Anticancer Effect by Combined Treatment of Artemisia annua L. Polyphenols and Docetaxel in DU145 Prostate Cancer Cells and HCT116 Colorectal Cancer Cells.

Docetaxel (DTX), a semi-synthetic analogue of paclitaxel (taxol), is known to exert potent anticancer activity in various cancer cells by suppressing normal microtubule dynamics. In this study, we examined how the anticancer effect of DTX is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in DU145 prostate cancer cells (mutant p53) and HCT116 colorectal cancer cells (wild-type p53). Here, we show that the anticancer effect of DTX was enhanced more significantly by pKAL in HCT116 cells than in DU145 cells via phase-contrast microscopy, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/propidium iodide-stained cells. Notably, mutant p53 was slightly downregulated by single treatment of pKAL or DTX in DU145 cells, whereas wild-type p53 was significantly upregulated by pKAL or DTX in HCT116 cells. Moreover, the enhanced anticancer effect of DTX by pKAL in HCT116 cells was significantly associated with the suppression of DTX-induced p53 upregulation, increase of DTX-induced phospho-p38, and decrease of DTX-regulated cyclin A, cyclin B1, AKT, caspase-8, PARP1, GM130, NF-κB p65, and LDHA, leading to the increased apoptotic cell death and plasma membrane permeability. Our results suggest that pKAL could effectively improve the anticancer effect of DTX-containing chemotherapy used to treat various cancers expressing wild-type p53.

ß-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

ß-lapachone (ß-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of ß-Lap associated with OxPt resistance, 5 µM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, ß-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that ß-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.

Artemisia annua L. Polyphenols Enhance the Anticancer Effect of ß-Lapachone in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

Recent studies suggest that the anticancer activity of ß-lapachone (ß-Lap) could be improved by different types of bioactive phytochemicals. The aim of this study was to elucidate how the anticancer effect of ß-Lap is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in parental HCT116 and oxaliplatin-resistant (OxPt-R) HCT116 colorectal cancer cells. Here, we show that the anticancer effect of ß-Lap is more enhanced by pKAL in HCT116-OxPt-R cells than in HCT116 cells via a CCK-8 assay, Western blot, and phase-contrast microscopy analysis of hematoxylin-stained cells. This phenomenon was associated with the suppression of OxPt-R-related upregulated proteins including p53 and ß-catenin, the downregulation of cell survival proteins including TERT, CD44, and EGFR, and the upregulation of cleaved HSP90, γ-H2AX, and LC3B-I/II. A bioinformatics analysis of 21 proteins regulated by combined treatment of pKAL and ß-Lap in HCT116-OxPt-R cells showed that the enhanced anticancer effect of ß-Lap by pKAL was related to the inhibition of negative regulation of apoptotic process and the induction of DNA damage through TERT, CD44, and EGFR-mediated multiple signaling networks. Our results suggest that the combination of pKAL and ß-Lap could be used as a new therapy with low toxicity to overcome the OxPt-R that occurred in various OxPt-containing cancer treatments.

Identification of Growth Factors, Cytokines and Mediators Regulated by Artemisia annua L. Polyphenols (pKAL) in HCT116 Colorectal Cancer Cells: TGF-ß1 and NGF-ß Attenuate pKAL-Induced Anticancer Effects via NF-κB p65 Upregulation.

The anticancer effects of natural phytochemicals are relevant to the modulation of cytokine signaling pathways in various cancer cells with stem-like properties as well as immune cells. The aim of this study was to elucidate a novel anticancer mechanism of Artemisia annua L. polyphenols (pKAL) involved in the regulation of growth factors, cytokines and mediators in stem-like HCT116 colorectal cancer cells. Through RayBiotech human L-1000 antibody array and bioinformatics analysis, we show here that pKAL-induced anticancer effects are associated with downregulation of growth factor and cytokine signaling proteins including TGFA, FGF16, PDGFC, CCL28, CXCR3, IRF6 and SMAD1. Notably, we found that TGF-ß signaling proteins such as GDF10, ENG and TGFBR2 and well-known survival proteins such as NGF-ß, VEGFD and insulin were significantly upregulated by pKAL. Moreover, the results of hematoxylin staining, cell viability assay and Western blot analysis demonstrated that TGF-ß1 and NGF-ß attenuated pKAL-induced anticancer effects by inhibiting pKAL-induced downregulation of caspase-8, NF-κB p65 and cyclin D1. These results suggest that certain survival mediators may be activated by pKAL through the TGF-ß1 and NGF-ß signaling pathways during pKAL-induced cell death and thus, strategies to inhibit the survival signaling are inevitably required for more effective anticancer effects of pKAL.

