Meiling Jin

Potential mechanism of interleukin-8 production from lung cancer cells: An involvement of EGF–EGFR–PI3K–Akt–Erk pathway

Tumor inflammatory microenvironment is considered to play the role in the sensitivity of tumor cells to therapies and prognosis of lung cancer patients. Interleukin‐8 (IL‐8) is one of critical chemo‐attractants responsible for leukocyte recruitment, cancer proliferation, and angiogenesis. The present study aimed at investigating potential mechanism of IL‐8 production from human non‐small cell lung cancer (NSCLC) SPC‐A1 cells. We initially found that EGF could directly stimulate IL‐8 production, proliferation, and bio‐behaviors of lung cancer cells through the activation of EGFR, PI3K, Akt, and Erk signal pathway. EGF‐stimulated IL‐8 production, phosphorylation of Akt and Erk, and cell proliferation and movement could be inhibited by EGFR inhibitor (Erlotinib), PI3K inhibitor (GDC‐0941 BEZ‐235 and SHBM1009), and ERK1/2 inhibitor (PD98059). Our data indicate that IL‐8 production from lung cancer cells could be initiated by their own produced factors, leading to the recruitment of inflammatory cells in the cancer tissue, and the formation of inflammatory microenvironment. Thus, it seems that the signal pathway of EGFR–PI3K–Akt–Erk can be the potential target of therapies for inflammatory microenvironment in lung cancer. J. Cell. Physiol. 227: 35–43, 2012.

The effects of Th2 cytokines on the expression of ADAM33 in allergen-induced chronic airway inflammation☆

A disintegrin and metalloprotease domain 33 (ADAM33) has been identified as an asthma susceptibility gene, which is associated with small-airway remodeling. However, the role of ADAM33 in the development of allergic airway inflammation is unclear. The present study used an established murine model of allergen-induced chronic airway inflammation, which was sensitized and then challenged by nebulized 2.5% ovalbumin (OVA) for 8 weeks (30 min/day, three times a week). The expression of ADAM33 mRNA detected by real time RT-PCR was significantly enhanced in the lung tissue of mice with OVA challenge, as compared with the group challenged with saline. This OVA-challenged model showed significant Th2-biased airway inflammation as well as airway remodeling with features of sub-epithelial fibrosis and mucus hyper-secretion. Furthermore, in vitro studies showed that IL-4 and IL-13 could significantly up-regulate the expression of ADAM33 mRNA in human fibroblasts in a concentration- and time-dependent manner as compared to normal controls. These results support the note that Th2 cytokines can up-regulate the expression of ADAM33 mRNA and ADAM33 may play an important role in the development of airway remodeling in allergen-induced chronic airway inflammation.

Role of aquaporin 5 in antigen‐induced airway inflammation and mucous hyperproduction in mice

Airway inflammation and mucus hyperproduction play the central role in the development of asthma, although the mechanisms remain unclear. The aquaporin (AQP)‐5 may be involved in the process due to its contribution to the volume of liquid secreted from the airways. The present study firstly found the overexpression of AQP5 in the airway epithelium and submucosal glands of asthmatics. Furthermore, we aimed at evaluating the role of AQP5 in airway inflammation and mucous hyperproductions during chronic allergic responses to house dust mite (HDM). Bronchoalveolar lavage levels of interleukin (IL)‐2, IL‐4, IL‐10, interferon‐γ and Mucin 5AC (MUC5AC), and number of peribronchial and perivascular cells were measured in AQP5 wild‐type and AQP5 knockout (KO) mice. We found that HDM induced airway inflammation, lung Th2 cell accumulation and mucin hypersecretion in C57BL/6 mice rather than AQP5 KO mice. Expression of MUC5AC and MUC5B proteins and genes in the lung tissue was significantly lower in AQP5 KO mice. Thus, our results implicate involvement of AQP5 in the development of airway inflammation and mucous hyperproduction during chronic asthma.

ADAM33 Gene Polymorphisms Associate with Asthma Susceptibility and Severity in East China Han Population

