Axenic Long-Term Cultivation of Pneumocystis jirovecii.

Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.

Design, synthesis and biological evaluation of a novel colchicine-magnolol hybrid for inhibiting the growth of Lewis lung carcinoma in Vitro and in Vivo.

Colchicine is a bioactive alkaloid originally from Colchicum autumnale and possesses excellent antiproliferative activity. However, colchicine-associated severe toxicity, gastrointestinal side effects in particular, limits its further therapeutic use. In the current study, we thus designed and synthesized a novel hybrid (CMH) by splicing colchicine and magnolol, a multifunctional polyphenol showing favorable gastrointestinal protection. The antitumor activity of CMH in Lewis lung carcinoma (LLC) was then evaluated in vitro and in vivo. Biologically, CMH inhibited the growth of LLC cells with an IC50 of 0.26 µM, 100 times more potently than cisplatin (26.05 µM) did. Meanwhile, the cytotoxicity of CMH was 10-fold lower than that of colchicine in normal human lung cells (BEAS-2B). In C57BL/6 mice xenograft model, CMH (0.5 mg/kg) worked as efficacious as colchicine (0.5 mg/kg) to inhibit tumor growth and 2 times more potently than cisplatin (1 mg/kg). In terms of mortality, 7 out of 10 mice died in colchicine group (0.75 mg/kg), while no death was observed in groups receiving CMH or cisplatin at 0.75 mg/kg. Mechanistic studies using Western blot revealed that CMH dose-dependently suppressed the protein expression of phosphorylated ERK. Molecular docking analysis further indicated that CMH was well fitted in the colchicine binding site of tubulin and formed several hydrogen bonds with tubulin protein. These results enable our novel hybrid CMH as a potential antineoplastic agent with lower toxicity, and provide perquisites for further investigation to confirm the therapeutic potentiality of this novel hybrid.

D­allose enhances the efficacy of hydroxychloroquine against Lewis lung carcinoma cell growth by inducing autophagy.

Various cancer cells require massive amounts of glucose as an energy source for their dysregulated growth. Although D­allose, a rare sugar, inhibits tumor cell growth via inhibition of glucose uptake, a few cells can survive after treatment. However, the mechanism by which D­allose­resistant cells are generated remains unclear. Here, we investigated the properties of D­allose­resistant cells and evaluated the efficacy of combined treatment with this rare sugar and antitumor drugs. To this end, we established a D­allose­resistant tumor cell line and prepared a C57BL/6J mouse tumor xenograft model using Lewis lung carcinoma (LLC) cells. Xenograft­bearing mice were treated with D­allose (9 g/kg) and/or hydroxychloroquine (HCQ, 60 mg/kg), an autophagy inhibitor, for two weeks. Although D­allose inhibited LLC cell growth in a dose­dependent manner, a few cells survived. The upregulation of LC3­II, a classical autophagy marker, and the downregulation of mTOR and its downstream molecule Beclin1 were observed in established D­allose­resistant LLC cells, which were more sensitive to cell death induced by HCQ. Similarly, in the tumor xenograft model, the tumor volume in mice co­treated with D­allose and HCQ was considerably smaller than that in untreated or HCQ­treated mice. Importantly, the administration of D­allose induced autophagy selectively at the tumor site of the xenograft­bearing mice. These results provide a new therapeutic strategy targeting autophagy which is induced in tumor cells by D­allose administration, and may be used to improve therapies for lung cancer.

Evodiamine suppresses the progression of non-small cell lung carcinoma via endoplasmic reticulum stress-mediated apoptosis pathway in vivo and in vitro.

