Kazunori Fujitaka

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.

SGLT Gene Expression in Primary Lung Cancers and Their Metastatic Lesions

Cancer cells show increased glucose uptake and utilization in comparison with their normal counterparts. Glucose transporters play an important role in glucose uptake. We previously reported the differential gene expression of the GLUT family in primary and metastatic lesions of lung cancer. To investigate the role of Na+/glucose cotransporter (SGLT) genes in cancers, we examined the levels of expression of SGLT1 and SGLT2 genes in primary lung cancers and their metastatic lesions. Ninety‐six autopsy samples (35 primary lung cancers, 35 corresponding normal lung tissues, 10 metastatic liver lesions, and 16 metastatic lymph nodes) from 35 patients were analyzed for SGLT1 and SGLT2 expression by reverse transcription (RT)‐polymerase chain reaction (PCR). There were no significant differences in the level of expression of either gene between the primary lung cancers and normal lung tissues. The level of SGLT1 expression in the metastatic lesions and primary lung cancers did not differ significantly. The level of SGLT2 expression was, however, significantly higher in the metastatic lesions of both the liver and lymph node than in the primary lung cancers. These results suggest that SGLT2 plays a role in glucose uptake in the metastatic lesions of lung cancer.

Association between expression of the MRP3 gene and exposure to platinum drugs in lung cancer

To investigate the roles played by the multidrug resistance‐associated protein (MRP1) homologues MRP3 and MRP4 in resistance to platinum drugs, we examined steady‐state levels of mRNA for both MRP3 and MRP4 in normal lung and lung cancer specimens as well as peripheral mononuclear cells (PMN) after platinum drug exposure. MRP3 and MRP4 gene expression levels were monitored in the PMN of 10 previously untreated lung cancer patients within 24 hr after carboplatin (CBDCA) administration. Expression levels for both genes were also examined in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients with lung cancer. MRP3 and MRP4 gene expression levels were assessed by quantitative reverse transcription‐polymerase chain reaction. MRP3 expression levels in the PMN rose rapidly within 24 hr after administration of CBDCA, whereas MRP4 expression levels remained the same. Furthermore, MRP3 expression levels in normal lung and tumor tissues from autopsy samples that had been exposed to platinum drugs while the patients were alive were significantly higher than those in unexposed tissues, but again MRP4 expression levels remained the same. These results suggest that platinum drugs and/or the physiological stress response to xenobiotics induce expression of the MRP3 gene.

Usefulness of monitoring the circulating Krebs von den Lungen-6 levels to predict the clinical outcome of patients with advanced nonsmall cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors

Krebs von den Lungen‐6 (KL‐6) is a high molecular weight glycoprotein classified in the category of human MUC1 mucin. KL‐6 has been reported to serve as a sensitive marker for interstitial pneumonia; however, recent studies have suggested that it can also be used as a tumor marker as its origin shows. To further elucidate the clinicopathological significance of circulating KL‐6 in lung cancer, we monitored the circulating KL‐6 levels in advanced nonsmall cell lung cancer (NSCLC) patients and analyzed the association between these levels and the clinical outcome of EGFR‐TKI treatment. The pretreatment levels of circulating KL‐6 were found to be significantly higher in progressive disease (PD) patients than disease‐controlled (partial response (PR) and stable disease (SD)) patients. Multivariate analyses revealed the circulating KL‐6 level to be an independent prognostic factor for overall survival as well as progression‐free survival. In addition to these observations, we found that changes in circulating KL‐6 levels at 2 weeks after the start of EGFR‐TKI treatment from the baseline could quite precisely discriminate PD cases from PR or SD patients and the clinical outcome of EGFR‐TKI in NSCLC patients. These results indicate that the monitoring of circulating KL‐6 levels in NSCLC patients is effective for both selecting patients to be treated with EGFR‐TKI and predicting the clinical outcome of EGFR‐TKI. In addition, the findings suggest that the circulating KL‐6 level could be used as a clinically relevant biomarker in patients with NSCLC, particularly those who are candidates for EGFR‐TKI treatment.

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.

Induction of cytochrome P450 3A4 by docetaxel in peripheral mononuclear cells and its expression in lung cancer.

