Activity In A549 Cells Research Articles

MiRNA-125b regulates apoptosis of human non-small cell lung cancer via the PI3K/Akt/GSK3β signaling pathway

The present investigation demonstrated that regulation of microRNA (miR)-125b affected the apoptosis of human non-small cell lung cancer (NSCLC) through targeting of the PI3K/Akt and Wnt/β-catenin signaling pathways. The expression of miR-125b was assessed in patients with NSCLC, which demonstrated that miR-125b expression in NSCLC tissue was higher than that in para-carcinoma tissue. Furthermore, survival analysis of patients with NSCLC over 3years indicated that the overall survival (OS) and disease-free survival (DFS) rates of patients with low miR-125b expression were higher than those of patients with high miR-125b expression. Proliferation and apoptosis assays were subsequently conducted in the human NSCLC cell line A549 using MTT assay and AnnexinV-FITC/PI kits, respectively. Caspase-3 activity ELISA and western blot analysis were also used to assess caspase-3 activity and the protein expression of Bax, Akt, phosphorylated(p)-Akt, p-GSK3β, Wnt and β-catenin. It was observed that downregulation of miR-125b inhibited the proliferation and induced the apoptosis of A549 cells. Downregulation of miR-125b also suppressed the protein expression of p-Akt, Wnt and β-catenin, and increased caspase-3 activity and Bax protein expression in A549 cells. In addition, downregulation of miR-125b combined with the PI3K inhibitor LY294002 enhanced cell growth inhibition, suppression of p-GSK3β, Wnt and β-catenin protein expression and promotion of caspase-3 activity in A549 cells. These results revealed that the downregulation of miR-125b regulates apoptosis in human NSCLC through the suppression of the PI3K/Akt/GSK3β and Wnt/β-catenin signaling pathways.

Polyphenolic compounds from Korean Lonicera japonica Thunb. induces apoptosis via AKT and caspase cascade activation in A549 cells.

Lonicera japonica Thunb. (L. japonica T.) has historically been used in Korean herbal medicine due to its anticancer and protective effects on the respiratory system. In the present study, the polyphenolic compounds in L. japonica T. were investigated using high-performance liquid chromatography coupled with tandem mass spectrometry, and its anticancer effects on A549 non-small-cell lung cancer cells were studied. Polyphenolic compounds potentially inhibit A549 cells in a dose-dependent manner. Flow cytometry and western blot analysis demonstrated that polyphenolic compounds induce apoptosis by regulating the protein expression levels of caspases, poly-(ADP-ribose) polymerase and the B-cell lymphoma-2-associated X-protein/B-cell lymphoma-extra large ratio. Furthermore, polyphenolic compounds inhibited mitochondrial membrane potential activity. Caspase-3 activity was increased in a dose-dependent manner and polyphenolic compounds inhibited the activation of protein kinase B by dephosphorylation. These results suggest that polyphenolic compounds in A549 cells indicate the anticancer activity through the induction of apoptosis.

In vitro effects of tanshinone II A on autophagy of A375 melanoma cells and related signaling pathway

