benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Lung-Neoplasms

Lung cancer is a prevalent disease and is one of the leading causes of mortality worldwide. Despite the development of various anticancer drugs, the prognosis of lung cancer is relatively poor. Metastasis of lung cancer, as well as chemoresistance, is associated with a high mortality rate for patients with lung cancer. Camptothecin (CPT) is a well-known anticancer drug, which causes cancer cell apoptosis via the induction of DNA damage; however, the cytotoxicity of CPT easily reaches a plateau at a relatively high dose in lung cancer cells, thus limiting its efficacy. The present study demonstrated that CPT may induce autophagy in two human non‑small cell lung cancer cell lines, H1299 and H460. In addition, the results of a viability assay and Annexin V staining revealed that CPT-induced autophagy could protect lung cancer cells from programmed cell death. Conversely, the cytotoxicity of CPT was increased when autophagy was blocked by 3-methyladenine treatment. Furthermore, inhibition of autophagy enhanced the levels of CPT-induced DNA damage in the lung cancer cell lines. Accordingly, these findings suggested that autophagy exerts a protective role in CPT-treated lung cancer cells, and the combination of CPT with a specific inhibitor of autophagy may be considered a promising strategy for the future treatment of lung cancer.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Camptothecin; Carcinoma, Non-Small-Cell Lung; Caspase Inhibitors; Cell Line, Tumor; Cell Movement; DNA Damage; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Histones; Humans; Inhibitory Concentration 50; Lung Neoplasms

Ajoene, a garlic-derived organosulfur compound, exerts anti-tumorigenic effect against various cancers. However, little is known about the biological effect of ajoene on lung adenocarcinoma, an aggressive malignancy with dismal prognosis. We investigated the biological effect of ajoene on lung adenocarcinoma and the underlying pathway. Lung adenocarcinoma cells A549, NCI-H1373, and NCI-H1395, along with the noncancerous lung bronchus cells BEAS-2B, were used. MTT test showed that ajoene (25 μM) reduces viability of lung adenocarcinoma cells but not the noncancerous BEAS-2B cells. Bromodeoxyuridine incorporation assay revealed that ajoene inhibits proliferation of lung adenocarcinoma cells. Treatment of lung adenocarcinoma cells with ajoene enhances apoptosis and ROS generation in a time- and dose-dependent fashion. Abrogation of caspase activation by zVAD-fmk completely prevents the ajoene-induced apoptosis; whereas block of ROS generation by N-acetylcysteine partly abolishes the ajoene-induced apoptosis. ROS-mediated induction of apoptosis contributes partially to the anti-tumorigenic property of ajoene observed, a phenomenon also confirmed by xenograft tumor study. Mitogen activated protein kinases (MAPKs), pivots of ROS-mediated signaling pathway, are activated upon ajoene treatment; Jun-N-terminal kinase (JNK)/p38 activations are required for signaling pathway underlying the ajoene-induced apoptosis. Our results suggest that ROS-mediated activation of JNK/p38 contributes partially to the pro-apoptotic action of ajoene on cells of lung adenocarcinoma. Ajoene may be a promising chemotherapeutic agent for lung adenocarcinoma.

Topics: A549 Cells; Adenocarcinoma; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Cell Line, Tumor; Cell Survival; Disulfides; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Male; Mice, Nude; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Sulfoxides; Time Factors; Xenograft Model Antitumor Assays

Osimertinib (OSI, also known as AZD9291) is the newest FDA-approved epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for non-small cell lung cancer (NSCLC) patients with EGFR T790M mutation. However, resistance to OSI is likely to progress and the study of potential OSI-resistant mechanisms in advanced is necessary. Here, the OSI-resistant NCI-H1975/OSIR cells were established. After cells developed resistance to OSI, cell proliferation was decreased while cell migration and invasion were increased. The NCI-H1975/OSIR cells exhibited more resistance to gefitinib, erlotinib, afatinib, rociletinib, doxorubicin, and fluorouracil, meanwhile showing higher sensitivity to paclitaxel, when compared with NCI-H1975 cells. In addition, the NCI-H1975/OSIR cells did not display multidrug resistance phenotype. The activation and expression of EGFR were decreased after cells exhibited resistance. Compared with NCI-H1975 cells, the activation of ERK and AKT in NCI-H1975/OSIR cells could not be significantly inhibited by OSI treatment. Navitoclax (ABT-263)-induced cell viability inhibition and apoptosis were more significant in NCI-H1975/OSIR cells than that in NCI-H1975 cells. Moreover, these effects of navitoclax in NCI-H1975/OSIR cells could be reversed by pretreatment of Z-VAD-FMK. Collectively, loss of EGFR could pose as one of the OSI-resistant mechanisms and navitoclax might be the candidate drug for OSI-resistant NSCLC patients.