BACKGROUND: Evodiamine (EVO) is one of the major components isolated from Evodia rutaecarpa (Juss.). Recent studies have shown that EVO has an anti-cancer effect. However, the pharmacological mechanism by which EVO impacts cancer is still poorly understood. OBJECTIVES: This study focused on asking the anti-cancer effect of EVO in human non-small cell lung carcinoma (NSCLC), and in particular to investigate whether EVO acts via modulating the endoplasmic reticulum stress (ERS)-mediated apoptosis pathway. MATERIALS AND METHODS: A Lewis lung carcinoma (LLC) tumor-bearing mouse model was treated with low-dose EVO (5 mg/kg) and high-dose EVO (10 mg/kg) intraperitoneally for 14 d. The effects of EVO on tumor growth, apoptosis, and ERS were assessed. In addition, NSCLC A549 and LLC cells were treated with EVO in vitro. The effects of EVO on cell proliferation, apoptosis, and ERS were investigated. Finally, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was used to validate whether EVO induced apoptosis of NSCLC cells by modulating ERS. RESULTS: EVO treatment significantly inhibited tumor growth in LLC tumor-bearing mice. H&E staining indicated that EVO treatment reduced the number of tumor cells and the nucleo-plasmic ratio. Immunostaining showed that EVO treatment significantly decreased the expression of Ki-67. TUNEL staining revealed that EVO induced apoptosis in the tumor. Likewise, EVO treatment up-regulated the expression of apoptosis-related genes and proteins and increased activation of the ERS pathway in the tumor. Additionally, EVO inhibited cell proliferation and increased cell apoptotic rates in A549 and LLC cells. EVO also increased the expression levels of genes and proteins associated with ERS-mediated apoptosis pathway in vitro. The effects of EVO on apoptosis were abolished by 4-PBA treatment. CONCLUSIONS: Our study demonstrated that EVO suppresses the progression of NSCLC by modulating the ERS-mediated apoptosis pathway.

The ethanol extract of Garcinia subelliptica Merr. induces autophagy.

BACKGROUND: Garcinia subelliptica Merr. is a multipurpose coastal tree, the potential medicinal effects of which have been studied, including cancer suppression. Here, we present evidence that the ethanol extract of G. subelliptica Merr. (eGSM) induces autophagy in human lung adenocarcinoma cells. METHODS: Two different human lung adenocarcinoma cell lines, A549 and SNU2292, were treated with varying amounts of eGSM. Cytotoxicity elicited by eGSM was assessed by MTT assay and PARP degradation. Autophagy in A549 and SNU2292 was determined by western blotting for AMPK, mTOR, ULK1, and LC3. Genetic deletion of AMPKα in HEK293 cells was carried out by CRISPR. RESULTS: eGSM elicited cytotoxicity, but not apoptosis, in A549 and SNU2292 cells. eGSM increased LC3-II production in both A549 and, more extensively, SNU2292, suggesting that eGSM induces autophagy. In A549, eGSM activated AMPK, an essential autophagy activator, but not suppressed mTOR, an autophagy blocker, suggesting that eGSM induces autophagy by primarily activating the AMPK pathway in A549. By contrast, eGSM suppressed mTOR activity without activating AMPK in SNU2292, suggesting that eGSM induces autophagy by mainly suppressing mTOR in SNU2292. In HEK293 cells lacking AMPKα expression, eGSM increased LC3-II production, confirming that the autophagy induced by eGSM can occur without the AMPK pathway. CONCLUSION: Our findings suggest that eGSM induces autophagy by activating AMPK or suppressing mTOR pathways, depending on cell types.

TFF-1 Functions to Suppress Multiple Phenotypes Associated with Lung Cancer Progression.

INTRODUCTION: Trefoil Factor (TFF) is a member of a protein family comprised of three isoforms, of which TFF-1 exhibits antithetical functions; promotion or suppression of cell proliferation, survival and invasion, depending on the cancer type. However, the pathobiological function of TFF-1 in lung carcinoma has been still unclear. METHODS: We examined the expression and secretion of TFF-1 using cultured human lung carcinoma cells by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay and quantitative real-time PCR analyses. The effects of TFF-1 on various phenotypes were analyzed in two cell lines, including those transfected with cDNA encoding TFF-1. Cell proliferation and death were examined by hemocytometer cell counting and by colorimetric viability/cytotoxicity assay. Cell cycle profile, migration and invasion were also examined by flow cytometry, wound healing assay and Matrigel Transwell assay, respectively. The effect of TFF-1 overexpression was confirmed by additional transfection of TFF-1-specific siRNA. RESULTS: Endogenous TFF-1 protein expression and secretion into the media were observed exclusively in adenocarcinoma-derived cell lines. Forced overexpression of TFF-1 drove cell cycle transition, while the proliferation decreased by 19% to 25% due to increased cell death. This cell death was predominantly caused by apoptosis, as assessed by the activation of caspase 3/7. Cell migration was also suppressed by 71% to 82% in TFF-1-transfected cells. The suppressive effect of TFF-1 on proliferation and migration was restored by transfection of TFF-1 siRNA. Moreover, invasion was also suppressed to 77% to 83% in TFF-1-transfected cells. CONCLUSION: These findings reveal that TFF-1 functions as a suppressor of cancer proliferation by induction of apoptosis, cell migration and invasion and thus may provide a synergistic target for potential treatment strategies for human lung carcinoma.

Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway.

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

Tacrine-Coumarin Derivatives as Topoisomerase Inhibitors with Antitumor Effects on A549 Human Lung Carcinoma Cancer Cell Lines.

A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a-2c) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (hTOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (1c, 1d = tacrine-(CH2)8-9-coumarin). The most active of the tested compounds, derivatives 1c and 1d, both display longer chains.

Effect of Varying Expression of EpCAM on the Efficiency of CTCs Detection by SERS-Based Immunomagnetic Optofluidic Device.

The circulating tumor cells (CTCs) isolation and characterization has a great potential for non-invasive biopsy. In the present research, the surface-enhanced Raman spectroscopy (SERS)-based assay utilizing magnetic nanoparticles and solid SERS-active support integrated in the external field assisted microfluidic device was designed for efficient isolation of CTCs from blood samples. Magnetic nanospheres (Fe2O3) were coated with SERS-active metal and then modified with p-mercaptobenzoic acid (p-MBA) which works simultaneously as a Raman reporter and linker to an antiepithelial-cell-adhesion-molecule (anti-EpCAM) antibodies. The newly developed laser-induced SERS-active silicon substrate with a very strong enhancement factor (up to 108) and high stability and reproducibility provide the additional extra-enhancement in the sandwich plasmonic configuration of immune assay which finally leads to increase the efficiency of detection. The sensitive immune recognition of cancer cells is assisted by the introducing of the controllable external magnetic field into the microfluidic chip. Moreover, the integration of the SERS-active platform and p-MBA-labeled immuno-Ag@Fe2O3 nanostructures with microfluidic device offers less sample and analytes demand, precise operation, increase reproducibly of spectral responses, and enables miniaturization and portability of the presented approach. In this work, we have also investigated the effect of varying expression of the EpCAM established by the Western Blot method supported by immunochemistry on the efficiency of CTCs' detection with the developed SERS method. We used four target cancer cell lines with relatively high (human metastatic prostate adenocarcinoma cells (LNCaP)), medium (human metastatic prostate adenocarcinoma cells (LNCaP)), weak (human metastatic prostate adenocarcinoma cells (LNCaP)), and no EpCAM expressions (cervical cancer cells (HeLa)) to estimate the limits of detection based on constructed calibration curves. Finally, blood samples from lung cancer patients were used to validate the efficiency of the developed method in clinical trials.

Toxoplasma GRA16 Inhibits NF-κB Activation through PP2A-B55 Upregulation in Non-Small-Cell Lung Carcinoma Cells.

Nuclear factor kappa B (NF-κB) activation is a well-known mechanism by which chemoresistance to anticancer agents is reported. It is well-known that irinotecan as a chemotherapeutic drug against non-small-cell lung carcinoma (NSCLC) has limited anticancer effect due to NF-κB activation. In this study, we propose the novel role of GRA16, a dense granule protein of Toxoplasma gondii, as an anticancer agent to increase the effectiveness of chemotherapy via the inhibition of NF-κB activation. To demonstrate this, H1299 cells were stably transfected with GRA16. The anticancer effects of GRA16 were demonstrated as a reduction in tumor size in a mouse xenograft model. GRA16 directly elevated B55 regulatory subunit of protein phosphatase 2A (PP2A-B55) expression in tumor cells, thereby decreasing GWL protein levels and ENSA phosphorylation. This cascade, in turn, induced PP2A-B55 activation and suppressed AKT/ERK phosphorylation and cyclin B1 levels, suggesting reduced cell survival and arrested cell cycle. Moreover, PP2A-B55 activation and AKT phosphorylation inhibition led to NF-κB inactivation via the reduction in inhibitory kappa B kinase beta (IKKß) levels, de-phosphorylation of inhibitor of kappa B alpha (IκBα), and reduction in the nuclear transit of NF-κB p65. Furthermore, this molecular mechanism was examined under irinotecan treatment. The PP2A-B55/AKT/NF-κB p65 pathway-mediated anticancer effects were only induced in the presence of GRA16, but not in the presence of irinotecan. Moreover, GRA16 synergistically promoted the anticancer effects of irinotecan via the induction of the sub-G1 phase and reduction of cell proliferation. Collectively, irinotecan and GRA16 co-treatment promotes the anticancer effects of irinotecan via NF-κB inhibition and cell cycle arrest induced by GRA16, subsequently increasing the chemotherapeutic effect of irinotecan to NSCLC cells via NF-κB inhibition.

