Smoking-induced CCNA2 expression promotes lung adenocarcinoma tumorigenesis by boosting AT2/AT2-like cell differentiation.

Lung adenocarcinoma (LUAD), a type of non-small cell lung cancer (NSCLC), originates from not only bronchial epithelial cells but also alveolar type 2 (AT2) cells, which could differentiate into AT2-like cells. AT2-like cells function as cancer stem cells (CSCs) of LUAD tumorigenesis to give rise to adenocarcinoma. However, the mechanism underlying AT2 cell differentiation into AT2-like cells in LUAD remains unknown. We analyze genes differentially expressed and genes with significantly different survival curves in LUAD, and the combination of these two analyses yields 147 differential genes, in which 14 differentially expressed genes were enriched in cell cycle pathway. We next analyze the protein levels of these genes in LUAD and find that Cyclin-A2 (CCNA2) is closely associated with LUAD tumorigenesis. Unexpectedly, high CCNA2 expression in LUAD is restrictedly associated with smoking and independent of other driver mutations. Single-cell sequencing analyses reveal that CCNA2 is predominantly involved in AT2-like cell differentiation, while inhibition of CCNA2 significantly reverses smoking-induced AT2-like cell differentiation. Mechanistically, CCNA2 binding to CDK2 phosphorylates the AXIN1 complex, which in turn induces ubiquitination-dependent degradation of ß-catenin and inhibits the WNT signaling pathway, thereby failing AT2 cell maintenance. These results uncover smoking-induced CCNA2 overexpression and subsequent WNT/ß-catenin signaling inactivation as a hitherto uncharacterized mechanism controlling AT2 cell differentiation and LUAD tumorigenesis.

Inhibition of non-muscular myosin light chain kinase accelerates the clearance of inflammatory cells by promoting the lysosome-mediated cell death.

Infections like COVID-19 are the primary cause of death around the world because they can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis. Inflammatory cells serve as crucial protective barriers in these diseases. However, excessive accumulation of inflammatory cells is also one of the major causes of organ damage. The non-muscular myosin light chain kinase (nmMLCK) plays crucial of cytoskeletal components involved in endothelial cell-matrix and cell-cell adhesion, integrity, and permeability. Our previous investigations found that ML-7, a specific inhibitor of MLCK, promoted neutrophil apoptosis through various signaling pathways. In this study, we found that knockout of MLCK significantly promote apoptosis of neutrophils and macrophages in the BALF of the LPS-induced ALI, meanwhile it had no effect on the apoptosis of neutrophils in the circulatory system. RNA-sequencing revealed that the effect of MLCK knockout in inducing apoptosis of inflammatory cells was mediated through lysosomes. Administering ML-7 into the lungs significantly promoted neutrophil apoptosis, accelerating their clearance. In the LPS- or CLP-induced sepsis models, ML-7 administration significantly improves the apoptosis of inflammatory cells, especially neutrophils, at the infection site but had no impact on neutrophils in the circulatory system. ML-7 also significantly improved the survival rate of mice with LPS- or CLP-induced sepsis. Taken together, we found that MLCK plays a crucial role in the survival of inflammatory cells at the infection site. Inhibiting MLCK significantly induces apoptosis of inflammatory cells at the infection site, promoting inflammation resolution, with no impact of the circulatory system.

Control of mitochondria-associated endoplasmic reticulum membranes by protein S-palmitoylation: Novel therapeutic targets for neurodegenerative diseases.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) are dynamic coupling structures between mitochondria and the endoplasmic reticulum (ER). As a new subcellular structure, MAMs combine the two critical organelle functions. Mitochondria and the ER could regulate each other via MAMs. MAMs are involved in calcium (Ca2+) homeostasis, autophagy, ER stress, lipid metabolism, etc. Researchers have found that MAMs are closely related to metabolic syndrome and neurodegenerative diseases (NDs). The formation of MAMs and their functions depend on specific proteins. Numerous protein enrichments, such as the IP3R-Grp75-VDAC complex, constitute MAMs. The changes in these proteins govern the interaction between mitochondria and the ER; they also affect the biological functions of MAMs. S-palmitoylation is a reversible protein post-translational modification (PTM) that mainly occurs on protein cysteine residues. More and more studies have shown that the S-palmitoylation of proteins is closely related to their membrane localization. Here, we first briefly describe the composition and function of MAMs, reviewing the component and biological roles of MAMs mediated by S-palmitoylation, elaborating on S-palmitoylated proteins in Ca2+ flux, lipid rafts, and so on. We try to provide new insight into the molecular basis of MAMs-related diseases, mainly NDs. Finally, we propose potential drug compounds targeting S-palmitoylation.

