近藤 征史

Pleural plaques are asymptomatic focal thickenings of the pleura and considered the hallmark of asbestos exposure. However, it is often difficult to detect pleural plaques on chest x-rays (CXR). In a retrospective study, using chest CT scans of 140 Japanese asbestos-exposed construction workers who have probable or definite findings of pleural plaque on CXR; firstly, we proposed plaque morphology-based classification for CXR findings, and then we examined if those classified findings could be confirmed as pleural plaques on CT scans. Our morphology-based classification of pleural plaque findings included nine types. The percentages of confirmed pleural plaques on CT scans by type (number of confirmed pleural plaque on CT/number of observed on CXR) were 93% (40/43) for straight, 89% (56/63) for diamond, 88% (7/8) for double, 83% (19/23) for tapered medially, 80% (20/25) for parallel, 77% (23/30) for crescent, 79% (11/14) for tenting, 72% (18/25) for tapered-laterally (long type), and 0% (0/9) for tapered-laterally (short type). When added to the ILO classification, morphology-based classification of CXR pleural plaque findings makes its detection easier and hence chest radiograph continues to be a suitable tool for screening asbestos-related pleural plaques based on its simplicity, low radiation exposure, wide availability and cost-effectiveness.

疼痛緩和治療におけるオピオイドの適正使用の立案と実施を目的として、オピオイドによる消化器症状の副作用と、それらの発現頻度・程度を増加させる可能性のある因子との関連性について検討した。名古屋大学医学部附属病院呼吸器内科病棟において、オピオイドを新規に導入した肺がんの入院患者37名を対象とした。オピオイド導入時に全身状態の指標であるperformance status(PS)が3以上またはbest supportive care(BSC)の患者において、オピオイドによる便秘、悪心または嘔吐のいずれかが発現した患者の割合は、PS2以下またはBSCでない患者のそれに比べ、有意に増加していた。そのリスク比は、それぞれ3.94(p<0.01)、2.30(p<0.01)であった。オピオイドの導入時期が遅延するほど副作用発現頻度・程度が増悪するため、早期から適切に疼痛評価を行うことや、オピオイド導入が必要な場合は迅速にオピオイドを導入することが提唱されている。それらに加え、本調査結果から、全身状態が悪化あるいはBSCの患者にオピオイドを導入する場合は、副作用対策を強化する必要があると示唆される。(著者抄録)

The Calvert formula, that is, carboplatin dose (mg) = target area under the concentration versus time curve (AUC) x (glomerular filtration rate [GFR] + 25), has not been validated in Japanese subjects in whom the GFR was accurately measured. The purpose of this study is to evaluate the validity of this formula for Japanese patients with cancer and modify it for this population. The GFR was measured on the basis of inulin clearance, which is considered to reflect the accurate GFR. Inulin clearance was measured in 28 patients with cancer. The adjusted 24-h creatinine clearance (24-h Ccr) was unbiased (mean prediction error [MPE] +/- SE = -2.3 +/- 4.5%) and acceptably precise (root mean squared error = 23.7%) for GFR assessment. The pharmacokinetics of carboplatin were analyzed in 21 patients with a GFR of 17.2-91.4 mL/min. The original Calvert formula overestimated carboplatin clearance, resulting in an MPE of 14.3%. When we revised the Calvert formula for Japanese patients by substituting a non-renal clearance of 15 for 25, that is, dose = target AUC x (GFR + 15), the MPE decreased to -0.1% (P < 0.001). We conclude that the adjusted 24-h Ccr is acceptably precise for GFR assessment, and the non-renal clearance of carboplatin is suggested to be lower in Japanese patients with cancer than in their Western counterparts. (Cancer Sci 2010; 101: 2601-2605).

The aim of this phase II study was to evaluate the feasibility and safety of a carboplatin and gemcitabine combination regimen in the treatment of completely resected non-small cell lung cancer (NSCLC). Patients with completely resected pathologically documented stage IB, II or IIIA NSCLC were treated with carboplatin and gemcitabine. Chemotherapy consisted of 4 cycles of carboplatin at an area under the curve of 5 (level 1) or 4 (level 2) on day 1 combined with gemcitabine 1,000 mg/m(2) on days 1 and 8 every 3 weeks. The primary endpoint of this study was the completion rate of 4 cycles. Twenty patients were treated, and the patient's demographics were: median age 61 years (range 51-74), gender male (n = 13, 65%)/female (n = 7, 35%), stage IB (n = 8, 40%), IIA (n = 1, 5%), IIB (n = 6, 30%), IIIA (n = 5, 25%). Seventeen patients (85%, 95% confidence interval 64.0-94.8) received the planned 4 cycles of the chemotherapy regimen at level 1 every 3 weeks. Among the 3 patients who failed to complete 4 cycles, the reasons for stopping were refusal (n = 1), thrombocytopenia (n = 1) and rash (n = 1). The main adverse effects were hematological toxicity as well as grade 3/4 neutropenia and thrombocytopenia (which occurred in 65% and 40% of the patients, respectively). Adjuvant chemotherapy with a carboplatin and gemcitabine combination regimen has an acceptable toxicity profile, and the majority of patients completed 4 cycles of therapy.

