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Biomedical subjects

K Umezawa

Publications and source records attributed to K Umezawa.

At least 55 records · Page 3Linked to original sources
PubMed1998

Induction of hydrogen peroxide production and Bax expression by caspase-3(-like) proteases in tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma cells.

In our previous studies (S. Simizu, et al., 1996, Cancer Res. 56, 4978-4982), we reported that apoptosis of human small cell lung carcinoma (SCLC) cells induced by protein tyrosine kinase inhibitors, such as erbstatin and herbimycin A, was mediated by H2O2 via a newly synthesized protein(s). In the present study, we demonstrated that induction of apoptosis by erbstatin resulted in activation of caspase-3(-like) proteases, which are interleukin-1 beta-converting enzyme family proteases (caspases) and that inhibition of these protease activities reduced the extent of cell death and H2O2 generation. We also demonstrated that expression of apoptotic protein Bax was induced by erbstatin. Erbstatin-induced Bax expression was inhibited by the inhibitor of caspase-3(-like) proteases. These results indicate that generation of intracellular H2O2 and Bax expression in tyrosine kinase inhibitor-induced apoptosis were modulated by the activation of caspase-3(-like) proteases in SCLC cells.

Apoptosis↗
PubMed1998

Inhibition of cyclin D1 expression and induction of apoptosis by inostamycin in small cell lung carcinoma cells.

Previously, we demonstrated that inostamycin, an inhibitor of phosphatidylinositol turnover, caused cell cycle arrest at the G1 phase, inhibiting the expression of cyclins D1 and E in normal cells. In the present study, we examined the effects of inostamycin on cell cycle progression and apoptosis in human small cell lung carcinoma Ms-1 cells. Treatment of exponentially proliferating Ms-1 cells with low concentrations of inostamycin caused cells to accumulate in the G1 phase. We found that inostamycin decreased cyclin D1, and increased cyclin-dependent kinase inhibitors such as p21WAF1 and p27KIP1 in Ms-1 cells. On the other hand, higher concentrations of inostamycin induced morphological apoptosis and DNA fragmentation in Ms-1 cells without affecting the expression of p53, Bcl-2 and Bax. Inostamycin-induced apoptosis was suppressed by an inhibitor of caspase-3, and a 17 kDa fragment of activated caspase-3 was detected following inostamycin treatment. Therefore, caspase-3(-like) would appear to be involved in inostamycin-induced apoptosis. On the other hand, an inhibitor of caspase-3(-like) proteases did not affect the inhibitory effect of inostamycin on cyclin D1 expression, suggesting that caspase-3(-like) proteases were not responsible for inostamycin-induced G1 arrest.

Anti-Bacterial Agents↗
PubMed1998

Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung carcinoma Ms-1 cells.

Vinblastine arrests cells in the G2/M phase of the cell cycle and subsequently induces cell death by apoptosis. We found that treatment of cells with vinblastine induced phosphorylation of Bcl-2, resulting in the dissociation of Bcl-2 and Bax. Moreover, vinblastine-induced apoptosis was suppressed by an inhibitor of caspase-3, Ac-DEVD-CHO; and a 17-kDa active fragment of caspase-3 was detected following vinblastine treatment, suggesting that caspase-3 is involved in vinblastine-induced apoptosis. However, Ac-DEVD-CHO affected neither vinblastine-induced Bcl-2 phosphorylation nor vinblastine-induced G2/M arrest. Vinblastine caused G2/M arrest prior to apoptosis, whereas vinblastine-induced apoptosis was not dependent on the duration of the G2/M phase. Thus, vinblastine-induced apoptosis might be mediated by the phosphorylation of Bcl-2, resulting in Bcl-2 inactivation, and by subsequent activation of caspase-3.

Apoptosis↗
PubMed1998

Induction of apoptosis in human pancreatic carcinoma cells by a synthetic bleomycin-like ligand.

Histidine-pyridine-histidine-3 (HPH-3) is an oxygen-activating ligand based on the structure of bleomycin. HPH-3 induced the death of human pancreatic adenocarcinoma AsPC-1 cells in 24 h, causing apoptotic morphology and internucleosomal degradation of DNA. HPH-3-induced cell death was not inhibited by antioxidants such as reduced glutathione and N-acetylcysteine, whereas hydrogen peroxide-induced cell death was inhibited by them, indicating that hydrogen peroxide is not involved in the induction of apoptosis by HPH-3. Induction of apoptosis by HPH-3 was inhibited by zinc and copper ions, indicating that chelation with ferrous ion is responsible for induction of apoptosis, as is the case in chelation by bleomycin to cleave DNA. Bleomycin A2 and its fragment having no DNA-binding region, glycopeptide-3, did not induce apoptosis in AsPC-1 cells. Bleomycin A2 induced G2/M block in flow-cytometric analysis, but HPH-3 did not and instead induced an apoptotic pre-G1 peak. Thus, HPH-3 induced apoptosis in human pancreatic carcinoma cells, which is a unique characteristic among bleomycin-related compounds.

