Treatment Of A549 Cells Research Articles

Role of tumour derived exosomes in manuring the metastatic niche in patients with lung cancer liver metastasis: Beyond seed soil hypothesis

Metastatic lung cancer is often diagnosed at a late stage and is widely known to metastasize to the liver in the Asian population, but the underlying mechanism still remains unclear. Tumour derived exosomes (TDEs) play an important role in metastasis and its contributions to the development of the pre-metastatic niche formation is of utmost importance. In this study serum, derived tumour exosomes form lung cancer liver metastatic patients showed active incorporation by A549 cells in a concentration and time-dependent manner and induced migratory/invasive properties. Moreover, it was observed that cellular uptake of exosomes was increased during G2/M phase stimulating the cells to enter cell cycle phases leading to cell proliferation. Further, we observed that E-cadherin, beta catenin, VEGFA, CDKN2A and TGFBR2 were differentially expressed in treated A549 cells demonstrating an important role of these TDEs in altering the tumour microenvironment. In vivo model demonstrated an increase in serum SGPT and SGOT levels whereas the histopathological examination showed patches of pneumonitis in lungs and advanced inflammation in the liver. Conclusively, our results depict an undisputable role of these exosomes as key modulators in the formation of the pre-metastatic niche required for the colonization of circulating tumour cells (CTCs) ultimately leading to distant metastasis.

Antiviral activity of extracellular vesicles derived from respiratory syncytial virus-infected airway epithelial cells.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children and elderly. No vaccine or effective treatment is currently available for RSV. Extracellular vesicles (EVs) are microvesicles known to carry biologically active molecules, including RNA, DNA and proteins (i.e. cargo). Viral infections can induce profound changes in EV cargo, and the cargo can modulate cellular responses of recipient cells. We have recently shown that EVs isolated from RSV-infected cells were able to activate innate immune response by inducing cytokine and chemokine release from human monocytes and airway epithelial cells, however, we did not investigate the potential antiviral contribution of EVs to a subsequent infection. The objective of this study was to assess the presence of innate immune mediators, including type I and III interferons (IFNs) in EVs released from airway epithelial cells infected with RSV, and their potential role in modulating viral replication in recipient cells. EV-derived from cells infected with RSV were associated with significant amounts of cytokine and chemokines, as well as IFN-β and -λ, compared to EVs isolated from mock-infected cells. Cells treated with RSV-EVs showed significantly lower levels of viral replication compared to untreated or mock-EV-treated RSV infected cells. Cellular pretreatment with Cerdulatinib, an IFN receptor signaling inhibitor, inhibited the antiviral activity of RSV-EVs in recipient airway epithelial cells. Furthermore, treatment of A549 cells with RSV-EVs induced the expression of IFN-dependent antiviral genes, supporting the idea that RSV-EVs exerts their antiviral activity through an interferon-dependent mechanism. Finally, we determined the concentrations of soluble and EV-associated IFN-β and IFN-λ in five nasopharyngeal secretions (NPS) of children with viral infections. There were significant levels of IFN-λ in NPS and NPS-derived EVs, while IFN-β was not detected in either of the two types of samples. EVs released from RSV-infected cells could represent a potential therapeutic approach for modulating RSV replication in the airways.

8-Hydroxyquinoline a natural chelating agent from Streptomyces spp. inhibits A549 lung cancer cell lines via BCL2/STAT3 regulating pathways.

Biomolecules from Streptomyces spp. are emerging sources of natural drugs and have been focused on over the decade. The discovery of bioactive chemotherapeutic molecules from soil Streptomyces spp. has opened the medium for the search for natural drugs. In the current study, 8-HOQ was extracted and purified from soil Streptomyces spp. and was evaluated on A549 and BEAS cell lines. The apoptotic and caspase mediated pathways were evaluated using cell proliferation, dual fluorescent staining, migration, invasion and mRNA as well as protein quantification of apoptotic markers. In vitro cytotoxicity test revealed that 8-HOQ possesses potent cytotoxicity activities with IC50values of 26µM, 5µM, 7.2µM at 24h, 48h, and 72h respectively against A549 lung cancer cell lines. The result also demonstrated that 8-HOQ from Streptomyces spp significantly inhibited the A549 lung cancer cell lines and activated the intrinsic pathways of apoptosis. The caspase-3 and caspase-8 activities were potentially elevated in 8-HOQ treated A549 cell lines and confirmed that 8-HOQ mediated A549 cancer cell death through the intrinsic pathway. The results explored caspase-mediated apoptosis as a mechanism underlying the inhibition of cancer cell viability in a dose-dependent manner. The expression of P53, BCL2 and STAT3 were inhibited in A549 cell lines and confirmed the metastasis inhibitory potential of 8-HOQ by blocking migration and invasion in A549 cell lines. These results indicated that 8-HOQ from Streptomyces spp. potentially inhibited growth and migration of A549 lung cancer cell lines.

