Genes In A549 Cells Research Articles

In situ quantitative mapping of coding single nucleotide polymorphism on mRNA inside cells by SERS-fluorescence dual-mode probe

Surface Enhanced Raman Scattering (SERS) has potential in nucleic acid detection, but its ability on single nucleotide polymorphisms (SNPs) detection in the early diagnosis of diseases is still limited. In fact, there is few research about in situ detection of coding SNPs which are carried on mRNA. In this paper, we developed SERS-Fluorescence Dual-mode probes to achieve in situ quantitative mapping of intracellular cSNP. The probes were based on Au@Ag core-shell structure that possessed both excellent biocompatibility and localized surface plasmon resonance. Fluorescence can provide a rapid visualization of intracellular cSNP, while SERS can achieve its in situ quantification. The quantitation was conducted by comparing the SERS signals of the reporting molecule p-mercaptobenzoic acid and fluorescent molecule FAM, the detecting limitation of which can be achieved as low as 1.4 fM. This ratiometric detection is more accurate than traditional analysis based on signal intensity directly. It successfully achieved intracellular imaging of the G12D mutation site of KRAS gene in A549 cells, which can help to get the distribution of cSNP in single cell more clearly. And it also demonstrated the good mapping ability for biomolecules (1003 cm−1 for phenylalanine) simultaneously. In conclusion, this kind of probe could be used as a potential tool to monitor cSNP as well as biomolecules in multiple biological processes, which could provide reference for disease prediction.

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

Lnc-RPS6P3 Inhibits Influenza A Virus Replication and Attenuates the Inhibitory Effect of NS1 on Innate Immune Response.

Host factors play important roles in influenza A virus (IAV) replication. In order to identify novel host factors involved in IAV replication, we compared the differentially expressed genes in A549 cells after IAV infection. We found that lncRNA lnc-RPS6P3 was up-regulated upon viral infection and poly(I:C) and IFN-β treatment, indicating it was an interferon-stimulated gene. Functional analysis demonstrated that overexpression of lnc-RPS6P3 inhibited IAV replication while knockdown of lnc-RPS6P3 promoted viral infection in A549 cells. Lnc-RPS6P3 inhibited both transcription and replication of IAV. Further study showed that lnc-RPS6P3 interacted with viral NP and interfered with NP self-oligomerization and, consequently, inhibited vRNP activity. In addition, lnc-RPS6P3 interacted with viral NS1 and reduced the interaction of NS1 and RIG-I; it also attenuated the inhibitory effect of NS1 on IFN-β stimulation. In conclusion, we revealed that lnc-RPS6P3 is an interferon-stimulated gene that inhibits IAV replication and attenuates the inhibitory effect of NS1 on innate immune response.

TPX2 influences the regulation of macrophage polarization via the NF-κB pathway in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Xklp2 targeting protein (TPX2), a crucial oncogene exhibits high expression levels in various cancers including LUAD, may serve as a potential target for clinical intervention. Additionally, the growth of lung cancer is significantly influenced by the tumor microenvironment (TME). However, there have been no reports on experiments investigating TPX2 in tumor-infiltrating immune cells (TIICs) in LUAD. Therefore, we verified the effect of TPX2 on macrophage polarization both in vitro and in vivo. MethodsWe silenced TPX2 the gene in A549 cells and collected supernatants for macrophage culture. We then used flow cytometry and Western blot analysis to assess macrophage polarization. Additionally, we verified the expression of macrophage colony-stimulating factor (M-CSF), and CD163 by immunohistochemistry (IHC) in tissue specimens from LUAD patients. Finally, pathways related to TPX2's regulatory function in macrophage polarization were analyzed through whole genome sequencing, Western blotting, and immunofluorescence (IF). ResultsSilencing TPX2 can affect the ratio of CD80+ M1/CD163+ M2 and reduce the polarization of M0 macrophages to CD163+ M2 macrophages mainly by inhibiting the expression of M-CSF. In human LUAD tissues, the expression levels of TPX2, M-CSF and CD163 increased with the degree of differentiation. Silencing TPX2 inhibits the NF-κB signaling pathway, thereby reducing the expression of M-CSF, and affecting macrophage polarization. ConclusionSilencing TPX2 can inhibit the expression of M-CSF by blocking the NF-κB signal, thereby reducing CD163+ M2 macrophage polarization. The TPX2/NF-κB/M-CSF signaling axis may be involved in regulating macrophage polarization.