Adenocarcinoma↗
PubMed1998

Potentiation of paclitaxel cytotoxicity by inostamycin in human small cell lung carcinoma, Ms-1 cells.

In the present study, we found that inostamycin increased the ability of paclitaxel to induce apoptosis in Ms-1 cells. A considerably higher concentration of paclitaxel was required for the induction of apoptosis in Ms-1 cells than in other cell lines tested. Treatment of Ms-1 cells with inostamycin, an inhibitor of phoshatidylinositol (PI) synthesis, reduced the dosage of paclitaxel required to induce cell death by apoptosis. This effect of inostamycin is specific to Ms-1 cells, and inostamycin did not increase the cytotoxicity of other antitumor drugs such as adriamycin, vinblastine, methotrexate, cisplatin, etoposide, or camptothecin in Ms-1 cells. Addition of inostamycin to paclitaxel-treated cells caused a significant increase in the sub G1 peak, representing apoptosis, which was accompanied by a decrease in the G2/M peak seen in paclitaxel-treated Ms-1 cells, without affecting paclitaxel-inhibited tubulin depolymerization. Moreover, paclitaxel did not enhance inostamycin-inhibited PI synthesis. The expression levels of Bcl-2, Bax, and Bcl-XL were not changed following the co-treatment with inostamycin plus paclitaxel, whereas the activated form of caspase-3 was markedly increased. Thus, inostamycin is a chemosensitizer of paclitaxel in small cell lung carcinoma Ms-1 cells.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

[Rapid growth of glioblastoma during therapy for multiple myeloma: case report].

Rapid growth of a glioblastoma during therapy for multiple myeloma is reported. A 53-year-old man was admitted to our hospital with a right costal tumor, which was resected. The diagnosis was plasmocytoma. Urine protein electrophoresis showed a monoclonal peak in the region of gamma-globulin, and examination of the bone marrow revealed 17.8% of atypical plasma cells. Brain magnetic resonance (MR) imaging detected two small lesions, but these could not be identified as brain tumor. He received chemotherapy (melphalan 10 mg/day and predonin 30 mg/day for 4 days) and was discharged. Two weeks after discharge, he was readmitted because of left hemiparesis. T1-weighted MR imaging showed two large hypointense lesions in the right frontal lobe, with ring-like enhancement following Gd-DTPA infusion. 1H-MR spectroscopy showed typical findings of tumor with increased choline and lactic acid peaks. 201Tl SPECT revealed high accumulation in both early and delayed images. Right carotid angiography showed a hypervascular tumor with venous filling and mass effect. The lesions were resected via right frontal craniotomy, followed by intraoperative radiation and placement of an Ommaya reservoir. Histological examination showed the tumors were glioblastoma. The brain between the tumors also showed the typical appearance of glioblastoma, suggesting that the lesions were continuous. Postoperatively, the patient's left hemiparesis disappeared. He received local irradiation and chemotherapy and was then discharged. Coexistence of glioblastoma and multiple myeloma is rare. The cause may be genetic abnormality, but immunodeficiency due to multiple myeloma, surgical damage, or chemotherapy may have contributed to the rapid growth of the glioblastoma.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

GDNF-induced neurite formation was stimulated by protein kinase inhibitors and suppressed by Ras inhibitors.

The effects of various inhibitors on the glial cell line-derived neurotrophic factor (GDNF)-induced neurite formation in TGW human neuroblastoma cells were investigated. Treatment of cells with Ser/Thr protein kinase inhibitors such as staurosporine, H-7, H-8 and HA-1004, induced neurite formation without GDNF. On the other hand, tyrosine kinase inhibitors such as erbstatin, genistein and herbimycin A did not produce neurites per se, but effectively enhanced the GDNF-induced neurite formation. A phosphatase inhibitor, okadaic acid, and Ras inhibitors such as oxanosine, damnacanthal and conophylline strongly suppressed the effect of GDNF. These results suggest that a tyrosine protein kinase has a suppressive role in the neurite formation induced by GDNF and that Ras is necessary for the signaling initiated by GDNF.

Enzyme Inhibitors↗
PubMed1997

Nitric oxide mediates murine cytomegalovirus-associated pneumonitis in lungs that are free of the virus.