Artemisia annua L. Polyphenol-Induced Cell Death Is ROS-Independently Enhanced by Inhibition of JNK in HCT116 Colorectal Cancer Cells.

c-Jun N-terminal kinase (JNK) is activated by chemotherapeutic reagents including natural plant polyphenols, and cell fate is determined by activated phospho-JNK as survival or death depending on stimuli and cell types. The purpose of this study was to elucidate the role of JNK on the anticancer effects of the Korean plant Artemisia annua L. (pKAL) polyphenols in p53 wild-type HCT116 human colorectal cancer cells. Cell morphology, protein expression levels, apoptosis/necrosis, reactive oxygen species (ROS), acidic vesicles, and granularity/DNA content were analyzed by phase-contrast microscopy; Western blot; and flow cytometry of annexin V/propidium iodide (PI)-, dichlorofluorescein (DCF)-, acridine orange (AO)-, and side scatter pulse height (SSC-H)/DNA content (PI)-stained cells. The results showed that pKAL induced morphological changes and necrosis or late apoptosis, which were associated with loss of plasma membrane/Golgi integrity, increased acidic vesicles and intracellular granularity, and decreased DNA content through downregulation of protein kinase B (Akt)/ß-catenin/cyclophilin A/Golgi matrix protein 130 (GM130) and upregulation of phosphorylation of H2AX at Ser-139 (γ-H2AX)/p53/p21/Bak cleavage/phospho-JNK/p62/microtubule-associated protein 1 light chain 3B (LC3B)-I. Moreover, JNK inhibition by SP600125 enhanced ROS-independently pKAL-induced cell death through downregulation of p62 and upregulation of p53/p21/Bak cleavage despite a reduced state of DNA damage marker γ-H2AX. These findings indicate that phospho-JNK activated by pKAL inhibits p53-dependent cell death signaling and enhances DNA damage signaling, but cell fate is determined by phospho-JNK as survival rather than death in p53 wild-type HCT116 cells.

p53 Enhances Artemisia annua L. Polyphenols-Induced Cell Death Through Upregulation of p53-Dependent Targets and Cleavage of PARP1 and Lamin A/C in HCT116 Colorectal Cancer Cells.

Plant-derived natural polyphenols exhibit anticancer activity without showing any noticeable toxicities to normal cells. The aim of this study was to investigate the role of p53 on the anticancer effect of polyphenols isolated from Korean Artemisia annua L. (pKAL) in HCT116 human colorectal cancer cells. We confirmed that pKAL induced reactive oxygen species (ROS) production, propidium iodide (PI) uptake, nuclear structure change, and acidic vesicles in a p53-independent manner in p53-null HCT116 cells through fluorescence microscopy analysis of DCF/PI-, DAPI-, and AO-stained cells. The pKAL-induced anticancer effects were found to be significantly higher in p53-wild HCT116 cells than in p53-null by hematoxylin staining, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/PI-stained cells. In addition, expression of ectopic p53 in p53-null cells was upregulated by pKAL in both the nucleus and cytoplasm, increasing pKAL-induced cell death. Moreover, Western bot analysis revealed that pKAL-induced cell death was associated with upregulation of p53-dependent targets such as p21, Bax and DR5 and cleavage of PARP1 and lamin A/C in p53-wild HCT116 cells, but not in p53-null. Taken together, these results indicate that p53 plays an important role in enhancing the anticancer effects of pKAL by upregulating p53 downstream targets and inducing intracellular cell death processes.

