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1.
Bioorg Med Chem ; 23(21): 6807-14, 2015 Nov 01.
Article in English | MEDLINE | ID: mdl-26455657

ABSTRACT

Nimesulide analogs bearing a methoxy substituent either at the ortho-, meta- or para-position on the phenyl ring, were designed, synthesized, and evaluated for potential as radioligands for brain cyclooxygenase-2 (COX-2) imaging. The synthesis of nimesulide and regioisomeric methoxy analogs was based on the copper-mediated arylation of phenolic derivatives for the construction of diaryl ethers. These isomeric methoxy analogs displayed lipophilicity similar to that of nimesulide itself, as evidenced by their HPLC logP7.4 values. In vitro inhibition studies using a colorimetric COX (ovine) inhibitor-screening assay demonstrated that the para-methoxy substituted analog retains the inhibition ability and selectivity observed for parent nimesulide toward COX-2 enzyme, whereas the meta- and ortho-methoxy substituents detrimentally affected COX-2-inhibition activity, which was further supported by molecular docking studies. Bidirectional transport cellular studies using Caco-2 cell culture model in the presence of the P-glycoprotein (P-gp) inhibitor, verapamil, showed that P-gp did not have a significant effect on the efflux of the para-methoxy substituted analog. Further investigations using the radiolabeled form of the para-methoxy substituted analog is warranted for in vivo characterization.


Subject(s)
Contrast Media/chemical synthesis , Cyclooxygenase 2 Inhibitors/chemical synthesis , Cyclooxygenase 2/metabolism , Sulfonamides/chemistry , Brain/enzymology , Caco-2 Cells , Catalytic Domain , Contrast Media/chemistry , Contrast Media/metabolism , Cyclooxygenase 2/chemistry , Cyclooxygenase 2 Inhibitors/chemistry , Cyclooxygenase 2 Inhibitors/metabolism , Humans , Isomerism , Molecular Conformation , Molecular Dynamics Simulation , Protein Binding , Sulfonamides/chemical synthesis , Sulfonamides/metabolism
2.
J Toxicol Sci ; 40(4): 501-8, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26165647

ABSTRACT

Identification of substances with specific toxicity for carcinoma cells promises to facilitate the development of cancer chemotherapeutics that cause minimal side effects. Here, we show that knockdown of the farnesoid X receptor (FXR) effectively suppresses the proliferation of human hepatocellular carcinoma cell lines HepG2 and HLE accompanied by elevated expression of cyclin-dependent kinase (CDK) inhibitor p16/INK4a and p21/Cip1 proteins. On the other hand, the growth of the primary human hepatocyte-derived cell line Fa2N-4 is not affected by the treatment with FXR siRNA irrespective of marked increases in the mRNAs of p16/INK4a and p21/Cip1. Surprisingly, the expression levels of p16/INK and p21/Cip1 proteins are left unchanged in Fa2N-4 cells that are subjected to the FXR siRNA treatment. Since the expression levels of these CDK inhibitor proteins in FXR-knockdown Fa2N-4 cells were elevated in the presence of proteasomal inhibitor MG132, these CDK inhibitors may be subjected to the proteasomal degradation, thereby counteracting the increased expression of their cognate mRNAs, therefore similar levels of p16 and p21 proteins were observed in control and FXR-knockdown Fa2N-4 cells. These results suggest that FXR-knockdown is effective for inhibiting the proliferation of hepatocellular carcinoma cells, not interfering with the regulatory mechanism of normal hepatocyte growth.


Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Proliferation/genetics , Gene Knockdown Techniques , Hepatocytes/cytology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/physiology , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Expression , Hep G2 Cells , Humans , Leupeptins , Proteasome Endopeptidase Complex/metabolism , RNA, Messenger , RNA, Small Interfering
3.
Sci Rep ; 5: 8264, 2015 Feb 05.
Article in English | MEDLINE | ID: mdl-25652335

ABSTRACT

Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8(-/-) mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8(-/-) mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8(+/-) mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.