Transcriptome profiling of A549 non-small cell lung cancer cells in response to Trichinella spiralis muscle larvae excretory/secretory products.

Trichinella spiralis (T. spiralis) muscle-larva excretory/secretory products (ML-ESPs) is a complex array of proteins with antitumor activity. We previously demonstrated that ML-ESPs inhibit the proliferation of A549 non-small cell lung cancer (NSCLC) cell line. However, the mechanism of ML-ESPs against A549 cells, especially on the transcriptional level, remains unknow. In this study, we systematically investigated a global profile bioinformatics analysis of transcriptional response of A549 cells treated with ML-ESPs. And then, we further explored the transcriptional regulation of genes related to glucose metabolism in A549 cells by ML-ESPs. The results showed that ML-ESPs altered the expression of 2,860 genes (1,634 upregulated and 1,226 downregulated). GO and KEGG analysis demonstrated that differentially expressed genes (DEGs) were mainly associated with pathway in cancer and metabolic process. The downregulated genes interaction network of metabolic process is mainly associated with glucose metabolism. Furthermore, the expression of phosphofructokinase muscle (PFKM), phosphofructokinase liver (PFKL), enolase 2 (ENO2), lactate dehydrogenase B (LDHB), 6-phosphogluconolactonase (6PGL), ribulose-phosphate-3-epimerase (PRE), transketolase (TKT), transaldolase 1 (TALDO1), which genes mainly regulate glycolysis and pentose phosphate pathway (PPP), were suppressed by ML-ESPs. Interestingly, tricarboxylic acid cycle (TCA)-related genes, such as pyruvate dehydrogenase phosphatase 1 (PDP1), PDP2, aconitate hydratase 1 (ACO1) and oxoglutarate dehydrogenase (OGDH) were upregulated by ML-ESPs. In summary, the transcriptome profiling of A549 cells were significantly altered by ML-ESPs. And we also provide new insight into how ML-ESPs induced a transcriptional reprogramming of glucose metabolism-related genes in A549 cells.

Long non-coding RNA GNAS-AS1 knockdown inhibits proliferation and epithelial-mesenchymal transition of lung adenocarcinoma cells via the microRNA-433-3p/Rab3A axis.

The goal of this study was to demonstrate the functions and specific mechanism of long non-coding RNA (lncRNA) GNAS-AS1 in lung adenocarcinoma. Levels of lncRNA GNAS-AS1, microRNA (miR)-433-3p, and Rab3A were assessed by quantitative real-time PCR (qRT-PCR). The target-binding sites of lncRNA GNAS-AS1, miR-433-3p, and Rab3A were predicted and confirmed by bioinformatics tool (StarBase) and a dual-luciferase reporter system. Cell proliferation and apoptosis were checked using MTT and flow cytometry, respectively. Additionally, the levels of apoptosis-related and epithelial-mesenchymal transition (EMT)-associated genes in A549 cells were analyzed by qRT-PCR and western blot. We found that lncRNA GNAS-AS1 was upregulated, miR-433-3p was low-expressed, and Rab3A was overexpressed in lung adenocarcinoma tissues and cell lines. LncRNA GNAS-AS1 interacted with miR-433-3p and negatively regulated miR-433-3p levels. Rab3A was a direct target of miR-433-3p. Downregulation of lncRNA GNAS-AS1 remarkably suppressed cell proliferation, promoted cell apoptosis, decreased B-cell lymphoma-2 (Bcl-2) expression, enhanced the Bcl-2-Associated X (Bax) level, promoted E-cadherin expression, and reduced N-cadherin and Rab3A levels. However, the miR-433-3p inhibitor reversed all these findings. Similarly, the inhibitory effects of miR-433-3p mimic on A549 cells were reversed by the Rab3A-plasmid. In conclusion, lncRNA GNAS-AS1 downregulation suppressed lung adenocarcinoma cell proliferation and EMT through the miR-433-3p/Rab3A axis.

