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Biomedical subjects

A Tohgo

Publications and source records attributed to A Tohgo.

At least 19 recordsLinked to original sources
PubMed2001

Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding.

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.

Animals↗
PubMed2001

Excretion into gastrointestinal tract of irinotecan lactone and carboxylate forms and their pharmacodynamics in rodents.

PURPOSE: To investigate the excretion of irinotecan hydrochloride (CPT-11) and its active metabolite, SN-38, into the gastrointestinal lumen via the biliary and/or intestinal membrane route after dosing with lactone and carboxylate forms of CPT-11, and to evaluate the toxic and antitumor effects of the two forms. METHODS: The excretions of CPT-11 and SN-38 were investigated by the in situ perfusion technique using rats. The incidence of delayed diarrhea was evaluated after i.v. dosing (60 mg/kg) with CPT-11 lactone and carboxylate forms for 4 days. Antitumor activity and changes in body weight were investigated in mice with Meth A tumors. RESULTS: The excretion of CPT-11 into bile was greater in dosing with CPT-11 carboxylate than that with its lactone form, whereas the exsorption across intestinal membrane was greater in dosing with CPT-11 lactone than that with its carboxylate form. Dosing with CPT-11 lactone dose-dependently inhibited the increase in tumor weights in Meth A tumor mice, whereas the dosing with its carboxylate form reduced the antitumor effect. CONCLUSIONS: The decreased antitumor effect caused by dosing with the CPT-11 carboxylate form could be due to less accumulation in the tissue including tumor cells resulting from the rapid elimination of the form in the body.

Animals↗
PubMed2001

Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF120918.

This study was aimed at characterizing the role of BCRP/MXR/ABCP (BCRP) in resistance of the human ovarian tumor cell lines T8 and MX3 to camptothecins more extensively and investigating whether resistance can be reversed by inhibiting BCRP by GF120918. Camptothecins studied were topotecan, CPT-11, and its active metabolite SN-38, 9-aminocamptothecin, and the novel experimental camptothecins NX211, DX8951f, and BNP1350. Notably, DX8951f and BNP1350 appeared to be very poor substrates for BCRP, with much lower resistance factors observed both in T8 and MX3 cells than observed for the other camptothecins tested. In the presence of a nontoxic dose level of GF120918, the intracellular accumulation of topotecan in the T8 and MX3 cells was completely restored to the intracellular levels observed in the sensitive IGROV1 parental cell line. This resulted in almost complete reversal of drug resistance to topotecan and to most of the other topoisomerase I drugs tested in the T8 cell line and to complete reversal in the MX3 cells. However, coincubation of DX8951f or BNP1350 with GF120918 did not affect the cytotoxicity of either of these drugs significantly. From the combined data, we conclude that the affinities of topoisomerase I drugs for BCRP are, in decreasing order: SN-38 > topotecan > 9-aminocamptothecin approximately CPT-11 > NX211 > DX8951f > BNP1350. Furthermore, GF120918 appears to be a potent reversal agent of BCRP-mediated resistance to camptothecins, with almost complete reversal noted at 100 nM. Potential BCRP-mediated resistance to topoisomerase I inhibitors can also be avoided by using the BCRP-insensitive drugs DX8951f or BNP1350. This observation may have important clinical implications for future development of novel camptothecins.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2000

Growth inhibitory effect of a new camptothecin analog, DX-8951f, on various drug-resistant sublines including BCRP-mediated camptothecin derivative-resistant variants derived from the human lung cancer cell line PC-6.

DX-8951f, a new water-soluble camptothecin (CPT) derivative, has been reported to show potent antitumor effects against various tumors in vitro and in vivo. We further evaluated the cytotoxic effect of DX-8951f against eight drug-resistant sublines derived by stepwise exposure of human oat cell carcinoma PC-6 to various drugs. In paclitaxel-, adriamycin-, vincristine- and etoposide-resistant cells, overexpression of P-glycoprotein (P-gp) and a correlative reduction in drug accumulation and typical drug-sensitivity pattern were confirmed. The etoposide-resistant line with the highest P-gp level was cross-resistant also to SN-38, CPT-11 and topotecan (TPT), but not to 9-aminocamptothecin (9-AC), CPT and DX-8951f. SN-38- and CPT-11-resistant cells, of which topoisomerase I activities and levels were similar to those of the parent cells, showed cross-resistance clearly to TPT, 9-AC and mitoxantrone, but hardly to DX-8951f. In these two resistant sublines, the intracellular topotecan level was significantly lower than that in parental PC-6 and the reduced accumulation was found to be mediated by breast cancer resistant protein (BCRP). The cisplatin-resistant variant, which had a 2-fold increase in glutathione content, showed no cross-resistance and the 5-fluorouracil-resistant variant, which had a 50% decrease in glutathione content, exhibited collateral sensitivity to most of the other anticancer agents including DX-8951f. We concluded that DX-8951f showed a potent cytotoxic effect on various types of drug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Comparison of DX-8951f and topotecan effects on tumor colony formation from freshly explanted adult and pediatric human tumor cells.

