Biomedical subjects

Takashi Tsuruo

Publications and source records attributed to Takashi Tsuruo.

At least 19 recordsLinked to original sources

PubMed2004

Aggrus: a diagnostic marker that distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors.

Aggrus (also known as T1alpha/podoplanin) is a membrane sialoglycoprotein whose function in tumors is unknown. We recently determined that Aggrus possessed the ability of inducing platelet aggregation and that its expression was frequently upregulated in colorectal tumors. Thus, Aggrus expression might be associated with tumor-induced platelet aggregation and tumor metastasis. Here we show, by means of cancer profiling array and real-time PCR, that aggrus mRNA expression is frequently upregulated in testicular germ cell tumors when compared with the surrounding normal tissue. Immunohistochemical staining revealed that Aggrus protein expression was detected in 10 of 11 seminomas (90.9%), but its expression was not observed in embryonal carcinomas (0/4; 0%). Specific markers for seminomas have not been reported, and Aggrus is a potential diagnostic marker for seminomas and may be associated with malignancies of the testis.

Animals↗

PubMed2004

Transport of somatostatin and substance P by human P-glycoprotein.

P-glycoprotein is an efflux pump for a broad spectrum of hydrophobic agents. We found that bioactive peptides including somatostatin and substance P inhibit ATP-dependent vincristine binding to P-glycoprotein-overexpressing K562/ADM membrane vesicles. Some of these bioactive peptides including somatostatin stimulate basal ATPase activity of P-glycoprotein; in contrast, other peptides including substance P inhibit it. The K562/ADM membrane vesicles showed an ATP-dependent, osmotically sensitive uptake of somatostatin and substance P, which was inhibited by valspodar, an inhibitor of P-glycoprotein. These findings suggested that certain bioactive peptides such as somatostatin and substance P directly interact with human P-glycoprotein as endogenous substrates for P-glycoprotein-mediated transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Protein tyrosine phosphatase receptor-type O (PTPRO) exhibits characteristics of a candidate tumor suppressor in human lung cancer.

Previous study in our laboratory demonstrated suppression of the gene for protein tyrosine phosphatase receptor-type O (PTPRO) in primary and established rat hepatomas. The present study showed methylation-mediated silencing of this gene in primary human lung tumors and in several human lung cancer cell lines, one of the characteristics of many tumor-suppressor genes. The reduced expression of PTPRO in the primary lung tumors correlated with the methylation status of its CpG island. Demethylation of the gene by deoxy-5-azacytidine treatment led to its reactivation in a lung cancer line (A549). Overexpression of PTPRO in A549 cells inhibited anchorage-independent growth, delayed reentry of the cells into the cell cycle after release from cell-cycle arrest, and increased susceptibility of the cells to apoptosis. These data have demonstrated the growth-suppressor characteristics of PTPRO that are unique to a classical tumor suppressor.

Base Sequence↗

PubMed2004

Effect on tumor cells of blocking survival response to glucose deprivation.

BACKGROUND: Glucose deprivation, a feature of poorly vascularized solid tumors, activates the unfolded protein response (UPR), a stress-signaling pathway, in tumor cells. We recently isolated a novel macrocyclic compound, versipelostatin (VST), that inhibits transcription from the promoter of GRP78, a gene that is activated as part of the UPR. We examined the effect of VST on the UPR induced by glucose deprivation or other stressors and on tumor growth in vivo. METHODS: Human colon cancer HT-29, fibrosarcoma HT1080, and stomach cancer MKN74 cells were cultured in the absence of glucose or in the presence of glucose and a UPR-inducing chemical stressor (the N-glycosylation inhibitor tunicamycin, the calcium ionophore A23187, or the hypoglycemia-mimicking agent 2-deoxyglucose [2DG]). The effect of VST on UPR induction was determined by reverse transcription-polymerase chain reaction and immunoblot analysis of the UPR target genes GRP78 and GRP94; by immunoblot analysis of the UPR transcriptional activators ATF6, XBP1, and ATF4; and by analyzing reporter gene expression in cells transiently transfected with a GRP78 promoter-reporter gene. Cell sensitivity to VST was examined with a colony formation assay and flow cytometry. In vivo antitumor activity of VST was assessed with an MKN74 xenograft model. RESULTS: VST inhibited expression of UPR target genes in glucose-deprived or 2DG-treated cells but not in cells treated with tunicamycin or A23187. VST also inhibited the production of the UPR transcriptional activators XBP1 and ATF4 during glucose deprivation. The UPR-inhibitory action of VST was seen only in conditions of glucose deprivation and caused selective and massive killing of the glucose-deprived cells. VST alone and in combination with cisplatin statistically significantly (P =.004 and P<.001 for comparisons with untreated control, respectively) inhibited tumor growth of MKN74 xenografts. CONCLUSION: Disruption of the UPR may provide a novel therapeutic approach to targeting glucose-deprived solid tumors.

