Induced Apoptosis In A549 Cells Research Articles

Synthesis and Biological Evaluation of New 1,3,4-Oxadiazoles as Potential Anticancer Agents and Enzyme Inhibitors.

1,3,4-Oxadiazoles have been known with a wide variety of pharmacological activities including anticancer activity. In this study, novel 2,5-disubstituted 1,3,4-oxadiazole derivatives were synthesized and evaluated for determining their anticancer, anticholinesterase and lipoxygenase (LOX) inhibitory activity. All compounds were tested against C6 rat glioma, A549 human lung carcinoma and NIH/3T3 mouse embryo fibroblast cell lines to define cytotoxic concentrations and apoptosis induction levels which they cause. Many of the compounds exhibited remarkable potency that compounds 2a, 2b, 2e, 2h and 2j against C6 cells and compounds 2a, 2b, 2d, 2g, 2i, 2j against A549 cells were found more active than cisplatin. Compound 2d namely, 2-[(5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl)thio]-1-(4-chlorophenyl)ethan-1-one induced apoptosis of A549 cells with 74.9% whereas cisplatin caused 70.9% percentage. Among them, compounds 2d and 2j against A549 cell line, compounds 2b and 2e against both tumor cell lines showed selective cytotoxicity evaluating the inhibition concentration against NIH/3T3 cell line. None of the compounds showed significant acetylcholinesterase (AChE) and lipoxygenase inhibitory activities.

Diprophylline inhibits non-small cell lung cancer A549 cell proliferation and migration, and promotes apoptosis, by downregulating PI3K signaling pathway.

Diprophylline (DPL) is identified as a methylxanthine (MX) derivative. A number of MX derivatives are reported to have anti-tumor effects. However, it is not clear whether DPL has a therapeutic effect on non-small cell lung cancer (NSCLC). The aim of the present study was to investigate the effects of DPL on NSCLC and to elucidate the potential underlying mechanism. A Cell Counting Kit-8 assay was used to evaluate the potential effect of DPL on A549 cell proliferation. Transwell invasion and migration assays were performed to assess the effect of DPL on A549 cell migration and invasion. Furthermore, the percentage of apoptotic cells was detected by flow cytometric analysis, and proteins associated with apoptosis, including apoptosis regulator Bcl-2, apoptosis regulator BAX and active caspase-3, were examined by western blotting. Finally, the expression levels of molecules relevant to phosphoinositide 3-kinase (PI3K) signaling were detected by western blot analysis. The present study demonstrated that DPL may significantly inhibit A549 cell proliferation, migration and invasion. Furthermore, treatment with DPL may significantly induce A549 cell apoptosis. Finally, the protein expression levels associated with the PI3K signaling pathway were significantly inhibited in A549 cells following treatment with DPL. In conclusion, DPL may inhibit the proliferation and migration of NSCLC by inactivating the PI3K signaling pathway, and DPL is a promising novel therapeutic drug for NSCLC.

LATS2 promotes apoptosis in non-small cell lung cancer A549 cells via triggering Mff-dependent mitochondrial fission and activating the JNK signaling pathway

LATS2 is a classical tumor suppressor that affects non-small cell lung cancer proliferation and mobilization. However, its role in lung cancer cell apoptosis is unknown. The aim of our study is to explore whether LATS2 activates mitochondria-related apoptosis in lung cancer cells. In the present study, A549 non-small cell lung cancer cells were transfected with a LATS2 adenovirus to induce LATS2 overexpression. Cell apoptosis was evaluated via the MTT assay, TUNEL staining, western blotting, trypan blue staining and ELISA. Mitochondrial function was measured using an immunofluorescence assay, western blotting and ELISA. The results demonstrated that LATS2 was downregulated in A549 lung cancer cells. Overexpression of LATS2 induced A549 cell apoptosis via activating mitochondrial fission. Subsequently, we confirmed that LATS2 modulated mitochondrial fission via the JNK-Mff signaling pathway. Inhibition of the JNK pathway and/or knockdown of Mff abolished the pro-apoptotic effect of LATS2 on A549 cells. Taken together, our results identified LATS2 as a classical tumor suppressor of lung cancer via triggering mitochondrial fission and activating the JNK-Mff signaling pathway. Our results lay the foundation for detailed study of the molecular mechanisms of LATS2 overexpression and regulation of mitochondrial fission for lung cancer treatment.