Dexamethasone as an anti-cancer or hepatotoxic

The linkage between inflammation and oxidative stress in liver damage has been proven and is undeniable; dexamethasone with some antioxidants can reduce the toxicity of liver tissue. Due to the importance of cancer treatment, glucocorticoids' synergistic effect in inhibiting cancer cell growth is also investigated. Dexamethasone alone and combined with etoposide were tested at concentrations of 1, 5, and 10 μM to evaluate the potency of dexamethasone in inhibiting the growth of A549 cells using oxidative stress factors and DNA damage. Also, intraperitoneal injection of dexamethasone in rats was used to induce liver toxicity. Coenzyme Q10 at different concentrations (1, 10, and 50 mg/kg) was used as an antioxidant to assess the oxidative stress factors and measure Caspase-3 activity. The results showed that dexamethasone combined with etoposide could significantly inhibit the growth of cancer cells and induce apoptosis. Treatment of A549 cells using dexamethasone also inhibits cancer cells' growth by inducing oxidative stress and DNA damage. Dexamethasone also, by inducing oxidative stress and activation of caspase 3, ultimately causes hepatotoxicity. Treatment with different concentrations of CoQ10 showed improved mitochondrial function, antioxidant defense, and liver enzyme. The best effect of coenzyme Q10 on dexamethasone-induced hepatotoxicity is 50 mg/kg. As a result, dexamethasone (alone and combined with etoposide) has an anti-cancer effect by damaging DNA and inducing oxidative stress. Also, CoQ10 has antioxidant effects against dexamethasone-induced hepatotoxicity by improving mitochondrial function and reducing caspase-3 activity.

Ursolic acid represses influenza A virus-triggered inflammation and oxidative stress in A549 cells by modulating the miR-34c-5p/TLR5 axis

BackgroundUrsolic acid (UA) is a pentacyclic triterpenoid compound with a wide range of anti-tumor, anti-inflammatory, hypotensive and other pharmacological effects. Here, the biological roles and regulatory mechanisms of UA in influenza A virus (IAV)-treated A549 cells were investigated. MethodThe cytotoxic impacts of UA on A549 cells with or without IAV treatment were determined using MTT and LDH assays. The inflammatory responses and oxidative stress of IAV-treated A549 cells were measured by RT–qPCR, ELISA, DCFH-DA probe, and colorimetric assays. A dual luciferase assay was carried out to validate the molecular interaction between miR-34c-5p and TLR5. Promoter methylation was detected by MSP experiment. Methylation-related proteins were quantified by western blot. Virus replication was assessed by TCID50 and western blot assays. ResultsUA significantly ameliorated IAV-triggered cell injury and inflammatory response, virus replication and oxidative stress by elevating cell viability, ROS level and the activities of SOD and GSH-Px but reducing the LDH, MDA, and TCID50 values and the expression of virus-related proteins (NP) and cytokines (TNF-α, IL-1β, IL-6, and IL-18). Moreover, UA promoted miR-34c-5p expression by repressing DNMTs-mediated methylation. TLR5 was verified to be a direct target of miR-34c-5p and could be downregulated by UA. Rescue experiments revealed that silencing miR-34c-5p diminished the regulatory roles of UA in IAV-treated A549 cells. ConclusionOur data elucidated that UA attenuated IAV-triggered inflammatory responses and oxidative stress in A549 cells by regulating the miR-34c-5p/TLR5 axis, suggesting that UA plays a protective role in IAV-induced pneumonia.