Topics: Acrylamides; Amino Acid Chloromethyl Ketones; Aniline Compounds; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Caspase Inhibitors; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; ErbB Receptors; Humans; Lung Neoplasms; Neoplasm Invasiveness; Piperazines; Protein Kinase Inhibitors; Signal Transduction; Sulfonamides; Time Factors

Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Benzimidazoles; Carbocyanines; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; Connexins; Drug Synergism; Flow Cytometry; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Indoles; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Nerve Tissue Proteins; Organoplatinum Compounds; Oxaliplatin; Robotics; Staining and Labeling; Staurosporine; Workflow

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autophagy; Autophagy-Related Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Docetaxel; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fungal Proteins; Ganoderma; Humans; Lung Neoplasms; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred NOD; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Taxoids; Thapsigargin; TOR Serine-Threonine Kinases; Tunicamycin; Vincristine; Xenograft Model Antitumor Assays

The fungus-derived compound cephalochromin, isolated from the fermented broth of Cosmospora vilior YMJ89051501, shows growth-inhibitory and apoptotic activity against human lung cancer A549 cells in a concentration-dependent manner with an IC50 value of 2.8 μM at 48 h. Cephalochromin induced cell cycle arrest at the G0/G1 phase through down-regulation of cyclin D1, cyclin E, Cdk 2, and Cdk 4 expressions. Cephalochromin markedly increased the hypodiploid sub-G1 phase (apoptosis) of the cell cycle at 48 h as measured by flow cytometric analysis. Reactive oxygen species generation and loss of the mitochondrial membrane potential (MMP) were also markedly induced by cephalochromin. Moreover, the immunoblotting assays showed that cephalochromin reduced survivin and Bcl-xL expression and induced the activation of caspase-8, -9, and -3 and the cleavage of poly(ADP-ribose) polymerase, indicating the involvement of a caspase signaling cascade. The caspase inhibitor Z-VAD-fmk significantly suppressed cephalochromin-induced apoptosis. Cephalochromin also triggered LC3 II, autophagic marker, expression. Taken together, this is the first report that cephalochromin induced an antiproliferative effect on human lung cancer cells through mitochondrial disruption and down-regulation of survivin, leading to cell cycle arrest at the G0/G1 phase, loss of MMP, and subsequently apoptotic cell death.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cephalosporins; Dose-Response Relationship, Drug; Down-Regulation; G1 Phase; G1 Phase Cell Cycle Checkpoints; Humans; Inhibitory Concentration 50; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Poly(ADP-ribose) Polymerases

To infect lung adenocarcinoma A549 cells with recombinant LaSota strain Newcastle disease virus (NDV) vaccine expressing the rabies virus glycoprotein (rL-RVG), and explore the effect of rL-RVG on proliferation and apoptosis of lung cancer cells.. A549 cells were infected with the rL-RVG and then detected for the expressions of RVG and NDV proteins by Western blotting. The cell proliferation was examined by MTT assay and apoptosis index and cell early apoptosis were respectively detected by TUNEL and annexin V-FITC/PI staining combined with flow cytometry. The expression of pro-apoptotic protein caspase-3 was observed by Western blotting. The LaSota strain of NDV was used as control group and PBS was the blank control.. Both RVG and NDV proteins were stably expressed in A549 cells infected with rL-RVG. MTT assay results showed that cell proliferation was significantly inhibited, and the inhibition rate of the rL-RVG group was higher than that of LaSota group. The apoptosis of A549 cells were promoted by rL-RVG infection. Flow cytometry revealed that the early apoptotic cells of the rL-RVG group increased as compared with the other two groups (P<0.05). Consistently, TUNEL assay showed the apoptotic index increased in the rL-RVG group as compared with the other two groups (P<0.05). Western blotting demonstrated that the expression of the pro-apoptotic protein caspase-3 was up-regulated. However, when we added specific broad-spectrum caspase inhibitor Z-VAD-FMK, the expression of the caspase-3 protein was obviously reduced.. The rL-RVG is stably expressed in the infected A549 cells. The rL-RVG inhibits lung cancer cell growth and promote cell apoptosis, and the effect of rL-RVG is better than the wild LaSota strain.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Flow Cytometry; Glycoproteins; Host-Pathogen Interactions; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Newcastle disease virus; Rabies virus; Recombinant Fusion Proteins; Viral Proteins