Objective To investigate in vitro effects of tanshinone ⅡA on the autophagy of A375 melanoma cells and related signaling pathway. Methods Some cultured A375 cells were divided into 5 groups to be treated with tanshinone ⅡA at concentrations of 0.5, 1, 2 and 4 mg/L, and DMEM containing 0.1% dimethyl sulfoxide (DMSO), respectively, for 24, 48, 72 hours. Methyl thiazol tetrazolium (MTT) assay was performed to estimate the proliferative activity of A375 cells. Some cultured A375 cells were divided into 4 groups to be treated with 1, 2 and 4 mg/L tanshinone ⅡA (1-, 2- and 4-mg/L tanshinone group), and DMEM containing 0.1% DMSO (control group), respectively, for 48 hours. Then, flow cytometry was conducted to count autophagosome-positive cells, and Western blot analysis to determine protein expression of autophagy-associated proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1 (p70S6K1). Results MTT assay showed that 24-, 48-, 72-hour treatments with tanshinone Ⅱ A at concentrations of 0.5, 1, 2 and 4 mg/L all could inhibit the proliferative activity of A375 cells, and the inhibitory effects increased in a dose- and time-dependent manner (F = 2 564.12, 1 235.25, both P < 0.05). The percentage of autophagosome-positive cells and protein expression of Beclin-1 and LC3-Ⅱ increased gradually and significantly in the 1-, 2- and 4-mg/L tanshinone groups (autophagosome-positive cells: 6.91% ± 0.35%, 13.11% ± 0.73%, 25.51% ± 0.83%, respectively; Beclin-1: 0.33 ± 0.01, 0.53 ± 0.04, 0.63 ± 0.02, respectively; LC3-Ⅱ: 0.41 ± 0.01, 0.52 ± 0.02, 0.64 ± 0.02, respectively), after 48-hour treatment, which were significantly different between the tanshinone groups (all P < 0.05), and higher in the tanshinone groups than in the control group (0.41% ± 0.02%; 0.09 ± 0.02; 0.21 ± 0.01, all P < 0.05). However, the protein expression of PI3K, phosphorylated Akt (p-Akt), p-mTOR and p-p70S6K1 in the PI3K-Akt-mTOR-p70S6K1 signaling pathway decreased gradually and significantly with the increase in tanshinone concentrations after 48-hour treatment, and were significantly lower in all the tanshinone groups than in the control group (all P < 0.05). Conclusion Tanshinone Ⅱ A can promote the auophagy of A375 cells, likely by blocking the PI3K-Akt-mtTOR-p70S6K1 signaling pathway. Key words: Melanoma; Tanshinone; Autophagy; A375 cells

Trypsin inhibitors demonstrate antioxidant activities, inhibit A549 cell proliferation, and increase activities of reactive oxygen species scavenging enzymes.

OBJECTIVES:Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostical and pharmacological studies. This study aimed to investigate antineoplastic and antioxidant activity of trypsin inhibitors (TIs) isolated from three plant sources and their inhibitory role in the cell line.MATERIALS AND METHODOLOGY:TIs were obtained from different plant sources. Antineoplastic potential on adenocarcinoma human alveolar basal epithelial cell line (A549) and normal Human Embryonic Kidney (HEK) was determined using MTT assay. Activities of antioxidant enzyme, nitric oxide scavenger, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase were assessed in cell lines incubated with and without TIs. The outcome was analyzed by spectrophotometer.RESULTS:TIs showed the higher cytotoxicity on A549 cells as compared to normal HEK cell line. TIs exhibited fair increase in antioxidant enzyme activity in A549 cells as compared to control. This might be one of the strategies of antineoplastic effect in cancer cells.CONCLUSIONS:This study has reported the antioxidant and antineoplastic properties of these TIs for the first time in A549 cells (to the best of our knowledge). The results show that TIs possess ability to prevent cancer and diseases caused due to oxidative stress. Therefore, we conclude that TIs can be used as supplements along with the conventional drugs for increased efficacy in the treatment of diseases such as cardiovascular disease, atherosclerosis, and cancer.

Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB.

Background LBP and fractalkine are known to be involved in the pathogenesis of ARDS. This study investigated the relationship between LBP and fractalkine in LPS-induced A549 cells and rat lung tissue in an ARDS rat model. Methods A549 cells were transfected with LBP or LBP shRNA plasmid DNA or pretreated with SB203580 or SC-514 following LPS treatment. An ARDS rat model was established using LPS with or without LBPK95A, SB203580, or SC-514 treatment. RT-PCR, western blotting, ELISA, immunofluorescence, coimmunoprecipitation, and immunohistochemical staining were used to study the expression of fractalkine and LBP and p38 MAPK and p65 NF-κB activities. Results LPS increased LBP and reduced fractalkine. LBP overexpression further decreased LPS-induced downregulation of fractalkine and p38 MAPK and p65 NF-κB activation; LBP gene silencing, SB203580, and SC-514 suppressed LPS-induced downregulation of fractalkine and p38 MAPK and p65 NF-κB activation in A549 cells. LBP and fractalkine in lung tissue were increased and decreased, respectively, following LPS injection. LBPK95A, SB203580, and SC-514 ameliorated LPS-induced rat lung injury and suppressed LPS-induced downregulation of fractalkine by decreasing phospho-p38 MAPK and p65 NF-κB. Conclusions The results indicate that LBP downregulates fractalkine expression in LPS-induced A549 cells and in an ARDS rat model through activation of p38 MAPK and NF-κB.