Carthamus tinctorius L. Inhibits Proliferation of Lung Cancer A549 Cells and Tender’s Mitochondrial Protection

Abstract: Background: Plant-based products are well-known as long-lasting chemo preventive and chemotherapeutic medicines against cancer. Objectives: The purpose of this research is to identify out how safflower extract affects A549 cells. Materials and Methods: The antiproliferative activity was determined using the Trypan blue assay, while the cytotoxicity was determined using the MTT assay. The ethidium bromide/acridine orange (AO/EB) dual staining method was used to observe the apoptotic inducing effect and the morphological assessment of A549 cells using phase contrast microscopy was studied to discern the nuclear changes if any on treatment with safflower. The mitochondrial membrane potential was studied using a cationic lipophilic dye rhodamine 123 under a confocal microscope, and oxidative stress was assessed using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) labelling. Results: The trypan blue assay speculated that there was a decrease in cell viability at highest concentration and that the effect was dose-dependent. The MTT assay revealed that cytotoxicity increases in cells treated with higher concentration. The LC50 range of the sample was found at 250 µg/ml concentration at 52.2%, whereas the viability of the cell declined to 28.19% at 350µg/ml concentration. The morphological features like shrinkage, detachment, membrane blebbing, and distorted shape were observed on safflower treated A549 cells that supports the antiproliferative activity. The ethidium bromide and acridine orange staining substantiates that safflower extract induces apoptosis wherein the untreated A549 cells showed green fluorescence with intact nuclear morphology and cells treated with safflower extract (200 µg and 250 µg/ml), showed apoptotic bodies, clearly validating the apoptosis inducing effect by safflower on A549 cells. Also, the safflower extract protects the mitochondria by decreasing the oxidative stress and by altering the mitochondrial membrane potential. Conclusion: Thus, it is found that safflower extract exerts its antiproliferative activity by inducing apoptosis and protects mitochondria by combating oxidative stress. Keywords: Safflower, Oxidative stress, Cytotoxicity, Mitochondria, Apoptosis.

Cimigenoside Affects Cell Viability, Apoptosis and Metastasis Of A549 Cells Via The NF-κB Pathway

Cimigenoside, one of the main elements isolated from the aboveground plants of the Cimicifuga genus, has anti-tumour effects. However, the question of whether Cimigenoside had effects on lung cancer remains elusive. This study was aimed at investigating the effects of Cimigenoside on A549 lung cancer cells. The A549 cells were cultured in vitro, and an MTT assay was carried out to explore the cell proliferative abilities after a treatment with Cimigenoside. The wound healing assays and Transwell invasion assays were carried out to explore the effects of Cimigenoside on cell migration and invasion in the A549 cells. A cell apoptosis assay using flow cytometry was used to evaluate the effects on cell apoptosis in the A549 cells. The related protein expression levels were measured using a Western blot assay. It was hypothesised that Cimigenoside could restrain tumour cell proliferation, migration and invasion, while inducing cell apoptosis with apoptosis-related protein change levels in a time- and dose-dependent manner. The Western blot assay revealed that the Cimigenoside treated A549 cells had a reduced expression of p65, while having an increased expression of IκBα at the protein level. Taken together, this study demonstrated that Cimigenoside has the potential to repress cell proliferation, migration and invasion, while inducing apoptosis in A549 lung cancer cells through the NF-κB pathway. These results reveal that Cimigenoside may have a therapeutic potential for lung cancer treatment.

Abstract 1096: Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy

Abstract Aggressive lung cancers such as non-small cell lung carcinomas (NSCLC) commonly recur following first line therapy. Recurring tumors metastasize more rapidly. We hypothesize that drug-resistant phenotypes created by the selection pressure of first line treatments such as platinum-based cytotoxic agents contribute to the increased aggressivity of recurring NSCLC. We showed previously that an engineered model of cisplatin-resistant NSCLC tissues secretes more inflammatory cytokines compared to untreated controls and induces more severe muscle tissue injury in a microphysiological model of cachexia processes. Here, we report methods for the systematic generation of cisplatin-resistant A549 lung adenocarcinoma cell cultures and demonstrate their increased aggressivity in 3D spheroid culture and in 3D organotypic culture with patient-derived lung cancer-associated fibroblasts (CAF). We treated A549 cells with cisplatin for 96 hours at doses of 15-45µM, yielding cultures of 54.8-8.9% surviving cells as quantified via flow cytometric of cell size and permeability. Here we report characterization of spheroids and tissues composed of cells selected for resistance with 25 µM cisplatin (Cis-R) compared with untreated parent A549. This dose was selected because we were unable to recover proliferating cultures with lower percentages of surviving. Cis-R spheroids exhibited a significant reduction in circularity that is indicative of increased asymmetry. We measured a Ki67 index of 6.4 +/- 1.4% for parent A549 spheroids and 49.7 +/- 9.5% for Cis-R spheroids. The marked increase in cell proliferation of CisR spheroids correlated with increased distances of invasion and more mesenchymal-like cell morphologies of invading cells in 3D cultures of Cis-R spheroids without CAF. These data suggest that cisplatin resistant A549 created using our protocol are more proliferative and invasive in 3D culture environments due to cell intrinsic changes. Organotypic 3D cultures of Cis-R spheroids and CAF exhibited an increased density and alignment of myofibroblast-like CAF with smooth muscle actin cytoskeleton when compared to cultures of parent spheroids with the same CAF. Our results demonstrate proof of principle for modeling recurrent tumors with more aggressive phenotypes using this approach. Future experiments will translate these methods to patient-derived cultures and test the hypothesis that cisplatin-resistant recurrent NSCLC stimulates more severe local and distant stromal reactions as a driver of metastasis. Engineering validated models of drug-resistant recurring cancers will help accelerate the discovery of anti-metastasis therapies in human cell-based microphysiological systems. Citation Format: Omar M. Ahmad, Elisabet A. Olsen, Delia A. Carlino, Charles E. Byrne, Mark Mondrinos. Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1096.

Investigation of the Pharmacological Effect and Mechanism of Jinbei Oral Liquid in the Treatment of Idiopathic Pulmonary Fibrosis Using Network Pharmacology and Experimental Validation.

Overview: Idiopathic pulmonary fibrosis (IPF) is a disease caused by many factors, eventually resulting in lung function failure. Jinbei oral liquid (JBOL) is a traditional Chinese clinical medicine used to treat pulmonary diseases. However, the pharmacological effects and mechanism of the action of JBOL on IPF remain unclear. This study investigated the protective effects and mechanism of the action of JBOL on IPF using network pharmacology analysis, followed by in vivo and in vitro experimental validation. Methods: The components of JBOL and their targets were screened using the TCMSP database. IPF-associated genes were obtained using DisGeNET and Drugbank. The common targets of JBOL and IPF were identified with the STRING database, and a protein–protein interaction (PPI) network was constructed. GO and KEGG analyses were performed. Sprague–Dawley rats were injected with bleomycin (BLM) to establish an IPF model and treated orally with JBOL at doses of 5.4, 10.8, and 21.6 ml/kg. A dose of 54 mg/kg of pirfenidone was used as a control. All rats were treated for 28 successive days. Dynamic pulmonary compliance (Cdyn), minute ventilation volume (MVV), vital capacity (VC), and lung resistance (LR) were used to evaluate the efficacy of JBOL. TGF-β–treated A549 cells were exposed to JBOL, and epithelial-to-mesenchymal transition (EMT) changes were assessed. Western blots were performed. Results: Two hundred seventy-eight compounds and 374 targets were screened, and 103 targets related to IPF were identified. Core targets, including MAPK1 (ERK2), MAPK14 (p38), JUN, IL-6, AKT, and others, were identified by constructing a PPI network. Several pathways were involved, including the MAPK pathway. Experimentally, JBOL increased the levels of the pulmonary function indices (Cdyn, MVV, and VC) in a dose-dependent manner and reduced the RL level in the BLM-treated rats. JBOL increased the epithelial marker E-cadherin and suppressed the mesenchymal marker vimentin expression in the TGF-β–treated A549 cells. The suppression of ERK1/2, JNK, and p38 phosphorylation by JBOL was validated. Conclusion: JBOL had therapeutic effects against IPF by regulating pulmonary function and EMT through a systemic network mechanism, thus supporting the need for future clinical trials of JBOL.

Abstract 208: Peposertib-induced senescence primes irradiated p53 wild-type cancer cells for clearance by both immune cells and senolytic drugs

Abstract Peposertib (previously known as M3814) is a potent and selective DNA-PK inhibitor in early-stage clinical development. It effectively blocks the non-homologous end-joining pathway for repair of DNA double-strand breaks (DSB) and has shown strong synergy with ionizing radiation (IR) and DSB-inducing chemotherapy. We recently reported that M3814 overactivates the ATM/p53 signaling axis, reinforces cell cycle checkpoint arrest, and promotes durable premature senescence in irradiated p53 wild-type cancer cells. This unique property of peposertib permits to turn the bulk of irradiated cancer cells into nearly uniform senescent cell population. Using this experimental model, we examined the immunological effects and potential therapeutic applications of IR+peposertib induced senescence. Gene expression analyses of IR+peposertib treated A549 cells showed activation of multiple immune-related genes in the senescent cells, consistent with a strong STING-driven inflammatory response. Multiple secreted cytokines were found substantially elevated in culture media compared to IR controls. Co-culture of IR+peposertib induced senescent cells with PBMCs from healthy volunteers coupled with FACS analyses and live imaging revealed stimulation of the T-cell compartment and killing of senescent but not control cells. Further co-culture experiments with isolated NK cells showed accelerated killing of the senescent IR+peposertib treated A549 cells as compared to controls. These results indicated that IR+peposertib induced inflammatory signaling in senescent A549 cells can stimulate an immune response in vitro, resulting in the effective elimination of both treated and naive A549 cells. We also explored a pharmacological "two-hit" approach. Viability assays and time-lapse imaging showed that the senescent, IR+peposertib treated A549 cells were sensitive to several reported senolytic agents. Of those, the BCL-XL inhibitor A-1331852 was most effective at killing peposertib-promoted senescent A549 cells. Our experiments revealed that selective inhibition of DNA-PK in irradiated p53 wild-type cancer cells provides a powerful mechanism for induction of senescent cancer cells with an immunostimulatory secretory phenotype in vitro. In addition, these cells can be selectively targeted by senolytic agents, suggesting that triple combination radiotherapy with DNA-PK inhibitors and senolytics could offer a potential new approach to the treatment of locally advanced tumors. Citation Format: Michael Carr, Qing Sun, Chien-Fei Lee, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. Peposertib-induced senescence primes irradiated p53 wild-type cancer cells for clearance by both immune cells and senolytic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 208.

Chemopreventive role of Tin oxide-Chitosan-Polyethylene glycol-Crocin nanocomposites against Lung cancer: An in vitro and in vivo approach

BackgroundLung cancer (LC) is a foremost cause of cancer-associated mortalities worldwide and it causes 17 % of the total cancer incidences and 20 % of the cancer-associated mortalities annually. ObjectiveIn this exploration, we intended to formulate the Tin oxide-Chitosan-Polyethylene glycol-Crocin nanocomposites (SCP-Cr-NCs) and examine its anticancer role against the LC in both in vitro and in vivo models. MethodologyThe formulated SCP-Cr-NCs were characterized using several methods. The LC was initiated to the BALB/c mice via injecting the 2 × 106 of A549 cells into the posterior flank and treated with the 10 and 20 µM of formulated SCP-Cr-NCs for 19 days. The tumor weight and xenobiotic dysfunction markers status was examined by standard methods. The status of pro-inflammatory markers was studied using ELISA kits. The lung histology was examined microscopically. The viability of SCP-Cr-NCs treated A549 cells were investigated. The ROS and MMP status and apoptosis in the SCP-Cr-NCs treated A549 cells were inspected by fluorescence staining techniques. ResultsThe tumor weight and the status of xenobiotic dysfunction markers were remarkably reduced by the SCP-Cr-NCs. The SCP-Cr-NCs supplementation also diminished the IL-1β, TNF-α, IL-6, and CEA. The viability of SCP-Cr-NCs administered A549 cells were reduced drastically. The outcomes of fluorescence assay was demonstrated that the SCP-Cr-NCs were diminished the MMP and elevated the ROS status and apoptosis in the A549 cells. SCP-Cr-NCs also restrained the cell adhesion of A549 cells. ConclusionCollectively, our findings provided the evidence that the formulated SCP-Cr-NCs demonstrated the anticancer potential and it could be a talented chemopreventive candidate to treat the LC.

Rosmarinic Acid Ameliorates Pulmonary Ischemia/Reperfusion Injury by Activating the PI3K/Akt Signaling Pathway.

