Biomedical subjects

N Koshikawa

Publications and source records attributed to N Koshikawa.

At least 37 records · Page 2Linked to original sources

PubMed2001

Monitoring of extracellular dopamine levels in the dorsal striatum and the nucleus accumbens with 5-minute on-line microdialysis in freely moving rats.

We report a reliable 5-min on-line monitoring of dopamine released from the dorsal striatum and the nucleus accumbens of rats using in vivo brain microdialysis. The detection limit for dopamine was approximately 20 fg in a 10-microl injection sample using high-performance liquid chromatography with electrochemical detection set-up. Basal levels of dopamine in the dorsal striatum and the nucleus accumbens 4 h after probe insertion were 2.65 +/- 0.30 pg/5 min and 1.57 +/- 0.31 pg/5 min, respectively, whereas those of 20 h after probe insertion were lower: 0.97 +/- 0.21 pg/5 min and 0.51 +/- 0.09 pg/5 min, respectively. Infusion of the sodium channel blocker, tetrodotoxin (TTX; 2 microM), essentially suppressed levels of dopamine in both brain areas. At 4 h after probe insertion, TTX perfused for 4 h via dialysis probe reduced levels of dopamine to 0.47 +/- 0.08 pg/5 min (80% reduction) in the dorsal striatum and to 0.56 +/- 0.19 pg/5 min (65% reduction) in the nucleus accumbens. At 20 h after probe insertion, a similar TTX perfusion more rapidly reduced levels of dopamine to 0.05 +/- 0.01 pg/5 min (95% reduction) in the dorsal striatum and to 0.08 +/- 0.01 pg/5 min (85 % reduction) in the nucleus accumbens. These results suggest that relatively fast changes in extracellular dopamine levels in these two brain areas can reliably be followed by this in vivo microdialysis technique.

Analysis of Variance↗

PubMed2001

The error-prone DNA polymerase zeta catalytic subunit (Rev3) gene is ubiquitously expressed in normal and malignant human tissues.

Mutagenesis induced by UV light and chemical agents in yeast is largely dependent on the function of Rev3, the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. Human and mouse homologues of the yeast Rev3 gene have recently been identified, and inhibition of Rev3 expression in cultured human fibroblasts by Rev3 anti-sense was shown to reduce UV-induced mutagenesis, indicating that Rev3 also plays a crucial role in mutagenesis in mammalian cells. A common variant transcript with an insertion of 128-bp between nucleotides +139 and +140 is found in both human and mouse Rev3 cDNAs, but its biological significance has not been defined. We show here that the insertion variant is not translatable either under in vitro or in vivo conditions. We also found that the translational efficiency of Rev3 gene is enhanced by the 5' untranslated region that contains a putative stem-loop structure postulated to inhibit the translation. Since the human Rev3 gene is localized to chromosome 6q21, a region previously shown to contain genes involved in tumor suppression and cellular senescence, we examined its expression in various normal and malignant tissues. Rev3 and its insertion variant transcripts were ubiquitously detected in all 27 normal human tissues studied, with an additional variant species found in tissues with relatively high levels of Rev3 expression. Levels of Rev3 transcripts were similar in lung, gastric, colon and renal tumors compared to normal tissue counterparts. The data indicate that Rev3 expression is ubiquitous and is not dysregulated in malignancies.

5' Untranslated Regions↗

PubMed2000

Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells.

