The mitochondrial protein YME1 Like 1 is important for non-small cell lung cancer cell growth.

The expression and biological function of the mitochondrial inner membrane protease YME1L (YME1 Like 1 ATPase) in NSCLC are tested here. Bioinformatical analyses and results from local human tissues show that YME1L expression is elevated in NSCLC tissues. YME1L upregulation was observed in primary and immortalized NSCLC cells. In NSCLC cells, shRNA-mediated silence of YME1L or dCas9/sgRNA-induced knockout (KO) of YME1L robustly suppressed cell growth and migration, and provoking apoptosis. YME1L shRNA/KO resulted in mitochondrial dysfunctions in NSCLC cells, leading to mitochondrial depolarization, ROS accumulation and ATP depletion. Conversely, ectopic YME1L overexpression augmented NSCLC cell proliferation and motility. Akt-S6K1 phosphorylation was reduced after YME1L shRNA/KO in primary NSCLC cells, but augmented after YME1L overexpression. Importantly, YME1L KO-caused anti-NSCLC cell activity was attenuated by a constitutively-activate Akt1 (S473D) construct. In vivo, subcutaneous NSCLC xenograft growth was remarkably slowed following intratumoral YME1L shRNA AAV injection in nude mice. YME1L knockdown, Akt-mTOR inactivation and ATP reduction were detected in YME1L-silenced NSCLC xenografts. Taken together, overexpressed YME1L in NSCLC exerts pro-tumorigenic function.

Gene expression profile of epithelial-mesenchymal transition in tumours of patients with nonsmall cell lung cancer: the influence of COPD.

Epithelial-mesenchymal transition (EMT) is involved in the pathophysiology of lung cancer (LC) and COPD, and the latter is an important risk factor for LC. We hypothesised that the EMT gene expression profile and signalling cascade may differ in LC patients with COPD from those with no respiratory diseases. In lung tumour specimens obtained through video-assisted thoracoscopic surgery from LC (n=20, control group) and LC-COPD patients (n=30), gene expression (quantitative real-time PCR amplification) of EMT markers SMAD3, SMAD4, ZEB2, TWIST1, SNAI1, ICAM1, VIM, CDH2, MMP1 and MMP9 was detected. In lung tumours of LC-COPD compared to LC patients, gene expression of SMAD3, SMAD4, ZEB2 and CDH2 significantly declined, while no significant differences were detected for the other analysed markers. A significant correlation was found between pack-years (smoking burden) and SMAD3 gene expression among LC-COPD patients. LC-COPD patients exhibited mild-to-moderate airway obstruction and a significant reduction in diffusion capacity compared to LC patients. In lung tumour samples of patients with COPD, several markers of EMT expression, namely SMAD3, SMAD4, ZEB2 and CDH2, were differentially expressed suggesting that these markers are likely to play a role in the regulation of EMT in patients with this respiratory disease. Cigarette smoke did not seem to influence the expression of EMT markers in this study. These results have potential clinical implications in the management of patients with LC, particularly in those with underlying respiratory diseases.

Profile of Clinical and Analytical Parameters in Bronchiectasis Patients during the COVID-19 Pandemic: A One-Year Follow-Up Pilot Study.

Whether the COVID-19 pandemic may have modified the clinical planning and course in bronchiectasis patients remains to be fully elucidated. We hypothesized that the COVID-19 pandemic may have influenced the management and clinical outcomes of bronchiectasis patients who were followed up for 12 months. In bronchiectasis patients (n = 30, 23 females, 66 years), lung function testing, disease severity [FEV1, age, colonization, radiological extension, dyspnea (FACED), exacerbation (EFACED)] and dyspnea scores, exacerbation numbers and hospitalizations, body composition, sputum microbiology, and blood analytical biomarkers were determined at baseline and after a one-year follow-up. Compared to baseline (n = 27, three patients dropped out), in bronchiectasis patients, a significant increase in FACED and EFACED scores, number of exacerbations, and erythrocyte sedimentation rate (ESR) was observed, while FEV1, ceruloplasmin, IgE, IgG, IgG aspergillus, IgM, and IgA significantly decreased. Patients presenting colonization by Pseudomonas aeruginosa (PA) remained unchanged (27%) during follow-up. In bronchiectasis patients, FEV1 declined only after a one-year follow-up along with increased exacerbation numbers and disease severity scores, but not hospitalizations. However, a significant decrease in acute phase-reactants and immunoglobulins was observed at the one-year follow-up compared to baseline. Despite the relatively small cohort, the reported findings suggest that lung function impairment may not rely entirely on the patients' inflammatory status.