An electrochemiluminescence assay for sensitive detection of methyltransferase activity in different cancer cells by hybridization chain reaction coupled with a G-quadruplex/hemin DNAzyme biosensing strategy.

In this work, a highly sensitive electrochemiluminescence (ECL) assay was fabricated for the detection of human DNA (cytosine-5)-methyltransferase1 (DNMT1) activity in cancer cells. The ECL assay coupled hybridization chain reaction with a G-quadruplex/hemin DNAzyme biosensing strategy. The ECL intensity changes (ΔI) allowed detection of DNMT1 activity down to 0.09 U mL-1, and ΔI was proportional to the logarithm of the activity of DNMT1 within the range of 1.0 to 30.0 U mL-1 in buffer solution. It also showed high sensitivity to DNMT1 activity in A549 cells, with a detection limit of about 2 cells. This ECL assay provides a promising platform for profiling of the mutational cells of tumors and shows a great potential for application to DNA methylation-related clinical diagnostics.

Inhibitory effects of l-theanine on airway inflammation in ovalbumin-induced allergic asthma

l-theanine, a water-soluble amino acid isolated from green tea (Camellia sinensis), has anti-inflammatory activity, antioxidative properties, and hepatoprotective effects. However, the anti-allergic effect of l-theanine and its underlying molecular mechanisms have not been elucidated. In this study, we investigated the protective effects of l-theanine on asthmatic responses, particularly airway inflammation and oxidative stress modulation in an ovalbumin (OVA)-induced murine model of asthma. Treatment with l-theanine dramatically attenuated the extensive trafficking of inflammatory cells into bronchoalveolar lavage fluid (BALF). Histological studies revealed that l-theanine significantly inhibited OVA-induced mucus production and inflammatory cell infiltration in the respiratory tract and blood vessels. l-theanine administration also significantly decreased the production of IgE, monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor-alpha (TNF-α), and interferon-gamma in BALF. The lung weight decreased with l-theanine administration. l-theanine also markedly attenuated the OVA-induced generation of reactive oxygen species and the activation of nuclear factor kappa B (NF-κB) and matrix metalloprotease-9 in BALF. Moreover, l-theanine reduced the TNF-α-induced NF-κB activation in A549 cells. Together, these results suggest that l-theanine alleviates airway inflammation in asthma, which likely occurs via the oxidative stress-responsive NF-κB pathway, highlighting its potential as a useful therapeutic agent for asthma management.

72 - Regulation of Histone Deacetylase 6 (HDAC6) Activity via S-Nitrosylation

Nitric oxide (NO) is a gaseous molecule produced by NO synthase (NOS) and plays essential functions via S-nitrosylation. In the course of screening for the target proteins of NO, we identified histone deacetylase 6 (HDAC6). HDAC6 plays key roles in regulating cell motility and signaling through deacetylation of substrate proteins such as alpha-tubulin. In the present study, we attempted to elucidate the role of NO on HDAC6 activity. Using biotin-switch assay, we found that HDAC6 is S-nitrosylated by both NO donors and NO derive from the inducible type of NOS in A549 cells treated with inflammatory cytokines. Next, we analyzed whether NO influences on enzymatic activity of HDAC6. In vitro assays showed that treatment with S-nitrosocysteine, a physiological NO donor, significantly attenuated deacetylase activity, indicating that NO directly inhibits catalytic activity of HDAC6. Then, we examined whether NO acts on endogenous HDAC6 activity in A549 cells. The level of acetylated alpha-tubulin was significantly elevated upon treatment with cytokines, but this increment was strongly suppressed with L-NAME, a NOS inhibitor. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

Reduced apurinic/apyrimidinic endonuclease activity enhances the antitumor activity of oxymatrine in lung cancer cells.