Abstract. Several recent studies have demonstrated that the cytochrome p450 (CYP) family plays an important role in the metabolism of taxanes. However, the role of CYP gene expression in tumors and peripheral mononuclear cells (PMN) is unknown. We therefore investigated the levels of CYP3A4 and CYP2C gene expression using reverse transcription polymerase chain reaction (RT-PCR) in PMN from 16 previously untreated lung cancer patients to determine whether the expression of the two genes is induced by docetaxel (TXT). Neither the CYP3A4 nor the CYP2C gene was induced after administration of carboplatin (CBDCA) alone. Expression of the CYP3A4 gene was induced by the administration of TXT alone or TXT and CBDCA, but expression of the CYP2C gene was unaffected. We also measured the expression of both genes using RT-PCR in 20 autopsy samples (ten non-small-cell lung cancers and their corresponding normal lung tissues) obtained from patients who had not received any chemotherapy during life. The level of CYP2C gene expression in samples of lung cancer was significantly higher than in normal lung tissue, but the level of CYP3A4 gene expression was not. These results suggest that the CYP3A4 gene is induced by TXT, and that it plays an important role in intracellular TXT metabolism.

Glutathione S-transferase-π gene expression and platinum drug exposure in human lung cancer

Abstract We examined the association between the gene expression levels of glutathione S -transferase-π (GST-π) and platinum drug exposure in human lung cancer. First we monitored GST-π gene expression levels in two lung cancer cell lines and in peripheral mononuclear cells of ten previously untreated lung cancer patients after platinum drug exposure. Next we examined GST-π gene expression levels in 40 lung cancer autopsy specimens. The GST-π gene expression levels were assessed by the quantitative reverse transcription–polymerase chain reaction or Northern blot analysis. The GST-π gene expression was not induced by platinum drugs either in vitro and in vivo within 24 h of exposure. In contrast, GST-π gene expression levels in lung cancer tissues of patients who had been exposed to platinum drugs at least 1 month before death were significantly higher than that in those of patients who had not been exposed. These results suggest that GST-π gene expression is associated with chronic exposure to platinum drugs in lung cancer and/or the stress response to xenobiotics.

Pattern of Lung Cancer in Turkey, 1994–1998

Background: Lung cancer is the most common neoplasm in Turkey, but there is not enough data on the characteristics of this mortal illness in our country. Objectives and Methods: The Turkish Thoracic Society, Lung and Pleural Malignancies Study Group (TTS-LPMSG) conducted a national retrospective hospital-based study to determine the pattern of lung cancer in Turkey. Results: A total of 11,849 lung cancer patients were studied between 1994 and 1998, 90.4% were male and 9.6% were female. The majority of patients were smokers (77.9%) or ex-smokers (10.8%). The mean age at the time of diagnosis was 58.4 years (20–84) and 56.7% of the patients were aged between 46 and 65 years. The most common histological types were squamous cell (45.4%), small cell (SCLC; 20.5%) and adenocarcinoma (20.2%). The majority of patients with non-small-cell lung cancer were diagnosed with metastatic disease (40.4%). Of the patients with SCLC patients, 37.9% had limited stage disease and 62.7% extensive stage disease at diagnosis. Conclusion: The results of the largest data so far collected in Turkey show that the vast majority of patients with lung cancer are male, squamous cell is the most common histological type, and only a small proportion of patients are diagnosed at an early stage.

Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts

Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

Intra-airway administration of small interfering RNA targeting plasminogen activator inhibitor-1 attenuates allergic asthma in mice.

Recent studies suggest that plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of the fibrinolytic system, may promote the development of asthma. To further investigate the significance of PAI-1 in the pathogenesis of asthma and determine the possibility that PAI-1 could be a therapeutic target for asthma, this study was conducted. First, PAI-1 levels in induced sputum (IS) from asthmatic subjects and healthy controls were measured. In asthmatic subjects, IS PAI-1 levels were elevated, compared with that of healthy controls, and were significantly higher in patients with long-duration asthma compared with short-duration asthma. PAI-1 levels were also found to correlate with IS transforming growth factor-β levels. Then, acute and chronic asthma models induced by ovalbumin were established in PAI-1-deficient mice and wild-type mice that received intra-airway administrations of small interfering RNA against PAI-1 (PAI-1-siRNA). We could demonstrate that eosinophilic airway inflammation and airway hyperresponsiveness were reduced in an acute asthma model, and airway remodeling was suppressed in a chronic asthma model in both PAI-1-deficient mice and wild-type mice that received intra-airway administration of PAI-1-siRNA. These results indicate that PAI-1 is strongly involved in the pathogenesis of asthma, and intra-airway administration of PAI-1-siRNA may be able to become a new therapeutic approach for asthma.