Akihito Yokoyama

Airflow Limitation in Smokers Is Associated with Subclinical Atherosclerosis

RATIONALEnChronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality from cardiovascular disease. Although a close association between COPD and atherosclerosis has been speculated, such scientific information is limited.nnnOBJECTIVESnTo evaluate subclinical atherosclerosis in smokers with airflow limitation.nnnMETHODSnThe subjects of this study were healthy middle-aged men. Smokers with airflow limitation (n = 61) and age-matched control smokers (n = 122) and control never-smokers (n = 122) without airflow limitation were included in the present study. Subjects with diabetes, acute infection, and respiratory disease other than COPD were excluded beforehand. All subjects underwent chest radiogram, spirometry, blood sampling, and carotid ultrasonography. We determined carotid intima-media thickness and focal atheromatous plaque as indicators of subclinical atherosclerosis.nnnMEASUREMENTS AND MAIN RESULTSnMean carotid intima-media thickness was greater in smokers with airflow limitation than in control smokers (P < 0.01) and control never-smokers (P < 0.005). Focal carotid plaque was significantly more prevalent in smokers with airflow limitation than in control never-smokers (P < 0.005). Multivariate analyses showed significant associations between thickened intima-media thickness and decreased percent predicted FEV(1) (P = 0.001) and between plaque and log(10) C-reactive protein (P = 0.013) independent of age, pack-years of smoking, body mass index, peripheral mean arterial pressure, heart rate, glucose, and low-density lipoprotein cholesterol.nnnCONCLUSIONSnSmokers with airflow limitation had exaggerated subclinical atherosclerosis. This study suggests that middle-aged men who are susceptible to COPD may also be susceptible to vascular atherosclerosis by smoking, and atherosclerotic change starts early in the disease process of COPD.

Suppression of plasminogen activator inhibitor-1 by RNA interference attenuates pulmonary fibrosis

Background and aim There is a growing body of evidence demonstrating that plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. In fact, PAI-1 knockout mice are protected from bleomycin-induced pulmonary fibrosis. This study was conducted to determine whether the intrapulmonary administration of small interfering RNA (siRNA) targeting PAI-1 (PAI-1-siRNA) limits the development of bleomycin-induced pulmonary fibrosis. Methods Lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) were stained for PAI-1. The distribution of siRNA in the lung, the PAI-1 level in bronchoalveolar (BAL) fluid and the extent of fibrotic changes in the lung were evaluated following the intranasal administration of PAI-1-siRNA in a mouse model of bleomycin-induced pulmonary fibrosis. The effect of PAI-1-siRNA on the epithelial to mesenchymal transition (EMT) was also evaluated using a mouse lung epithelial cell line, LA-4. Results PAI-1 was overexpressed in the hyperplastic type 2 pneumocytes lining the honeycomb lesions of patients with IPF. The single intranasal instillation of PAI-1-siRNA resulted in the diffuse uptake of siRNA into the epithelial cells lining the dense fibrotic lesions. The repeated administration of PAI-1-siRNA initiated during either the inflammatory or the fibrotic phase into bleomycin-injured mice reduced the PAI-1 level in BAL fluid and limited the accumulation of collagen in the lungs. EMT induced by transforming growth factor β (TGFβ) in LA-4 cells was inhibited by transfection with PAI-1-siRNA. Conclusions The direct suppression of PAI-1 in the lung by the intrapulmonary administration of PAI-1-siRNA attenuated the development and progression of pulmonary fibrosis. The inhibition of EMT may be, at least in part, involved in this effect.

A novel treatment strategy targeting polo-like kinase 1 in hematological malignancies

Objectives:u2003Polo-like kinase1 (PLK1) belongs to the family of serine/threonine kinases and plays an important role in centrosome maturation, bipolar spindle formation, and cytokinesis during mitosis. We found in this study that PLK1 was aberrantly highly expressed in a variety of human leukemia cell lines (n=20), as well as, freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n=50) and acute lymphoblastic leukemia (n=15) compared with bone marrow mononuclear cells from healthy volunteers (n=13) (acute myelogenous leukemia, P=0.016; acute lymphoblastic leukemia, P=0.008), as measured by real-time RT–PCR. Downregulation of PLK1 by a small interfering RNA in NB4 acute myelogenous leukemia cells inhibited their proliferation. GW843682X is a novel selective PLK1 inhibitor. The compound-induced growth inhibition, caused accumulation of cells in the G2/M phase of the cell cycle and mediated apoptosis of human leukemia cells. Pre-treatment of cells with the caspase inhibitor Z-VAD-FMK attenuated the action of GW843682X in leukemia cells, indicating the involvement of the caspase pathway in the PLK1 inhibitor-mediated apoptosis. Furthermore, we found that the PLK1 inhibitor synergistically potentiated the growth inhibition and apoptosis of leukemia cells when combined with tubulin-depolymerizing agent vincristine. Taken together, targeting PLK1 may be a promising treatment strategy for individuals with leukemia.