Cancer cachexia in a mouse model of oxidative stress.

BACKGROUND: Cancer is associated with muscle atrophy (cancer cachexia) that is linked to up to 40% of cancer-related deaths. Oxidative stress is a critical player in the induction and progression of age-related loss of muscle mass and weakness (sarcopenia); however, the role of oxidative stress in cancer cachexia has not been defined. The purpose of this study was to examine if elevated oxidative stress exacerbates cancer cachexia. METHODS: Cu/Zn superoxide dismutase knockout (Sod1KO) mice were used as an established mouse model of elevated oxidative stress. Cancer cachexia was induced by injection of one million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (saline) into the hind flank of female wild-type mice or Sod1KO mice at approximately 4 months of age. The tumour developed for 3 weeks. Muscle mass, contractile function, neuromuscular junction (NMJ) fragmentation, metabolic proteins, mitochondrial function, and motor neuron function were measured in wild-type and Sod1KO saline and tumour-bearing mice. Data were analysed by two-way ANOVA with Tukey-Kramer post hoc test when significant F ratios were determined and α was set at 0.05. Unless otherwise noted, results in abstract are mean ±SEM. RESULTS: Muscle mass and cross-sectional area were significantly reduced, in tumour-bearing mice. Metabolic enzymes were dysregulated in Sod1KO mice and cancer exacerbated this phenotype. NMJ fragmentation was exacerbated in tumour-bearing Sod1KO mice. Myofibrillar protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.00847 ± 0.00205; wildtype LLC, 0.0211 ± 0.00184) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0180 ± 0.00118; Sod1KO LLC, 0.0490 ± 0.00132). Muscle mitochondrial oxygen consumption was reduced in tumour-bearing mice compared with saline-injected wild-type mice. Mitochondrial protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.0204 ± 0.00159; wild-type LLC, 0.167 ± 0.00157) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0231 ± 0.00108; Sod1 KO LLC, 0.0645 ± 0.000631). Sciatic nerve conduction velocity was decreased in tumour-bearing wild-type mice (wild-type saline, 38.2 ± 0.861; wild-type LLC, 28.8 ± 0.772). Three out of eleven of the tumour-bearing Sod1KO mice did not survive the 3-week period following tumour implantation. CONCLUSIONS: Oxidative stress does not exacerbate cancer-induced muscle loss; however, cancer cachexia may accelerate NMJ disruption.

Enhancement of membrane lipid peroxidation in lung cancer cells irradiated with monoenergetic X-rays at the K-shell resonance absorption peak of phosphorus.

The aim of this study was to determine whether membrane lipid peroxidation in mammalian cells is enhanced by X-ray irradiation at the K-shell resonance absorption peak of phosphorus. A549 and wild-type p53-transfected H1299 (H1299/wtp53) cell lines derived from human lung carcinoma were irradiated with monoenergetic X-rays at 2.153 keV, the phosphorus K-shell resonance absorption peak, or those at 2.147 or 2.160 keV, which are off peaks. Immunofluorescence staining for 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, was used as marker for protein modification. In both cell lines, the HNE production was significantly enhanced after irradiation at 2.153 keV compared to sham-irradiation. The enhancement (E) was calculated as the ratio of the fluorescence intensity of irradiated cells to that of sham-irradiated cells. In both the cell lines, E2.153 was significantly larger than E2.147 and no significant difference between E2.147 and E2.160 was observed. The extra enhancement at 2.153 keV was possibly caused by energy transition within the phosphorus K-shell resonance absorption. Our results indicate that membrane lipid peroxidation in cells is enhanced by the Auger effect after irradiation at the K-shell resonance absorption peak of phosphorus rather than by the photoelectric effect of the constituent atoms in the membrane lipid at 2.147 keV.

