant coronary artery stenosis.

Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.

Neural network-based analysis of thiol proteomics data in identifying potential selenium targets.

Abstract Generation of a monomethylated selenium metabolite is critical for the anticancer activity of selenium. Because of its strong nucleophilicity, the metabolite can react directly with protein thiols to cause redox modification. Here, we report a neural network‐based analysis to identify potential selenium targets. A reactive thiol specific reagent, BIAM, was used to monitor thiol proteome changes on 2D gel. We constructed a dynamic model and evaluated the relative importance of proteins mediating the cellular responses to selenium. Information from this study will provide new clues to unravel mechanisms of anticancer action of selenium. High impact selenium targets could also serve as biomarkers to gauge the efficacy of selenium chemoprevention.

Inhibitory effect of a naphthazarin derivative, S64, on heat shock factor (Hsf) activation and glutathione status following hypoxia.

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment. Resistance of hypoxic cells to ionizing radiation and anticancer drugs has in part been attributed to changes in altered gene expression by hypoxia. We previously reported an activation of heat shock factor (Hsf) in murine tumor RIF cells following hypoxia and suggested that a subsequent accumulation of heat shock protein(s) (Hsp) is likely to contribute to the malignant progression of hypoxic tumor cells (Baek et al., 2001). In this study, we showed that hypoxia induced a DNA-binding activity of Hsf and activation ofhsp70 gene expression in colon cancer Clone A cells, and that a naphthazarin derivative, S64, significantly inhibited the hypoxia-induciblehsp70 gene expression in Clone A cells. We also showed that S64 significantly reduced the cellular glutathione levels in this cell line. Considering the proposed effects of Hsp and glutathione on radiation and chemotherapy sensitivity, we suggest that the inhibitory effects of S64 on Hsf activation and cellular glutathione levels have potentially important clinical implications. We believe that the previously reportedin vitro andin vivo anti-tumor effect of S64 (Song et al., 2000a, 2001) might be attributed, at least in part, to its effect on Hsf activation and/or glutathione depletion. We also believe that the detailed molecular mechanisms underlying the effects of S64 on Hsf and glutathione level following hypoxia deserve a more rigorous future study, the results of which could offer novel strategy to manipulate the resistance mechanisms of solid tumors.