Polyphenols Extracted from Artemisia annua L. Exhibit Anti-Cancer Effects on Radio-Resistant MDA-MB-231 Human Breast Cancer Cells by Suppressing Stem Cell Phenotype, ß-Catenin, and MMP-9.

Artemisia annua L. has been reported to show anti-cancer activities. Here, we determined whether polyphenols extracted from Artemisia annua L. (pKAL) exhibit anti-cancer effects on radio-resistant MDA-MB-231 human breast cancer cells (RT-R-MDA-MB-231 cells), and further explored their molecular mechanisms. Cell viability assay and colony-forming assay revealed that pKAL inhibited cell proliferation on both parental and RT-R-MDA-MB-231 cells in a dose-dependent manner. The anti-proliferative effects of pKAL on RT-R-MDA-MB-231 cells were superior or similar to those on parental ones. Western blot analysis revealed that expressions of cluster of differentiation 44 (CD44) and Oct 3/4, matrix metalloproteinase-9 (MMP-9) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation were significantly increased in RT-R-MDA-MB-231 cells compared to parental ones, suggesting that these proteins could be associated with RT resistance. pKAL inhibited the expression of CD44 and Oct 3/4 (CSC markers), and ß-catenin and MMP-9 as well as STAT-3 phosphorylation of RT-R-MDA-MB-231. Regarding upstream signaling, the JNK or JAK2 inhibitor could inhibit STAT-3 activation in RT-R-MDA-MB-231 cells, but not augmented pKAL-induced anti-cancer effects. These findings suggest that c-Jun N-terminal kinase (JNK) or Janus kinase 2 (JAK2)/STAT3 signaling are not closely related to the anti-cancer effects of pKAL. In conclusion, this study suggests that pKAL exhibit anti-cancer effects on RT-R-MDA-MB-231 cells by suppressing CD44 and Oct 3/4, ß-catenin and MMP-9, which appeared to be linked to RT resistance of RT-R-MDA-MB-231 cells.

Proteomic analysis of SP600125-controlled TrkA-dependent targets in SK-N-MC neuroblastoma cells: inhibition of TrkA activity by SP600125.

The c-Jun N-terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin-related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125-controlled TrkA-dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA-mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α-tubulin, ß-tubulin homolog, actin homolog, and eIF-5A-1 protein spots were upregulated by ectopic expression of TrkA, whereas α-enolase, peroxiredoxin-6, PROS-27, HSP70, PP1-gamma, and PDH E1-alpha were downregulated by TrkA, and these TrkA-dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125-controlled TrkA-dependent targets. Moreover, SP600125 strongly suppressed TrkA-mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125-controlled TrkA-dependent targets.

Proteomic analysis of novel targets associated with TrkA-mediated tyrosine phosphorylation signaling pathways in SK-N-MC neuroblastoma cells.

Tropomyosin-related kinase A (TrkA) is a receptor-type protein tyrosine kinase and exploits pleiotypic roles via nerve growth factor (NGF)-dependent or NGF-independent mechanisms in various cell types. Here, we showed that the inhibition of TrkA activity by GW441756 resulted in the suppression of tyrosine phosphorylation of cellular proteins including extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). To find novel targets associated with TrkA-mediated tyrosine phosphorylation signaling pathways, we investigated GW441756 effects on TrkA-dependent targets in SK-N-MC neuroblastoma cells by proteomic analysis. The major TrkA-dependent protein spots controlled by GW441756 were determined by PDQuest image analysis, identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS, and verified by 2DE/Western blot analysis. Thus, we found that most of the identified protein spots were modified forms in a normal condition, and their modifications were regulated by TrkA activity. Especially, our results demonstrated that the modifications of α-tubulin and heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) were significantly upregulated by TrkA, whereas α-enolase modification was downregulated by TrkA, and it was suppressed by GW441756, indicating that TrkA activity is required for their modifications. Taken together, we suggest here that the major novel TrkA-dependent targets such as α-tubulin, hnRNP C1/C2, and α-enolase could play an essential role in TrkA-mediated tyrosine phosphorylation signaling pathways via regulation of their posttranslational modifications.

