Protein Kinase A Inhibits Lysophosphatidic Acid-Induced Migration of Airway Smooth Muscle Cells

ABSTRACT Lysophosphatidic acid (LPA) is a bioactive phospholipid that is released from activated platelets and affects contractile properties of airway smooth muscle cells. However, possible roles of LPA on cell migration, one of the initial events of airway remodeling, are not clarified. This study aimed to examine the effects of LPA on migration and actin fiber formation in bovine tracheal smooth muscle cells (BTSMCs). Random and oriented cell migrations were examined with wound assay and Boyden chamber assay, respectively. Cytosolic actin fibers were stained with rhodaminephalloidin. Membrane translocation of RhoA, a hallmark of RhoA activation, was assessed by Western blotting. LPA augmented the migration of BTSMCs from wounded confluent monolayer but did not accelerate the chemotactic migration toward LPA. LPA also induced a transient actin reorganization and RhoA activation. Dense actin fibers were observed mainly in the wound edge but not in migrated cells, thereby suggesting the role of actin reorganization in the initiation of cell migration. LPA-induced actin fiber formation was blocked by Y27632 [R-(ϩ)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide], an inhibitor of Rho kinase. Effects of LPA on migration and actin fiber formation were also inhibited by cAMP-elevating agents, i.e., dibutyryl cAMP, forskolin, isoproterenol, and theophylline. KT5720 (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester], a protein kinase A (PKA) inhibitor, reversed the inhibitory actins of cAMP on LPA-induced responses. These results indicate that LPA induces cAMP/PKAsensitive, RhoA-mediated random migration of BTSMCs. Regulation of this mechanism would be beneficial for the control of airway remodeling. Proliferation and hypertrophy of airway smooth muscle cells cause airway remodeling Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that is generated in serum This work was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org

NC-6301, a polymeric micelle rationally optimized for effective release of docetaxel, is potent but is less toxic than native docetaxel in vivo

Drug release rate is an important factor in determining efficacy and toxicity of nanoscale drug delivery systems. However, optimization of the release rate in polymeric micellar nanoscale drug delivery systems has not been fully investigated. In this study NC-6301, a poly(ethylene glycol)-poly(aspartate) block copolymer with docetaxel (DTX) covalently bound via ester link, was synthesized with various numbers of DTX molecules bound to the polymer backbone. The number of DTX molecules was determined up to 14 to achieve an optimal release rate, based upon the authors’ own pharmacokinetic model using known patient data. Efficacy and toxicity of the formulation was then tested in animals. When administered three times at 4-day intervals, the maximum tolerated doses of NC-6301 and native DTX were 50 and 10 mg/kg, respectively, in nude mice. Tissue distribution studies of NC-6301 in mice at 50 mg/kg revealed prolonged release of free DTX in the tumor for at least 120 hours, thus supporting its effectiveness. Furthermore, in cynomolgus monkeys, NC-6301 at 6 mg/kg three times at 2-week intervals showed marginal toxicity, whereas native DTX, at 3 mg/kg with the same schedule, induced significant decrease of food consumption and neutrophil count. NC-6301 at 50 mg/kg in mice also regressed a xenografted tumor of MDA-MB-231 human breast cancer. Native DTX, on the other hand, produced only transient and slight regression of the same tumor xenograft. NC-6301 also significantly inhibited growth of OCUM-2MLN human scirrhous gastric carcinoma in an orthotopic mouse model. Total weight of metastatic lymph nodes was also reduced. In conclusion, NC-6301 with an optimized release rate improved the potency of DTX while reducing its toxicity

Advanced small cell carcinoma of the uterine cervix treated by neoadjuvant chemotherapy with irinotecan and cisplatin followed by radical surgery

Small cell carcinoma of the uterine cervix is a rare form of cervical cancer characterized by extreme aggressiveness and poor prognosis because of its rapid growth, frequent distant metastases, and resistance to conventional treatment modalities. We report here a case of advanced-stage small cell carcinoma of the uterine cervix treated by neoadjuvant chemotherapy, followed by radical surgery, resulting in locoregional disease control. A 39-year-old Japanese woman was diagnosed as having stage IIIb small cell carcinoma of the uterine cervix. She was treated by neoadjuvant chemotherapy with irinotecan/cisplatin, followed by extended radical hysterectomy with pelvic and paraaortic lymphadenectomy. The patient was further treated by adjuvant chemotherapy with irinotecan/cisplatin. Intrapelvic recurrence has not been detected throughout the postoperative course. However, the patient died with distant metastases of the disease, 27 months following the initial treatment. It has been suggested that neoadjuvant chemotherapy therapy followed by radical surgery is a treatment option for advanced-stage small cell carcinoma of the uterine cervix for the locoregional disease control. Further studies are necessary to obtain information regarding multimodal treatment including sequence, duration, frequency, and type of effective chemotherapy agents to be used in the treatment of small cell carcinoma of the uterine cervix