Lung cancer is the leading cause of cancer-related deaths worldwide and is associated with a very poor outcome. Oxymatrine exerts antitumor effects by inducing apoptosis and inhibiting the proliferation of different cancer cells; however, the anticancer effects and mechanism of action of oxymatrine have not been evaluated sufficiently in human lung cancer cells. Thus, the present study aimed to investigate the anticancer effects of oxymatrine in human lung cancer cells and identify the molecular mechanisms underlying these effects. MTT assays demonstrated that oxymatrine significantly inhibited the proliferation of A549 and H1299 cells in a time- and dose-dependent manner. In addition, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays suggested that oxymatrine treatment may induce lung cancer cell apoptosis in a dose-dependent manner. Furthermore, we detected that oxymatrine induced a significant increase in DNA damage and the expression of PARP and phosphorylated H2AX, and a significant decrease in that of nuclear APE1 and AP endonuclease activity in A549 cells. APE1 knockdown cells (APE1shRNA) plus oxymatrine treatment reduced cells proliferation and induced apoptosis more seriously than control shRNA cells. This appeared to be a consequence of an increase in the number of apurinic/apyrimidinic (AP) sites, DNA damage, PARP and H2AX phosphorylation, which together resulted in the induction of apoptosis. In contrast, the sensitizing effects of APE1 overexpression plus oxymatrine treatment did not occur in APEOE cells. These findings reveal a potential mechanism of action for oxymatrine-induced apoptosis and suggest that oxymatrine is a promising potential therapeutic agent for the treatment of lung cancer.

In vitro inflammatory effects of polyhexamethylene biguanide through NF-κB activation in A549 cells

Polyhexamethylene biguanide (PHMB) is a member of the polymeric guanidine family, which is used as a biocide and preservative in industrial, medicinal, and consumer products. Some studies reported that polyhexamethylene guanidine phosphate, which is also a member of the guanidine family, induced severe inflammation and fibrosis in the lungs. However, limited studies have evaluated the pulmonary toxicity of PHMB associated with inflammatory responses. The aim of this study was to elucidate the inflammatory responses and its mechanisms induced by PHMB in lung cells. A549 cells exposed to PHMB showed decreased viability, reactive oxygen species (ROS) generation, inflammatory cytokine secretion, and nuclear factor kappa B (NF-κB) activation. The cells showed dose-dependent cytotoxicity and slight generation of ROS. PHMB triggered inflammatory cytokine secretion and NF-κB activation by modulating the degradation of IκB-α and the accumulation of nuclear p65. TNF-α plays important roles in IL-8 expression as well as NF-κB activation. Moreover, IL-8 production induced by PHMB was completely suppressed by a NF-κB inhibitor, but not by a ROS scavenger. In conclusion, we suggest that PHMB induces the inflammatory responses via the NF-κB signaling pathway.

APRIL, BCMA and TACI proteins are abnormally expressed in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-associated mortality. Non-small cell lung cancer (NSCLC) accounts for >80% of lung cancers. The overall survival for NSCLC is dismal, with a 5-year survival of <5% for patients. Thus, identifying an effective biomarker for early diagnosis of lung cancer is the first essential step to reduce mortality. It has been recognized that certain inflammatory and immune responses are important in lung cancer development and prevention. The present study demonstrated that, in NSCLC, a proliferation-inducing ligand (APRIL), B-cell maturation antigen (BCMA)and transmembrane activator and CAML interactor (TACI) proteins are abnormally expressed by immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blotting. In addition, the expression of APRIL, BCMA and TACI were observed to be involved in extracellular signal-regulated kinase (ERK)1/2 activation in A549 cells. Overall, the present study provides evidence that APRIL and its receptors, BCMA and TACI, may play roles in the biological processes of NSCLC tumors through the ERK1/2 signaling pathway.