In this study, we investigated the anti-cancer effects of luteolin, a member of the flavonoid family, in NCI-H460 human lung carcinoma cells. It was shown that luteolin induces apoptotic cell death through modulating both the extrinsic pathway and intrinsic pathways, which are suppressed by z-VAD-fmk, indicating that luteolin triggers caspase-dependant apoptosis. Furthermore, we found that the α subunit of the eukaryotic initiation factor 2 (eIF2α/C/EBP homologous protein pathway, but not the c-Jun N-terminal kinase pathway, played a critical role in induction of apoptosis by luteolin. The data indicated that luteolin also induces autophagy; evidence for this is the accumulation of microtubule-associated protein light chain-3 (LC3) II protein, the increase of LC3 puncta as well as an enhanced autophagy flux. In addition, inhibiting autophagy by bafilomycin A1 reduced apoptotic cell death, suggesting that luteolin-induced autophagy functions as a cell death mechanism. Notably, the activated caspases that appeared with luteolin treatment cleaved Beclin-1, and the expression of LC3II remained the same even after cells were challenged with Beclin-1 siRNA, demonstrating that luteolin induces Beclin-1-independent autophagy. Taken together, our findings showed that luteolin triggers both endoplasmic reticulum stress-related apoptosis and non-canonical autophagy, which function as a cell death mechanism in NCI-H460 human lung cancer cells.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; DNA Fragmentation; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Luteolin; Macrolides; Membrane Proteins; Microtubule-Associated Proteins; RNA, Small Interfering

Herein, inhibition of hepatocyte growth factor receptor, c-Met, significantly increased cytochrome c release and Bax/Bcl-2 ratio, indicating that c-Met played an anti-apoptotic role. The following experiments are to elucidate this anti-apoptotic mechanism, then the effect of c-Met on autophagy has also been discussed.. Investigated was the influence of c-Met on apoptosis, autophagy and loss of mitochondrial transmembrane potential (Δψm), and the relevant proteins were examined.. First, we found that activation of extracellular signal-regulated kinase (ERK), p53 was promoted by c-Met interference. Subsequent studies indicated that ERK was the upstream effector of p53, and this ERK-p53 pathway mediated release of cytochrome c and up-regulation of Bax/Bcl-2 ratio. Secondly, the inhibition of c-Met augmented oridonin-induced loss of mitochondrial transmembrane potential (Δψm), resulting apoptosis. Finally, the inhibition of c-Met increased oridonin-induced A549 cell autophagy accompanied by Beclin-1 activation and conversion from microtubule-associated protein light chain 3 (LC3)-I to LC3-II. Activation of ERK-p53 was also detected in autophagy process and could be augmented by inhibition of c-Met. Moreover, suppression of autophagy by 3-methyladenine (3-MA) or small interfering RNA against Beclin-1 or Atg5 decreased oridonin-induced apoptosis. Inhibition of apoptosis by pan-caspase inhibitor (z-VAD-fmk) decreased oridonin-induced autophagy as well and Loss of Δψm also occurred during autophagic process.. Thus, inhibiting c-Met enhanced oridonin-induced apoptosis, autophagy and loss of Δψm in A549 cells.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Beclin-1; Cytochromes c; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Humans; Isodon; Lung Neoplasms; Membrane Potential, Mitochondrial; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-met; RNA, Small Interfering; Tumor Suppressor Protein p53

The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Autophagy; Autophagy-Related Proteins; Carcinoma, Non-Small-Cell Lung; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Enzyme Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Macrolides; Microtubule-Associated Proteins; NF-E2-Related Factor 2; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Saponins; Survivin; Ubiquitin-Conjugating Enzymes

Anacardic acid (AA) is a constituent of the cashew nut shell and is known as an inhibitor of nuclear factor-κB (NF-κB). We investigated the cytotoxicity of AA on cancer cells and more experiments to reveal the cell death mechanism focused on A549 lung adenocarcinoma cells for our interest in lung cancer. To examine the molecular mechanism of cell death in AA treated A549 cells, we performed experiments such as transmission electron microscopy (TEM), western blot analysis, fluorescence-activated cell sorting (FACS), genomic DNA extraction and staining with 4',6-diamidino-2-phenylindole (DAPI). For the first time we revealed that AA induces caspase-independent apoptosis with no inhibition of cytotoxicity by pan-caspase inhibitor, Z-VAD-fmk, in A549 cells. Our results showed the possibility of mitochondrial-mediated apoptosis through the activation of apoptosis-inducing factor (AIF) and an intrinsic pathway executioner such as cytochrome c. This study will be helpful in revealing the cell death mechanisms and in developing potential drugs for lung cancer using AA.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Amino Acid Chloromethyl Ketones; Anacardic Acids; Apoptosis; Apoptosis Inducing Factor; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; HEK293 Cells; Hep G2 Cells; Humans; Lung Neoplasms; Mitochondria; NF-kappa B; Signal Transduction

