cytochrome-c-t and Carcinoma--Non-Small-Cell-Lung

Pendulone, an isoflavone compound, is known to act against human cancer cells. However, its role in human non-small cell lung cancer (NSCLC) and the exact molecular mechanisms of action have never been reported.. We investigated the effects of pendulone on cell proliferation and apoptosis in human NSCLC H1299 cells. Cell viability was examined using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay. Flow cytometry was employed to evaluate apoptotic indices such as the cell cycle, mitochondrial membrane potential, cytochrome c release, caspase activity, and death receptor expression. The expression of proteins related to the cell cycle and apoptosis were analyzed by Western blot analysis.. Pendulone significantly decreased H1299 cell viability by inducing endoplasmic reticulum (ER) stress through the accumulation of reactive oxygen species (ROS). Pendulone induced the expression of ER stress-associated proteins, such as ATF4 and CHOP, which promoted the expression of death receptors. Activation of caspase 8 induced extrinsic pathway apoptosis. Pendulone also caused the loss of mitochondrial membrane potential, inhibited the anti-apoptotic proteins BCL-2 and activated the pro-apoptotic protein BAX, which promoted the release of cytochrome c to activate caspase 9. Antioxidant N-acetylcysteine (NAC), with its ROS-suppressive property, reversed pendulone-induced ER stress and cell apoptosis.. Our findings provide evidence that pendulone induces apoptosis by inducing ER stress through ROS accumulation and mitochondrial dysfunction in NSCLC cell lines.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum Stress; Humans; Isoflavones; Lung Neoplasms; Membrane Potential, Mitochondrial; Quinones; Reactive Oxygen Species

Lung adenocarcinoma (LUAD) is the most common subtype of nonsmall cell lung cancer. Cytochrome c (Cyt c), which is produced from mitochondria, interacts with a protein called Apaf-1 to form the heptameric apoptosome. This heptameric apoptosome then activates the caspase cascade, which ultimately results in the execution of apoptosis. The purpose of our research was to discover a new prognostic model that is based on cytochrome c-related genes (CCRGs) for LUAD patients. Through LASSO regression analysis conducted on the LUAD datasets included in the TCGA datasets, a CCRGs signature was created. The diagnostic accuracy of the multigene signature was verified by an independent source using the GSE31210 and GSE72094 datasets. The GO and KEGG enrichment analysis were performed. In this study, there were 159 differentially expressed CCRGs in the TCGA dataset, while there were 68 differentially expressed CCRGs in the GSE31210 dataset. Additionally, there were 57 genes that overlapped across the two datasets. Using LASSO and Cox regression analysis, a signature consisting of 12 differentially expressed CCRGs was developed from the total of 57 such genes. On the basis of their risk ratings, patients were categorized into high-risk and low-risk categories, with low-risk patients having lower risk scores and a greater likelihood of surviving the disease. Univariate and multivariate analyses both concluded that this signature is an independent risk factor for LUAD. ROC curves demonstrated that this risk signature is capable of accurately predicting the 1-year, 2-year, 3-year, and 5-year survival rates of patients who have LUAD. The infiltration of antigen-presenting cells was higher in the low-risk group, such as aDCs, DCs, pDCs, and iDCs. The expression of multiple immune checkpoints was significantly higher in the low-risk group, such as BTLA, CD28, and CD86. Finally, we showed that the signature can be used to predict the drug sensitivity of already available or under investigational drugs. Overall, patient classification and individualized therapy options may benefit from this study's development of a powerful gene signature with high value for prognostic prediction in LUAD.

Topics: Adenocarcinoma of Lung; Apoptosomes; Carcinoma, Non-Small-Cell Lung; Caspases; CD28 Antigens; Cytochromes c; Drugs, Investigational; Humans; Lung Neoplasms; Prognosis

N-heterocyclic carbenes-modified half-sandwich iridium(III) complex [(η

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Coordination Complexes; Cytochromes c; Drug Screening Assays, Antitumor; HeLa Cells; Heterocyclic Compounds; Humans; Iridium; Lung Neoplasms; Lysosomes; Methane; Microscopy, Confocal; Mitochondria; Organometallic Compounds

Non‑small cell lung cancer (NSCLC) is the most common type of lung cancer, and numerous oncogenes are associated with this disease. Oxysterol‑binding protein‑related protein 8 (ORP8) is essential for cell growth, migration and the modulation of mitochondrial respiration and morphology. However, the underlying role of ORP8 in NSCLC remains unclear. In the present study, it was reported that the expression of ORP8 was low in NSCLC cells and tissues. The ORP8 expression levels were analyzed by immunohistochemistry (IHC), quantitative real‑time PCR (qPCR) and western blot analysis. ORP8 overexpression inhibited cell growth and induced apoptosis in NSCLC cells with MTS, anchorage‑independent growth and Hoechst 33342 staining assay. Further experiments demonstrated that ORP8 overexpression induced the apoptosis of NSCLC cells via the release of cytochrome c from mitochondria into the cytoplasm with western blot analysis and confocal microscopy results. In addition, qPCR analysis showed that miR‑421 was upregulated in NSCLC cell lines, with the bioinformatics analysis, western blot analysis and Dual‑Luciferase reporter assay, it was determined that miR‑421 could target ORP8. The inhibition of cell proliferation via ORP8 overexpression was rescued by a miR‑421 mimic, which aided in maintaining the proliferative potential of the cells. Overall, the present study revealed that ORP8 may be a candidate target in the prevention and treatment of NSCLC.

Topics: A549 Cells; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Cytoplasm; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Mitochondria; Receptors, Steroid

Non-small cell lung cancer (NSCLC) is one of the most prevailing malignancies worldwide. It has been previously shown that wogonoside exerts anti-tumor activities in various kinds of human cancers. But its role in NSCLC remains elusive. In the present study, we determined the anti-tumor effect of wogonoside in human NSCLC A549 cells. We found that wogonoside effectively inhibits A549 cell viability through inducing cell cycle arrest and apoptosis. Moreover, administration of wogonoside by intraperitoneal injection inhibits the growth of A549 cell xenografts in athymic nude mice. Additionally, mitochondrial membrane potential was disrupted and cytochrome c was released to cytosol in the wogonoside-treated A549 cells. Finally, we found that AMPK/mTOR signaling might be implicated in the anti-NSCLC efficacy of wogonoside. Therefore, we may assume that wogonoside may be considered as a potential therapeutic agent for the treatment of NSCLC.

Topics: A549 Cells; AMP-Activated Protein Kinases; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Flavanones; Glucosides; Humans; Lung Neoplasms; Male; Membrane Potential, Mitochondrial; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Artocarpin has been shown to exhibit cytotoxic effects on different cancer cells, including non-small cell lung carcinoma (NSCLC, A549). However, the underlying mechanisms remain unclear. Here, we explore both p53-dependent and independent apoptosis pathways in artocarpin-treated NSCLC cells. Our results showed that artocarpin rapidly induced activation of cellular protein kinases including Erk1/2, p38 and AktS473. Inhibition of these protein kinases prevented artocarpin-induced cell death. Moreover, artocarpin-induced phosphorylation of these protein kinases and apoptosis were mediated by induction of reactive oxygen species (ROS), as pretreatment with NAC (a ROS scavenger) and Apocynin (a Nox-2 inhibitor) blocked these events. Similarly, transient transfection of p47Phox or p91Phox siRNA attenuated artocarpin-induced NADPH oxidase activity and cell death. In addition, p53 dependent apoptotic proteins including PUMA, cytochrome c, Apaf-1 and caspase 3 were activated by artocarpin, and these effects can be abolished by antioxidants, MAPK inhibitors (U0126 and SB202190), but not by PI3K inhibitor (LY294002). Furthermore, we found that artocarpin-induced Akt phosphorylation led to increased NF-κB activity, which may act as an upstream regulator in the c-Myc and Noxa pathway. Therefore, we propose that enhancement of both ERK/ p38/ p53-dependent or independent AktS473/NF-κB/c-Myc/Noxa cascade by Nox-derived ROS generation plays an important role in artocarpin-induced apoptosis in NSCLC cells.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Disease Models, Animal; Gene Expression; Humans; Lung Neoplasms; Male; Mannose-Binding Lectins; Mice; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Plant Lectins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Ginsenoside Rk3 (Rk3) is present in the roots of processed Panax notoginseng herbs and it exerts anti-platelet aggregation, pro-immunogenic and cardioprotective effects. However, little is known regarding the anticancer activities of this compound, especially in lung cancer. This study was designed to investigate the anticancer effects of Rk3 on non-small cell lung cancer (NSCLC) cells and in an H460 xenograft tumor model. Our results showed that Rk3 reduced cell viability, inhibited both cell proliferation and colony formation, and induced G1 phase cell cycle arrest by downregulating the expression of cyclin D1 and CDK4 and upregulating the expression of P21. Rk3 also induced apoptosis in a concentration-dependent manner in H460 and A549 cells by Annexin V/PI staining, TUNEL assay and JC-1 staining, resulting in a change in the nuclear morphology. Moreover, Rk3 induced the activation of caspase-8, -9, and -3, promoted changes in mitochondrial membrane potential, decreased the expression of Bcl-2, increased the expression of Bax, and caused the release of cytochrome c, which indicated that the apoptosis-inducing effects of Rk3 were triggered via death receptor-mediated mitochondria-dependent pathways. Furthermore, Rk3 significantly inhibited the growth of H460 xenograft tumors without an obvious effect on the body weight of the treated mice. Histological analysis indicated that Rk3 inhibited tumor growth by altering the proliferation and morphology of tumor cells. In addition, we confirmed that Rk3 inhibited angiogenesis via CD34 staining and chick embryo chorioallantoic membrane (CAM) assay in vivo. Taken together, our findings revealed not only the anticancer effect of Rk3 on NSCLC cells but also a new promising therapeutic agent for human NSCLC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chickens; Chorioallantoic Membrane; Cytochromes c; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Ginsenosides; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Mitochondria

