Human Alveolar Adenocarcinoma Cells Research Articles

Design, synthesis and antitumor activity of triphenylphosphonium-linked derivatives of quinazolinone

Cancer is the leading cause of human death. Quinazolinone heterocyclic compounds have a variety of biological activities and have been extensively studied in recent years, especially for their potential anticancer activity. The triphenylphosphonium moiety (TPP+) has become a very important lipophilic cation, especially concerning its application the development of anticancer agents. In this work, we designed and synthesised 24 new TPP+ -conjugated quinazolinone derivatives, which have alkylated TPP+ at the N-3 position and different small group substitutions at the C-6 position, and their antiproliferative activity was evaluated in three cancer cell lines (human alveolar adenocarcinoma cells (A549), human hepatoblastoma cells (HepG2) and human breast cancer cells (MCF-7)) and human normal liver cells (QSG-7701). The cytotoxicity screening results showed that some derivatives exhibited effective inhibitory effects in cancer cells. Among them, the compound 5k–o showed better antiproliferative activity than the positive control drug gefitinib on MCF-7 and A549 cells. The most active compounds being 5o, with IC50 values of 6.56, 14.52 and 7.51 µM in MCF-7 cells, HepG2 cells and A549 cells, respectively. Compound 5o may be a promising compound for cancer treatment worthy of further study.

Synthesis of carboxymethyl chitosan–guar gum–poly(vinylpyrrolidone) ternary blended hydrogels with antibacterial/anticancer efficacy and drug delivery applications

Biopolymers have the utmost significance in biomedical applications and blending synthetic polymers has shown favorable characteristics versus individual counterparts. The utilization of the blends can be restricted through the use of toxic chemical agents such as initiators or crosslinkers. In this regard, a chemical agent-free ionizing irradiation is a beneficial alternative for preparing the hydrogels for biomedical applications. In this study, carboxymethyl chitosan (CM-CS), guar gum (GG), and poly(vinylpyrrolidone) (PVP) based ternary blends (TB) were crosslinked using various doses of ionizing irradiation to fabricate hydrogels. The prepared hydrogels were characterized for physicochemical properties, swelling analysis, biological assays, and drug delivery applications. Swelling analysis in distilled water revealed that the hydrogels exhibit excellent swelling characteristics. An in vitro cytocompatibility assay showed that the hydrogels have greater than 90% cell viability for the human epithelial cell line and a decreasing cell viability trend for the human alveolar adenocarcinoma cell line. In addition, the prepared hydrogels possessed excellent antibacterial characteristics against gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli). Finally, the release studies of anti-inflammatory Quercus acutissima (QA) loaded hydrogels exhibited more than 80% release in phosphate-buffered saline (pH = 7.4). These findings suggest that TB hydrogels can be used as suitable carrier media for different release systems and biomedical applications.

Modulation of Heat Shock Protein Expression in Alveolar Adenocarcinoma Cells through Gold Nanoparticles and Cisplatin Treatment.

Heat-shock proteins (HSPs) are stress-responsive molecules belonging to the family of evolutionary molecular chaperones known to be crucial in many cancer types, including human alveolar adenocarcinoma cells (A549). These proteins are highly overexpressed in cancers to support their ability to accommodate imbalances in cell signalling, DNA alterations, proteins, and energy metabolism associated with oncogenesis. The current study evaluated the effects of gold nanoparticles (AuNPs) combined with cisplatin (CDDP) on molecular chaperone HSPs in A549 cells. It was found that AuNPs:CDDP decreased the percentage of cell viability (38.5%) measured using the modified lactated dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. AuNPs:CDDP exposure caused a significant (p < 0.05) increase in reactive oxygen species (ROS) generation by 1.81-fold, apoptosis induction, and a decrease in the mitochondrial membrane potential (MMP) compared to AuNPs or CDDP alone. Similarly, exposure to the AuNPs:CDDP combination had pronounced cytotoxic effects on the expression of HSPs and PI3K/AKT/mTOR, as well as apoptosis-related proteins. The results demonstrate that the combination of AuNPs with CDDP might enhance the anticancer efficacy of CDDP.