Study on effects of total saponins from Lilii Bulbus on proliferation, apoptosis, invasion and metastasis of lung cancer cells and its preliminary mechanism

The potential role of total saponins extracted from Lilium lancifolium bulbs (TSLL) on the proliferation, apoptosis, migration and invasion of human lung cancer A549 cells and its possible mechanism were discussed. Effect of TSLL on proliferation of A549 cells were detected by CCK-8, clone formation assay and EdU staining. Effect of TSLL on apoptosis morphology of A549 cells was observed by fluorescence microscope using Annexin V/PI double staining and Hoechst 33342 staining. Effect of TSLL on cell migration and invasion was detected by Transwell migration test and Transwell invasion test, respectively. Western blot was used to detect TSLL on the expression change of intracellular associated proteins. Results showed that TSLL intervention in A549 cells within 24, 48 or 72 h significantly inhibited cell growth, and its IC₅₀values were about 229, 173 and 71 mg·L⁻¹, respectively. TSLL significantly reduced the clone formation rate of A549 cells and decreased the DNA synthesis rate of A549 cells in a concentration dependent manner. TSLL induced A549 cells apoptosis and reduced the migratory behavior of A549 cells. TSLL decreased invasion of A549 cells to the artificial basement membrane. The expression level of intracellular PCNA and the ratio of Bcl-2/Bax protein were down-regulated and procaspase 3 was activated. In addition, TSLL had no obvious effect on epithelial mesenchymal transition (EMT) related marker proteins E-cadherin and vimentin expression. The above results indicated that TSLL possess inhibitory effects against proliferation, migration and invasion of lung cancer A549 cells and apoptosis-induced effect. The anti-proliferation effect of TSLL is very likely by inhibiting intracellular DNA synthesis through reducing the expression of PCNA in lung cancer cells. And the apoptosis induction of TSLL on lung cancer cells is associated with the regulation of Bcl-2 and Bax proteins expression. Nevertheless, there is no incontestable correlation between anti-invasion and metastasis effects of TSLL and EMT in lung cancer cells.

Butein induces lung adenocarcinoma A549 cell apoptosis via activating forkhead box O 3a/p27 kinase inhibitor protein 1 and cell oxidative stress

Objective To investigate the role of Butein on lung adenocarcinoma A549 cells and the role of forkhead box O 3a (FOXO3a)/p27 kinase inhibitor protein 1 (p27kip1) and cell oxidative stress in this process. Methods A549 cells were treaded with 25, 50 and 100 μmol/L Butein, then the cell viability, cell migration, Caspase-3 activity, NADPH oxidase activity, Reactive oxygen species (ROS) and total glutathione (GSH) were detected. Further expressions of FOXO3a, p27kip1, B-cell lymphoma-2 (bcl-2) and bcl-2 associated X protein (bax) were detected by Western blotting. In addition, small interference RNA was used to knock down FOXO3a expression in A549 cell. Then the cells were divided into four groups, which were the control group, the FOXO3a siRNA group, the Butein 100 μmol/L group and FOXO3a siRNA + Butein 100 μmol/L group. Expression of FOXO3a, p27kip1, bcl-2 and bax were detected. Results After treatment of Butein (25, 50, 100 μmol/L), cell viability were reduced from 100% to (78.3±4.6)%, (63.5±4.8)% and (40.1±4.1)% (t=10.330, P=0.000) and the distance between cell boundary were increased from 100% to (120.0±8.4)%, (150.0±10.6)% and (175.0±11.1)% (t=4.777, P=0.001). In addition, Butein treatment increased Caspase-3 activity, NADPH oxidase activity and ROS production, and reduced total GSH concentration. Furthermore, Butein treatment increased FOXO3a, p27kip1 and bax expression and reduced bcl-2 expression. FOXO3a siRNA treatment blocked FOXO3a expression and p27kip1 transcription, and reversed Butein induced A549 cell apoptosis by increasing bcl-2 and reducing bax expression. Conclusion Butein can induce A549 cell apoptosis via activating FOXO3a/p27kip1 and cell oxidative stress. Key words: Butein; Lung adenocarcinoma; Forkhead box O 3a; P27 kinase inhibitor protein 1; Oxidative Stress; Cell apoptosis

Synthesis and biological evaluation of 2-(3-aminophenyl)-benzothiazoles as antiproliferative and apoptosis-inducing agents