Isolation, identification, and characterization of α- asarone, from hydromethanolic leaf extract of Acorus calamus L. and its apoptosis-inducing mechanism in A549 cells

Due to the presence of several active secondary metabolites, the traditional Indian and Chinese medicinal herb Acorus calamus L. has been utilized for both medical and culinary purposes since ancient times. A recent report has underscored the promising cytotoxic effect of A. calamus leaves extract against non-small cell lung cancer A549 cells. Thus, we want to separate the bioactive substance from the hydromethanolic extract of A. calamus leaves in the current investigation. Thin-layer chromatography was used to separate the compounds and different spectroscopic methods (UV, FTIR, NMR, and LCMS/MS) were used for the structure prediction. α-asarone was found to be the main bioactive compound present and it was isolated from A. calamus leaves extract. It exerted a good cytotoxic effect with an IC50 value of 21.43 ± 1.27 μM against A549 cells and IC50 value of 324.12 ± 1.32 μM against WI-38 cells. The induction of apoptosis in A549 cells by α-asarone was reaffirmed by the diverse differential staining methods including DAPI, Acridine Orange/Ethidium Bromide, and Giemsa staining. Additionally, α-asarone induced mitochondrial membrane potential (ΔΨm) dissipation with a concomitant increase in the production of ROS. Furthermore, it also increased expressions of caspase-3, caspase-9, caspase-8, DR4, and DR5 genes in A549 cells. In conclusion, α-asarone-induced apoptotic cell death in non-small lung cancer cells (A549) as a result of loss of mitochondrial function, increased ROS production, subsequent activation of an internal and extrinsic caspase pathway, and altered expression of genes controlling apoptosis. As a whole, α-asarone is a plausible therapeutic agent for managing lung cancer. HIGHLIGHTS Isolation of bioactive compound from hydromethanolic leaves extract of Acorus calamus L. by thin layer chromatography. Structural elucidation of the bioactive compound was carried out using different methods like UV analysis, FTIR, NMR, and LC-MS/MS analysis. A plausible mode of action revealed that α-asarone can induce apoptosis in lung cancer cells (A549). Communicated by Ramaswamy H. Sarma

Diffractaic acid exhibits thioredoxin reductase 1 inhibition in lung cancer A549 cells.

Lung cancer is the leading cause of cancer-related deaths all over the world. Therefore, it has gained importance in the development of new chemotherapeutic strategies to identify anticancer agents with low side effects, reliable, high anticancer potential, and specific to lung cancer cells. Thioredoxin reductase 1 (TrxR1) is an important therapeutic target for lung cancer treatment because of its overexpression in tumor cells. Here, we aimed to examine the anticancer effect of diffractaic acid, a lichen secondary metabolite, in A549 cells by comparing it with the commercial chemotherapeutic drug carboplatin and also to investigate whether the anticancer effect of diffractaic acid occurs via TrxR1-targeting. The IC50 value of diffractaic acid on A549 cells was determined as 46.37 μg/mL at 48 h, and diffractaic acid had stronger cytotoxicity than carboplatin in A549 cells. qPCR results revealed that diffractaic acid promoted the intrinsic apoptotic pathway through the upregulation of the BAX/BCL2 ratio and P53 gene in A549 cells, which is consistent with the flow cytometry results. Furthermore, migration analysis results indicated that diffractaic acid impressively suppressed the migration of A549 cells. While the enzymatic activity of TrxR1 was inhibited by diffractaic acid in A549 cells, no changes were seen in the quantitative expression levels of gene and protein. These findings provide fundamental data on the anticancer effect of diffractaic acid on A549 cells targeting TrxR1 activity, suggesting that it could be considered a chemotherapeutic agent for lung cancer therapy.