Respiratory and Peripheral Muscle Weakness and Body Composition Abnormalities in Non-Cystic Fibrosis Bronchiectasis Patients: Gender Differences.

As demonstrated in COPD, bronchiectasis patients may experience respiratory and peripheral muscle dysfunction. We hypothesized that respiratory and peripheral (upper and lower limbs) muscle function and nutritional status may be more significantly altered in female than in males for identical age and disease severity. In mild-to-moderate bronchiectasis patients (n = 150, 114 females) and 37 controls (n = 37, 21 females), radiological extension, maximal inspiratory and expiratory pressures (MIP and MEP), sniff nasal inspiratory pressure (SNIP), hand grip and quadriceps muscle strengths, body composition, and blood analytical biomarkers were explored. Compared to the controls, in all bronchiectasis patients (males and females), BMI, fat-free mass index (FFMI), fat tissue, upper and lower limb muscle strength, and respiratory muscle strength significantly declined, and FFMI, fat tissue, and quadriceps muscle function were significantly lower in female than male patients. In patients with mild-to-moderate bronchiectasis, respiratory and peripheral muscle function is significantly impaired and only partly related to lung disease status. Quadriceps muscle strength was particularly weakened in the female patients and was negatively associated with their exercise tolerance. Muscle weakness should be therapeutically targeted in bronchiectasis patients. Body composition and peripheral muscle function determination should be part of the comprehensive clinical assessment of these patients.

Do Redox Balance and Inflammatory Events Take Place in Mild Bronchiectasis? A Hint to Clinical Implications.

We hypothesized that in mild bronchiectasis patients, increased systemic inflammation and redox imbalance may take place and correlate with clinical parameters. In plasma samples from patients with very mild bronchiectasis, inflammatory cells and molecules and redox balance parameters were analyzed. In the patients, lung function and exercise capacity, nutritional status, bacterial colonization, and radiological extension were assessed. Correlations between biological and clinical variables were determined. Compared to healthy controls, levels of acute phase reactants, neutrophils, IgG, IgA, myeloperoxidase, protein oxidation, and GSH increased and lung function and exercise capacity were mildly reduced. GSH levels were even greater in ex-smoker and Pseudomona-colonized patients. Furthermore, radiological extension inversely correlated with airway obstruction and, disease severity, and positively correlated with neutrophil numbers in mild bronchiectasis patients with no nutritional abnormalities. In stable patients with mild bronchiectasis, several important inflammatory and oxidative stress events take place in plasma. These findings suggest that the extension of bronchiectasis probably plays a role in the development of redox imbalance and systemic inflammation in patients with mild bronchiectasis. These results have therapeutic implications in the management of bronchiectasis patients.

The Anti-Non-Small Cell Lung Cancer Cell Activity by a mTOR Kinase Inhibitor PQR620.

In non-small-cell lung carcinoma (NSCLC), aberrant activation of mammalian target of rapamycin (mTOR) contributes to tumorigenesis and cancer progression. PQR620 is a novel and highly-potent mTOR kinase inhibitor. We here tested its potential activity in NSCLC cells. In primary human NSCLC cells and established cell lines (A549 and NCI-H1944), PQR620 inhibited cell growth, proliferation, and cell cycle progression, as well as cell migration and invasion, while inducing significant apoptosis activation. PQR620 disrupted assembles of mTOR complex 1 (mTOR-Raptor) and mTOR complex 2 (mTOR-Rictor-Sin1), and blocked Akt, S6K1, and S6 phosphorylations in NSCLC cells. Restoring Akt-mTOR activation by a constitutively-active Akt1 (S473D) only partially inhibited PQR620-induced cytotoxicity in NSCLC cells. PQR620 was yet cytotoxic in Akt1/2-silenced NSCLC cells, supporting the existence of Akt-mTOR-independent mechanisms. Indeed, PQR620 induced sphingosine kinase 1 (SphK1) inhibition, ceramide production and oxidative stress in primary NSCLC cells. In vivo studies demonstrated that daily oral administration of a single dose of PQR620 potently inhibited primary NSCLC xenograft growth in severe combined immune deficient mice. In PQR620-treated xenograft tissues, Akt-mTOR inactivation, apoptosis induction, SphK1 inhibition and oxidative stress were detected. In conclusion, PQR620 exerted potent anti-NSCLC cell activity via mTOR-dependent and -independent mechanisms.