Linear Energy Transfer Measurements and Estimation of Relative Biological Effectiveness in Proton and Helium Ion Beams Using Fluorescent Nuclear Track Detectors

PurposeTo develop a methodology for assessing the linear energy transfer (LET) and relative biological effectiveness (RBE) in clinical proton and helium-ion beams using fluorescent nuclear track detectors (FNTDs). Methods and materialsFNTDs were exposed behind solid water to proton and helium- (4He-) ion spread-out Bragg peaks (SOBPs). Detectors were imaged with a confocal microscope, and the LET spectra were derived from the fluorescence intensity. The track- and dose-averaged LET (LETF and LETD, respectively) were calculated from the LET spectra. LET measurements were used as input on RBE-models to estimate the RBE. Human alveolar adenocarcinoma cells (A549) were exposed at the same positions as the FNTDs. The RBE was calculated from the resulting survival curves. All measurements were compared with Monte Carlo simulations. ResultsFor protons, average relative differences between measurements and simulations were 6% and 19% for LETF and LETD, respectively. For helium ions the same differences were 11% for both quantities. The position of the experimental LET spectra primary peaks agreed with the simulations within 9% and 14% for protons and helium-ions, respectively. For the RBE-models using LETD as input, FNTD-based RBE values ranged from 1.02 ± 0.01 to 1.25 ± 0.04 and from 1.08 ± 0.09 to 2.68 ± 1.26 for protons and helium-ions, respectively. The average relative differences between these values and simulations were 2% and 4%. For A549 cells, the RBE ranged from 1.05 ± 0.07 to 1.47 ± 0.09 and from 0.89 ± 0.06 to 3.28 ± 0.20 for protons and helium-ions, respectively. Regarding the RBE-weighted dose (2.0 Gy at the SOBP), the differences between simulations and measurements were below 0.10 Gy. ConclusionThis study demonstrates for the first time that FNTDs can be used to perform direct LET measurements and to estimate the RBE in clinical proton and helium-ion beams.

Functionalized Gold Nanoparticles Suppress the Proliferation of Human Lung Alveolar Adenocarcinoma Cells by Deubiquitinating Enzymes Inhibition.

Functionalized gold nanoparticles (AuNPs) are widely used in therapeutic applications, but little is known regarding the impact of their surface functionalization in the process of toxicity against cancer cells. This study investigates the anticancer effects of 5 nm spherical AuNPs functionalized with tannate, citrate, and PVP on deubiquitinating enzymes (DUBs) in human lung alveolar adenocarcinoma (A549) cells. Our findings show that functionalized AuNPs reduce the cell viability in a concentration- and time-dependent manner as measured by modified lactate dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. An increased generation of intracellular reactive oxygen species (ROS) and depletion of glutathione (GSH/GSSG) ratio was observed with the highest AuNP concentration of 10 μg/mL. The expression of DUBs such as ubiquitin specific proteases (USP7, USP8, and USP10) was slightly inhibited when treated with concentrations above 2.5 μg/mL. Moreover, functionalized AuNPs showed an inhibitory effect on protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and wingless-related integration site (Wnt) signaling proteins, and this could further trigger mitochondrial related-apoptosis by the upregulation of caspase-3, caspase-9, and PARP in A549 cells. Furthermore, our study shows a mechanistic understanding of how functionalized AuNPs inhibit the DUBs, consequently suppressing cell proliferation, and can be modulated as an approach toward anticancer therapy. The study also warrants the need for future work to investigate the effect of functionalized AuNPs on DUB on other cancer cell lines both in vitro and in vivo.

Abstract 3947: S100A8/A9-RAGE pathway and chronic airway inflammation in cigarette smoke-induced lung carcinogenesis

