benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Carcinoma--Non-Small-Cell-Lung

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

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

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

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

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

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

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

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

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