Akihiro Yoshii

A major role for the Rho‐associated coiled coil forming protein kinase in G‐protein‐mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea

G protein‐mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho‐associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y‐27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐induced contraction in α‐toxin‐ or β‐escin‐permeabilized rabbit trachea. Although pre‐treatment with Y‐27632 dose‐dependently inhibited GTPγS (10 μM)‐induced Ca2+ sensitization of α‐toxin‐permeabilized trachea, a Y‐27632‐insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPγS response in the presence of Y‐27632 (100 μM). GF 109203X (5 μM) abolished 1 μM 4β‐phorbol 12, 13‐dibutyrate (PDBu)‐induced, but only partially inhibited the GTPγS‐induced Ca2+ sensitization. A combination of Y‐27632 (100 μM) and GF 109203X (5 μM) totally abolished the GTPγS response. GTPγS caused only a small contraction in the absence of Ca2+. Wortmannin (30 μM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+‐induced contraction. ATP‐triggered contraction of the strip which had been treated with calyculin A (1 μM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 μM), but not by Y‐27632 (100 μM), in the presence of GTPγS and Ca2+. GTPγS, but not PDBu, contracted the β‐escin‐permeabilized trachea in the absence of Ca2+, but the presence of Ca2+‐independent MLCK. We conclude that ROCK plays a primary role in G‐protein‐mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.

Evaluation of Y-27632, a Rho-kinase inhibitor, as a bronchodilator in guinea pigs

To evaluate (+)-(R)-trans-4-(l-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632), a selective Rho-kinase inhibitor, as a novel bronchodilator in vivo and in vitro, we investigated the effect of Y-27632 on the acetylcholine- or ovalbumin-induced increase in lung resistance (R(L)) in non-sensitized or passively sensitized guinea pigs, and the relaxant effects of salbutamol, Y-27632 and theophylline on acetylcholine- or ovalbumin-induced contraction of isolated trachea. Y-27632 inhalation (1 mM, 2 min) inhibited acetylcholine- or ovalbumin-induced increase in R(L) without changes in mean blood pressure, and the effect persisted for at least 3 h. Salbutamol, Y-27632 and theophylline each completely reversed the acetylcholine- or ovalbumin-induced contraction of isolated trachea with rank order of potency, salbutamol>Y-27632>theophylline. The relaxant effect of Y-27632 was not affected by propranolol. We conclude that, although Y-27632 is not as potent as a beta-adrenoceptor agonist, Y-27632 may become an alternative inhaled bronchodilator, because Y-27632 is more potent than theophylline, and the relaxant effect is independent of beta-adrenoceptors.

A phase II study of amrubicin, a synthetic 9-aminoanthracycline, in patients with previously treated lung cancer.

PURPOSE This study was designed to confirm the efficacy and safety of amrubicin, a new anthracycline agent, in patients with previously treated non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). METHODS Eligible patients were required to have recurrent or refractory NSCLC and SCLC after one or two previous chemotherapy regimens. All patients received intravenous amrubicin 35 mg/m(2) on days 1-3 every 3 weeks. Overall response rate (ORR), progression-free survival (PFS), and overall survival (OS) were evaluated. RESULTS Sixty-six patients (37 NSCLC and 29 SCLC) were assessable for efficacy and safety evaluation. Grade 3 or 4 neutropenia was observed in 39.4% of all patients (NSCLC, 37.8%; SCLC, 41.4%). Nonhematological toxicities were mild. No treatment-related death was observed. The ORRs were 13.5% (95% CI, 4.5-28.8%) in NSCLC and 44.8% (95% CI, 26.4-64.3%) in SCLC. In SCLC, ORRs were 60.0% in the sensitive relapse and 36.8% in the refractory relapse (p=0.2332). In NSCLC, the PFS, OS, and 1-year survival were 3.3 months, 12.0 months, and 35.3%, respectively. In SCLC, the PFS, OS, and 1-year survival were 4.0 months, 12.0 months, and 46.7%, respectively. CONCLUSIONS Amrubicin is an active and well-tolerated regimen in patients with previously treated lung cancer. Amrubicin 35 mg/m(2) seems to achieve similar efficacy with less toxicity than amrubicin 40 mg/m(2) in this patient population. These results warrant further evaluation in previously treated lung cancer.

