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Biomedical subjects

Takashi Tsuruo

Publications and source records attributed to Takashi Tsuruo.

At least 19 recordsLinked to original sources
PubMed2003

Survival-signaling pathway as a promising target for cancer chemotherapy.

The serine/threonine kinase AKT, also known as PKB or RAC-PK, is a key molecule for protecting cells from undergoing apoptosis. Several studies have suggested that the AKT-mediated survival-signaling pathway is an attractive target for cancer chemotherapy: (1) the AKT pathway is relatively inactive in resting cells; (2) amplification of the AKT gene occurs in some tumors; (3) loss of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) is common in tumors and its loss constitutively activates AKT; (4) AKT is activated at the cancer invasion front. To clarify which drugs exhibit their cytotoxicity by inhibiting the AKT pathway, we screened anticancer drugs that could downregulate phospho-AKT levels and AKT kinase activity. We found that UCN-01 (7-hydroxystaurosporine), heat-shock protein 90 (HSP90) inhibitors, and topotecan (10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) possessed the ability to interfere with the AKT pathway. UCN-01 directly suppressed upstream AKT kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) (IC(50) <33 nM) both in vitro and in tumor xenografts. HSP90 inhibitors and topotecan suppressed AKT activity via indirectly downregulating PDK1 and phosphatidylinositide-3-OH kinase activities. Transfection of the constitutively active AKT complementary DNA into cells attenuated the cytotoxic effects of the drugs, indicating that inhibition of the AKT pathway plays an important role in exerting their cytotoxic effects. These results strongly suggest that the AKT-mediated survival-signaling pathway is a promising and attractive target for cancer chemotherapy.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2003

Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow.

Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.

Animals↗
PubMed2003

Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: involvement of Na+/Ca2+ exchanger.

The molecular regulatory mechanisms and the characterization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hypoxia were studied in a mouse brain capillary endothelial cell line, MBEC4. Activation of GAPDH gene expression by hypoxia was suppressed by an intracellular Ca(2+) chelator and inhibited by a non-selective cation channel blocker or a Na(+)/Ca(2+) exchanger (NCX) blocker. Sequencing of reverse transcription-PCR products demonstrated that MBEC4 expressed an mRNA encoding NCX3, which functions even under cellular ATP-depleted conditions, in addition to mRNAs encoding NCX1 and NCX2. The inhibition of Ca(2+)/calmodulin-dependent protein kinases or c-Jun/AP-1 activation caused a significant decrease in the activation of GAPDH mRNA by hypoxia. These results suggest that hypoxia stimulates Ca(2+) influx through non-selective cation channels and causes the reverse operation of the three NCX isoforms, and consequently, increased intracellular Ca(2+) up-regulates GAPDH gene expression through an AP-1-dependent pathway. Furthermore, subcellular fractionation experiments showed that hypoxia increased GAPDH proteins not only in the cytosolic fraction, but also in the nuclear and particulate fractions, in which GAPDH should play no roles in glycolysis. However, the GAPDH activity did not rise in proportion to the increase of GAPDH protein by hypoxia even in the cytosolic fraction. These results suggest that not all hypoxia-induced GAPDH molecules contribute to glycolysis.

Animals↗
PubMed2003

Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in vitro and in vivo.

Animal and epidemiological studies reveal that consuming food and beverages rich in polyphenols (e.g., catechins, flavones, and antocyanines) is associated with a lower incidence of cancer, and several molecular mechanisms have been proposed for explaining this effect. However, because most of these mechanisms were observed only under specific and nonphysiological conditions, and in most cases, with practically irrelevant concentrations, there is still no clear-cut or universal explanation for the major events that underlie the anticancer effects of polyphenols. In this study we present clear in vitro and in vivo evidence that the inhibition of the cancer-associated enzyme telomerase is a key mechanism involved in cancer inhibition by epigallocatechin gallate (EGCG), a major tea polyphenol. We demonstrate that EGCG and other selected polyphenols undergo structural rearrangements at physiologically permissible conditions that result in remarkably increased telomerase inhibition. In nude mice models bearing both telomerase-dependent and -independent xenograft tumors cloned from a single human cancer progeny, only the telomerase-dependent tumors responded to prolonged oral administration of EGCG. Thus, EGCG and likely other structurally related dietary polyphenols seem to act as prodrug-like molecules that, once ingested and distributed, undergo structural changes that favor potent activity against telomerase.