We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes (ZEB1, SIP1, Snail, and Slug) ZEB1expression was most significantly correlated with the mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore. ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

We conducted a phase II trial to evaluate the safety and efficacy of weekly paclitaxel in patients with resistant or relapsed non-small cell lung cancer (NSCLC) treated with docetaxel and carboplatin. Thirty-two NSCLC patients at a median age of 58.0 years (range 33-75) were enrolled. The Eastern Cooperative Oncology Group performance status scores (0/1/2) were 18/9/5, respectively. The majority of patients had adenocarcinoma (84%) and stage IV disease (81%). The response rate for the first-line chemotherapy was 28%. Paclitaxel was administered at a dose of 80 mg/m(2) as an intravenous infusion 60 min weekly for 6 consecutive weeks of an 8-week cycle. All patients were assessable for response and toxicity. The median number of cycles administered was two (range 1-8), and the overall response rate was 15.6%. The median survival time (MST) was 10.6 months (95% CI = 8.2-12.5), while the 1-year survival rate was 37.5%, and the median progression-free survival was 4.9 months (95% CI = 3.0-7.1). Hematological toxicities (grade 3 or 4) were observed in 15 patients (46.9%) with leukopenia, and in 4(12.5%) with anemia. Non-hematological toxicity was generally mild, though grade 3 anorexia was observed in 3 patients (9.3%). No treatment-related deaths were observed. In conclusion, second-line weekly paclitaxel is effective in NSCLC patients treated with docetaxel plus carboplatin and is associated with a tolerable toxicity profile. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca(2+) concentration ([Ca(2+)](i)), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca(2+)](i), as measured by fura-2 fluorescence, was transiently increased in a strain amplitude-dependent manner. The elevation of [Ca(2+)](i) induced by stretch was not evident in the Ca(2+)-free solution and was blocked by Gd(3+), a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca(2+)](i). In contrast, increases in [Ca(2+)](i) induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca(2+)](i). A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca(2+) channels. In conclusion, mechanical stretch activates Ca(2+) influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton different from other Ca(2+) influx pathways such as receptor-operated and store-operated Ca(2+) entries in HPMVECs. These results suggest that abnormal Ca(2+) homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.

The present study concerns previously unreported effects of menthol, a cyclic terpene alcohol produced by the peppermint herb, on anion transporters in polarized human airway Calu-3 epithelia. Application of menthol (0.01-1 mM) attenuated transepithelial anion transport, estimated as short-circuit currents (I(SC)), after stimulation by forskolin (10 mu M) but not before. In contrast, menthol potentiated forskolin-stimulated and -unstimulated apical Cl(-) conductance, which reflected the cystic fibrosis transmembrane conductance regulator (CFTR: the cAMP-regulated Cl(-) channel)-mediated conductance, without correlation to changes in cytosolic cAMP levels. These results indicate that menthol-induced attenuation of forskolin-induced I(SC) despite CFTR up-regulation was due to cAMP-independent inhibition of basolateral anion uptake, which is the rate-limiting step for transepithelial anion transport. Analyses of the responsible basolateral anion transporters revealed that forskolin increased both bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2Cl(-) cotransporter [NKCC1])- and DNDS (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters [NBC1/ AE2])-sensitive I(SC) in the control whereas only the former was prevented by the application of menthol. Neither the bumetanide- nor DNDS-sensitive component was, however, reduced by menthol without forskolin. These heterologous effects of menthol were reproduced by latrunculin B. an inhibitor of actin polymerization. F-actin staining showed that menthol prevented forskolin-stimulated rearrangements of actin microfilaments without affecting the distribution of forskolin-unstimulated microfilaments. Collectively, menthol functions as an activator of CFTR and prevents activation of NKCC1 without affecting NBC1/AE although all of these transporters are commonly cAMP-dependent. The heterologous effects may be mediated by the actin cytoskeleton, which interacts with CFTR and NKCC1. (C) 2010 Elsevier B.V. All rights reserved.