Abstract Background: Cigarette smoke-induced chronic airway inflammation increases the risk of lung cancer, and plays a multifaceted role in lung cancer initiation and progression, and RAGE-ligand axis (HMGB1, S100A8/A9) contributes Cigarette smoke-induced persistent and progressive inflammation. The aim of the present study was to determine the inflammatory effect on S100A8/A9-RAGE pathway in smoke-induced carcinogenesis in lung cancer. Methods: Human alveolar adenocarcinoma cells (A549) and normal bronchial epithelial cells (BEAS-2B) were co-cultured for 24 h with the PBMC and then, cells were incubated in the presence or absence of 1.5% cigarette smoke extract (CSE) for 24 h. The cells transfected with siRNA targeting S100A8/9 or RAGE. The cell viability, colony-forming ability, migration, invasion, metastasis and morphological changes were assessed. Female A/J mice were given a benzo(a)pyrene (100mg/kg) in 0.1 ml corn oil via oral gavage at once a week for 3 weeks, and administrated CSE (1.25 ul/g) via intratracheal (IT) at twice a week for 4 weeks. Tumor load was determined by averaging the total tumor volume in each group. BAL differential cells were counted to analyze inflammatory cells. Results: Cellular growth and colony-forming were promoted by exposure to CSE, and significantly inhibited by S100A8/9 and RAGE-siRNA transfection, especially in A549 cells than BEAS-2B. Cell migration, invasion, MMP-2/9 activities, and the mRNA and protein expression of TLR4, NF-κB, RAGE, S100A8/9 and HMBG1 were higher in CSE exposed A549 cells. S100A8/9 and RAGE-siRNA transfection attenuated those expression, significantly. B(a)P induced an average tumor volume 7.28 mm3 per mouse, and CSE significantly increased tumor volume (8.10 mm3) compared to the B(a)P group. The total cell number and lymphocytes in BAL fluids tended to increase after CSE administration in lung cancer mouse group. Moreover, CSE significantly induced the levels of S100A8/9 and RAGE, p38 and p-ERK, and decreased JNK in serum and tumors with adjacent lung tissues (p &amp;lt; 0.05). Conclusions: We identified cigarette smoke-induced S100A8/A9-RAGE pathway promoted airway inflammation and lung carcinogenesis in vitro and in vivo. Although S100A8/A9 protein contributes to many types of disease, this study will help us gain a better understanding of the complexities of cancer progression. Further studies are needed for therapeutic target of cigarette smoke-induced inflammation and immunosuppression in tumor microenviornment. Citation Format: Hye Seon Kang, In Kyoung Kim, Chang Dong Yeo, Jin Woo Kim, Sang Haak Lee. S100A8/A9-RAGE pathway and chronic airway inflammation in cigarette smoke-induced lung carcinogenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3947.

Gold Nanoparticles Induced Size Dependent Cytotoxicity on Human Alveolar Adenocarcinoma Cells by Inhibiting the Ubiquitin Proteasome System.

Gold nanoparticles (AuNPs) are widely used in biomedicine due to their remarkable therapeutic applications. However, little is known about their cytotoxic effects on the ubiquitin proteasome system (UPS). Herein, the cytotoxicity of different sizes of AuNPs (5, 10, and 80 nm) on the UPS was investigated with a particular focus on deubiquitinating enzymes (DUBs) such as ubiquitin-specific proteases (USP) and ubiquitin carboxyl-terminal hydrolases (UCHL-1) in human alveolar epithelial adenocarcinoma (A549). It was found that all sizes of AuNPs reduced the percentage of viable A549 cells and increased lactate dehydrogenase (LDH) release, measured using the MTT and LDH assays, respectively. Furthermore, the 5 nm AuNPs were found to exhibit greater cytotoxicity than the 10 and 80 nm AuNPs. In addition, apoptosis and necrosis were activated through reactive oxygen species (ROS) generation due to AuNPs exposure. The internalisation of AuNPs in A549 cells increased with increasing particle size (80 > 10 > 5 nm). Interestingly, the expression of USP7, USP8, USP10, and UCHL-1 was significantly (p < 0.001) downregulated upon treatment with 5-30 µg/mL of all the AuNPs sizes compared to control cells. Moreover, the inhibition of these proteins triggered mitochondrial-related apoptosis through the upregulation of poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9. Collectively, these results indicate that AuNPs suppress the proliferation of A549 cells and can potentially be used as novel inhibitors of the proteasome.