We examined whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the promoter of the vascular endothelial growth factor (VEGF) gene that is activated by hypoxia is effective in killing highly metastatic Lewis lung carcinoma A11 cells under hypoxic conditions. We isolated the promoter region encompassing the hypoxia response element (HRE) of the mouse VEGF gene. To assess the hypoxia responsiveness of the VEGF promoter, A11 cells were transiently transfected with luciferase reporter plasmids. Exposure of the transfectants to hypoxia resulted in a 2-3-fold induction of luciferase activity. Deletion of the HRE site abolished VEGF promoter activity under both normoxic and hypoxic conditions. We constructed a retroviral vector harboring the HSV-TK or green fluorescence protein (GFP) gene under the control of the VEGF promoter. A11 cells transfected with vector harboring the VEGF promoter fused to the HSV-TK gene [A11(HRE/TK) cells] were more sensitive to ganciclovir than cells transfected with the control vector harboring the VEGF promoter alone, and the sensitivity of the A11(HRE/TK) cells was increased by exposure to hypoxia followed by reoxygenation. Culturing A11 cells transfected with vector harboring the VEGF promoter fused to the GFP gene under hypoxic conditions resulted in an increase in the expression of GFP. Monitoring GFP expression and vascularity in the A11 transfectant tumors revealed up-regulation of GFP expression in poorly vascularized regions. Administration of ganciclovir to mice bearing s.c. tumors formed by A11(HRE/TK) cells resulted in regression of the tumors. These results suggest a possible application of the suicide gene driven by the VEGF promoter to cancer gene therapy that efficiently targets hypoxic tumor cells.

Animals↗

PubMed2000

Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2.

Trypsin is widely expressed in various non-pancreatic tissues at low levels and overexpressed in some types of human cancers. In the present study, we found that trypsin stimulates integrin-dependent adhesion and growth of MKN-1 human gastric carcinoma cells. MKN-1 cells expressed both proteinase-activated receptor-1 (PAR-1) and PAR-2, which are activated by thrombin and trypsin, respectively. Both trypsin and the PAR-2 ligand SLIGKV promoted integrin alpha(5)beta(1)-mediated adhesion of MKN-1 cells to fibronectin, and less effectively integrin alpha(v)beta(3)-mediated cell adhesion to vitronectin, but not that to type IV collagen or laminin-1 at all. Thrombin and the PAR-1 ligand SFLLRN promoted the cell adhesion to vitronectin more strongly than trypsin or the PAR-2 ligand, but not the cell adhesion to fibronectin at all. The cell adhesion-stimulating effect of the PAR-2 ligand was significantly reduced by the pre-treatment of cells with trypsin, indicating that the effect of trypsin is mediated by PAR-2 activation. The trypsin-stimulated cell adhesion to vitronectin, but not to fibronectin, was effectively inhibited by the G(i) protein blocker pertussis toxin, and both cell adhesions were completely inhibited by the Src kinase inhibitor herbimycin A. Furthermore, trypsin and the PAR-2 ligand stimulated growth of MKN-1 cells more strongly than thrombin or the PAR-1 ligand. These results show that trypsin regulates cellular adhesion and proliferation by inducing PAR-2/G protein signalings, and that the integrin alpha(5)beta(1)- and integrin alpha(v)beta(3)-dependent cell adhesions are regulated by different PAR/G protein signalings.

Cell Adhesion↗

PubMed2000

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5.

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.

Animals↗

PubMed2000

Characterization of a zinc finger protein ZAN75: nuclear localization signal, transcriptional activator activity, and expression during neuronal differentiation of P19 cells.

The ZAN75 cDNA was first identified in NIH 3T3 cells and codes for a DNA-binding protein with two zinc finger motifs. In this study, we characterized the nuclear localization signal of ZAN75, tested if ZAN75 regulates transcription, and examined its expression during embryonic development and neuronal differentiation of P19 mouse embryonal carcinoma cells. By examining the cellular localization of deletion mutants of ZAN75 fused to green fluorescence protein, ZAN75 was revealed to have a bipartite nuclear localization signal sequence upstream of the zinc finger domains. The N-terminal region of ZAN75, when fused to the GAL4 DNA-binding domain, strongly activated transcription. The expression of ZAN75 mRNA was found to be developmentally regulated, showing the highest expression in E11.5 embryos. In situ hybridization experiments using E11.5 embryos showed a high expression of the transcripts in neuronal tissues such as brain and neural tube. The expression of ZAN75 was transiently increased at both the mRNA and the protein levels when P19 cells were treated with retinoic acid to induce neuronal differentiation. Taken together, these results indicate that ZAN75 is a transcriptional activator with a bipartite nuclear localization signal and may play a role in neuronal differentiation.

