Biomedical subjects

Naotoshi Sugimoto

Publications and source records attributed to Naotoshi Sugimoto.

At least 19 recordsLinked to original sources

PubMed2006

Essential role for class II phosphoinositide 3-kinase alpha-isoform in Ca2+-induced, Rho- and Rho kinase-dependent regulation of myosin phosphatase and contraction in isolated vascular smooth muscle cells.

The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells, combined with the RNA interference-mediated gene-silencing technique, allowed us to determine the role of phosphoinositide 3-kinase (PI3K) class II alpha-isoform (PI3K-C2alpha) as a novel, Ca2+-dependent regulator of myosin light-chain phosphatase (MLCP) and contraction. The Ca2+-ionophore ionomycin induced a robust contractile response with an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The PI3K-C2alpha-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2alpha protein expression. The siRNA-mediated knockdown of PI3K-C2alpha, but not class I PI3K p110alpha, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. PI3K-C2alpha is uniquely less sensitive to the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) than the other PI3K members, including p110alpha. Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations showing that ionomycin and membrane depolarization induced Rho activation in vascular smooth muscle tissues in a Ca2+-dependent manner, ionomycin-induced contraction was dependent on Rho and Rho-kinase. Ionomycin induced phosphorylation of the MLCP-regulatory subunit myosin targeting protein 1(MYPT1) at Thr850 and the 20-kDa myosin light chain (MLC) in a Rho kinase-dependent manner. Knockdown of PI3K-C2alpha suppressed phosphorylation of both MYPT1 and MLC. The receptor agonist noradrenaline, which induced a rapid increase in the [Ca2+]i and Ca2+-dependent contraction, stimulated phosphorylation of MYPT1 and MLC, which was also dependent on Ca2+, PI3K-C2alpha, and Rho-kinase. These observations indicate that PI3K-C2alpha is necessary for Ca2+-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction.

Animals↗

PubMed2006

Ca2+-independent, inhibitory effects of cyclic adenosine 5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle.

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.

Animals↗

PubMed2006

RhoA-dependent PAI-1 gene expression induced in endothelial cells by monocyte adhesion mediates geranylgeranyl transferase I and Ca2+ signaling.

We investigated the role of RhoA activation and its mechanism in plasminogen activator inhibitor-1 (PAI-1) gene expression induced in endothelial cells by monocyte adhesion. Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased PAI-1 expression at the transcriptional level and activated RhoA which was accompanied by an increase in the activity of geranylgeranyl transferase I (GGTase I), an enzyme responsible for geranylgeranylation, and actin stress fiber formation. Inhibition of RhoA by C3 exoenzyme or by adenovirus-mediated expression of N19RhoA, as well as by pravastatin, prevented the upregulation of PAI-1 induced by monocyte adhesion. GGTI-286, an inhibitor of GGTase I, prevented the monocyte-induced RhoA activation and PAI-1 expression in endothelial cells. Monocyte attachment induced an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells and Ca(2+) chelation prevented the increased promoter activity and expression of PAI-1 induced by monocyte adhesion. C3 exoenzyme and GGTI-286 also suppressed endothelial intracellular Ca(2+) mobilization and Ca(2+) entry induced by monocytes. The present study shows that GGTase I plays a role in the RhoA activation in endothelial cells induced by monocyte adhesion and that GGTase I-mediated Ca(2+) signaling may contribute to RhoA-dependent PAI-1 gene expression.

Actins↗

PubMed2006

Negative regulation of endothelial morphogenesis and angiogenesis by S1P2 receptor.

We speculated that the sphingosine-1-phosphate (S1P) receptor S1P(2), which uniquely inhibits cell migration, might mediate inhibitory effects on endothelial cell migration and angiogenesis, different from S1P(1) and S1P(3). Mouse vascular endothelial cells, which endogenously express S1P(2) and S1P(3), but not S1P(1), responded to S1P and epidermal growth factor (EGF) with stimulation of Rac, migration, and the formation of tube-like structures on the Matrigel. The S1P(3)-antagonist VPC-23019 abolished S1P-induced, G(i)-dependent Rac stimulation, cell migration, and tube formation, whereas the S1P(2)-antagonist JTE-013 enhanced these S1P-induced responses, suggesting that S1P(2) exerts inhibitory effects on endothelial Rac, migration, and angiogenesis. S1P(2) overexpression markedly augmented S1P-induced, G(i)-independent inhibition of EGF-induced migration and tube formation. Finally, the blockade of S1P(2) by JTE-013 potentiated S1P-induced stimulation of angiogenesis in vivo in the Matrigel implant assay. These observations indicate that in contrast to S1P(1) and S1P(3), S1P(2) negatively regulates endothelial morphogenesis and angiogenesis most likely through down-regulating Rac.