A major concern in cancer therapy is resistance of tumors such as human non-small cell lung cancer and esophageal cancer to radiotherapy. Intrinsic radioresistance of these cancer cells limits therapeutic efficiency. Here, we determined in two cancer cell lines the potential radiosensitizing activity of Tat-SmacN7, a small molecule compound, which mimics the activity of Smac, a mitochondrial protein released during apoptosis. We found that Tat-SmacN7 can enter the cells and promote RNA expression and the activity of caspase-3, -8 and -9 and sensitized the cancer cells to radiation with a sensitization enhancement ratio (SER) of 1.5-1.6. Tat-SmacN7 radiosensitization was mediated by both extrinsic and intrinsic apoptosis pathways through activation of caspases. Consistently, blockage of caspase activation, through treatment with a caspase inhibitor, z-VAD-fmk, inhibited apoptosis and abrogated Tat-SmacN7 radiosensitization. Our study demonstrates that Tat-SmacN7 also has radiosensitization effects in vivo, so it could be further developed as a novel class of radiosensitizers for the treatment of radioresistant human non-small cell lung cancer and esophageal cancer.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Gene Products, tat; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Nude; Mitochondrial Proteins; Neoplasm Transplantation; Peptides; Radiation Tolerance; Radiation-Sensitizing Agents; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

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

This study was performed to elucidate the anticancer mechanism of a lipid-soluble ginseng extract (LSGE) by analyzing induction of apoptosis and arrest of cell cycle progression using the NCI-H460 human lung cancer cell line. Proliferation of NCI-H460 cells was potently inhibited by LSGE in a dose-dependent manner. The cell cycle arrest at the G0/G1 phase in NCI-H460 cells was induced by LSGE. The percentage of G0/G1 phase cells significantly increased, while that of S phase cells decreased after treatment with LSGE. The expression levels of cyclin-dependent kinase2 (CDK2), CDK4, CDK6, cyclin D3 and cyclin E related to G0/G1 cells progression were also altered by LSGE. In addition, LSGE-induced cell death occurred through apoptosis, which was accompanied by increasing the activity of caspases including caspase-8, caspase-9 and caspase-3. Consistent with enhancement of caspase activity, LSGE increased protein levels of cleaved caspase-3, caspase-8, caspase-9, and poly-ADP-ribose polymerase (PARP). These apoptotic effects of LSGE were inhibited by the pan-caspase inhibitor Z-VAD-fmk. These findings indicate that LSGE inhibits NCI-H460 human lung cancer cell growth by cell cycle arrest at the G0/G1 phase and induction of caspase-mediated apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Cycle; Cell Death; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; G1 Phase; Humans; Lipids; Lung Neoplasms; Panax; Plant Extracts; Poly(ADP-ribose) Polymerases; Resting Phase, Cell Cycle; S Phase; Solubility

Several studies have shown that curcumin can induce apoptosis and inhibit growth in human tumor cell lines. However, the mechanism is not completely understood yet. The present studies were designed to investigate the effects of curcumin on human A549 lung adenocarcinoma cells lines to better understand its effect on apoptosis and apoptosis-related genes in vitro. Apoptosis induction, mitochondria membrane potential, mitochondria structure, and apoptotic associated gene expression were examined by flow cytometric assay, confocal microscopy, Western blotting and electron microscopy. After treatment with curcumin, percentage of apoptotic cells increased dose- and time-dependently, and morphology observation revealed typical apoptotic features. Our data further indicated that the expression of Bax proteins in A549 cells was increased in a dose-dependent manner, whereas the expression of Bcl-2 was significantly decreased, thus the ratio of Bax/Bcl-2 was increased. The apoptotic process was accompanied by the change of mitochondrial function and structure which led to release of the cytochrome c, and activation of caspase-9 and caspase-3. Furthermore, curcumin also induced a dose-dependent cleavage of PARP. Caspases activation during the course of curcumin-induced apoptosis was additionally confirmed by using a broad-spectrum caspases inhibitor, Z-VAD-fmk. As expected, the inhibitor was able to decrease curcumin-induced apoptosis on A549 cells. These results suggested that mitochondria played an important role in the curcumin-induced apoptosis, and mitochondria membrane potential loss initiated apoptosis via the activation of caspases.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Shape; Curcumin; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Time Factors

Sanguinarine is a plant-derived benzophenanthridine alkaloid and has been shown to possess anti-tumor activities against various cancer cells. In this study, we investigated whether sanguinarine induces apoptosis in A549 human lung cancer cells. Treatment of A549 cells with sanguinarine induced apoptosis in a dose- and time-dependent manner. Treatment with sanguinarine led to activation of caspases and MAPKs as well as increased MKP-1 expression. Importantly, pretreatment with z-VAD-fmk, a pan caspase inhibitor suppressed the sanguinarine-induced apoptosis in A549 cells. Moreover, pretreatment with NAC, a sulfhydryl group-containing reducing agent strongly suppressed the apoptotic response and caspase activation to sanguinarine. However, the sanguinarine-mediated cytotoxicity in A549 cells was not protected by pharmacological inhibition of MAPKs or MKP-1 siRNA-mediated knockdown of MKP-1. These results collectively suggest that sanguinarine induces apoptosis in A549 cells through cellular glutathione depletion and the subsequent caspase activation.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Benzophenanthridines; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Dual Specificity Phosphatase 1; Enzyme Activation; Enzyme Inhibitors; Gene Silencing; Glutathione; Humans; Isoquinolines; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Neuroprotective Agents; RNA, Small Interfering

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.