Receptor-dependent G protein-mediated Ca2+ sensitization in canine airway smooth muscle.

To determine the mechanisms of receptor-dependent Ca2+ sensitization in airway smooth muscle, canine tracheal smooth muscle (CTSM) was permeabilized with alpha-toxin or beta-escin. Although the effects of 5-hydroxytryptamine (100 microM), histamine (100 microM), and the thromboxane A2 analogue U-46619 (100 microM) were negligible, carbachol (100 microM) and endothelin-1 (ET-1, 1 microM) evoked additional contractions of 47.0 +/- 5.90% and 25.0 +/- 5.37% (n = 6) at pCa 6.7 with GTP (3 microM) (normalized to the maximum contraction at pCa 4.5) in alpha-toxin-permeabilized CTSM. GDP-beta-S (1 mM) reversed the carbachol and ET-1 responses completely. GTP-gamma-S (30 microM) and 4 beta-phorbol 12,13-dibutyrate (PDBu, 3 microM) increased the Ca2+ sensitivity (median effective pCa) of contraction by 1.8- and 4.4-fold, respectively (n = 4-11, P < 0.05). The effects of saturating concentrations of GTP-gamma-S and PDBu were additive. A synthetic peptide (T2) corresponding to the actin-binding site of calponin caused a dose-dependent contraction of beta-escin permeabilized CTSM, with the peak effect (25 +/- 4%, n = 4) at 1200 microM, PDBu (3 microM) caused contraction of the T2 peptide-treated CTSM. In conclusion, Ca2+ sensitization of CTSM depends on receptor type and is mediated by G proteins and protein kinase C whose effects are additive, with a partial contribution by calponin.

InsP3, but not novel Ca2+ releasers, contributes to agonist-initiated contraction in rabbit airway smooth muscle

1 To examine the contributions of the putative Ca2+ releasers, inositol 1,4,5‐trisphosphate (InsP3), cyclic ADP ribose (cADPR), and nicotinate adenine dinucleotide phosphate (NAADP), to carbachol (CCh)‐induced contraction in airway smooth muscle, we measured force development of permeabilized rabbit tracheal smooth muscle, human bronchial smooth muscle and guinea‐pig ileum longitudinal smooth muscle. 2 In the presence of 50 μm GTP, CCh and InsP3 contracted α‐toxin‐permeabilized tracheal smooth muscle dose dependently; the EC50 values for CCh and InsP3 were 1.84 μm and 363 μm, and the maximum responses (normalized to the 30 mM caffeine response) to 100 μm CCh and to 800 μm InsP3 were 206 ± 13.4 % (mean ± s.e.m.) and 84.4 ± 5.3 %, respectively. 3 However, cADPR (10‐300 μm), β‐NAD+ (2.5 mM), FK506 (30 μm) and NAADP (100 μm) neither contracted the strip by themselves nor affected the subsequent CCh (1 μm) response. α‐Toxin‐permeabilized bronchial smooth muscle and ileum smooth muscle also responded to caffeine, InsP3 and CCh but not to cADPR. 4 Both 100 μm 8‐amino‐cADPR, a selective cADPR antagonist, and 100 μm thionicotinamide‐NADP, a selective NAADP antagonist, failed to inhibit the CCh response, although procaine abolished the caffeine, InsP3 and CCh responses in the permeabilized tracheal smooth muscle. 5 Although inhibition of the caffeine response by 30 μm ryanodine was nearly complete, approximately 30 % of the InsP3 (300 μm) plus GTP (50 μm) response was retained, and the resultant response disappeared after the caffeine response was evoked in the presence of ryanodine. 6 Heparin (300 μg ml−1) blocked InsP3 (300 μm) and CCh (3 μm) responses in β‐escin‐permeabilized tracheal smooth muscle, while Ruthenium Red (100 μm) partially inhibited the CCh response. 7 Collectively, InsP3 but not cADPR or NAADP plays a key role in CCh‐initiated contraction, and InsP3 utilizes a single compartment of the caffeine/ryanodine‐sensitive stored Ca2+ in airway smooth muscle.