Activating Transcription Factor 4↗

PubMed2004

Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function.

Apollon (also known as BRUCE or BIRC6) is a large protein containing baculoviral-IAP-repeat (BIR) and ubiquitin-conjugating enzyme (UBC) domains at the amino- and carboxy termini, respectively. Apollon inhibits apoptosis, but its molecular and physiological function remains unclear. Here we report that Apollon binds to, ubiquitinates and facilitates proteasomal degradation of SMAC and caspase-9, which both contain IAP-binding motifs. Targeted disruption of Apollon in mice caused embryonic and neonatal lethality. Notably, SMAC induced apoptosis in Apollon-deficient cells, but not in Apollon-expressing cells. Furthermore, the IAP-binding motif of SMAC was required to induce apoptosis in Apollon-deficient cells. These results suggest that Apollon has an essential function in preventing SMAC-induced apoptosis.

Animals↗

PubMed2004

Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms.

We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominant-negative mutants) or beta1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants. Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on beta1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on beta1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation state-specific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.

Anoikis↗

PubMed2004

Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin) molecules expressed in Chinese hamster ovary cells.

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.

Animals↗

PubMed2004

Involvement of 3-phosphoinositide-dependent protein kinase-1 in the MEK/MAPK signal transduction pathway.

The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2004

Interaction between glucose-regulated destruction domain of DNA topoisomerase IIalpha and MPN domain of Jab1/CSN5.

DNA topoisomerase (topo) IIalpha, an essential enzyme for cell proliferation, is targeted to a proteasome-dependent degradation pathway when human tumor cells are glucose-starved. Here we show that the topo IIalpha destabilization depends on the newly identified domain, GRDD (glucose-regulated destruction domain), which was mapped to the N-terminal 70-170 amino acid sequence. Indeed, the deletion of GRDD conferred a stable feature on topo IIalpha, whereas the fusion of GRDD rendered green fluorescent protein unstable under glucose starvation conditions. Nuclear localization was a prerequisite for GRDD function, because the inhibition of nuclear translocation resulted in the suppression of GRDD-mediated topo IIalpha degradation. Further, GRDD was identified as an interactive domain for Jab1/CSN5, which promoted the degradation of topo IIalpha in a manner dependent on the MPN (Mpr1p/Prd1p N terminus) domain. Depleting Jab1/CSN5 by antisense oligonucleotide and treating cells with the CSN-associated kinase inhibitor, curcumin, inhibited topo IIalpha degradation induced by glucose starvation. These findings demonstrate that GRDD can act as a stress-activated degron for regulating topo IIalpha stability, possibly through interaction with the MPN domain of Jab1/CSN5.

Adenosine Triphosphatases↗

PubMed2004

Hypoxic up-regulation of triosephosphate isomerase expression in mouse brain capillary endothelial cells.

A protein with a molecular mass of 27kDa was induced by hypoxia in a mouse brain capillary endothelial cell line and identified as triosephosphate isomerase (TPI) by amino-terminal sequencing. Hypoxia caused an elevation of the TPI protein level, concomitant with an increase of the TPI mRNA level. However, hypoxia resulted in an insufficient elevation of TPI activity level, compared to an increase of TPI protein level. When cells expressing the recombinant TPI protein with histidine tag were exposed to hypoxia and the TPI protein was affinity-purified, the catalytic activity (specific activity) of the TPI protein purified from hypoxic cells was substantially lower than that obtained from normoxic cells. In addition, three TPI isoforms with an electrophoretic multiplicity were found; two of the three isoforms were substantially increased in response to the hypoxia, but the level of the most acidic isoform was barely changed. The induction of TPI gene expression by hypoxia was suppressed by (1) a chelator of intracellular Ca(2+), (2) a blocker of non-selective cation channels, (3) a blocker of Na(+)/Ca(2+) exchangers, (4) an inhibitor of Ca(2+)/calmodulin-dependent protein kinases, and (5) an inhibitor of c-jun/AP-1 activation.