A series of new 2-(3-aminophenyl)-benzothiazole derivatives were synthesized and evaluated for their in vitro antiproliferative activity against various human cancer cell lines including A549, HeLa, HepG2, MCF-7, MV4-11, and DB. Among the tested compounds, N-[3-(benzo[d]thiazol-2-yl)phenyl]nicotinamide displayed significantly improved antiproliferative activity toward A549 and MV4-11 cells with IC50 values of 5.42 ± 1.33 and 7.51 ± 0.98 μM, respectively, much stronger than the hit 3-(benzo[d]thiazol-2-yl)-N-(4-bromobenzyl)aniline. Furthermore, flow cytometric analysis indicated that N-[3-(benzo[d]thiazol-2-yl)phenyl]nicotinamide induced A549 cell apoptosis with cell cycle arrest at G1 phase in a concentration-dependent manner.

Nanoscale Melittin@Zeolitic Imidazolate Frameworks for Enhanced Anticancer Activity and Mechanism Analysis.

The cytolytic peptide melittin (MLT) is an important candidate of anticancer drug owing to its hemolytic properties. Nevertheless, its clinical applications are severely restricted as a result of its nonspecific toxicities like hemolysis. In this work, we reported MLT-loaded zeolitic imidazolate framework-8 (MLT@ZIF-8) nanoparticles (NPs). The formed MLT@ZIF-8 NPs not only possess excellent stability but also efficiently inhibit the hemolysis bioactivity of MLT. Confocal scanning imaging and cytotoxicity experiments revealed that as-synthesized MLT@ZIF-8 NPs exhibit enhanced cellular uptake and cytotoxicity toward cancer cells compared to MLT. The mechanism is well investigated by a series of transcriptome analysis, which indicates that MLT@ZIF-8 NPs can regulate the expression of 3383 genes, and the PI3K/Akt-regulated p53 pathway is involved in MLT@ZIF-8 NPs induced A549 cells apoptosis. Finally, MLT@ZIF-8 NPs exhibit enhanced antitumor activity than free MLT in vivo, while no obvious systemic toxicity has been found. This work emphasizes the great potential of utilizing MOF as a simple and efficient nanoplatform for deliverying cytolytic peptides in cancer treatment, and also the investigation on the antitumor mechanism could provide theoretical support for clinical usage of MLT.

Synthesis and biological evaluation of podophyllotoxin derivatives as selective antitumor agents

To improve podophyllotoxin's cytotoxicity and selective effect, twenty-two podophyllotoxin derivatives had been designed and synthesized. The cytotoxicity of these compounds was evaluated on A549, MCF-7, HepG2 and L-02 cell lines. As a result, most of the compounds were more potent than the positive drugs Etoposide (VP-16) and Doxorubicin which were widely used in clinical for antitumor. There were no magnitude differences about these de-protected (without Boc group) podophyllotoxin amino acid derivatives' cytotoxicity between three tumor cell lines and normal hepatic L-02 cells. Interestingly, some protected (with Boc group) amino acid derivatives and some ligustrazine derivatives showed high selectivity, especially the compound 2 (sarcosine derivative with Boc group). It exhibited highly selectivity both on the cancer cells and the normal cells. The IC50 of compound 2 was 9.5 ± 0.03 nM, 132.6 ± 24.1 nM, 96.4 ± 1.3 nM and 160.2 ± 4.7 nM against A549, MCF-7, HepG2 and L-02 cells, respectively. The SI (IC50L−02/IC50A549) value of compound 2, Doxorubicin and Etoposide was 16.9, 0.2 and 0.5, respectively. Meanwhile, SI (IC50MCF−7/IC50A549) value and SI (IC50HepG2/IC50A549) value of compound 2 were 14.0 and 10.1, respectively. In summary, compound 2 showed high selectivity especially on A549 cells. Further research on cell apoptosis indicated that compound 2 could induce apoptosis of A549 cells through nuclei fragmentation and had lower toxicity to normal hepatic L-02 cells. The detection of apoptosis and cell cycle analysis indicated that compound 2 induced A549 cells apoptosis and prevented A549 cells transition from S to G2 phase while there were no obvious changes on L-02 cells. Moreover, the structure-activity relationships of these derivatives were briefly discussed.