Subject(s)
Fucosyltransferases/deficiency , Liver Regeneration , Animals , Cell Proliferation/drug effects , Fucose/administration & dosage , Fucose/metabolism , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Gene Expression , Genotype , Hepatectomy , Hepatocytes/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Liver Regeneration/genetics , Mice , Mice, Knockout , Models, Animal , Receptors, Growth Factor/metabolism , Signal Transduction
4.
Cell Biochem Funct ; 33(2): 73-9, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25663666

ABSTRACT

Acquired radioresistance of cancer cells interferes with radiotherapy and increases the probability of cancer recurrence. HepG2-8960-R, which is one of several clinically relevant radioresistant (CRR) cell lines, has a high tolerance to the repeated clinically relevant doses of X-ray radiation. In this study, HepG2-8960-R had slightly lower cell proliferation ability than HepG2 in the presence of FBS. In particular, epidermal growth factor (EGF) hardly enhanced cell proliferation and DNA synthesis in HepG2-8960-R. Additionally, EGF could not induce the activation of Erk1/2, because the expression of EGF receptor (EGFR) protein decreased in HepG2-8960-R in accordance with the methylation of the EGFR promoter region. Therefore, cetuximab did not inhibit HepG2-8960-R cell proliferation. Our study showed that HepG2-8960-R had radioresistant and cetuximab-resistant abilities.


Subject(s)
Carcinoma, Hepatocellular/radiotherapy , Cell Proliferation , Epidermal Growth Factor/metabolism , Liver Neoplasms/radiotherapy , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Cetuximab , Hep G2 Cells , Humans , Liver Neoplasms/pathology , Methylation , Promoter Regions, Genetic , Radiation Tolerance
5.
Cancer Sci ; 105(10): 1351-9, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25098609

ABSTRACT

Standard fractionated radiotherapy for the treatment of cancer consists of daily irradiation of 2-Gy X-rays, 5 days a week for 5-8 weeks. To understand the characteristics of radioresistant cancer cells and to develop more effective radiotherapy, we established a series of novel, clinically relevant radioresistant (CRR) cells that continue to proliferate with 2-Gy X-ray exposure every 24 h for more than 30 days in vitro. We studied three human and one murine cell line, and their CRR derivatives. Guanine nucleotide-binding protein 1 (GBP1) gene expression was higher in all CRR cells than their corresponding parental cells. GBP1 knockdown by siRNA cancelled radioresistance of CRR cells in vitro and in xenotransplanted tumor tissues in nude mice. The clinical relevance of GBP1 was immunohistochemically assessed in 45 cases of head and neck cancer tissues. Patients with GBP1-positive cancer tended to show poorer response to radiotherapy. We recently reported that low dose long-term fractionated radiation concentrates cancer stem cells (CSCs). Immunofluorescence staining of GBP1 was stronger in CRR cells than in corresponding parental cells. The frequency of Oct4-positive CSCs was higher in CRR cells than in parental cells, however, was not as common as GBP1-positive cells. GBP1-positive cells were radioresistant, but radioresistant cells were not necessarily CSCs. We concluded that GBP1 overexpression is necessary for the radioresistant phenotype in CRR cells, and that targeting GBP1-positive cancer cells is a more efficient method in conquering cancer than targeting CSCs.


Subject(s)
GTP-Binding Proteins/physiology , Neoplasms/radiotherapy , Neoplastic Stem Cells/radiation effects , Radiation Tolerance , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , Female , GTP-Binding Proteins/analysis , Humans , Immunohistochemistry , Male , Mice , Middle Aged , Neoplasms/pathology , Octamer Transcription Factor-3/analysis , Oligonucleotide Array Sequence Analysis
6.
Cancer Med ; 3(2): 310-21, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24464839

ABSTRACT

Radiotherapy is widely used to treat cancer because it has the advantage of physically and functionally conserving the affected organ. To improve radiotherapy and investigate the molecular mechanisms of cellular radioresistance, we established a clinically relevant radioresistant (CRR) cell line, SAS-R, from SAS cells. SAS-R cells continue to proliferate when exposed to fractionated radiation (FR) of 2 Gy/day for more than 30 days in vitro. A xenograft tumor model of SAS-R was also resistant to 2 Gy/day of X-rays for 30 days. The density of blood vessels in SAS-R tumors was higher than in SAS tumors. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, sensitized microvascular endothelial cells to radiation, but failed to radiosensitize SAS and SAS-R cells in vitro. Everolimus with FR markedly reduced SAS and SAS-R tumor volumes. Additionally, the apoptosis of endothelial cells (ECs) increased in SAS-R tumor tissues when both Everolimus and radiation were administered. Both CD34-positive and tomato lectin-positive blood vessel densities in SAS-R tumor tissues decreased remarkably after the Everolimus and radiation treatment. Everolimus-induced apoptosis of vascular ECs in response to radiation was also followed by thrombus formation that leads to tumor necrosis. We conclude that FR combined with Everolimus may be an effective modality to overcome radioresistant tumors via targeting tumor ECs.