Analysis of Aurora B kinase in non-Hodgkin lymphoma

This study explored the levels of Aurora B, a key regulator of mitosis, in 71 lymph nodes and tumor specimens excised operatively from individuals with various types of non-Hodgkin lymphoma (NHLs). Immunohistochemical examination found that diffuse large B-cell lymphoma (10/21, 48%) and Burkitt lymphoma (BL) (6/7, 86%) cells highly (percentage of positive cells, >20%) expressed Aurora B in their nuclei. On the other hand, none of the low-grade B-cell lymphoma (n=20), except for one case of follicular lymphoma, highly expressed this protein kinase, suggesting that levels of Aurora B correlated with histological grade in B-cell NHLs (P<0.01). Exposure of BL/leukemia cells to AZD1152-HQPA in vitro, a selective inhibitor of Aurora B kinase, potently induced growth arrest and apoptosis in a caspase-dependent, as well as -independent manner. Moreover, AZD1152 synergistically enhanced the effects of vincristine (VCR) to induce growth arrest of these cells. Further experiments found that VCR increased levels of the p-Aurora B through the activation of c-Jun N-terminal kinase, which was blocked in the presence of AZD1152-HQPA.

Long-term exposure of leukemia cells to multi-targeted tyrosine kinase inhibitor induces activations of AKT, ERK and STAT5 signaling via epigenetic silencing of the PTEN gene

Imatinib induces complete molecular response in patients with chronic myeloid leukemia (CML) and chronic eosinophilic leukemia (CEL). However, development of resistance to imatinib has emerged as an important clinical problem for molecular-targeted therapy in CML and CEL. In this study, we have established the imatinib-resistant CEL EOL-1 sub-lines (designated as EOL-1R) by culturing cells with increasing concentrations of imatinib for 6 months. Interestingly, EOL-1R cells showed epigenetic silencing of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene. Exposure of EOL-1R cells to imatinib failed to dephosphorylate AKT, ERK and STAT5, although PDGFRα was effectively inactivated. The forced expression of PTEN negatively regulated these signal pathways and sensitized EOL-1R cells to imatinib. Notably, hypermethylation of the promoter region of the PTEN gene in association with the downregulation of this genes transcripts was identified in imatinib-resistant leukemia cells isolated from individuals with CEL, CML and Philadelphia-positive acute lymphoblastic leukemia. In addition, anti-epigenetic agents restored PTEN expression, resulting in the sensitization of EOL-1R cells to imatinib. Taken together, epigenetic silence of PTEN is one of the mechanisms that cause drug resistance in individuals with leukemia after exposure to imatinib. Anti-epigenetic agents may be useful for overcoming drug resistance in such a case.

Long‐term exposure of gastrointestinal stromal tumor cells to sunitinib induces epigenetic silencing of the PTEN gene

Although sunitinib possesses significant clinical effects on imatinib‐resistant gastrointestinal stromal tumors (GISTs), the individuals with GIST eventually become resistant to treatment with this tyrosine kinase inhibitor. The mechanism of resistance to sunitinib is still under investigation. To address this issue, we have established sunitinib‐resistant GIST‐T1 sublines (designated as GIST‐T1R) by culturing cells with increasing concentrations of sunitinib. GIST‐T1R cells were also resistant to imatinib‐mediated growth inhibition. Examination of intracellular signaling found that Akt/ mammalian target of rapamycin (mTOR) signaling remained activated in GIST‐T1R but not in parental GIST‐T1 cells, after exposure of these cells to sunitinib, as measured by immunoblotting. Further study found that the phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene was silenced by methylation of the promoter region of the gene. Notably, forced‐expression of PTEN in GIST‐T1R cells negatively regulated the Akt/mTOR pathways and sensitized these cells to sunitinib‐mediated growth arrest and apoptosis. Taken together, epigenetic silence of PTEN might be one of the mechanisms which cause drug‐resistance in individuals with GIST after exposure to tyrosine kinase inhibitors. Blockade of the PI3K/Akt signaling with the specific inhibitors could be useful in such a case.

Perceived Stress, Severity of Asthma, and Quality of Life in Young Adults with Asthma