Design, synthesis and biological evaluation of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)benzoyl hydrazide derivatives as thymidylate synthase (TS) inhibitors and as potential antitumor drugs

The Inhibition of cellular nucleotide metabolism to promote apoptosis is a key principle of cancer therapy. Thymidylate synthase (TS) is a key rate-limiting enzyme in the initiation of DNA synthesis in cell. Here, we presented two types of thymidylate synthase inhibitors, and, the key pharmacological properties of these two types of thymidylate synthase inhibitor were extracted and combined to design new compounds with inhibitory activity. Therefore, two series of 42 new compounds with the common biological effect of promoting apoptosis are designed and synthesized by combination principle. Most of the compounds had good anti-proliferative activity on A549, OVCAR-3, SGC7901 and MDA-MB-231 cells. The IC50 of compound 10l on A549 cells was 1.26 μM, which was better than that of pemetrexed (PTX, IC50 = 3.31 μM), furthermore, the selection index of compound 10l was higher than PTX. Flow cytometry analysis showed that compound 10l (the apoptosis rate is 39.4%) could induce A549 cell apoptosis and effectively inhibit tumor cell proliferation. Further western blot analysis showed that compound 10l could induce intrinsic apoptosis by activating caspase-3, increasing expression of cleaved caspase-3 and reducing the ratio of bcl-2/bax. All of this makes compound 10l to be a promising compound in future animal tumor models.

Paclitaxel controlled delivery using a pH-responsive functional-AuNP/block-copolymer vesicular nanocarrier composite system

Paclitaxel (PTX)-loaded gold nanoparticles functionalized with mercaptooctanoic acid (MOA) and folic acid (FA) (AuMOA-FA) were encapsulated within pH-sensitive poly(2-vinylpyridine)-b-poly(ethylene oxide) (P2VP-PEO) vesicles with the aim to develop a more selective injectable nano-formulation for PTX, lacking the side effects of the conventional PTX delivery system. The size of the resulting composite vesicles was lower than 200 nm, i.e. it is suitable for tumor targeting applications taking advantage of the enhanced permeability and retention (EPR) effect. The vesicles did not aggregate in the presence of high electrolyte concentrations, indicating the colloidal stability of the vesicles. The vesicles did not leak their AuMOA-FA or PTX content at physiological pH of 7.4. However, AuMOA-FA and PTX release were significantly accelerated at acidic pHs resembling tumor environment and acidic intracellular compartments. PTX release from the vesicles at acidic pH apparently follows AuMOA-FA release from the vesicles. Flow cytometry measurements and confocal laser scanning microscopy images showed that the vesicles could enter A549 cancer cells in culture and that cellular uptake increased with time. Blank vesicles did not exhibit cytotoxicity and did not induce apoptosis in A549 cancer cells. The PTX currying vesicles exhibited comparable or a little higher cytotoxicity than free PTX. Both the PTX currying vesicles and free PTX induced A549 cells apoptosis, however the vesicle-encapsulated PTX induced a higher percentage of late apoptotic cells than free PTX.

The inhibitory effect of gypenoside stereoisomers, gypenoside L and gypenoside LI, isolated from Gynostemma pentaphyllum on the growth of human lung cancer A549 cells

Ethnopharmacological relevanceGypenosides are major constituents in Gynostemma pentaphyllum (Thunb.) Makino. Previous studies have shown that gypenosides isolated from G. pentaphyllum possess inhibitory effect on the growth of cancer cells, especially A549 cells, with structure-activity relationship (SAR). However, the underlying mechanism of gypenoside-induced A549 cell death remains to be clarified. Aim of the studyTo further investigate SAR and the underlying mechanism of gypenosides in A549 cells. Materials and methodsGypenosides were isolated from G. pentaphyllum using chromatography methods and identified using MS and NMR data. The cytotoxicity was determined with CCK-8 assay. The effects of gypenosides on apoptosis, cell cycle and migration were investigated through cell morphology observation, flow cytometry analysis and key proteins detection. ResultsThree gypenosides, 2α,3β,12β,20(S)-tetrahydroxydammar-24-ene-3-O-β-D-glucopyranoside-20-O-β-D-glucopyranoside, gypenoside L and gypenoside LI were isolated from G. pentaphyllum. Gypenoside stereoisomers, gypenoside L (S configuration at C20) and gypenoside LI (R configuration at C20) showed stronger activity against A549 cells. Furthermore, both induced A549 cell apoptosis through intrinsic and extrinsic pathways evidenced by reducing mitochondrial membrane potential (MMP), generating reactive oxygen species (ROS), releasing more cytochrome c and down-regulating procaspase 8. However, gypenoside L blocked A549 cells in G0/G1, while gypenoside LI induced G2/M arrest, which was further verified by different expression of CDK1, CDK2 and CDK4. In addition, both inhibited A549 cell migration, which was evidenced by down-regulation of MMP-2/9 as well as scratch wound assay and transwell assay. ConclusionC20 of gypenoside played an important role in A549 cell cytotoxicity and gypenoside stereoisomers could be used as potential multi-target chemopreventive agents for cancer.