A 55-year-old woman treated with infliximab for rheumatoid arthritis was admitted due to progressive ascites. A CT scan showed massive ascites, bilateral pleural effusion, disseminated granular shadows in the lung, and multiple swollen mediastinal lymph nodes. A FDG-PET/CT scan showed increases of FDG uptake in the mesentery and peritoneum, mimicking peritoneal carcinomatosis. Subsequent pleural and peritoneal fluid analysis showed elevated adenosine deaminase activity levels with no malignant cells. A right pleural biopsy specimen revealed Langhans giant cells and granulomas. Finally, a diagnosis of miliary tuberculosis was established because cultures of the sputum and gastric fluid were positive for Mycobacterium tuberculosis. Several weeks after a standard anti-tuberculosis regimen with 4 drugs was initiated, her clinical condition and radiological findings ameliorated. Since the initial manifestations of tuberculosis tend to be more severe during treatment of rheumatoid arthritis with tumor necrosis factor-alpha inhibitors such as infliximab due to immune suppression, we should pay closer attention to the possibility of tuberculosis infection in these patients.

In order to promote the appropriate control of cancer pain by opioids, we distributed a pocket-sized protocol pamphlet on cancer pain control and opioid prescription to the medical staff of Nagoya University Hospital. In this study, we examined whether the prescription rate of opioids for rescue use, antiemetics for preventing adverse effects, and the rate of increase of regular opioid dosage were increased after distribution to evaluate its utility. Rescue opioid prescriptions, increase of regular opioid dosage and antiemetic prescription rate after distribution were all significantly increased, compared with before distribution. Furthermore, the frequency of nausea and vomiting was reduced by the use of prophylactic antiemetics. These results suggest that distribution of this protocol for cancer pain control may contribute to appropriate pain management.

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073-82]

In patients with acute respiratory distress syndrome, mechanical over-distension of the lung by a large tidal volume causes further damage and inflammation, called ventilator-induced lung injury (VILI), however, it is unclear how mechanical stretch affects the cellular functions or morphology in human pulmonary microvascular endothelial cells (HPMVECs). IL-8 has been proposed to play an important role in the progression of VILI by activating neutrophils. We demonstrated that HPMVECs exposed to cyclic uni-axial stretch produce IL-8 protein with p38 activation in strain- and time-dependent manners. The IL-8 synthesis was not regulated by other signal transduction pathways such as ERK1/2, JNK, or stretch-activated Ca(2+) channels. Moreover, cyclic stretch enhanced IL-6 and monocyte chemoattractant protein-1 production and reoriented cell perpendicularly to the stretch axis accompanied by actin polymerization. Taken together, IL-8 production by HPMVECs due to excessive mechanical stretch may activate neutrophilic inflammation, which leads to VILI. (C) 2009 Elsevier Inc. All rights reserved.

Endothelin-1 is considered to be an important mediator in the pathophysiology of asthma because it induces contraction, hypertrophy, and proliferation in airway smooth muscle cells as well as inflammatory responses in the airway. Airway smooth muscle cells have been suggested to contribute to airway inflammation in asthma by producing cytokines. Nevertheless, the role of intracellular Ca2+ signal in cytokine production in human airway smooth muscle cells is still unclear. We investigated the mechanisms by which endothelin-1 induces production of interleukin (IL)-6, a pleiotropic cytokine, in primary cultured human airway smooth muscle cells. Levels of IL-6 protein and mRNA were significantly increased by endothelin-1 in dose- and time-dependent manners. Endothelin-1-induced IL-6 production was markedly attenuated by EGTA and various Ca2+ channel inhibitors such as 3,5-bis(trifluoromethyl)-1H-pyrazole derivative (BTP-2), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365), and nifedipine. Endothelin-1-induced increases in intracellular Ca2+ concentrations were significantly inhibited in Ca2+-free solution and by BTP-2, SKF96365, and nifedipine. The IL-6 synthesis was also inhibited by the extracellular signal-regulated kinase (ERK)1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate (U0126) and the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4pyridyl)1H-imidazole (SB203580), but not by the c-Jun NH2-terminal kinase inhibitor anthra[1,9-cd]-pyrazol-6- (2H)-one (SP600125). Endothelin-1 significantly upregulated phosphorylation of ERK1/2 and p38 but blocking Ca2+ influx pathways did not inhibit either upregulation. These findings demonstrate that endothelin-1-induced IL-6 synthesis in airway smooth muscle cells occurs via two parallel but independent events that include Ca2+ influx and activation of ERK1/2 and p38. (C) 2009 Elsevier B.V. All rights reserved.