Abstract 4454: Loss of CX3CL1 expression mediates immune evasion in STK11 mutated lung adenocarcinomas

Abstract Immunotherapy has improved survival for many cancer patients, especially for immunogenic malignancies such as lung cancer. However, even in lung cancer, the response rate for anti-PD1 therapy (for example) does not exceed 20%. Thus, it is critical to identify mechanisms that block the response to immune checkpoint therapies. STK11 (also known as LKB1) is encoded by the serine threonine kinase 11 gene (STK11). Patients harboring tumors with STK11 mutations have reduced infiltrates of cytotoxic T-cells and clinical studies have shown that they respond poorly to anti-PD1 or anti-PDL-1 therapies regardless of PDL-1 status, which otherwise predicts benefit. Herein, we have used gene expression data from a cohort of 442 lung adenocarcinoma patients to identify CX3CL1 (fractalkine) as a gene silenced in STK11 mutant tumors with potential to be a key direct modulator of the immune system relevant to STK11 loss. To further explore this hypothesis, we have edited the STK11 gene in A549 cells back to wild type (STK11 corrected). As predicted, restoration of STK11 function resulted in modest expression of CX3CL1 as measured by Western blotting. Unexpectedly, exposure of STK11 corrected cells to human immune cells isolated from the blood of healthy donors resulted in a 5 to 10-fold increase in CX3CL1 suggesting interactive signaling between tumor and immune cells. Using transwell assays and STK11 corrected A549 cells we find that restoration of functional STK11 increases immune cell migration, adhesion and invasion in vitro. Finally, cytotoxicity assays demonstrate that STK11 corrected A549 cells are 3 to 5-fold more sensitive to immune cell killing. Taken together these results suggest the hypothesis that CX3CL1 loss mediates immune evasion in STK11 mutant lung adenocarcinomas. Future work will further explore the mechanisms underlying this pathway in preclinical models. Citation Format: Eria Eksioglu, Gabriela M. Wright, Trent R. Percy, Kenneth L. Wright, W. Douglas Cress. Loss of CX3CL1 expression mediates immune evasion in STK11 mutated lung adenocarcinomas. [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 4454.

MiR-183-5p regulates ECM and EMT to promote non-small cell lung cancer progression by targeting LOXL4.

Non-small cell lung cancer (NSCLC) progression is mediated by changes in gene expression induced by microRNAs. However, the underlying mechanisms remain to be elucidated. In this study, we investigated the roles of miR-183-5p and its target gene in lung cancer development. Relative levels of miR-183-5p and lysyl oxidase-like 4 (LOXL4) expression in lung cancer cells or tissues were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence or Western blotting as appropriate. The binding of miR-183-5p to LOXL4 sequences was verified by a dual luciferase reporter assay, and cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and Edu staining. The cell cycle stage and apoptosis were detected by flow cytometry, and Transwell assays were performed to evaluate cell migration and invasion capabilities. The tumorigenic capability of cancer cells was analyzed using a cancer cell line-based xenograft nude mouse model. miR-183-5p expression was decreased in the lung cancer tissues and cell lines and was negatively correlated with elevated LOXL4 expression. Treatment with miR-183-5p mimics suppressed LOXL4 expression, while treatment with an miR-183-5p inhibitor promoted LOXL4 expression in A549 cells. miR-183-5p was found to directly bind to the 3' UTR of the LOXL4 gene in A549 cells. Overexpression of LOXL4 enhanced cell proliferation, cell cycle progression, migration, and invasion, but repressed their apoptosis, and activated extracellular matrix (ECM) and the epithelial mesenchymal transition (EMT) process in A549 cells, while LOXL4 knockdown produced the opposite effects. Treatment with an miR-183-5P inhibitor promoted the proliferation, cell cycle progression, migration, and invasion of A549 cells but suppressed their apoptosis, and activated the ECM and EMT process, while all these effects were abrogated by LOXL4 knockdown. The tumorigenic capability of A540 cells in nude mice was greatly impaired by treatment with miR-183-5p mimics. miR-183-5p repressed the proliferation, migration, invasion, ECM formation, and EMT processes, and promoted the apoptosis of lung cancer cells by targeting LOXL4 expression.