A highly selective inhibitor of rho-associated coiled-coil forming protein kinase, Y-27632. Prolongs cardiac allograft survival of the BALB/c-to-c3H/He mouse model

BACKGROUND Current studies provide evidence that a small G protein, RhoAp21, and its target protein, Rho-associated coiled-coil forming protein kinase (ROCK), regulate not only cell shape but also cell migration. However, contribution of Rho/ROCK signaling to graft rejection is unknown. The purpose of this study was to evaluate the inhibitory effect of Y-27632, a highly selective ROCK inhibitor, on rejection of heterotopic cardiac transplantation in mice. METHODS BALB/c (H-2(d)) hearts were transplanted into C3H/He (H-2(k)) as allografts that were full histoincompatibility combinations. The recipients received several doses of Y-27632, commencing 1 day before cardiac transplantation until rejection. We used immunohistochemical study to detect the expression of myocardial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and we immunoenzymatically measured serum interleukin (IL)-6. Furthermore, we evaluated cardiac allograft vasculopathy treated with either FK506 or Y-27632 at Day 100. RESULTS The Y-27632-treated (2 mg/kg/day) allografts prolonged the mean survival time (49.6 +/- 10.1 days, n = 12) as compared with the untreated allografts (8.1 +/- 0.4 days, n = 7, p < 0.001). Histologic examinations of the Y-27632-treated allografts at Day 7 showed greatly reduced leukocyte infiltration compared with the untreated allografts. The Y-27632-treated allografts revealed faint expression of myocardial ICAM-1 and VCAM-1 at Day 7. The serum IL-6 levels also decreased in the Y-27632-treated mice. In the long-surviving Y-27632-treated allografts at Day 100, we saw neither active rejection nor apparent thickening of vascular intima. CONCLUSION Our results suggest that ROCK plays a major role in cardiac rejection in the BALB/c-to-C3H/He mouse model. Inhibition of this Rho/ROCK signaling may be an alternative therapeutic option for managing acute and chronic rejection.

Interferon-γ rescues TNF-α-induced apoptosis mediated by up-regulation of TNFR2 on EoL-1 cells

Recent studies show that apoptosis is important for the resolution of chronic inflammation. Using a human myeloblastic leukemia cell line, EoL-1, we investigated the effect of interferon-gamma (IFN-gamma), which differentiates EoL-1 into monocyte/macrophage-like cells on Fas antigen (Fas)- and tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. Both TNF and anti-Fas monoclonal antibody (CH-11) induced apoptosis of EoL-1 cells. Pretreatment with IFN-gamma for 72 hours enhanced the CH-11-induced apoptosis with up-regulation of Fas. However, the treatment markedly inhibited the TNF-induced apoptosis. In flow cytometric analysis, EoL-1 expressed two types of tumor necrosis factor receptors (TNFR1 and TNFR2), and the expression of TNFR2 but not of TNFR1 was up-regulated significantly after the IFN-gamma treatment. The TNF-induced apoptosis was mimicked by a TNFR1 stimulating antibody (htr-9), and was reversed by a TNFR1 blocking antibody (H398). Although the TNFR1-mediated cytotoxic signal was not affected by IFN-gamma pretreatment, blocking TNFR2 by a specific antagonistic antibody (utr-1) canceled the inhibitory effect of IFN-gamma. In conclusion, TNF-induced apoptosis was mediated preferentially by TNFR1, and the anti-apoptotic effect of IFN-gamma was result from up-regulated TNFR2 in EoL-1 cell line. This cell line is a useful model to provide new insights into crosstalk among Fas/FasL-, TNF-, and IFN-gamma-mediated signaling.

