A549 Non-small Cell Lung Carcinoma Research Articles

Human Noxin is an anti‐apoptotic protein in response to DNA damage of A549 non‐small cell lung carcinoma

Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in non-small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti-tumor efficacy. Thus, hNoxin is a DNA damage-induced anti-apoptotic protein and potential therapeutic target in cancer.

Cytotoxic activity of gypenosides and gynogenin against non-small cell lung carcinoma A549 cells

The activity of gypenosides and gynogenin of Gynostemma pentaphyllum against non-small cell lung carcinoma (NSCLC) A549 cells was investigated to identify the structural characteristics of gypenosides and gynogenin to have anti-NSCLC activity. Of the tested dammarane-type compounds, 20S-dammar-24-en-2α,3β,12β,20-tetrol showed the strongest activity against A549 cells. Based on the structure and cytotoxic activity relationships of gypenosides and gynogenin, the OH group in C-2, the connected sugar number and the configuration in C-20 were important for cytotoxic activity against A549 cells.

Sensitive and Specific Antibody Probes Directed Against The Erythropoietin Receptor – From Basic Studies To Clinical Implementation

Recombinant erythropoietin (Epo) is an effective anti-anemic agent in most cancer patients and improves their quality of life. Yet, concerns of disease progression and reduced survival in recombinant human Epo (rhuEpo)-treated patients have been raised by Phase III clinical trial data showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Epo has pleiotropic actions and its receptor, EpoR, is expressed by many different cell types outside the erythroid compartment. It was thus proposed that a major possible mechanism for this potentially harmful effect of Epo in cancer patients is the activation of EpoRs on cancer cells. The original clinical studies have been criticized because they deployed polyclonal antibodies later shown to lack specificity for EpoR.Furthermore, multiple isoforms of EpoR caused by differential splicing have been reported, but only at the RNA level, in different cancer cell lines. Investigations into whether these spliced versions actually result in abnormal EpoR forms at the protein level, alter Epo responsiveness and have an impact on tumor progression in vivo, have been hampered by a lack of well characterized monoclonal antibodies.The ‘EpoCan' Consortium, funded by the EU, is directed to promote improved pathological testing of EpoR in patient samples, leading to safer clinical use of rHuEpo and other Erythropoietic Stimulating Agents (ESAs). To date, 25 murine and rat monoclonal antibodies have been produced against the EpoR, using novel genetic and traditional peptide immunization protocols. Of these antibodies, several were found to specifically recognize the receptor in various assays including Western blot (WB), immunoprecipitation (IP), immunofluorescence (IF), flow cytometry (FACS) and immunohistochemistry (IHC). Table 1 lists the antibodies that were selected for further analysis, and their experimental applications. The antibodies were tested on EpoR transfected cells (HEK 293 cells and COS cells) and Epo-dependent UT7 cells which endogenously express EpoR. In addition the antibodies were tested on non-erythroid cells viz. the non-small cell lung carcinoma A549, and breast cancer MDA-MB 231 cell lines. Specificity of the antibodies towards the EpoR in these two latter cell lines was ensured by the lack of reactivity with the corresponding EpoR silenced cells.Table 1Anti EpoR Antibodies - immunizing antigens and applicationsImmunogenSubclone nameSystematic nameIsotypeEpitope locationApplicationsPeptideBCO-3H2-D3GM1201rIgG2bhEpoR cytoplasmic domainWB, IP, IF, IHCPeptideBCO-4B5-C9GM1202rIgG2ah/mEpoR cytoplasmic domainWB, IF, IHCDNAVP-2E8-B6GM1204mIgG1hEpoR extracellular domainIP, IF, FACSDNAVP-4D8-C4GM1205mIgG1hEpoR extracellular domainIP, IF, FACSDNABBQ-9C4-D8GM1206rIgG2ah/mEpoR extracellular domainFACSDNABBQ-10E10-F2GM1207rIgG2ah/mEpoR extracellular domainWBImmunohistochemical analysis was performed on a panel of human non-small cell lung carcinoma sections. Two rat monoclonal antibodies, BCO-3H2-D3 which recognizes full length EpoR and BCO-4B5-C9 which recognizes full length and truncated isoforms, have proved valuable in comparative immunohistochemical and Western blot studies in a range of human tumors and cell lines. These well characterized monoclonal antibodies will enable a careful dissection of EpoR function in cancer cells and the detection of EpoR isoforms in tumor tissue.In the longer term such studies should allow clinicians to balance the benefits and risks of ESA treatment in cancer.THIS WORK WAS SUPPORTED BY THE FP7-HEALTH EUROPEAN COMMISSION EPOCAN GRANT (282551). Disclosures:Grunert:Aldevron Freiburg GmBH: Employment. Thompson:Aldevron Freiburg GmbH: Employment.