Anti-proliferative action, molecular investigation and computational studies of novel fused heterocyclic cellulosic compounds on human cancer cells

Reactivity of MCEC (3) with diazonium chloride, DMF-DMA and NH2NH2 to afford fused heterocyclic cellulosic derivative which confirmed through spectral analysis. Also, the cellulosic compounds were evaluated their growth inhibitory activity contra cancer cells A549 and Caco2 using neutral red uptake assay. Compounds, MCEN(6), MCPT(5a), MCPT(5b) showed better growth inhibitory activity on A549 and Caco2 growth compared to control values. While compound MCPY(7) exerted the best cytotoxic activity against A549 cells after 48 h. The expression levels of HIF-1α, β-Catenin, MYC, Cyclin D1 and MMP7 genes in A549 cells were examined using QRT-PCR. The compounds MCEN(6) and MCPY(7) down regulated levels of β-Catenin, MYC, Cyclin D1 and MMP7 genes and up-regulated levels of HIF-1α when treated with A549 cells compared to control values. Also, these biological studies confirmed through docking stimulation with different proteins and showed least binding energy with amino acids and attached with NH2 and OH of cellulose with hydrogen bond interaction. Moreover, optimization of compounds using DFT/6-311(G) showed the stability of them and identifies their physical descriptors which showed excellent correlation with experimental results. Noteworthy, compounds, MCEN(6) and MCPY(7) were most promising anticancer agents against lung cancer through the regulation of apoptosis, cell cycle, proliferation, progression, chemo-resistance, and angiogenesis.

Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets.

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs) that are able to separate anti-inflammatory effects via gene repression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene repression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms running both in academia and industry may therefore boost chances to find novel GR ligands with an actual improved therapeutic benefit.

APEX1 regulates alternative splicing of key tumorigenesis genes in non-small-cell lung cancer

BackgroundAberrant alternative splicing (AS) contributes to tumor progression. Previous studies have shown that apurinic-apyrimidinic endonuclease-1 (APEX1) is involved in tumor progression. It is unknown whether APEX1 functions in tumor progression by regulation of AS. It is also unknown whether APEX1 can regulate non-small-cell lung cancer (NSCLC) proliferation and apoptosis. We analyzed APEX1 expression levels in 517 lung NSCLC samples from the TCGA (Cancer Genome Atlas) database. The impact of APEX1 over expression on A549 cell proliferation and apoptosis was detected by the methyl thiazolyl tetrazolium assay and by flow cytometry. The transcriptome of A549 cells with and without APEX1 over expression was determined by Illumina sequencing, followed by analysis of AS. RT-qPCR validated expression of APEX1-related genes in A549 cells. We have successfully applied RNA-seq technology to demonstrate APEX1 regulation of AS.ResultsAPEX1 expression was shown to be upregulated in NSCLC samples and to reduce cell proliferation and induce apoptosis of A549 cells. In addition, APEX1 regulated AS of key tumorigenesis genes involved in cancer proliferation and apoptosis within MAPK and Wnt signaling pathways. Each of these pathways are involved in lung cancer progression. Furthermore, validated AS events regulated by APEX1 were in key tumorigenesis genes; AXIN1 (axis inhibition protein 1), GCNT2 (N-acetyl glucosaminyl transferase 2), and SMAD3 (SMAD Family Member 3). These genes encode signaling pathway transcription regulatory factors.ConclusionsWe found that increased expression of APEX1 was an independent prognostic factor related to NSCLC progression. Therefore, APEX1 regulation of AS may serve as a molecular marker or therapeutic target for NSCLC treatment.

In Vitro Effect of Δ9-Tetrahydrocannabinol and Cannabidiol on Cancer-Associated Fibroblasts Isolated from Lung Cancer.