Amino Acid Sequence↗

PubMed2000

Effects of saiko-ka-ryukotsu-borei-to on spontaneous locomotor activity in mice. Oriental Medicine Research Group.

The effects of the Japanese Kampo (herbal) medicine, Saiko-ka-ryukotsu-borei-to, on spontaneous locomotor activity were studied in mice. Saiko-ka-ryukotsu-borei-to (60 mg, 150 mg and 300 mg/kg/day) was administered for 14 consecutive days in the drinking water and spontaneous locomotor activity was measured for 60 min by a photocell ambulometer. Saiko-ka-ryukotsu-borei-to (60 mg/kg/day) significantly increased the total activity count on the 11th day after the start of administration when compared to vehicle control, whereas failed to significantly affect the activity on the 2nd, 5th, 8th and 14th days. A similar significant increase was also found with a higher dose (150 mg/kg/day) on the 8th day after the start of administration. However, the highest dose (300 mg/kg/day) did not significantly affect locomotor activity throughout the experimental period. We have previously reported that Saiko-ka-ryukotsu-borei-to, at a dose of 60 mg/kg/day, enhances escape attempts assessed by water-wheel rotations in a mouse model of despair, particularly on the 8th, 11th and 14th days after the start of chronic treatment. However, at higher doses (150 and 300 mg/kg/day), Saiko-ka-ryukotsu-borei-to decreases the escaped attempts on the 5th and 8th days after the treatment. It is therefore concluded that the previously reported changes in escape attempts of mice are not associated with the changes in their spontaneous locomotor activity.

Administration, Oral↗

PubMed2000

Marked increase of trypsin(ogen) in serum of linitis plastica (gastric cancer, borrmann 4) patients.

Linitis plastica, or Borrmann 4 gastric cancer, shows very poor prognosis, and the reason has not been understood. In the present study, we examined serum levels of trypsin(ogen) in 44 gastric cancer patients, including 17 early gastric cancer, 18 non-Borrmann 4 advanced gastric cancer, and 9 Borrmann 4 gastric cancer, by using the RIA gnost Trypsin kit (Hoechst Japan, Tokyo, Japan), which was expected to detect trypsin-1, trypsin-2, trypsinogen-1, and trypsinogen-2 in sera. The trypsin(ogen) concentration was much higher in the patients with linitis plastica than in the other gross types of gastric cancer. Hypertrypsinemia was identified in approximately 60% of advanced gastric cancer cases. Lymph node involvement, liver metastasis, or poorly differentiated adenocarcinoma is an important factor of hypertrypsinemia. The serum trypsin(ogen) level in linitis plastica patients was 3484.4 +/- 2319.7 ng/ml (mean +/- SD), which was significantly higher not only than that of the early gastric cancer (384.1 +/- 92.1) but also the stage IV gastric cancer patients (578 +/- 440.4), excluding those with linitis plastica. The elevated serum trypsinogen level in linitis plastica patients may be related to the malignant behavior of this type of cancer cell. Serum trypsin(ogen) of linitis plastica shows significantly higher concentrations than do the other types of advanced gastric cancer. Therefore, serum concentration of trypsin(ogen) might be a good marker of gastric cancer of linitis plastica.

Humans↗

PubMed1999

Overexpression of laminin gamma2 chain monomer in invading gastric carcinoma cells.

Laminin (LN)-5, a heterotrimer of alpha3, beta3, and gamma2 chains, has been suggested to be involved in tumor cell invasion. The present immunohistochemical study investigated the distribution of the LN gamma2 chain in 48 different human gastric adenocarcinomas. The immunohistochemical analysis showed two distinct patterns of LN gamma2 chain expression: (a) extracellular deposition; and (b) cytoplasmic accumulation. The extracellular deposition of the LN gamma2 chain was typically observed at neoplastic basement membranes of well-differentiated adenocarcinomas. The immunoreactivity was continuous along tumor basement membranes in these tumors but was irregular and diffuse in poorly differentiated carcinomas. These tumor cells coexpressed the LN alpha3 and beta3 chains, suggesting that the LN gamma2 chain was deposited as the LN-5 complex. In contrast, tumor cells at the invading fronts showed strong cytoplasmic staining for the LN gamma2 chain without any detectable signal for the LN alpha3 or beta3 chain in both well- and poorly differentiated carcinomas. On the other hand, in vitro analysis by two-dimensional SDS-PAGE demonstrated that human gastric carcinoma cells secrete a high level of LN gamma2 chain monomer in addition to the LN-5 complex into culture medium. These results indicate that the LN gamma2 chain can be secreted as a single subunit and might be involved in tumor cell invasion.