Dicopper(II) and Dizinc(II) Complexes with Nonsymmetric Dinucleating Ligands Based on Indolo[3,2-c]quinolines: Synthesis, Structure, Cytotoxicity, and Intracellular Distribution

Dicopper(II) and dizinc(II) complexes [Cu2(MeOOCLCOO)(CH3COO)2] (1) and [Zn2(MeOOCLCOO)(CH3COO)2] (2) were synthesized by reaction of Cu(CH3COO)2·H2O and Zn(CH3COO)2·2H2O with a new nonsymmetric dinucleating ligand EtOOCHLCOOEt prepared by condensation of 6-hydrazinyl-11H-indolo[3,2-c]quinoline with diethyl-2,2′-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate. The design and synthesis of this elaborate ligand was performed with the aim of increasing the aqueous solubility of indolo[3,2-c]quinolines, known as biologically active compounds, and investigating the antiproliferative activity in human cancer cell lines and the cellular distribution by exploring the intrinsic fluorescence of the indoloquinoline scaffold. The compounds have been comprehensively characterized by elemental analysis, spectroscopic methods (IR, UV–vis, 1H and 13C NMR spectroscopy), ESI mass spectrometry, magnetic susceptibility measurements, and UV–vis complex formation studies (for 1) as well as by X-ray crystallography (1 and 2). The antiproliferative activity of EtOOCHLCOOEt, 1, and 2 was determined by the MTT assay in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma), yielding IC50 values in the micromolar concentration range and showing dependence on the cell line. The effect of metal coordination on cytotoxicity of EtOOCHLCOOEt is also discussed. The subcellular distribution of EtOOCHLCOOEt and 2 was investigated by fluorescence microscopy, revealing similar localization for both compounds in cytoplasmic structures.

Effect of oxymatrine on apoptosis and regulatory mechanism in human non-small cell lung carcinoma A549 cells

Objective To investigate the effects of oxymatrine (OXY) on apoptosis and the regulatory mechanism in the human non-small cell lung carcinoma A549 cells.Methods A549 cells were cultured.Methyl thiazol tetrazolium (MTT) assay was used to determine cell proliferation.The apoptosis rate was detected by using flow cytometry.Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blotting were used to examine OXY mRNA and protein expression.Extracellular signal-regulated kinase (ERK),C-Jun N-terminal kinase (JNK),p38 mitogen-activated protein kinase (p38MAPK),p-ERK,p-JNK and p-p38MAPK protein expression levels were tested by using Western blotting.Results OXY could inhibit the proliferation of A549 cells.As compared with control group [(5.63 ±0.76)％],the apoptosis rate was increased [(13.26 ± 2.15)％] in OXY group (P ＜ 0.01).As compared with control group (1.02 ±0.27,0.23 ±0.04),B lymphocytes/leukemia-2 (bcl-2) associated X protein (bax) mRNA (1.61 ±0.19) and protein (0.71 ±0.10) expression levels were increased in OXY group (P ＜ 0.05,P ＜ 0.01).As compared with control group (0.94 ± 0.14,0.64 ± 0.09),bcl-2 mRNA (0.42 ± 0.05) and protein (0.21 ± 0.04) expression levels were decreased in OXY group (P ＜0.01).As compared with control group (0.29 ± 0.03),the expression of p-JNK was significantly increased in OXY group (0.66 ±0.06) (P ＜0.01).bax expression (0.39 ±0.05) and p-JNK (0.26 ±0.04) were reduced,and bcl-2 expression (0.50 ± 0.08) was increased in OXY + SP600125 group as compared with those in OXY group (0.66 ± 0.07,0.28 ± 0.03 and 0.65 ± 0.08 respectively) (P ＜0.01).Conclusion OXY could induce apoptosis of A549 cells by up-regulating p-JNK pathway. Key words: Oxymatrine; Non-small cell lung carcinoma; Apoptosis; Phosphorylated c-Jun N-terminal kinase

Striking difference in antiproliferative activity of ruthenium- and osmium-nitrosyl complexes with azole heterocycles.