There is evidence that demonstrates the effect of cannabinoid agonists inhibiting relevant aspects in lung cancer, such as proliferation or epithelial-to-mesenchymal transition (EMT). Most of these studies are based on evidence observed in in vitro models developed on cancer cell lines. These studies do not consider the complexity of the tumor microenvironment (TME). One of the main components of the TME is cancer-associated fibroblasts (CAFs), cells that are relevant in the control of proliferation and metastasis in lung cancer. In this work, we evaluated the direct effects of two cannabinoid agonists, tetrahydrocannabinol (THC) and cannabidiol (CBD), used alone or in combination, on CAFs and non-tumor normal fibroblasts (NFs) isolated from adenocarcinoma or from healthy lung tissue from the same patients. We observed that these compounds decrease cell density in vitro and inhibit the increase in the relative expression of type 1 collagen (COL1A1) and fibroblast-specific protein 1 (FSP1) induced by transforming growth factor beta (TGFβ). On the other hand, we studied whether THC and CBD could modulate the interactions between CAFs or NFs and cancer cells. We conditioned the culture medium with stromal cells treated or not with THC and/or CBD and cultured A549 cells with them. We found that culture media conditioned with CAFs or NFs increased cell density, induced morphological changes consistent with EMT, inhibited cadherin-1 (CDH1) gene expression, and induced an increase in the relative expression of cadherin-2 (CDH2) and vimentin (VIM) genes in A549 cells. These changes were inhibited or decreased by THC and CBD administered alone or in combination. In another series of experiments, we conditioned culture media with A549 cells treated or not with THC and/or CBD, in the presence or absence of TGFβ. We observed that culture media conditioned with A549 in the presence of TGFβ induced an increase in the expression of COL1A1 and VIM, both in CAFs and in non-tumor NFs. Both THC and CBD ameliorated these effects. In summary, the results presented here reinforce the usefulness of cannabinoid agonists for the treatment of some relevant aspects of lung cancer pathology, and demonstrate in a novel way their possible effects on CAFs as a result of their relationship with cancer cells. Likewise, the results reinforce the usefulness of the combined use of THC and CBD, which has important advantages in relation to the possibility of using lower doses, thus minimizing the psychoactive effects of THC.

Exploration of Potential Targets and Mechanisms of Fisetin in the Treatment of Non-Small-Cell Lung Carcinoma via Network Pharmacology and In Vitro Validation.

Purpose The morbidity and fatality rates of non-small-cell lung cancer (NSCLC) were high, although a combination of multiple treatments was used. Fisetin, a small flavonoid compound, had shown anticancer activities. Thus, we aimed at exploring the mechanisms of Fisetin in the treatment of NSCLC. Methods TCMSP and Swiss target tools were used to screen the targets of Fisetin, and GeneCards was used to collect the genes related to NSCLC. The genes common to Fisetin and NSCLC were obtained by Venn analysis, whose possible functions were further annotated. A “Compound-Target-Disease” network was then constructed and hub genes were filtered. Also, molecular docking was performed to predict the binding abilities between Fisetin and the hub genes. Then, the effects of Fisetin on the expression of hub genes in lung adenocarcinoma cells were preliminarily evaluated in vitro. Results A total of 131 genes common to Fisetin and NSCLC were filtered out, which might be enriched in several biological processes including antioxidation, cell proliferation, and various signaling pathways, such as PI3K-Akt and IL-17 signaling pathways. Among them, PIK3R1, CTNNB1, JUN, EGFR, and APP might be the hub genes. Molecular docking indicated the close bond between Fisetin and them. Experiments implied a possible effect of Fisetin on the expression of hub genes in A549 cells. Conclusion The present study found a series of novel targets and pathways for Fisetin treating NSCLC. Multiple angles, targets, and pathways were involved in the biological processes, which need to be verified in further experiments.