AMPK activation by ASP4132 inhibits non-small cell lung cancer cell growth.

Activation of adenosine monophosphate-activated protein kinase (AMPK) is able to produce significant anti-non-small cell lung cancer (NSCLC) cell activity. ASP4132 is an orally active and highly effective AMPK activator. The current study tested its activity against NSCLC cells. In primary NSCLC cells and established cell lines (A549 and NCI-H1944) ASP4132 potently inhibited cell growth, proliferation and cell cycle progression as well as cell migration and invasion. Robust apoptosis activation was detected in ASP4132-treated NSCLC cells. Furthermore, ASP4132 treatment in NSCLC cells induced programmed necrosis, causing mitochondrial p53-cyclophilin D (CyPD)-adenine nucleotide translocase 1 (ANT1) association, mitochondrial depolarization and medium lactate dehydrogenase release. In NSCLC cells ASP4132 activated AMPK signaling, induced AMPKα1-ACC phosphorylation and increased AMPK activity. Furthermore, AMPK downstream events, including mTORC1 inhibition, receptor tyrosine kinases (PDGFRα and EGFR) degradation, Akt inhibition and autophagy induction, were detected in ASP4132-treated NSCLC cells. Importantly, AMPK inactivation by AMPKα1 shRNA, knockout (using CRISPR/Cas9 strategy) or dominant negative mutation (T172A) almost reversed ASP4132-induced anti-NSCLC cell activity. Conversely, a constitutively active AMPKα1 (T172D) mimicked and abolished ASP4132-induced actions in NSCLC cells. In vivo, oral administration of a single dose of ASP4132 largely inhibited NSCLC xenograft growth in SCID mice. AMPK activation, mTORC1 inhibition and EGFR-PDGFRα degradation as well as Akt inhibition and autophagy induction were detected in ASP4132-treated NSCLC xenograft tumor tissues. Together, activation of AMPK by ASP4132 potently inhibits NSCLC cell growth in vitro and in vivo.

Resibufogenin suppresses growth and metastasis through inducing caspase-1-dependent pyroptosis via ROS-mediated NF-κB suppression in non-small cell lung cancer.

The purpose of this study is to explore the antitumor properties of resibufogenin (RB) in non-small cell lung cancer (NSCLC) and elucidate its underlying mechanism. A549 and H520 cells were treated with various concentrations of RB with or without NLRP3 inhibitor (MCC950), caspase-1 inhibitor (VX765), or N-acetyl-l-cysteine (an ROS scavenger). Cell counting kit-8 and colony formation assays were conducted to determine cell viability. Cell invasion was detected by using the transwell assay. The release of lactate dehydrogenase (LDH) was determined by the LDH detection assay. The protein expression levels of related genes were measured by western blotting and immunohistochemistry. The reactive oxygen species (ROS) level was detected by using a 2,7-dichlorodihydrofluorescein diacetate ROS Assay Kit. The in vivo effects of RB were evaluated in a xenograft mouse model. RB treatment reduced cell viability and invasion in a dose-dependent manner. Furthermore, RB also enhanced pyroptosis levels in A549 and H520 cells, as indicated by the increased release of LDH and pyroptosis-related proteins. Interestingly, we also found that the antiproliferative and antimetastatic effects of RB were alleviated by the blockade of pyroptosis using NLRP3 inhibitor MCC950. Further study demonstrated that RB induced pyroptosis in a caspase-1-dependent manner, as evidenced by the finding that VX765 effectively reversed the effects of RB on A549 and H520 cells. We also found that RB could trigger caspase-1-dependent pyroptosis through ROS-mediated NF-κB suppression. In summary, our findings provide a potential antitumor agent and a novel insight into the mechanism of RB treatment of NSCLC.

JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor.

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.

Experimental study on inhibition effects of the XAF1 gene against lung cancer cell proliferation.

OBJECTIVE: To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. METHODS: 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/ F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT- PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/ XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. RESULTS: The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. CONCLUSION: Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.