Subject(s)
Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/radiotherapy , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/radiotherapy , Radiation-Sensitizing Agents/pharmacology , Sirolimus/analogs & derivatives , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/radiotherapy , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Endothelial Cells/drug effects , Endothelial Cells/radiation effects , Everolimus , Female , HeLa Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Sirolimus/pharmacology , Squamous Cell Carcinoma of Head and Neck , Xenograft Model Antitumor Assays
7.
Nucl Med Biol ; 40(3): 387-94, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23347829

ABSTRACT

INTRODUCTION: With the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named "Lactosome", which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the (18)F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out. METHODS: (18)F-labeled Lactosomes were prepared by a film hydration method under sonication in water at 50°C from a mixture of 4-[(18)F]fluoro-benzoyl poly-L-lactic acid ((18)F-BzPLLA30) and the amphiphilic polydepsipeptide. For biodistribution studies, BALB/cA Jcl-nu/nu mice bearing HeLa cells in the femur region were used. We took both PET and near-infrared fluorescence (NIRF) images of tumor bearing mice after co-injection of (18)F-labeled Lactosome and NIRF-labeled Lactosome. RESULTS: (18)F-labeled Lactosomes were prepared at good yields (222-420MBq) and more than 99% of (18)F-BzPLLA30 was incorporated into (18)F-labeled Lactosome. The radioactivity of (18)F-labeled Lactosome was found to be stable and maintained at high level for up to 6h after injection into the blood stream. Tumor uptake increased gradually after the injection. The uptake ratio of tumor/muscle was 2.7 at 6h from the time of injection. Tumor PET imaging with (18)F-labeled Lactosome was as capable as tumor NIRF imaging with NIRF-labeled Lactosome. CONCLUSION: Tumor PET imaging using Lactosome as a nanocarrier may be therefore a potential candidate for a facile and general solid tumor imaging technique.


Subject(s)
Depsipeptides/chemistry , Fluorine Radioisotopes , Hydrophobic and Hydrophilic Interactions , Nanostructures/chemistry , Polyesters/chemistry , Radiochemistry , Animals , Female , HeLa Cells , Humans , Mice , Micelles , Positron-Emission Tomography
8.
J Biochem ; 152(6): 577-86, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22969091

ABSTRACT

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.


Subject(s)
Cell Proliferation , Receptors, Cytoplasmic and Nuclear/physiology , Butadienes/pharmacology , Carcinoma, Hepatocellular , Cell Nucleus Shape , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Hep G2 Cells , Hepatocyte Growth Factor/physiology , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Humans , Liver Neoplasms , MAP Kinase Signaling System , Nitriles/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA, Small Interfering/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism
9.
Am J Physiol Gastrointest Liver Physiol ; 303(7): G861-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22837346

ABSTRACT

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.


Subject(s)
Cell Proliferation , ErbB Receptors , GTP-Binding Proteins/metabolism , Hepatocytes/physiology , Norepinephrine , Transglutaminases/metabolism , Age Factors , Animals , Cells, Cultured , DNA Replication , Down-Regulation , Epidermal Growth Factor/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Norepinephrine/genetics , Norepinephrine/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Rats , Rats, Wistar , Receptors, Adrenergic/genetics , Receptors, Adrenergic/metabolism
10.
Am J Physiol Gastrointest Liver Physiol ; 299(1): G106-14, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20448147

ABSTRACT

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the alpha(1)-adrenergic receptor coupled with G protein, Galpha(h). However, the molecular mechanism is not well understood. Galpha(h) is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [(3)H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Galpha(h).