We report a case of idiopathic pulmonary veno-occlusive disease (PVOD). The patient experienced progressively worsening dyspnea. Heart catheterization revealed severe pulmonary hypertension. High-resolution computed tomography (HRCT) showed diffuse, poorly identified centrilobular ground-glass opacities. Surgical lung biopsy led to the diagnosis of PVOD. A microscopic examination revealed occlusions of pulmonary veins and venules over a wide area with prominent loop-like capillary dilatations. These pathological findings may be correlated with the radiological characteristics of HRCT in this case.

Menthol, known as a cold receptor agonist, has widely been used in the relief of respiratory symptoms such as coughing and chest congestion. Previous studies have demonstrated that menthol reduces bronchoconstriction and airway hyperresponsiveness. The aim of this study was to examine the effects of menthol and icilin, another cold receptor agonist, on airway smooth muscle contraction. Isometric force was monitored using epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. Intracellular Ca(2+) concentrations were assessed by fura-2 fluorescence. (-)Menthol (0.01-1 mM) inhibited contraction induced by methacholine (MCh, 0.01-10 mu M) and high extracellular K(+) concentrations (20-60 mM) in a concentration-dependent manner. Moreover, the increases of intracellular Ca(2+) concentrations induced by MCh or high K(+) were significantly reduced by (-)menthol. Icilin (100 mu M) also significantly attenuated contraction induced by MCh or high K(+). The inhibitory effect of 1 mM (-)menthol on MCh-induced contraction was significantly higher at cool temperature (24-26 degrees C) than at 37 degrees C. The present results demonstrate that inhibition of Ca(2+) influx plays an important role in the menthol-mediated inhibition of contraction in airway smooth muscle. Furthermore, our findings indicate that stimulation of unknown cold receptors may be involved in these mechanisms. These findings suggest that the use of menthol is beneficial for reducing respiratory symptoms because of its inhibitory effects on airway smooth muscle contraction. (c) 2008 Elsevier Ltd. All rights reserved.

In response to mechanical stretch, airway smooth muscle exhibits various cellular functions such as contraction, proliferation, and cytoskeletal remodeling, all of which are implicated in the pathophysiology of asthma. We tested the hypothesis that mechanical stretch of airway smooth muscle cells increases intracellular Ca2+ concentration ([Ca2+](i)) by activating stretch-activated (SA) nonselective cation channels. A single uniaxial stretch (3 s) was given to human bronchial smooth muscle cells cultured on an elastic silicone membrane. After the mechanical stretch, a transient increase in [Ca2+], was observed. The [Ca2+](i) increase was significantly dependent on stretch amplitude. The augmented [Ca2+](i) due to stretch was completely abolished by removal of extracellular Ca2+ and was markedly attenuated by an application of Gd3+, an inhibitor of SA channels, or ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor. In contrast, the stretch-induced rises of [Ca2+](i) were not altered by other Ca2+ channel inhibitors such as nifedipine, BTP-2, and SKF-96365. Moreover, the [Ca2+](i) increases were not affected by indomethacin, a cyclooxygenase inhibitor, U-73122, a phospholipase C inhibitor, or xestospongin C, an inhibitor of the inositol-trisphosphate receptor. These findings demonstrate that a novel Ca2+ influx pathway activated by mechanical stretch, possibly through the Ca2+-permeable SA channel activated directly by stretch rather than by indirect mechanisms via intracellular messenger production, is involved in human airway smooth muscle cells. A molecular candidate for the putative SA channel may be one of the members of the TRPV channel family. Thus, abnormal Ca2+ homeostasis in response to excessive mechanical strain would contribute to the pathogenesis of asthma.

