Search

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient’s survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study’s aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2Y15, p-cdc2T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, the expression and association of p21Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21Waf1/Cip1-dependent signaling pathway in the NSCLC cells.

Intraperitoneal (IP) injection of mesenchymal stem cells (MSCs) has been reported to treat colitis in mice. Complications such as abdominal organ injury and infection are significant concerns. We studied a single injection of human umbilical cord MSCs (hUCMSCs) via the subcutaneous (SC) and IP routes in mice with colitis. Male C57BL/6Ncrl mice were divided into control, phosphate-buffered saline (PBS), hUCMSCs SC, and hUCMSCs IP injection groups. Colitis was induced by 3% dextran sulfate sodium (DSS). The disease activity index (DAI), colon length, histology, inflammation score, cytokine and chemokine staining and in vivo stem cell images were recorded. The DAI in the SC group was significantly lower than that in the IP group during late acute colitis. The colon was significantly shorter, and the colon inflammation score was significantly higher in the PBS group than the control group. There were no significant differences in the colon length and inflammation score between the control group and the SC and IP groups. The expressions of IL17A and Gro-α decreased in the SC group compared with those in the IP group on the 8th and 25th days. hUCMSCs via SC injection accumulated in the subcutaneous tissue to the 25th day. hUCMSCs via SC and IP injection reduced DSS-induced acute colitis and decreased progression to chronic colitis. The anti-inflammatory effects of hUCMSCs were better in the SC injection group than the IP injection group. In clinical practice in humans, SC injection of hUCMSCs is relatively safer and more convenient than IP injection.

BACKGROUND The biologic and cellular IL-6-related therapies have been used to treat the autoimmune diseases (AID), which prompting us to furtherly explore the IL-6 role in human umbilical cord mesenchymal stem cells (hUCMSCs) therapy.MATERIALS & METHODS Peripheral blood mononuclear cells (PBMCs) were responder co-cultured with hUCMSCs or exogenous IL-6. PBMC suppression assay was used to analyze the anti-inflammatory effects by using the MTT assay. The IL-6 concentration in supernatant was measured using ELISA. The correlation between anti-inflammation effect of hUCMSCs and IL-6 levels, and the relevant roles of IL-6, IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was furtherly evaluated.RESULTS Initially, hUCMSCs did not exert any inhibitory effect on PBMCs, however, a potent inhibitory effect on PBMCs was observed and the IL-6 concentration reached about 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 could inhibit PBMCs inflammations but less than that of hUCMSCs. The hUCMSCs exerts excellent anti-inflammation effect by inducing higher IL-6 level which is different from TCZ IL-6 antagonistCONCLUSIONS High concentration IL-6 cytokine secretion plays an important role in the anti-inflammation effect of hUCMSCs cell therapy.

Staphylococcus aureus (S. aureus) can lead to many life-threatening diseases. It has the ability to invade normal endovascular tissue. The molecular mechanisms and pathological changes of endothelial cells after S. aureus infection are of interest, but the basic understanding of how S. aureus destroys this barrier is not clear. Here, we showed that S. aureus enhanced COX-2 expression and prostaglandin E2 (PGE2) secretion in human aortic endothelial cells (HAECs). In addition, S. aureus induced PGE2/interleukin-6 (IL-6)/matrix metallopeptidase-9 (MMP-9)-dependent cell migration. S. aureus-induced COX-2, IL-6, and MMP-9 levels were inhibited by transfection with siRNA of Toll-like receptor 2 (TLR2), p38, p42, p44, p50, or p65. S. aureus also induced p38 MAPK, ATF2, ERK1/2, and NF-κB p65 activation. Interestingly, we proved that S. aureus decreased intracellular generation of reactive oxygen species (ROS), which suggests that the inhibition of ROS production promoted inflammatory responses. Finally, we showed that S. aureus enhanced a variety of biomarkers of inflammation in cardiovascular diseases. However, the free radical scavenger (MCI-186) or antioxidant (N-acetyl-L-cysteine, NAC) markedly enhanced S. aureus-induced COX-2 mRNA levels in the aorta tissues. Taken together, these findings established that S. aureus promoted aorta inflammation via activation of p38 MAPK, ERK1/2, and NF-κB and inhibition of ROS generation.