MicroRNA 34a contributes to virus-mediated apoptosis through binding to its target gene Bax in influenza A virus infection

Influenza A virus is a cytolytic virus that induces apoptosis in numerous cell types. However, molecular mechanism of cell apoptosis induced by influenza A virus is not well understood. The present study was aimed to investigate the molecular mechanism of miR-34a involved in influenza A virus induced apoptosis. Firstly, microRNA array was used to select miRNAs involved in influenza A virus infection. Then, Luciferase reporter assay and western blot were carried to determine the target gene of miR-34a. Finally, miR-34a mimic was transfected in influenza A virus-infected A549 cells, and western blot was used to test the function of miR-34a and its target gene in in influenza A virus infection. miR-34a was found significantly down-regulated in influenza A virus-infected A549 cells and was further confirmed by qRT-PCR in vitro and in vivo (P<0.01). Next, transfection miR-34 mimic/inhibitor in influenza A virus-infected A549 cells showed that overexpression of miR-34a could inhibit influenza virus-induced apoptosis. Furthermore, target prediction analysis revealed that miR-34a complemented to the 3'-UTR of Bax mRNA. Luciferase reporter assay and western blot confirm that miR-34a directly targeted Bax. Moreover, we detected influenza virus infection enhanced the luciferase reporter translational activity in A549 cells transfected with the construct with Bax 3'-UTR encoding miR-34a binding site. However, western blot analysis showed that the up-regulation of Bax protein level was significantly reversed by overexpression miR-34a. It is, therefore, concluded that influenza virus infection regulated pro-apoptotic Bax expression through miR-34a downregulation, which might be the partial mechanism of influenza virus-induced cell apoptosis.

Repair activity of oxidatively damaged DNA and telomere length in human lung epithelial cells after exposure to multi-walled carbon nanotubes

One type of carbon nanotubes (CNTs) (MWCNT-7, from Mitsui) has been classified as probably carcinogenic to humans, however insufficient data does not warrant the same classification for other types of CNTs. Experimental data indicate that CNT exposure can result in oxidative stress and DNA damage in cultured cells, whereas these materials appear to induce low or no mutagenicity. Therefore, the present study aimed to investigate whether in vitro exposure of cultured airway epithelial cells (A549) to multi-walled CNTs (MWCNTs) could increase the DNA repair activity of oxidatively damaged DNA and drive the cells toward replicative senescence, assessed by attrition of telomeres. To investigate this, H2O2 and KBrO3 were used to induce DNA damage in the cells and the effect of pre-exposure to MWCNT tested for a change in repair activity inside the cells or in the extract of treated cells. The effect of MWCNT exposure on telomere length was investigated for concentration and time response. We report a significantly increased repair activity in A549 cells exposed to MWCNTs compared to non-exposed cells, suggesting that DNA repair activity may be influenced by exposure to MWCNTs. The telomere length was decreased at times longer than 24h, but this decrease was not concentration dependent. The results suggest that the seemingly low mutagenicity of CNTs in cultured cells may be associated with an increased DNA repair activity and a replicative senescence, which may counteract the manifestation of DNA lesions to mutations.

Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly.

Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells. A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis. The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome β1 subunit and no binding affinity to the β2 and β5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome β1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the β1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity. BF211 inhibits proteasome activity in A549 cells by decreasing β1 subunit expression and disrupting proteasome assembly.

Construction of a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene and its effects on apoptosis and autophagy of A375 melanoma cells