Animals↗
PubMed2003

Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide.

The inhibitor of apoptosis proteins (IAPs) plays a central role in repressing caspase-mediated cell death. However, little is known about the actual role of endogenously expressed IAPs in cancer cells. We found that the cytochrome c/apoptotic protease-activating factor-1 (apoptosome)-dependent caspase activation is deficient in human non-small cell lung cancer (NSCLC) NCI-H460 cells. This dysfunctional apoptosome activity was not correlated with any decrease of apoptosome component factors, but it was linked to an increased X-linked inhibitor of apoptosis protein (XIAP). In H460 cells, the overexpressed XIAP, but not c-IAP1, bound to the processed form of caspase-9 and suppressed the activation of downstream effector caspases. Moreover, the defect in apoptosome activity in H460 cells was dramatically restored by the IAP-targeting SmacN7 peptide, which disrupted XIAP-caspase-9 binding, indicating an essential role of the IAP in the apoptosome inhibition. However, the SmacN7 did not show any striking effect on the apoptosome activity of normal lung fibroblast cells, although these cells also expressed modest amounts of IAP. To explore the therapeutic approach, we additionally developed SmacN7(R)8, a newly designed cell permeable peptide. The SmacN7(R)8 selectively reversed the apoptosis resistance of H460 cells, and when in combination with chemotherapy, regressed the tumor growth in vivo with little toxicity to the mice. Our results indicate that IAP-dependent suppression of apoptosome predominantly occurs in IAP-overexpressing tumor, and the IAP-targeting Smac peptide is an effective molecule to increase tumor cell death induced by chemotherapy in vitro and in vivo.

Animals↗
PubMed2003

Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.

BACKGROUND: Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells. METHODS: A bicistronic retrovirus vector, Ha-MDR-IRES-gp91, was constructed for the co-expression of MDR1 and gp91, a gene responsible for X-linked chronic granulomatous disease (X-CGD). Drug-selected co-expression of P-glycoprotein and gp91 was evaluated in transduced cells. RESULTS: Epstein-Barr virus-transformed B cells from X-CGD patients transduced with Ha-MDR-IRES-gp91 co-expressed human P-glycoprotein and gp91, and acquired superoxide-generating activity. Human CD34-positive cells from an X-CGD patient were transduced with Ha-MDR-IRES-gp91 and subsequently treated with 2 ng/ml vincristine. After 13 days, 20% of Ha-MDR-IRES-gp91-transduced cells were P-glycoprotein- and gp91-positive by FACS analysis. The superoxide-generating activity of the transduced population was 27% of that of normal cells. Mice transplanted with Ha-MDR-IRES-gp91-transduced bone marrow cells showed co-expression of P-glycoprotein and gp91 in peripheral blood mononuclear cells. By administering paclitaxel, the proportions of P-glycoprotein- and gp91-positive cells were increased in all the four mice examined. When mice transplanted with Ha-MDR-IRES-gp91-transduced cells were repeatedly administered paclitaxel, the ratios of P-glycoprotein- and gp91-positive cells were maintained for over 1 year. CONCLUSIONS: These results suggest that MDR1-bicistronic vectors may be useful to select the transduced hematopoietic cells in vivo. This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal.

Recent progress in the development of molecular cancer therapeutics has revealed new types of antitumor drugs, such as Herceptin, Gleevec, and Iressa, as potent therapeutics for specific tumors. Our work has focused on molecular cancer therapeutics, mainly in the areas of drug resistance, apoptosis and apoptosis resistance, and survival-signaling, which is related to drug resistance. In this review, we describe our research on molecular cancer therapeutics, including molecular mechanisms and therapeutic approaches. Resistance to chemotherapeutic drugs is a principal problem in the treatment of cancer. P-Glycoprotein (P-gp), encoded by the MDR1 gene, is a multidrug transporter and has a major role in multidrug resistance (MDR). Targeting of P-gp by small-molecular compounds and/or antibodies is an effective strategy to overcome MDR in cancer, especially hematologic malignancies. Several P-gp inhibitors have been developed and are currently under clinical phased studies. In addition to the multidrug transporter proteins, cancer cells have several drug resistance mechanisms. Solid tumors are often placed under stress conditions, such as glucose starvation and hypoxia. These conditions result in topo II poison resistance that is due to proteasome-mediated degradation of DNA topoisomerases. Proteasome inhibitors effectively prevent this stress-induced drug resistance. Glyoxalase I, which is often elevated in drug- and apoptosis-resistant cancers, offers another possibility for overcoming drug resistance. It plays a role in detoxification of methylglioxal, a side product of glycolysis, which is highly reactive with DNA and proteins. Inhibitors of glyoxalase I selectively kill drug-resistant tumors that express glyoxalase I. Finally, the susceptibility of tumor cells to apoptosis induced by antitumor drugs appears to depend on the balance between pro-apoptotic and survival (anti-apoptotic) signals. PI3K-Akt is an important survival signal pathway, that has been shown to be the target of various antitumor drugs, including UCN-01 and geldanamycin, new anticancer drugs under clinical evaluation. Our present studies provide novel targets for future effective molecular cancer therapeutics.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2003