4 wk after intraperitoneal inoculation of 0.2 LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable in the salivary glands, but not in the lungs or other organs. When the T cells of these mice were activated in vivo by a single injection of anti-CD3 monoclonal antibody, interstitial pneumonitis was induced in the lungs that were free of the virus with an excessive production of the cytokines. In the lungs of such mice persistently infected with MCMV, the mRNA of the cytokines such as IL-2, IL-6, TNF-alpha, and IFN-gamma were abundantly expressed 3 h after the anti-CD3 injection, and the elevated levels continued thereafter. A marked expression of inducible nitric oxide synthetase (iNOS) was then noted in the lungs, suggesting that such cytokines as TNF-alpha and IFN-gamma may have induced iNOS. Although the increase in NO formation was demonstrated by the significant elevation of the serum levels of nitrite and nitrate, the interstitial pneumonitis was not associated with either increased superoxide formation or peroxynitrite-induced tyrosine nitration. Nevertheless, the administration of an NO antagonist also alleviated the interstitial pneumonitis provoked by anti-CD3 mAb. Based on these findings, it was concluded that MCMV-associated pneumonitis is mediated by a molecule of cytokine-induced NO other than peroxynitrite.

Animals↗
PubMed1997

Enhancement or induction of neurite formation by a protein tyrosine phosphatase inhibitor, 3,4-dephostatin, in growth factor-treated PC12h cells.

We studied the effect of the 3,4-dihydroxy analogue of dephostatin (3,4-dephostatin), an inhibitor of protein-tyrosine phosphatase (PTPase), on the differentiation of rat pheochromocytoma PC12 cells. 3,4-Dephostatin accelerated NGF-induced neurite formation in PC12h cells, a subline of PC12 cells, whereas the inhibitor alone did not induce neurite formation. It sustained the NGF-induced tyrosine phosphorylation of several proteins, most prominently that of mitogen-activated protein (MAP) kinase. EGF alone did not induce differentiation in PC12h cells, but it induced neurite formation in the presence of 3,4-dephostatin. The inhibitor also prolonged EGF-induced tyrosine phosphorylation and activation of MAP kinase. An inactive analogue of dephostatin, 2'-O-methyl-dephostatin showed no effect on either neurite formation or MAP kinase tyrosine phosphorylation in NGF or EGF-treated PC12h cells. Thus, we demonstrated that the PTPase inhibitor could enhance growth factor-induced differentiation in PC12 cells possibly by sustaining the MAP kinase activity.

Animals↗
PubMed1997

Induction of cellular differentiation and apoptosis by signal transduction inhibitors.

We have isolated signal transduction inhibitors of low molecular weight from microorganisms and plants. Since inducers of differentiation and apoptosis may be developed as new anticancer agents, we have studied induction of differentiation and apoptosis in neoplastic cells by our signal transduction inhibitors. Aristeromycin isolated as an Abl function inhibitor induced erythroid differentiation in human CML K562 cells. Aristeromycin may induce differentiation by inhibition of methylating reactions in the cell. We isolated dephostatin from Streptomyces as a tyrosine phosphatase inhibitor, and synthesized its stable analogue, 3,4-dephostatin. The stable analogue, 3,4-dephostatin, potentiated NGF-induced morphological differentiation in rat pheochromocytoma PC12h cells, possibly by inhibition of tyrosine dephosphorylation of MAPK. Erbstatin, a tyrosine kinase inhibitor, induced morphological apoptosis and internucleosomal DNA fragmentation in mouse leukemia L1210 and human SCLC cells. Erbstatin was shown to induce apoptosis by hydrogen peroxide formation. Thus, these signal transduction inhibitors appear to be useful tools for the mechanistic study of cellular differentiation and apoptosis.

Adenosine↗
PubMed1997

Hirudin suppresses the invasion of inflammatory cells and the appearance of vimentin-positive astrocytes in the rat cerebral ablation model.