Animals↗

PubMed2006

Rho-dependent, Rho kinase-independent inhibitory regulation of Rac and cell migration by LPA1 receptor in Gi-inactivated CHO cells.

Lysophosphatidic acid (LPA) is a major serum lysophospholipid that stimulates cell migration in diverse cell types including ovarian cancer cells. We report here that in the absence of Gi function, LPA induces inhibition, rather than stimulation, of cellular Rac activity, lamellipodium formation, and cell migration in response to insulin like growth factor I (IGF-I) in Chinese hamster ovary (CHO) cells, which solely express LPA1 as a LPA receptor. The inhibitory effects of LPA are abrogated by the expression of either Galpha13 C-terminal peptide or C3 toxin pretreatment, but not a Rho kinase inhibitor. Without PTX pretreatment, LPA stimulates Rac and cell migration yet similarly activates Rho, indicating that Rho activation by itself is not sufficient for inhibition of cell migration. Conversely, the expression of a dominant negative Rac mutant sufficiently mimics the LPA inhibition of cell migration. LPA inhibits IGF I-induced Akt activation by only 40% in a manner dependent on Rho kinase. These results demonstrate that inhibition of Gi function converts LPA regulation on Rac and cell migration to an inhibitory mode, which is mediated by G13 and Rho but not Rho kinase, and raise a possibility of Gi as a new therapeutic target for LPA-dependent tumor progression.

Actins↗

PubMed2006

Class II phosphoinositide 3-kinase alpha-isoform regulates Rho, myosin phosphatase and contraction in vascular smooth muscle.

We demonstrated previously that membrane depolarization and excitatory receptor agonists such as noradrenaline induce Ca2+-dependent Rho activation in VSM (vascular smooth muscle), resulting in MP (myosin phosphatase) inhibition through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1. In the present study, we show in de-endothelialized VSM strips that the PI3K (phosphoinositide 3-kinase) inhibitors LY294002 and wortmannin inhibited KCl membrane depolarization- and noradrenaline-induced Rho activation and MYPT1 phosphorylation, with concomitant inhibition of MLC (20-kDa myosin light chain) phosphorylation and contraction. LY294002 also augmented de-phosphorylation of MLC and resultantly relaxation in KCl-contracted VSM, whereas LY294002 was much less effective or ineffective under the conditions in which MP was inhibited by either a phosphatase inhibitor or a phorbol ester in Rho-independent manners. VSM express at least four PI3K isoforms, including the class I enzymes p110alpha and p110beta and the class II enzymes PI3K-C2alpha and -C2beta. The dose-response relationships of PI3K-inhibitor-induced inhibition of Rho, MLC phosphorylation and contraction were similar to that of PI3K-C2alpha inhibition, but not to that of the class I PI3K inhibition. Moreover, KCl and noradrenaline induced stimulation of PI3K-C2alpha in a Ca2+-dependent manner, but not of p110alpha or p110beta. Down-regulation of PI3K-C2alpha expression by siRNA (small interfering RNA) inhibited contraction and phosphorylation of MYPT1 and MLC in VSM cells. Finally, intravenous wortmannin infusion induced sustained hypotension in rats, with inhibition of PI3K-C2alpha activity, GTP-loading of Rho and MYPT1 phosphorylation in the artery. These results indicate the novel role of PI3K-C2alpha in Ca2+-dependent Rho-mediated negative control of MP and thus VSM contraction.

Animals↗

PubMed2006

[Combination chemotherapy of S-1 +CPT-11 (q 4-5 w) for metastatic gastric cancer].

The effectiveness of systemic chemotherapy for metastatic gastric cancer has already been established. However, a standard chemotherapy still remains uncertain. New agents such as S-1, CPT-11 and taxanes are markedly improving the response rates for gastric cancer. Including these new drugs, several randomized phase III trials are ongoing in Japan. In the near future, the candidate for standard regimen to treat gastric cancer will be reported. In this article, we described the current state of S-1 +CPT-11 combination chemotherapy for gastric cancer. Among various CPT-11 based chemotherapy, S-1 +CPT-11 appears to be the most effective and less toxic treatment.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Involvement of the βγ subunits of G proteins in the cAMP response induced by stimulation of the histamine H1 receptor.