Design and fabrication of a dual-drug loaded nano-platform for synergistic anticancer and cytotoxicity effects on the expression of leptin in lung cancer treatment

Lung cancer is progressive and fatal malignancy and is one of the main causes of cancer-related death worldwide. Leptin (LEP) is a cytokine derived from adipocyte that is known to be a key mitogenic agent in lung carcinogenesis. Excessive amounts of systemic and pulmonary LEP are contributed with an enhanced risk of lung cancer. Currently, combinatorial treatments in cancer therapy are of intense interest. Metformin (Met) and Curcumin (Cur) are bioactive compounds that have several anti-cancer properties in pre-clinical and clinical studies. Herein, a dual-drug loaded nano-platform was designed for targeted drug delivery to A549 lung carcinoma cells. Met/Cur-loaded poly (D, L lactide-co-glycolide)-b-poly (ethylene glycol) (PLGA-PEG) nanoparticles (NPs) were synthesized. The inhibitory property of dual drug-loaded PLGA/PEG NPs on LEP and LEP receptor genes expression was determined compared to Met and Cur. DLS, FTIR, XRD, SEM and TEM techniques were employed for the characterization of synthesized NPs as well as nano-formulated drugs. The pattern of in-vitro drugs released from PLGA-PEG NPs was investigated through dialysis method. Cytotoxicity effects of free, nanocapsulated, and dual-loaded drugs on A549 cells were assessed by MTT assay. In addition, the expression of LEP and LEP receptor genes in A549 cells after treatments were evaluated through real-time PCR assay. According to the results, free and nanocapsulated forms of Cur and Met exhibited a cytotoxic effect on A549 cells in a dose-dependent pattern. In particular, Met-Cur-PLGA/PEG NPs posse synergistic anti-cancer properties than free and nanocapsulated forms of the drugs. Likewise, expression of LEP and LEP receptor genes in Met-Cur-PLGA/PEG NPs treated cells was much more suppressed compared to cells, which were incubated with free and nanocapsulated forms of the drugs. Our findings suggested that the co-capsulation of Met and Cur in PLGA/PEG NPs could significantly increase the efficiency of targeted drug delivery for cancer treatment proposes.

The effect of the iron chelator Deferasirox in combination with Cisplatin chemotherapy against lung carcinoma

The preferred treatment for patients with lung carcinoma (LC) is platinum‐based regimens, eg., Cisplatin (CisPt). Unfortunately, despite improvements in LC outcomes, many of these tumors metastasize and develop resistance against such Pt‐drugs, inducing abysmally low survival prognosis. Due to this, there is an urgent need to develop alternative therapies for LC mostly focused on overcome or diminish chemoresistance and inhibit the metastatic spread. FDA‐approved drugs which target cancer metabolic pathways are considered as repurposing drugs that could diminish these adverse effects. Deferasirox (Def), an FDA‐approved iron chelation therapy, is a drug under study for cancer therapy due to iron plays a key role in cell growth and energy production in cancer. Herein, we determined the effect of deferasirox (Def) alone and in combination with CisPt on the genes related to chemoresistance and metastasis in LC cells. Viability assay results showed that Def has synergistic cytotoxic effects in combination with CisPt after 24 h of incubation in A549 LC cells. qPCR gene expression studies showed that Def induced a significant downregulation of EGFR, VEGF, MMP9, Pgp, P53, NDRG1, CHD4 genes in A549 cells. In addition, we determined that Def induce the production of reactive oxygen species in LC. Furthermore, in in vivo studies performed in Lewis Lung Carcinoma mouse model, Def co‐administered with CisPt showed tumor size reduction. The results obtained from this study will impact the field of lung cancer therapy adding knowledge on the Def cellular mechanisms and genetic regulation against chemoresistance and metastasis processes.

ACC2 is under-expressed in lung adenocarcinoma and predicts poor clinical outcomes.