Pseudomonas aeruginosa (P. aeruginosa) infection in the lung is common in patients with cystic fibrosis (CF). Intercellular adhesion molecule-1 (ICAM-1) is known to play a key role in lung inflammation. Acute inflammation and its timely resolution are important to ensure bacterial clearance and limit tissue damage. Carbon monoxide (CO) has been shown to exert anti-inflammatory effects in various tissues and organ systems. Here, we explored the protective effects and mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on P. aeruginosa-induced inflammatory responses in human pulmonary alveolar epithelial cells (HPAEpiCs). We showed that P. aeruginosa induced prostaglandin E2 (PGE2)/interleukin-6 (IL-6)/ICAM-1 expression and monocyte adherence to HPAEpiCs. Moreover, P. aeruginosa-induced inflammatory responses were inhibited by transfection with siRNA of Toll-like receptor 4 (TLR4), PKCα, p47phox, JNK2, p42, p50, or p65. P. aeruginosa also induced PKCα, JNK, ERK1/2, and NF-κB activation. We further demonstrated that P. aeruginosa increased intracellular ROS generation via NADPH oxidase activation. On the other hand, P. aeruginosa-induced inflammation was inhibited by pretreatment with CORM-2. Preincubation with CORM-2 had no effects on TLR4 mRNA levels in response to P. aeruginosa. However, CORM-2 inhibits P. aeruginosa-induced inflammation by decreasing intracellular ROS generation. P. aeruginosa-induced PKCα, JNK, ERK1/2, and NF-κB activation was inhibited by CORM-2. Finally, we showed that P. aeruginosa induced levels of the biomarkers of inflammation in respiratory diseases, which were inhibited by pretreatment with CORM-2. Taken together, these data suggest that CORM-2 inhibits P. aeruginosa-induced PGE2/IL-6/ICAM-1 expression and lung inflammatory responses by reducing the ROS generation and the inflammatory pathways., Graphical abstract fx1, Highlights • CORM-2 inhibits P. aeruginosa-induced PGE2/IL-6/ICAM-1 expression. • CORM-2 reduced PKCα phosphorylation in response to P. aeruginosa. • We provide molecular mechanisms for antibacterial effects of CORM-2.

Multiple injections of bone marrow mesenchymal stem cells (BMMSCs) have been used for treatment of chronic colitis in mice. We aimed to report the therapeutic effects of a single injection of human umbilical cord mesenchymal stem cells (hUCMSCs) on acute and chronic colitis. Male C57BL/6JNarl mice were divided into control, phosphate-buffered saline (PBS), and hUCMSCs treated groups, respectively. Acute and chronic colitis were induced in the mice (except controls) using 3% dextran sulfate sodium (DSS). The mice in the hUCMSCs group underwent a single injection of hUCMSCs. The disease activity index (DAI), colon length, histology, colon inflammation score, in vivo stem cells images, and blood cytokine levels were recorded. The DAI was significantly higher in the hUCMSCs group than in the control group and lower than in the PBS group on all days. The colon length was significantly longer and the colon inflammation score was significantly lower in the hUCMSCs group than in the PBS group on days 8 and 25. IL17A, Gro-α, MIP-1α, MIP-2, and eotaxin were significantly lower in the hUCMSCs group than in the PBS group on days 8 and 25. Single-injection hUCMSCs improved DSS–induced acute colitis and decreased progression of acute colitis to chronic colitis.

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).

Staphylococcus aureus (S. aureus) can often lead to many life-threatening diseases. It has the ability to invade normal endovascular tissue. Acute inflammation and its resolution are important to ensure bacterial clearance and limit tissue injury. Carbon monoxide (CO) has been shown to exert anti-inflammatory effects in various tissues and organ systems. In our study, we investigated the effects and the mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on S. aureus-induced inflammatory responses in human aortic endothelial cells (HAECs). We proved that S. aureus induced cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2)/interleukin-6 (IL-6)/matrix metallopeptidase-9 (MMP-9) expression and cell migration, which were decreased by CORM-2. Moreover, CORM-2 had no effects on TLR2 mRNA levels in response to S. aureus. Interestingly, we proved that S. aureus decreased intracellular ROS generation, suggesting that the inhibition of ROS further promoted inflammatory responses. However, CORM-2 significantly inhibited S. aureus-induced inflammation by increasing intracellular ROS generation. S. aureus-induced NF-κB activation was also inhibited by CORM-2. Finally, we proved that S. aureus induced levels of the biomarkers of inflammation in cardiovascular diseases, which were inhibited by CORM-2. Taken together, these results suggest that CORM-2 inhibits S. aureus-induced COX-2/PGE2/IL-6/MMP-9 expression and aorta inflammatory responses by increasing the ROS generation and reducing the inflammatory molecules levels.