Objective To construct a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene, to investigate its expression in A375 melanoma cells after transfection, and to evaluate its effects on apoptosis and autophagy of A375 cells. Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR (RT-PCR)with total RNA extracted from A375 cells as the template, then inserted into the eukaryotic expression plasmid PCDNA3.1(+)to construct a recombinant plasmid, PCDNA3.1(+)-PKCI-1/HINT1. Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid (PCDNA3.1 (+)-PKCI-1/HINT1 group)or the empty plasmid PCDNA3.1(+)(control group). After additional 48-hour culture, RT-PCR and Western blot analysis were performed to quantify the mRNA and protein expressions of PKCI-1/HINT1 respectively, Hoechst 33342 staining was conducted to detect apoptosis of A375 cells, Western blot analysis to detect the expressions of intracellular caspase-3 and autophagyassociated protein beclin1, and cell autophagy was observed by using the green fluorescent protein (GFP)microtubule-associated protein 1 light chain 3 (LC3)labelling method combined with confocal laser scanning microscopy. Methyl thiazolyl tetrazolium (MTT)assay was performed to evaluate the proliferative activity of A375 cells at 24, 48, 72 and 96 hours after transfection. Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic expression plasmid PCDNA3.1(+)-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells. MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells, and the number of live cells was decreased by 17.0%, 25.6% and 29.4% in the PCDNA3.1(+)-PKCI-1/HINT1 group at 48, 72 and 96 hours, respectively, compared with the control group (all P < 0.05). Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cells. Confocal laser scanning microscopy demonstrated that the overexpression of PKCI-1/HINT1 increased GFP-LC3 puncta formation in A375 cells. In addition, Western blot analysis indicated that PKCI-1/HINT1 upregulated the protein expressions of caspase-3 and beclin1 in A375 cells. Conclusions The eukaryotic expression plasmid PCDNA3.1 (+ )-PKCI-1/HINT1 was successfully constructed, and PKCI-1/HINT1 could be effectively expressed in A375 cells. High-level expression of PKCI-1/HINT1 could suppress cellular proliferation, promote apoptosis, and induce autophagy, of A375 cells. Key words: Melanoma; Cell line, tumor; Apoptosis; Autophagy; PKCI-1/HINT1

Clonal heterogeneity in osteogenic potential of lung cancer-associated fibroblasts: promotional effect of osteogenic progenitor cells on cancer cell migration.

Cancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified. CAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed. The nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential. Our studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.

A novel small molecule, Rosline, inhibits growth and induces caspase-dependent apoptosis in human lung cancer cells A549 through a reactive oxygen species-dependent mechanism.

Chemical screening using synthetic small molecule libraries has provided a huge amount of novel active molecules. It generates lead compound for drug development and brings focus on molecules for mechanistic investigations on many otherwise intangible biological processes. In this study, using non-small cell lung cancer cell A549 to screen against a structurally novel and diverse synthetic small molecule library of 2,400 compounds, we identified a molecule named rosline that has strong anti-proliferation activity on A549 cells with a 50% cell growth inhibitory concentration (IC50 ) of 2.87 ± 0.39 µM. We showed that rosline treatment increased the number of Annexin V-positive staining cell, as well as G2/M arrest in their cell cycle progression. Further, we have demonstrated that rosline induces a decrease of mitochondrial membrane potential (Δφm ) and an increase of caspases 3/7 and 9 activities in A549 cells, although having no effect on the activity of caspase 8. Moreover, we found that rosline could induce the production of reactive oxygen species (ROS) and inhibit the phosphorylation of signaling molecule Akt in A549 cells. Alternatively, an antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated rosline's effects on the mitochondrial membrane potential, caspases 3/7 and 9 activities, cell viabilities and the phosphorylation of Akt. Our results demonstrated that ROS played an important role in the apoptosis of A549 cells induced by rosline.

Apo‐10′‐Lycopenoic acid Inhibits Cancer Cell Migration and Angiogenesis by Suppressing Lamellipodia Formation

Epidemiologic studies have shown that an increase in the dietary intake of tomato and tomato‐based products or higher blood levels of lycopene, is associated with a reduced risk of cancer. Apolycopenoids can be produced by either lycopene cleavage through carotenoid cleavage enzymes, such as beta‐carotene 9′,10′‐oxygenase or the consumption of tomato and tomato products, such as tomato juice. We have previously shown that apo‐10′‐lycopenoic acid, a relative stable derivative of apo‐10′‐lycopenoids, could inhibit tumorigenesis in both lung cancer and liver cancer animal models. To further elucidate the potential mechanisms for the chemopreventive effect of apo‐10′‐lycopenoic acid against cancer development, we examined the ability of apo‐10′‐lycopenoic acid on migration of cancer cells (A549 and HuH7 cells) and endothelial cells (HMEC‐1) using cell migration assays, and angiogenesis using endothelial tube formation and aortic ring assays. We demonstrated that apo‐10′‐lycopenoic acid, in dose‐dependent manner, is effective at inhibiting migration and invasion of both cancer and endothelial cells by suppressing actin remodeling and ruffling/lamellipodia formation. In addition, apo‐10‐lycopenoic acid treatment results in suppression of angiogenesis in both tube formation and aortic ring assays. Apo‐10‐lycopenoic acid also inhibits matrix metalloproteinase‐2 (MMP‐2) expression and activation in A549 cells. Overall, these data demonstrate that apo‐10′‐lycopenoic acid can inhibit cancer cell motility and angiogenesis which could be one of the potential mechanisms for the chemopreventive effect of apo‐10′‐lycopenoic acid against tumorigenesis.Support or Funding InformationSupported by USDA/ARS (58‐1950‐0014)