Acetyl-CoA Carboxylases (ACCs) are key fatty acid metabolic enzymes responsible for catalyzing the carboxylation of acetyl-CoA to malonyl-CoA. The role of ACC1 has been associated with tumor biology, but the role of ACC2 in cancer remains largely uncharacterized. We conducted a transcriptomic analysis using GEPIA and Oncomine to study the expression of ACC2 in different cancers. Immunohistochemistry was used to examine the expression of ACC2 in lung cancer tissue microarray, and the correlation between ACC2 expression and clinical parameters was analyzed. Following ACC2 knockdown by RNA interference in A549 and HCC827 cells, Cell Counting Kit‑8 and transwell assays were used to detect cell proliferation and migration. Real-time PCR was used to detect cell cycle-related genes in A549 cells. GEO dataset and KM-plotter database were used to analyze the relationship between ACC2 expression and the prognosis in lung cancer patients. We found that ACC2 is under-expressed in cancerous tissue and the expression of ACC2 is negatively correlated with tumor size, regional lymph-node metastases, and clinical stage of lung adenocarcinoma patients. In addition, knocking down ACC2 in A549 cells and HCC827 cells can promote cell proliferation and migration, and cell cycle-related genes MAD2L1 and CCNB2 were up-regulated after ACC2 was knockdown in A549 cells. Finally, we found that lung adenocarcinoma patients with under-expressed ACC2 have a worse prognosis. Our results suggest that ACC2 is a potential diagnostic and prognostic marker that negatively correlated with clinical outcomes in lung adenocarcinoma.

DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway.

The purpose of the present study was to examine the epigenetic mechanism by which microRNA (miR)-152-3p regulates proliferation in non-small cell lung cancer A549 cells via neural cell adhesion molecule 1 (NCAM1). Bisulfite sequencing PCR (BSP), the gold standard for methylation detection, uses bisulfite-treated DNA to determine its pattern of methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. It was conducted and demonstrated a relatively high level of methylation in the miR-152-3p promoter region. Chromatin immunoprecipitation was combined with PCR to detect the binding of DNA methyltransferase 3B (DNMT3B) protein to miR-152-3p, which tends to occur in the core region of the miR-152-3p gene in A549 cells. Luciferase assay identified NCAM1 as the target gene of miR-152-3p. MTT, colony formation and Transwell assays indicated that miR-152-3p could decrease cell proliferation and invasion and in addition to reducing the expression level of NCAM1. Overexpression of NCAM1 could attenuate the effect of miR-152-3p. DNMT3B knockdown decreased the proliferative ability of A549 cells and increased the expression of miR-152-3p, while decreased that of NCAM1. After treatment with miR-152-3p inhibitor, these effects were attenuated and the NCAM1 expression level was upregulated. The results indicated that miR-152-3p may suppress the proliferation of A549 cells by downregulating NCAM1. In addition, DNMT3B negatively regulated the expression of miR-152-3p via modulation of the methylation level in the miR-152-3p core region, thus mediating the proliferation of lung tumor cells.

Identifying PIF1 as a Potential Target of Wenxia Changfu Formula in Promoting Lung Cancer Cell Apoptosis: Bioinformatics Analysis and Biological Evidence.

Lung cancer remains the leading cause of cancer-related deaths worldwide. Traditional Chinese medicine (TCM) is a valuable resource of active natural products and plays an important role in cancer treatment with the advantages of high efficiency and safety. Wenxia Changfu formula (WCF) is modified from Dahuang Fuzi decoction from Han Dynasty and has been used for treating lung cancer in China. Our previous research showed that WCF had an antitumor effect in vivo and in vitro, while the mechanism has not been well illustrated. In this study, the effect of WCF on the proliferative ability in three lung cancer cells and one noncancerous human cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. WCF suppressed A549, H460, and PC-9 cell viability in a dose-dependent manner, with no inhibition of noncancerous MRC-5 cells after 48 h treatment with WCF (0–50 mg/mL). Furthermore, we screened for genes in A549 cells using four WCF-treated samples and four control samples on a gene expression profile microarray. 21 genes were significantly downregulated by WCF, which may potentially play an important role in the proliferation of A549 cells. High-content screening evaluated whether silencing the 21 genes affected A549 cell growth. The results showed that PIF1 knockdown exhibited the most potent inhibition of cell proliferation compared with the other genes. Downregulation of PIF1 increased A549 cell apoptosis and the activity of caspase 3/7. Besides, RT-PCR showed that the expression levels of PIF1 mRNA decreased significantly in A549, H460, and PC-9 cells after WCF treatment. In conclusion, the present observations indicate that WCF may inhibit lung cancer cell proliferation by promoting apoptosis via regulating the expression of PIF1.