Molecular cancer therapeutics: recent progress and targets in drug resistance.

Recent progress in development of molecular cancer therapeutics revealed new types of antitumor drugs, such as Herceptin, Gleevec, and Iressa as potent therapeutics for each specific tumor. We have been working on molecular cancer therapeutics, and in particular, those related to drug resistance, Here, I describe several resistance mechanisms, including apoptosis regulation, cellular stress response and cellular survival signals which have show close relevance to drug resistance. P-glycoprotein (P-gp) is the key molecule in multidrug resistance (MDR) and a good target for chemotherapy. Proteasome is involved in the resistance mechanism to topo II-targeted chemotherapy in solid tumors. Apoptosis program in tumor cells plays a critical role in chemotherapy-induced tumor cell killing, and the blockade of the apoptosis-inducing pathway could be another mechanism for drug resistance. Glyoxalase I is a molecule involved in apoptosis resistance mechanism in tumors. Survival (antiapoptosis) signals are the good targets for various antitumor drugs to overcome innate drug resistance. Our present studies provide novel targets for effective molecular cancer therapeutics in future.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Reversal of breast cancer resistance protein-mediated drug resistance by estrogen antagonists and agonists.

Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer agents such as SN-38, mitoxantrone, and topotecan. In a previous study, we found that estrogens reverse drug resistance of BCRP-expressing cells. In this study, estrogen antagonists, estrogen agonists, and their derivatives were evaluated for BCRP-reversing activity. First, compounds were tested for effects on the cellular accumulation of topotecan in BCRP-transduced K562 cells (K562/BCRP). Next, these compounds were examined for their ability to reverse SN-38 and mitoxantrone resistance in K562/BCRP cells. Among commercially available estrogen antagonists and agonists tested, diethylstilbestrol showed the strongest BCRP-reversing activity. Diethylstilbestrol increased the cellular accumulation of topotecan and reversed drug resistance in K562/BCRP cells but showed marginal or no effect in parental K562 cells. The reversal activities of estrone and diethylstilbestrol were more prominent for mitoxantrone than for SN-38. Tamoxifen and toremifene were also found to enhance topotecan uptake in K562/BCRP cells. Next, various tamoxifen derivatives were screened for anti-BCRP activity. In the first cycle of screening with 14 compounds, TAG-11 showed the strongest effect. In the second cycle of screening of 25 TAG-11-related compounds, TAG-139 showed the strongest effect. Reversal of SN-38 and mitoxantrone resistance in K562/BCRP cells by TAG-139 was 5-fold stronger than that by estrone. Dose-dependent characteristics of drug resistance reversal with estrone and TAG-139 were very similar, suggesting that estrone and tamoxifen derivatives interact with the same drug-binding site of BCRP. Derivatives of antiestrogens that exhibit no other biological effects promise to be useful in overcoming BCRP-mediated drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

[Translational research for cancer therapeutics].

The importance and necessity of promoting translational research in Japan are well recognized. Successful development of molecular cancer therapeutics is strongly dependent on the promotion of such translational research. Obviously, the success of translational research in the field of molecular cancer therapeutics is essential for the development of new drugs for cancer treatment in the 21st century. For this purpose, the formation of research teams composed of basic molecular biologists, basic pharmacological scientists and clinical physicians is important. At the same time, the research must be conducted on a scientific basis with the reliable and speedy development of supporting systems in Japan.

Antineoplastic Agents↗
PubMed2003

[Globalization of clinical trials].