(一般演題(口演)9 癌性胸膜炎・心膜炎,第48回日本肺癌学会総会号)

Background The release of adenosine triphosphate (ATP) from the airway epithelial cells during the inflammatory process is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. Objective This study was designed to determine whether extracellular ATP is involved in the bronchial hyperresponsiveness as an interaction between epithelium and smooth muscle in the airways. Methods We examined the contractile response to methacholine (MCh) before and after exposure to low concentrations (<= 10 mu m) of ATP in isolated, epithelium-denuded guinea-pig tracheal smooth muscle by measuring isometric tension. Intracellular Ca2+ concentrations ([Ca2+](i)) were assessed by fluorescent intensities of fura-2. Results MCh-induced contractile force was increased with no change in [Ca2+](i) after exposure to 10 pm ATP for 15 min. The ability of ATP to enhance the MCh-induced contraction was markedly attenuated by suramin, a non-selective P2 receptor inhibitor. Pre-incubation with ATP gamma S, a non-hydrolysable analogue of ATP and alpha,beta-meATP, a P2X agonist, also enhanced the MCh-induced contraction. In contrast, uracil triphosphate, a P2Y agonist, did not affect the MCh-induced contraction. Y-27632, a Rho-kinase inhibitor, suppressed the ability of ATP to enhance the MCh-induced contraction. Moreover, PP1 and PP2, Src tyrosin kinase inhibitors, suppressed the enhancement of MCh-induced contraction by ATP. Conclusion Pre-treatment with ATP induces hyperresponsiveness to MCh mediated by Ca2+ sensitization via the P2X receptor in airway smooth muscle. The present findings suggest the possible involvement of both the Rho-kinase and Src pathways in the intracellular mechanism of this phenomenon.

(第90回 日本肺癌学会中部支部会,支部活動)

Identification of a homozygous deletion in cancer cells provides strong evidence for the location of a tumor suppressor gene (TSG). We analyzed the 2p24 homozygous deletion of a non-small-cell lung cancer (NSCLC) cell line, NCI-H2882, and found that the deletion size was 3.7 Mbp. Since RhoB, which has been suggested to be a candidate TSG, was located in this region, we analyzed RhoB for alterations in NSCLC. Although we found no mutations in 48 cell lines including 20 NSCLCs, a loss of heterozygosity (LOH) analysis in 128 primary NSCLCs showed that 25 of 62 informative samples had LOH at the RhoB locus. Northern blot analysis of 28 cell lines (including 15 NSCLCs) indicated that RhoB expression was downregulated in 27. We analyzed RhoB expression in 112 primary NSCLCs with immunohistochemistrv and found no or a weak RhoB expression in 33 (42%) of 78 adenocarcinomas, whereas we found it in 29 (94%) of 31 squamous cell carcinomas. No or a weak expression of RhoB was more frequently observed in poorly- or moderately-differentiated adenocarcinomas than in well-differentiated ones (p = 0.0014). Furthermore, no or a weak expression of RhoB indicated a tendency to poor patient prognosis. Although hypermethylation was not to found at the promoter region, the RhoB expression in NSCLC cell lines was induced by histone deacetylase inhibition, suggesting that RhoB downregulation may be due to histone modification. The present study demonstrates that RhoB expression is frequently downregulated in NSCLCs by multiple mechanisms, suggesting that RhoB is a candidate TSG or NSCLC. (c) 2006 Wiley-Liss, Inc.

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyperreactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F-340/F-380). A 15-min pretreatment with S1P (> 100 nM) markedly enhanced methacholine-induced contraction without elevating F-340/F-380. This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-transN-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca2+ mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (< 100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (< 100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca2+ sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.

Reactive oxidant species are implicated in the chronic airway inflammation related to asthma and chronic obstructive pulmonary disease. This study was designed to determine mechanisms underlying contraction induced by hydrogen peroxide (H2O2), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca2+ concentrations ([Ca2+](i)), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H2O2 (0-01-1 mM) caused contraction with an augmentation of [Ca2+](i) in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca2+ concentrations. The contractile force and [Ca2+](i) by H2O2 (I mM) were approximately half of those in response to 1 mu M methacholine. However, contraction by H2O2 was not generated under the condition that extracellular Ca2+ concentrations were less than 0.15 mM. Verapamil (10 mu M), an inhibitor of voltage-operated Ca2+ channels, partially but significantly inhibited the H2O2-induced contraction. In contrast, SKF-96365 (1-beta-[3-(4-methoxyphenyl)propoxy]-4methoxyphenethyl)-1H-imidazole hydrochloride) (100 mu M), a non-selective inhibitor of Ca2+ channels, completely abolished both the contraction and the increase in [Ca2+](i) elicited by H2O2. Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03-10 mu M), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H2O2-induced contraction. In conclusion, both the Ca2+ influx from the extracellular side and the Ca2+ sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H2O2. An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress. (c) 2006 Elsevier B.V All rights reserved.