Fluorinated 3,6,9-trisubstituted acridine derivatives as DNA interacting agents and topoisomerase inhibitors with A549 antiproliferative activity.

A series of new 3,6,9-trisubstituted acridine derivatives with fluorine substituents on phenyl ring were synthesized and their interaction with calf thymus DNA was investigated. Analysis using UV-Vis absorbance spectra provided valuable information about the formation of the acridine-DNA complex. In addition, compounds 8b and 8d were found to display an increased binding affinity (K = 2.32 and 2.28 × 106 M-1, respectively). Topo I/II inhibition mode assays were also performed, and the results verify that the novel compounds display topoisomerase I and II inhibitory activity; compounds 8a, 8b and 8c completely inhibited topoisomerase I activity at a concentration of 60 × 10-6 M, but only compound 8d showed partial ability to inhibit topoisomerase II at concentrations of 30 and 50 × 10-6 M. The ability of the derivatives to impair cell proliferation was tested through an analysis of cell cycle distribution, quantification of cell number, viability studies, metabolic activity measurement and clonogenic assay. The content and localization of the derivatives in cells were analyzed using flow cytometry and fluorescence microscopy. The compounds 8b and 8d altered the physiochemical properties and improved antiproliferative activity in A549 human lung carcinoma cells (compound 8d displayed the highest level of activity, 4.25 × 10-6 M, after 48 h).

Toxicity of C60 fullerene-cisplatin nanocomplex against Lewis lung carcinoma cells.

Cisplatin (Cis-Pt) is the cytotoxic agent widely used against tumors of various origin, but its therapeutic efficiency is substantially limited by a non-selective effect and high toxicity. Conjugation of Cis-Pt with nanocarriers is thought to be one option to enable drug targeting. The aim of this study was to estimate toxic effects of the nanocomplex formed by noncovalent interaction of C60 fullerene with Cis-Pt against Lewis lung carcinoma (LLC) cells in comparison with free drug. Scanning tunneling microscopy showed that the minimum size of C60-Cis-Pt nanoparticles in aqueous colloid solution was 1.1 nm whereas that of C60 fullerene was 0.72 nm, thus confirming formation of the nanocomplex. The cytotoxic effect of C60-Cis-Pt nanocomplex against LLC cells was shown to be higher with IC50 values 3.3 and 4.5 times lower at 48 h and 72 h, respectively, as compared to the free drug. 12.5 µM Cis-Pt had no effect on LLC cell viability and morphology while C60-Cis-Pt nanocomplex in Cis-Pt-equivalent concentration substantially decreased the cell viability, impaired their shape and adhesion, inhibited migration and induced accumulation in proapoptotic subG1 phase. Apoptosis induced by the C60-Cis-Pt nanocomplex was confirmed by caspase 3/7 activation and externalization of phosphatidylserine on the outer surface of LLC cells with the double Annexin V-FITC/PI staining. We assume that C60 fullerene as a component of the C60-Cis-Pt nanocomplex promoted Cis-Pt entry and intracellular accumulation thus contributing to intensification of the drug's toxic effect against lung cancer cells.

Continuous, label-free, 96-well-based determination of cell migration using confluence measurement.

Cellular migration is essential in diverse physiological and pathophysiological processes. Here, we present a protocol for quantitative analysis of migration using confluence detection allowing continuous, non-endpoint measurement with minimal hands-on time under cell incubator conditions. Applicability was tested using substances which enhance (EGF) or inhibit (cytochalasin D, ouabain) migration. Using a gap-closure assay we demonstrate that automated confluence detection monitors cellular migration in the 96-well microplate format. Quantification by % confluence, % cell free-area or % confluence in cell-free area against time, allows detailed analysis of cellular migration. The study describes a practicable approach for continuous, non-endpoint measurement of migration in 96-well microplates and for detailed data analysis, which allows for medium/high-throughput analysis of cellular migration in vitro.

Omics approach reveals metabolic disorders associated with the cytotoxicity of airborne particulate matter in human lung carcinoma cells.