Stimulation of the histamine H1 receptor has been shown to enhance adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation in various cell types but, to date, the mechanism by which this occurs is still unclear. In the present study, we examined the possibility that the betagamma subunits of G proteins (G betagamma) are involved in this process in cultured Chinese hamster ovary cells transfected with the human histamine H1 receptor (CHO-H1). Histamine increased intracellular cAMP levels in a concentration-dependent manner in CHO-H1 cells, and this histamine action was abolished by pyrilamine (1 microM). Inhibition of histamine H1 receptor-G(q) protein coupling by stable expression of the C-terminal peptide of G alpha(q) protein significantly attenuated the cAMP accumulation induced by histamine. By comparison, neither BAPTA/AM (50 microM), an intracellular Ca2+ chelator, nor GF 109203X (1 microM), an inhibitor of protein kinase C, influenced the cAMP response. Histamine H1 receptor-mediated cAMP accumulation was significantly inhibited by transient transfection of CHO-H1 cells with the C-terminal peptide of beta-adrenoceptor kinase I (residues 542-685), a scavenger of G betagamma. Stable expression of the C-terminal peptide of the G alpha(s) protein, but not treatment with pertussis toxin (200 ng/ml for 24 h), attenuated the histamine H1 receptor-mediated cAMP accumulation. These results suggest that stimulation of histamine H1 receptors activates adenylyl cyclase through the release of G betagamma subunits from G proteins, thereby elevating intracellular cAMP levels.

Adenylyl Cyclases↗

PubMed2005

Endogenous NO blockade enhances tissue factor expression via increased Ca2+ influx through MCP-1 in endothelial cells by monocyte adhesion.

OBJECTIVE: Ca2+ plays an important role in tissue factor (TF) gene expression. We investigated the role of endogenous nitric oxide (NO) in the induction of TF expression in endothelial cells (ECs) by monocyte adhesion and the mechanisms of NO action. METHODS AND RESULTS: Inhibition of endogenous NO by Nomega-nitro-L-arginine methyl ester (L-NAME) enhanced TF promoter activity and protein expression induced in human coronary ECs by monocyte adhesion, as well as EC surface TF activity. L-NAME also induced monocyte chemoattractant protein-1 (MCP-1) expression, which was blocked by an NO donor, NOC18. Exogenous MCP-1 enhanced TF expression induced by monocyte adhesion, whereas adenovirus-mediated expression of the mutant MCP-1, 7ND, abolished the L-NAME enhancement of TF expression induced by monocyte adhesion. Monocyte attachment to L-NAME-treated ECs increased Ca2+ influx, which was prevented by NOC18, anti-MCP-1 antibody or 7ND. These results indicate that the binding of increased MCP-1 induced by endogenous NO blockade to CCR2 mediated the enhancement of Ca2+ influx only when monocytes adhered to ECs, which upregulated TF expression in ECs triggered by monocyte adhesion. CONCLUSIONS: MCP-1/CCR2 may play a role in Ca2+ influx-dependent TF regulation in the monocyte-EC interaction in the impairment of NO synthesis.

Calcium↗

PubMed2005

Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP.

cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10(-8) M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10(-8) M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3',5'-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis.

Actins↗

PubMed2005

Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells.

RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 +/- 8 and 69 +/- 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA.

Actins↗

PubMed2005

A novel videoendoscopy system by using autofluorescence and reflectance imaging for diagnosis of esophagogastric cancers.

BACKGROUND: Image quality of the prior autofluorescence (AF) imaging systems, including the fiber-optic endoscope, was not feasible for general clinical use. The use of AF image alone resulted in low specificity. The objective of the study was to evaluate the resolution and the sensitivity of the novel videoendoscopy system by using AF and reflectance imaging (AFI) in the diagnosis of early esophagogastric cancers. METHODS: This was a case series study. The setting was a pretreatment examination at a cancer center. Five patients with superficial esophageal cancers (SEC) and 21 patients with 22 early gastric cancers (EGC) were included in the study. The extent of the tumors was diagnosed by white light (WL), AF and chromoendoscopic observations. The main outcome measurement was the diagnostic accuracy of each observation in relation to the histologic mapping as a criterion standard. RESULTS: Two of 5 SECs (40%) were correctly diagnosed in the WL image and all (100%) in the AF image as purple or magenta color in a green background. EGCs in atrophic mucosa were observed as purple or magenta areas in a green background, while diffuse-type EGCs in fundic mucosa were observed as green areas in a purple background. Of the 22 EGCs, diagnostic accuracy of WL, AF, and chromoendoscopic observations were 36%: 95% CI [16%, 56%], 68%: 95% CI [49%, 88%], and 91%: 95% CI [79%, 100%], respectively. AFI could reveal flat or isochromatic extensions that were not detected in the WL images. The limitations of the study were ulcerations or inflammation that caused overdiagnosis in the AF observation. CONCLUSIONS: The resolution of the AFI at present is limited, but the image quality was acceptable. The current system of AFI does not equal to chromoendoscopy in sensitivity but has an advantage over standard WL videoendoscopy.

Aged↗

PubMed2004

Blood lipid mediator sphingosine 1-phosphate potently stimulates platelet-derived growth factor-A and -B chain expression through S1P1-Gi-Ras-MAPK-dependent induction of Kruppel-like factor 5.

Platelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P(1)/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a G(i)-dependent manner, and activated ERK and p38 MAPK in G(i)- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn(17)Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SB203580 markedly suppressed PDGF mRNA and protein up-regulation, indicating the involvement of G(i)-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on G(i), Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P(1)-triggered, G(i)-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells.

Adenoviridae↗

PubMed2004

A mechanism underlying STAT4-mediated up-regulation of IFN-gamma induction inTCR-triggered T cells.

IL-12 promotes T(h)1 development/IFN-gamma expression by activating STAT4. However, it is still unclear how STAT4 elicits IFN-gamma promoter activation. Here, we investigated the mechanism by which IL-12-activated STAT4 functions for IFN-gamma induction in TCR-triggered T cells. TCR stimulation induced high levels of IFN-gamma production depending on co-stimulation with IL-12. IL-12 stimulation greatly enhanced the promoter-binding activity of c-Jun/AP-1, a critical transcription factor for IFN-gamma gene expression in wild-type T cells, but not in STAT4-deficient (STAT4(-/-)) T cells. Comparable amounts of c-Jun were induced by TCR stimulation in both wild-type and STAT4(-/-) T cells irrespective of IL-12 co-stimulation. However, c-Jun bound to STAT4 in IL-12-co-stimulated wild-type T cells. c-Jun forming a complex with STAT4 efficiently interacted with the AP-1-related sequence of the IFN-gamma promoter. Such an enhanced c-Jun binding did not occur in STAT4(-/-) T cells. These results show that STAT4 contributes to enhancing IFN-gamma expression by up-regulating the binding of TCR signal-induced AP-1 to the relevant promoter sequence.

Animals↗

PubMed2004

Endogenous cryogens existing in the blood of a hypothermic patient.

We treated a young female patient who suffered from severe hypothermia. The etiology of the hypothermia was not identified, though various medical examinations were performed. Intraperitoneal (i.p.) injections of the patient's serum produced a significant and profound hypothermia in rats that was not associated with circulatory shock. The patient's serum was separated into 4 fractions by the use of various types of centrifugal filtration units according to molecular weights (MW), i.e., below 10 kDa, 10-30 kDa, 30-100 kDa, and beyond 100 kDa. The first two fractions never induced hypothermia in rats, but the i.p. injections of the fraction with MW from 30 to 100 kDa consistently produced hypothermia. The fraction with MW beyond 100 kDa caused marked hypothermia in one out of 3 rats tested. The results suggest that the patient was excessively producing endogenous cryogenic substances of which MW may be greater than 30 kDa.

Adolescent↗

PubMed2004

[CPT-11 combined chemotherapy for metastatic gastric cancer].

The effectiveness of chemotherapy for metastatic gastric cancer has been already revealed. But a standard chemotherapy has not been established yet. New agents such as TS-1, CPT-11 and taxanes are improving the response rates and also the survivals for gastric cancer. Including these new drugs, several randomized phase III trials are ongoing in Japan. In the near future, the candidate for standard resume will be sent abroad. In this article, we described the current state of CPT-11 combined chemotherapy for gastric cancer. Among various CPT-11- combined chemotherapy, CPT-11 + TS-1 is the most effective and less toxic treatment.

Antineoplastic Agents, Phytogenic↗

PubMed2003

Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction.

Ca2+ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase-dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca2+-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca2+ or the addition of a dihydropyridine Ca2+ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N19RhoA suppressed Ca2+-induced Thr695 phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca2+ removal and depletion of the intracellular Ca2+ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A2 analogue-induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca2+-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.

Adrenergic alpha-Agonists↗

PubMed2003

Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils.

Like neutrophilic leukocytes, differentiated HL-60 cells respond to chemoattractant by adopting a polarized morphology, with F-actin in a protruding pseudopod at the leading edge and contractile actin-myosin complexes at the back and sides. Experiments with pharmacological inhibitors, toxins, and mutant proteins show that this polarity depends on divergent, opposing "frontness" and "backness" signals generated by different receptor-activated trimeric G proteins. Frontness depends upon Gi-mediated production of 3'-phosphoinositol lipids (PI3Ps), the activated form of Rac, a small GTPase, and F-actin. G12 and G13 trigger backness signals, including activation of a second GTPase (Rho), a Rho-dependent kinase, and myosin II. Functional incompatibility causes the two resulting actin assemblies to aggregate into separate domains, making the leading edge more sensitive to attractant than the back. The latter effect explains both the neutrophil's ability to polarize in uniform concentrations of chemoattractant and its response to reversal of an attractant gradient by performing a U-turn.

Cell Polarity↗