Up-Regulation of Human Inducible Nitric Oxide Synthase by p300 Transcriptional Complex.

p300, a ubiquitous transcription coactivator, plays an important role in gene activation. Our previous work demonstrated that human inducible nitric oxide synthase (hiNOS) expression can be highly induced with the cytokine mixture (CM) of TNF-α + IL-1β + IFN-γ. In this study, we investigated the functional role of p300 in the regulation of hiNOS gene expression. Our initial data showed that overexpression of p300 significantly increased the basal and cytokine-induced hiNOS promoter activities in A549 cells. Interestingly, p300 activated cytokine-induced hiNOS transcriptional activity was completely abrogated by deleting the upstream hiNOS enhancer at -5 kb to -6 kb in the promoter. Furthermore, p300 over-expression increased cytokine-induced transcriptional activity on a heterologous minimal TK promoter with the same hiNOS enhancer. Site-directed mutagenesis of the hiNOS AP-1 motifs revealed that an intact upstream (-5.3kb) AP-1 binding site was critical for p300 mediated cytokine-induced hiNOS transcription. Furthermore, our ChIP analysis demonstrated that p300 was binding to Jun D and Fra-2 proteins at -5.3 kb AP-1 binding site in vivo. Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site, and ChIP loop assay confirmed that p300 binds to AP-1 and RNA pol II proteins. Overall, our results suggest that coactivator p300 mediates cytokine-induced hiNOS transactivation by forming a distant DNA loop between its enhancer and core promoter region.

Effects of 5,6-Dihydroxy-2,4-Dimethoxy-9,10-Dihydrophenanthrene on G2/M Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells.

5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) is an active compound isolated from the rhizome extracts of Dioscorea membranacea Pierre, a Thai medicinal plant. This study aimed to investigate the growth-inhibitory and apoptosis-inducing effects of HMP in human lung cancer A549 cells. The antiproliferative and cytotoxic effects of HMP were analyzed by a Sulforhodamine B assay. Cell division, cell cycle distribution and membrane asymmetry changes were each performed with different fluorescent dyes and then analyzed by flow cytometry. Real-time PCR and immunoblotting were used to detect cell cycle- and apoptosis-related mRNA levels and proteins, respectively. The nuclear morphology of the cells stained with DAPI and DNA fragmentation were detected by fluorescence microscopy and gel electrophoresis, respectively. The results showed that HMP exerted strong antiproliferative and cytotoxic activities in A549 cells with the highest selectivity index. It halted the cell cycle in [Formula: see text]/M phase via down-regulation of the expression levels of regulatory proteins Cdc25C, Cdk1 and cyclinB1. In addition, HMP induced early apoptotic cells with externalized phosphatidylserine and subsequent apoptotic cells in sub-[Formula: see text] phase. HMP increased caspase-3 activity and levels of the cleaved (active) form of caspase-3 whose actions were supported by the cleavage of its target PARP, nuclear condensation and DNA apoptotic ladder. Moreover, HMP significantly increased the mRNA and protein levels of proapoptotic Bax as well as promoted subsequent caspase-9 activation and BID cleavage, indicating HMP-induced apoptosis via both intrinsic and extrinsic pathways. These data support, for the first time, the potential role of HMP as a cell-cycle arrest and apoptosis-inducing agent for lung cancer treatment.