Adenocarcinoma↗

PubMed1999

SK&F 83959 and non-cyclase-coupled dopamine D1-like receptors in jaw movements via dopamine D1-like/D2-like receptor synergism.

This study compared the effects of the dopamine D1-like receptor agents SK&F 83959 (3-methyl-6-chloro-7,8-dihydroxy-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro- 1 H-3-benzazepine), which inhibits the stimulation of adenylyl cyclase, and A 68930 ([1R,3S]-1-aminomethyl-5,6-dihydroxy-3-phenyl-isochroman), a full efficacy agonist, in regulating jaw movements in the rat by synergism with dopamine D2-like receptor agonism. When SK&F 83959 and A 68930 were given in combination with quinpirole, there was a synergistic induction of jaw movements. Responsivity to SK&F 83959 + quinpirole was antagonised by the dopamine D1-like receptor antagonists SCH 23390 ([R]-3-methyl-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-ben zaz epine) and BW 737C ([S]-6-chloro-1-[2,5-dimethoxy-4-propylbenzyl]-7-hydroxy-2-methyl- 1,2,3,4-tetrahydroisoquinoline); synergism was antagonised also by the dopamine D2-like receptor antagonist YM 09151-2 (cis-N-[1-benzyl-2-methyl-pyrrolidin-3-yl]-5-chloro-2-methoxy-4-++ +methyl-aminobenzamide). Responsivity to A 68930 + quinpirole was enhanced by low doses of SCH 23390, BW 737C and YM 09151-2, and antagonised by higher doses of SCH 23390 and YM 09151-2. These results implicate a novel, dopamine D1-like receptor that is coupled to a transduction system other than/additional to adenylyl cyclase, and suggest that its functional role extends to the regulation of jaw movements by synergistic interactions with dopamine D2-like receptors.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1999

Matrilysin as a target for chemotherapy for colon cancer: use of antisense oligonucleotides as antimetastatic agents.

Matrilysin (MMP-7) is the smallest member of the matrix metalloproteinase (MMP) family. It is frequently expressed in various types of cancer including colon, stomach, prostate, and brain cancers. Previous studies have suggested that matrilysin plays important roles in the progression and metastasis of colon cancer. Recently, we have examined the effects of a matrilysin-specific antisense phosphorothioate oligodeoxyribonucleotide on in vitro invasion and liver metastasis in nude mice of two human colon carcinoma cell lines (CaR-1 and WiDr). In culture, the antisense oligonucleotide effectively inhibited both the secretion of matrilysin by CaR-1 cells and their in vitro invasion through a reconstituted basement membrane. In a nude mouse model, the antisense oligonucleotide potently suppressed the experimental liver metastasis of WiDr cells from the spleen. These results suggest that matrilysin has an important role in the liver metastasis of human colon cancer and that matrilysin antisense oligonucleotides have therapeutic potential for the prevention of metastasis.

Animals↗

PubMed1999

Effects of chronic haloperidol and clozapine on vacuous chewing and dopamine-mediated jaw movements in rats: evaluation of a revised animal model of tardive dyskinesia.