Based on reviews of the Japanese clinical trial situation in lung cancer, gastric cancer, prostate cancer and breast cancer, it was clear that much progress has been made in short time. There are considerable differences between Japan and the West and also differences between clinical areas in Japan. For regulatory purposes bridging studies have become increasingly important. Use of identical protocols are required for effective bridging. Participations in global phase III trials is the best way of achieving registration in Japan. For successful global trials in Japan it is important to include Japanese investigators in the preparation of the protocol and to recognise the challenges facing such a project. Clinical practice in diagnosis and treatment have many differences, thus it is recommended to have clear and detailed information in the protocol. Hard end points like survival are important since they are not biased by cultural differences. There are clear difficulties with HE or QOL outcomes. The emergence of focus on evidence based medicine is also happening in Japan and will help to harmonize documentation across the world. For large adjuvant or prevention cancer global trials are essential. To facilitate global studies further development of infrastructure is necessary in Japan. Use of electronic data capture web based communication etc. will help overcome communication difficulties. Other improvements that will make Japanese participation in global trials easier and better include establishment of clinical trial centre at each hospital, introduction of trial coordinators or study nurses and an improved collaboration with company staff. A critical issue that also need addressing is agreement of centre target recruitment. We need to introduce a new flexible system in Japan if participation in global trial is to be optimised. If we can address these issues Japanese investigators and collaborative groups should be able to initiate and lead global trials in the future.

Antineoplastic Agents↗
PubMed2002

SUMO-1 conjugation to intact DNA topoisomerase I amplifies cleavable complex formation induced by camptothecin.

DNA topoisomerase I (Topo1) manages the topological state of DNA. Cleavable complexes, the covalent Topo1-DNA intermediates, become DNA damaged when the catalytic cycles are inhibited by the anti-tumor drug camptothecin (CPT). Intriguingly, Topo1 is modified rapidly and extensively with SUMO-1, a ubiquitin-like protein, in response to CPT. This study shows that the sumoylation enhances the cleavable complex formation and apoptosis induced by CPT. Indeed, substitutions of Lys117 and Lys153, identified as Topo1 sumoylation sites, reduced the CPT-induced cleavable complexes without influencing its in vitro catalytic activity. Consistent with this observation, CPT-induced cleavable complexes of wild-type Topo1 increased in a sumoylation-dependent manner. We also found that Topo1 sumoylation occurred independently of CPT when Topo1 was inactivated by mutation of the catalytic Tyr723. These findings suggested that Topo1 inactivation by CPT treatment can trigger Topo1 sumoylation, leading to enhanced cleavable complex formation.

Binding Sites↗
PubMed2002

Modulation of heat-shock protein 27 (Hsp27) anti-apoptotic activity by methylglyoxal modification.

Methylglyoxal (MG) is one of the side-products in glycolysis, and it reacts with proteins under physiological conditions. Here, we identified heat-shock protein 27 (Hsp27) as a major MG-modified protein in cells. MG modification of Hsp27 selectively occurs at Arg-188 to form argpyrimidine, and mutation in the residue represses the formation of a large oligomer. This modification process is essential to its repressing activity for cytochrome c-mediated caspase activation. Inhibition of MG modification of Hsp27 causes sensitization of the cells to anti-tumor drug-induced apoptosis. Thus, MG is a novel modulator of cell survival by directly incorporating with the specific protein residue.

Amino Acid Sequence↗
PubMed2002

Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B.

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.

Animals↗
PubMed2002

Nuclear localization of proteasomes participates in stress-inducible resistance of solid tumor cells to topoisomerase II-directed drugs.

Physiological cell conditions of solid tumors, such as glucose starvation and hypoxia,induce cellular resistance to topoisomerase II-directed drugs. Here, we show that the induction of drug resistance is mediated by nuclear accumulation of proteasomes, large multicatalytic protease complexes. We found that the nuclear proteasome accumulation during glucose starvation was attenuated by stable expression of a mutant type of proteasome subunit, XAPC7, that lacked the nuclear localization signal (NLS). It is important that the expression of NLS-defective XAPC7 also diminished the induction of resistance to etoposide and doxorubicin, typical topoisomerase II-directed drugs. Under normal conditions, however, the NLS-defective XAPC7 had little effect on either nuclear proteasome distribution or etoposide sensitivity. Our findings demonstrate that stress-induced nuclear proteasome accumulation occurs through up-regulation of the NLS-dependent transport. Inhibition of the nuclear proteasome accumulation can be a novel approach to circumventing resistance to topoisomerase II-directed drugs.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitro through the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3 in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241.

14-3-3 Proteins↗