Hirudin is a specific and direct-acting thrombin inhibitor superior to heparin as an anticoagulant. Thrombin is a multifunctional molecule that acts as a serine protease locally generated from prothrombin during blood coagulation related to injury and/or inflammation. We previously reported that thrombin might be involved in the inflammatory response, glial reaction, and scar formation that occurred in central nervous system (CNS). Here we studied the suppressive effects of hirudin on the inflammation, vimentin-positive astrocytes, and glial fibrillary acidic protein (GFAP)-positive astrocytes using rat cerebral ablation models. Hirudin and vehicle solution soaked in Gelform were administered to the cavity of the traumatic brain defect. Brains were examined by conventional histologic and immunohistologic technique. Antibodies for monocytes/macrophages, GFAP, and vimentin were used to assess the infiltration of inflammatory cells and reaction of astrocytes. The number of the inflammatory cells, vimentin-positive astrocytes, and GFAP-positive astrocytes were quantitatively analyzed. Hirudin suppressed the infiltration of inflammatory cells and the increase in vimentin-positive astrocytes, but had no effects on the increase in GFAP-positive astrocytes. These data suggest that thrombin may play an important role in inflammatory and glial responses to CNS injury, and that hirudin can be a candidate for the therapeutic agent that minimizes the secondary brain damage following the inflammation, and the glial reaction mediated by vimentin-positive astrocytes near the lesion site.

Animals↗
PubMed1997

Induction of nitric oxide synthesis and xanthine oxidase and their roles in the antimicrobial mechanism against Salmonella typhimurium infection in mice.

The role of superoxide anion (O2-) and nitric oxide (NO) in the host defense mechanism against Salmonella typhimurium (LT-2) was examined by focusing on xanthine oxidase (XO) as an O2(-)-generating system and on inducible NO synthase (iNOS). When ICR mice were infected with a 0.1 50% lethal dose (2 x 10(5) CFU) of S. typhimurium, bacterial growth in the liver reached a peak value 3 days after infection (10(4.32) CFU/g of liver) and decreased thereafter. XO activity in the liver became maximum at 7 days after infection; the value was 34.6 +/- 1.4 mU/g of liver at 7 days (compared with 11.0 +/- 1.3 mU/g of liver before infection). The time profile of NO production in the liver as determined by electron spin resonance spectroscopy was consistent with that of XO activity. Histological examination of infected liver showed the formation of multiple microabscesses with granulomatous lesions consisting of polymorphonuclear cells and mononuclear cells, and iNOS-expressing cells were localized in the confined areas of the microabscesses. When XO inhibitors such as allopurinol and 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) were administered to the infected mice, the mortality of the mice was significantly increased (10 of 21 and 11 of 20 for the allopurinol- and AHPP-treated groups, respectively, versus 2 of 20 for control mice), and bacterial growth was significantly enhanced. A similar exacerbation of the infection was obtained with N(omega)-monomethyl-L-arginine (L-NMMA) treatment of the mice. Of considerable importance is that granuloma formation in the liver was poorly developed by treatment with either XO inhibitors or L-NMMA. These results suggest that XO and NO play an important role in the antimicrobial mechanism against S. typhimurium in mice.

Allopurinol↗
PubMed1997

Polymorphonuclear cells in chronic hemodialysis patients have intact phagocytotic and impaired bactericidal activities.

Although it has been well documented that chronic hemodialysis (HD) patients are highly susceptible to infectious diseases, the reasons for this have yet to be clarified. The present study was thus designed in order to better define this issue. Fifty-eight stable chronic HD patients without any evidence of infection were selected for the study. Blood samples were collected before and after HD from the same patient to determine the effect of HD. Reactive oxygen species (ROS) production in polymorphonuclear cells (PMNC) was measured by chemiluminescence using luminol. When the PMNC collected after HD were stimulated in vitro with a calcium ionophore (A23187), they produced a larger amount of ROS than that obtained from healthy volunteers [mean 7.4 x 10(5) photon counts (n = 58) vs. 3.0 x 10(5) photon counts (n = 17); p < 0.01]. A higher production of ROS after HD was seen in patients using membranes such as cellulose triacetate, polymethylmetacrylate and cellulose diacetate, whereas cuprophane did not seem to augment ROS production at all. On the other hand, when the PMNC after HD were stimulated with phorbol myristate acetate, their photon counts (mean 4.3 x 10(7)) were comparable to those before HD (mean 3.5 x 10(7)), and to those of PBMC obtained from healthy volunteers (mean 4.1 x 10(7)). It was thus suggested that the enhanced ROS production of PMNC was related to some stimuli, possibly even to the assay used to measure ROS. The phagocytotic activity and bactericidal effect of PBMC were measured by coculturing 1 x 10(5) PMNC with 1 x 10(5) CFU of Escherichia coli. Similar phagocytotic activities were noted in the PMNC from healthy volunteers and chronic HD patients before and after HD: the mean number of phagocytosed bacteria (log10 CFU) was 3.3, 3.3, and 3.3, respectively. However, in the case of a bactericidal effect, only the PMNC from healthy volunteers, but not the PMNC from HD patients, could effectively kill the bacteria, since the number of bacteria in PMNC decreased from 10(3.3) to 10(2.1). The PMNC from HD patients could not kill the bacteria-regardless of the characteristics of the membranes. It was thus concluded that the PMNC of chronic HD patients possess an intact phagocytotic activity which impaired bacterial killing, and was probably due to an abnormality occurring in the ROS production pathway.

Adult↗
PubMed1997

[Molecular design of apoptosis inducing agents derived from bleomycin].

Reactive oxygen species (ROS) are often involved in the mechanism of apoptosis. Bleomycin consists of an oxygen activating domain and a DNA binding domain, and is an antitumour agent that induces double-strand scission in DNA by oxygen activation. However, bleomycin only weakly induces apoptosis in limited conditions. Previously we have reported efficient oxygen activation by iron complexes of synthetic models of bleomycin namely PYML designed by the direct analogy to the BLM metal core. Recently, novel ligands having symmetrized coordination environment consisting of two histidine units and a pyridine (HPH) were prepared. Oxygen activating efficiency of the iron complexes of the synthetic ligands increased by introducing electron donating substituent into the pyridine ring. HPH compounds have no DNA binding region which is present in bleomycin. HPH compounds but not bleomycin induced apoptosis in mouse leukemia L1210 cells.

Animals↗
PubMed1997

[Screening of phosphatidylinositol turnover inhibitors and regulation of cell cycle progression].

Phosphatidylinositol (PI) turnover is considered to be involved in the regulation of cell growth. The enzymes for PI turnover include phospholipase C (PLC), PI4-kinase and PI synthase. We have isolated pholipeptin and fluvirucin B2 from microorganisms and akaterpin from a marine sponge as PLC gamma inhibitors. We also isolated echiguanines from Streptomyces as PI4-kinase inhibitors. Since echiguanines did not inhibit the enzyme in situ, we synthesized their ribosylated derivatives that were effective in cultured cells. We previously isolated inostamycin from Streptomyces as an inhibitor of PI synthase. We found that inostamycin induced G1 block in cycling NRK cells. Inostamycin inhibited the serum-induced S-phase induction in quiescent NRK cells. Inostamycin was found to decrease serum-induced expression of cyclin D and cyclin E, without inhibiting the activation of MAP kinase. It also inhibited serum-induced activation of CDK2 and phosphorylation of pRB. Thus, PI synthesis was suggested to be involved in regulation of serum-induced S-phase induction by modulating G1 cyclin expression.

Animals↗
PubMed1997

Induction of flat morphology in K-ras-transformed fibroblasts by lycorine, an alkaloid isolated from the tropical plant Eucharis grandiflora.

In the course of screening for Ras function inhibitors from plant extracts, we isolated lycorine from a chloroform extract of Eucharis grandiflora leaves. Lycorine induced flat morphology in K-ras-NRK cells after treatment for 2-3 days, whereas its morphological effect on NRK cells was weaker. Lycorine was found to inhibit protein synthesis specifically in cultured K-ras-NRK cells. It also lowered the cellular amount of Ras in 2-3 days.

Alkaloids↗
PubMed1997

Preferential inhibition of glioblastoma cells with wild-type epidermal growth factor receptors by a novel tyrosine kinase inhibitor ethyl-2,5-dihydroxycinnamate.

Epidermal growth factor receptor (EGFR) gene overexpression and mutations play an important role in the pathogenesis of a variety of malignant human cancers. In this study, we tested the effects of a novel EGFR tyrosine kinase inhibitor, ethyl-2,5-dihydroxycinnamate (EtDHC), against related human glioblastoma cell lines expressing specific forms of EGFR gene mutations. EtDHC more potently inhibited cell growth and DNA synthesis in glioblastoma cells with endogenous or overexpressed wild-type EGFR compared with those with truncated EGFR, by preferentially inhibiting the tyrosine kinase activity and autophosphorylation of the wild-type EGFR. Higher concentrations of EtDHC were required to inhibit cells expressing the truncated EGFR. These findings are the reverse of another highly specific tyrosine kinase inhibitor, tyrphostin AG 1478, which preferentially inhibited glioblastoma cells with truncated EGFR compared with those with wild-type EGFR. The differential susceptibility of various glioblastoma cells to highly specific tyrosine kinase inhibitors is significant because human gliomas are composed of heterogeneous cells with subsets of cells expressing specific gene mutations. This cellular heterogeneity could be one of the reasons why tumor cells are resistant to chemotherapy. Thus, EtDHC, especially when in combination with drugs targeting other specific gene mutations (such as tyrphostin AG 1478), holds a significant potential for chemotherapy for human glioblastomas.

Cell Division↗