Rats received haloperidol (1.0 mg/kg i.p.) or clozapine (10 mg/kg i.p.), twice daily for 4 weeks: vacuous chewing--recorded 26 h after the final injection--similarly increased in both groups. Three h later, the rats were challenged with dopaminomimetics, and automatically recorded jaw movements were analysed. Both apomorphine and a mixture of D1 and D2 receptor agonists (SKF 38393 resp. quinpirole) increased jaw movements in haloperidol-treated, but not clozapine-treated rats; SKF 38393 or quinpirole remained ineffective, when given alone. A fixed dose of quinpirole together with increasing doses of SKF 38393, but not a fixed dose of SKF 38393 together with increasing doses of quinpirole, produced a dose-dependent increase in jaw movements in otherwise non-treated rats, suggesting that the noted haloperidol-induced increase was due to a shift in the D1-D2 receptor balance towards a predominance of D1 receptors. This study presents a new animal model of tardive dyskinesia with predictive validity, good reliability and, especially, great efficiency.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1999

Behavioural and neurochemical effects of cholinergic and dopaminergic agonists administered into the accumbal core and shell in rats.

The first goal of this study was to investigate whether turning behaviour elicited by unilateral injections of the cholinergic agonist carbachol into the shell of the nucleus accumbens differs from that elicited by similar injections into the core of this nucleus, and to compare the behavioural effects with the known effects of such injections of the mixture of the dopamine D1 and D2 receptor agonists SKF 38393 (5 microg) and quinpirole (10 microg). The second goal was to investigate whether these injections of carbachol produce neurochemical alterations in the ventrolateral striatum that differ from similar injections of the mixture of the dopamine D1 and D2 receptor agonists into these brain regions. Injections of carbachol into the shell produced predominantly (a) contralateral circling marked by normal stepping and running in wide circles during the initial 50 min and (b) postural asymmetry during the following 75 min; similar injections into the core produced (a) contralateral pivoting, namely pathological head-to-tail turning marked by abnormal hindlimb stepping during the initial 50 min and (b) postural asymmetry during the next 75 min. The postural asymmetry seen after the carbachol injections was closely associated with the drug-induced increase in the dopamine release measured by microdialysis in the ipsilateral striatum. Injections of the mixture of dopamine agonists into the shell, but not core, also produced pivoting. These shell injections increased the dopamine release in the ipsilateral striatum, and decreased it in the contralateral striatum. The relative increase in the ipsilateral striatum was closely associated with the drug-induced pivoting. The data show that stimulation of cholinergic and dopaminergic receptors in the shell and core elicit effects that vary according to the subregion of the nucleus accumbens. It is concluded that the accumbens-specific, cholinergic effects are mediated via substrates that differ from those involved in the shell-specific, dopaminergic effects.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1999

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.

Animals↗

PubMed1999

High and low responders to novelty: effects of a catecholamine synthesis inhibitor on novelty-induced changes in behaviour and release of accumbal dopamine.

The purpose of the present study was two-fold: (i) to investigate to what extent novelty, i.e. a novel cage with slightly larger dimensions than the home cage and lacking the floor covering that was originally present, produced behavioural effects in high responders to novelty and low responders to novelty that could be correlated with the extracellular amount of accumbal dopamine, using the microdialysis technique, and (ii) to establish the ability of the catecholamine synthesis inhibitor alpha-methyl-p-tyrosine to inhibit the novelty-induced responses in high and low responders. The difference in the behavioural response to novelty between the high and low responders was limited to walking, which lasted significantly longer in high responders than in low responders. The novelty-induced increase in extracellular concentration of accumbal dopamine was significantly greater in high responders than in low responders; moreover, the shape of the growth curves differed between high and low responders. The behavioural changes did not correlate with the neurochemical effects, which outlasted the duration of the novelty-induced behavioural arousal. It is hypothesized that this long-lasting increase in accumbal dopamine produces "adaptive changes" that help and/or allow the animal to incorporate knowledge about the condition that it experienced. When the nucleus accumbens was perfused with alpha-methyl-p-tyrosine for a period of 40 min, given at the same time as the transfer of the rat to the novel cage, it reduced the novelty-induced increase in walking in the high responders, but did not alter the novelty-induced behaviour of low responders. Finally, alpha-methyl-p-tyrosine reduced the novelty-induced increase in the release of accumbal dopamine in high responders, but enhanced it in low responders. The present neurochemical data are discussed in view of the outcome of earlier reported pharmacobehavioural studies on the neurochemical state of the nucleus accumbens of non-challenged versus challenged high and low responders. It is hypothesized that, in the high responder, exposure to novelty enhances the release of accumbal dopamine from reserpine-resistant, alpha-methyl-p-tyrosine-sensitive pools that are under the stimulatory control of beta-adrenergic receptors in the nucleus accumbens, and that, in the low responder, exposure to novelty enhances the release of accumbal dopamine from reserpine-sensitive, alpha-methyl-p-tyrosine-resistant pools that are under the inhibitory control of alpha-adrenergic receptors in the nucleus accumbens.