Enhanced proliferation of smooth muscle cells contributes to airway remodeling of bronchial asthma. Recently, statins, inhibitors of 3-hydroxy-3-methylglutaryi-coenzyme A reductase, have been shown to inhibit proliferation of both vascular and airway smooth muscle cells independently of lowering cholesterol. However, the mechanisms remain to be elucidated. The purpose of this study was to determine molecular processes by which statins inhibit proliferation of human bronchial smooth muscle cells. Sirrivastatin (0.1-1.0 mu M) significantly inhibited cell proliferation and DNA synthesis induced by FBS in a concentration-dependent manner. The inhibitory effects of simvastatin were antagonized by mevalonate and geranylgeranylpyrophosphate, whereas the effects were not affected by squalene and farnesylpyrophosphate. The antiproliferative effects of simvastatin were mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, and Y-27632, an inhibitor of Rho-kinase, a target protein of RhoA. Western blot analysis showed that the level of membrane localization of RhoA (active Rho) was markedly increased by FBS, and that the level of active RhoA increased by FBS was reduced by simvastatin. Moreover, the inhibitory effect of simvastatin on FBS-induced RhoA activation was also antagonized by geranylgeranylpyrophosphate, but not by farnesylpyrophosphate. Because these isoprenoids are required for prenylation of small G proteins RhoA and Ras, respectively, the present results demonstrate that an inhibition in airway smooth muscle cell proliferation by simvastatin is due to prevention of geranylgeranylation of RhoA, not farnesylation of Ras. Therefore, statins may have therapeutic potential for prohibiting airway remodeling in bronchial asthma.

In guinea pigs, it is well-known that mechanical stretch of airway smooth muscle exhibits spontaneous tone which is mediated by cyclooxygenase (COX) activation. We tested the hypothesis that this spontaneous contraction of airway smooth muscle is mediated by stretch-activated non-selective cation channels and the Rho/Rho-kinase pathway, as well as COX-2 using a pharmacological approach. Isometric force and intracellular Ca2+ concentrations ([Ca2+](i)) were assessed in isolated guinea pig tracheal smooth muscle tissues. The samples were stretched to a given level and the muscle behavior was monitored under isometric conditions. We observed an increase in [Ca2+](i) and subsequent force generation over a 15-min period. The augmented [Ca2+](i) and spontaneous contraction due to the stretch were markedly attenuated by application of Gd3+, an inhibitor of stretch-activated channels, and removal of extracellular Ca2+. In contrast, nifedipine only had a mild inhibitory effect on the contraction. (R)-(+)trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide (Y-27632; a Rho-kinase inhibitor) abolished the spontaneous contraction with no changes in [Ca2+](i). Simvastatin, which down-regulates Rho activity, also significantly inhibited the contraction. Moreover, indomethacin, an inhibitor of COX-1 and -2, and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; a COX-2 inhibitor) abolished the stretch-induced contraction without affecting [Ca2+](i), whereas the inhibitory effect of 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC560; a COX-1 inhibitor) on the contraction was much less. These findings demonstrated that Ca2+ entry via stretch-activated channels, the Rho/Rho-kinase pathway, and COX-2 are involved in the mechanotransduction in guinea pig tracheal smooth muscle. Additionally, while the Rho/Rho-kinase pathway and COX-2 regulate the spontaneous contraction independently of [Ca2+](i), COX-1 is not involved in the stretch-induced force generation. (c) 2006 Elsevier B.V. All rights reserved.

(症例6, 第47回日本肺癌学会総会)

Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription-polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild-type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second-hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light-smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC.

The Hedgehog (Hh) signaling pathway and the Wnt signaling pathway are known to play important roles in carcinogenesis and the progression of various human malignant tumors. Although a relationship between these two pathways has recently been reported, the mechanism by which β-catenin, one of the key molecules of the Wnt signaling pathway, influences the Hh pathway has not yet been revealed in detail. To clarify the role of β-catenin in relation to the Hh signaling pathway, we transfected GLI1 and β-catenin expression constructs into human malignant cells, including stomach, colon, and lung cancers, and evaluated the luciferase activity of GLI-responsive reporter constructs. While exogenous GLI1 increased the luciferase activity, exogenous β-catenin also enhanced the activity under overexpression of GLI1. However, co-transfection with T-cell factor (TCF)-4 or lymphocyte enhancer factor (LEF)-1 did not influence the activity, indicating that the enhancement of β-catenin in relation to the Hh signaling pathway is not TCF/LEF-dependent. Our results suggest that β-catenin might be involved in the Hh signaling pathway via enhancement of the transcriptional activity of GLI.