benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Melanoma

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP-binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration- and time-dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase-3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP-bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin-induced cytotoxic effects in a variety of canine tumour cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dog Diseases; Dogs; Gene Expression Regulation, Neoplastic; Hemangiosarcoma; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lymphoma; Melanoma; ras Proteins; Simvastatin

Ursodeoxycholic acid (UDCA) is a type of hydrophilic bile acid extracted from animal bile with a wide range of biological functions. The present results demonstrated that UDCA could effectively inhibit the proliferation of two human melanoma cell line (M14 and A375) with time‑ and concentration‑dependence. Following exposure to various concentrations of UDCA, M14 cells exhibited typical morphological changes and weaker ability of colony forming. Flow cytometry analysis demonstrated that UDCA could induce a decrease of mitochondrial membrane potential and an increase in reactive oxygen species (ROS) levels in M14 cells. The cell cycle was arrested in the G2/M phase, which was confirmed by the decrease of cyclin‑dependent kinase 1 and cyclinB1 at the protein level. However, when M14 cells were treated with UDCA and Z‑VAD‑FMK (caspase inhibitor) synchronously, the apoptosis rate of the cells was reduced significantly. In addition, it was demonstrated that UDCA induced apoptosis of human melanoma M14 cells through the ROS‑triggered mitochondrial‑associated pathway, which was indicated by the increased expression of cleaved‑caspase‑3, cleaved‑caspase‑9, apoptotic protease activating factor‑1, cleaved‑poly (ADP‑ribose) polymerase 1 and the elevation of B cell lymphoma‑2 (Bcl‑2) associated X protein/Bcl‑2 ratio associated with apoptosis. Therefore, UDCA may be a potential drug for the treatment of human melanoma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Humans; Melanoma; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Reactive Oxygen Species; Signal Transduction; Skin Neoplasms; Ursodeoxycholic Acid

We have efficiently synthesized albiziabioside A (1) together with its six disaccharide analogues through a linear synthesis, and evaluated their cytotoxicity against six different skin cancer cells. All of the analogues showed weak cytotoxicity, with the exception of compound 1, which exhibited strong cytotoxicity against A375 cells. Albiziabioside A can induce cell cycle arrest in both the S and G2/M phases. Moreover, albiziabioside A can induce A375 cell apoptosis via mitochondrial pathways involving a caspase cascade. These results provide for the first time a basic mechanism for the anticancer activity of 1.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Saponins; Signal Transduction

The aim of the present study was to compare the effect of realgar nanoparticles and arsenic trioxide (ATO) on viability, DNA damage, proliferation, autophagy and apoptosis in the human melanoma cell lines BOWES and A375. The application of various flow cytometric methods for measurements cell viability, DNA cell cycle, mitochondrial potential, lysosomal activity, and intracellular content of glutathione was used. In addition, quantitative PCR, western blotting and multiplex bead array analyses were applied for evaluation of redox stress, autophagic flux, and cell signaling alterations.The results showed that realgar treatment of studied cells caused modulation of cell proliferation, induced a block in G2/M phase of the cell cycle and altered phosphorylation of IκB, Akt, ERK1/2, p38, and JNK kinases, as well as decreased mitochondrial membrane potential. Additionally, it appeared that induction of cell death by both realgar and ATO was dose-dependent, when lower (0.3 µM) dosage increased lysosomal activity and induced autophagy and higher (1.25 µM) concentration resulted in the appearance of apoptosis, while pan-caspase inhibitor attenuated more efficiently realgar- than ATO-induced cell death. Furthermore, low concentrations of ATO and realgar nanoparticles increased the content of intracellular glutathione and elevated γ-H2AX expression confirmed DNA damage preferentially at higher concentrations of both drugs used. Further analysis revealed slight differences in time-dependent phosphorylation pattern due to both realgar and ATO treatments, while significant differences were noticed between cell lines. In conclusion, realgar nanoparticles and ATO treatment induced dose-dependent activation of autophagy and apoptosis in both melanoma cell lines, when autophagy flux was determined at lower drug concentrations and the switch to apoptosis occurred at higher concentrations of both arsenic forms.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Autophagy; Cell Line, Tumor; Cell Proliferation; Chloroquine; DNA Damage; Glutathione; Humans; Melanoma; Nanoparticles; Oxides; Phosphorylation; Sulfides

The mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. This article shows that parthenolide stimulates in human osteosarcoma MG63 and melanoma SK-MEL-28 cells a mechanism of cell death, which is not prevented by z-VAD-fmk and other caspase inhibitors. In particular treatment with parthenolide rapidly stimulated (1-2 h) reactive oxygen species (ROS) generation by inducing activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, cell detachment from the matrix, and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca(2+) also favored dissipation of Δψm, which seemed primarily determined by permeability transition pore opening, since Δψm loss was partially prevented by the inhibitor cyclosporin A. Staining with Hoechst 33342 revealed in most cells, at 3-5 h of treatment, chromatin condensation, and fragmentation, while only few cells were propidium iodide (PI)-positive. In addition, at this stage apoptosis inducing factor (AIF) translocated to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15 h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by N-acetylcysteine, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis Inducing Factor; Caspase Inhibitors; Caspases; Cell Death; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Melanoma; NADPH Oxidases; NF-kappa B; Osteosarcoma; Reactive Oxygen Species; Sesquiterpenes

Oncolytic influenza A viruses with deleted NS1 gene (delNS1) replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15) coding sequence into the viral NS gene segment (delNS1-IL-15). DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1) infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected) melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Cells, Cultured; Chlorocebus aethiops; Cisplatin; Cysteine Proteinase Inhibitors; Gene Deletion; Humans; Influenza A Virus, H1N1 Subtype; Interleukin-15; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oncolytic Viruses; RNA Interference; Vero Cells; Viral Nonstructural Proteins; Virus Replication

Search for new substances with antiproliferative activity towards melanoma cells is important since malignant melanoma is notoriously resistant to conventional chemotherapy. Benzo[c]phenanthridine alkaloids (BAs) are natural products with significant anti-proliferative activities, therefore they are considered as agents promising for cancer therapy.. The effects of five BAs (sanguinarine, chelerythrine, chelidonine, sanguilutine, and chelilutine) on human malignant melanoma cell lines were compared. The study focused on BAs effects on DNA, anti-apoptotic and p53 protein levels; and the involvement of p53 in cellular responses to alkaloids treatment.. Melanoma cell lines, two wild types and two with dysfunctional p53 derived from one of them were used. The mechanism of anti-proliferative and pro-apoptotic effects and the effect on DNA was investigated using MTT assay, flow cytometry, Western blot analysis, fluorescence and electron microscopy.. All tested alkaloids exhibit strong anti-proliferative activity. CHL, CHE and SA induced apoptosis, which was probably mediated by decreasing levels of anti-apoptotic proteins (Bcl-xL, Mcl-1, XIAP) and was accompanied by mitochondrial membrane potential decrease as well as caspase-3 and PARP cleavage. Although all alkaloids caused DNA damage, which was demonstrated by induction of H2AX phosphorylation, none of the tested alkaloids stabilised p53 and their toxicity in cells with non-functional p53 was comparable to wild type cells.. Despite the profound similarity of BAs molecular structures, it is clear that the mechanism of cell death induction is different for each alkaloid. Our results indicate that BAs could be effective in malignant melanoma treatment, including tumours which have lost wild type p53.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Apoptosis; Benzophenanthridines; Caspases; Cell Proliferation; Cell Survival; DNA; DNA Damage; Genes, p53; Humans; Melanoma; Models, Biological; Models, Chemical; Skin Neoplasms; Tumor Suppressor Protein p53

Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound, 1,4-diselenophene-1,4-diketone (DSeD), has been synthesized by Double Friedel-Crafts reaction and identified as a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was relatively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initiation of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis pathway.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Free Radicals; Humans; Ketones; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Organoselenium Compounds; Reactive Oxygen Species

High mobility group box 1 (HMGB1) is one of the recently defined damage-associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis. We induced melanoma cell death with cytolytic lymphokine-activated killing (LAK) cells, tumor-specific cytolytic T lymphocytes, TRAIL, or granzyme B delivery and assessed intracellular HMGB1 retention or release to investigate the mechanism of HMGB1 release by cytolytic cells. HMGB1 release from melanoma cells (451Lu, WM9) was detected within 4 h and 24 h following incubation with IL-2-activated PBMC (LAK activity). HLA-A2 and MART1 or gp100-specific cytolytic T lymphocytes induced HMGB1 release from HLA-A2-positive and MART1-positive melanoma cells (FEM X) or T2 cell-loaded, gp100-specific peptides. TRAIL treatment, however, induced HMGB1 release, and it is interesting that this extrinsic pathway-mediated cell death was blocked with the pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Conversely, granzyme B delivery did not induce HMGB1 release. HMGB1, along with other intracellular factors released from tumor cells induced by cytolysis, may be important components of the disordered tumor microenvironment. This has important implications for the immunotherapy of patients with cancer. Specifically, HMGB1 may promote healing or immune reactivity, depending on the nature of the local inflammatory response and the presence (or absence) of immune effectors.