Animals↗

PubMed2004

Reconstitution of caspase-3 confers low glucose-enhanced tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity and Akt cleavage.

PURPOSE AND EXPERIMENTAL DESIGN: We have previously observed that glucose deprivation enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death as well as caspase activation (caspase-3, -9, and -8) in human prostate adenocarcinoma DU-145 cells. In this study, we used caspase-3-deficient MCF-7 breast cancer cells to examine the possible role of caspase-3 in glucose deprivation-enhanced TRAIL cytotoxicity. RESULTS: Combined glucose deprivation and 200 ng/ml TRAIL treatment markedly induced cytotoxicity in caspase-3 cDNA transfected cells (MCF-7/casp-3) but not in control vector transfected cells (MCF-7/vector). We also observed that the level of Akt, an antiapoptotic protein, was reduced by treatment with TRAIL in MCF-7/casp-3 cells but not in MCF-7/vector cells. The reduction of Akt by TRAIL was promoted in the absence of glucose in MCF-7/casp-3 cells. However, pretreatment with 20 micro M Z-LEHD-FMK, a caspase-9 inhibitor, protected MCF-7/casp-3 cells from the combinatorial treatment of TRAIL and glucose deprivation-induced cytotoxicity. This compound also prevented the reduction of Akt level during the combinatorial treatment. Moreover, this Akt reduction was not inhibited by treatment with MG-132, a proteosome inhibitor. Data from site-directed mutagenesis show that Akt was cleaved at amino acid 108, but not 119, during treatment with TRAIL and glucose deprivation. CONCLUSIONS: Our results suggest that caspase-3 is involved in the reduction of Akt level, and its involvement is mediated through caspase-9 activation. The reduction of Akt level is also due to cleavage of Akt rather than degradation of Akt.

Breast Neoplasms↗

PubMed2004

Alteration in copy numbers of genes as a mechanism for acquired drug resistance.

Chemoresistance is a major obstacle for successful treatment of cancer. To identify regions of the genome associated with acquired resistance to therapeutic drugs, we conducted molecular cytogenetic analyses of 23 cancer-cell lines, each resistant to either camptothecin, cisplatin, etoposide (VP-16), Adriamycin, or 1-beta-D-arabinofuranosylcytosine, although the parental tumor lines were not. Subtractive comparative genomic hybridization studies revealed regions of gain or loss in DNA-copy numbers that were characteristic of drug-resistant cell lines; i.e., differences from their drug-sensitive parental cell lines. Thirteen ATP-binding cassette (ABC) transporter genes [ABCA3, ABCB1 (MDR1), ABCB6, ABCB8, ABCB10, ABCB11, ABCC1 (MRP1), ABCC4, ABCC9, ABCD3, ABCD4, ABCE1, and ABCF2] were amplified among 19 of the resistant cell lines examined. Three genes encoding antiapoptotic BCL-2 proteins (BCL2L2, MCL1, and BCL2L10) were also amplified and consequently overexpressed in three of the derivative lines. Down-regulation of BCL2L2 with an antisense oligonucleotide sensitized a VP-16 resistant ovarian-cancer cell line (SKOV3/VP) to VP-16. A decrease in copy numbers of genes encoding deoxycytidine kinase, DNA topoisomerase I, and DNA topoisomerase II alpha reduced their expression levels in one cytosine arabinoside-resistant line, two of three camptothecin-resistant lines, and two of five VP-16-resistant cell lines, respectively. Our results indicated that changes in DNA-copy numbers of the genes mentioned can activate or down-regulate them in drug-resistant cell lines, and that such genomic alterations might be implicated in acquired chemoresistance.