Caveolin-1 inhibits TrkA-induced cell death by influencing on TrkA modification associated with tyrosine-490 phosphorylation.

Caveolin-1, a main structural protein constituent of caveolae, plays an important role in the signal transduction, endocytosis, and cholesterol transport. In addition, caveolin-1 has conflictive role in the regulation of cell survival and death depending on intracellular signaling pathways. The receptor tyrosine kinase TrkA has been known to interact with caveolin-1, and exploits multiple functions such as cell survival, death and differentiation. In this report, we investigated how TrkA-induced cell death signaling is regulated by caveolin-1 in both TrkA and caveolin-1 overexpressing stable U2OS cells. Here we show that TrkA co-localizes with caveolin-1 mostly as a large aggresome around nucleus by confocal immunofluorescence microscopy. Interestingly, TrkA-mediated Bak cleavage was suppressed by caveolin-1, indicating an inhibition of TrkA-induced cell death signaling by caveolin-1. Moreover, caveolin-1 altered TrkA modification including tyrosine-490 phosphorylation and unidentified cleavage(s), resulting in the inhibition of TrkA-induced apoptotic cell death. Our results suggest that caveolin-1 could suppress TrkA-mediated pleiotypic effects by altering TrkA modification via functional interaction.

Cytosolic accumulation of gammaH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells.

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal that gammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.

Functional characterization of the promoter region of the chicken elongation factor-2 gene.

Elongation factor 2 (EF-2) plays a key role in the essential process of protein synthesis by translocating tRNAs from the ribosomal A- and P-sites to the P- and E-sites. EF-2 regulates the outcome of protein synthesis in mammalian cells. This report demonstrates that chicken EF-2 protein levels are dependent on transcription in 8-bromo-cAMP, insulin and phorbol ester-treated cells. In order to delineate functional domains that control chicken EF-2 gene transcription, the 5'-flanking region of the chicken EF-2 promoter was analyzed. Deletion constructs from -550 and -86 had the same basal level promoter activity as the whole EF-2 promoter. The sequence between nucleotides -700 and -550 was determined to be a regulatory region for the chicken EF-2 basal promoter activity. The region between -700 and -550 has a negative regulatory region and two regulatory proteins (I, II). 8-bromo-cAMP increased chicken EF-2 promoter activity (-700/+102) in Rat 1 HIR fibroblast cells more than insulin and phorbol ester treatment. Binding of protein I and II were decreased by 8-bromo-cAMP but restored by a protein kinase A inhibitor (KT5720). GATA consensus sequence oligonucleotide and fragment -86/-50 prevented protein II binding of fragment -700/-550. This result suggested that protein II is a GATA-like protein. These observations provide a novel regulatory mechanism for the EF-2 promoter.

Identification of simvastatin-regulated targets associated with JNK activation in DU145 human prostate cancer cell death signaling.

The results of this study show that c-Jun N-terminal kinase (JNK) activation was associated with the enhancement of docetaxel-induced cytotoxicity by simvastatin in DU145 human prostate cancer cells. To better understand the basic molecular mechanisms, we investigated simvastatin-regulated targets during simvastatin-induced cell death in DU145 cells using two-dimensional (2D) proteomic analysis. Thus, vimentin, Ras-related protein Rab-1B (RAB1B), cytoplasmic hydroxymethylglutaryl-CoA synthase (cHMGCS), thioredoxin domain-containing protein 5 (TXNDC5), heterogeneous nuclear ribonucleoprotein K (hnRNP K), N-myc downstream-regulated gene 1 (NDRG1), and isopentenyl-diphosphate Delta-isomerase 1 (IDI1) protein spots were identified as simvastatin-regulated targets involved in DU145 cell death signaling pathways. Moreover, the JNK inhibitor SP600125 significantly inhibited the upregulation of NDRG1 and IDI protein levels by combination treatment of docetaxel and simvastatin. These results suggest that NDRG1 and IDI could at least play an important role in DU145 cell death signaling as simvastatinregulated targets associated with JNK activation. [BMB Reports 2017; 50(9): 466-471].