Long non-coding RNA CASC2 improved acute lung injury by regulating miR-144-3p/AQP1 axis to reduce lung epithelial cell apoptosis

Background and objectiveApoptosis of lung epithelial cell is implicated in the pathogenesis of acute lung injury (ALI). To study the protective effect and mechanism of cancer susceptibility candidate 2 (CASC2) on reducing lung epithelial cell apoptosis after LPS inducing acute lung injury in mice.Methods and resultsThe ALI mice model was performed by intratracheally instilling with lipopolysaccharide (LPS). The CASC2 expression detected by quantitative real-time polymerase chain reaction was significantly decreased in LPS-induced A549 cell and ALI mice model. LPS induced A549 cell apoptosis, while transfection with pcDNA-CASC2 reversed the increased cell apoptosis, suggesting overexpression of CASC2 inhibited LPS-induced A549 cell apoptosis. In addition, we found that miR-144-3p expression were opposite to CASC2, while Aquaporin-1 (AQP1) expression was opposite to miR-144-3p in LPS-induced A549 cell and ALI mice model. The RNA immunoprecipitation and RNA pull-down assay demonstrated that CASC2 could function as a miR-144-3p decoy. The luciferase reporter assay revealed that AQP1 was a target of miR-144-3p in A549 cell. And then, further in vitro studied showed that CASC2 controlled AQP1 expression by regulating miR-144-3p, and LPS induced A549 cell apoptosis by regulating CASC2/miR-144-3p/AQP1 axis. At last, after injection with lentivirus-expressing CASC2 or control lentivirus, the mice were intratracheally instilled with LPS. Comparing to the mice injected with pcDNA, the mice injected with pcDNA-CASC2 had a significantly reduced lung wet–dry weight ratio.ConclusionsLong non-coding RNA CASC2 improved acute lung injury by regulating miR-144-3p/AQP1 axis to reduce lung epithelial cell apoptosis.

Hesperidin induces apoptosis and G0/G1 arrest in human non-small cell lung cancer A549 cells.

Lung cancer has high incidence and mortality rates worldwide. In the present study, the mechanisms by which hesperidin decreases the viability and induces the apoptosis of human non-small cell lung cancer(NSCLC) A549 cells were investigated. Initially, MTT and flow cytometric assays were performed to evaluate the effects of hesperidin on the viability and apoptosis of A549 cells and human normal lung epithelial BEAS-2B cells. The results revealed that hesperidin has no negative effects on the human normal lung epithelial BEAS-2B cells and the viability of cells treated with various concentrations of hesperidin was inhibited in a time- and dose-dependent manner compared with the control groups. Subsequently, the expression levels of proteins involved in the mitochondria-associated apoptotic pathway were studied by western blot analysis. Hesperidin was identified to induce A549 cell apoptosis by downregulating the levels of B-cell lymphoma-2(Bcl-2) and Bcl extra large protein and simultaneously upregulating the levels of Bcl-2-associated Xprotein, BH3 interacting-domain death agonist(Bid), tBid, cleaved caspase-9, cleaved caspase-3 and cleaved poly(adenosine diphosphate ribose)polymerase. The effect of hesperidin on the cell cycle was assessed by flow cytometry. Hesperidin was observed to cause G0/G1 arrest of A549 cells by decreasing the expression of cyclinD1 and increasing the expression of p21 and p53. In summary, it was demonstrated that hesperidin induced apoptosis through the mitochondrial apoptotic pathway and induced G0/G1 arrest in human NSCLC A549 cells. Therefore, hesperidin may be developed as a potential therapeutic drug for the treatment of NSCLC.

Oxymatrine induces A549 human non‑small lung cancer cell apoptosis via extrinsic and intrinsic pathways.