Phase II study of oral S-1 and cisplatin with concurrent radiotherapy for locally advanced non-small-cell lung cancer

PURPOSE To determine the efficacy and safety of oral S-1 in combination with cisplatin and thoracic radiotherapy in patients with unresectable stage III non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS S-1 (50mg/m(2)) was administered orally twice daily for 14 days, with cisplatin (40 mg/m(2)) on days 1 and 8 of each cycle every 3 weeks, for 2-4 cycles. Thoracic radiation therapy was administered in 2 Gy fractions five times weekly for a total dose of 60 Gy. The primary endpoint was the response rate, and secondary endpoints included progression-free survival, overall survival and safety. RESULTS Forty-one patients were enrolled in this study. The objective response rate was 87.8% (98% CI: 77.8-97.8%). The median progression-free survival was 467 days (15.4 months), and the median survival time was 904 days (29.7 months). The overall survival rates at 1- and 2-years were 85.7% and 52.9%, respectively. Hematological toxicities included grade 3/4 neutropenia (17%) and grade 3/4 leukopenia (27%). No grade 3 febrile neutropenia was detected, and grade 3/4 non-hematological toxicities were also mild. A grade 3 gastrointestinal hemorrhage was observed in one patient. CONCLUSIONS The combination of oral S-1 plus cisplatin with concurrent radiotherapy is a promising treatment with a high efficacy and lower toxicity in patients with locally advanced NSCLC.

Phase II study of oral S-1 plus cisplatin with bevacizumab for advanced non-squamous non-small cell lung cancer

BACKGROUND We conducted a phase II study to evaluate the efficacy and safety of S-1 plus cisplatin with bevacizumab followed by maintenance bevacizumab in patients with advanced non-squamous non-small cell lung cancer (NSCLC). PATIENTS AND METHODS Chemotherapy-naïve patients received S-1 plus cisplatin with bevacizumab. S-1 (80 mg/m(2)) was administered orally twice daily for 14 days, cisplatin (60 mg/m(2)) on day 1, and bevacizumab (15 mg/kg) on day 1 and every 3 weeks for 4-6 cycles. Patients with an objective response or stable disease received maintenance bevacizumab every 3 weeks until disease progression. RESULTS Thirty patients were enrolled in this study. The median number of chemotherapy was four (range, 1-6 cycles), and the median number of bevacizumab alone was three (range, 1-31 cycles). The grade 3/4 toxicities were neutropaenia (23%), thrombocytopaenia (10%), febrile neutropaenia (3%), hypertension (17%), pneumonia (7%), and bowel perforation (3%). The objective response rate was 71% (95% CI, 55-88%) for a disease control rate of 100%. The median progression-free and overall survival times were 7.0 months and 20.0 months, respectively. CONCLUSIONS S-1 plus cisplatin with bevacizumab is an active and well-tolerated regimen in patients with chemotherapy-naïve non-squamous NSCLC.

Clinical impact of postprogression survival for overall survival in elderly patients (aged 75 years or older) with advanced nonsmall cell lung cancer.

INTRODUCTION The effects of first-line single-agent chemotherapy on overall survival (OS) might be confounded by subsequent treatments in elderly patients with nonsmall cell lung cancer (NSCLC). We, therefore, aimed to evaluate whether progression-free survival (PFS), postprogression survival (PPS), or tumor response might be a valid surrogate endpoint for OS in this patient population. PATIENTS AND METHODS We retrospectively reviewed the clinical data of 58 elderly patients with advanced NSCLC, who received first-line single-agent cytotoxic chemotherapy at our institution between October 2003 and November 2013. The relationships of PFS, PPS, and tumor response with OS were individually analyzed. RESULTS The study cohort included 46 men and 12 women with a median age of 79 years (range: 75-87 years). There were 30 adenocarcinomas, 22 squamous cell carcinomas, and 6 other histologic types with 1 stage IIIA, 9 IIIB, and 48 IV cases. The performance status (PS) scores were 0, 1, and 2 in 18, 35, and 5 patients, respectively. The median PFS and OS were 2.8 and 5.4 months, respectively. Our analyses revealed a strong correlation of PPS and PFS with OS, whereas that between tumor shrinkage and OS was weak. Tumor stage and PS after initial treatment were significantly associated with PPS. Individual analysis indicated that PPS might serve as a surrogate for OS in elderly patients with advanced NSCLC receiving first-line single-agent chemotherapy. CONCLUSION Our findings suggested that the disease course after progression following first-line single-agent chemotherapy might influence the OS of elderly patients with advanced NSCLC.