Subject(s)
Cell Proliferation , GTP-Binding Proteins/metabolism , Hepatocytes/enzymology , Liver Regeneration , Norepinephrine/metabolism , Transglutaminases/metabolism , Adrenergic Antagonists/pharmacology , Animals , Cell Proliferation/drug effects , Cells, Cultured , DNA Replication , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Hepatocytes/drug effects , Hydrolysis , Liver Regeneration/drug effects , Male , Phospholipase C delta/metabolism , Phosphorylation , Protein Binding , Protein Glutamine gamma Glutamyltransferase 2 , Protein Multimerization , Rats , Rats, Wistar , Signal Transduction , Time Factors
11.
Radiat Res ; 173(5): 620-8, 2010 May.
Article in English | MEDLINE | ID: mdl-20426661

ABSTRACT

Exposure to X radiation is associated with a decline in the proliferative activity of the liver, but the molecular mechanism(s) is not well understood. We investigated whether exposure to X radiation is involved in functional changes in the epidermal growth factor (EGF) receptor (EGFR), thereby causing a reduction of EGF-induced DNA synthesis using periportal hepatocytes (PPH) and perivenous hepatocytes (PVH), which differ in their proliferative activity. X radiation dose-dependently decreased DNA synthesis in both subpopulations. The rate of decline in the DNA synthesis was greater in PPH than in PVH, but the zonal difference disappeared after exposure to 10 Gy X radiation. [(125)I]EGF binding studies indicated that high-affinity EGFRs in both subpopulations were down-regulated after X irradiation. Furthermore, EGF-induced EGFR dimerization and phosphorylation at Y1173 in both subpopulations were down-regulated after X irradiation, and the rate of decline was greater in PPH than in PVH. In contrast, phosphorylation at Y845 after EGF treatment was dose-dependently up-regulated after X irradiation in both subpopulations. These results suggest that the X-radiation-related decline in EGF-induced DNA synthesis is caused at least partly by the modification of EGFR function.


Subject(s)
Down-Regulation/radiation effects , ErbB Receptors/metabolism , Hepatocytes/radiation effects , Animals , Cells, Cultured , DNA Replication/radiation effects , Dimerization , Dose-Response Relationship, Radiation , Hepatocytes/cytology , Hepatocytes/metabolism , Male , Phosphorylation , Rats , Rats, Wistar , X-Rays
12.
J Radiat Res ; 51(3): 297-302, 2010.
Article in English | MEDLINE | ID: mdl-20410675

ABSTRACT

The high-density survival (HDS) assay was originally elaborated to assess cancer cell responses to therapeutic agents under the influence of intercellular communication. Here, we simplified the original HDS assay and studied its applicability for the detection of cellular radioresistance. We have recently defined clinically relevant radioresistant (CRR) cells, which continue to proliferate with daily exposure to 2 gray (Gy) of X-rays for more than 30 days in vitro. We established human CRR cell lines, HepG2-8960-R from HepG2, and SAS-R1 and -R2 from SAS, respectively. In an attempt to apply the HDS assay to detect radioresistance with clinical relevance, we simplified the original HDS assay by scoring the total number of surviving cells after exposure to X-rays. The modified HDS assay successfully detected radioresistance with clinical relevance. The modified HDS assay detected CRR phenotype, which is not always detectable by clonogenic assay. Therefore, we believe that the modified HDS assay presented in this study is a powerful tool to predict the effectiveness of fractionated radiotherapy against malignant tumors.


Subject(s)
Cell Line, Tumor/radiation effects , Dose Fractionation, Radiation , Radiation Tolerance , Radiotherapy/methods , Animals , Cell Survival/radiation effects , Dose-Response Relationship, Radiation , Humans , K562 Cells , Mice , Mice, Nude , Mitosis , Neoplasm Transplantation , Tetrazolium Salts/pharmacology , Thiazoles/pharmacology , U937 Cells , X-Rays
13.
Cancer Sci ; 100(4): 747-52, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19215227

ABSTRACT

Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy and radiochemotherapy. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5-8 weeks. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we established a novel radioresistant cell line, HepG2-8960-R with clinical relevance from parental HepG2 cells by long-term fractionated exposure to 2 Gy of X-rays. HepG2-8960-R cells continued to proliferate with daily exposure to 2 Gy X-rays for more than 30 days, while all parental HepG2 cells ceased. After exposure to fractionated 2 Gy X-rays, induction frequencies of micronuclei and remaining foci of gamma-H2AX in HepG2-8960-R were less than those in HepG2. Flow cytometric analysis revealed that the proportion of cells in S- and G2/M-phase of the cell cycle was higher in HepG2-8960-R than in HepG2. These suggest that the response of clinically relevant radioresistant (CRR) cells to fractionated radiation is not merely an accumulated response to each fractionated radiation. This is the first report on the establishment of a CRR cell line from an isogenic parental cell line.