ATP-Binding Cassette Transporters↗

PubMed2004

Cullin 3 promotes proteasomal degradation of the topoisomerase I-DNA covalent complex.

DNA topoisomerase I (TOP1)-DNA covalent complexes are the initial lesions produced by antitumor camptothecins (CPTs). The TOP1-directed drugs stimulate degradation of TOP1 via the ubiquitin-proteasome pathway. We found that proteasome inhibition prevents degradation of DNA-bound TOP1 and sustains high levels of covalent complexes, thus enhancing CPT-induced cell death. Consistent with this, increased degradation of TOP1-DNA covalent complexes was seen in acquired CPT-resistant cells. We found that the resistant cells showed elevated expressions of Cul3, a member of the cullin family of E3 ubiquitin ligases. The reduction in Cul3 expression by small interfering RNA decreased degradation of TOP1-DNA covalent complexes. Conversely, Cul3 overexpression by stable transfection promoted covalent complex degradation and reduced CPT-induced cell death without affecting basal TOP1 expression levels. These results indicate that Cul3, by promoting proteasomal degradation of TOP1-DNA covalent complexes, becomes an important regulator for cellular CPT sensitivity.

Camptothecin↗

PubMed2004

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways.

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model. Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells. Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied. For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed. These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs.

Apoptosis↗

PubMed2004

Pharmacokinetic characterization of dehydroevodiamine in the rat brain.

The objective of this study was to examine the kinetics of the distribution of dehydroevodiamine (DHED) in the rat brain. After an intravenous infusion of 15 min (dose of 1-10 mg/kg), the temporal profiles of the plasma levels of DHED declined in a multiexponential manner. Moment analysis indicated that the clearance and steady-state volume of distribution for DHED were not statistically different with the dose, indicating that the pharmacokinetics for DHED is linear in the range examined. Nonlinear regression analysis of DHED concentrations in the plasma and the brain revealed that the linear kinetics into and out from the brain reasonably described the data and that the clearances for influx into and efflux from the brain were comparable. Transport clearances for DHED across MBEC4 monolayers, an in vitro model of the blood-brain barrier, were also comparable for influx and efflux, and were independent of the medium concentration. The concentration of DHED in cerebrospinal fluid was negligible compared with that found in plasma, indicating that the drug is not primarily distributed to the brain via the blood-cerebrospinal fluid barrier. These observations indicate that DHED is transported from the systemic circulation to the brain via the blood-brain barrier by linear kinetics.

Algorithms↗

PubMed2004

Production of biologically active taxoids by a callus culture of Taxus cuspidata.

Ten known taxoids, paclitaxel, 7-epi-taxol, taxol C, baccatin VI, taxayuntin C, taxuyunnanine C and its analogues (2-5), and yunnanxane (6), and an abietane, taxamairin A, were produced in the callus culture of Taxus cuspidata cultivated on a modified Gamborg's B5 medium in the presence of 0.5 mg/L NAA. After stimulation with 100 microM methyl jasmonate, five more taxoids, cephalomannine, 1beta-dehydroxybaccatin VI, taxinine NN-11 (1), baccatin I, and 2alpha-acetoxytaxusin, and one more abietane, taxamairin C, were found in addition to the above-mentioned compounds. It was also observed that the content of the products increased over three times. Taxinine NN-11 (1) is a new taxane whose structure was assigned as 5alpha,13alpha-diacetoxy-9alpha-cinnamoyloxy-4(20),11-taxadien-10beta-ol by analysis of its spectral data. Taxinine NN-11 (1) exhibited significant MDR reversal activity toward 2780 AD tumor cells. The results of primary screening based on 39 human cancer cell lines suggest that 1 also belongs to a new mechanistic class. Efficient production of 1 was investigated using the callus culture of T. cuspidata.

Antineoplastic Agents, Phytogenic↗

PubMed2004

Uptake and efflux of quinacrine, a candidate for the treatment of prion diseases, at the blood-brain barrier.

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Transforming growth factor-beta1 upregulates the tight junction and P-glycoprotein of brain microvascular endothelial cells.

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.

ATP Binding Cassette Transporter, Subfamily B, Mem↗