Ruthenium nitrosyl complexes of the general formulas (cation)+[cis-RuCl4(NO)(Hazole)]−, where (cation)+ = (H2ind)+, Hazole = 1H-indazole (Hind) (1c), (cation)+ = (H2pz)+, Hazole = 1H-pyrazole (Hpz) (2c), (cation)+ = (H2bzim)+, Hazole = 1H-benzimidazole (Hbzim) (3c), (cation)+ = (H2im)+, Hazole = 1H-imidazole (Him) (4c) and (cation)+[trans-RuCl4(NO)(Hazole)]−, where (cation)+ = (H2ind)+, Hazole = 1H-indazole (1t), (cation)+ = (H2pz)+, Hazole = 1H-pyrazole (2t), as well as osmium analogues of the general formulas (cation)+[cis-OsCl4(NO)(Hazole)]−, where (cation)+ = (n-Bu4N)+, Hazole =1H-indazole (5c), 1H-pyrazole (6c), 1H-benzimidazole (7c), 1H-imidazole (8c), (cation)+ = Na+; Hazole =1H-indazole (9c), 1H-benzimidazole (10c), (cation)+ = (H2ind)+, Hazole = 1H-indazole (11c), (cation)+ = H2pz+, Hazole = 1H-pyrazole (12c), (cation)+ = (H2im)+, Hazole = 1H-imidazole (13c), and (cation)+[trans-OsCl4(NO)(Hazole)]−, where (cation)+ = n-Bu4N+, Hazole = 1H-indazole (5t), 1H-pyrazole (6t), (cation)+ = Na+, Hazole = 1H-indazole (9t), (cation)+ = (H2ind)+, Hazole = 1H-indazole (11t), (cation)+ = (H2pz)+, Hazole = 1H-pyrazole (12t), have been synthesized. The compounds have been comprehensively characterized by elemental analysis, ESI mass spectrometry, spectroscopic techniques (IR, UV–vis, 1D and 2D NMR) and X-ray crystallography (1c·CHCl3, 1t·CHCl3, 2t, 3c, 6c, 6t, 8c). The antiproliferative activity of water-soluble compounds (1c, 1t, 3c, 4c and 9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c) in the human cancer cell lines A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma) has been assayed. The effects of metal (Ru vs Os), cis/trans isomerism, and azole heterocycle identity on cytotoxic potency and cell line selectivity have been elucidated. Ruthenium complexes (1c, 1t, 3c, and 4c) yielded IC50 values in the low micromolar concentration range. In contrast to most pairs of analogous ruthenium and osmium complexes known, they turned out to be considerably more cytotoxic than chemically related osmium complexes (9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c). The IC50 values of Os/Ru homologs differ by factors (Os/Ru) of up to ∼110 and ∼410 in CH1 and SW480 cells, respectively. ESI-MS studies revealed that ascorbic acid may activate the ruthenium complexes leading to hydrolysis of one M–Cl bond, whereas the osmium analogues tend to be inert. The interaction with myoglobin suggests nonselective adduct formation; i.e., proteins may act as carriers for these compounds.

Abstract 445: shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation.

Abstract Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which, if not repaired, augment sensitivity to subsequent ionizing radiation (IR). DNA mismatches were not required to elicit cytotoxicity but were necessary for radiosensitization to occur. We therefore hypothesized that RNAi mediated suppression of RR activity would also radiosensitize cells. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. To determine whether the suppression of either R2 or p53R2 produced a similar extent of radiosensitization compared to dFdCyd, two different shRNAs were used to suppress each subunit in wild-type p53 MCF7 breast carcinoma and A549 non-small cell lung carcinoma cells, producing a profound (≥ 90%) suppression of the target protein, R2 or p53R2, with little effect (≤20%) on expression of its homolog. shRNAs for both R2 and p53R2 produced effects similar to those observed with dFdCyd on dATP depletion (% control value) (MCF7: 66 ± 6% (shRNA) vs. 85 ± 10 % (dFdCyd); A549: 75 ± 20% (shRNA) vs. 80 ± 5% (dFdCyd), and cytotoxicity (MCF7: 35-60% (shRNA) vs. 40-50% (FdCyd); A549: 40-65% (shRNA) vs. 40-60% (dFdCyd)). When shRNA suppression was followed by IR, radiosensitivity was similar with the shRNAs (R2 and p53R2) vs. dFdCyd (MCF7: radiation enhancement ratio (RER)) = 1.45 ± 0.08 (shRNA) vs. 1.7 ± 0.14 (dFdCyd); A549: RER= 1.51 ± 0.09 (shRNA) vs. 1.55 ± 0.14(dFdCyd)), and a similar increase in DNA mismatches compared to untreated wild type cells (MCF7: ≥ 7-fold (dFdCyd) vs. 5-7 fold (shRNA); A549: ≥ 3 fold (dFdCyd) vs. 3-5 fold (shRNA)) was observed. Although p53R2 is thought to be induced primarily in response to DNA damage, its effects on dATP depletion and DNA mismatches prior to IR suggest that it may play a previously unidentified role in normal maintenance of DNA replication. These results reinforce our prior finding that the mechanism of radiosensitization with dFdCyd is the decrease in dATP and not its incorporation into DNA or its cytotoxicity. Excellent radiosensitization with suppression of either R2 or p53R2, compared to dFdCyd, suggests that this targeted approach merits in vivo evaluation. Elevated expression of R2 is known to enhance the invasiveness of cancer cells, and increase resistance to dFdCyd, thus its suppression would further enhance anticancer activity in addition to its effect on radiosensitization. Citation Format: Sheryl A. Flanagan, Kristin Cooper, Sudha Mannava, Mikhail Nikiforov, Donna S. Shewach. shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2013-445

Phosphotyrosine Profiling of NSCLC Cells in Response to EGF and HGF Reveals Network Specific Mediators of Invasion

Growth factor signaling is deregulated in cancer and often leads to invasion, yet receptor tyrosine kinase signaling pathways driving invasion under different growth factor conditions are not well understood. To identify specific signaling molecules regulating invasion of A549 non-small cell lung carcinoma (NSCLC) cells downstream of the epidermal growth factor receptor (EGFR) and Met, quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation was performed following epidermal growth factor (EGF) or hepatocyte growth factor (HGF) stimulation, at three different growth factor concentrations and at two time points. Through this analysis, the temporal and concentration dependent phosphorylation profiles were obtained for 131 and 139 sites downstream of EGF and HGF stimulation, respectively. To characterize the effect of these signaling network alterations, we quantified 3D cell migration/invasion through Matrigel. Partial least-squares regression (PLSR) analysis was performed to identify the tyrosine phosphorylation sites most strongly correlated with EGF and/or HGF mediated invasion. Potential common and specific signaling events required for driving invasion downstream of EGFR and Met were identified using either a combined or two independent PLSR models, based on the quantitative EGF or HGF data. Our data highlight the integration and compartmentalization of signaling required for invasion in cancer.

Abstract PR16: Genetically engineered T cells redirected against cancer-associated fibroblasts to potentiate immunotherapeutic effects.

Abstract Adoptive T-cell therapy has had considerable success in effectuating anti-tumor responses, however complete eradication of bulky solid tumors is rarely observed. This limited efficacy can in part be attributed to the tumor associated stroma; an immunosuppressive microenvironment that greatly decreases the efficacy of the T-cell product. Specifically, cancer associated fibroblasts (CAFs); the central component of the tumor stroma, secrete inhibitory factors and nutrient depleting enzymes that are detrimental to effector T cell function. In addition, CAFs secrete components of the extra-cellular matrix that contributes to desmoplasia and tumor growth. Recent reports also suggest a more pivotal role of the CAFs in regulating the self-renewing cancer stem cell (CSC) niche. CAFs express fibroblast activation protein alpha (FAP); a membrane bound serine protease, in a number of a solid tumors making it an attractive immunotherapeutic target. We hypothesized that targeting CAFs with FAP-specific T cells will destroy the ‘tumor promoting haven’, resulting in significant antitumor effects. To test this hypothesis, we successfully generated FAP-specific T cells using a second-generation chimeric antigen receptor (CAR) specific for FAP. A prototypical CAR combines the antigen specificity of an antibody with the signaling function of a T cell. The resulting genetically engineered FAP-specific T cells recognized and killed human as well as murine FAP-positive target cells ex vivo. To analyze the effects of targeting the tumor associated stroma in vivo we utilized 1) a locoregional model using the FAP negative lymphoblastoid cell line (LCL) tumor and 2) a systemic model using the FAP negative A549 lung adenocarcinoma. In the local model, mice receiving a subcutaneous admixture of FAP-specific T cells and LCL showed a significant decrease in tumor engraftment and growth. Both untreated and control T cell treated mice showed progressive tumor growth with a concomitant induction of murine FAP rich supportive stroma. In our systemic model, the FAP negative non-small cell lung carcinoma (NSCLC) A549 cell line induced the expression of a murine FAP positive reactive stroma in the lungs when injected intravenously through the tail vein. Treatment with FAP-specific T cells showed a significant decrease in murine FAP expression in the lungs with a concomitant decrease in tumor growth and improved survival. Thus targeting FAP in the tumor stroma alone, resulted in a significant anti-tumor effect both in our local and systemic models. Given the reciprocal relationship between tumor cells and CAFs, we hypothesized that co-targeting the tumor cells and CAFs would result in enhanced anti-tumor response than targeting either alone. Erythropoietin-producing hepatocellular carcinoma-A2 (EphA2) CAR positive T cells were used to target the A549 tumor cells. EphA2-specific T cells when administered together with FAP-T cells, resulted in a significant decrease in tumor growth and increased survival compared to mice that received either EphA2 or FAP-T cells alone. Our research therefore underscores the importance of targeting the tumor associated stroma in addition to tumor targeting for a more efficacious therapeutic response. This abstract is also presented as Poster A91. Citation Format: Sunitha Kakarla, Kevin Chow, Melinda Mata, Xiao-Tong Song, Meng-Fen Wu, Hao Liu, Lisa Wang, David Rowley, Klaus Pfizenmaier, Stephen Gottschalk. Genetically engineered T cells redirected against cancer-associated fibroblasts to potentiate immunotherapeutic effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR16.

Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro

To study the possible anticancer and antiproliferative activities of ethanolic leaf extract of Thuja occidentalis (TO) on A549 non-small lung carcinoma cells in vitro. Cell viability was ascertained through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after deployment of TO in different doses. The half maximal inhibitory concentration (IC50) dose (282 μg/mL) was determined, and two other doses for dose-dependence study, one below the IC50 dose (IC35=188 μg/mL) and one above the IC50 dose (IC65=376 μg/mL) were selected. Bromodeoxyuridine (BrdU) incorporation assay and migration studies were performed to elucidate antiproliferative activity of the drug, if any. Fluorescence-activated cell sorting analysis after annexin V-fluorescein isothiocyanate and propidium iodide (annexin V-FITC-PI) dual staining method was done to ascertain early stage of apoptosis, if any. DNA fragmentation assay was done through Hoechst 33258 and acridine orange-ethidium bromide staining. DNA damage was quantified through comet assay. Bax-Bcl2 regulation and expression studies were performed through indirect enzyme-linked immunosorbent assay (ELISA). Caspase 3 activity was measured at gene level through reverse transcription-polymerase chain reaction (RT-PCR) analysis. Its activation at protein level was analyzed through indirect ELISA and Western blot analysis. TO demonstrated a dose-dependent decrease in viability of A549 cells after 24 h of exposure. Cell proliferation was reduced in a time-dependent manner of drug exposure as revealed from BrdU incorporation and migration studies. Annexin-V-FITC positivity of cells up to 11.72% as compared to the untreated control revealed early state of TO-induced apoptosis. Occurrence of comet tail and increased fluorescence of Hoechst after 24 h of drug exposure revealed significant DNA nick generation and chromatin condensation. Bax up-regulation and Bcl-2 down-regulation suitably altered ratio of Bax/Bcl-2 in favor of apoptosis. From RT-PCR, indirect ELISA and Western blot studies, caspase 3 activity was also found to be significantly increased along with cleaved poly ADP-ribose polymerase expression. Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.

Independent transcriptional reprogramming and apoptosis induction by cisplatin

Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part “private,” i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance

Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence

Mutations of oncogenes and tumor suppressor genes which activate mTOR through several downstream signaling pathways are common to cancer. Activation of mTOR when combined with inhibition of cell cycle progression or DNA replication stress has previously been shown to promote cell senescence. In the present study, we examined the conditions under which human non-small cell lung carcinoma A549 cells can undergo senescence when treated with the DNA alkylating agent mitomycin C (MMC). While exposure of A549 cells to 0.1 or 0.5 µg/ml of MMC led to their arrest in S phase of the cell cycle and subsequent apoptosis, exposure to 0.01 or 0.02 µg/ml for 6 d resulted in induction of cell senescence and near total (0.01 µg/ml) or total (0.02 µg/ml) elimination of their reproductive potential. During exposure to these low concentrations of MMC, the cells demonstrated evidence of DNA replication stress manifested by expression of γH2AX, p21WAF1 and a very low level of EdU incorporation into DNA. The data are consistent with the notion that enduring DNA replication stress in cells known to have activated oncogenes leads to their senescence. It is reasonable to expect that tumors having constitutive activation of oncogenes triggering mTOR signaling may be particularly predisposed to undergoing senescence following prolonged treatment with low doses of DNA damaging drugs.

Synthesis, structure, spectroscopic studies and cytotoxic effect of novel palladium(II) complexes with 2-formylpyridine-4-Nethyl-thiosemicarbazone: Potential antitumour agents

New palladium(II) complexes have been synthesized by the reaction of Pd(II) with 2-formylpyridine-4-Nethyl-thiosemicarbazone, HFo4NEt, 1. The complexes [Pd(Fo4NEt)Cl], 2, [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 and [Pd(Fo4NEt)2], 4 have been characterised by elemental analyses and spectroscopic studies. The crystal structure of the complex [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 and the protonated ligand [(H2Fo4NEt)Cl], 5 have been solved by single-crystal X-ray diffraction. The cytotoxic activities of 1–4 have been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L). The compound 3 display IC50 values in a μM range better than that of the antitumor drug cis-platin against MCF-7 and T-24 cell lines and is considered as agent with potential antitumor activity candidates for further stages of screening in vitro and/or in vivo.

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells.

The purpose of this study was to evaluate the level of mitotic cyclin B1 in the context of senescence and cell death in A549 non-small cell lung carcinoma cells. This was performed through analysis of the cell cycle, the percentage of SA-β-galactosidase-positive, as well as TUNEL-positive cells. Morphological alterations were studied using a transmission electron microscope. Changes in the intracellular level and the presence of cyclin B1 in the nucleus and cytoplasm areas were detected by flow cytometry and confocal fluorescence microscopy, respectively. In the cells exposed to various concentrations of doxorubicin, different kinds of cell death and senescent phenotype were observed. Alterations in the cell cycle and increased polyploidy may be indicative of mitotic catastrophe execution. Changes in cyclin B1 may also be strictly related to its different regulation at mitotic catastrophe and senescence programs.

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

The purpose of this study was to evaluate the level of mitotic cyclin B1 in the context of senescence and cell death in A549 non-small cell lung carcinoma cells. This was performed through analysis of the cell cycle, the percentage of SA-β-galactosidase-positive, as well as TUNEL-positive cells. Morphological alterations were studied using a transmission electron microscope. Changes in the intracellular level and the presence of cyclin B1 in the nucleus and cytoplasm areas were detected by flow cytometry and confocal fluorescence microscopy, respectively. In the cells exposed to various concentrations of doxorubicin, different kinds of cell death and senescent phenotype were observed. Alterations in the cell cycle and increased polyploidy may be indicative of mitotic catastrophe execution. Changes in cyclin B1 may also be strictly related to its different regulation at mitotic catastrophe and senescence programs.

Abstract 192: MiR-34c has an anti-apoptotic role in non-small-cell lung carcinoma

Abstract MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that negatively regulate the expression of their target genes. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and are considered as promising new therapeutic targets for cancer. However, the identity of miRNAs involved in apoptosis and their respective targets remain largely unknown. Given the elevated complexity of miRNA regulation of gene expression, we performed a functional screening as an alternative strategy to identify those miRNAs that in lung cancer cells may interfere with the apoptotic process. To this aim we generated a derivative of the non-small cell lung carcinoma A549 cell line in which caspase-8, a critical upstream initiator of apoptosis, can be activated by the administration of the small dimerizer drug AP20187. We found a number of miRNAs that may rescue cell viability from caspase-8 activation. They included miRNAs already described as oncogenic such as miR-17, miR-135, miR-520, but also some miRNAs such as miR-124a and miR-34c for which a tumor suppressive role has been instead described or expected. Among them, miR-34c-5p markedly increased resistance to paclitaxel induced apoptosis. We demonstrate that Bmf (Bcl-2 modifying factor) is a target of miR-34c-5p and that its silencing, together with that of c-myc, a known target of miR-34c-5p, contributes to resistance to apoptosis induced by paclitaxel via p53 downregulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 192. doi:1538-7445.AM2012-192

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

Acquisition of an enhanced aggressive phenotype in human lung cancer cells selected by suboptimal doses of cisplatin following cell deattachment and reattachment

Chemotherapy is one major approach for treating non-small cell lung carcinoma (NSCLC). However, the progression-free survival rate depends on whether there is tumor metastasis after drug treatment. The biological behavior for its characteristics remains to be clarified. Here, we treated A549 and H1299 NSCLC cell lines with cisplatin, doxorubicin and gemcitabine at the IC50 dose. Most attached cells were surviving cells (A549-A and H1299-A), whereas only a small portion of detached cells survived and reattached to tissue culture plates (A549-R and H1299-R) for further growth. Using cisplatin, a series of H1299 sublines (H1299-R2∼H1299-R5) were also generated by the same selection procedure. Drug treatment increased the migratory ability of A549-R and H1299-R cells. A serial selection could enhance the invasiveness of cells. Cisplatin treatment inhibited the adhesion ability of H1299-R cells compared with their H1299 and H1299-A counterparts. H1299-R cells exhibited increased drug resistance to cisplatin and increased expression of ABCG2, CD133 and CD44. Compared with mice subcutaneously injected with H1299 cells, mice subcutaneously injected with H1299-R cells showed an increase in the number of metastatic lung nodules. We conclude that H1299-R cells selected by suboptimal doses of cisplatin following detachment from and reattachment to the tissue culture plate acquire an enhanced malignant phenotype. Therefore, they provide a more faithful lung cancer model associated with biological aggressiveness for studying clinically recurrent cancers after chemotherapy.

MiR-34c may protect lung cancer cells from paclitaxel-induced apoptosis

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that negatively regulate the expression of their target genes. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and are considered as promising new therapeutic targets for cancer. However, the identity of miRNAs involved in apoptosis and their respective targets remain largely unknown. Given the elevated complexity of miRNA regulation of gene expression, we performed a functional screening as an alternative strategy to identify those miRNAs that in lung cancer cells may interfere with the apoptotic process. To this aim, we generated a derivative of the non-small cell lung carcinoma A549 cell line in which caspase-8, a critical upstream initiator of apoptosis, can be activated by administration of the small dimerizer drug AP20187. We found a number of miRNAs that may rescue cell viability from caspase-8 activation. They included miRNAs already described as oncogenic such as miR-17, miR-135 and miR-520, but also some miRNAs such as miR-124-1 and miR-34c for which a tumor-suppressive role has instead been described or expected. Among them, miR-34c-5p markedly increased resistance to paclitaxel-induced apoptosis. We demonstrate that Bmf (Bcl-2-modifying factor) is a target of miR-34c-5p, and that its silencing, together with that of c-myc, a known target of miR-34c-5p, contributes to resistance to apoptosis induced by paclitaxel through p53 downregulation.

Platinum(II) complexes with 5,7-disubstituted-1,2,4-triazolo [1,5-a]pyrimidines: Spectroscopical characterization and cytotoxic activity in vitro

Complexes of the types: cis-[PtI2(dptp)2] (1), cis-[PtI2(NH3)(dptp)] (2), trans-[PtI2(dptp)(dmso)] (3) and trans-[PtI2(dbtp)(dmso)] (4), where dptp=5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp), dbtp=5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine have been synthesized and characterized by infrared and multinuclear magnetic resonance spectroscopic techniques (1H, 13C, 15N, 195Pt). In 195Pt NMR, the cis-diiodo complexes were observed between –2601ppm and –3261ppm, while the trans coordination compounds were found at higher field (ca. −4389ppm). In all cases significant 15N NMR shielding (92–95ppm) were observed for N(3) atom indicating this nitrogen atom as a coordination site. The cis complexes have been assayed for antitumor activity in vitro against two human cell lines: A549 (non-small cell lung carcinoma) and T47D (breast cancer). The results indicate a moderate antiproliferative activity of (2) against human cancer lines.