Subject(s)
DNA Breaks, Double-Stranded , DNA Damage , DNA Repair , DNA, Neoplasm/radiation effects , Radiation Tolerance/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle/genetics , Cell Line, Tumor , Cell Survival/radiation effects , Colony-Forming Units Assay , Dose-Response Relationship, Radiation , Humans , Kinetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , X-Rays/adverse effects
14.
Protein Pept Lett ; 16(2): 138-42, 2009.
Article in English | MEDLINE | ID: mdl-19200036

ABSTRACT

We attempted to develop a stable radiolabeled transferrin (Tf) useful in experimental studies related to Tf receptor. 67Ga and 111In were used as labeling radioisotopes. The results from gel chromatography, dialysis, and electrophoresis showed that 111In-DTPA-Tf was the most stable among the radiolabeled Tfs examined in the present study. 111In-DTPA-Tf was also the most stable radiolabeled transferrin in the blood.


Subject(s)
Gallium Radioisotopes/chemistry , Indium Radioisotopes/chemistry , Transferrin/chemistry , Animals , Dialysis , Electrophoresis , Gallium Radioisotopes/blood , Hydrogen-Ion Concentration , Indium Radioisotopes/blood , Pentetic Acid/chemistry , Protein Stability , Rats , Transferrin/analysis
15.
Appl Radiat Isot ; 66(9): 1245-9, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18353660

ABSTRACT

Indium-111 ((111)In) has a strong binding affinity for transferrin (Tf), and the (111)In-Tf complex binds to Tf receptor in various tissues. In partial hepatectomy (PH), a part of blood in circulation is lost along with removed liver tissues; consequently, the number of blood cells and the amount of Tf in circulation decreases. These decreases should greatly affect the uptake of (111)In in the liver and bone marrow. In order to investigate this effect, we compared the uptake in partially hepatectomized rats with that in venesectioned rats, in which only the volume of blood in circulation had been decreased. Our data show that fewer blood cells and smaller amount of Tf in circulation due to venesection increased the uptake of (111)In in bone marrow, but not in the liver, whereas PH increased the uptake of (111)In in both bone marrow and liver. The higher bone marrow uptake of (111)In must be related to the increase of the hematopoietic function resulted from the smaller amount of blood; the higher uptake in liver may be closely related to liver regeneration.


Subject(s)
Bone Marrow/metabolism , Hepatectomy , Indium Radioisotopes/pharmacokinetics , Liver/metabolism , Phlebotomy , Animals , Citrates/pharmacokinetics , Gallium/pharmacokinetics , Gallium Radioisotopes/pharmacokinetics , Liver Regeneration/physiology , Male , Rats , Transferrin/metabolism
16.
J Radiat Res ; 49(1): 29-40, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18049034

ABSTRACT

The liver is one of the target organs of radiation-induced cancers by internal exposures. In order to elucidate radiation-induced liver cancers including Thorotrast, we present a new approach to investigate in vivo effects of internal exposure to alpha-particles. Adopting boron neutron capture, we separately irradiated Kupffer cells and endothelial cells in mouse liver in vivo and analyzed the changes in gene transcriptions by an oligonucleotide microarray. Differential expression was defined as more than 3-fold for up-regulation and less than 1/3 for under-regulation, compared with non-irradiated controls. Of 6,050 genes examined, 68 showed differential expression compared with non-irradiated mice. Real-time polymerase chain reaction validated the results of the microarray analysis. Exposure to alpha-particles and gamma-rays produced different patterns of altered gene expression. Gene expression profiles revealed that the liver was in an inflammatory state characterized by up-regulation of positive acute phase protein genes, irrespective of the target cells exposed to radiation. In comparison with chemical and biological hepatotoxicants, inductions of Metallothionein 1 and Hemopexin, and suppressions of cytochrome P450s are characteristic of radiation exposure. Anti-inflammatory treatment could be helpful for the prevention and protection of radiation-induced hepatic injury.


Subject(s)
Down-Regulation/radiation effects , Gene Expression Profiling , Liver/radiation effects , Up-Regulation/radiation effects , Alpha Particles , Animals , Boron , Down-Regulation/genetics , Endothelial Cells/metabolism , Endothelial Cells/radiation effects , Gamma Rays , Kupffer Cells/metabolism , Kupffer Cells/radiation effects , Liposomes , Liver/cytology , Liver/metabolism , Male , Mice , Mice, Inbred C3H , Microarray Analysis , Neutrons , Polymerase Chain Reaction , Radioisotopes , Up-Regulation/genetics , Whole-Body Irradiation
17.
J Hepatol ; 48(2): 246-54, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18006107

ABSTRACT

BACKGROUND/AIMS: Aging relates to declined proliferative capacity of the liver, but the molecular mechanism is not well understood. We examined whether functional changes of epidermal growth factor (EGF) receptor (EGFR) are involved in age-related decline in EGF-induced DNA synthesis using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in the proliferative capacity. METHODS: Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) in 7-, 30-, and 90-week-old rats were isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [methyl-(3)H]thymidine incorporation. EGFR binding affinity to EGF was analyzed by Scatchard analysis using [(125)I]EGF. EGFR dimerization and phosphorylation were determined by Western blot analysis. RESULTS: EGF-induced DNA synthesis was greater in PPH than in PVH from rats of 7 weeks, but the zonal difference disappeared with aging. [(125)I]EGF binding studies indicated that high-affinity EGFR in both subpopulations also disappeared with aging. Furthermore, EGF-induced dimerization in both subpopulations was down-regulated with aging, and the pattern of EGFR phosphorylation was parallel to that of dimerization. CONCLUSIONS: These data suggest that age-related decline in EGF-induced DNA synthesis of PPH and PVH is caused by down-regulation of EGFR dimerization through the decrease of high-affinity EGFR.


Subject(s)
Aging/pathology , Epidermal Growth Factor/pharmacology , Hepatocytes/drug effects , Aging/metabolism , Animals , Cell Proliferation/drug effects , Cell Separation , Cells, Cultured , DNA/biosynthesis , Dimerization , Epidermal Growth Factor/metabolism , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Hepatocytes/cytology , Male , Phosphorylation , Rats , Rats, Wistar
18.
Cell Biochem Funct ; 26(3): 359-65, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18008394

ABSTRACT

Liver regeneration is regulated by several factors, including growth factors, cytokines, and post-translational modifications of several proteins. It is suggested that transglutaminase 2 (TG2) and ornithine decarboxylase (ODC) are involved in liver regeneration. To investigate the role of TG2 and ODC activities in regenerating liver, we used retinoic acid (RA), an inducer of TG2 and a suppressor of ODC. Regenerating rat liver was prepared by 70% partial hepatectomy (PH). Rats were sacrificed at 1, 2, 3, 4, and 6 days after surgery. RA was intraperitoneally injected immediately after PH. TG2 and ODC activities and products (epsilon-(gamma-glutamyl) lysine isopeptide (Gln-Lys) and polyamines, respectively) were examined at the indicated times. In RA-treated rat, DNA synthesis and ODC activity declined and the peak shifted to 2 days after PH, whereas TG2 activity increased at 1 day after PH. At that time, protein-polyamine, especially the protein-spermidine (SPD) bond, transiently decreased, whereas the formation of the Gln-Lys bond increased after PH. These results suggested that in regenerating liver, enhanced the formation of Gln-Lys bonds catalyzed by TG2 led to reduced DNA synthesis, whereas when ODC produced newly synthesized SPD, the inhibition of Gln-Lys bond production by the preferential formation of protein-SPD bonds led to an increase in DNA synthesis.