Chlorophyllin (CHL), a sodium-copper-salt derived from chlorophyll, has been widely used as a food-dye, also reportedly having some anti-cancer effect. We tested if PLGA-loaded CHL (NCHL) could have additional protective abilities through its faster and targeted drug delivery in cancer cells. Physico-chemical characterization of NCHL was done through atomic-force microscopy and UV-spectroscopy. NCHL demonstrated greater ability of drug uptake and strong anti-cancer potentials in non-small cell lung cancer cells, A549, as revealed from data of% cell viability, generation of reactive-oxygen-species and expression of bax, bcl2, caspase3, p53 and cytochrome c proteins. Circular dichroic spectral data indicated strong binding of NCHL with calf-thymus-DNA, causing a conformational/structural change in DNA. Further, NCHL could cross the blood-brain-barrier in mice and showed greater efficacy in recovery process of tissue damage, reduction in chromosomal aberrations and% of micronuclei in co-mutagens (Sodiumarsenite+Benzo[a]Pyrene)-treated mice at a much reduced dose, indicating its use in therapeutic oncology.

Topics: Animals; Antineoplastic Agents; Antioxidants; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chlorophyllides; Chromosome Aberrations; Circular Dichroism; Cytochromes c; DNA; Humans; Lactic Acid; Lung Neoplasms; Mice, Inbred BALB C; Mitochondria; Nanostructures; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Reactive Oxygen Species; Signal Transduction

Non-small cell lung cancer has a devastating prognosis, and markers enabling a precise prediction of therapy response have long remained scarce. Better treatment monitoring would allow an individual's more effective patient adjusted therapy with lesser side effects and good clinical outcomes. In the present study, we monitored the serum cytochrome c levels pre- and post-chemotherapy of non-small cell lung cancer patients. Using highly sensitive enzyme-linked immunosorbent assay, we evaluated cytochrome c levels in serum of 100 non-small cell lung cancer and 100 healthy controls. We observed about threefold lower serum cytochrome c level in newly diagnosed non-small cell lung cancer patients than healthy individuals. Patients in advanced stages and grade 3 histological differentiation showed significantly low level of serum cytochrome c, and the lower levels were associated with worse survival outcome of non-small cell lung cancer patients. In addition, serum cytochrome c level was observed to be more than 13-fold higher after first cycle of conventional chemotherapy, wherein patients with higher level of serum cytochrome c before any therapy showed better response to chemotherapy in terms of significantly higher level of serum cytochrome c after first cycle of chemotherapy than patients with low level of serum cytochrome c at the time of diagnosis. Detection of serum cytochrome c levels at the time of diagnosis may be useful in suggesting disease severity and prognosis of the non-small cell lung cancer patients. Monitoring of serum cytochrome c might also serve as a sensitive apoptotic marker in vivo reflecting chemotherapy-induced cell death burden in patients with non-small cell lung cancer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Neoplasm Staging; Prognosis

Pemetrexed is a multitargeted antifolate used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). However, the mechanism by which pemetrexed induces apoptosis remains unclear. In the present study, we investigated the involvement of reactive oxygen species (ROS) and sirtuin 1 (SIRT1) in pemetrexed-induced apoptosis in MSTO-211 malignant mesothelioma cells and A549 NSCLC cells. Pemetrexed enhanced caspase-dependent apoptosis, induced intracellular ROS generation, and downregulated SIRT1 in the MSTO-211 and A549 cells. Pemetrexed-induced apoptosis, which was prevented by pretreatment with N-acetyl-cysteine (NAC), was mediated by effects on the mitochondria, including mitochondrial membrane potential transition (MPT) and cytosolic release of cytochrome c, and also involved regulation of SIRT1 expression. Interference with SIRT1 expression using siRNA enhanced pemetrexed-induced apoptosis through mitochondrial dysfunction and ROS generation, whereas resveratrol, an activator of SIRT1, protected against pemetrexed-induced apoptosis. These results show that pemetrexed induces apoptosis in MSTO-211 mesothelioma cells and A549 NSCLC cells through mitochondrial dysfunction mediated by ROS accumulation and SIRT1 downregulation.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cytochromes c; Down-Regulation; Free Radical Scavengers; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mesothelioma; Mesothelioma, Malignant; Mitochondria; Pemetrexed; Reactive Oxygen Species; Sirtuin 1

So-Cheong-Ryong-Tang (SCRT), a traditional Korean medicine containing eight species of medicinal plant, has been used to treat patients with bronchial asthma and allergic rhinitis for hundreds of years; however, its anti-cancer potential is poorly understood. The present study was designed to evaluate the apoptotic effect of SCRT against human non-small-cell lung cancer (NSCLC) A549 cells.. The effects of SCRT on cell growth and viability were evaluated by trypan blue dye exclusion and 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assays, respectively. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay.. SCRT treatment resulted in significantly decreased A549 cell growth and viability by induction of apoptosis. SCRT induced the translocation of pro-apoptotic Bax to the mitochondria, mitochondrial membrane permeabilization, cytochrome c release from mitochondria to cytosol, and activated caspase-9 and caspase-3. SCRT also increased death receptor-associated ligands and enhanced the activation of caspase-8 and cleavage of its substrate Bid. However, the pan-caspases inhibitor significantly blocked the SCRT-induced apoptosis, suggesting that it is a caspase-dependent pathway. In addition, SCRT suppressed the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt, and treatment with a potent inhibitor of PI3K further increased the apoptotic activity of SCRT.. These findings suggest that SCRT may play its anti-cancer actions partly through a suppression of the PI3K/Akt signal pathway in A549 cells, and further in vivo studies on the potential of SCRT for prevention and therapy of NSCLCs are warranted.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 9; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Humans; Mitochondria; Phosphatidylinositol 3-Kinases; Phosphorylation; Phytotherapy; Proto-Oncogene Proteins c-akt; Signal Transduction

Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

Topics: Adenine; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Gene Expression; Humans; Hydroxychloroquine; Lung Neoplasms; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Quercetin; Xenograft Model Antitumor Assays

Combretastatin A-4 (CA-4) is one of the most effective agents used in chemotherapy. Nevertheless, the contribution of p53 and Bim proteins in the CA-4-induced apoptosis in non-small lung cancer cells (NSCLC) remains unresolved, specifically on involving of p53 in the mitochondrial pathway activation by a transcription-independent mechanism. In this context, the p53-null H1299 and wt-p53 H460 NSCLC cells, in the absence and presence of pifithrin-µ (PFTµ), an inhibitor of p53 mitochondrial-translocation, were treated with CA-4 and different cellular endpoints were analysed. In contrast to previous observations in H460 cells, CA-4 failed in the activation of an apoptotic response in H1299 cells, thus indicating an involvement of p53 in the cell death induced by the drug. We found that CA-4 led to p53 cellular re-localisation in H460 cells; in particular, p53 was released from the microtubular network and accumulated at mitochondria where it interacts with Bim protein and other proteins of the Bcl-2 (B-cell leukaemia-2) family, leading to cytochrome c release, alteration in the mitochondrial membrane polarisation, cell cycle arrest at the G2/M-phase, and cell death. Interestingly, the cytosolic and the mitochondrial accumulation of protein Bim was strictly dependent on p53 status. The extent of cell death was not reduced in H460 after combined treatment of PFTµ with CA-4. Overall, the data support a model of CA-4-induced apoptosis in NSCLC, for which the expression of p53 protein is essential, but its mitochondrial function, linked to p53-transcription independent apoptosis pathway, is negligible.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bibenzyls; Carcinoma, Non-Small-Cell Lung; Cytochromes c; Humans; Lung Neoplasms; Mitochondria; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Bortezomib is a novel proteasome inhibitor that has promising antitumor activity against various cancer cells. We have assessed its antitumor activity in non-small cell lung cancer (NSCLC) A549 and H157 cells in vitro where it inhibited cell growth and induced apoptosis, which was associated with cytochrome c release and caspase-3 activation. Bortezomib upregulated autophagic-related proteins, the Atg12-Atg5 complex and LC3-II, which indicated autophagy had occurred. The combination of bortezomib with autophagic inhibitor 3-methyladenine or chloroquine significantly enhanced suppression of cell growth and apoptosis induced by bortezomib in A549 and H157 cells. Our study indicated that inhibition of both proteasome and autophagy has great potential for NSCLC treatment.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Boronic Acids; Bortezomib; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Epithelial Cells; Humans; Pyrazines