Topics: Active Transport, Cell Nucleus; Amino Acid Chloromethyl Ketones; Cell Death; Cell Line, Tumor; Cell Nucleus; Cells, Cultured; Cytotoxicity Tests, Immunologic; Flow Cytometry; Granzymes; HMGB1 Protein; Humans; Killer Cells, Lymphokine-Activated; Melanoma; T-Lymphocytes; TNF-Related Apoptosis-Inducing Ligand

Malignant melanoma is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the past years a huge variety of new compounds has been developed with potential chemotherapeutic activity which needs to be tested in vitro and in vivo. We investigated the in vitro action of three new experimental antifolate substances (MR7, MR21 and MR36) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. The response of two melanoma cell lines (SK-MEL-2 derived from malignant melanoma metastasis and SK-MEL-28 derived from primary malignant melanoma) was examined after treatment with these substances. The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by the TUNEL technique and Comet Assay. Western-blot analysis showed a down-regulation of Bcl-2 protein level and PARP cleavage, otherwise p53 and Bax expressions were not modulated. Moreover, these antifolate-induced apoptosis was accompanied by both pro-caspase-9 and -8 activations. These results were supported by the use of the pan-caspases inhibitor Z-VAD-FMK that almost completely decreased the amount of apoptosis in both the melanoma cell lines treated with antifolate. In conclusion our results show that TS inhibitors are able to induce apoptosis through a caspase-mediated pathway, but without the involvement of the p53/Bax signalling.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspase 9; Cell Line, Tumor; DNA Fragmentation; Enzyme Inhibitors; Folic Acid Antagonists; Humans; In Situ Nick-End Labeling; Melanoma; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Thymidylate Synthase; Tumor Suppressor Protein p53

Taxanes have a broad spectrum of activity against various human cancers, including melanoma. In this study, we have examined the molecular mechanism of docetaxel-induced apoptosis of human melanoma. We report that docetaxel induced varying degrees of apoptosis in a panel of melanoma cell lines but not in normal fibroblasts. Induction of apoptosis was caspase dependent and associated with changes in mitochondrial membrane potential that could be inhibited by overexpression of Bcl-2. Docetaxel induced changes in Bax that correlated with sensitivity to docetaxel-induced apoptosis. These changes in Bax were not inhibited by overexpression of Bcl-2. Kinetic studies of caspase-2 activation by Western blotting and fluorogenic assays revealed that activation of caspase-2 seemed to be the initiating event. Inhibition of caspase-2 with z-VDVAD-fmk or by small interfering RNA knockdown inhibited changes in Bax and mitochondrial membrane potential and events downstream of mitochondria. Activation of caspase-8 and Bid seemed to be a late event, and docetaxel was able to induce apoptosis in cells deficient in caspase-8 and Bid. p53 did not seem to be involved as a p53 null cell line was sensitive to docetaxel and an inhibitor of p53 did not inhibit apoptosis. Small interfering RNA knockdown of PUMA and Noxa also did not inhibit apoptosis. These results suggest that docetaxel induces apoptosis in melanoma cells by pathways that are dependent on activation of caspase-2, which initiates mitochondrial dependent apoptosis by direct or indirect activation of Bax.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspase 2; Caspase Inhibitors; Cell Nucleus; Cysteine Endopeptidases; Cytochromes c; Docetaxel; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Humans; Melanoma; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; RNA, Small Interfering; Taxoids; Transfection; Tumor Cells, Cultured

G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Humans; Melanoma; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Thionucleotides; Transfection