Pulmonary ischemia/reperfusion (IR) injury is the leading cause of acute lung injury, which is mainly attributed to reactive oxygen species (ROS) induced cell injuries and apoptosis. Since rosmarinic acid (RA) has been identified as an antioxidant natural ester, this natural compound might protect against pulmonary IR injury. In this study, the mice were given RA daily (50, 75, or 100 mg/kg) by gavage for 7 days before the pulmonary IR injury. We found that hypoxemia, pulmonary edema, and serum inflammation cytokines were aggravated in pulmonary IR injury. RA pretreatment (75 and 100 mg/kg) effectively reversed these parameters, while 50 mg/kg RA pretreatment was less pronounced. Our data also indicated RA pretreatment mitigated the upregulation of pro-oxidant NADPH oxidases (NOX2 and NOX4) and the downregulation of anti-oxidant superoxide dismutases (SOD1 and SOD2) upon IR injury. In vitro studies showed RA preserved the viability of anoxia/reoxygenation (AR)-treated A549 cells (a human lung epithelial cell line), and the results showed the protective effect of RA started at 5 μM concentration, reached its maximum at 15 μM, and gradually decreased at 20–25 μM. Besides, RA pretreatment (15 μM) greatly reduced the lactate dehydrogenase release levels subjected to AR treatment. Moreover, the results of our research revealed that RA eliminated ROS production and reduced alveolar epithelial cell apoptosis through activating the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway, which was supported by using wortmannin, because in the presence of wortmannin, the RA-mediated protection was blocked. Meanwhile, wortmannin also reversed the protective effects of RA in mice. Together, our results demonstrate the beneficial role of RA in pulmonary IR injury via PI3K/Akt-mediated anti-oxidation and anti-apoptosis, which could be a promising therapeutic intervention for pulmonary IR injury.

Biofabrication of silver nanoparticles using Praecitrullus fistulosus fruit extract exhibits in vitro antibacterial and anticancer activity

The silver nanoparticles were synthesized using green mediated approach aided by the fruit of extract of Praecitrullus fistulosus to evaluate its biological properties. The synthesized silver nanoparticle (hereafter named as Pf@AgNPs) was characterized using UV–visible spectroscopy, FTIR, DLS, Zeta potential followed by TEM. The average size of the synthesized nanoparticles was found to be 24 nm. In this work, spherical morphology of the nanoparticles was identified by TEM with a range between 18 and 30 nm. The zeta potential of AgNPs was found to be −5.2 eV. The antibacterial activity of Pf@AgNPs was evaluated against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Shigella flexineri and Pseudomonas aeruginosa using disc diffusion and minimum inhibitory concentration assay with proven activities. The dye degradation property of Pf@AgNPs was tested and effectively acts on Rhodamine compared to that of methylene blue and trypan blue dyes. Further, in vitro cytotoxic potential of Pf@AgNP's was tested against various human cancer cell lines using an MTT assay. Among tested cell lines, an A549 cells was found to be more sensitive and IC50 was found to be 23.45 ± 1.27 μg/mL. Further evaluation using clonogenic assay, scratch assay, DNA fragmentation, microscopy and western blot analysis supports the cytotoxic potential of Pf@AgNP's. Results showed that fragmented DNA and altered expression of Bax/Bcl2 protein ratio clearly indicated the activation of apoptosis in treated A549 cells. Further, angiosuppressive potential of Pf@AgNP's using CAM assay using in ovo model showed that significant decrease in the microvessel density indicating the angiosuppressive activity of Pf@AgNP's.

Atorvastatin suppressed proliferation and facilitated apoptosis of A549 cells through mediating recruitment of Fas and CD59 in lipid raft