The intrinsic apoptosis pathway represents an important mechanism of stress-induced death of cancer cells. To gain insight into the functional status of the apoptosome apparatus in non-small cell lung carcinoma (NSCLC), we studied its sensitivity to activation, the assembly of apoptosome complexes and stability of their precursors, and the importance of X-linked inhibitor of apoptosis (XIAP) in the regulation of apoptosome activity, using cell-free cytosols from NSCLC cell lines and NSCLC tumours and lungs from 62 surgically treated patients. Treatment of cytosol samples with cytochrome c (cyt-c) and dATP induced proteolytic processing of procaspase-9 to caspase-9, which was followed by procaspase-3 processing to caspase-3, and by generation of caspase-3-like activity in 5 of 7 studied NSCLC cell lines. Further analysis demonstrated formation of high-Mr Apaf-1 complexes associated with cleaved caspase-9 in the (cyt-c + dATP)-responsive COLO-699 and CALU-1 cells. By contrast, in A549 cells, Apaf-1 and procaspase-9 co-eluted in the high-Mr fractions, indicating formation of an apoptosome complex unable of procaspase-9 processing. Thermal pre-treatment of cell-free cytosols in the absence of exogenous cyt-c and dATP lead to formation of Apaf-1 aggregates, unable to recruit and activate procaspase-9 in the presence of cyt-c and dATP, and to generate caspase‑3‑like activity. Further studies showed that the treatment with cyt-c and dATP induced a substantially higher increase of caspase-3-like activity in cytosol samples from NSCLC tumours compared to matched lungs. Tumour histology, grade and stage had no significant impact on the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity. Upon addition into the cytosol, the XIAP-neutralizing peptides AVPIAQK and ATPFQEG only moderately heightened the (cyt-c + dATP)-induced caspase‑3‑like activity in some NSCLC tumours. Taken together, the present study provides evidence that the apoptosome apparatus is functional in the majority of NSCLCs and that its sensitivity to the (cyt-c + dATP)-mediated activation is often enhanced in NSCLCs compared to lungs. They also indicate that XIAP does not frequently and effectively suppress the activity of apoptosome apparatus in NSCLCs.

Topics: Aged; Apoptosis; Apoptotic Protease-Activating Factor 1; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Cytosol; Deoxyadenine Nucleotides; Female; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Male; Middle Aged; X-Linked Inhibitor of Apoptosis Protein

Tanshinone IIA (TSIIA), a natural diterpene quinone in the traditional Chinese medicinal herb Dan-Shen (Salvia miltiorrhiza), has extensively exerted antitumor activity in cellular and animal models. However, the molecular mechanisms underlying the antitumor effects of TSIIA remain largely unknown. The in vitro effects of TSIIA on apoptosis were investigated in A549 non-small cell lung cancer (NSCLC) cells. The data showed that TSIIA significantly suppressed the proliferation of A549 cells in a dose-dependent manner, with IC50 values of 16.0±3.7 and 14.5±3.3 µM at 48 h as determined by Cell Counting Kit-8 (CCK-8) assay and clone formation assay, respectively. The change of mitochondrial morphology and the loss of mitochondrial membrane potential (MMP) were observed during the induction. Furthermore, TSIIA induced A549 cell apoptosis as confirmed by typical morphological changes, with cytochrome c release from the mitochondria and Bax translocation to the mitochondria. Caspase activity data indicated that TSIIA activated caspase-9 and caspase-3 of mitochondria-mediated apoptosis, but not caspase-8 of receptor-mediated apoptosis, which could be largely rescued by SP600125 (JNK inhibitor). Taken together, these findings provide the first evidence that TSIIA inhibits growth of NSCLC A549 cells, induces activation of JNK signaling and triggers caspase cascade apoptosis mediated by the release of cytochrome c, which provides a better understanding of the molecular mechanisms of TSIIA on lung cancer.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Fluorescent Antibody Technique; Humans; Lung Neoplasms; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Microscopy, Fluorescence

Interferon-gamma (IFN-γ) has been used to treat various malignant tumors. However, the molecular mechanisms underlying the direct anti-proliferative activity of IFN-γ are poorly understood. In the present study, we examined the in vitro antitumor activity of IFN-γ on two human non-small-cell lung carcinoma (NSCLC) cell lines, H322M and H226. Our findings indicated that IFN-γ treatment caused a time-dependent reduction in cell viability and induced apoptosis through a FADD-mediated caspase-8/tBid/mitochondria-dependent pathway in both cell lines. Notably, we also postulated that IFN-γ increased indoleamine 2,3-dioxygenase (IDO) expression and enzymatic activity in H322M and H226 cells. In addition, inhibition of IDO activity by the IDO inhibitor 1-MT or tryptophan significantly reduced IFN-γ-induced apoptosis and death receptor 5 (DR5) expression, which suggests that IDO enzymatic activity plays an important role in the anti-NSCLC cancer effect of IFN-γ. These results provide new mechanistic insights into interferon-γ antitumor activity and further support IFN-γ as a potential therapeutic adjuvant for the treatment of NCSLC.

Topics: Adenocarcinoma; Antiviral Agents; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Cytochromes c; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Lung Neoplasms; Membrane Potential, Mitochondrial; Receptors, TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

The antineoplastic agent cis-diammineplatinum(II) dichloride (cisplatin, CDDP) is part of the poorly effective standard treatment of non-small cell lung carcinoma (NSCLC). Here, we report a novel strategy to improve the efficacy of CDDP. In conditions in which CDDP alone or either of two PARP inhibitors, PJ34 hydrochloride hydrate or CEP 8983, used as standalone treatments were inefficient in killing NSCLC cells, the combination of CDDP plus PJ34 or that of CDDP plus CEP 8983 were found to kill a substantial fraction of the cells. This cytotoxic synergy could be recapitulated by combining CDDP and the siRNA-mediated depletion of the principal PARP isoform, PARP1, indicating that it is mediated by on-target effects of PJ34 or CEP 8983. CDDP and PARP inhibitors synergized in inducing DNA damage foci, mitochondrial membrane permeabilization leading to cytochrome c release, and dissipation of the inner transmembrane potential, caspase activation, plasma membrane rupture and loss of clonogenic potential in NSCLC cells. Collectively, our results indicate that CDDP can be advantageously combined with PARP inhibitors to kill several NSCLC cell lines, independently from their p53 status. Combined treatment with CDDP and PARP inhibitors elicits the intrinsic pathway of apoptosis.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Membrane; Cisplatin; Cytochromes c; DNA Damage; Drug Synergism; Enzyme Inhibitors; Humans; Lung Neoplasms; Mitochondrial Membranes; Phenanthrenes; Phthalimides; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; RNA Interference; RNA, Small Interfering

Chemotherapy is an important option for the treatment of various cancers including lung cancer. However, tumor resistance towards cytotoxic chemotherapy has become more common. It has been reported that autophagy is one of the processes contributing to this resistance. In the present study, we found that the anti-cancer drug 5-fluorouraci(5-FU) could induce autophagy in A549 cells. 5-FU treatment could lead to the conversion of LC3 I/II, the up-regulation of Beclin-1, the down-regulation of p62 and the formation of acidic vesicular organelles (AVOs) in A549 cells. Pre-treatment of cancer cells with 3-MA or siAtg7 could enhance 5-FU-induced apoptosis through the activation of caspases, and the caspase inhibitor z-VAD-fmk rescued the cell viability reduction. Furthermore, the inhibition of autophagy also stimulated ROS formation and scavenging of ROS by antioxidant NAC inhibited caspase-3 activity, prevented the release of cyt-c from mitochondria and eventually rescued cancer cells from 5-FU-mediated apoptosis. These results suggest that 5-FU-elicited autophagic response plays a protective role against cell apoptosis and the inhibition of autophagy could sensitize them to 5-FU-induced caspase-dependent apoptosis through the stimulation of ROS formation.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Fluorouracil; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species

Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Humans; Iridoids; Mitochondria; Molecular Targeted Therapy; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured

To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.. Human non-small-cell lung carcinoma cell lines H460, A549 and H157 were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cells death was examined by flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, -8 and -9 were measured via ELISA. Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential. Bcl-2/Bcl-XL/XIAP/Bid/DR5 and DR4 proteins were analyzed using western blot.. The concentrations required for a 50% decrease in cell growth (IC(50)) ranged from 1.8 to 3.2 μM. Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway, including the depolarization of the mitochondrial membrane, release of cytochrome c from mitochondria, activation of procaspase-9 and -3, and increase of DNA fragments. Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zDEVD-FMK suppressed casticin-induced apoptosis. In addition, casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage. In H157 cell line, casticin increased expression of DR5 at protein levels but not affect the expression of DR4. The pretreatment with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells. No correlation was found between cell sensitivity to casticin and that to p53 status, suggesting that casticin induce a p53-independent apoptosis.. Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Flavonoids; Humans; Lung Neoplasms; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Up-Regulation

The present study examines grape seed extract (GSE) efficacy against a series of non-small-cell lung cancer (NSCLC) cell lines that differ in their Kras and p53 status to establish GSE potential as a cytotoxic agent against a wide range of lung cancer cells. GSE suppressed growth and induced apoptotic death in NSCLC cells irrespective of their k-Ras status, with more sensitivity toward H460 and H322 (wt k-Ras) than A549 and H1299 cells (mutated k-Ras). Mechanistic studies in A549 and H460 cells, selected, based on comparative efficacy of GSE at higher and lower doses, respectively, showed that apoptotic death involves cytochrome c release associated caspases 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, strong phosphorylation of ERK1/2 and JNK1/2, downregulation of cell survival proteins, and upregulated proapoptotic Bak expression. Importantly, GSE treatment caused a strong superoxide radical-associated oxidative stress, significantly decreased intracellular reduced glutathione levels, suggesting, for the first time, the involvement of GSE-caused oxidative stress in its apoptotic inducing activity in these cells. Because GSE is a widely-consumed dietary agent with no known untoward effects, our results support future studies to establish GSE efficacy and usefulness against NSCLC control.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Down-Regulation; Grape Seed Extract; Humans; MAP Kinase Signaling System; Oxidative Stress; Phosphorylation; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Seeds; Tumor Suppressor Protein p53; Vitis

Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer biology and therapeutics.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Respiration; Cytochromes c; Disease Progression; Energy Metabolism; Glucose; Heme; Hemeproteins; Humans; Lung Neoplasms; Membrane Transport Proteins; Mitochondria; Neoplasm Proteins; Oxygen; Oxygen Consumption; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Berberine; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclooxygenase 2; Cytochromes c; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; NF-kappa B; Signal Transduction; Telomerase; Transcription Factor AP-2; Vascular Endothelial Growth Factor A

In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells.

Topics: Animals; Annexin A5; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Comet Assay; Cytochromes c; DNA Damage; Down-Regulation; Humans; Mice; Mice, Nude; RNA, Small Interfering; Triazines; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

We intended to study the mechanism of the inhibitory action of curcumin on human non-small cell lung cancer A549 cell. The cell growth was determined by CCK-8 assay, and the results indicated that curcumin inhibited the cell proliferation in a concentration dependent manner. And to further confirm the relative anti-cancer mechanism of curcumin, RT-PCR was carried out to analysis the expression of relative apoptotic proteins Bax, Bcl-2. We found that curcumin could up-regulate the expression of Bax but down-regulate the expression of Bcl-2 in A549 cells. In addition, curcumin affect the mitochondrial apoptosis pathway. These results suggested that curcumin inhibited cancer cell growth through the regulation of Bcl-2/Bax and affect the mitochondrial apoptosis pathway.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Curcumin; Cytochromes c; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Serine/threonine protein kinase B (PKB/Akt) is involved in cell survival and growth. Carboxyl-terminal modulator protein (CTMP), a novel Akt binding partner, prevents Akt activation at the plasma membrane in response to various stimuli, and thus possesses a tumor suppressor-like function. In a previous study, we have demonstrated that CTMP inhibits tumor progression by facilitating apoptosis in a mouse lung cancer model. However, the precise mechanism of CTMP-induced apoptosis remains to be elucidated. The present study was performed to examine the role of CTMP in mitochondrial-mediated apoptosis and regulation of mitochondrial function in human lung carcinoma cells. Our results showed that CTMP altered mitochondrial morphology and caused the release of cytochrome c by inhibiting OPA1 expression. Additionally, CTMP facilitated mitochondrial-mediated apoptosis by inhibiting heat-shock protein 27 and preventing cytochrome c interaction with Apaf-1. Our data suggest that CTMP may therefore play a critical role in mitochondrial-mediated apoptosis in lung cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptotic Protease-Activating Factor 1; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cytochromes c; Dactinomycin; GTP Phosphohydrolases; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Molecular Chaperones; RNA Interference; Signal Transduction; Staurosporine; Thiolester Hydrolases; Time Factors; Transfection

Baohuoside I (also known as Icariside II) is a flavonoid isolated from Epimedium koreanum Nakai. Although Baohuoside I exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human lung cancer cells are poorly understood. Therefore, in the present study, we investigated the usefulness of Baohuoside I as a potential apoptosis-inducing cytotoxic agent using human adenocarcinoma alveolar basal epithelial A549 cells as in vitro model. The apoptosis induced by Baohuoside I in A549 cells was confirmed by annexin V/propidium iodide double staining, cell cycle analysis and dUTP nick end labeling. Further research revealed that Baohuoside I accelerated apoptosis through the mitochondrial apoptotic pathway, involving the increment of BAX/Bcl-2 ratio, dissipation of mitochondrial membrane potential, transposition of cytochrome c, caspase 3 and caspase 9 activation, degradation of poly (ADP-ribose) polymerase and the over-production of reactive oxygen species (ROS). A pan-caspase inhibitor, Z-VAD-FMK, only partially prevented apoptosis induced by Baohuoside I, while NAC, a scavenger of ROS, diminished its effect more potently. In addition, the apoptotic effect of Baohuoside I was dependent on the activation of ROS downstream effectors, JNK and p38(MAPK), which could be almost abrogated by using inhibitors SB203580 (an inhibitor of p38(MAPK)) and SP600125 (an inhibitor of JNK). These findings suggested that Baohuoside I might exert its cytotoxic effect via the ROS/MAPK pathway.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Flavonoids; Humans; Lung Neoplasms; MAP Kinase Kinase 4; Membrane Potential, Mitochondrial; Mitochondria; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species

The mammalian target of rapamycin (mTOR) is a key kinase acting downstream of growth factor receptor PI3K and AKT signaling, leading to processes resulting in increased cell size and proliferation through translation control. Rapamycin, a specific inhibitor of mTOR, results predominately in G1 cell cycle arrest through translation control and occasionally, cell type-dependent apoptosis by an unknown mechanism. In this study, we investigated the effect and mechanism of action of rapamycin on non-small cell lung cancer (NSCLC) cell lines with p53 mutations. Cell proliferation was evaluated by modified MTT assay. The apoptotic effect of rapamycin was measured by caspase-3 activation and flow cytometric analysis of Annexin V binding. The expression of Bcl-2 and the release of cytochrome c from mitochondria were evaluated by western blotting. We found that rapamycin induced apoptosis in NSCLC cell lines with p53 mutations. Western blot analysis demonstrated that rapamycin downregulates the expression levels of Bcl-2, which leads to increased cytochrome c release from mitochondria and subsequent activation of caspase cascades. These findings suggest that rapamycin induces p53-independent apoptosis through downregulation of Bcl-2 and the mitochondrial pathway in NSCLC cell lines as a novel antitumor mechanism.

Topics: Antibiotics, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Lung Neoplasms; Mitochondria; Mutation; Sirolimus; Tumor Suppressor Protein p53

Flavokawain B (FKB) possesses strong anti-neoplastic activity against many cancer cells. Here we assessed its antitumor activity and molecular mechanisms in lung cancer H460 cells in vitro. FKB significantly inhibited cell proliferation and caused arrest of the cell cycle G2-M of H460 cells in a dose-dependent manner. FKB also inducted apoptosis, which was associated with cytochrome c release, caspase-7 and caspase-9 activation and Bcl-xL/Bax dys-regulation. FKB significantly down-regulated survivin and XIAP, and the inhibitory effect induced by FKB was greatly attenuated by through over-expression of survivin or Bax(-/-) MEFs. Furthermore, FKB activated the mitogen-activated protein kinases and the JNK inhibitor SP600125 significantly decreased the growth-inhibitory and apoptotic effects of FKB. Together, these results suggest the anti-lung cancer potential of flavokawain B for the prevention and treatment of lung cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Flavonoids; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; MAP Kinase Signaling System; Mitochondria; Poly(ADP-ribose) Polymerases; Survivin

Celastrol is a natural compound extracted from the traditional Chinese medicinal herb, Tripterygium wilfordii Hook. It has attracted interests for its potential anti-inflammatory and antitumor effects. However, the molecular mechanisms of celastrol-induced apoptosis in cancer cells remain unclear. In this study, we investigated the effects of celastrol on the human non-small-cell lung cancer (NSCLC) cell line A549 in vitro. Celastrol caused a dose- and time-dependent growth inhibition of A549 cells with an IC(50) of 2.12 μM at 48 h treatment. Celastrol induced A549 cells apoptosis as confirmed by annexin V/propidium iodide staining and DNA fragmentation. Celastrol-induced apoptosis was characterized by cleavage of caspase-9, caspase-8, caspase-3, and PARP protein, increased Fas and FasL expression, and a reduction in the mitochondrial membrane potential. Furthermore, celastrol induced the release of cytochrome c. Celastrol also up-regulated the expression of pro-apoptotic Bax, down-regulated anti-apoptotic Bcl-2, and inhibited Akt phosphorylation. These results demonstrate that celastrol can induce apoptosis of human NSCLC A549 cells through activation of both mitochondria- and FasL-mediated pathways.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Down-Regulation; Fas Ligand Protein; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Pentacyclic Triterpenes; Propidium; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Triterpenes; Up-Regulation

Transcription-dependent apoptosis triggered by p53 hardly occurs in alternative reading frame (ARF)-null cancer cells. Loss of ARF leads to hyperactivation of murine double minute 2 (MDM2), resulting in the degradation of p53. In the present study, A549 (ARF-null) human non-small lung cancer cells were transfected with a plasmid DNA encoding human wild-type p53 and the mitochondrial transmembrane domain of Tom5 (p53-Tom5) for delivering p53 to mitochondria. As a result, p53-Tom5 exclusively localized at mitochondria in A549 cells and suppressed the proliferation of them, whereas wild-type p53 did not. In addition, mitochondrial dysfunction and release of cytochrome c were induced by p53-Tom5 in A549 cells. These data suggest that p53-Tom5 suppressed the proliferation of A549 cells through direct mitochondrial dysfunction.

Topics: Apoptosis; Biological Transport; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Proliferation; Cytochromes c; DNA; Drug Resistance, Neoplasm; Gene Transfer Techniques; Genetic Therapy; Humans; Lung Neoplasms; Membrane Transport Proteins; Mitochondria; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Plasmids; Reading Frames; Transfection; Tumor Suppressor Protein p53

A major focus of cancer research is to identify compounds that sensitize resistant cancer cells to radiation treatment. Lung cancer cells, in particular, have high rates of radioresistance that lead to treatment failure. We have previously shown that the autophagy induced in the context of decreased apoptosis confers radiosensitivity to prostate and lung cancer cells. Zinc supplementation has antiapoptotic effects in cell culture. In addition, the accumulation of zinc in response to oxidative stress has been associated with increased autophagy in astrocyte and breast cancer cells.. In this study, we hypothesized that the zinc ionophore PCI-5002 radiosensitizes lung cancer cells by inducing autophagic cell death. To test this hypothesis, we used a combination of in vitro and in vivo approaches, including clonogenic assays to test for radiosensitivity, biochemical analyses of apoptosis and autophagy, and a xenograft mouse model of tumor growth.. We found that PCI-5002 reduced clonogenic survival in treated cells compared with untreated cells (0.03% versus 0.1% surviving fraction, p < 0.001). The increased radiosensitive fraction of PCI-5002-treated cells was accompanied by increased autophagy. PCI-5002 treatment also reduced caspase-3 cleavage. In an irradiated xenograft mouse model, the tumor growth of irradiated, PCI-5002-treated mice was slower than untreated, irradiated mice (25 days versus 22 days to reach a 1.0 cm tumor size).. PCI-5002 treatment sensitizes lung cancer cells to radiation, both in vitro and in vivo. This data suggest that PCI-5002 could potentially treat radioresistant/locally advanced lung cancer by amplifying the effects of radiotherapy.

Topics: Animals; Apoptosis; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cesium Radioisotopes; Coordination Complexes; Cytochromes c; Embryo, Mammalian; Female; Fibroblasts; Gamma Rays; Humans; Immunoblotting; Ionophores; Lung Neoplasms; Mice; Mice, Knockout; Mice, Nude; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays; Zinc

Trilinolein has been identified as one of the active constituents isolated from Panax notoginseng used widely in traditional Chinese medicine. Protective actions of Panax notoginseng against cerebral ischemia, beneficial effects on the cardiovascular system, and hemostatic, antioxidant, hypolipidemic, hepatoprotective, renoprotective and estrogen-like activities have been illustrated. In the present study, the effects of trilinolein on the growth of non-small cell lung carcinoma A549 were investigated. It was found that the exposure of A549 cells to trilinolein resulted in the growth inhibition and the induction of apoptosis in a dose- and time- dependent manner. Trilinolein treatment induced the upregulation of pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2 expression, which was associated with the proteolytic activation of caspases and the concomitant degradation of poly(ADP-ribose) polymerase (PARP) protein. Intracellular reactive oxygen species seem to play a role in the trilinolein-induced apoptosis, since ROS were produced early in the trilinolein treatment. Moreover, the activity of PI3K/Akt was downregulated in trilinolein-treated cells. Our results demonstrated that the most important regulators of trilinolein-induced apoptosis are Bcl-2 family and caspase-3, which are associated with cytochrome c release and dephosphorylation on the Akt signaling pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Humans; Panax notoginseng; Phosphatidylinositol 3-Kinases; Phosphorylation; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Triglycerides

Although AKT ⁄ protein kinase B is constitutively active in nonsmall cell lung cancer (NSCLC) cells and is an attractive target for enhancing the cytotoxicity of therapeutic agents, the distinct roles of the AKT isoforms in NSCLC are largely unknown. In the present study, we investigated the roles of AKT1 and AKT2 in NSCLC cells using RNAi. The siRNA targeting of AKT1 or AKT2 effectively decreased protein levels of AKT1 and AKT2, respectively, in A549 and H460 cells. Cisplatin treatment of these cells increased apoptotic cell death compared with control. The siRNA-induced knockdown of AKT1 in H460 cells significantly decreased basal MEK⁄ ERK1 ⁄ 2 activity, resulting in nuclear factor-κB activation, whereas knockdown of AKT2 resulted in anti-apoptotic Bcl-2 family protein MCL-1 (MCL-1) cleavage, the collapse of mitochondrial membrane potential, cytochrome c release, and activation of the caspase cascade. Consequently, both siRNA treatments enhanced the chemosensitivity of H460 cells to cisplatin. However, neither AKT1 nor AKT2 siRNA treatment had any effect of p27 expression, and although both treatments tended to induced G₂ ⁄M phase arrest, the effect was not statistically significant. Treatment with AKT1 siRNA markedly decreased colony formation growth and migration, but AKT2 siRNA had no significant effects on these parameters. These data suggest that AKT1 and AKT2 both contribute to cell survival, albeit via different mechanisms, and that the effects on cell growth and migration are predominantly regulated by AKT1. These findings may aid in refining targeted strategies for the inhibition of AKT isoforms towards the sensitization of NSCLC cells to therapeutic agents.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cisplatin; Cytochromes c; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinases; Myeloid Cell Leukemia Sequence 1 Protein; NF-kappa B; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Signal Transduction

PGD2 (prostaglandin D2) is a mediator in various pathophysiological processes, including inflammation and tumorigenesis. PGD2 can be converted into active metabolites and is known to activate two distinct receptors, DP (PGD2 receptor) and CRTH2/DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). In the past, PGD2 was thought to be involved principally in the process of inflammation. However, in recent years, several studies have shown that PGD2 has anti-proliferative ability against tumorigenesis and can induce cellular apoptosis via activation of the caspase-dependent pathway in human colorectal cancer cells, leukaemia cells and eosinophils. In the lung, where PGD2 is highly released when sensitized mast cells are challenged with allergen, the mechanism of PGD2-induced apoptosis is unclear. In the present study, A549 cells, a type of NSCLC (non-small cell lung carcinoma), were treated with PGD2 under various conditions, including while blocking DP and CRTH2/DP2 with the selective antagonists BWA868C and ramatroban respectively. We report here that PGD2 induces A549 cell death through the intrinsic apoptotic pathway, although the process does not appear to involve either DP or CRTH2/DP2. Similar results were also found with H2199 cells, another type of NSCLC. We found that PGD2 metabolites induce apoptosis effectively and that 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2) is a likely candidate for the principal apoptotic inducer in PGD2-induced apoptosis in NSCLC A549 cells.

Topics: Apoptosis; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Cytochromes c; Cytosol; DNA Fragmentation; Humans; Hydantoins; Mitochondria; Prostaglandin D2; Receptors, Immunologic; Receptors, Prostaglandin; Sulfonamides

Molecular mechanism(s) responsible for drug resistance of non-small cell lung cancer (NSCLC) cells to cisplatin was investigated. Results showed that cisplatin (50muM)-induced cell death (apoptosis) was more significant in CH27 and A549 cell lines than in H460. The high protein levels of X-linked inhibitor-of-apoptosis protein (XIAP) observed in H460 cells appeared to play a key role in the regulation of cisplatin resistance of H460 cells. XIAP can bind to and suppress the activities of caspase 3 in H460 cells and lead to apoptosis inhibition of these cells. Blockade of XIAP activity by Embelin (XIAP inhibitor) or siRNA has increased caspase 3 activities and promoted cisplatin-induced cell death of H460 cells. The results indicate a therapeutic value of Embelin and/or XIAP siRNA in the control of cisplatin-resistant NSCLC cells (H460).

Topics: Apoptosis; Benzoquinones; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cisplatin; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; RNA, Small Interfering; Time Factors; X-Linked Inhibitor of Apoptosis Protein

Although Ocimum sanctum has been used extensively for its medicinal values in India and China, its antitumor activity against human nonsmall cell lung carcinoma (NSCLC) A549 cells has not been investigated until now. Therefore, the antitumor mechanism of ethanol extracts of Ocimum sanctum (EEOS) was elucidated in A549 cells in vitro and the Lewis lung carcinoma (LLC) animal model. EEOS exerted cytotoxicity against A549 cells, increased the sub-G1 population and exhibited apoptotic bodies in A549 cells. Furthermore, EEOS cleaved poly(ADP-ribose)polymerase (PARP), released cytochrome C into cytosol and simultaneously activated caspase-9 and -3 proteins. Also, EEOS increased the ratio of proapoptotic protein Bax/antiapoptotic protein Bcl-2 and inhibited the phosphorylation of Akt and extracellular signal regulated kinase (ERK) in A549 cancer cells. In addition, it was found that EEOS can suppress the growth of LLC inoculated onto C57BL/6 mice in a dose-dependent manner. Overall, these results demonstrate that EEOS induces apoptosis in A549 cells via a mitochondria caspase dependent pathway and inhibits the in vivo growth of LLC, suggesting that EEOS can be applied to lung carcinoma as a chemopreventive candidate.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Lewis Lung; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Ocimum; Phosphorylation; Phytotherapy; Plant Extracts; Plant Leaves; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2

In this study, we have examined the molecular events induced by parthenolide, a sesquiterpene lactone, and explored possible mechanisms of resistance and sensitization of tumor cells to Taxol. We showed that parthenolide could antagonize Taxol-mediated nuclear factor-kappaB (NF-kappaB) nuclear translocation and activation and Bcl-xl up-regulation by selectively targeting I-kappaB kinase activity. In A549 cells, inhibition of nuclear factor-kappaB by parthenolide resulted in activation of the mitochondrial death pathway to promote cytochrome c release and caspase 3 and 9 activation. In contrast, Taxol alone induced apoptosis via a pathway independent of mitochondria cytochrome c cascade. In addition, depletion of Bcl-xl rescued the apoptotic response to Taxol. Moreover, treatment with parthenolide increased the efficacy of the Taxol-induced inhibition of A549 tumor xenografts in mice. This study elucidated the cellular responses induced by parthenolide that decrease the threshold of mitochondria-dependent apoptosis in the treatment of non-small cell lung cancer cells.

Topics: Analysis of Variance; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Synergism; Histocytochemistry; I-kappa B Kinase; Kaplan-Meier Estimate; Lung Neoplasms; Mice; Mitochondrial Membranes; NF-kappa B; Paclitaxel; Sesquiterpenes; Xenograft Model Antitumor Assays

Gene therapy and virotherapy are among the approaches currently being used to treat lung cancer. The success of cancer gene therapy depends on treatments where different types of tumors can be selectively targeted and destroyed without affecting normal cells and tissue. Previously, we described a promoter system (TTS) that we designed that is specifically targeted to lung cancer cells but which does not affect other types of cells including stem cells. In our study, we have enhanced the utility of the TTS system by inserting the pro-apoptotic gene BH3 domain interacting death agonist (Bid) into the TTS promoter system (TTS/Bid) to create a drug regulatable lung cancer-specific gene therapy. A recombinant adenoviral vector was used to introduce TTS/Bid (Ad-TTS/Bid) into lung cancer cells. BID expression and apoptosis occurred in A549 pulmonary adenocarcinoma cells but little Bid expression or apoptosis occurred in MCF7 breast cancer cells or in normal human lung fibroblasts. The use of cisplatin enhanced the processing of full length BID to t-BID which significantly increased lung cancer-specific cell death. In in vivo experiments, intraperitonal injection of cisplatin enhanced the antitumor effects of the vector in a lung cancer xeno-graft mouse model. Moreover, dexamethasone effectively suppressed exogenous BID expression and the antitumor effect of Ad-TTS/Bid both in vitro and in vivo. Here, we describe the efficacy of the use of cisplatin and dexamethasone with the anti lung cancer promoter system (Ad-TTS/Bid) for a safe and effective gene therapy against advanced lung cancer.

Topics: Adenocarcinoma; Adenoviridae; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Caspases; Cisplatin; Colony-Forming Units Assay; Cytochromes c; Dexamethasone; Female; Fibroblasts; Flow Cytometry; Humans; Immunoblotting; Lung; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Promoter Regions, Genetic; Xenograft Model Antitumor Assays

Combretastatin A-4 (CA-4), a natural stilbenoid isolated from Combretum caffrum, is a new vascular targeting agent (VTA) known for its antitumor activity due to its anti-tubulin properties. We investigated the molecular mechanisms leading to cell death in non-small cell lung cancer H460 cells induced by natural (CA-4) and synthetic stilbenoids (ST2151) structurally related to CA-4. We found that both compounds induced depolymerization and rearrangement of spindle microtubules, as well as an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Prolonged exposition to ST2151 led cells to organize multiple sites of tubulin repolymerization, whereas tubulin repolymerization was observed only after CA-4 washout. H460 cells were arrested at a pro-metaphase stage, with condensed chromosomes and a triggered spindle assembly checkpoint, as evaluated by kinetochore localization of Bub1 and Mad1 antibodies. Persistent checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, cytochrome c release, activation of caspase-9 and -3, PARP cleavage and DNA fragmentation. On the other hand, caspase-2, and -8 were not activated by the drug treatment. The ability of cells to reassemble tubulin in the presence of an activated checkpoint may be responsible for ST2151-induced multinucleation, a recognized sign of mitotic catastrophe. In conclusion, we believe that discovery of new agents able to trigger mitotic catastrophe cell death as a result of mitotic block and prolonged spindle checkpoint activation is particularly worthwhile, considering that tumor cells have a high proliferative rate and mitotic failure occurs irrespective of p53 status.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Humans; Lung Neoplasms; Microscopy, Electron; Microtubules; Mitochondria; Mitosis; Spindle Apparatus; Stilbenes

Defects in the apoptotic machinery may contribute to chemoresistance of non-small cell lung cancer (NSCLC) cells. We have previously showed a deficiency in mitochondria-dependent caspase-9 activation in NSCLC H460 cells after exposure to cisplatin, a drug widely used to treat NSCLC. Here we show that, unlike cisplatin, the novel anticancer agent bortezomib efficiently induces caspase-9 activation and apoptosis in H460 cells. A comparative analysis of molecular events underlying cell death in bortezomib-treated versus cisplatin-treated H460 cells revealed that bortezomib, but not cisplatin, caused a rapid and abundant release of cytochrome c and Smac/DIABLO from mitochondria. This was associated with a marked increase in levels of the BH3-only proapoptotic protein Noxa and the antiapoptotic protein Mcl-1. Taken together, our data show that bortezomib, by promoting a proapoptotic shift in the levels of proteins involved in mitochondrial outer-membrane permeabilization, is a potent activator of the mitochondrial pathway of apoptosis in NSCLC cells. Our preclinical results support further investigation of bortezomib-based therapies as a possible new treatment modality for NSCLC.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Carcinoma, Non-Small-Cell Lung; Caspase Inhibitors; Caspases; Cisplatin; Cytochromes c; Humans; Lung Neoplasms; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Tumor Cells, Cultured; Up-Regulation

Lung cancer is one of the most common causes of cancer death worldwide. Although recent advances in chemotherapy and radiation therapy have yielded modest improvements in patient outcomes, overall survival remains poor. Therefore, new therapeutic targets are needed. Phosphoinositide-dependent kinase-1 (PDK1) is one potential target. The aim of the present studies was to investigate the potential of a celecoxib-derived PDK1 inhibitor (OSU03013), that does not inhibit cyclooxygenase-2, to kill lung cancer cells in vitro. Using human non-small-cell lung cancer A549 cells, OSU03013 dose-dependently induced apoptosis. After 6 h of treatment with 7.5 microM OSU03013, 26% of the cells were apoptotic, compared to 4% of the control cells as determined by measuring the sub-G1 peak of propidium iodide stained cells with flow cytometry. A similar increase in apoptosis was evident using the Cell Death ELISA assay. OSU03013-induced apoptosis was accompanied by a reduction in the mitochondrial membrane potential, the release of cytochrome c and the cleavage of caspase-3. Surprisingly, the phosphorylation of Akt at serine 473 was increased in A549 cells treated with 7.5 microM OSU03013. However, the toxicity of OSU03013 was reduced in A549 cells expressing a constitutively active form of Akt. These data demonstrate that OSU03013 induces apoptosis in A549 cells via the mitochondrial pathway. Inhibition of the Akt pathway appears uninvolved in this toxicity, although Akt can provide protection. These results also suggest the potential of celecoxib-derived agents to treat some forms of lung cancer.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspases; Celecoxib; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytochromes c; Humans; Lung Neoplasms; Membrane Potentials; Mitochondria; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; Sulfonamides; Tumor Cells, Cultured

Sensitivity of tumor cells to treatment with anticancer drugs depends on expression and function of antiapoptotic and antioxidant proteins. The goal of our study was to determine the functional role of the novel antioxidant protein Peroxiredoxin V (PrxV), in protection of human lung carcinoma cell lines against apoptosis. Analysis of expression of PrxV in multiple lung carcinoma cell lines revealed a positive correlation between the expression of PrxV and radioresistance in vitro. Clones of the lung carcinoma cells U1810 with down-regulated expression of PrxV, or with its impaired enzymatic function (expression of redox-negative PrxV), demonstrated increased sensitivity to treatment with anticancer drugs etoposide and adriamycin. Pre-treatment of these clones with antioxidant N-acetyl-cysteine did not change their sensitivity to adriamycin, suggesting the involvement of a non-redox activity of PrxV. Expression of the redox-negative PrxV mainly affected the mitochondrial pathway of apoptosis, as assessed by cytochrome c release assay. Impairment of the PrxV enzymatic function also affected transmembrane potential and calcium loading capacity of mitochondria, as well as mitochondrial morphology. Altogether, these findings suggest that PrxV is a multifunctional protein, which is essential for protection against apoptosis induced by anticancer drugs.

Topics: Apoptosis; Calcium; Carcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cytochromes c; Down-Regulation; Humans; Lung Neoplasms; Membrane Potentials; Mitochondria; Peroxidases; Peroxiredoxins

Activation of caspase-9 in response to treatment with cytotoxic drugs is inhibited in NSCLC cells, which may contribute to the clinical resistance to chemotherapy shown in this type of tumor. The aim of the present study was to investigate the mechanism of caspase-9 inhibition, with a focus on a possible role of TUCAN as caspase-9 inhibitor and a determinant of chemosensitivity in NSCLC cells.. Caspase-9 processing and activation were investigated by Western blot and by measuring the cleavage of the fluorogenic substrate LEHD-AFC. Proteins interaction assays, and RNA interference in combination with cell viability and apoptosis assays were used to investigate the involvement of TUCAN in inhibition of caspase-9 and chemosensitivity NSCLC.. Analysis of the components of the caspase-9 activation pathway in a panel of NSCLC and SCLC cells revealed no intrinsic defects. In fact, exogenously added cytochrome c and dATP triggered procaspase-9 cleavage and activation in lung cancer cell lysates, suggesting the presence of an inhibitor. The reported inhibitor of caspase-9, TUCAN, was exclusively expressed in NSCLC cells. However, interactions between TUCAN and procaspase-9 could not be demonstrated by any of the assays used. Furthermore, RNA interference-mediated down-regulation of TUCAN did not restore cisplatin-induced caspase-9 activation or affect cisplatin sensitivity in NSCLC cells.. These results indicate that procaspase-9 is functional and can undergo activation and full processing in lung cancer cell extracts in the presence of additional cytochrome c/dATP. However, the inhibitory protein TUCAN does not play a role in inhibition of procaspase-9 and in determining the sensitivity to cisplatin in NSCLC.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; CARD Signaling Adaptor Proteins; Caspase 9; Caspase Inhibitors; Caspases; Cisplatin; Cytochromes c; Deoxyadenine Nucleotides; Down-Regulation; Gene Expression Profiling; Humans; Neoplasm Proteins; Protein Binding; RNA Interference; Tumor Cells, Cultured

The role of the peroxisome proliferator-activated receptor-gamma (PPARgamma) in cell differentiation, cell-cycle arrest, and apoptosis has attracted increasing attention. We have recently demonstrated that PPARgamma ligands-troglitazone (TGZ) induced apoptosis in lung cancer cells. In this report, we further studied the role of ERK1/2 in lung cancer cells treated by TGZ. The result demonstrated that TGZ induced PPARgamma and ERK1/2 accumulation in the nucleus, in which the co-localization of both proteins was found. The activation of ERK1/2 resulted in apoptosis via a mitochondrial pathway. Both PPARgamma siRNA and U0126, a specific inhibitor of ERK1/2, were able to block these effects of TGZ, suggesting that apoptosis induced by TGZ was PPARgamma and ERK1/2 dependent. Inhibition of ERK1/2 by U0126 also led to a significant decrease in the level of PPARgamma, indicating a positive cross-talk between PPARgamma and ERK1/2 or an auto-regulatory feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and apoptosis. In addition to ERK1/2, TGZ also activated Akt. Interestingly, inhibition of ERK1/2 prevented the activation of Akt whereas the suppression of Akt had no effect on ERK1/2, suggesting that Akt was not necessary for TGZ-PPARgamma-ERK pathway. However, the inhibition of Akt promoted the release of cytochrome c, suggesting the activation of Akt may have a negative effect on apoptosis induced by TGZ. In conclusion, our study has demonstrated that TGZ, a synthetic PPARgamma ligand, induced apoptosis in NCI-H23 lung cancer cells via a mitochondrial pathway and this pathway was PPARgamma and ERK1/2 dependent.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Differentiation; Cell Nucleus; Chromans; Cytochromes c; Enzyme Activation; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Potentials; Mitochondrial Membranes; Models, Biological; Phosphatidylinositol 3-Kinases; Phosphorylation; PPAR gamma; Thiazolidinediones; Troglitazone; Tumor Cells, Cultured

The effects of different marine derived agents were studied in A549 cell growth. These drugs induced cell cycle arrest at the G2-M phase associated with the up-regulation of GADD45alpha-gamma and down-regulation of c-Myc. In treated cells, GADD45alpha-gamma and c-Myc were up- and down-regulated, respectively. A cascade of events leading to apoptotic mitochondrial 'intrinsic' pathway was observed in treated cells: (1) dephosphorylation of BAD serine136; (2) BAD dissociation from 14-3-3 followed by its association with BCL-XL; (3) cytochrome c release; (4) caspase-3 activation, and (5) cleavage of vimentin. Caspase(s) inhibitor prevented the formation of cleavage products and, in turn, apoptosis was inhibited through a p53-independent mechanism. Moreover, these compounds did not activate NF-kappaB. Our findings may offer new insights into the mechanisms of action of these agents in A549 cells. The better understanding of their effects might be important to fully exploit the potential of these new drugs.

Topics: 14-3-3 Proteins; Antineoplastic Agents; Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cytochromes c; Gene Expression Regulation; Humans; Lung Neoplasms; Marine Toxins; Mitochondria; NF-kappa B; Nuclear Proteins; Phosphorylation; Proto-Oncogene Proteins c-myc; Vimentin

Beta-elemene is a novel anticancer drug, which was extracted from the ginger plant. However, the mechanism of action of beta-elemene in non-small-cell lung cancer (NSCLC) remains unknown. Here we show that beta-elemene had differential inhibitory effects on cell growth between NSCLC cell lines and lung fibroblast and bronchial epithelial cell lines. In addition, beta-elemene was found to arrest NSCLC cells at G2-M phase, the arrest being accompanied by decreases in the levels of cyclin B1 and phospho-Cdc2 (Thr-161) and increases in the levels of p27(kip1) and phospho-Cdc2 (Tyr-15). Moreover, beta-elemene reduced the expression of Cdc25C, which dephosphorylates/activates Cdc2, but enhanced the expression of the checkpoint kinase, Chk2, which phosphorylates/ inactivates Cdc25C. These findings suggest that the effect of beta-elemene on G2-M arrest in NSCLC cells is mediated partly by a Chk2-dependent mechanism. We also demonstrate that beta-elemene triggered apoptosis in NSCLC cells. Our results clearly show that beta-elemene induced caspase-3, -7 and -9 activities, decreased Bcl-2 expression, caused cytochrome c release and increased the levels of cleaved caspase-9 and poly(ADP-ribose) polymerase in NSCLC cells. These data indicate that the effect of beta-elemene on lung cancer cell death may be through a mitochondrial release of the cytochrome c-mediated apoptotic pathway.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Checkpoint Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Enzyme Activation; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Lung Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Sesquiterpenes; Tumor Suppressor Proteins

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. In order to understand the molecular alterations leading to heterogeneous cisplatin sensitivity and apoptosis inducibility in NSCLC cells, we analyzed various apoptotic pathways, including the activation of caspase-8, -9 and -3, the release of cytochrome c from mitochondria and the expression levels of pro- and anti-apoptotic proteins such as Bax, Bad, Bcl-2, Bcl-xL, Fas and p53 using heterogeneously apoptosis-sensitive cells (Ma-10, Ma-31 and Ma-46). Cisplatin treatment induced the activation of caspase-8, -9 and -3 and the release of cytochrome c in apoptosis-sensitive Ma-46. The expression of Bcl-xL was the highest and p53 was not expressed in apoptosis-resistant Ma-31, and Fas was not expressed in Ma-46. These expression levels were not correlated with the apoptosis inducibility of the three cell lines. These results suggest that blockage of the apoptotic signal from mitochondria is responsible for apoptosis resistance in NSCLC cell lines. Our findings also indicate that anti-apoptotic Bcl-xL and pro-apoptotic p53 are necessary but not sufficient for resistance to cisplatin-induced apoptosis in NSCLC cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspases; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; fas Receptor; Humans; Lung Neoplasms; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Beauvericin (BEA), a cyclic hexadepsipeptide from Codyceps cicadae, possesses anti-convulsion, anti-arrhythmia, sedation, and anti-tumor activities. It has been reported that BEA induces apoptosis in several cancer cell lines. However, the molecular mechanism underlying the BEA-induced apoptotic process is not yet clearly understood. In the present study, the intracellular signaling pathways of BEA-induced apoptosis in human non-small cell lung cancer (NSCLC) A549 cells were investigated using morphological analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique. In this study, BEA-induced apoptosis in human NSCLC A549 cells demonstrated a BEA concentration- and treatment time-dependent manner. This BEA-induced apoptosis in human NSCLC A549 cells was also accompanied by the up-regulation of Bax, Bak, and p-Bad and down-regulation of p-Bcl-2, but no effect on the levels of Bcl-X(L) or Bad proteins. Moreover, the BEA treatment resulted in a significant reduction of mitochondrial membrane potential, increase in the release of mitochondrial cytochrome c (cyt c), and activation of caspase 3. Furthermore, treatment with caspase 3 inhibitor (z-DEVD-fmk) was capable to prevent the BEA-induced caspase 3 activity and cell death. These results clearly demonstrate that the induction of apoptosis by BEA involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins, mitochondrial membrane potential, mitochondrial cyt c, and caspase 3, they all participate in BEA-induced apoptotic process in human NSCLC A549 cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Depsipeptides; DNA Fragmentation; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins c-bcl-2

The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes.

Topics: Adenoviridae; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Biological Assay; Buthionine Sulfoximine; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspases; Cell Line, Tumor; Combined Modality Therapy; Cytochromes c; Deoxyribonucleases; Gene Transfer Techniques; Genes, bcl-2; Genetic Vectors; Glutathione; Humans; Lung Neoplasms; Mitochondria; Oxidation-Reduction; Poly-ADP-Ribose Binding Proteins; Proto-Oncogene Proteins c-bcl-2

The intrinsic apoptosis apparatus plays a significant role in generating and amplifying cell death signals. In this study we examined whether there are differences in the expression of its components and in its functioning in non-small cell lung carcinoma (NSCLC) and the lung. We show that NSCLC cell lines express Apaf-1 and procaspase-9 and -3 proteins and that the expression of Apaf-1 and procaspase-3, but not of procaspase-9 and -7, is frequently up-regulated in NSCLC tissues as compared to the lung. NSCLC tissues and lungs and some NSCLC cell lines expressed also caspase-9S(b) and displayed a high caspase-9S(b)/procaspase-9 expression ratio. Procaspase-3 from NSCLCs and lungs was readily processed to caspase-3 by granzyme B or caspase-8, and the granzyme B-generated caspase-3-like activity was significantly higher in tumor tissues and cells than in lungs. By contrast, cytochrome c plus dATP could induce a significant increase of caspase-3-like activity in cytosol only in some NSCLC cell lines and in subsets of studied NSCLC tissues and lungs, while procaspase-3 and -7 were detectably processed only in NSCLC tissues which showed a high (cytochrome c+dATP)-induced caspase-3-like activity. Taken together, the present study provides evidence that the expression of Apaf-1 and procaspase-3 is up-regulated in NSCLCs and indicates that the tumors have a capability to suppress the apoptosome-driven caspase activation in their cytosol.

Topics: Actins; Adult; Aged; Apoptosis; Apoptotic Protease-Activating Factor 1; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 7; Caspases; Cell Line, Tumor; Cytochromes c; Cytosol; Deoxyadenine Nucleotides; Enzyme Activation; Enzyme Precursors; Female; Gene Expression; Granzymes; Humans; Immunoblotting; Lung Neoplasms; Male; Middle Aged; Protein Biosynthesis; Proteins; RNA, Messenger; Serine Endopeptidases; Transcription, Genetic

Cytotoxic agents eliminate tumor cells via different mechanisms including apoptosis, although this process is not equally efficient in all kinds of cancer cells. Thus, small cell lung carcinomas (SCLCs) are more sensitive than non-small cell lung carcinomas (NSCLCs) to therapy-induced killing. During apoptosis, several apoptogenic proteins release from the mitochondria. Among these proteins is Smac/DIABLO. Overexpression of Smac effectively potentiates apoptosis by neutralizing the caspase-inhibitory function of the inhibitors of apoptosis proteins (IAPs). However, the physiological relevance of endogenously released Smac in the promotion of malignant cell death is still unclear. Analysis of a panel of human lung cancer cell lines revealed that there is no altered Smac expression in NSCLC and SCLC that might initially impair the drug-induced cell death. Upon engagement of the mitochondrial pathway of apoptosis, etoposide provoked cytosolic accumulation of Smac along with cytochrome c and loss of the mitochondrial membrane potential. Most of these events as well as nuclear apoptotic changes required caspase activation in SCLC, but not in NSCLC. Unexpectedly, pan-caspase inhibition had no effect on Smac release. Co-treatment of SCLC with the IAP-binding peptide Smac-N7 enhanced etoposide-induced apoptosis in a concentration-dependent manner, whereas Smac downregulation by small interfering RNA (siRNA) did not influence caspase-3/-7 activities, nuclear morphological changes, DNA fragmentation, and plasma membrane integrity. Release of cytochrome c and mitochondrial protease Omi/HtrA2 is still detectable at these conditions. These data suggest that Smac deficiency may be compensated for by action of redundant determinants to kill cancer cells. Thus, translocation of endogenous Smac into cytosol does not play a critical role in cell death of human lung carcinoma after etoposide treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Membrane; Cytochromes c; DNA Fragmentation; Down-Regulation; Etoposide; HeLa Cells; High-Temperature Requirement A Serine Peptidase 2; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Peptides; Protein Transport; Proteins; RNA, Small Interfering; Serine Endopeptidases

The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.

Topics: Acid Anhydride Hydrolases; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 8; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Neoplasm Proteins