Induction of programmed cell death in lung cancer cells by secondary metabolites of Nocardia carnea UTMC 863 as soil actinomycetes

Background: Apoptosis induction is one of the effective mechanisms in cancer therapy. So far, various natural sources have been identified for inducing apoptosis in cancer cells. This study proposed identifying promising active drug pharmacophores of soil actinomycetes with the capability of apoptosis induction in A549 cells, a human alveolar adenocarcinoma cell line.&#x0D; Methods: The crude extract of Nocardia carnea UTMC 863 was obtained from UTBC (University of Tehran Biocompound Collection). After 48 hours of exposure, cell viability, gene expression, and apoptosis rate were determined using MTT, quantitative real-time-PCR, and flow cytometry.&#x0D; Results: The MTT assay exhibited that the effective concentrations of UTMC863 and doxorubicin (positive control) were 24 µg/ml and 1 µM, respectively. UTMC 863 with a 24 µg/ml concentration and doxorubicin could induce apoptosis in the A549 cell line. Also, apoptosis-related gene expression increased in the UTMC863 group compared to the untreated group (p&lt;0.01).&#x0D; Conclusions: The crude extract of Nocardia carnea UTMC 863 can induce apoptosis in A549 cells, and it may be one of the promising pharmacophores for cancer therapy.

Global regulatory factor VeA upregulates the production of antitumor substances in endophytic Fusarium solani.

A number of studies have demonstrated that endophytic fungi have the potential to produce antitumor active substances with novel structures and significant activities. In our previous studies, we isolated a Fusarium strain from the stem of the medicinal plant Nothapodytes pittosporoides (Oliv.). In this study, we identified this strain as Fusarium solani and found that its crude extract has significant antitumor activity against human alveolar adenocarcinoma cells (A549). We overexpressed the global regulatory factor VeA in F. solani (VeAOE), resulting in a significant increase in antitumor activity. The MTT assay results showed that the inhibition rate of the VeAOE mutant extract on A549 cancer cells was significantly higher than that of the WT extract, as the IC50 decreased from 369.22 to 285.89μg/mL, and the apoptosis ratio was significantly increased by approximately 4.86-fold. In VeAOE, accumulation of alkaloids, terpenoids, carboxylic acid derivatives, phenols and flavonoid metabolites with potential antitumor activity was significantly increased compared with WT based on metabolomic analysis. Additionally, transcriptome analysis found that the expression patterns of 48 genes related to antitumor activity were significantly changed in VeAOE, mainly involving glycosyl hydrolases, the Zn(2)-Cys(6) class, cytochrome P450 monooxygenase, 3-isopropylmalate dehydratase, and polyketide synthases. These results suggested that VeA mediated the antitumor activity of the metabolites in F. solani HB1-J1 by regulating multiple metabolic pathways.

Stretchable electrical cell-substrate impedance sensor platform for monitoring cell monolayers under strain

Stretchable microelectrodes paired with an ultra-elastic substrate can be used for electrical sensing of mechanically stretched cells and cell monolayers. Here, we present the development of a cell-stretching platform with thin-film interdigitated microelectrodes. Up to 35 % cyclic stretch are feasible with a novel interlaced meander design connected to the microelectrodes and using Poly(dimethylsiloxane) (PDMS) with a Young’s modulus of 50 kPa as an ultra-elastic substrate. Reliable electrical contacting of the microelectrodes under stretch was achieved by perforation of the contact pads. The novel platform enables label-free, real-time electrical cell-substrate impedance (ECIS) monitoring of cell monolayers. Proof-of-concept experiments indicated that electrical impedance of Madin-Darby canine kidney (MDCK) cell monolayers increased sharply by uniaxial mechanical strain above 20 %. For comparison, human alveolar basal epithelial adenocarcinoma (A549) cell monolayers, which are known to lack mature cell junctions, showed a continuous decrease of electrical impedance over the whole applied strain range of 35 %. The data reveal that impedance changes upon stretching depend on epithelial cell types and existence of tight cellular junctions. The system provides the basis for reliable continuous long-term monitoring of electrical properties of cell monolayers under strain by electrical impedance spectroscopy, e.g., to monitor epithelial permeability changes in real time and under label-free conditions to screen the influence of pharmacological substances.