DX-8951f, which shows great therapeutic potential, was tested in the human tumor cloning system in adult and pediatric tumor types against which topotecan has been active. In 47 tumors from adults, DX-8951f had definite cytotoxic activity in a concentration-dependent manner with both 1 h and continuous exposures. Topotecan was minimally effective using a 1 h exposure and showed concentration-dependent inhibition with continuous exposure. In head-to-head comparisons at 1 h exposure against adult tumors, DX-8951f was significantly more effective at 0.1 and 1.0 microg/ml than topotecan. In head-to-head comparisons (continuous exposure), 1.0 microg/ml DX-8951f was more effective than topotecan at 1.0 microg/ml in adult tumors, including three of four head and neck, one of two kidney, two of five liver, six of 10 non-small cell lung, five of eight ovarian, four of eight prostate tumors, and in single specimens of breast, mesothelioma, colon and small cell lung tumors. With continuous exposure, DX-8951f and topotecan were equally effective at equimolar concentrations. The maximum tolerated dose for DX-8951f is 3 times that of topotecan, so higher doses of DX-8951f could be administered to patients. DX-8951f is a promising new antineoplastic agent with significant activity against tumors taken directly from patients.

Adult↗
PubMed1998

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P </= 0.05). Insulin secretion from pancreatic islets by glucose is significantly inhibited by the addition of the NIDDM sera with anti-CD38 antibodies (P </= 0.04-0.0001), and the inhibition of insulin secretion is abolished by the addition of recombinant CD38 (P </= 0.02). The increase of cADPR levels in pancreatic islets by glucose was also inhibited by the addition of the sera (P </= 0.05). These results strongly suggest that the presence of anti-CD38 autoantibodies in NIDDM patients can be one of the major causes of impaired glucose-induced insulin secretion in NIDDM.

ADP-ribosyl Cyclase↗
PubMed1998

Cyclic ADP-ribose and inositol 1,4,5-trisphosphate as alternate second messengers for intracellular Ca2+ mobilization in normal and diabetic beta-cells.

Intracellular Ca2+ mobilization occurs in a variety of cellular processes and is mediated by two major systems, the inositol 1,4, 5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR) systems. cADPR has been proposed to be a second messenger for insulin secretion induced by glucose in pancreatic beta-cells (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). Here we show that the cADPR signal system for insulin secretion is replaced by the IP3 system in diabetic beta-cells such as ob/ob mouse islets and RINm5F cells. We measured the cADPR content in these beta-cells by radioimmunoassay and found that the increase of the cADPR content by glucose did not occur in ob/ob mouse islets and RINm5F cells, whereas the increased cADPR level by glucose was observed in normal rat and mouse islets. Microsomes of these diabetic beta-cells released Ca2+ in response to IP3 but not to cADPR. In the diabetic beta-cells, CD38 (ADP-ribosyl cyclase/cADPR hydrolase) and type 2 ryanodine receptor mRNAs were scarcely detected and, in contrast, an increased expression of IP3 receptor mRNAs was observed. The diabetic beta-cells secreted insulin rather by carbamylcholine than by glucose.

ADP-ribosyl Cyclase↗
PubMed1998

A missense mutation in the CD38 gene, a novel factor for insulin secretion: association with Type II diabetes mellitus in Japanese subjects and evidence of abnormal function when expressed in vitro.

Cyclic adenosine 5'diphosphate-ribose (cADPR) is thought to have a second messenger role in insulin secretion through mobilisation of Ca2+. As human lymphocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activity, it may be important in glucose-induced insulin secretion in islets. Thirty one randomly selected Japanese patients with Type II diabetes mellitus who had first-degree and/or second-degree relative(s) with Type II diabetes mellitus were screened for mutations of this gene using single-stranded conformation polymorphism. Two variant patterns in exon 3 and exon 4 of the CD38 gene were identified. The variant in exon 3 resulted in an amino acid substitution from Arg140 (CGG) to Trp (TGG). The Arg140Trp mutation was observed in 4 of 31 patients, and allele frequencies were significantly different in patients and the control subjects (p = 0.004). One patient with this mutation has two missense mutations on beta cell/liver glucose transporter (GLUT2) gene; her mother, who has impaired glucose tolerance, also has this mutation on the CD38 gene and one missense mutation on the GLUT2 gene. Enzyme activity studies using COS-7 cells expressing the Arg140Trp mutation showed a reduction in ADP-ribosyl cyclase and cADPR hydrolase activity of around 50%. The Arg140Trp mutation on CD38 thus appears to contribute to the development of Type II diabetes mellitus via the impairment of glucose-induced insulin secretion in the presence of other genetic defects.