The Lectures on the Development of Teaching Plans and Teaching Materials Tried in the Science Education Class in Master's Course, Okayama University

岡山大学大学院教育学研究科の理科教育専攻の講義として、教材開発・授業案開発をテーマとした新しい講義を試みたのでその報告を行う。本講義は、理科教育講座に所属する大学院生、理科教育講座の大学教員、附属学校の理科関係の教諭が三者協働で進めることが特徴である。課題設定、教材開発を含めた実践準備および実践を院生チームを組み遂行させ、さらに経験や専門性の異なる人材と論議を重ねて活動を進めることを通じ、将来、協働で学校現場の課題提案・解決を行うことのできる能力を養うことを目標とした。This is a report of the new type of lectures on the development of teaching plans and teaching materials attempted in the science class in the Master's Course, Okayama University. The lectures were conducted in collaboration of the graduate students and the academic staffs in the science education course, and the science teachers of the attached school of Okayama University. The graduate students were grouped into two teams of five or six people, assigned to decide on themes, prepare and practice the development of teaching plans and teaching materials. They conducted their activities discussing problems with people of different experience and specialty, thus developing the ability to jointly propose and solve problems at schools in future

Mutations in TGFbeta-RII and BAX mediate tumor progression in the later stages of colorectal cancer with microsatellite instability

Abstract Background Microsatellite instability (MSI) occurs in 15% of colorectal cancers (CRC). The genetic targets for mutation in the MSI phenotype include somatic mutations in the transforming growth factor beta receptor typeII (TGFbetaRII), BAX, hMSH3 and hMSH6. It is not clear how mutations of these genes mediate tumor progression in the MSI pathway, and the temporal sequence of these mutations remains uncertain. In this study, early stage CRCs were examined for frameshift mutations in these target genes, and compared with late stage tumors and CRC cell lines. Methods We investigated 6 CRC cell lines and 71 sporadic CRCs, including 61 early stage cancers and 10 late stage cancers. Mutations of repetitive mononucleotide tracts in the coding regions of TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR and Fas antigen were identified by direct sequencing. Results Thirteen (18.3%) of 71 CRC, including 9/61 (14.7%) early stage cancers and 4/10 (40%) late stage cancers, were identified as MSI and analyzed for frameshift mutations. No mutation in the target genes was observed in any of the 9 early stage MSI CRCs. In contrast, frameshift mutations of TGFbetaRII, BAX, hMSH3 and hMSH6 were present in 3/4 late stage MSI tumors. There is a statistical association (p = 0.014) between mutation in any one gene and tumor stage. Conclusions TGFbetaRII, BAX, hMSH3 and hMSH6 mutations are relatively late events in the genesis of MSI CRCs. The frameshift mutations in these target genes might mediate progression from early to late stage cancer, rather than mediating the adenoma to carcinoma transition.</p

Phosphorylated Smad2 in Advanced Stage Gastric Carcinoma

<p>Abstract</p> <p>Background</p> <p>Transforming growth factor β (TGFβ) receptor signaling is closely associated with the invasion ability of gastric cancer cells. Although Smad signal is a critical integrator of TGFβ receptor signaling transduction systems, not much is known about the role of Smad2 expression in gastric carcinoma. The aim of the current study is to clarify the role of phosphorylated Smad2 (p-Smad2) in gastric adenocarcinomas at advanced stages.</p> <p>Methods</p> <p>Immunohistochemical staining with anti-p-Smad2 was performed on paraffin-embedded specimens from 135 patients with advanced gastric adenocarcinomas. We also evaluated the relationship between the expression levels of p-Smad2 and clinicopathologic characteristics of patients with gastric adenocarcinomas.</p> <p>Results</p> <p>The p-Smad2 expression level was high in 63 (47%) of 135 gastric carcinomas. The p-Smad2 expression level was significantly higher in diffuse type carcinoma (p = 0.007), tumours with peritoneal metastasis (p = 0.017), and tumours with lymph node metastasis (p = 0.047). The prognosis for p-Smad2-high patients was significantly (p = 0.035, log-rank) poorer than that of p-Smad2-low patients, while a multivariate analysis revealed that p-Smad2 expression was not an independence prognostic factor.</p> <p>Conclusion</p> <p>The expression of p-Smad2 is associated with malignant phenotype and poor prognosis in patients with advanced gastric carcinoma.</p

Myelin Basic Protein as a Novel Genetic Risk Factor in Rheumatoid Arthritis—A Genome-Wide Study Combined with Immunological Analyses

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10−4 by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10−8, OR 1.23, 95% CI: 1.14–1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease

Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line

<p>Abstract</p> <p>Background</p> <p>Management of peritoneal dissemination is the most critical problem in gastric cancer. This study was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA.</p> <p>Methods</p> <p>The effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA <it>in vivo </it>were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay.</p> <p>Results</p> <p>OCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (<it>P </it>< 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl-α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (<it>P </it>< 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (<it>P </it>< 0.001).</p> <p>Conclusions</p> <p>VPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer.</p