Cigarette smoke extract stimulates bronchial epithelial cells to undergo a SUMOylation turnover.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) characterized by the airway and lung inflammation, is a leading cause of morbidity and mortality worldwide, especially among smokers over 40 years of age and individuals exposed to biomass smoke. Although the detailed mechanisms of this disease remain elusive, there is feasible evidence that protein posttranslational modifications (PTMs) may play a role in its pathoetiology. We thus conducted studies to dissect the effect of cigarette smoke extracts (CSE) on the change of SUMOylated substrates in human bronchial epithelial cells (HBEs). METHODS: Samples were collected in HBEs with or without 24 h of CSE insult and then subjected to Western-blot and LC-MS/MS analysis. Subsequently, bioinformatic tools were used to analyze the data. The effect of SUMOylation on cytochrome P450 1A1 (CYP1A1) was evaluated by flow cytometry. RESULTS: It was noted that CSE stimulated HBEs to undergo a SUMOylation turnover as evidenced by the changes of SUMOylated substrates and SUMOylation levels for a particular substrate. The SUMOylated proteins are relevant to the regulation of biological processes, molecular function and cellular components. Particularly, CSE stimulated a significant increase of SUMOylated CYP1A1, a critical enzyme involved in the induction of oxidative stress. CONCLUSIONS: Our data provide a protein SUMOylation profile for better understanding of the mechanisms underlying COPD and support that smoking induces oxidative stress in HBEs, which may predispose to the development of COPD in clinical settings.

Chrysophanol protects human bronchial epithelial cells from cigarette smoke extract (CSE)-induced apoptosis.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by the persistent airflow obstruction. Chrysophanol, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, has been reported to be protective for some inflammatory diseases. The present report aimed to dissect its effect on cigarette smoke extract (CSE)-induced apoptosis in 16HBECs, a human bronchial epithelial cell line. METHODS: CCK8 cell viability assay was conducted to evaluate the protective effect of chrysophanol on 16HBECs after CSE induction. Western blot analysis, Annexin V/PI staining and TUNEL assay were conducted to test the effect of chrysophanol on 16HBECs apoptosis induced by CSE. Then the western blot assay measured associated molecular pathways to dissect the mechanisms underlying protective effect of chrysophanol on 16HBECs. RESULTS: Chrysophanol protects 16HBECs against CSE-induced apoptosis in a dose dependent manner. Specifically, pre-treatment of 16HBECs with 20 mmol/l of chrysophanol, reduced CSE-induced apoptosis by almost 10%. Mechanistically, chrysophanol manifested high potency to attenuate CSE-induced expression of apoptotic markers, Bax and cleaved caspase 3. In particular, chrysophanol not only represses CSE-induced oxidative stress by inhibiting CYP1A1 expression, but also suppresses CSE-induced ER stress by inhibiting pPERK, ATF4 and ATF6 expression. CONCLUSION: Chrysophanol showed protective effect on CSE-induced epithelial injuries in cell line 16HBECs. And our data support that chrysophanol could be employed to reduce the toxicity of cigarette smoke in bronchial epithelial cells, which may have the potential to decrease the risk for developing COPD in smoking subjects.

Analysis of methyl DNA adducts and metabolites in BEAS-2B cells induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is classified as a Group 1 human carcinogen. It is metabolically activated by P450 enzymes to intermediate methylate and pyridyloxobutylate DNA, resulting in the formation of DNA adduct that is critical for the carcinogenicity of NNK. To directly and objectively examine the DNA adduct formation profiles without the complexity of factors in vivo, in the present study, five kinds of methyl DNA adducts were first identified in the incubation model of NNK established with human lung epithelial cells (BEAS-2B). The level of methyl DNA adducts and metabolites of NNK were quantitatively analyzed, respectively. With the increase of exposure time and dose, the level of methyl DNA adducts and metabolites increased. Furthermore, with the changes of the activity of P450 enzymes, which is the main enzyme regulating the α-hydroxylation of NNK, we found the levels of both methyl adducts and metabolites formed via α-hydroxylation in experimental groups showed the same trend compared with those in control group, while the metabolites formed via other pathways changed in the opposite trend. The result proves that the methyl adducts induced by NNK generate via α-hydroxylation pathway in BEAS-2B cells.

The impact of tobacco smoking on physical activity and metabolism in mice.