ADP-ribosyl Cyclase↗
PubMed1998

Potent and broad antitumor effects of DX-8951f, a water-soluble camptothecin derivative, against various human tumors xenografted in nude mice.

PURPOSE: We have previously reported that DX-8951f, a water-soluble and nonprodrug camptothecin (CPT) derivative, exhibits both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The purpose of this study was to evaluate the therapeutic efficacy of DX-8951f against human tumor xenografts in nude mice and to compare its activity with those of CPT-11 and other current CPT derivatives. METHODS: The antitumor activity of DX-8951f against xenografts of several different types of human tumors was determined in nude mice using a schedule in which DX-8951f was administered intravenously every 4th day for a total of four injections. RESULTS: Against both gastric adenocarcinoma SC-6 and its CPT-11-resistant variant, SC-6/CPT-11, DX-8951f demonstrated superior antitumor activity and antitumor activity over a broader range of doses than did CPT-11, SK&F104864 (hycamtin, topotecan) and GG-211 (GI147211). DX-8951f at 75 mg/kg was effective (growth inhibition rate IR > or = 58%) against 15 of 16 lines of human cancers examined (6 colon cancers, 5 lung cancers, 2 breast cancers, 1 renal cancer and the above 2 gastric cancers), and exhibited excellent antitumor activity (IR > or = 80%) against 14 of these lines. CPT-11 exhibited antitumor activity with IR values of 58% and higher against 11 lines and IR values of 80% and higher against only eight of the same 16 human tumors. DX-8951f was effective in inhibiting the growth of an SN-38-resistant tumor and some P-glycoprotein-expressing tumors, but CPT-11 was not. CONCLUSIONS: DX-8951f exhibited potent antitumor activity against various types of human tumor xenografts. Its in vivo antitumor effects were superior to those of current camptothecin analogs against certain tumors.

Animals↗
PubMed1998

Antitumour activity of DX-8951f: a new camptothecin derivative.

DX-8951f is a water-soluble camptothecin analogue with a unique hexacyclic structure. Compared to other current camptothecin derivatives, DX-8951f is the most effective topoisomerase I (topo I) inhibitor and has the most potent cytotoxic activity against various tumour cell lines in vitro. Of particular interest is DX-8951f's significant effect on certain tumour cell lines resistant to other camptothecin derivatives, as well as on multi-drug resistant variants that overexpress P-glycoprotein. In addition, in in vivo xenograft systems using nude mice, DX-8951f strongly inhibits the growth of human solid tumours, including resistant tumours. Its antitumour effects and resulting life prolongation in tumour-bearing mice have also been confirmed in several metastasis models. DX-8951f provides greater therapeutic efficacy and broader effective dose ranges using multiple injections than with a bolus injection and simple intermittent applications. The in vivo effects of the compound are superior to those of CPT-11 and SK&F104864, suggesting that DX-8951f is a promising therapeutic agent for the treatment of cancer patients. Phase I clinical trials are ongoing in Europe, the USA and Japan.

Journal Article↗
PubMed1997

DX-8951f, a water-soluble camptothecin analog, exhibits potent antitumor activity against a human lung cancer cell line and its SN-38-resistant variant.

We previously reported that DX-8951f, a novel water-soluble camptothecin analog, significantly inhibits the growth of various human and murine tumors in vitro and in vivo. The antitumor effects and topoisomerase I inhibitory activity of DX-8951f are stronger than those of other current camptothecin analogs. In this study, we established an SN-38-resistant cell line, PC-6/SN2-5, from the human oat cell carcinoma PC-6 cell line by a stepwise selection system, investigated the mechanism of resistance of this cell line and then compared the antitumor activity of camptothecin analogs against the cell line. PC-6/SN2-5 cells were resistant to SN-38 (32-fold) and SK&F 104864 (topotecan; 14-fold), but barely resistant to CPT-11 (3-fold) and DX-8951f (2-fold). Topoisomerase I protein levels and topoisomerase I activities of parental cells were similar to those of resistant cells. Determination of the cellular drug concentration by either flow cytometric analysis or the high-performance liquid chromatography method confirmed that the cellular accumulation of SN-38 and topotecan was significantly reduced in PC-6/SN2-5 cells, whereas that of DX-8951f was only slightly reduced. Furthermore, DX-8951f stabilized the cleavable complex formations in intact PC-6/SN2-5 cells as well as in parental cells, but SN-38 and topotecan did not in the resistant cells. Our data suggest that PC-6/SN2-5 cells may have acquired resistance to camptothecin analogs by a decrease in intracellular drug accumulation and that DX-8951f may have the potency to overcome such a type of resistance mechanism induced by camptothecin compounds.