Proteomic analysis of novel targets associated with the enhancement of TrkA-induced SK-N-MC cancer cell death caused by NGF.

Nerve growth factor (NGF) is known to regulate both cancer cell survival and death signaling, depending on the cellular circumstances, in various cell types. In this study, we showed that NGF strongly upregulated the protein level of tropomyosin-related kinase A (TrkA) in TrkA-inducible SK-N-MC cancer cells, resulting in increases in various TrkA-dependent cellular processes, including the phosphorylation of c-Jun N-terminal kinase (JNK) and caspase-8 cleavage. In addition, NGF enhanced TrkA-induced morphological changes and cell death, and this effect was significantly suppressed by the JNK inhibitor SP600125, but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. To investigate novel targets associated with the enhancement of TrkA-induced SK-N-MC cell death caused by NGF, we performed Coomassie Brilliant Blue staining and two-dimensional (2D) proteomic analysis in TrkA-inducible SK-N-MC cells. We identified 31 protein spots that were either greatly upregulated or downregulated by TrkA during NGF treatment using matrix-associated laser desorption/ionization time of flight/time of flight mass spectrometry, and we analyzed the effects of SP600125 and wortmannin on the spots. Interestingly, 11 protein spots, including heterogeneous nuclear ribonucleoprotein K (hnRNP K), lamin B1 and TAR DNA-binding protein (TDP43), were significantly influenced by SP600125, but not by wortmannin. Moreover, the NGF/TrkA-dependent inhibition of cell viability was significantly enhanced by knockdown of hnRNP K using small interfering RNA, demonstrating that hnRNP K is a novel target associated with the regulation of TrkA-dependent SK-N-MC cancer cell death enhanced by NGF.

Hypertensive left ventricular hypertrophy: relation to beta-adrenergic receptor kinase-1 (betaARK1) in peripheral lymphocytes.

BACKGROUND: Left ventricular hypertrophy (LVH) is associated with increased cardiovascular risk and altered sympathetic regulation in hypertension. OBJECTIVES: To determine whether the level of beta-adrenergic receptor kinase-1 (betaARK1) in lymphocytes is related to LVH in patients with hypertension. METHODS: Forty-nine patients with untreated essential hypertension were recruited to the study and classified into two groups: left ventricular hypertrophy (LVH: left ventricular mass index > or =134 g/m in men and > or =110 g/m in women; ages 52.4 +/- 12.8 years, n = 25) and non-LVH (NLVH: left ventricular mass index < 134 g/m in men and < 110 g/m in women; ages 50.8 +/- 13.1 years, n = 24). Lymphocytes were isolated from patients and quantitative-competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were used to estimate the expression of betaARK1 in the lymphocytes. G-protein-coupled receptor kinase activity was assessed by rhodopsin phosphorylation assay. RESULTS: The expression of betaARK1 in lymphocytes was greater in the LVH group than in the NLVH group (0.0069 +/- 0.002 ng compared with 0.0048 +/- 0.0017 ng, P < 0.01) and correlated well with left ventricular mass index (r = 0.527, P < 0.001) and relative wall thickness (r = 0.627, P < 0.001). The concentration of betaARK1 protein in lymphocytes from individuals with LVH was increased two-fold compared with that in the NLVH group (both n = 7). Lymphocyte G-protein-coupled receptor kinase activity from LVH was enhanced 1.7-fold compared with NLVH (1.03 +/- 2.16 and 1.79 +/- 1.87 pmol phosphate/min per mg protein, respectively; P < 0.05, n = 7 for each group). CONCLUSIONS: The concentration of betaARK1 in lymphocytes is greater in hypertensive individuals with LVH than in those without LVH and parallels the degree of hypertrophy. Generalized alterations in beta-adrenergic signalling, including betaARK1, could be a major contributory factor in the development of LVH in hypertension, and the concentration of betaARK1 in lymphocytes can reflect the development of LVH in a patient with hypertension.

Interaction between chicken protein tyrosine phosphatase 1 (CPTP1)-like rat protein phosphatase 1 (PTP1) and p60(v-src) in v-src-transformed Rat-1 fibroblasts.