Cigarette smoking can increase the risk of many respiratory and chronic systemic diseases. Particularly, cigarette smoke produces toxic particulate matter (PM), which is harmful to the smokers. Although previous studies have demonstrated the toxicity of cigarette smoke PM and its relationship with disease pathogenesis, systematic data for the impact of cigarette smoke PM on physical activity and metabolism in animals are still lacking. In this report, the C57/B6 mice were exposed to cigarette smoke PM in a smoking chamber coupled with the analysis of metabolic changes and physical activity in metabolic cages at indicated time, for a period up to 12-month-old of age. The mice became excited following short period (e.g., 3 months) but listless after long-term cigarette smoke PM exposures (e.g., 9 or 10 months), as manifested by the changes of drink/food intake and daily activities along with increased oxygen consumption and CO2 accumulation. Our data suggest that particulate matter originated from cigarette smoke impairs metabolism and physical activities.

Highly sensitive photoelectrochemical biosensor for kinase activity detection and inhibition based on the surface defect recognition and multiple signal amplification of metal-organic frameworks.

A turn-on photoelectrochemical (PEC) biosensor based on the surface defect recognition and multiple signal amplification of metal-organic frameworks (MOFs) was proposed for highly sensitive protein kinase activity analysis and inhibitor evaluation. In this strategy, based on the phosphorylation reaction in the presence of protein kinase A (PKA), the Zr-based metal-organic frameworks (UiO-66) accommodated with [Ru(bpy)3]2+ photoactive dyes in the pores were linked to the phosphorylated kemptide modified TiO2/ITO electrode through the chelation between the Zr4+ defects on the surface of UiO-66 and the phosphate groups in kemptide. Under visible light irradiation, the excited electrons from [Ru(bpy)3]2+ adsorbed in the pores of UiO-66 injected into the TiO2 conduction band to generate photocurrent, which could be utilized for protein kinase activities detection. The large surface area and high porosities of UiO-66 facilitated a large number of [Ru(bpy)3]2+ that increased the photocurrent significantly, and afforded a highly sensitive PEC analysis of kinase activity. The detection limit of the as-proposed PEC biosensor was 0.0049UmL-1 (S/N!=!3). The biosensor was also applied for quantitative kinase inhibitor evaluation and PKA activities detection in MCF-7 cell lysates. The developed visible-light PEC biosensor provides a simple detection procedure and a cost-effective manner for PKA activity assays, and shows great potential in clinical diagnosis and drug discoveries.

Gem-difluoromethylated and trifluoromethylated derivatives of DMDP-related iminosugars: synthesis and glycosidase inhibition.

Gem-difluoromethylated and trifluoromethylated derivatives of DMDP-related iminosugars have been synthesized from cyclic nitrones 12, 13, 18, ent-18 or 23 and nitrone-derived aldehydes 20 or ent-20. The fluorinated iminosugars were assayed against various glycosidases, and ent-8 showed moderate but selective α-l-rhamnosidase inhibition. Difluoro or trifluoro units influenced the inhibitory activities of iminosugars in a more complex manner than single fluoro substitution. This may be correlated with their highly hydrophobic character and strong electron-withdrawing effect.

[Multivariate analysis of pharyngo cutaneou fistulas after larynx cancer and lower pharynx cancer surgery].

OBJECTIVE: To investigate the relative factors of pharyngo cutaneou fistulas after larynx cancer and lower pharynx cancer surgery. METHOD: The clinical datas of 87 larynx cancer patients and lower pharynx cancer patients admitted were retrospectively analyzed. According to the type of postoperative complications all cases could be divided into pharyngo cutaneou fistulas group and no pharyngo cutaneou fistulas group. Thirty-eight kinds of factors,including age, clinical stage, plasma electrolytes level and type of procedure are in the multivariate analysis, and the variability indicators are in binary-regression analysis. RESULT: Eleven patients had pharyngo cutaneou fistulas (12.64%). Univariate analysis indicated that BMI, pre-operative serum potassium, operation time, cervical lymph dissection, post-operative prealbumin, post-operative hemoglobin, infection and delayed union of incision were the risk factors of pharyngo cutaneou fistulas (P < 0.05). Logistic stepwise regression analysis indicated that post-operative prealbumin and operation time were the independent risk factors. CONCLUSION: To avoid pharyngo cutaneou fistulam, it is very necessary to correct electrolyte disorder and negative nitrogen balance. To shorten the operation time, to avoid incision infection and delayed union were helpfulness, too.

[Kinetic study on pyrolysis of psoralen].

In this study, products of psoralen pyrolysis were detected using a solid pyrolysis apparatus and synchrotron radiation vacuum ultraviolet photoionization mass spectrum (SVUV-PIMS). The pyrolytic kinetics of psoralen was also studied by calculating its initial pyrolytic route in quantum chemistry. According to the findings with SVUV-PIMS, three pyrolytic products were observed, CO, C9H6O and C10H6O2. Theoretically, three fragment pathways were calculated for psoralen, in which the major primary decomposition route was de-CO, and the major secondary decomposition reaction was de-CO reaction of de-CO products.