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

In the present study, we describe the cytotoxicity of the new drug prodigiosin (PG) in two small cell lung carcinoma (SCLC) cell lines, GLC4 and its derived doxorubicin-resistant GLC4/ADR cell line, which overexpresses multidrug-related protein 1 (MRP-1). We observed through Western blot that PG mediated cytochrome c release, caspase cascade activation and PARP cleavage, thereby leading to apoptosis in a dose-response manner. MRP-1 expression increased after PG treatment, although that does not lead to protein accumulation. The MTT assay showed no difference in sensitivity to PG between the two cell lines. Our results support PG as a potential drug for the treatment of lung cancer as it overcomes the multidrug resistance phenotype produced by MRP-1 overexpression.

Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Small Cell; Caspases; Cytochromes c; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Lung Neoplasms; Poly(ADP-ribose) Polymerases; Prodigiosin; Tumor Cells, Cultured

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carcinoma, Small Cell; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Drug Synergism; Humans; Lymphoma; Magnetic Resonance Spectroscopy; Mice; Mitochondria; Models, Molecular; Neoplasms; Nitrophenols; Paclitaxel; Piperazines; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship; Sulfonamides; Survival Rate

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 effect of the depletion or oxidation of cellular GSH on cytotoxicity of MG132 was assessed. Viability loss and decrease in GSH contents in small cell lung cancer (SCLC) cells treated with MG132 was attenuated by caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk). Thiol compounds (N-acetylcysteine and N-(2-mercaptopropionyl)glycine) and free radical scavengers reduced MG132-induced cell death. Antioxidants, including N-acetylcysteine, inhibited the MG132-induced nuclear damage, loss in mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c and caspase-3 activation. Depletion of GSH due to buthionine sulfoxime did not affect the cell viability loss, ROS formation and GSH depletion due to MG132 in SCLC cells. A thiol oxidant monochloramine, p-chloromercuribenzoate and N-ethylmaleiamide also did not affect cytotoxicity of MG132. The results suggest that the toxicity of MG132 on SCLC cells is mediated by activation of caspase-8, -9 and -3. Removal of free radicals and recovery of GSH contents may attenuate MG132-induced apoptotic cell death. Nevertheless, depletion or oxidation of cellular GSH may not affect toxicity of MG132.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Survival; Cytochromes c; DNA Damage; Drug Interactions; Enzyme Activation; Glutathione; Humans; Leupeptins; Membrane Potentials; Mitochondria; Oxidation-Reduction; Tumor Cells, Cultured

The effect of GSH depletion on mitochondrial damage and cell death due to mitomycin c (MMC) was assessed in small cell lung cancer (SCLC) cells. Cytotoxicity of MMC was attenuated by Tempol and dicumarol, inhibitors of the enzymatic reduction, and increased by xanthine oxidase. The MMC-induced cell death and decrease in the GSH contents in SCLC cells were inhibited by caspase inhibitors (z-DQMD.fmk, z-IETD.fmk and z-LEHD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol and N-(2-mercaptopropionyl)glycine, melatonin, rutin and carboxy-PTIO). Thiol compounds, melatonin and rutin attenuated the MMC-induced nuclear damage, decrease in mitochondrial transmembrane potential, release of cytochrome c and activation of caspase-3. Treatment of MMC caused a significant decrease in GSH contents in SCLC cells, which was followed by increase in the formation of reactive oxygen species. Depletion of GSH due to L-buthionine sulfoximine enhanced the MMC-induced activation of caspase-3 and cell death in SCLC cells. Antioxidants, including N-acetylcysteine, depressed formations of nitric oxide, malondialdehyde and carbonyls due to MMC in SCLC cells. The results show that the reductive activation of MMC may cause cell death in SCLC cells by inducing mitochondrial dysfunction, leading to caspase-3 activation, and by activation of caspase-8. The MMC-induced change in the mitochondrial membrane permeability, followed by cell death, in SCLC cells may be significantly enhanced by decrease in the intracellular GSH contents due to oxidative attack of free radicals.

Topics: Antibiotics, Antineoplastic; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Survival; Cytochromes c; Glutathione; Humans; Lung Neoplasms; Membrane Potentials; Mitochondria; Mitomycin; Nitric Oxide; Oxidation-Reduction; Reactive Oxygen Species; Tumor Cells, Cultured

The mitochondrial release of cytochrome c and Smac/DIABLO has been implicated in the activation of apoptosis in response to cell stress. Smac promotes cytochrome c-induced activation of caspases by sequestering the inhibitor of apoptosis protein (IAP) family of potent caspase suppressors. Differential release from mitochondria of cytochrome c and Smac can occur, but the underlying mechanism and physiological significance of this are unclear. Here we show that the mechanism by which fibroblast growth factor 2 (FGF-2) protects small cell lung cancer (SCLC) cells from etoposide-induced cell death involves inhibition of Smac release but not of cytochrome c release. This process is MEK dependent and correlates with an increased expression of XIAP and cellular IAP-1, mediated principally through translational regulation. Exogenous expression of XIAP is sufficient to inhibit caspase 9 activation, Smac release, and cell death induced by etoposide. Prevention of the FGF-2-promoted increase in levels of functional IAPs by RNA interference or the cell-permeant Smac amino-terminal peptide blocked FGF-2-induced protection. FGF-2 can thus protect SCLC cells from chemotherapeutic drugs by modulating IAP levels via posttranscriptional regulation, providing a mechanism for postmitochondrial survival signaling by the MEK/mitogen-activated protein kinase pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Small Cell; Carrier Proteins; Caspases; Cell Death; Cell Line, Tumor; Culture Media, Serum-Free; Cytochromes c; Enzyme Activation; Etoposide; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase 1; Mitochondria; Mitochondrial Proteins; Mitogen-Activated Protein Kinase Kinases; Nucleic Acid Synthesis Inhibitors; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proteins; X-Linked Inhibitor of Apoptosis Protein

Prodigiosin (PG) is a secondary metabolite, isolated from a culture of Serratia marcescens, which has shown potent cytotoxicity against various human cancer cell lines as well as immunosuppressive activity. The purpose of this study was to evaluate the role of mitochondria in PG-induced apoptosis. Therefore, we evaluated the apoptotic action of PG in GLC4 small cell lung cancer cell line by Hoechst 33342 staining. In these cells, we examined mitochondrial apoptosis-inducing factor (AIF) and cytochrome c (cyt c) release to the cytosol in PG time-response studies. These findings suggest that PG induces apoptosis in both caspase-dependent and caspase-independent pathways.

Topics: Apoptosis; Carcinoma, Small Cell; Cell Line, Tumor; Cytochromes c; Humans; Lung Neoplasms; Microscopy, Fluorescence; Mitochondria; Prodigiosin