Topics: Amino Acid Chloromethyl Ketones; Apoptotic Protease-Activating Factor 1; Azepines; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Membrane Permeability; DNA Damage; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Mitochondria; Peptoids; RNA, Small Interfering; S Phase; Transfection

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood.. In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (DeltaPsim) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis.. Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of DeltaPsim and the activation of caspase-3.. These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

Topics: Amino Acid Chloromethyl Ketones; Antimalarials; Apoptosis; Artemisinins; Caspase 3; Cell Line, Tumor; Cell Shape; Cell Survival; Cysteine Proteinase Inhibitors; Enzyme Activation; Fluorescence Resonance Energy Transfer; Humans; Lung; Lung Neoplasms; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria

Commonly used regimens in cancer therapy rely on the induction of apoptotic cell death, and drug resistance can be attributed, at least in part, to a disabled apoptotic program. Non-small cell lung carcinomas (NSCLC), exhibit an intrinsic resistance to chemotherapy. Here, we show that co-treatment with etoposide (VP16) and the pan-histone deacetylase (HDAC) inhibitor trichostatin A (TSA), but not valproic acid (VPA), induced apoptotic cell death in drug-resistant NSCLC cells. Co-treatment, but not single treatment, with VP16 and TSA induced apoptosis in a caspase-dependent manner accompanied by a crucial decrease in Bcl-xL expression allowing Bax activation and subsequent initiation of the apoptosis inducing factor (AIF)-dependent death pathway. Importantly, AIF proved to be required for the effects of TSA/VP16 as RNA knockdown of AIF resulted in a complete abolishment of TSA/VP16-induced apoptotic cell death in drug-resistant NSCLC cells. Our results thus provide evidence for the requirement of both caspase-dependent and caspase-independent apoptotic pathways in TSA/VP16-mediated death of drug-resistant NSCLC cells, and extend previous suggestions that HDAC inhibitors in combination with conventional chemotherapeutic drugs could be valuable in the treatment of NSCLC cancer and other malignancies in which Bcl-xL is overexpressed.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Caspase Inhibitors; Caspases; Drug Evaluation, Preclinical; Etoposide; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Models, Biological; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; Caspase 3; Caspase Inhibitors; Cell Count; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Oncogene Protein v-akt; Phenanthrenes

Histone deacetylase inhibitors (HDIs) are a promising new class of antineoplastic agents with the capacity to induce growth arrest and/or apoptosis of cancer cells. However, their precise mechanism of action is uncertain; particularly, the role of caspases in the apoptotic response to HDIs is controversial. Here, we show that the HDIs explored, suberoylanilide hydroxamic acid, sodium butyrate and trichostatin A, activated caspase-3 in A549 and PC-3 carcinoma cells. Additionally, the poly-caspase inhibitor z-VAD-fmk prevented HDI-induced apoptosis, as judged by determining mitochondrial membrane potential and by quantifying internucleosomal DNA fragmentation. Importantly, z-VAD-fmk also significantly inhibited HDI-elicited cell death, as assessed by measuring propidium iodide uptake. As an accessory finding, with the inhibition of caspases, a HDI-induced G2-M arrest became evident. Taken together, these results provide evidence that HDIs require activated caspases to induce apoptosis of carcinoma cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Line, Tumor; DNA Fragmentation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Lung Neoplasms; Male; Prostatic Neoplasms

Malignant mesothelioma (MM) is a cancer with uniformly poor responses to current therapeutic regimens. This study evaluates whether taurolidine, a novel antineoplastic agent, is effective against human MM cell lines and a murine model of human MM.. Cell growth inhibition and viability assays were performed on REN, LRK, and H28 cell lines after 24-72-h exposure to 0-200 microm taurolidine. Cell cycle analysis with annexin-V binding, terminal deoxynucleotidyl transferase-mediated nick end labeling assay, electron microscopy, and response to the general caspase inhibitor z-VAD-fmk were performed on MM cell lines after 24-72-h exposure to 50-150 microm taurolidine. Athymic mice were given i.p. injections of 20 x 10(6) REN cells, followed by i.p. taurolidine (17.5 or 20 mg), 3 days/week for up to 3 weeks. Tumors were assessed at day 30. All statistical tests were two-sided.. A 72-h exposure of MM cells to taurolidine showed IC50 of 28-42.7 microm and 50% viability at 49.8-135 microm. Annexin V assay for apoptosis revealed significant increases in annexin binding after 24-72-h exposure to 50-150 microm taurolidine (P <0.05), which was significantly inhibited by z-VAD (P <0.05). MM cells exposed to 50-150 microm taurolidine for 24-72 h showed terminal deoxynucleotidyl transferase-mediated nick end labeling staining consistent with apoptosis, as well as structural evidence of apoptosis via electron microscopy. In vivo, there were significant tumor reductions (62 to >99% reduction) for all dosage regimens compared with untreated controls (P <0.001). In addition, all control animals exhibited ascites and diaphragmatic tumors while treated animals did not.. Taurolidine has significant antineoplastic activity against MM in vitro and in vivo, in part, due to tumor cell apoptosis. These findings warrant further study for potential clinical usefulness.