Antineoplastic Agents↗
PubMed1997

Human gene encoding CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase): organization, nucleotide sequence and alternative splicing.

We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as a second messenger for glucose-induced insulin secretion [Takasawa et al. (1993a) Science 259, 370-373] and that CD38 has both ADP-ribosyl cyclase (ADRC) and cADPR hydrolase activities [Takasawa et al. (1993b) J. Biol. Chem. 268, 26052-26054]. In this study, we determined the structure of the human CD38 gene, and showed that two mRNA forms originated by alternative splicing from the CD38 gene. The human CD38 gene consists of 8 exons that extend more than 77 kb on the human genome. Exon 1 encoded the 5'-untranslated region of the mRNA, the N-terminal end of CD38 and the putative transmembrane domain, and exon 2-8 encoded the remainder of CD38: the exon-intron organization of the human CD38 gene is similar to that of the Aplysia ADRC gene [Nata et al. (1995) Gene 158, 213-218]. This structural conservation between human and Aplysia genes suggests that both genes may have evolved from a common ancestral gene.

ADP-ribosyl Cyclase↗
PubMed1997

Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase.

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.

ADP-ribosyl Cyclase↗
PubMed1997

Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes.

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

Adenosine Diphosphate Ribose↗
PubMed1997

Antitumor effect of DX-8951, a novel camptothecin analog, on human pancreatic tumor cells and their CPT-11-resistant variants cultured in vitro and xenografted into nude mice.

DX-8951 is a novel water-soluble derivative of camptothecin. We evaluated the effects of DX-8951 on the growth of several pancreatic tumor cell lines in vitro and in vivo. In vitro cytotoxic activity of DX-8951 against SUIT-2 and KP-1N cells, as indicated by IC50 value, was several times more potent than that of SN-38, an active metabolite of CPT-11, and dozens of times more potent than that of SK&F104864 (topotecan). DX-8951 also showed the greatest cytotoxicity against CPT-11-resistant variants, SUIT-2/CPT-11 and KP-1N/CPT-11 cells, and the cross-resistance of these cells to DX-8951 was lower than that to SN-38 and SK&F104864. Topoisomerase I inhibitory activity of DX-8951 was about three-fold stronger than that of SN-38, as measured in crude nuclear extract obtained from SUIT-2 cells. DX-8951 induced DNA fragmentation, a specific feature of apoptosis, in SUIT-2 cells more effectively than SN-38. DX-8951 exhibited potent antitumor effects against SUIT-2 in a solid tumor model and in a liver metastasis model, in which tumor cells were xenografted subcutaneously and intrasplenically, respectively, into nude mice. The in vivo effects were closely similar to or somewhat superior to those of CPT-11. DX-8951 also showed significant antitumor effects against SUIT-2/CPT-11 solid tumors, against which CPT-11 had no effect. These results suggest that, on the basis of its strong antitumor activity and effectiveness against CPT-11-resistant tumors, DX-8951 may be a useful therapeutic agent in the treatment of human cancer. The potent cytotoxicity of DX-8951 may result from strong inhibition of topoisomerase I, which may then trigger apoptotic cell death.

Animals↗
PubMed1997

Antitumor effect of DT-5461, a lipid A derivative, against human tumor xenografts is mediated by intratumoral production of tumor necrosis factor and affected by host immunosuppressive factors in nude mice.

We previously reported that DT-5461, a synthetic low-toxic lipid A analog, inhibits growth of various murine tumors through activation of host immune systems. In the present study, DT-5461 also exhibited significant antitumor effects against 5 out of 6 human tumor xenografts in nude mice. The antitumor activity was similar to or greater than those of chemotherapeutics. Antitumor effects of DT-5461 significantly correlated with intratumoral levels of tumor necrosis factor (TNF) induced by the compound (r = 0.701, p < 0.05). In vitro TNF production by DT-5461-stimulated macrophages was augmented by tumor cells, and the augmentative effect correlated with TNF activity detected in these tumor tissues. Meanwhile, a weaker therapeutic efficacy of DT-5461 was observed against certain tumors that caused a significant increase in the level of immunosuppressive factors in host blood. These findings support the idea that intratumoral TNF plays a crucial role in the antitumor mechanisms of DT-5461 and suggest that its antitumor action is influenced by an augmentative effect of tumor cells on TNF production and by blood levels of immunosuppressive factors.

Adjuvants, Immunologic↗