Objective. Multiple genetic and environmental factors impact the pathogenesis of asthma. ADAM33 (a disintegrin and metalloproteinase domain 33) represents a novel susceptibility gene for asthma in several diverse populations. The aim of this study was to determine whether single nucleotide polymorphisms (SNPs) of the ADAM33 gene associate with asthma susceptibility and severity in the Chinese Han population. Methods. A total of 224 subjects were enrolled, including 74 normal controls and 150 asthmatic patients. The asthmatic enrollees were further categorized into high- or low-severity groups according to the percentage of forced expiratory volume in 1 second of predicted value, symptoms, nighttime awakening, requirement for short-acting β2-agonist, and interference with normal activities. Six SNPs (F + 1, ST + 4, S1, S2, T1, and T2) in ADAM33 were genotyped using the polymerase chain restriction fragment length polymorphism method. Results. Three SNPs (F + 1, T1, and T2) of ADAM33 were found to have significant associations with asthma in the study population (p = .0058−.0067). The allele frequencies of two SNPs (F + 1, T1) in both the low- and high-severity groups were significantly different from the allele frequency in the control group. The allelic frequency of the T2 SNP was significantly different from that of the control group only in the high-severity group (p = .0081). Haplotype analysis demonstrated that the frequency of 7575G, 12433T, and 12462C (GTC haplotype) is higher in healthy controls than amongst asthma patients (78.4% vs. 61.8%, p = .0004). Conclusions. Polymorphisms of the ADAM33 gene associate with asthma susceptibility in the east China Han population, and the genetic association is stronger in high-severity asthmatics.

Lipopolysaccharide decreases aquaporin 5, but not aquaporin 3 or aquaporin 4, expression in human primary bronchial epithelial cells

Background and objective:  The aim of this study was to investigate the changes in expression of aquaporins (AQP) during differentiation of human bronchial epithelial cells and the role of lipopolysaccharide (LPS) in AQP expression.

Aquaporin 5 expression inhibited by LPS via p38/JNK signaling pathways in SPC-A1 cells.

Proper H(2)O to mucin ratio of airway mucus is important for mucociliary clearance. Recent studies suggest that decreased aquaporin 5 (AQP5) is correlated with increased staining of MUC5AC in submucosal glands of COPD patients. Lipopolysaccharide (LPS) is one of the major insults in airway mucin secretion in COPD. In this study, changes in both AQP5 and MUC5AC expression levels in SPC-A1, a human airway submucosal gland cell line, were quantified after exposure of the cells to LPS. AQP5 transcription and protein expression were decreased while MUC5AC expression was increased by LPS exposure in SPC-A1 cells. Further studies revealed that AQP5 expression was down-regulated via the p38/JNK signaling pathway, while MUC5AC was up-regulated through the EGFR-p38/JNK pathway. Therefore, p38 and JNK may become promising targets to preserve AQP5 expression and prevent MUC5AC over-expression to restore proper H(2)O to mucin ratio of the airway mucus, which may be beneficial to the clinical management of COPD patients.

Urban particulate matter triggers lung inflammation via the ROSMAPK- NF-κB signaling pathway

Background Particulate matter (PM) is a high risk factor for various respiratory diseases and triggers an inflammatory response in lung tissues. However, the molecular mechanism of the PM-induced inflammatory response is incompletely understood. Methods Human bronchial epithelial cells (HBECs) were treated with the urban PM 1649b for assessment of the inflammatory response. The intracellular level of reactive oxygen species (ROS) was measured by flow cytometry. PM-activated signaling pathways were addressed with specific inhibitors. In vivo, the C57 mice model of PM-induced acute lung inflammation was established with intratracheal instillation of PM for 2 consecutive days. The oxidant stress in lung tissues was assessed with dihydroethidium (DHE) staining, and malondialdehyde (MDA) activity and hydrogen peroxide (H2O2) assays. The histopathologic changes in lung tissues and number of inflammatory cells in bronchoalveolar lavage fluid (BALF) were examined. Expression of pro-inflammatory cytokines in BALF was measured by ELISA. Results PM increased the expression of interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-9 and cyclooxygenase (COX)-2 in a dose-dependent manner. ROS generation and activation of MAPK (ERK, JNK, p38 MAPK) and NF-κB pathways were detected in PM-exposed HBECs. Pretreatment with N-acetylcysteine (NAC) led to the inflammatory response, ROS level and activation of the MAPK and NF-κB pathways to be attenuated. Blockade of ERK, JNK or p38 MAPK pathway with specific inhibitor prevented the release of pro-inflammatory cytokines and activation of the NF-κB pathway. Inhibition of the NF-κB pathway reduced the expression of pro-inflammatory cytokines. In vivo, PM exposure increased oxidant stress in lung tissues, infiltration of inflammatory cells around PM in lung tissues, the number of total cells and inflammatory cells in BALF, and the concentrations of IL-1β, IL-6, IL-8 and MMP-9 in BALF, all of which were reversed partially upon NAC treatment. Conclusions PM exposure enhanced the airway inflammatory response significantly through ROS-mediated activation of MAPK (ERK, JNK, p38 MAPK) and downstream NF-κB signaling pathways. Oxidative stress appeared to be the key regulator for PM-induced lung inflammation. These results suggested the molecular mechanism of lung inflammation caused by PM.

Therapeutic roles of telocytes in OVA-induced acute asthma in mice

Telocytes (TCs) newly discovered as the mesenchyme‐derived interstitial cells were found to have supportive effects on mesenchymal stem cells (MSCs). The present study aimed at investigating effects of TCs or TCs gathered with MSCs on experimental airway inflammation and hyper‐responsiveness. The TCs were isolated from the lung tissue of the female BALB/c mice. The ovalbumin (OVA)‐induced asthma model was established. TCs (1 × 106/2 × 106) and/or MSCs (1 × 106) were injected through mice tail vein for consecutive three days before OVA excited the mice. This study at first demonstrated that the transplantation of TCs could improve allergen‐induced asthma by obviously inhibiting airway inflammation and airway hyper‐responsiveness preclinically, with the down‐regulation of Th2‐related cytokine IL‐4, transcription factor GATA‐3 and Th2 cell differentiation, while up‐regulation of Th1‐related cytokine IFN‐γ, transcription factor T‐bet and Th1 cells proliferation in asthma, just like MSCs. Co‐transplantation of TCs with MSCs showed better therapeutic effects on experimental asthma, even though the therapeutic effects of TCs alone were similar to those of MSCs alone. TCs and the combination of TCs with MSCs could improve the airway inflammation and airway hyper‐responsiveness and can be a new alternative for asthma therapy.

Peroxiredoxin 6 suppresses Muc5ac overproduction in LPS-induced airway inflammation through H2O2-EGFR-MAPK signaling pathway

Mucus hypersecretion is a prominent mechanism in airway inflammation. Muc5ac is a major component of mucus and can be activated by reactive oxygen species (ROS). Peroxiredoxin 6 (Prdx6) highly expresses in airway epithelium and protects the airway from oxidative stress. In this study, we investigated the roles of Prdx6 in lipopolysaccharide (LPS)-induced mucin production in mice. We found that the levels of H2O2 and the Muc5ac mRNA were significantly increased in Prdx6 (-/-) mice compared to those in C57BL/6J mice after LPS instillation, which were markedly inhibited by epithelial growth factor receptor (EGFR) inhibitor Elrotinib. In vitro studies showed that mRNA levels of Prdx6 were decreased while H2O2 and Muc5ac were increased in a dose-dependent manner after LPS exposure, with significant increase in Prdx6 knockdown bronchial epithelial cells compared with those in normal epithelial cells. LPS-induced Muc5ac release was significantly inhibited by EGFR inhibitor, p38 inhibitor and JNK inhibitor, but not ERK1/2 inhibitor, indicating that the H2O2-EGFR-MAPK pathway is likely involved in the responses. This study indicated that Prdx6 decreased LPS-induced Muc5ac increase and played important roles in mucin hypersecretion after LPS exposure.

Clinical characterization of ERBB2 exon 20 insertions and heterogeneity of outcomes responding to afatinib in Chinese lung cancer patients

Purpose ERBB2 exon 20 insertions (20ins) have been identified as oncogenic drivers in lung cancers. Lung cancer patients with 20ins benefit from afatinib. However, response heterogeneity was observed in patients harboring different 20ins subtypes. In this study, we interrogated clinical characteristics in ERBB2-mutated Chinese lung cancer and investigated the clinical outcomes of specific ERBB2 20ins in response to afatinib. Experimental design In this study, we retrospectively collected genomic profiling data of 7,520 lung cancer patients sequenced using next-generation sequencing in a Clinical Laboratory Improvement Amendments-certified laboratory. We analyzed the clinical and molecular features of patients harboring ERBB2 20ins and evaluated clinical outcomes of 19 patients with clinical records after afatinib treatment. Results ERBB2 20ins were identified in 2.27% (171/7,520) of this lung cancer cohort. It occurred with a high proportion in females with adenocarcinoma histology. ERBB2 20ins was mutually exclusive with other well-established lung cancer oncogenic driver mutations. The most frequently appearing subtype was Y772_A775dup (69.6%) and several novel insertion subtypes were also identified. The correlations of specific 20ins subtypes and survival were investigated. The presence of a glycine at position 778 in ERBB2 was suggested to be a common feature of drug sensitivity mutations. Patients harboring G778_P780dup (G778) subtype achieved longer median progression-free survival and median overall survival than other 20ins (non-G778) subtypes (median progression-free survival, 10 vs 3.3 months, P=0.32; median overall survival, 19.7 vs 7 months, P=0.16). Moreover, we presented the first clinical case of a lung squamous cell carcinoma patient harboring ERBB2 20ins who achieved partial response to afatinib. Conclusion This study interrogated the characteristics of ERBB2 20ins in a large cohort from single ethnicity and demonstrated the response heterogeneity to afatinib among different ERBB2 20ins subtypes. Further studies in a larger cohort are needed to investigate the underlying molecular mechanisms and clinical response of different ERBB2 20ins subtypes.