Staphylococcus aureus (S. aureus) can lead to many life-threatening diseases. It has the ability to invade normal endovascular tissue. The molecular mechanisms and pathological changes of endothelial cells after S. aureus infection are of interest, but the basic understanding of how S. aureus destroys this barrier is not clear. Here, we showed that S. aureus enhanced COX-2 expression and prostaglandin E

Expression of transforming growth factor (TGF)-β1 and increases in angiogenesis and deposition of extracellular matrix are the key features of tracheal granulation formation. The aim of this study was to investigate the potential role of thalidomide in preventing granulation tissue formation from the aspect of cellular effects in vitro, including fibroblast proliferation, vascular endothelial growth factor (VEGF) release, and collagen production. Human lung fibroblasts were obtained from bronchus and cultured. The effects of thalidomide on cell proliferation, migration, TGF-β1-induced VEGF, and signal pathway were investigated. Thalidomide (20 μM) not only inhibited cell proliferation after 24 h [fold increase of cell number, 0.85 ± 0.09 vs. 1.47 ± 0.14 (treatment vs. control group); P

Aims Matrix metalloprotease (MMP)-9 is present in abundance in various chronic airway disorders and is involved in lung remodeling. MMP may cleave membrane-bound precursor proteins and release epidermal growth factor-like ligands that subsequently bind to epidermal growth factor receptor (EGFR). We hypothesized that MMP-9 may stimulate the airway epithelium to produce fibrogenic mediators through activation of membrane-bound receptors. Main methods Human airway epithelial cells were grown on air–liquid interface culture inserts. MMP-9 was employed to stimulate the cells. Conditioned medium following MMP-9 stimulation was co-incubated with human lung fibroblasts. Key findings MMP-9 stimulated human airway epithelial cells to produce transforming growth factor (TGF)-β 1 at both the mRNA and protein level. Using a microarray, increased phosphorylation of EGFR tyrosine kinase (TK) was identified and further confirmed by immunoprecipitation and Western blot analysis. A significant increase in EGF and TGF-α release was observed after MMP-9 had been added for 30 min. Protease inhibitor, EGFR monoclonal antibody and EGFR-TK inhibitor blocked this action and subsequent TGF-β 1 production. Neutralizing antibodies against EGF and TGF-α substantially inhibited TGF-β 1 production following MMP-9 stimulation. MMP-9-induced TGF-β 1 production occurred through MAP kinase p44/42 phosphorylation. Selective p44/42 kinase inhibitor UO126 successfully inhibited TGF-β 1 production. Conditioned medium from epithelial cells treated with MMP-9 significantly induced Smad3 phosphorylation and subsequent fibroblast proliferation after 24 h culture. Significance These data indicate that MMP-9 induces TGF-β 1 production in the airway epithelium through the cleavage of EGF and EGF-like ligands and activating EGFR, suggesting potential targets of therapeutic intervention in airway fibrotic disorders.