Purpose: Lipid raft facilitated progression of NSCLC and atorvastatin could break cholesterol. The purpose of this study was to determine the potential mechanism of atorvastatin through lipid raft mediation in NSCLC.Methods: A549 cells were first grouped as NC, methyl-β-cyclodextrin (MβCD, lipid raft inhibitor, 5mM and 10mM), atorvastatin (0, 5mM, 10mM and 15mM) and MβCD (10mM) with atorvastatin (15mM). Later 10mM MβCD treated A549 cells were divided into three groups: NC, 3BDO (mTOR agonist) (60μM) and 3BDO (60μM) with atorvastatin (15mM) group. Thereafter, FLOT-2, SLP-2 expressions were assessed with RT-qPCR，mTOR proteins were measured by western blot and cell viability by CCK-8 method. Meanwhile, apoptosis was analyzed by flow cytometry. Moreover, lipid raft isolation was performed for acquiring Fas and CD59 and concentrations were detected by ELISA.Results: MβCD treatment significantly inhibited FLOT-2 and SLP-2 RNA expressions and cell viability of A549 cells but up-regulated apoptosis. Besides that, Fas protein level was promoted and CD59 was suppressed. Atorvastatin also repressed FLOT-2 and SLP-2 RNA levels. Meanwhile, atorvastatin downregulated cell viability and accelerated apoptosis. Moreover, Fas was increased and CD59 was inhibited by atorvastatin and MβCD enhanced functions of atorvastatin. MβCD inhibited mTOR RNA expression in A549 cells was increased by 3BDO but atorvastatin restored 3BDO caused up-regulation of mTOR. Furthermore, up regulated cell viability of A549 by 3BDO was declined with atorvastatin and decreased apoptosis by 3BDO was reversed through atorvastatin. Fas suppressed by 3BDO and promotion of CD59 were also resumed by atorvastatin.Conclusion: Atorvastatin suppressed FLOT-2 and SLP-2 and mediated recruitments of Fas and CD59 with suppressing cell viability and facilitating apoptosis of A549 cells via mTOR signaling pathway.

The cytotoxic effects of sertraline on ovarian (A2780) cancer cells in vitro

A number of studies have shown that antidepressants can affect cancer cells viability, however, there are few studies focusing on anticancer effects of sertraline on ovarian cancer cells. Therefore, the aim of this study was to investigate the effects of sertraline on cell viability in ovarian (A2780) cancer cells. A2780 cells were divided into control group and groups treated with 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 μg/ml of sertraline. 24 and 48 hours after treatment, cell viability was assessed by MTT assay method. Data were analyzed using one-way analysis of variance. Treatment with 6.25, 12.5, 25, 50 and 100 μg/ml of sertraline led to significant decrease in cell viability compared to control group 24 and 48 hours after treatment. Although treatment of A2780 cells with 3.125 μg/ml of sertraline did not significantly change the cell viability 24h after treatment, however, treatment of A2780 cells with 3.125 μg/ml of sertraline for 48h significantly decreased the cell viability compared to control group. IC50 value was 11.6 and 5.8 μg/ml for sertraline 24 and 48 hours after treatment, respectively, showing a significant decrease in IC50 (48h) compared to IC50 (24h). The results of this study revealed that sertraline has cytotoxic effects on ovarian cancer cells in vitro. Increased treatment duration led to enhanced cytotoxicity of sertraline on ovarian cancer cells.

Vesicle transport related protein Synaptotagmin-1 mediates paraquat transport to antagonize paraquat toxicity

In this study, A549/PQ cells with moderate resistance to paraquat (PQ) were obtained by treating A549 cells with PQ, their growth rate was slowed down, the accumulation concentration of PQ and the levels of growth inhibition, injury and early apoptosis induced by PQ were significantly lower than those of parental A549 cells. Microarray screening and RT-qPCR detection found that Synaptotagmin-1 (SYT1) expression in drug-resistant cells was significantly increased, and PQ further enhanced its expression. After inhibiting SYT1 expression in A549/PQ cells, cell viability, intracellular PQ concentration and the expression of Bcl-2, SNAP25 and RAB26 were significantly reduced, while the mortality, early apoptosis rate and Bax expression were significantly increased. In vivo experiments also further showed that PQ promoted the expression of SYT1, SNAP25 and RAB26 in PQ-poisoned mice; when inhibiting SYT1 expression, PQ concentration in lung tissues was significantly increased, and the levels of lung injury and apoptosis were also significantly enhanced, while the expression of SNAP25 and RAB26 was significantly reduced. This indicates that PQ poisoning leads to compensatory up-regulation of vesicle transport related proteins such as SYT1 in vivo, thereby promoting PQ transmembrane transport, and then reducing the pulmonary accumulation of PQ and PQ-caused lung injury.

Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2.

Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-β1 and TGFβ type I receptor (TGFβRI), and protein expressionwas assessed by immunoblotting and immunofluorescence. Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-β1 expression, and silencing of TGF-β1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFβRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

Evaluation of in vivo Lethality and in vitro Cytotoxic Effect of Odontobuthus bidentatus Scorpion Venom.