Exposure to airborne particulate matter (PM) 2.5 induced various adverse health effects, such as metabolic syndrome, systemic inflammation and respiratory infection. However, a global influence of PM2.5-induced metabolic and proteomic disorders remains confusing, and the underlying mechanism is still under-explored. Herein, LC-MS/MS-based metabolomics, lipidomics and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics were applied to analyze the toxicological characteristics of PM2.5 from Taiyuan City in China (Taiyuan-PM2.5) on human lung carcinoma cells (A549) after the 24-h treatment. Metabolites, lipids and proteins that have distinctive differences were screened by SIEVE, LipidSearch and Proteome Discoverer, respectively. The abundance of 56 metabolites (40 increased and 16 decreased), 22 lipids (19 increased and 3 decreased) and 81 proteins (55 up-regulated and 26 down-regulated) were significantly changed upon the PM2.5 treatment. Among the proteomics analysis, 16 proteins were specifically related to RNA splicing, mainly including up-regulated serine/arginine-rich splicing factor 1 (SRSF1), SRSF2, small nuclear ribonucleoprotein 70 kDa (snRNP70), small nuclear ribonucleoprotein polypeptide B (SNRPB), SNRPC, SNRPE and down-regulated heterogeneous nuclear ribonucleoprotein U-like 2 (hnRNP UL2). At the metabolic level, PM2.5 exposure significantly altered the sphingolipid metabolism, including ceramide, serine, sphingosine and sphingomyelin. It was proposed that excessive accumulation of ceramide and expression of key enzymes (ceramide synthases, phingomyelinase, sphingosine kinase types 2 and protein phosphatase-1) induced the secretion of pro-inflammatory cytokines, generation of lipotoxicity and alterations of RNA splicing in PM2.5-treated A549  cells. In general, our results demonstrated that ceramide accumulation and altered RNA splicing could becritical contributors to PM2.5-induced cytotoxicity at metabolic and proteomic level, which might be considered as potential markers for toxicological evaluation of PM2.5 samples.

Deletion of Adipose Triglyceride Lipase Links Triacylglycerol Accumulation to a More-Aggressive Phenotype in A549 Lung Carcinoma Cells.

Adipose triglyceride lipase (ATGL) catalyzes the rate limiting step in triacylglycerol breakdown in adipocytes but is expressed in most tissues. The enzyme was shown to be lost in many human tumors, and its loss may play a role in early stages of cancer development. Here, we report that loss of ATGL supports a more-aggressive cancer phenotype in a model system in which ATGL was deleted in A549 lung cancer cells by CRISPR/Cas9. We observed that loss of ATGL led to triacylglycerol accumulation in lipid droplets and higher levels of cellular phospholipid and bioactive lipid species (lyso- and ether-phospholipids). Label-free quantitative proteomics revealed elevated expression of the pro-oncogene SRC kinase in ATGL depleted cells, which was also found on mRNA level and confirmed on protein level by Western blot. Consistently, higher expression of phosphorylated (active) SRC (Y416 phospho-SRC) was observed in ATGL-KO cells. Cells depleted of ATGL migrated faster, which was dependent on SRC kinase activity. We propose that loss of ATGL may thus increase cancer aggressiveness by activation of pro-oncogenic signaling via SRC kinase and increased levels of bioactive lipids.

Comparative study of cyto- and genotoxic potential with mechanistic insights of tungsten oxide nano- and microparticles in lung carcinoma cells.

The exigency of semiconductor and super capacitor tungsten oxide nanoparticles (WO3 NPs) is increasing in various sectors. However, limited information on their toxicity and biological interactions are available. Hence, we explored the underlying mechanisms of toxicity induced by WO3 NPs and their microparticles (MPs) using different concentrations (0-300 µg ml-1 ) in human lung carcinoma (A549) cells. The mean size of WO3 NPs and MPs by transmission electron microscopy was 53.84 nm and 3.88 µm, respectively. WO3 NPs induced reduction in cell viability, membrane damage and the degree of induction was size- and dose-dependent. There was a significant increase in the percentage tail DNA and micronuclei formation at 200 and 300 µg ml-1 after 24 hours of exposure. The DNA damage induced by WO3 NPs could be attributed to increased oxidative stress and inflammation through reactive oxygen species generation, which correlated with the depletion of reduced glutathione content, catalase and an increase in malondialdehyde levels. Cellular uptake studies unveiled that both the particles were attached/surrounded to the cell membrane according to their size. In addition, NP inhibited the progression of the cell cycle in the G2 /M phase. Other studies such as caspase-9 and -3 and Annexin-V-fluorescein isothiocyanate revealed that NPs induced intrinsic apoptotic cell death at 200 and 300 µg ml-1 concentrations. However, in comparison to NPs, WO3 MPs did not incite any toxic effects at the tested concentrations. Under these experimental conditions, the no-observed-significant-effect level of WO3 NPs was determined to be ≤200 µg ml-1 in A549 cells.