Introduction: Four phase III randomized trials adding epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors to standard chemotherapeutics in patients with advanced non-small cell lung cancer (NSCLC) have failed to show benefits. The mechanism of these failures was examined. Methods: Fifteen previously untreated NSCLC cell lines were simultaneously treated with gefitinib plus cisplatin. Three exhibited sensitizing-EGFR mutations. Three selected lines were further tested with paclitaxel/cisplatin, paclitaxel/gefitinib, and paclitaxel/cisplatin/gefitinib combinations. The tetrazolium colorimetric assay with application of the classic isobole method was used, and dose-versus-log-response curves (DRCs) were analyzed to evaluate possible resistance mechanisms. Results: Of the 15 cell lines tested, combined gefitinib/cisplatin was significantly antagonistic in 10 wild-type and three sensitizing-EGFR mutant cell lines (group mean combination index=1.184, 95% confidence interval=1.12–1.24, p = 0.001). The mean combination index values of paclitaxel/cisplatin/gefitinib were higher than or comparable with those of paclitaxel/cisplatin and paclitaxel/gefitinib. DRC analysis consistently showed nonsaturable passive resistance, suggesting that gefitinib at 0.001 to 0.3 μM can interfere with cisplatin cell entry (at concentrations >1–3 μM) in a dose-dependent manner and lead to antagonism. This antagonism may or may not be schedule dependent in different cell lines. Conclusions: In most EGFR wild-type or sensitizing-mutant NSCLC cells, the concomitant gefitinib/cisplatin combination showed antagonism, likely because gefitinib interfered with cisplatin entry into the cell. The findings that three-drug combination was not better than the two-drug combinations are in accordance with the results of the randomized trials. The EGFR-tyrosine kinase inhibitor/platinum antagonism is a possible reason for the failure of those randomized trials.

Background Acquired tracheal stenosis is common in patients with a long-term tracheostomy and granulation is one of the most commonly observed lesions in benign airway stenosis. The aim of this study was to investigate the mechanisms of tracheal granulation formation and find the potential therapeutic targets to prevent the granulation formation. Results In granulation tissue obtained from patients during interventional bronchoscopy for the relief of airway obstruction, increased expression of transforming growth factor (TGF)- β 1 and vascular endothelial growth factor (VEGF), as well as increased numbers of fibroblasts, was found by immunohistochemical staining. TGF- β 1 expression was detected in both the epithelial and submucosal layers. The highest levels of VEGF and vimentin expression occurred in the submucosal layers. In comparison with the control, significantly increased numbers of small vessels were observed in the submucosal layers of the granulation tissue. In vitro , TGF- β 1 stimulated production of VEGF by cultured fibroblasts at both the mRNA and protein level. VEGF siRNA treatment resulted in a significant decrease of TGF- β 1 -induced VEGF production. SIS3, a selective Smad3 inhibitor, and UO126 both inhibited p44/42 MAP kinase phosphorylation and attenuated subsequent VEGF production by fibroblasts. A low concentration of erythromycin (1 μg/ml), but not dexamethasone (100 μM), inhibited TGF- β 1 -induced VEGF production. Conclusion This study provides important information that facilitates an understanding, at least in part, of the mechanisms of granulation formation. Targeting these mediators and cells may help to prevent the formation of granulation tissue in long-term tracheostomy or prolonged endotracheal intubation patients.

Rationale Gastroesophageal reflux (GER) is common in patients with various airway diseases. Airway epithelial cells can release growth factors that promote fibroblast proliferation. Exposure of airway epithelium to bile acids may induce a fibrotic response. Objectives To determine how bile acids interact with airway epithelium; particularly, whether transforming growth factor-β 1 secretion and fibroblast proliferation are affected. Methods Induced sputum from patients with asthma, GER, or asthma associated with GER symptoms, or from healthy control subjects was collected. Total bile acids were measured by a spectrophotometric enzymatic assay. The major components of bile acids, chenodeoxycholic acid (CD) and glycochenodeoxycholic acid (GCD), were used to stimulate primary airway epithelial cells. Quantitative polymerase chain reaction and Western blotting were applied for messenger RNA expression and signal pathway analysis, respectively. Conditioned medium following CD stimulation was coincubated with fibroblasts for proliferation study. Results The amount of total bile acids in induced sputum was significantly higher in patients with GER and asthma-associated GER symptoms compared to that of healthy control subjects (p 1 production. TGF-β 1 messenger RNA expression was 2.5-fold increased compared to unstimulated cells. This occurred via p38 mitogen-activated protein (MAP) kinase and activating transcription factor-2 activation. Pretreatment with dexamethasone inhibited TGF-β 1 production at both messenger RNA and protein levels by inhibiting p38 MAP kinase phosphorylation. Conditioned medium from CD-treated epithelial cells enhanced fibroblast proliferation. Conclusions Aspiration of bile acids may induce airway fibrosis through the production of TGF-β 1 and fibroblast proliferation. Early intervention to attenuate these processes may reduce fibrogenesis in various airway diseases associated with GER.