The results of numerous studies have revealed that some deadly scorpion venoms are composed of various bioactive molecules that have significant cytotoxic effects on cancer cells. In this study, the in vivo lethality and cytotoxic effect of Odontobuthus bidentatus venom were evaluated in different cancer cell lines. Through MTT assay, the cytotoxic effects of O. bidentatus scorpion venom were analyzed on the MCF-7, A549, AGS, HepG2, and Ht-29 cancer cell lines and Hu02 normal cells. To this end, six venom fractions were obtained through a Sephadex G-50 column, and the cytotoxic effects of isolated fractions were evaluated on A549 lung cancer cells. The median lethal dose of O. bidentatus scorpion venom was determined at 0.73 mg/kg by intravenous administration of different venom doses in male BALB/c mice according to the Spearman-Karber method. The O. bidentatus scorpion whole venom had a significant cytotoxic effect on MCF-7, A549, and AGS cells. The treatment of A549 cells with various concentrations of fraction F1 showed that this fraction significantly induced growth inhibitory effect on the cells in a dose-dependent manner, compared to untreated cells.

Cassia auriculata Linn. extracts induce apoptosis and cell cycle arrest of A549 lung cancer cell lines: An in vitro approach

Cassia auriculata is an ethnobotanically important traditional medicinal plant of tropical Asia and recently, phytochemicals and their derivatives have gained scientific attention for their use as agents in cancer treatment and prevention. The aerial parts of Cassia auriculata plant were subjected to successive cold maceration to yield four fractions viz., n-hexane, dichloromethane, ethyl acetate, and ethanol extract. The extracts were fractionated by GC–MS (Gas Chromatography-Mass Spectrometry) and HPLC (High-Performance Liquid Chromatography) analysis for their phytochemical content. The chromatographic techniques revealed the presence of mainly the flavonoid compounds like flavone, chrysin, and quercetin. The anti-proliferative properties of ethanolic extract of C. auriculata on A549 human lung cancer cells have been reported. The cancer cell survival rate (MTT 3- [4, 5- dimethythiazol-2-yl] 2, 5-diphenyl tetrazolium bromide) was found to be reduced with lung cancer cells (IC50 26.24 μg/ml) when treated with C. auriculata ethanol extract (CAEE) when compared to the non-cancerous cells L132. The toxicity of the extract on L132 cells was low (IC50 55.78 μg/ml). The mode of cell death induced by CAEE on A549 cells was examined by various fluorescent staining techniques and characteristic nuclear and morphological changes for apoptosis were evaluated. The expression of caspase 9 activity was documented, which might have triggered cell death through apoptosis by the activation of caspase 9 in the ethanolic fraction treated A549 cells. Cell cycle analysis of treated A549 cells revealed a promising S phase cell cycle arrest. The flavonoid compounds identified in the present study might be responsible for the remarkable anticancer and other activities of the plant. From these findings, the plant C. auriculata can be further exploited to develop promising candidate molecules, particularly for lung cancer chemotherapy.

Chitosan based encapsulation increased the apoptotic efficacy of thymol on A549 cells and exhibited non toxic response in swiss albino mice

Thymol is a plant-derived natural phenolic compound abundantly present in Thymus vulgaris species. In the present study, we developed a chitosan-based drug delivery system to deliver thymol to A549 cells. The physicochemical properties of thymol-loaded chitosan nanoparticles (thymol-NP) were characterized using polyphasic techniques viz., FTIR, XRD, DLS, and SEM. Thymol-NP exhibited a size of 282.5 nm and encapsulation efficiency of 74.08 ± 0.73%. The IC50 of thymol-NP against A549 cells was 99.57 μg/ml at 24 h, which was lower than that of the pure form. Clear apoptotic features such as cellular morphology, cell shrinkage, and augmentation of dead cells were observed in both the thymol and thymol-NP treated A549 cells. The percentage of apoptotic cells in the thymol-NP IC50 treated cells was >90% which was considerably higher than the group treated with thymol alone. In vivo toxicity study showed that the swiss albino mice treated up to a concentration of 1000 mg/kg of thymol-NP neither showed signs of toxicity nor death up to 14 days. Also, no significant influence was observed on behavior, body weight, organ weight, and organ histology. Overall, the data concluded that thymol-NP can be considered a safe and potent drug candidate against A549 cells.