Anti-Proliferative Activity of Triterpenoids and Sterols Isolated from Alstonia scholaris against Non-Small-Cell Lung Carcinoma Cells.

(1) Background: In China and South Asia, Alstonia scholaris (Apocynaceae) is an important medicinal plant that has been historically used in traditional ethnopharmacy to treat infectious diseases. Although various pharmacological activities have been reported, the anti-lung cancer components of A. scholaris have not yet been identified. The objective of this study is to evaluate the active components of the leaf extract of A. scholaris, and assess the anti-proliferation effects of isolated compounds against non-small-cell lung carcinoma cells; (2) Methods: NMR was used to identify the chemical constitutes isolated from the leaf extract of A. scholaris. The anti-proliferative activity of compounds against non-small-cell lung carcinoma cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (3) Results: Eight triterpenoids and five sterols were isolated from the hexane portion of A. scholaris, and structurally identified as: (1) ursolic acid, (2) oleanolic acid, (3) betulinic acid, (4) betulin, (5) 2ß,3ß,28-lup-20(29)-ene-triol, (6) lupeol, (7) ß-amyrin, (8) α-amyrin, (9) poriferasterol, (10) epicampesterol, (11) ß-sitosterol, (12) 6ß-hydroxy-4-stigmasten-3-one, and (13) ergosta-7,22-diene-3ß,5α,6ß-triol. Compound 5 was isolated from a plant source for the first time. In addition, compounds 9, 10, 12, and 13 were also isolated from A. scholaris for the first time. Ursolic acid, betulinic acid, betulin, and 2ß,3ß,28-lup-20(29)-ene-triol showed anti-proliferative activity against NSCLC, with IC50 of 39.8, 40.1, 240.5 and 172.6 µM, respectively.; (4) Conclusion: These findings reflect that pentacyclic triterpenoids are the anti-lung cancer chemicals in A. scholaris. The ability of ursolic acid, betulinic acid, betulin, and 2ß,3ß,28-lup-20(29)-ene-triol to inhibit the proliferative activity of NSCLC can constitute a valuable group of therapeutic agents in the future.

Targeted Delivery System Based on Gemcitabine-Loaded Silk Fibroin Nanoparticles for Lung Cancer Therapy.

Here, a targeted delivery system was developed based on silk fibroin nanoparticles (SFNPs) for the systemic delivery of gemcitabine (Gem) to treat induced lung tumor in a mice model. For targeting the tumorigenic lung tissue, SP5-52 peptide was conjugated to Gem-loaded SFNPs. Different methods were used to characterize the structural and physicochemical properties of the SFNPs. The prepared nanoparticles (NPs) showed suitable characteristics in terms of size, zeta potential, morphology, and structural properties. Moreover, the targeted Gem-loaded SFNPs showed higher cytotoxicity, cellular uptake, and accumulation in the lung tissue in comparison to the nontargeted SFNPs and control groups. Afterward, a mice model with induced lung tumor was developed by intratracheal injection of Lewis lung carcinoma (LL/2) cells into the lungs for assessing the therapeutic efficacy of the prepared drug delivery system. The histopathological assessments and single-photon-emission computed tomography-CT radiographs showed successful lung tumor induction. Moreover, the obtained results showed higher potential of targeted Gem-loaded SFNPs in treating induced lung tumor compared with that of the control groups. Higher survival rate, less mortality, and no sign of metastasis were also observed in those animals treated with targeted NPs based on the histological and radiological analyses. This study presented an effective anticancer drug delivery system for specific targeting of induced lung tumor that could be useful in treating malignant lung cancers in future.