Oxymatrine is one of the primary natural compounds extracted from the Sophora flavescens, and has been reported to exhibit numerous pharmacological properties including cancer‑preventive and anti‑cancer effects, however the mechanisms as to how oxymatrine exhibits anti‑proliferative activity in non‑small cell lung carcinoma cells remains uncertain. The present study aimed to explore the mechanism of its anti‑cancer effect, and whether it is due to apoptosis induction and anti‑migration in the A549 lung cancer cell line. Detection of morphological alterations, MTT analysis, Hoechst/propidium iodide dual staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assays verified that oxymatrine induced A549 cell apoptosis. The caspase pan‑inhibitor z‑VAD‑FMK resulted in disappearance of oxymatrine‑elicited nuclei fragmentation via Hoechst 33342 staining. JC‑1 staining demonstrated a decrease in mitochondrial membrane potential which further verified the induction of apoptosis by oxymatrine. The caspase‑3, 8 and 9 activities of oxymatrine‑treated cells were activated, which suggested that extrinsic and intrinsic apoptotic pathways were involved in the anti‑proliferative effects of oxymatrine in A549 cells. Furthermore, the wound healing assay verified the anti‑migratory effects of oxymatrine in A549 cells.

Discovery of Novel Bromophenol Hybrids as Potential Anticancer Agents through the Ros-Mediated Apoptotic Pathway: Design, Synthesis and Biological Evaluation.

A series of bromophenol hybrids with N-containing heterocyclic moieties were designed, and their anticancer activities against a panel of five human cancer cell lines (A549, Bel7402, HepG2, HCT116 and Caco2) using MTT assay in vitro were explored. Among them, thirteen compounds (17a, 17b, 18a, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, and 23b) exhibited significant inhibitory activity against the tested cancer cell lines. The structure-activity relationships (SARs) of bromophenol derivatives were discussed. The promising candidate compound 17a could induce cell cycle arrest at G0/G1 phase and induce apoptosis in A549 cells, as well as caused DNA fragmentations, morphological changes and ROS generation by the mechanism studies. Furthermore, compound 17a suppression of Bcl-2 levels (decrease in the expression of the anti-apoptotic proteins Bcl-2 and down-regulation in the expression levels of Bcl-2) in A549 cells were observed, along with activation caspase-3 and PARP, which indicated that compound 17a induced A549 cells apoptosis in vitro through the ROS-mediated apoptotic pathway. These results might be useful for bromophenol derivatives to be explored and developed as novel anticancer drugs.

Berberine and cinnamaldehyde together prevent lung carcinogenesis.

Starving tumor cells by restricting nutrient sources is a promising strategy for combating cancer. Because both berberine and cinnamaldehyde can activate AMP-activated protein kinase (AMPK, a sensor of cellular energy status), we investigated whether the combination of berberine and cinnamaldehyde could synergistically prevent lung carcinogenesis through tumor cell starvation. Urethane treatment induced lung carcinogenesis in mice, downregulated AMPK and mammalian target of rapamycin (mTOR) while upregulating aquaporin-1 (AQP-1) and nuclear factor kappa B (NF-κB). Together, berberine and cinnamaldehyde reduced mouse susceptibility to urethane-induced lung carcinogenesis, and reversed the urethane-induced AMPK, mTOR, AQP-1, and NF-κB expression patterns. In vitro, berberine and cinnamaldehyde together induced A549 cell apoptosis, prevented cell proliferation, autophagy, and wound healing, upregulated AMPK, and downregulated AQP-1. The effects of the combined treatment were reduced by rapamycin (a mTOR inhibitor) or HgCL2 (an AQP inhibitor), but not Z-VAD-FMK (a caspase inhibitor). The berberine/cinnamaldehyde combination also prevented A549 cell substance permeability and decreased intracellular ATP concentrations. These results suggest the combination of berberine and cinnamaldehyde limited both primary and adaptive nutrient acquisition by lung tumors via AMPK-reduced AQP-1 expression, which ultimately starved the tumor cells.

Targeting ROS-rich cancer cells with thermo/oxidation-responsive polymeric nanoaggregates: synthesis, morphological and in vitro studies

Paclitaxel (PTX)-loaded gold nanoparticles functionalized with mercaptooctanoic acid (MOA) and folic acid (FA) (AuMOA-FA) were encapsulated within pH-sensitive poly(2-vinylpyridine)-b-poly(ethylene oxide) (P2VP-PEO) vesicles with the aim to develop a more selective injectable nano-formulation for PTX, lacking the side effects of the conventional PTX delivery system. The size of the resulting composite vesicles was lower than 200 nm, i.e. it is suitable for tumor targeting applications taking advantage of the enhanced permeability and retention (EPR) effect. The vesicles did not aggregate in the presence of high electrolyte concentrations, indicating the colloidal stability of the vesicles. The vesicles did not leak their AuMOA-FA or PTX content at physiological pH of 7.4. However, AuMOA-FA and PTX release were significantly accelerated at acidic pHs resembling tumor environment and acidic intracellular compartments. PTX release from the vesicles at acidic pH apparently follows AuMOA-FA release from the vesicles. Flow cytometry measurements and confocal laser scanning microscopy images showed that the vesicles could enter A549 cancer cells in culture and that cellular uptake increased with time. Blank vesicles did not exhibit cytotoxicity and did not induce apoptosis in A549 cancer cells. The PTX currying vesicles exhibited comparable or a little higher cytotoxicity than free PTX. Both the PTX currying vesicles and free PTX induced A549 cells apoptosis, however the vesicle-encapsulated PTX induced a higher percentage of late apoptotic cells than free PTX.

Mechanism of Poly(I: C)-induced apoptosis in lung adenocarcinoma A549 cells

Objective To study the effects of Poly(I: C) on lung adenocarcinoma A549 cells viability and illuminate the mechanism of Poly(I: C)-induced apoptosis in A549 cells. Methods A549 cells were transfected with the complex of Poly(I: C) and lipofectamine 3000. The viability of A549 cells was tested by methyl thiazolyl tetrazolium (MTT) method. The apoptotic cells were tested by flow cytometry. The caspase proteins were tested by Western blotting and the expressions of interferon-β (IFN-β) and CXCL-10 were assayed by real-time PCR. After employing the pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, the variation of Poly(I: C) proapoptosis in A549 cells was observed. RNA interfering experiments were employed to knock down melanoma differentiation related antigen 5 (MDA5) or retinoic aci-dinduced gene Ⅰ (RIG-Ⅰ), and the above indexes were tested. Results The viability of A549 cells was significantly reduced to 74.92%±6.24% after 200 ng/ml Poly(I: C) transfection compared with that before transfection (95.32%±3.05%, t=2.883, P=0.041). The apoptotic rates induced by 100, 200, 400 ng/ml Poly(I: C) were 9.97%±0.88%, 23.63%±1.41%, 32.57%±2.39%, respectively. All of them were higher than that in the control group (0.74%±0.15%), with significant differences (t=4.489, P=0.002; t=11.616, P=0.000; t=16.932, P=0.000). Besides, the death receptor pathway proteins such as TNF-related apoptosis inducing ligand (TRAIL), cleaved-caspase-8 and cleaved-caspase-3 increased obviously. MDA5/RIG-Ⅰpathway was also activated dramatically and the expressions of IFN-β, CXCL-10 were significantly up-regulated. The apoptotic rates reduced to 3.17%±0.66%, 5.35%±0.64% with pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK pretreatment, compared with the control group (15.87%±0.93%), and the differences were statistically significant (t=8.643, P=0.001; t=6.824, P=0.002). Moreover, the expressions of TRAIL, IFN-β and CXCL-10 induced by Poly(I: C) were inhibited with MDA5 or RIG-Ⅰ depletion. Conclusion Poly(I: C) can reduce the survival rate of A549 cells and promote the apoptosis mainly by activating the death-receptor pathway mediated by MDA5/RIG-Ⅰprobably, which may involve in IFN-β, CXCL-10. Key words: Lung neoplasms; Apoptosis; Poly(I: C)

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

Design of antitumor agents containing carbohydrate based on GLUT1, and evaluation of antiproliferative activity

A series of novel carbohydrate-modified antitumor compounds were designed based on glucose transporter 1 (GLUT1), and evaluated for their anticancer activities against four cancer cell lines. The ribose derivatives (compound 9 and 10) exhibited modest inhibitory activity. The compound 9 significantly inhibited the migration of A549 cell and induced A549 cell apoptosis in a concentration-dependent manner. Moreover, compound 9 blocked A549 cells at the G0/G1 phase. The cellular uptake studies suggested that ribose-modified compound 9 could be taken through GLUT1 in A549 cell line.