Interleukin-6 (IL-6) has been shown to regulate both growth and neuroendocrine (NE) differentiation in some types of human cancer cells, and erbB2 may be a critical component of IL-6 signaling. Non-small cell lung cancer (NSCLC) tumors that demonstrate NE properties have been suggested to have biological characteristics similar to small cell lung cancers with initial responsiveness to chemotherapy. We investigated whether IL-6 is implicated in the cell growth, NE differentiation, and chemosensitivity of NSCLC-NE cells. NSCLC-NE cells were treated with exogenous IL-6, and a subclone of an IL-6-transfected NSCLC cell line that constitutively expressed IL-6 receptor was also generated. These cells were assessed for cell proliferation by cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays, chemosensitivity to cisplatin and etoposide by MTT assays, and NE differentiation by observing morphological changes and immunoblotting for neuron-specific enolase (NSE). The IL-6-treated cells and the IL-6-transfected cells showed enhanced cell proliferation and downregulated NSE expression, but little change in chemosensitivity. In the culture medium, IL-6-transfected cells grew as looser aggregates than the parental cells. IL-6 could not activate the erbB genes. In conclusion, IL-6 can induce cell proliferation and NE dedifferentiation but has little effect on chemosensitivity in IL-6 receptor-expressing NSCLC-NE cells. The status of NSE expression is unlikely to be a crucial factor for chemosensitivity in NSCLC cells.

Purpose: Mutations in epidermal growth factor receptor (EGFR) can be used to predict the tumor response of patients receiving gefitinib for non–small cell lung cancer (NSCLC). We investigated the association between mutations in EGFR tyrosine kinase domain and tumor response and survival in gefitinib-treated NSCLC patients. Experimental Design: EGFR mutations in exons 18 to 21 were analyzed by DNA sequencing of paraffin-embedded tumor tissues from gefitinib-treated NSCLC patients. The results were correlated with clinical variables. Results: EGFR mutations were found in 61.1% (33 of 54) of cases; response rate and disease control rate were 56.8% and 68.5%, respectively. There was no significant difference in mutation rates between adenocarcinoma (29 of 43) and nonadenocarcinoma (4 of 11; P = 0.085). However, all four nonadenocarcinomas with EGFR mutations had no response to gefitinib. Presence of EGFR mutations was the only independent predictor for disease control (P = 0.003) and tumor response (P = 0.017) in multivariate analysis; positive predictive values were 87.9% and 70.8% and negative predictive values were 61.9% and 69.2%, respectively. In comparison with patients whose tumor was negative for EGFR mutations, patients with EGFR mutations had better progression-free survival (median, 7.6 versus 1.7 months; P = 0.011) and overall survival (median, 14.7 versus 4.7 months; P = 0.046). Conclusions: Mutations in EGFR tyrosine kinase correlate with treatment response and survival in gefitinib-treated NSCLC patients and can be used as a predictive and prognostic factor. Thus, analysis of EGFR tyrosine kinase mutations in lung adenocarcinoma is of clinical significance, as it can permit the customization of treatment with EGFR tyrosine kinase inhibitors.

Nucleotide excision repair (NER) is a major repair mechanism for DNA lesions induced by cisplatin. Overexpressions of epidermal growth factor receptor (EGFR) and HER‐2/neu have been reported to affect the sensitivity of certain human cancer cells to cisplatin, presumably by modification of DNA repair activity through interference with NER. Using an in vitro repair assay, we investigated NER activity of cisplatin‐induced DNA lesions in a panel of 16 non‐small cell lung cancer (NSCLC) cell lines. The interrelationships between NER activity, cisplatin sensitivity, HER‐2/neu expression and EGFR level, were also analyzed. The results showed that high NER activity was closely correlated with cisplatin resistance and high levels of HER‐2/neu expression (P < 0.05). Analysis of the relationships between EGFR level and each of the other three parameters revealed no statistically significant correlations (all P values were > 0.05 by Spearman rank correlation), but a trend of association (all the values of proportion of accordance were ≥62.5% by using a 2x2 contingency table). These results suggest that NER activity may play an important role in the cisplatin resistance of NSCLC cells and there may be an association between enhanced NER activity and high levels of p185neu and probably EGFR in NSCLC cells. The finding that high levels of EGFR showed very little influence on the relationship between p185neu and cisplatin resistance suggests that EGFR may be a less crucial factor in modulating the chemoresistance of NSCLC cells when compared with HER‐2/neu.

Background Combined epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) with chemotherapy is believed to be more effective in treating non-small-cell lung cancer (NSCLC) with sensitizing-EGFR mutation (SEM). This hypothesis failed to be realized clinically and needs to be examined in vitro. Materials and methods Using the tetrazolium colorimetric assay and classical isobole method, we investigated the combination effects of 6 gefitinib-chemotherapeutic doublets (gefitinib/cisplatin, gemcitabine, pemetrexed, paclitaxel, docetaxel, or vinorelbine) in a panel of 15 NSCLC cell lines. Results Upon treatment with the 6 gefitinib-chemotherapeutic doublets, the 12 cell lines that did not harbor SEM displayed a broad spectrum of group results, from obvious synergism to robust antagonism. The values of group mean combination index (mCIs) ranged from 0.769 to 1.201. In contrast, the 3 cell lines with SEM showed a tendency toward consistent antagonism to the tested doublets, impressively, with a narrow range of higher group mCIs (0.993–1.141). In the presence of gefitinib, the SEM or gefitinib-sensitive group was more chemo-refractory than the non-SEM (index of chemo-refractoriness (RI): 69.33 versus 42.67; P =0.036) or gefitinib-resistant group (68.25 versus 40.64, P =0.0108), respectively. The results of using the gefitinib/drug combinations with the gefitinib-sensitive non-SEM cell line H322 and the gefitinib-resistant EGFR mutant H820 shared patterns similar to those with the SEM and non-SEM cell lines, respectively. Conclusion Gefitinib-treated EGFR-TKI-sensitive NSCLC cells showed a wide spectrum of chemo-refractoriness, suggesting that concomitantly combined EGFR-TKI-chemotherapy might not be a good treatment strategy for NSCLC harboring SEM.

Acquired tracheal stenosis is common in patients with a long-term tracheostomy and granulation is one of the most commonly observed lesions in benign airway stenosis. The aim of this study was to investigate the mechanisms of tracheal granulation formation and find the potential therapeutic targets to prevent the granulation formation.In granulation tissue obtained from patients during interventional bronchoscopy for the relief of airway obstruction, increased expression of transforming growth factor (TGF)-β₁ and vascular endothelial growth factor (VEGF), as well as increased numbers of fibroblasts, was found by immunohistochemical staining. TGF-β₁ expression was detected in both the epithelial and submucosal layers. The highest levels of VEGF and vimentin expression occurred in the submucosal layers. In comparison with the control, significantly increased numbers of small vessels were observed in the submucosal layers of the granulation tissue. In vitro, TGF-β₁ stimulated production of VEGF by cultured fibroblasts at both the mRNA and protein level. VEGF siRNA treatment resulted in a significant decrease of TGF-β₁-induced VEGF production. SIS3, a selective Smad3 inhibitor, and UO126 both inhibited p44/42 MAP kinase phosphorylation and attenuated subsequent VEGF production by fibroblasts. A low concentration of erythromycin (1 μg/ml), but not dexamethasone (100 μM), inhibited TGF-β₁-induced VEGF production.This study provides important information that facilitates an understanding, at least in part, of the mechanisms of granulation formation. Targeting these mediators and cells may help to prevent the formation of granulation tissue in long-term tracheostomy or prolonged endotracheal intubation patients.