Osteopontin silencing attenuates bleomycin-induced murine pulmonary fibrosis by regulating epithelial–mesenchymal transition

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients’ lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial–mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-β1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-β1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-β1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

Straightforward synthesis of novel spiroether derivatives

We report the first full synthesis of novel spiroether derivatives via reaction of 4-oxobut-2-enoates and alloxan. Our synthesis features the single-step assembly, mild reaction conditions, good yields, high diastereoselectivity, operational simplicity, and the absence of tedious separation procedures. Most of the prepared compounds possessed moderate growth inhibitory effects on human alveolar adenocarcinoma (A549), malignant melanoma (A375), ovarian adenocarcinoma (SK-OV-3), and fibrosarcoma (HT-1080) cell lines.

N'-((3-(substituted phenyl)-1-phenyl-1H-Pyrazol-4-yl)methylene)-(substituted) benzhydrazide: Synthesis, characterization and pharmacological evaluation

A series of new N'-((3-(substitutedphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-(substituted benzo hydrazones (7a-j) were synthesized by the reaction of substituted benzohydrazones with 1-phenyl-3-(substitutedphenyl)-pyrazole-4-carbaldehydes. Structures of these compounds were established by IR, 1 H, 13 C NMR and HRMS spectral data. Anticancer screening tests were performed against A549 adenocarcinomic human alveolar basal epithelial and MCF-7 human breast adenocarcinoma cell lines. In vitro antimicrobial activity was studied against two bacterial and two fungal strains. Compound 7b emerged as potent anticancer agent against A549 cell line with IC 50 13.48 µM and against MCF-7 cell line with IC 50 25.23 µM. Compound 7c exhibited anticancer activity with IC 50 13.49 µM against A549 cell line and against MCF-7 cell line with IC 50 27.48 µM respectively. Compounds 7a-j were found to be moderately active against tested microbial organisms. Molecular docking studies were performed on B-RAFV600E and EGFR kinases which showed similar binding mode to standards employed.

Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells.

Epithelial-mesenchymal transition (EMT), a biological process through which epithelial cells transdifferentiate into mesenchymal cells, is involved in several pathological events, such as cancer progression and organ fibrosis. So far, we have found that methotrexate (MTX), an anticancer drug, induced EMT in the human A549 alveolar adenocarcinoma cell line. However, the relationship between EMT and the cytotoxicity induced by MTX remains unclear. In this study, we compared the processes of MTX-induced EMT and apoptosis in A549 cells. Q-VD-Oph, a caspase inhibitor, suppressed MTX-induced apoptosis, but not the increase in mRNA expression of α-smooth muscle actin (SMA), a representative EMT marker. In addition, SB431542, an EMT inhibitor, did not inhibit MTX-induced apoptosis. By using isolated clonal cells from wild-type A549 cells, the induction of EMT and apoptosis by MTX in each clone was analyzed, and no significant correlation was observed between the MTX-induced increase in α-SMA mRNA expression and the proportion of cells undergoing apoptosis. Furthermore, the increase in the mRNA expression of α-SMA was well correlated with cyclin-dependent kinase inhibitor 1A, a cell cycle arrest marker, but not with BCL-2 binding component 3 and Fas cell surface death receptor, which are both pro-apoptotic factors, indicating that the MTX-induced EMT may be related to cell cycle arrest, but not to apoptosis. These findings suggested that different mechanisms were involved in the MTX-induced EMT and apoptosis.

Targeting hub genes and pathways of innate immune response in COVID-19: A network biology perspective

The current pandemic of 2019 novel coronavirus disease (COVID-19) caused by a novel virus strain, 2019-nCoV/SARS-CoV-2 have posed a serious threat to global public health and economy. It is largely unknown how the human immune system responds to this infection. A better understanding of the immune response to SARS-CoV-2 will be important to develop therapeutics against COVID-19. Here, we have used transcriptomic profile of human alveolar adenocarcinoma cells (A549) infected with SARS-CoV-2 and employed a network biology approach to generate human-virus interactome. Network topological analysis discovers 15 SARS-CoV-2 targets, which belongs to a subset of interferon (IFN) stimulated genes (ISGs). These ISGs (IFIT1, IFITM1, IRF7, ISG15, MX1, and OAS2) can be considered as potential candidates for drug targets in the treatments of COVID-19. We have identified significant interaction between ISGs and TLR3 agonists, like poly I: C, and imiquimod, and suggests that TLR3 agonists can be considered as a potential drug for drug repurposing in COVID-19. Our network centric analysis suggests that moderating the innate immune response is a valuable approach to target COVID-19.