Animals↗

PubMed1999

Fentanyl increases dopamine release in rat nucleus accumbens: involvement of mesolimbic mu- and delta-2-opioid receptors.

The effects of the mu-receptor agonist fentanyl on extracellular levels of dopamine in rat nucleus accumbens were studied in awake animals by in vivo brain microdialysis. Fentanyl dose-dependently increased the levels of dopamine when given intravenously (microg/kg) or via a microdialysis probe placed into the ventral tegmental area or the nucleus accumbens (nmol). The effect of fentanyl given into the nucleus accumbens was blocked by systemic administration of the non-selective opioid receptor antagonist naloxone and by accumbens administration of D-Phe-Cys-Tyr-D-Trp-Om-Thr-Phe-Thr-NH2 (nmol), a mu-opioid receptor antagonist, and naltrindole (nmol), a non-selective delta-opioid receptor antagonist, in a dose-dependent manner. The delta2-opioid receptor antagonist, naltriben (nmol), also blocked the effects of fentanyl, whereas the delta1-opioid receptor antagonist, (E)-7-benzylidenenaltrexone (nmol), was ineffective. When marginally effective doses of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 and naltriben were given simultaneously, the effect of fentanyl was nearly fully blocked; the pretreatment itself had no effect. Administration of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (nmol), the delta1-opioid receptor agonist [D-Pen2,5]-enkephalin (nmol) or the delta2-opioid receptor agonist [D-Ala2,Glu4]-deltorphin (nmol) into the nucleus accumbens enhanced the amount of accumbal dopamine. This study provides evidence that not only activation of delta1- and delta2-opioid receptors, but also activation of mu-opioid receptors in the nucleus accumbens increases the release of accumbal dopamine in freely moving rats. We suggest that the effect of intra-accumbens administration of fentanyl upon accumbal release of dopamine is either due to the simultaneous activation of mu-opioid receptors and delta2-opioid receptors or due to activation of mu-opioid receptors that interact with delta2-opioid receptors in a complex manner.

Analgesics, Opioid↗

PubMed1999

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.

Amyloid beta-Protein Precursor↗

PubMed1998

Matrilysin-specific antisense oligonucleotide inhibits liver metastasis of human colon cancer cells in a nude mouse model.

Human colon cancer frequently develops liver metastasis. Matrilysin (MMP-7), the smallest member of the matrix metalloproteinase (MMP) family, is commonly produced by human colon carcinoma cells and has been suggested to be involved in the progression and metastasis of this type of cancer. In the present study, we tested the effect of a matrilysin-specific antisense phosphorothioate oligonucleotide on liver metastasis of the human colon carcinoma cell line WiDr in nude mice. In culture, the antisense oligonucleotide moderately inhibited the secretion of matrilysin by WiDr cells. Injection of WiDr cells into the spleen of nude mice produced many metastatic tumor nodules in the liver. When the antisense oligonucleotide was injected daily into the mice for 11 days, the formation of the metastatic tumor nodules was strongly inhibited in a dose-dependent manner. An inhibition of liver metastasis of over 70% was obtained at a dose of 120 micrograms of the oligonucleotide per mouse. The antisense oligonucleotide did not inhibit tumor growth in spleen and in liver. A scrambled control oligonucleotide had no effect on liver metastasis of WiDr cells. Our results demonstrate an important role of matrilysin in liver metastasis of human colon cancer and the therapeutic potential of matrilysin antisense oligonucleotides for the prevention of metastasis.

Aged↗