Topics: Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Antineoplastic Agents; Apoptosis; Body Weight; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Models, Animal; Enzyme Inhibitors; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Male; Mesothelioma; Mice; Mice, Nude; Microscopy, Electron; Phosphatidylserines; Taurine; Thiadiazines; Time Factors

We have found that omega-hydroxy palmitic acid (16-hydroxy palmitic acid, omega-HPA) has both cell growth inhibiting and cell death inducing actions on human lung adenosquamous carcinoma cell line H596 and adenocarcinoma cell line A549. Further, these effects were dose- and time-dependent in both cell lines. However, in squamous carcinoma cell line H226, omega-HPA had no cytotoxic effect. On the other hand, in the human small cell lung carcinoma (SCLC) cell line H128, this compound showed weak cytotoxicity. The sensitivity toward omega-HPA was higher in H596 cells than in A549 cells. In both H596 and A549 cells, cell growth was inhibited to 24.4 and 9.4%, respectively, by treatment with 100 microM omega-HPA for 12 h. In the 24 h treatment cells, growth inhibition was increased to 100 and 38.1%, respectively. In cytotoxicity experiments, the number of dead cells increased with incubation times in the presence of omega-HPA: on three days incubation with 100 microM omega-HPA, viability was 0 and 13.5%, respectively, in H596 and A549 cells. Further, the fragmentation of DNA to oligonucleosomal-sized ladder fragments, which is an index of apoptosis, was observed in both cell lines on treatment with omega-HPA. Therefore, it is assumed that these cell deaths induced by omega-HPA, were apoptosis in these cell lines. Since the number of dead cells following treatment with omega-HPA decreased by treatment with omega-HPA in combination with Z-VAD-fmk, a caspase family inhibitor, it is thought that apoptotic cell death was related to caspase activity.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Caspase Inhibitors; Cell Division; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Palmitic Acids; Time Factors; Tumor Cells, Cultured

Apoptosis, a tightly controlled multi-step mechanism of cell death, is important for anti-cancer therapy-based elimination of tumor cells. However, this process is not always efficient. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) cells display different susceptibility to undergo apoptosis induced by anticancer treatment. In contrast to SCLC, NSCLC cells are cross-resistant to a broad spectrum of apoptotic stimuli, including receptor stimulation, cytotoxic drugs and gamma-radiation. Since resistance of tumor cells to treatment often accounts for the failure of traditional forms of cancer therapy, in the present study attempts to find a potent broad-range apoptosis inductor, which can kill therapy-resistant NSCLC cells were undertaken and the mechanism of apoptosis induction by this drug was investigated in detail. We found that staurosporine (STS) had cell killing effect on both types of lung carcinomas. Release of cytochrome c, activation of apical and effector caspases followed by cleavage of their nuclear substrates and morphological changes specific for apoptosis were observed in STS-treated cells. In contrast to treatment with radiation or chemotherapy drugs, STS induces mitochondrial dysfunction followed by translocation of AIF into the nuclei. These events preceded the activation of nuclear apoptosis. Thus, in lung carcinomas two cell death pathways, caspase-dependent and caspase-independent, coexist. In NSCLC cells, where the caspase-dependent pathway is less efficient, the triggering of an AIF-mediated caspase-independent mechanism circumvents the resistance of these cells to treatment.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Caspase 3; Caspase 7; Caspases; Cell Cycle; Cell Nucleus; Cysteine Proteinase Inhibitors; Cytochrome c Group; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Flavoproteins; Gamma Rays; Humans; Jurkat Cells; Lung Neoplasms; Membrane Potentials; Membrane Proteins; Mitochondria; Poly(ADP-ribose) Polymerases; Protein Transport; Radiation Tolerance; Reactive Oxygen Species; Recombinant Fusion Proteins; Staurosporine; Superoxides; Tumor Cells, Cultured

Here we report on the role of mitochondria, death receptors (DRs), and caspases in exerting the cytotoxic effect of clinically relevant concentrations of paclitaxel in the non-small cell lung cancer cell line NCI-H460.. We have characterized paclitaxel-induced cell death with annexin V, propidium iodide staining, and poly(ADP-ribose) polymerase cleavage assays. The involvement of the mitochondria pathway was studied by monitoring cytochrome c release and using H460 cells stable in overexpressing Bcl-2 or Bcl-x(L). DR dependency was analyzed in FADD dominant-negative or cytokine response modifier A-overexpressing cells, and a possible role for DR4 and DR5 was investigated by antagonistic antibodies. Caspase activity and cleavage assays and treatment with the synthetic inhibitor zVAD-fmk were used to determine the involvement of caspases.. Paclitaxel-treated cells displayed several features of apoptosis, including annexin V staining and poly(ADP-ribose) polymerase cleavage. The sequence of events suggested the involvement of a DR, as indicated by an early role for Fas-associated death domain and caspase-8, followed by cleavage of Bid and the disruption of mitochondria; nonetheless, we failed to demonstrate the involvement of DR4 and DR5. Interestingly, inhibition of either one of these routes only resulted in a 30% reduction of cell death that was in line with the observed small effect of caspase inhibition by zVAD-fmk on H460 cell survival.. Paclitaxel triggers cell death in H460 cells mainly via a currently unidentified caspase-independent mechanism in which the basic apoptotic machinery is merely coactivated. This finding is in sharp contrast with the largely caspase-dependent response elicited by DNA-damaging agents in these cells. We speculate on therapeutic implications.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Arabidopsis Proteins; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Caspase 8; Caspase 9; Caspases; Cell Death; Cell Line; Cell Survival; Cytochrome c Group; Cytosol; DNA Damage; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Fatty Acid Desaturases; Genes, Dominant; Humans; Lung Neoplasms; Mitochondria; Models, Biological; Paclitaxel; Poly(ADP-ribose) Polymerases; Propidium; Protein Structure, Tertiary; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Spectrometry, Fluorescence; Time Factors; Transfection; Tumor Cells, Cultured

The antiarrhythmic amiodarone (AM) and its metabolite desethylamiodarone (Des) are known to cause AM-induced pulmonary toxicity, but the mechanisms underlying this disorder remain unclear. We hypothesized that AM might cause AM-induced pulmonary toxicity in part through the induction of apoptosis or necrosis in alveolar epithelial cells (AECs). Two models of type II pneumocytes, the human AEC-derived A549 cell line and primary AECs isolated from adult Wistar rats, were incubated with AM or Des for 20 h. Apoptotic cells were determined by morphological assessment of nuclear fragmentation with propidium iodide on ethanol-fixed cells. Necrotic cells were quantitated by loss of dye exclusion. Both AM and Des caused dose-dependent necrosis starting at 2.5 and 0.1 microg/ml, respectively, in primary rat AECs and at 10 and 5 microg/ml in subconfluent A549 cells (P < 0.05 and P < 0.01, respectively). AM and Des also induced dose-dependent apoptosis beginning at 2.5 microg/ml in the primary AECs (P < 0.05 for both compounds) and at 10 and 5 microg/ml, respectively, in the A549 cell line (P < 0.01). The two compounds also caused significant net cell loss (up to 80% over 20 h of incubation) by either cell type at drug concentrations near or below the therapeutic serum concentration for AM. The cell loss was not due to detachment but was blocked by the broad-spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Furthermore, the angiotensin-converting enzyme inhibitor captopril (500 ng/ml) and the angiotensin-receptor antagonist saralasin (50 microg/ml) significantly inhibited both the induction of apoptosis and net cell loss in response to AM. These results are consistent with recent work from this laboratory demonstrating potent inhibition of apoptosis in human AECs by captopril (Uhal BD, Gidea C, Bargout R, Bifero A, Ibarra-Sunga O, Papp M, Flynn K, and Filippatos G. Am J Physiol Lung Cell Mol Physiol 275: L1013-L1017, 1998). They also suggested that the accumulation of AM and/or its primary metabolite Des in lung tissue may induce cytotoxicity of AECs that might be inhibitable by angiotensin-converting enzyme inhibitors or other antagonists of the renin-angiotensin system.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Amiodarone; Angiotensin-Converting Enzyme Inhibitors; Animals; Apoptosis; Captopril; Cysteine Proteinase Inhibitors; Cytotoxins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Humans; In Vitro Techniques; Lung Neoplasms; Male; Pulmonary Alveoli; Rats; Rats, Wistar; Renin-Angiotensin System; Tumor Cells, Cultured