Bis(α-hydroxycycloalkyl)phosphine Oxides Obtained from White Phosphorus via Phosphine Oxide H3 PO: Synthesis, Molecular Structure, Coordination Properties and Biological Activity.

Reaction of the electrochemically in situ from elemental white phosphorus generated phosphine oxide H3 PO in a single electrochemical cell, supplied with lead cathode and aluminium anode, with cyclic ketones (cyclopentanone and cyclohexanone) results in formation of secondary phosphine oxides (bis(α-hydroxycyclopentyl)phosphine oxide 2 a, isolated yield 15 %, and bis(α-hydroxycyclohexyl)phosphine oxide 2 b, isolated yield 12 %) with two α-hydroxycycloalkyl substituents at the phosphorus atom. Bis(α-hydroxycyclopentyl)phosphine oxide reacts with [PdCl2 (COD)] (COD=1,5-cyclooctadiene) to give a new palladium complex trans-[PdCl2 {P(OH)(cyclo-C5 H8 -1-OH)2 }2 ] (3 a, isolated yield 11 %) bearing phosphinous acid as a ligand formed via tautomerization of the phosphine oxide. Finally, the cytotoxicity of the synthesized secondary phosphine oxides on tumor and healthy human cell lines was studied. It was found that at a concentration of 10-6 -10-4 M, phosphine oxides 2 a,b exhibit similar IC50 values for the M-Hela cell line (ca. 50 mM), but are non-toxic for MCF-7 cells. For human alveolar adenocarcinoma cells (A-549), only 2 a is active (ca. 35 mM), while 2 b is not toxic.

TGF-β1 increases sialidase 3 expression in human lung epithelial cells by decreasing its degradation and upregulating its translation

Purpose: We previously found extensive desialylation of glycoconjugates and upregulation of the sialidase enzyme NEU3 in fibrotic lesions in human and mouse lungs. However, studies using microarray analysis of whole lung tissue mRNA and single cell RNA-seq found no significant difference in levels of NEU3 mRNA between IPF patients and controls. This study aimed to elucidate how NEU3 was upregulated in fibrotic lungs.Materials and methods: Transforming growth factor-β1 (TGF-β1), a key driver of fibrosis, was added to A549 human alveolar basal epithelial adenocarcinoma cells and human small airway epithelial cells (HSAEpC). NEU3 expression in A549 cells and HSAEpC was detected by immunofluorescence staining. NEU3 translation and degradation were assessed by polysome profiling (polysomes efficiently translate mRNAs; monosomes poorly translate mRNAs) and cycloheximide chase after treating cells with or without TGF-β1 for 48 h.Results: TGF-β1 increased NEU3 expression and secretion in A549 cells and HSAEpC but did not change total (nuclear + cytosolic) NEU3 mRNA levels. TGF-β1 decreased the degradation rate of NEU3 in A549 cells. TGF-β1 decreased NEU3 mRNA levels in monosomes and increased NEU3 mRNA level in polysomes.Conclusion: TGF-β1 upregulates levels of NEU3 in epithelial cells by both decreasing NEU3 degradation and by increasing the translation of NEU3 mRNA, explaining the apparent paradox of high levels of NEU3 protein in pulmonary fibrosis without a concomitant increase in the expression of NEU3 mRNA.

Chlorin e6 Conjugated Methoxy-Poly(Ethylene Glycol)-Poly(D,L-Lactide) Glutathione Sensitive Micelles for Photodynamic Therapy.