Gastroesophageal reflux (GER) is common in patients with various airway diseases. Airway epithelial cells can release growth factors that promote fibroblast proliferation. Exposure of airway epithelium to bile acids may induce a fibrotic response.To determine how bile acids interact with airway epithelium; particularly, whether transforming growth factor-beta1 secretion and fibroblast proliferation are affected.Induced sputum from patients with asthma, GER, or asthma associated with GER symptoms, or from healthy control subjects was collected. Total bile acids were measured by a spectrophotometric enzymatic assay. The major components of bile acids, chenodeoxycholic acid (CD) and glycochenodeoxycholic acid (GCD), were used to stimulate primary airway epithelial cells. Quantitative polymerase chain reaction and Western blotting were applied for messenger RNA expression and signal pathway analysis, respectively. Conditioned medium following CD stimulation was coincubated with fibroblasts for proliferation study.The amount of total bile acids in induced sputum was significantly higher in patients with GER and asthma-associated GER symptoms compared to that of healthy control subjects (p0.005). CD, but not GCD, significantly induced TGF-beta1 production. TGF-beta1 messenger RNA expression was 2.5-fold increased compared to unstimulated cells. This occurred via p38 mitogen-activated protein (MAP) kinase and activating transcription factor-2 activation. Pretreatment with dexamethasone inhibited TGF-beta1 production at both messenger RNA and protein levels by inhibiting p38 MAP kinase phosphorylation. Conditioned medium from CD-treated epithelial cells enhanced fibroblast proliferation.Aspiration of bile acids may induce airway fibrosis through the production of TGF-beta1 and fibroblast proliferation. Early intervention to attenuate these processes may reduce fibrogenesis in various airway diseases associated with GER.

Cysteinyl leukotrienes (CysLTs) play an important role in the pathogenesis of airway remodeling. We investigated the interaction between epithelium and CysLTC4, and the contribution of this interaction to airway fibrosis. Human airway epithelial cells were grown on air-liquid interface culture inserts. CysLTC4 was employed to stimulate the cells. Conditioned medium following CysLTC4 stimulation was coincubated with human lung fibroblasts. Our results have demonstrated that CysLTC4 stimulates airway epithelial cells, through a p38 mitogen-activated protein kinase (MAPK) activation mechanism, to produce transforming growth factor beta1 (TGF-beta1), which results in fibroblast proliferation. The selective p38 MAPK inhibitor S203580 successfully inhibits p38 MAPK phosphorylation and subsequent TGF-beta1 production. CysLT1 receptor antagonist montelukast and corticosteroid inhibit TGF-beta1 production at the mRNA and protein levels. When treated with LTC4, the conditioned medium from epithelial cells enhances fibroblast proliferation, this mitogenic effect being attributed to TGF-beta1 and LTC4 remaining in the culture medium. In addition, LTC4 itself acts as a potential growth factor for lung fibroblasts. These data indicate that interactions between LTC4 and airway epithelial cells may contribute to the pathogenesis of airway remodeling. Early intervention to stop these processes may be useful in preventing airway fibrosis in chronic allergic inflammation.

Interleukin-6 (IL-6) has been identified as an important growth regulator of lung cancer cells. Elevation of serum levels of IL-6 has been found in a subpopulation of lung cancer patients, but rarely in patients with benign lung diseases. Approximately 15% of non-small cell lung cancer (NSCLC) tumors exhibit neuroendocrine (NE) properties (NSCLC-NE) and have been suggested to have the biological characteristics similar to small cell lung cancer (SCLC) with early metastasis and initial responsiveness to chemotherapy. We recently showed that IL-6 promotes cell proliferation and downregulates the expression of neuron-specific enolase (NSE, one of the major NE markers) in NSCLC-NE cells. In this study, we show that IL-6 stimulates a transient increase of tyrosine phosphorylation of STAT3 in a dose-dependent fashion. Inhibition of STAT3 signaling pathway by either AG-490 (JAK2-specific inhibitor) or overexpression of STAT3Y705F (a dominant-negative STAT3) reverses NSE expression in IL-6- treated NSCLC-NE cells. In addition, IL-6 induces phosphorylation and activation of p38 MAPK. SB-203580, a p38 MAPK-specific inhibitor, inhibits IL-6-induced p38 MAPK phosphorylating activity and suppresses IL-6-stimulated cell proliferation. Together, our results indicate that STAT3 signaling pathway is involved in IL-6-induced NE differentiation and that p38 MAPK is associated with IL-6-stimulated growth regulation in NSCLC-NE cells. These data suggest that both kinase pathways play critical roles in the pathogenesis of NSCLC-NE malignancies, providing new molecular targets for future therapeutic approaches.