Several inhibitor of apoptosis proteins (IAPs) are cleaved during apoptosis. Studies of the melanoma-associated IAP (ML-IAP) Livin, using recombinant molecules, have implicated both caspases 3/7 and the serine protease Omi/HtrA2 in its proteolytic cleavage.. To characterize the apoptotic cleavage of Livin in melanocytic cells, and evaluate the role of known proteases.. We assessed the capacity of a variety of stimuli to induce Livin cleavage in human melanoma cell lines and normal human melanocytes. The role of caspases and Omi was examined using caspase inhibitors and RNAi, respectively. A potential caspase substrate was further examined by site-directed mutagenesis. Deletion mapping was used to identify the cleavage site.. Livin cleavage was observed in multiple human melanoma cell lines in response to a variety of apoptotic stimuli (UVB, 4-TBP, cisplatin, TNF, Bax), and not affected by the addition of various protease inhibitors or RNAi-mediated silencing of Omi/HtrA2. Livin cleavage induced by 4-TBP, but not UVB or cisplatin, was blocked by the pan-caspase inhibitor zVAD-fmk. Mutation of Asp52 to Glu in Livin did not affect cleavage, while either mutation of Asp52 to Ala, deletion of Asp52, or deletion of the adjacent region (residues 53-61) abrogated cleavage.. Livin cleavage, induced by multiple apoptotic stimuli in melanoma cells, likely occurs in an Omi-independent fashion at residue 52 within its potential caspase substrate (DHVD52). However, relative insensitivity of the apoptotic cleavage to zVAD-fmk, or Asp52 to Glu mutation, suggests the involvement of a non-canonical caspase.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Amino Acid Substitution; Apoptosis; Caspase Inhibitors; Caspases; Endopeptidases; High-Temperature Requirement A Serine Peptidase 2; Humans; Inhibitor of Apoptosis Proteins; Melanocytes; Melanoma; Mitochondrial Proteins; Mutation; Neoplasm Proteins; Protease Inhibitors; Protein Interaction Mapping; Serine Endopeptidases; Substrate Specificity

TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Antibiotics, Antineoplastic; Cell Death; Chromatography, High Pressure Liquid; Cyclohexanes; DNA, Neoplasm; Fluorescein-5-isothiocyanate; Humans; Melanoma; Membrane Potential, Mitochondrial; Mice; O-(Chloroacetylcarbamoyl)fumagillol; Propidium; Sesquiterpenes; U937 Cells

Histone deacetylase (HDAC) inhibitors have attracted much interest because of their ability to arrest cell growth, induce cell differentiation, and in some cases, induce apoptosis of cancer cells. In the present study, we have examined a new HDAC inhibitor, suberic bishydroxamate (SBHA), for its effect on a panel of human melanoma cell lines. We report that it induces varying degrees of apoptosis in the melanoma lines but not in melanocytes and fibroblasts. Induction of apoptosis was caspase dependent and was associated with induction of changes in mitochondrial membrane permeability, which could be inhibited by overexpression of Bcl-2. The changes in mitochondria were independent of caspase activation and were associated with changes in conformation of Bax. SBHA down-regulated several key antiapoptotic proteins including X-linked inhibitor of apoptosis and the Bcl-2 family proteins, Bcl-XL and Mcl-1. In contrast, it induced up-regulation of the Bcl-2 family proapoptotic proteins, Bim, Bax, and Bak. In addition, SBHA induced relocation of the protein Bim to mitochondria and its association with Bcl-2. De novo protein synthesis was required for initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide, inhibited SBHA-induced conformational changes in Bax as well as changes in mitochondrial membrane permeability and activation of caspase-3. These results suggest that SBHA induces apoptosis by changing the balance between proapoptotic and antiapoptotic proteins in melanoma cells. The protein Bim may be a key initiator of apoptosis in cells treated with SBHA.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Intracellular Membranes; Melanoma; Membrane Proteins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Oligopeptides; Permeability; Protein Conformation; Protein Transport; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; X-Linked Inhibitor of Apoptosis Protein

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine-induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase-3, but not that of caspase-1, in vitro. Both evodiamine-induced DNA fragmentation and caspase-3 activity were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl-2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl-2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Division; Cysteine Proteinase Inhibitors; Dactinomycin; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Evodia; Fibrosarcoma; Fluorouracil; Furans; Gene Expression Regulation, Neoplastic; Genes, bcl-2; HeLa Cells; Hepatocytes; Heterocyclic Compounds, 4 or More Rings; Humans; Indole Alkaloids; Leukemia, Monocytic, Acute; Leukocytes, Mononuclear; Melanoma; Mice; Mitochondria; Molecular Structure; Neoplasm Proteins; Oligopeptides; Plant Extracts; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Rats; Rats, Inbred BUF; Sarcoma 180; Tumor Cells, Cultured

As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Survival; Chelating Agents; Colforsin; Cysteine Proteinase Inhibitors; Disulfiram; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Melanoma; Melanoma, Experimental; Mesoporphyrins; Mice; Models, Biological; Photochemotherapy; Tumor Cells, Cultured

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.