In this study, we developed a polymeric micellar system for glutathione-mediated intracellular delivery of a photosensitizer, chlorin e6 (Ce6) by synthesizing an amphiphilic polymer, methoxy-poly(ethylene glycol)-poly(D,L-lactide)-disulfide-Ce6 (mPEG-PLA-S-S-Ce6), which self-assembled in aqueous environment to form micelles. The polymer-drug conjugate was characterized by NMR. The singlet oxygen (2O1) generation and in vitro release of Ce6 micelles were evaluated. Further, glutathione-mediated intracellular drug delivery was assessed in human alveolar adenocarcinoma cells (A549), mouse mammary carcinoma cells (4T1) and 3D A549 spheroids. The micellar system protected Ce6 from aggregation leading to improved 2O1 generation compared to free Ce6. Due to the availability of glutathione, the disulfide bonds in the micelles were cleaved resulting in rapid release of Ce6 evident from the in vitro study. The Ce6 micelles displayed quicker drug release in presence of glutathione monoester (GSH-OEt) pre-treated A549 and 4T1 cells compared to without pre-treated cells. In vitro phototoxicity of micelles displayed enhanced toxicity in 10mM GSH-OEt pre-treated A549 and 4T1 cells compared to untreated cells. As anticipated, Ce6 micelles showed lower drug release in presence of 0.1mM of buthionine sulfoximine (BSO) pretreated A549 and 4T1 cells exhibiting lower phototoxicity. Further, A549 3D spheroids treated with Ce6 micelles showed significant inhibition in growth, enhanced phototoxicity, and cellular apoptosis in comparison to free Ce6. The above results showed that the developed strategy could be effective in improving the PDT efficacy of Ce6, and the developed polymeric micellar system could be utilized as a PDT regimen for cancer.

MicroRNA-31/184 is involved in transforming growth factor-β-induced apoptosis in A549 human alveolar adenocarcinoma cells

AimsTGF-β-induced alveolar epithelial cells apoptosis were involved in idiopathic pulmonary fibrosis (IPF). This study aimed to explore potential targets and mechanisms of IPF. Main methodsmRNA and microRNA arrays were used to analyze differentially expressed genes and miRNAs. Several essential targets of TGF-β-SMADs and TGF-β-PI3K-AKT pathways were detected. Key findingsmiR-31 and miR-184 expression levels were positively correlated with smad6 and smad2/akt expression levels in IPF patients. TGF-β could induce miR-31 and suppress miR-184 levels in A549 cells. miR-31 was confirmed to bind to the smad6-3′UTR and functionally suppress its expression. Down-regulated SMAD6 enhanced SMAD2/SMAD4 dimer formation and translocation due to its failure to prevent SMAD2 phosphorylation. In contrast, anti-fibrotic functions of miR-184 were abolished due to TGF-β directly suppressing miR-184 levels in A549 cells. When A549 was stimulated by TGF-β combined with or without miR-31 inhibitor/miR-184 mimic, it was showed that depleted miR-31 and/or increased miR-184 significantly ameliorated TGF-β-induced viability of A549 cells, as well as inhibited the expression of profibrotic factors, MMP7 and RUNX2. SignificanceInhibiting miR-31 and/or promoting miR-184 protect against TGF-β-induced fibrogenesis by respectively repressing the TGF-β-SMAD2 and TGF-β-PI3K-AKT signaling pathways, implying that miR-31/184 are potential targets and suggesting a new management strategy for IPF.

Liposome mediated-CYP1A1 gene silencing nanomedicine prepared using lipid film-coated proliposomes as a potential treatment strategy of lung cancer

The occurrence of lung cancer is linked with tobacco smoking, mainly through the generation of polycyclic aromatic hydrocarbons (PAHs). Elevated activity of cytochrome P4501A1 (CYP1A1) plays an important role in the metabolic processing of PAHs and its carcinogenicity. The present work aimed to investigate the role of CYP1A1 gene in PAH-mediated growth and tumor development in vitro and using an in vivo animal model. RNAi strategy was utilized to inhibit the overexpression of CYP1A1 gene using cationic liposomes generated using a lipid film-coated proliposome microparticles. Treatment of PAH-induced human alveolar adenocarcinoma cell line with cationic liposomes carrying CYP1A1 siRNA resulted in down regulation of CYP1A1 mRNA, protein as well as its enzymatic activity, triggering apoptosis and inhibiting multicellular tumor spheroids formation in vitro. Furthermore, silencing of CYP1A1 gene in BALB/c nude xenografts inhibited tumor growth via down regulation of CYP1A1 expression. Altogether, our findings showed that liposome-based gene delivery technology is a viable and stable approach for targeting cancer causing genes such as CY1PA1. This technology facilitated by the use of sugar particles coated with lipid films has demonstrated ability to generate anticancer effects that might be used in the future for therapeutic intervention and treatment of lung cancer.