BACKGROUNDnPeople suffering from asthmatic symptoms have a lower quality of life than those without. The aim of this study is to clarify the association of quality of life with the severity of asthma, perceived stress, and other factors such as the comorbidity of allergic diseases among young adults with asthma.nnnMETHODSnThe study participants were 695 asthma patients, aged 20-44 years, from 29 medical centers in Japan. We excluded from the analysis of the result of the study 116 patients with complications of serious diseases, cough-variant asthma or aspirin-intolerant asthma. The patients completed the 8-Item Short-Form Health Survey (SF-8), the Airways Questionnaire 20 (AQ20), and the Japanese version of the Perceived Stress Scale (JPSS) and their doctors also provided clinical information including diagnosis, complications, severity of asthma, and results of pulmonary function and immunological tests.nnnRESULTSnThere was a weak correlation between the generic quality of life (SF-8) and the disease-specific quality of life (AQ20). The AQ 20 revealed almost no association with the results of pulmonary function and immunological tests, and only a slight association with comorbidity of allergic rhinitis and food allergy. The AQ20 showed a moderate relation with perceived stress (JPSS) but a weak association with the severity of asthma. The multiple logistic models demonstrated that there was no relationship between the severity of asthma and the AQ20 in females, and in the age group of 20-34 years.nnnCONCLUSIONSnA major variable related to the disease-specific quality of life was perceived stress, followed by the severity of asthma. Stress management of patients with asthma may improve their quality of life.

CD34+/CD38− acute myelogenous leukemia cells aberrantly express CD82 which regulates adhesion and survival of leukemia stem cells†

To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated CD34+/CD38− cells with that of CD34+/CD38+ counterparts from individuals with acute myelogenous leukemia (n = 2, AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in CD34+/CD38− AML cells compared with their CD34+/CD38+ counterparts. Proteins overexpressed in CD34+/CD38− AML cells included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, antiapoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in CD34+/CD38− AML cells was noted in additional clinical samples (n = 12) by flow cytometry. Importantly, down‐regulation of CD82 in CD34+/CD38− AML cells by a short hairpin RNA (shRNA) inhibited adhesion to fibronectin via up‐regulation of matrix metalloproteinases 9 (MMP9) and colony forming ability of these cells as assessed by transwell assay, real‐time RT‐PCR, and colony forming assay, respectively. Moreover, we found that down‐regulation of CD82 in CD34+/CD38− AML cells by an shRNA significantly impaired engraftment of these cells in severely immunocompromised mice. Taken together, aberrant expression of CD82 might play a role in adhesion of LSCs to bone marrow microenvironment and survival of LSCs. CD82 could be an attractive molecular target to eradicate LSCs.

Genetic ablation of the Bach1 gene reduces hyperoxic lung injury in mice: Role of IL-6

Bach1 is a transcriptional repressor of the heme oxygenase (HO)-1 gene. Bach1-null (Bach1(-/-)) mice are reported to be protected from myocardial ischemia/reperfusion injury; however, the effect of Bach1 disruption on another oxidative stress model of hyperoxic lung injury has yet to be determined. To investigate the role of Bach1 in hyperoxic lung injury, Bach1(-/-) mice and wild-type (WT) mice were exposed to 90% O(2). During hyperoxic exposure, the survival of Bach1(-/-) mice was significantly longer than that of WT mice. However, the administration of zinc protoporphyrin, an inhibitor of HO-1 activity, did not change the mortality in either of the mice, thus suggesting that this protective effect was not mediated by an HO-1 overexpression in Bach1(-/-) mice. The indices of lung injury in the lungs of Bach1(-/-) mice were lower than those of WT mice; unexpectedly, however, the levels of IL-6 in bronchoalveolar lavage (BAL) fluid from Bach1(-/-) mice were significantly higher than those of WT mice. Interestingly, the intrapulmonary administration of small interfering RNA against IL-6 was shown to reduce the IL-6 levels in BAL fluids and shorten the survival in Bach1(-/-) mice during hyperoxic exposure. In addition, a chromatin immunoprecipitation analysis revealed the binding of Bach1 to the IL-6 promoter and its detachment after oxidative stress. Considering the previous observation that the transgenic mice overexpressing IL-6 are protected from hyperoxic lung injury, these results therefore indicate that IL-6 mediates an increased survival in Bach1(-/-) mice during hyperoxic exposure.

AZ960, a Novel Jak2 Inhibitor, Induces Growth Arrest and Apoptosis in Adult T-Cell Leukemia Cells

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease in which the Jak2/Stat5 pathway is constitutively activated. This study found that AZ960, a novel inhibitor of Jak2 kinase, effectively induced growth arrest and apoptosis of human T-cell lymphotropic virus type 1, HTLV-1–infected T cells (MT-1 and MT-2) in parallel with downregulation of the phosphorylated forms of Jak2 and Bcl-2 family proteins including Bcl-2 and Mcl-1. Interestingly, AZ960 increased levels of Bcl-xL in MT-1 and MT-2 cells in association with accumulation of cAMP response element-binding protein bound to the Bcl-xL promoter as measured by chromatin immunoprecipitation assay. Importantly, genetic inhibition of Bcl-xL by a small interfering RNA potentiated antiproliferative effects of AZ960 in MT-1 cells. Taken together, Jak2 is an attractive molecular target for treatment of ATL. Concomitant blockade of Jak2 and Bcl-xL may be a promising treatment strategy for this lethal disease. Mol Cancer Ther; 9(12); 3386–95. ©2010 AACR.