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kDa in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60(v-src) in vivo and CPTP1 also can associate with p60(v-src) in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60(v-src) was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60(v-src) in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kDa protein which has a possibility to be tyrosine- phosphorylated by p60(v-src) in v-src-transformed Rat-1 fibroblasts. These results suggest that HPTP1B-type PTPs may play an important role in p60(src) dependent signal pathway in eucaryotic cells.

Disease-specific proteins from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that mainly destroys cartilages or bones at the joints. This inflammatory disorder is initiated by self-attack using own immune system, but the detail of pathological mechanism is unclear. Features of autoantigens leading to autoimmune disease are also under veil although several candidates including type II collagen have been suggested to play a role in pathogenesis. In this report, we tried to identify proteins responding to antibodies purified from RA patients and screen proteins up-regulated or down-regulated in RA using proteomic approach. Fibronectin, semaphorin 7A precursor, growth factor binding protein 7 (GRB7), and immunoglobulin mu chain were specifically associated with antibodies isolated from RA synovial fluids. In addition, some metabolic proteins such as adipocyte fatty acid binding protein, galectin-1 and apolipoprotein A1 precursor were overexpressed in RA synovium. Also, expression of peroxiredoxin 2 was up-regulated in RA. On the contrary, expression of vimentin was severely suppressed in RA synoviocytes. Such findings might give some insights into understanding of pathological mechanism in RA.

The expression of ketohexokinase is diminished in human clear cell type of renal cell carcinoma.

For identification and targeting of tumor-associated marker proteins, the proteome of clear cell type of renal cell carcinoma (RCC) and normal kidney tissues was analyzed by 2-DE. Ketohexokinase (also called fructokinase), which catalyzes the phosphorylation of fructose to fructose 1-phosphate, was identified by MALDI-TOF MS and found to be expressed at low rates in the renal tumor tissues. We found a decreased amount of ketohexokinase mRNA in RCC compared to that observed in the normal kidney tissues by Northern blot. The activity of ketohexokinase in 20 clear cell RCC specimens and the 20 corresponding normal kidneys was investigated, and its activity was shown to be approximately 1.4-fold lower in the RCC specimens than in the normal kidney. Ketohexokinase activity in tumor stage pT3 RCC was 1.5-fold lower than in pT1 RCC. The level of ketohexokinase activity in histological grade 3 RCC was 1.8-fold lower than that in grade 1 cancer. In addition, using in situ hybridization, it was revealed that ketohexokinase in the normal kidney tissue was confined to the proximal tubular epithelial cells, while the expression of ketohexokinase in RCC tissues was extremely low. Our research results show that the expression of human ketohexokinase was diminished in clear cell RCC.

Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin isotype II that enhances HeLa cell resistance to H2O2-induced cell death.

Although biochemical properties of 2-Cys peroxiredoxins (Prxs) have been extensively studied, their real physiological functions in higher eukaryotic cells remain obscure and certainly warrant further study. Here we demonstrated that human (h) PrxII, a cytosolic isotype of human 2-Cys Prx, has dual functions as a peroxidase and a molecular chaperone, and that these different functions are closely associated with its adoption of distinct protein structures. Upon exposure to oxidative stress, hPrxII assumes a high molecular weight complex structure that has a highly efficient chaperone function. However, the subsequent removal of stressors induces the dissociation of this protein structure into low molecular weight proteins and triggers a chaperone-to-peroxidase functional switch. The formation of a high molecular weight hPrxII complex depends on the hyperoxidation of its N-terminal peroxidatic Cys residue as well as on its C-terminal domain, which contains a "YF motif" that is exclusively found in eukaryotic 2-Cys Prxs. A C-terminally truncated hPrxII exists as low and oligomeric protein species and does not respond to oxidative stress. Moreover, this C-terminal deletion of hPrxII converted it from an oxidation-sensitive to a hyperoxidation-resistant form of peroxidase. When functioning as a chaperone, hPrxII protects HeLa cells from H(2)O(2)-induced cell death, as measured by a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay and fluorescence-activated cell sorting analysis.

Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis.

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).