Most cell culture and in vitro drug sensitivity assays utilize serum-supplemented media (SSM). However, fully defined serum-free media (SFM) offer several advantages and are being used increasingly for initiation and maintenance of cell cultures. Because serum inhibits the in vitro cytotoxicity of certain antineoplastic agents, we investigated the inter-relationships between medium type, cell proliferation and cytotoxic effect. Twenty-four human lung cancer cell lines were tested with nine anticancer agents in both media types. A semi-automated tetrazolium (MTT) colorimetric assay was used for assaying cell survival. Cell lines initiated and maintained in SFM preferentially proliferated in that medium type or proliferated equally well in both media types. In contrast, cell lines established in SSM varied considerably in their medium of preference. There appeared to be a direct correlation or trend between cell proliferative rate and cytotoxicity of all drugs with the possible exceptions of methotrexate and carmustine. In general, the cell lines were more sensitive to anticancer agents when they were exposed in the culture medium in which they preferentially proliferated. Therefore, to determine the influence of culture media on cytotoxicity, we analyzed the data only from lines that replicated equally efficiently in both media. After correction for cell proliferative rate, SSM had a negative effect on the cytotoxic action of some drugs (especially methotrexate, 5-fluorouracil and, to a lesser extent, mitomycin-C). Our results demonstrate that fully defined SFM may be suitable for initiating cell lines and for use in in vitro cytotoxicity assays for selection of individualized therapy or for screening of new anti-neoplastic agents, and thus may increase the number of antineoplastic agents that can be tested satisfactorily.

BACKGROUND At diagnosis, most small-cell lung cancers (SCLCs) are chemosensitive, whereas non-small-cell lung cancers (NSCLCs) are usually chemoresistant. Activation of ras genes and HER-2/neu genes (also known as ERBB2) is encountered in subpopulations of NSCLC but not in SCLC and has been linked to shortened survival. Therefore, activation of these genes may be associated with intrinsic chemoresistance in NSCLC. Studies have also suggested that the multidrug-resistant phenotype expressed by the MDR1 gene (also known as PGY1) does not correlate with the in vitro chemosensitivity of NSCLC cells or with clinical response to therapy and does not explain the spectrum of cross-resistance to drugs. PURPOSE The purpose of this study was to investigate the relationships between chemoresistance and the presence of ras gene point mutations and overexpression of the HER-2/neu gene in NSCLC cell lines, which indicates gene activation. METHODS Using a panel of 20 NSCLC cell lines established from untreated patients, we assessed the differences in HER-2/neu messenger RNA (mRNA) expression in the cell lines with or without ras mutations. We performed in vitro drug sensitivity testing by the tetrazolium-based MTT [i.e., 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide] assay with doxorubicin, carmustine, cisplatin, melphalan, mitomycin, and etoposide, and we determined the differences in IC50 values (i.e., the drug concentrations required to inhibit cell growth by 50%) for the cell lines. RESULTS We found a statistically significant correlation between the IC50 values for all six drugs and the degree of HER-2/neu gene expression in all 20 cell lines (r = .67-.86; P < .005) as well as in the subpopulation of eight cell lines with ras mutations (r = .83-.98; P < .05). The IC50 values for doxorubicin, carmustine, cisplatin, and melphalan were not significantly different in the cell lines with or without ras mutations, but the values for mitomycin and etoposide in lines with ras mutations were slightly lower than in those without ras mutations (borderline significance, P = .031). Levels of HER-2/neu expression in cell lines with ras mutations were lower than those without ras mutations, but the difference was not statistically significant. CONCLUSION Our findings indicate that overexpression of HER-2/neu is a marker for intrinsic multidrug resistance in NSCLC cell lines. IMPLICATIONS If the clinical relevance of our findings is confirmed, HER-2/neu gene expression can be used as a predictor of therapeutic failure in NSCLCs. The relationships between HER-2/neu gene expression, cell proliferation, and chemoresistance in NSCLC require further investigation.