Subject(s)
GTP-Binding Proteins/metabolism , Liver Regeneration/drug effects , Ornithine Decarboxylase/metabolism , Transglutaminases/metabolism , Tretinoin/pharmacology , Animals , DNA/biosynthesis , Enzyme Induction/drug effects , Flow Cytometry , GTP-Binding Proteins/biosynthesis , Hepatectomy , Liver/drug effects , Liver/enzymology , Male , Ornithine Decarboxylase/biosynthesis , Polyamines/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Rats , Rats, Wistar , Time Factors , Transglutaminases/biosynthesis
19.
Life Sci ; 81(7): 577-84, 2007 Jul 26.
Article in English | MEDLINE | ID: mdl-17673261

ABSTRACT

Transglutaminase 2 (TG2) has been reported to be involved in cell growth through the formation of epsilon-(gamma-glutamyl) lysine (Gln-Lys) or N-(gamma-glutamyl) polyamine (Gln-polyamine). We have recently reported that the inhibition of Gln-Lys cross-linking by the formation of Gln-spermidine led to the increase of DNA synthesis in regenerating rat liver. TG2 may catalyze the replacement reaction between Lys residues in protein and polyamines. In the present study, we attempted to develop an experimental model for ascertaining this replacement reaction. We examined whether or not TG2 exhibited the association and dissociation reaction of Gln-polyamine bond in protein, using N,N-dimethylcasein (DC). The dissociated polyamines were identified by autoradiography. The dissociation of [(14)C] polyamines from DC bond [(14)C] polyamines complex by TG2 could occur in the presence of non-radioactive polyamines as second amine donor, whereas in the absence, could not almost occur. Moreover, it was indicated that this release of old [(14)C] polyamine bonded to DC was due to binding of added new [(14)C] polyamine to Gln residues in DC. These results demonstrate that TG2 catalyzes the replacement reaction between added [(14)C] polyamine and DC bond [(14)C] polyamine. The dissociation and association reaction may both occur together, the new DC-polyamine complex being formed at the same time as the dissociation of old DC-polyamine complex, since readying a second amine donor is necessary to dissociate DC-polyamine complex. These results indicate that this experimental model is successful in the study of TG2-catalyzed dissociation and association reaction of Gln-polyamine bond in protein.


Subject(s)
GTP-Binding Proteins/metabolism , Polyamines/metabolism , Proteins/metabolism , Transglutaminases/metabolism , Animals , Autoradiography , Calcium/pharmacology , Caseins/chemistry , Catalysis , Chromatography, Thin Layer , Cross-Linking Reagents , Densitometry , Dithiothreitol/pharmacology , Dose-Response Relationship, Drug , In Vitro Techniques , Liver/drug effects , Liver/enzymology , Protein Glutamine gamma Glutamyltransferase 2 , Rats , Sulfhydryl Reagents/pharmacology
20.
Int J Radiat Biol ; 83(7): 433-42, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17538793

ABSTRACT

PURPOSE: In order to identify supportive evidence of radiation exposure to cells, we analyzed the relationship between exposure to ionizing radiation and the induction of deletions in mitochondrial DNA (mtDNA). MATERIALS AND METHODS: Using human hepatoblastoma cell line, HepG2 and its derivatives, HepG2-A, -89 and -400, established after long term exposure to X-ray, mtDNA deletions were analyzed by polymerase chain reaction (PCR) and real-time PCR after cells were subjected to radiation and genotoxic treatments. RESULTS: Common Deletion (CD), the most extensively studied deletion of mtDNA, was induced within 24 h after exposure to 5 Gray (Gy) of X-rays and was associated with replication of mtDNA. CD became undetectable several days after the exposure due to the death of cells containing mitochondria within which CD had been induced. Furthermore, we found a novel mtDNA deletion that consisted of a 4934 base-pair deletion (4934del) between nucleotide position 8435 and 13,368. A lower dose of ionizing radiation was required to induce the 4934del than for CD and this was independent of the quality of radiation used and was not induced by treatments with hydrogen peroxide (H(2)O(2)) and other genotoxic reagents including bleomycin. CONCLUSION: CD is induced by ionizing radiation, however, the amount of CD detected at a certain point in time after radiation exposure is dependent on the initial frequency of CD induced and the death rate of cells with mtDNA containing CD. The novel mtDNA deletion found in this study, therefore, will be used to determine whether cells were exposed to ionizing radiation.


Subject(s)
DNA, Mitochondrial/genetics , DNA, Mitochondrial/radiation effects , Gene Deletion , Radiation, Ionizing , Base Sequence , Bleomycin/pharmacology , Cell Death , Cell Line, Tumor , DNA Primers/chemistry , Dose-Response Relationship, Radiation , Humans , Hydrogen Peroxide/pharmacology , Molecular Sequence Data , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
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