Topics: Amino Acid Chloromethyl Ketones; Amino Acids, Aromatic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carrier Proteins; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Fas Ligand Protein; fas Receptor; Humans; Ligands; Melanoma; Membrane Glycoproteins; Mitochondria; Models, Biological; Phenylalanine; Time Factors; Tumor Cells, Cultured; Tyrosine; Ubiquinone

Induction of apoptosis in cells by TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is believed to be regulated by expression of two death-inducing and two inhibitory (decoy) receptors on the cell surface. In previous studies we found no correlation between expression of decoy receptors and susceptibility of human melanoma cells to TRAIL-induced apoptosis. In view of this, we studied the localization of the receptors in melanoma cells by confocal microscopy to better understand their function. We show that the death receptors TRAIL-R1 and R2 are located in the trans-Golgi network, whereas the inhibitory receptors TRAIL-R3 and -R4 are located in the nucleus. After exposure to TRAIL, TRAIL-R1 and -R2 are internalized into endosomes, whereas TRAIL-R3 and -R4 undergo relocation from the nucleus to the cytoplasm and cell membranes. This movement of decoy receptors was dependent on signals from TRAIL-R1 and -R2, as shown by blocking experiments with Abs to TRAIL-R1 and -R2. The location of TRAIL-R1, -R3, and -R4 in melanoma cells transfected with cDNA for these receptors was similar to that in nontransfected cells. Transfection of TRAIL-R3 and -R4 increased resistance of the melanoma lines to TRAIL-induced apoptosis even in melanoma lines that naturally expressed these receptors. These results indicate that abnormalities in "decoy" receptor location or function may contribute to sensitivity of melanoma to TRAIL-induced apoptosis and suggest that further studies are needed on the functional significance of their nuclear location and TRAIL-induced movement within cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Brefeldin A; Cell Nucleus; Cysteine Proteinase Inhibitors; DNA, Complementary; GPI-Linked Proteins; Humans; Leupeptins; Ligands; Melanoma; Membrane Glycoproteins; Protein Synthesis Inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Member 10c; Signal Transduction; Subcellular Fractions; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor Decoy Receptors; Tumor Necrosis Factor-alpha

Past studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis in a high proportion of cultured melanoma by caspase-dependent mechanisms. In the present studies we have examined whether TRAIL-induced apoptosis of melanoma was mediated by direct activation of effector caspases or whether apoptosis was dependent on changes in mitochondrial membrane potential (MMP) and mitochondrial-dependent pathways of apoptosis. Changes in MMP were measured by fluorescent emission from rhodamine 123 in mitochondria. TRAIL, but not TNF-alpha or Fas ligand, was shown to induce marked changes in MMP in melanoma, which showed a high correlation with TRAIL-induced apoptosis. This was associated with activation of proapoptotic protein Bid and release of cytochrome c into the cytosol. Overexpression of B cell lymphoma gene 2 (Bcl-2) inhibited TRAIL-induced release of cytochrome c, changes in MMP, and apoptosis. The pan caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and the inhibitor of caspase-8 (z-Ile-Glu-Thr-Asp-fluoromethylketone; zIETD-fmk) blocked changes in MMP and apoptosis, suggesting that the changes in MMP were dependent on activation of caspase-8. Activation of caspase-9 also appeared necessary for TRAIL-induced apoptosis of melanoma. In addition, TRAIL, but not TNF-alpha or Fas ligand, was shown to induce clustering of mitochondria around the nucleus. This process was not essential for apoptosis but appeared to increase the rate of apoptosis. Taken together, these results suggest that TRAIL induces apoptosis of melanoma cells by recruitment of mitochondrial pathways to apoptosis that are dependent on activation of caspase-8. Therefore, factors that regulate the mitochondrial pathway may be important determinants of TRAIL-induced apoptosis of melanoma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Nucleus; Cytochrome c Group; Cytosol; Enzyme Activation; Enzyme Inhibitors; Humans; Immunity, Innate; Intracellular Membranes; Ligands; Melanoma; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha