Activation of astrocytes by ANXA2-derived extracellular vesicles from lung cancer cells affects aggressiveness, immunotherapy response and microenvironment of lung cancer.

Background: ANXA2 has been extensively documented in relation to cancer. Nevertheless, the involvement of ANXA2 in lung carcinoma remains uncertain. Methods: Data from The Cancer Genome Atlas (TCGA) database was downloaded using open-access methods. The examination of publicly available data was conducted utilizing the R software. The mRNA level of specific molecules was detected using Real-time Quantitative PCR (qPCR). The protein level of specific molecules was detected using the Western blot assay. The cell proliferation ability of cancer cells was assessed using the CCK8 assay. The invasion and migration capability of cancer cells was assessed using the Transwell assay. Validation of exosomes extraction was conducted using electron microscopy and particle size analysis. Results: In this study, based on series experiments, we found that ANXA2 can promote the activation of neuroastrocytes cells CP-H122 through the exosome pathway. Also, we found that ANXA2 can be transmitted from A549 cells to CP-H122 through the exosomes pathway and further promote the activation of CP-H122. Activated CP-H122 cells further enhance the proliferation, invasion and metastasis of A549 cells. Meanwhile, we performed transcriptome sequencing to explore the downstream genes of ANXA2 to screen potential targets for follow-up studies. Analysis based on public data showed that ANXA2 was related to the worse survival performance and clinical features of lung cancer. Gene set enrichment analysis based on the Hallmark gene set indicated that the patient with high ANXA2 expression might have a higher activity of the apical surface, reactive oxygen species pathway, angiogenesis, TGF-ß signaling, MYC target, but lower activity of pancreas-ß cells. More important, our results showed that ANXA2 can affects immunotherapy response and reshape immune microenvironment of lung cancer. Conclusions: This study demonstrates that ANXA2 activates CP-H122 cells, affects A549 cell behavior, and impacts lung cancer prognosis and immunotherapy response.

Simultaneous online SPE-HPLC-MS/MS quantification of gefitinib, osimertinib and icotinib in dried plasma spots: Application to therapeutic drug monitoring in patients with non-small cell lung cancer.

Gefitinib, osimertinib and icotinib are the most commonly used tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with EGFR mutation. Therapeutic drug monitoring (TDM) for these TKIs has become a standard and essential procedure. Dried plasma spots (DPS) was choosen for microsampling strategies for TDM, allowing easy and cost-effective logistics in many settings. This study developd and validated an assay for the simultaneous quantitative determination of gefitinib, osimertinib and icotinib in DPS by online solid-phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS) system. The TKIs were extracted from DPS with methanol and enriched on a Welch Polar-RP SPE column (30 × 4.6 mm, 5 µm), followed by separation on Waters X Bridge C18 analytical column(4.6 × 100 mm, 3.5 µm). The method achieved LLOQ of 2 ng mL-1 for gefitinib and osimertinib (4 ng mL-1 for icotinib), respectively (r2 > 0.99). Precision (within-run 1.54-7.41 % RSD; between-run 3.03-12.84 % RSD), accuracy (range from 81.47 % to 105.08 %; between-run bias 87.87-104.13 %). Osimertinib and icotinib were stable in DPS stored at - 40 °C for 30 days, 4 °C, 42 °C and 60 °C for 5 days and well-sealed 37 °C,75 % humidity (except gefitinib). Lastly, the assay was applied to TDM of TKIs in 46 patients and the results were compared to SALLE assisted LC-MS analysis, it could be confirmed that the developed method achieves similarly good results as the already established one and no bias could be detected. It implies that this method capable of supporting clinical follow-up TDM of TKIs in DPS from poor medical environment.

(-)-Epicatechin ameliorates cigarette smoke-induced lung inflammation via inhibiting ROS/NLRP3 inflammasome pathway in rats with COPD.

Chronic obstructive pulmonary disease (COPD) with increased morbidity and mortality is a worldwide healthcare challenge closely associated with cigarette smoking (CS). Currently, there is no effective therapeutic strategy to control inflammation in COPD patients. The present study tested the protective effects of (-)-Epicatechin (EC), a type of flavonoid, on CS-induced COPD and the underlying mechanism. Also, EC repressed the production of reactive oxygen species (ROS) and improved human bronchial epithelial cell viability after cigarette smoke extract (CSE) treatment. Further studies demonstrated that EC promotes ubiquitin-mediated Keap1 degradation by upregulating tripartite motif-containing protein 25 (TRIM25) expression and enhances the nuclear localization of Nrf2 protein. Also, EC dramatically inhibits the activation of NLRP3 inflammasome and reduces the CSE-induced pyroptosis, as indicated by decreasing lactate dehydrogenase release and the number of caspase-1-positive cells. Importantly, Nrf2 knockdown reversed the protective effect of EC on human bronchial epithelial cells, at least partially. Consistent with the results in vitro, EC inhibits the activation of NLRP3 inflammasome and relieves the CS-induced lung inflammation, as evident from decreased interleukin (IL)-1ß and IL-18 secretion in a COPD rat model. In conclusion, this study revealed the protective effect of EC on experimental COPD rats and elucidated the mechanism of EC promoting Nrf2 activity, which might provide a novel therapeutic strategy for COPD.

Simultaneous and rapid determination of 12 tyrosine kinase inhibitors by LC-MS/MS in human plasma: Application to therapeutic drug monitoring in patients with non-small cell lung cancer.

In recent years, more than 50 tyrosine kinase inhibitors (TKIs) was indicated against numerous cancers, especially outstanding advantages in the treatment of non-small cell lung cancer (NSCLC), and several studies have shown that therapeutic drug monitoring (TDM) of TKIs can improve treatment efficacy and safety. The present study aimed to develop and validate a LC-MS/MS method for the TDM of 12 TKIs (gefitinib, erlotinib, afatinib, dacomitinib, icotinib, osimertinib, crizotinib, ceritinib, alectinib, dabrafenib, trametinib, anlotinib) in patients with NSCLC. The analytes of interest and internal standard were extracted from human plasma. Salting-out assisted liquid-liquid extraction (SALLE) with 5 M ammonium acetate solution was optimized for method validation and compared to simple protein precipitation (PPT). Chromatographic separation was conducted on Waters X bridge C18 column (100 × 4.6 mm, 3.5 µm) using a gradient elution of acetonitrile/5mM ammonium acetate in pure water with 0.1% (v/v) formic acid at 40 °C within 6 min. The total flow was maintained at 1 mL/min, 30% of the post column flow was split into the mass spectrometer and the rest to waste via a 3-way tee. The mass analysis was performed by positive ion electrospray ionization (ESI) in multiple-reaction monitoring (MRM) mode. The assay was validated based on the guidelines on bioanalytical methods by FDA. This quantification method was proved to be satisfactory in selectivity, accuracy, precision, linearity (r2 > 0.995), recovery, matrix effect and stability and the accuracy was further assessed in plasma with a degree of hemolysis of 4%. The described method to simultaneously quantify the 12 selected anticancer drugs in human plasma was successfully validated and applied to routine TDM of gefitinib, erlotinib, icotinib, osimertinib, crizotinib and anlotinib in cancer patients. TKIs plasma monitoring helps to individualize dose adjustment and manage adverse effects in NSCLC patients.

MiR-222 inhibition alleviates Staphylococcal Enterotoxin B-induced inflammatory acute lung injury by targeting Foxo3.

Acute lung injury (ALI) is a common acute and severe disease in clinical practice. Staphylococcal Enterotoxin B (SEB) is a superantigen that can cause inflammatory ALI. MiR-222 has been demonstrated to be upregulated in SEB-induced inflammatory ALI, but its exact roles and functions remain ill-defined. In this study, SEB exposure led to inflammatory ALI and high expression of miR-222 in model mice and lung infiltrating mononuclear cells, but the inflammatory response and high expression of miR-222 were restored in miR-222-/- mice. Moreover, we investigated the roles of miR-222 in vitro and observed that the concentrations of inflammatory cytokines and the expression of miR-222 were all elevated in SEB-activated splenocytes and miR-222 inhibition reversed the effects. Foxo3 was confirmed as a direct target of miR-222. Interestingly, SEB exposure led to a decrease of Foxo3 expression, and Foxo3 knockdown partially reversed the promotion of Foxo3 and the inhibition of inflammatory cytokines induced by miR-222 inhibitor in SEB-activated splenocytes. Our data indicated that miR-222 inhibition could alleviate SEB-induced inflammatory ALI by directly targeting Foxo3, shedding light on the potential therapeutic of miR-222 for SEB-induced inflammation in the lung.

The release of tryptase from mast cells promote tumor cell metastasis via exosomes.

BACKGROUND: Cancer cells release exosomes and can be taken up by mast cells (MCs), but the potential functional effects of MCs on tumor metastasis remain unknown. METHOD: Exosomes were isolated from the lung adenocarcinoma cell line A549, and the uptake of PKH26-labeled exosomes by bone marrow MCs was examined via flow cytometry and fluorescence microscopy. Cytokines and tryptase in MC supernatant were analyzed using an ELISA kit, and the presence of tryptase was evaluated by Western blotting. Cell proliferation and migration were determined through CCK-8 and transwell assays. Proteins in the tryptase-JAK-STAT signaling pathway were detected by Western blotting. RESULTS: In this study, we show that exosomes from A549 cells can be taken up by MCs. Moreover, A549 exosomes contain stem cell factor (SCF) to MCs and subsequently induce the activation of MCs through SCF-KIT signal transduction, which leads to MC degranulation and the release of tryptase. Tryptase accelerates the proliferation and migration of human umbilical vein endothelial cells (HUVECs) through the JAK-STAT signaling pathway. CONCLUSIONS: Our results reveal a mechanism for metastasis in which exosomes can transfer SCF to and activate MCs, which can affect the release of tryptase and the angiogenesis of HUVECs.

CXCR4 knockdown prevents inflammatory cytokine expression in macrophages by suppressing activation of MAPK and NF-κB signaling pathways.

BACKGROUND: Recent evidence has shown that C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in acute lung injury (ALI). Macrophages are key factors in the pathogenesis of ALI. The aim of this study was to investigate the role of CXCR4 in macrophages after lipopolysaccharide (LPS) stimulation and confirm that CXCR4 knockdown can inhibit inflammatory cytokines by suppressing mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathway activation. RESULTS: In this study, we found that CXCR4 expression in lung tissue of ALI was significantly increased using immunofluorescence. We also found that the expression of CXCR4 in macrophages sorted from bronchoalveolar lavage fluid (BALF) of ALI was obviously upregulated through RT-qPCR. After CXCR4 knockdown using siRNA, we found that the expression of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) was obviously down regulated in macrophages. Additionally, the phosphorylation of p38, Erk, and p65 was significantly decreased after CXCR4 knockdown through western blotting. CONCLUSIONS: Taken together, the present study suggests that CXCR4 knockdown may inhibit inflammatory cytokine expression in macrophages by suppressing MAPK and NF-κB signaling pathway activation. Therefore, CXCR4 knockdown may have potential clinical value in treating ALI.

LPS-induced MMP-9 expression is mediated through the MAPKs-AP-1 dependent mechanism in BEAS-2B and U937 cells.

AIM OF THE STUDY: Matrix metalloproteinases (MMPs) play a critical role in chronic obstructive pulmonary disease (COPD). This study investigated the role of mitogen-activated protein kinases (MAPKs) in MMP-9 secretion of BEAS-2B cells, a human bronchial epithelial cell line and U937 cells, a human myeloid leukaemia cell line, which could differentiate into macrophage, after LPS stimulation, and some details of involved signaling. MATERIALS AND METHODS: MTT assay was used to measure cell viability. U937 cells were incubated for 48h with 100ng/ml PMA, and had a resting period of 24h with culture medium without PMA for differentiation of U937 cells into macrophages. For the experiments, U937 cells or BEAS-2B cells were pretreated with several inhibitors and then stimulated by LPS. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and DNA binding activity assay were used for measuring the protein expression, RNA expression, cytokine production and DNA binding activity, respectively. RESULTS: We found LPS induced MMP-9 expression and secretion were completely blocked by stress-activated protein kinase/jun kinase (SAPK/JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors but not by p38 inhibitor. LPS-induced transactivation of AP-1 was also inhibited by JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059. CONCLUSIONS: The present study suggests that in BEAS-2B cells and U937 cells, LPS probably activates ERK1/2 pathway and JNK pathway, which in turn initiate AP-1 activity, and leading to MMP-9 expression. Thus the ERK1/2 inhibitor and JNK inhibitor may have potential clinical value in treating COPD.

Angiotensin-Converting Enzyme 2 Inhibits Apoptosis of Pulmonary Endothelial Cells During Acute Lung Injury Through Suppressing MiR-4262.

BACKGROUND/AIMS: Angiotensin converting enzyme 2 (ACE2) treatment suppresses the severity of acute lung injury (ALI). The effects of ACE2 in ALI have been shown to not only result from its antagonizing hydrolyzing angiotensin II (AngII), which is responsible for reduction in the vascular tension and pulmonary accumulation of inflammatory cells, but also result from a role of ACE2 in suppressing the ALI-induced apoptosis of pulmonary endothelial cells (PECs). Nevertheless, the underlying mechanisms of the role of ACE2 on PEC apoptosis are not completely understood. METHODS: Here, we used a bleomycin-induced mouse model for ALI that has been published in our previous studies. We analyzed the mRNA and protein levels of an anti-apoptotic protein Bcl-2 in the ALI-mice that have been treated w/o ACE2. We analyzed miR-4262 levels in the mouse lung in these mice. Bcl-2-targeting miRNAs were predicted using bioinformatics algorithms and a luciferase reporter assay was applied to examine the effects of miR-4262 on the Bcl-2 protein translation upon their binding to 3'-UTR of Bcl-2 mRNA. Adeno-associated viruses carrying either miR-4262 mimics or antisense were injected into ALI-mice without ACE2, and their effects on the apoptosis in mouse lung cells were analyzed by Western blot. RESULTS: ACE2 inhibited the ALI-induced apoptosis of pulmonary cells in vivo partially through upregulation of Bcl-2 protein, but not Bcl-2 mRNA. ACE2 appeared to significantly suppress the upregulation of miR-4262 in mouse lung after ALI. MiR-4262 was found to target 3'-UTR of Bcl-2 mRNA to inhibit its protein translation in PECs. In vivo administration of antisense of miR-4262 decreased apoptosis of pulmonary cells and severity of the ALI in mice. CONCLUSION: ACE2-induced suppression of miR-4262 partially contribute to the inhibition of the PEC apoptosis after ALI through Bcl-2. MiR-4262 may be a novel promising treatment target for ALI and ARDS.