Chemotherapy-induced apoptosis is generally thought to be dependent on a pathway headed by caspase-9. This model is primarily based on studies performed in leukemia cells; however, little is known about caspase cascades in relatively resistant solid tumor cells, including non-small cell lung cancer (NSCLC) cells. Using the NSCLC cell line NCI-H460 (H460), here, we studied the effect of stable expression of various caspase inhibitors on apoptosis induced by the anticancer drugs cisplatin, topotecan, and gemcitabine. Interestingly, overexpression of caspase-9S and X-linked inhibitor of apoptosis (XIAP), both able to inhibit caspase-9 activity, failed to block apoptosis. In contrast, stable expression of caspase-8 inhibitors, such as cytokine response modifier A (CrmA) and dominant-negative caspase-8, almost completely abrogated apoptosis and also enhanced clonogenic survival. Caspase-8 activation in H460 cells was not mediated by death receptors, inasmuch as overexpression of dominant-negative Fas-associated death domain (FADD-DN) did not prevent procaspase-8 cleavage and subsequent apoptosis. However, stable expression of Bcl-2 and Bcl-xL did suppress these apoptotic events, including the release of cytochrome c from mitochondria, which was observed in drug-treated H460 cells. In the NSCLC cell line H460, we, thus, provide evidence for the existence of a novel drug-inducible apoptotic pathway in which activation of caspase-8, and not of caspase-9, forms the apical and mitochondria-dependent step that subsequently activates the downstream caspases.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; bcl-X Protein; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cisplatin; Cytochrome c Group; Cytosol; Deoxycytidine; DNA, Complementary; Fas-Associated Death Domain Protein; Gemcitabine; Genes, Dominant; Humans; Inhibitory Concentration 50; Jurkat Cells; Lung Neoplasms; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Spectrometry, Fluorescence; Topotecan; Transfection; Tumor Cells, Cultured

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been shown to induce apoptosis in various tumor cell lines including human non-small cell lung carcinoma (NSCLC) cells, which are resistant to the natural all-trans retinoic acid and to many synthetic receptor-selective retinoids. Although the mechanism of this effect was not elucidated, it was found to be independent of nuclear retinoid receptors. In the present study, we analysed the mechanisms by which CD437 induces apoptosis in two human NSCLC cell lines: H460 with wild-type p53 and H1792 with mutant p53. Both cell lines underwent apoptosis after exposure to CD437, although the cell line with wild-type p53 (H460) was more sensitive to the induction of apoptosis. CD437 increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the H460 cells. The caspase inhibitors (Z-DEVD-FMK and Z-VAD-FMK) suppressed CD437-induced CPP32-like caspase activation and apoptosis in both cell lines. CD437 induced the expression of the p53 gene and its target genes, p21, Bax, and Killer/DR5, only in the H460 cells. These results suggest that CD437-induced apoptosis is more extensive in NSCLC cells that express wild-type p53, possibly due to the involvement of the p53 regulated genes Killer/DR5, and Bax although CD437 can also induce apoptosis by means of a p53-independent mechanism. Both pathways of CD437-induced apoptosis appear to involve activation of CPP32-like caspase.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase Inhibitors; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Lung Neoplasms; Neoplasm Proteins; Oligopeptides; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoids; Tumor Cells, Cultured

Resistance to chemotherapy is a principal problem in the treatment of small cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites. Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as a result of beta1 integrin-stimulated tyrosine kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create a specialized microenvironment, and the survival of cells bound to ECM could explain the partial responses and local recurrence of SCLC often seen clinically after chemotherapy. Strategies based on blocking beta1 integrin-mediated survival signals may represent a new therapeutic approach to improve the response to chemotherapy in SCLC.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Bradykinin; Carcinoma, Small Cell; Caspase 3; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Division; Collagen; Cyclophosphamide; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Inhibitors; Etoposide; Extracellular Matrix Proteins; Fibronectins; Galanin; Humans; Integrin beta1; Laminin; Lung Neoplasms; Protein-Tyrosine Kinases; Tenascin; Tumor Cells, Cultured; Tyrphostins

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been recently identified to be a potent inducer of apoptosis in human non-small cell lung carcinoma (NSCLC) cells through a nuclear retinoic acid receptor independent mechanism. To approach the mechanism by which CD437 induces apoptosis in NSCLC cells, we investigated the involvement of c-Myc in CD437-induced apoptosis. CD437 (1 microM) up-regulated the expression of c-Myc and of its downstream target genes ornithine decarboxylase (ODC) and cdc25A in all three NSCLC cell lines (i.e., H460, SK-MES-1 and H1792) used. These effects were correlated with cellular susceptibilities to induction of apoptosis by CD437. Furthermore, CD437-induced apoptosis could be blocked by the ODC inhibitor difluoromethylornithine, the caspase inhibitors Z-VAD FMK and Z-DEVD FMK, and c-Myc antisense oligodeoxynucleotide, respectively. These data indicate that c-Myc gene plays an important role in mediating CD437-induced apoptosis in human NSCLC cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Non-Small-Cell Lung; cdc25 Phosphatases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Eflornithine; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Lung Neoplasms; Oligonucleotides, Antisense; Oligopeptides; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-myc; Retinoids; Tumor Cells, Cultured

Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 microg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensinogen; Animals; Antibodies; Antisense Elements (Genetics); Apoptosis; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Fibrosis; Gene Expression; Humans; Lisinopril; Lung Neoplasms; Male; Membrane Glycoproteins; Neutralization Tests; Peptidyl-Dipeptidase A; Pulmonary Alveoli; Rats; Rats, Wistar; Receptors, Angiotensin; Renin-Angiotensin System; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured