cytochrome-c-t and Melanoma

The concept of "vasculogenic mimicry" (VM) was introduced to describe the unique ability of highly aggressive tumor cells to form capillary-like structure (CLS) and matrix-rich patterned network in three-dimensional cultures that mimic embryonic vasculogenic network. Here, we provide the experimental evidence that CLS structure formation requires apoptotic cell death through activation of caspase-dependent mechanism. Our results indicate that the formation of CLS is also related to the reactive oxygen species (ROS) levels.

Topics: Antioxidants; Apoptosis; Capillaries; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Melanoma; Models, Biological; Neovascularization, Pathologic; Reactive Oxygen Species; Skin Neoplasms; Time Factors

Melanoma is a malignant tumor that originates from the skin's melanocytes and has the highest death rate from skin cancer. Developing more efficacious anticancer medications with fewer adverse effects is the key to effective cancer management. Natural products are considered relevant and cost-effective sources of treatment. The plant (Polypodium vulgare) is a small and evergreen fern. One of the most important chemical compounds in the extract of this herb is flavonoids, which are thought to have beneficial effects in the treatment of melanoma through antioxidant properties.. Due to the limitations of current cancer management and cytotoxic drugs available in the country, the need to study drugs of natural origin has become more prominent. In this regard, the present study aims to investigate the cytotoxic effects of the ethanolic extract of Polypodium vulgare on A375 melanoma cells.. Polypodium vulgare was extracted in 80% ethanol by the maceration. Then, its effects on the cell death of the melanoma cell line A375 compared to the AGO-1522 cell line as control were measured using the MTT-assay technique. The amount of cellular lipid peroxidation was estimated by TBARS assay. The amount of cellular ROS was calculated by fluorescent reagent 2,7-dichlorofluorescein diacetate. Cytochrome c concentration was measured by a cytochrome c immunoassay kit.. In this experiment, the anticancer effects of Polypodium vulgare ethanolic extract on human melanoma cell lines were investigated for the first time. Herb extract with a concentration of 0.123 mg/ml significantly increased the death of A375 melanoma cells (p < 0.001), lipid peroxidation (p < 0.01), and reactive oxygen species (ROS) (p < 0.01) and cytochrome c concentration (p < 0.001). Meanwhile, the same amount was ineffective and safe on AGO-1522 normal fibroblast cells.. A 0.123 mg/ml concentration of Polypodium vulgare increases apoptosis in melanoma cells. Meanwhile, the same amount was safe on healthy cells. So, it could be considered an effective treatment without side effects in human melanoma.

Topics: Antineoplastic Agents; Cell Line; Cell Line, Tumor; Cytochromes c; Ethanol; Humans; Melanoma; Melanoma, Cutaneous Malignant; Plant Extracts; Polypodium; Reactive Oxygen Species; Skin Neoplasms

The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome

Topics: Apoptosis; Caspases; Cell Death; Cell Line, Tumor; Chalcones; Cytochromes c; Humans; Indoles; Melanoma; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Mitotic catastrophe induced by mictotubule-targeting drugs such as benzimidazole carbamates has been demonstrated to be an efficient mechanism for suppression of tumor cells growth and proliferation, with variable resulting endpoints. The present study was designed to explore some of these endpoints; i.e. the apoptosis as well as autophagy and their related signaling in several stabilized cell lines as well as human explant melanoma cells treated with flubendazole (FLU). FLU-induced mitotic catastrophe resulted in mitochondrial and caspase-dependent apoptosis, which occurred at various rates in all treated cells during 96 h of treatment. The process was characterized by enhanced transcriptional activity of TP53 and NF-κB as well as upregulated Noxa expression. Also, inactivation of Bcl-2, BclXL and Mcl-1 proteins by JNK mediated phosphorylation was observed. Although increased autophagic activity took place in treated cells too, no discernible functional linkage with ongoing cell death process was evidenced. Together these results advance our evidence over the effectiveness of FLU cytotoxicity-related killing of melanoma cells while calling for more extensive testing of melanoma samples as a prerequisite of further preclinical evaluation of FLU antineoplastic potential.

Topics: Aged; Antineoplastic Agents; Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Cytochromes c; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mebendazole; Melanoma; Membrane Potential, Mitochondrial; Middle Aged; Mitosis; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Hydnocarpin (Hy) is a flavonoid isolated and purified from the seeds of

Topics: Antineoplastic Agents; Apoptosis; Cell Proliferation; Cycloaddition Reaction; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Flavonolignans; Humans; Lung Neoplasms; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Structure-Activity Relationship; Tumor Cells, Cultured

This study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2).. Following GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting.. G361 cells treated with GNP-HER2 showed condensation of nuclei, which is an apoptotic phenomenon, and translocation of apoptosis-inducing factor and cytochrome c from mitochondria into the nucleus and cytoplasm, respectively. Increases in BAX in cells undergoing apoptosis, activation of caspase-3 and -9, and fragmentation of poly (ADP-ribose) polymerase and DNA fragmentation factor 45 (inhibitor of caspase-activated DNase) were observed upon GNP-HER2 treatment. Following GNP-HER2 treatment, an increase of cells in sub-G1 phase, which is a signal of cell apoptosis, was observed. This resulted in the down-regulation of cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and cdc2 and the up-regulation of p21. Thus, GNP-HER2 treatment was confirmed to induce the cessation of cell cycle progression. Also, decreases in phospho-focal adhesion kinase and phospho-human epidermal growth factor receptor, which activate cellular focal adhesion, and decreases in phospho-paxillin, which stimulates the disassembly of filamentous actin, were observed. Reduced cell adhesion and disassembly of the intracellular structure indicated cell deactivation.. GNP-HER2 can selectively kill G361 melanoma cells without affecting normal cells. The mechanism of G361 cell death upon treatment with GNP-HER2 was apoptosis accompanied by activation of caspases.

Topics: Actins; Antibodies; Apoptosis; Apoptosis Inducing Factor; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cytochromes c; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; G1 Phase; Gold; Humans; Melanoma; Metal Nanoparticles; Receptor, ErbB-2

Nanomaterials (NM) exhibit novel anticancer properties.. The toxicity of three nanoparticles that are currently being produced in high tonnage including single-walled carbon nanotube (SWCNT), multi-walled carbon nanotube (MWCNT) and Fe. All tested nanoparticles induced selective toxicity and caspase 3 activation through mitochondria pathway in melanoma cells and mitochondria cause the generating of reactive oxygen species (ROS), mitochondrial membrane potential decline (MMP collapse), mitochondria swelling, and cytochrome c release. The pretreatment of butylated hydroxytoluene (BHT), a cell-permeable antioxidant and cyclosporine A (Cs. A), a mitochondrial permeability transition (MPT), pore sealing agent decreased cytotoxicity, caspase 3 activation, ROS generation, and mitochondrial damages induced by SWCNT, MWCNT, and IONPs.. Our promising results provide a potential approach for the future therapeutic use of SWCNT, MWCNT, and IONPs in melanoma through mitochondrial targeting.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cytochromes c; Ferric Compounds; Melanoma; Membrane Potential, Mitochondrial; Mice; Mitochondria; Nanoparticles; Nanotubes, Carbon; Reactive Oxygen Species; Succinate Dehydrogenase; Tumor Cells, Cultured

The purpose of this study was to investigate the effects of 5-aminolaevulinic acid mediated photodynamic therapy (ALA-PDT) on the survival activity and apoptosis of human melanoma cell line A375 and non-melanoma skin carcinoma cell line A431 cells. The mechanism for cellular apoptosis was explored.. The cell survival activity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the proportion of apoptotic cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression levels of Bcl-2, Bax, caspase-3, caspase-8 and caspase-9 protein were assessed by western blot. The subcellular localization of cytochrome c was comparatively investigated by immunohistochemistry between pre-ALA-PDT and post- ALA-PDT.. ALA-PDT significantly inhibited the survival activity of A375 cells and A431 cells in a dose- and time-dependent manner. The optimum inhibition efficiencies for A375 cells and A431 cells were obtained at 0.6 mM ALA at 4 h and 8 h after ALA-PDT, respectively. The phenomena of apoptosis were observed in ALA-PDT treated cells by TUNEL assay. The apoptotic rates of A375 cells and A431 cells were 90.0% and 61.5% at 6 h after ALA-PDT, respectively. Apoptosis induced by ALA-PDT involved in down-regulation of Bcl-2 protein, up-regulation of Bax protein and cleaved-PARP protein. It was observed that the expression of cleaved- caspase-3, caspase-8 and caspase-9 proteins in A375 cells and A431 cells gradually increased in 2 h and 4 h but decreased at 4-6 h and 6-8 h after ALA-PDT, respectively. In apoptosis cells immunohistochemical localization show that cytochrome C diffused from the mitochondria into the cytosol.. ALA-PDT could significantly inhibit the survival activity of A375 and A431 cells. The apoptosis induced by ALA-PDT in A375 and A431 cells was related to the caspase-dependent death-receptor pathway and Cytochrome c-dependent mitochondrial pathway.

Topics: Aminolevulinic Acid; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Down-Regulation; Humans; Melanoma; Mitochondria; Photochemotherapy; Photosensitizing Agents; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Up-Regulation

Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Cytochromes c; Flavonoids; Flavonols; Humans; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Uveal Neoplasms

Iron has been shown to trigger oxidative stress by elevating reactive oxygen species (ROS) and to participate in different modes of cell death, such as ferroptosis, apoptosis and necroptosis. However, whether iron-elevated ROS is also linked to pyroptosis has not been reported. Here, we demonstrate that iron-activated ROS can induce pyroptosis via a Tom20-Bax-caspase-GSDME pathway. In melanoma cells, iron enhanced ROS signaling initiated by CCCP, causing the oxidation and oligomerization of the mitochondrial outer membrane protein Tom20. Bax is recruited to mitochondria by oxidized Tom20, which facilitates cytochrome c release to cytosol to activate caspase-3, eventually triggering pyroptotic death by inducing GSDME cleavage. Therefore, ROS acts as a causative factor and Tom20 senses ROS signaling for iron-driven pyroptotic death of melanoma cells. Since iron activates ROS for GSDME-dependent pyroptosis induction and melanoma cells specifically express a high level of GSDME, iron may be a potential candidate for melanoma therapy. Based on the functional mechanism of iron shown above, we further demonstrate that iron supplementation at a dosage used in iron-deficient patients is sufficient to maximize the anti-tumor effect of clinical ROS-inducing drugs to inhibit xenograft tumor growth and metastasis of melanoma cells through GSDME-dependent pyroptosis. Moreover, no obvious side effects are observed in the normal tissues and organs of mice during the combined treatment of clinical drugs and iron. This study not only identifies iron as a sensitizer amplifying ROS signaling to drive pyroptosis, but also implicates a novel iron-based intervention strategy for melanoma therapy.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; bcl-2-Associated X Protein; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Caspase 3; Cell Line, Tumor; Cytochromes c; HEK293 Cells; Humans; Iron; Melanoma; Membrane Transport Proteins; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Pyroptosis; Reactive Oxygen Species; Receptors, Cell Surface; Receptors, Estrogen; Signal Transduction; Xenograft Model Antitumor Assays

Urea Transporter B (UT-B) is a membrane channel protein that mediates the rapid transmembrane transport of urea and participates in urine concentration. Urea Transporter B is expressed in skin, but we found that there is little expression in human melanoma tissue. In this study, we examined the effects of UT-B overexpression in melanoma. The results indicated that there is no UT-B mRNA expression in B16 cells, and UT-B overexpression repressed B16 cell proliferation and induced apoptosis in vitro. We show that UT-B overexpression causes increased reactive oxygen species production, which may be caused by mitochondria dysfunction. The mitochondrial membrane potential (ΨΔm) was lower in UT-B-overexpressing B16 cells. The proteins involved in complexes I, III, IV and V of the respiratory chain were clearly downregulated in UT-B-overexpressing B16 cells, which would strongly reduce the activity of the electron transport chain. We found that mitochondrial release of cytochrome C into the cytoplasm also increased, indicating that apoptosis had been activated. In addition, UT-B overexpression reduced AKT phosphorylation and MDM2 expression and increased p53 expression; p53 activation may be involved in the anticancer effects of UT-B overexpression. Urea Transporter B overexpression also inhibited tumor growth in vivo. In conclusion, we demonstrated that UT-B may be related to the occurrence of melanoma and play a role in tumor development.

Topics: Aged; Animals; Cell Death; Cell Line, Tumor; Cytochromes c; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Melanoma; Melanoma, Experimental; Membrane Potential, Mitochondrial; Membrane Transport Proteins; Mice; Middle Aged; Mitochondria; Signal Transduction; Skin Neoplasms; Tumor Suppressor Protein p53; Urea Transporters

Objective: A variety of approaches such as surgery, chemotherapy, radiotherapy, hormonal therapy and\ immunotherapy are used to treat melanomas, but unfortunately in most case, the response is very weak and often side\ effects are serious. This study concerns selective toxicity of an extract of Turbo coronatus on cells and mitochondria from\ a syngeneic mouse model of melanoma. Methods: Cells and mitochondria isolated from extra tumoral and melanoma\ tissues were exposed toa T. coronatus crude extract and fractions obtained by gel-filtration chromatography and assayed\ for mitochondrial and cellular parameters. Result: Crude extract (375, 750 and 1,500 μg/ml) and fraction 1; F1; (275,\ 550 and 1100 μg/ml) of T. coronatus extract induced a significant (p<0.05) increase of the reactive oxygen species\ (ROS) level, swelling of mitochondria, collapse of mitochondrial membrane potential (MMP), release of cytochrome\ c and caspase-3 activation only in the mitochondria and cells obtained from melanoma but not extra tumoral tissues. In\ addition, the F1 fraction decreased the percentage of viable cells and induced apoptosis in melanoma cells. Conclusion:\ For the first time we could demonstrate that the F1 fraction of a T. coronatus extract, selectively induces ROS mediated\ cytotoxicity by directly targeting mitochondria in melanoma tissues and it may be a suitable candidate for novel drug\ treatment of malignant melanomas.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cytochromes c; Female; Indian Ocean; Melanoma; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Mollusca; Reactive Oxygen Species; Tumor Cells, Cultured

Topics: Anacardium; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cytochromes c; Humans; Melanoma; Mitochondria; Nuts; Phenols; Plant Extracts; Reactive Oxygen Species; Signal Transduction

The currently available data suggest that natural products may exert significant cytotoxic and immunomodulatory effects. Plant-derived chemotherapeutic agents such as taxol, etoposide or vincristine, currently used in cancer therapy, are prominent examples in this regard. However, there is a need for new and nat- ural anticancer compounds with low or without toxicity to normal cells. One of the active compounds responsible for the immune effects is β-glucan derived from cereals, fungi, seaweeds, yeasts and bacteria. The recent data suggest that β-glucans are potent immunomodulators with anticancer properties. Antitumor properties of fungi and yeast derived β-glucans have been widely recognized, but those polysaccharides are mostly insoluble, creating several problems especially in topical formulation. To overcome the issue of low water solubility, in the current study a more soluble β-glucan type from oats was chosen for the investigation of its antitumor activities. Cytotoxic effects were studied using a human melanoma cell line (Me45). The effect of electroporation on the antitumor activity of oat β-glucan was investigated as well. Cellular viability assessment, immuno-cytochemistry and immunofluochemistry were employed to evaluate biologic effects. Our results indicate strong anticancer properties of oat β-glucan, enhanced by electroporation.

Topics: Adult; Antineoplastic Agents, Phytogenic; Apoptosis; Avena; beta-Glucans; Caspase 12; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Electrochemotherapy; Humans; Male; Melanoma; Phytotherapy; Plants, Medicinal; Skin Neoplasms; Solubility

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod-induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c-Jun-N-terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA-like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca(2+) release, degradation of calpain and subsequent cleavage of caspase-4. Moreover, imiquimod triggers the activation of NF-κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X-linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod-induced apoptosis. They demonstrate that inhibition of NF-kB by the inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor Bay 11-782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.

Topics: Aminoquinolines; Apoptosis; Calcium; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Imiquimod; JNK Mitogen-Activated Protein Kinases; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; NF-kappa B; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

The aim of the present study was to examine the apoptotic effect of flavonoids in methanol extracts of Ginkgo biloba fallen leaves (MEGFL) on melanoma cells. Ginkgo biloba is a deciduous castle chaplain and its leaves include various types of flavonoids such as flavonol-O-glycosides. Ginkgo biloba is known to have therapeutic properties against a number of diseases such as cerebrovascular diseases, blood circulation disease and hypertension. In the present study MEGFL exhibited a higher cytotoxic effect on melanoma cells than Ginkgo biloba leaves (MEGL). It was also found that MEGFL induced apoptotic cell death which was characterized by DNA fragmentation. During the cell death process following treatment with MEGFL, the expression of a variety of death-associated proteins including p53, caspase-3, caspase-9, cytochrome c and Bax were analyzed in the cytosol of melanoma cells. MEGFL significantly increased the expression levels of caspase-3, caspase-9 and p53 in a dose-dependent manner. Our results indicate that MEGFL induced apoptotic cell death by increasing the expression of cell death-associated proteins in melanoma cells.

Topics: Acetylation; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Damage; DNA Fragmentation; Dose-Response Relationship, Drug; Flavonoids; Ginkgo biloba; Melanoma; Mice; Phosphorylation; Plant Leaves; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Artemisia species are important medicinal plants throughout the world. The present in vitro study, using a sesquiterpene lactone-bearing fraction prepared from Artemisia khorassanica (SLAK), sought to investigate anti-cancer properties of this plant and elucidate potential underlying mechanisms for the effects.. Anti-cancer potential was evaluated by toxicity against human melanoma and fibroblast cell lines. To explore the involved pathways, pattern of any cell death was determined using annexin-V/PI staining and also the expression of Bax and cytochrome c was investigated by Western blotting.. The results showed that SLAK selectively caused a concentration-related inhibition of proliferation of melanoma cells that was associated with remarkable increase in early events and over-expression of both Bax and cytochrome c.. The current experiment indicates that Artemisia may have anti-cancer activity. We anticipate that the ingredients may be employed as therapeutic candidates for melanoma.

Topics: Apoptosis; Artemisia; Blotting, Western; Caspases; Cell Cycle; Cell Proliferation; Cells, Cultured; Cytochromes c; Fibroblasts; Humans; Lactones; Melanoma; Plant Components, Aerial; Plant Extracts; Sesquiterpenes

Melanoma is one of the most aggressive and lethal cancers. Discovery and identification of novel therapeutic targets is urgently needed. In this study, we demonstrated that ribosomal protein S3 (RPS3) was a potential target involved in melanoma growth. Knockdown of RPS3 by siRNA suppressed cell growth and induced apoptosis in melanoma cells. Further mechanism studies showed that RPS3 knockdown in melanoma cells triggered the release of cytochrome C (Cyto C) from mitochondrial, increased the location of BID on mitochondrial membrane and the cleavage of the pro-apoptotic proteins (PARP, caspase-3 and -9), promoted the opening of mitochondrial permeability transition pore and the flooding of calcium ions (Ca(2+)) into the mitochondrial, and decreased the expression of the Ca(2+) gatekeeper MICU1 and its location on the mitochondrial. We also found that knockdown of RPS3 significantly inhibited tumor growth in a melanoma xenograft mouse model. Furthermore, we showed that RPS3 was highly expressed in melanoma cell lines and melanoma tumor tissues, and overexpression of RPS3 was associated with the poor prognosis of melanoma patients. Our results therefore demonstrate that RPS3 regulates melanoma growth through the modulation of the Cyto C/Ca(2+)/MICU1 dependent mitochondrial signaling and suggest that RPS3 is a potential therapeutic target for melanoma treatment.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Calcium; Calcium-Binding Proteins; Cation Transport Proteins; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Melanoma; Mice, Nude; Microscopy, Fluorescence; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Ribosomal Proteins; RNA Interference; RNAi Therapeutics; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin's cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide's effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening.

Topics: Adenine Nucleotide Translocator 1; Cell Death; Cell Line, Tumor; Curcumin; Cyclophilins; Cytochromes c; Humans; Lactones; Melanoma; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Peptidyl-Prolyl Isomerase F; Spiro Compounds

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Topics: Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Cell Respiration; Cells, Cultured; Cerulenin; Citrate (si)-Synthase; Cytochromes c; Fatty Acid Synthases; Fatty Acid Synthesis Inhibitors; Humans; Keratinocytes; Melanocytes; Melanoma; Membrane Potential, Mitochondrial; Mice; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spectrometry, Mass, Electrospray Ionization

Centratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called "Kayakalp", commonly applied to treat skin disorders in India and Southeast Asia. Despite its well known anti-inflammatory property on skin diseases, the anti-cancer effect of C. anthelminticum seeds on skin cancer is less documented. The present study aims to investigate the anti-cancer effect of Centratherum anthelminticum (L.) seeds chloroform fraction (CACF) on human melanoma cells and to elucidate the molecular mechanism involved.. A chloroform fraction was extracted from C. anthelminticum (CACF). Bioactive compounds of the CACF were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human melanoma cell line A375 was treated with CACF in vitro. Effects of CACF on growth inhibition, morphology, stress and survival of the cell were examined with MTT, high content screening (HSC) array scan and flow cytometry analyses. Involvement of intrinsic or extrinsic pathways in the CACF-induced A375 cell death mechanism was examined using a caspase luminescence assay. The results were further verified with different caspase inhibitors. In addition, Western blot analysis was performed to elucidate the changes in apoptosis-associated molecules. Finally, the effect of CACF on the NF-κB nuclear translocation ability was assayed.. The MTT assay showed that CACF dose-dependently inhibited cell growth of A375, while exerted less cytotoxic effect on normal primary epithelial melanocytes. We demonstrated that CACF induced cell growth inhibition through apoptosis, as evidenced by cell shrinkage, increased annexin V staining and formation of membrane blebs. CACF treatment also resulted in higher reactive oxygen species (ROS) production and lower Bcl-2 expression, leading to decrease mitochondrial membrane potential (MMP). Disruption of the MMP facilitated the release of mitochondrial cytochrome c, which activates caspase-9 and downstream caspase-3/7, resulting in DNA fragmentation and up-regulation of p53 in melanoma cells. Moreover, CACF prevented TNF-α-induced NF-κB nuclear translocation, which further committed A375 cells toward apoptosis.. Together, our findings suggest CACF as a potential therapeutic agent against human melanoma malignancy.

Topics: Apoptosis; Asteraceae; Caspases; Cell Proliferation; Cytochromes c; Female; Humans; Melanoma; Middle Aged; Mitochondria; NF-kappa B; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Seeds; Signal Transduction; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal microscopies. Circular dichroic spectral analysis and stability curve for Gibb's free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photostability studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formulations to have better ameliorative potentials in combating skin melanoma.

Topics: Apigenin; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Chemistry, Pharmaceutical; Cytochromes c; DNA Fragmentation; Humans; Lactic Acid; Melanoma; Melanoma, Cutaneous Malignant; Mitochondria; Nanoparticles; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Reactive Oxygen Species; Skin Neoplasms

Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Biomarkers, Tumor; Caspase 9; Cell Line, Tumor; Cytochromes c; Diclofenac; Enzyme Activation; Humans; Melanoma; Mitochondria; Reactive Oxygen Species; Superoxide Dismutase

Although chemotherapeutic drugs could theoretically target all metastatic sites, current treatments do not provide complementary therapeutics. Therefore, the development of an alternative approach replacing the traditional therapy is urgently needed. To assess the killing efficiency of the functionally identified apoptosis-related protein (APR)-1 in melanoma cells, we established a system for the regulated expression of APR-1. The induction of APR-1 expression caused apoptosis of melanoma cells via the interaction with the juxtamembrane region of p75 neurotrophin receptor (p75NTR), and possible also via the competition with tumour necrosis factor receptor-associated factor-6 (TRAF6) and the catalytic receptor of neurotrophin (Trk) for the same p75NTR interacting site. The accumulation of APR-1 in melanoma cells may block the physical association of p75NRT with TRAF6 and/or Trk, leading to the disruption of both NF-κB and extracellular signal-regulated kinase (ERK) pathways. Also, accumulation of APR-1 protein enhanced the activity of both c-Jun-N-terminal kinase (JNK) and p38 pathways. However, the analysis of APR-1-modulated pathways demonstrated the involvement of apoptosis-regulating kinase 1-JNK/p38 pathway in the induction of Bax expression leading to both mitochondrial dysregulation [as demonstrated by the loss of mitochondrial membrane potential, the release of both cytochrome c and apoptosis-inducing factor into cytoplasm, and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP)] and endoplasmic reticulum stress as demonstrated by the increase of intracellular Ca(2+) release. Thus, besides the analysis of its pro-apoptotic function, our data provide insight into the molecular mechanism of APR-1-induced apoptosis of melanoma cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum Stress; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Melanoma; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Mitochondria; Neoplasm Proteins; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Receptor, Nerve Growth Factor; Receptor, trkA; RNA Interference; RNA, Small Interfering; Signal Transduction; TNF Receptor-Associated Factor 6

Capsaicin and resveratrol are strong chemopreventive agents with promising human consumption safety records and anticarcinogenic activities. However, the mechanism by which they induce apoptosis in tumor cells remains to be defined. In this study, we examined the role of nitric oxide (NO•) during apoptosis induced by these agents in A375 human melanoma cells. Capsaicin and resveratrol, alone or in combination, inhibited cell growth and promoted apoptosis by the elevation of NO• in A375 cells. Increased NO• production following treatment stimulated p53 and triggered mitochondrial apoptotic events by inducing conformational changes in Bax and Bcl-2 with subsequent release of cytochrome c and activation of caspase 9 and 3. Caspase 8 activation concurrently appeared to be mediated by death receptor processing and downstream caspases. Collectively, our data suggest that capsaicin and resveratrol activate the mitochondrial and death receptor pathways, working together to induce apoptosis in A375 cells, and indicate that NO• could be considered a potential target for improvement of the effectiveness of melanoma treatment.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Capsaicin; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Humans; Melanoma; Mitochondria; Nitric Oxide; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Signal Transduction; Stilbenes; Tumor Suppressor Protein p53

To study the cytotoxic effects and related signaling pathways of butein on human uveal melanoma cells in vitro.. Three human uveal melanoma cell lines (M17, SP6.5, and C918), retinal pigment epithelial (RPE) cells and scleral fibroblasts were treated with butein at different dosages. The effects of butein on cell viability were assessed by using the MTT assay. Cell apoptosis was determined using annexin V-FITC/ethidium homodimer III flow cytometry. Mitochondrial transmembrane potential changes were assessed by using the JC-1 fluorescent reader, cytosol cytochrome c levels, and the activities of caspase-3, -8, and -9 were measured by using an enzyme-linked immunosorbent assay or colorimetric assay.. Butein reduced the cell viability of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 μM), with IC50 at 13.3 μM and 15.8 μM in SP6.5 and M17 cell lines, respectively. Similar effects were also found in a highly aggressive and metastatic C918 cell line (IC50 16.7 μM). Butein at lower concentrations (10-30 μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of RPE cells and fibroblasts. Butein-induced apoptosis of melanoma cells, increased mitochondrial permeability and the level of cytosol cytochrome c, caspase-9 and -3 activities (but not caspase-8) in a dose-dependent manner.. Butein has selectively potent pro-apoptotic effects on cultured human uveal melanoma cells via the intrinsic mitochondrial pathway.

Topics: Annexin A5; Apoptosis; Caspases; Cell Survival; Chalcones; Cytochromes c; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Retinal Pigment Epithelium; Tumor Cells, Cultured; Uveal Neoplasms

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.

Topics: Analysis of Variance; Animals; Apoptosis; Calcium; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cerulenin; Cytochromes c; DNA Primers; Enzyme-Linked Immunosorbent Assay; Fatty Acid Synthases; Fatty Acid Synthesis Inhibitors; Flow Cytometry; Lactones; Lipids; Melanoma; Mice; Orlistat; Reactive Oxygen Species; RNA Interference

Melanoma continues to be a therapeutic challenge for the medical community owing to the scarcity of effective agents available to treat the disease. Honokiol, a traditional Chinese herb, has been proven to have anti-cancer effects in various cell types, therefore we hypothesized it may have similar cytotoxic capabilities against melanoma cells in vitro.. Two cell lines, SK-MEL2 and MeWo, were grown in culture and exposed to increasing doses of Honokiol. Cell proliferation, cytochrome c release into the cytosol, intra-cellular caspase activity, and mitochondrial depolarization were then evaluated after treatment with honokiol.. Melanoma cells in culture underwent cell death, had increased cytosolic cytochrome c, showed greater caspase activity, and demonstrated increased mitochondrial depolarization after treatment when compared to controls.. It appears that honokiol is an effective inhibitor of cultured human melanoma cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Biphenyl Compounds; Blotting, Western; Caspases; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Humans; In Vitro Techniques; Lignans; Melanoma; Tumor Cells, Cultured

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine.

Topics: Adenine Nucleotides; Antineoplastic Agents, Phytogenic; Apoptosis; Berberine; Blotting, Western; Caspases; Cell Cycle; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cytochromes c; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; G1 Phase; G2 Phase; Humans; Melanoma; Microscopy, Confocal; Mitochondria; Proto-Oncogene Proteins B-raf; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 μg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells.

Topics: Actin Cytoskeleton; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; Cytoskeletal Proteins; Drugs, Chinese Herbal; Hedyotis; Humans; Melanoma; Mitochondria; Molecular Chaperones; Phalloidine; Photochemotherapy; Proteomics; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

Metastatic cancers including melanoma are frequently associated with increased resistance to apoptosis induced by various therapeutic modalities, and the success of systemic therapy for the treatment of metastatic melanoma is minimal. In the present study, we demonstrated the ability of apoptosis-related protein (APR)-2 to trigger cell death via mechanism mediated by both endoplasmic reticulum (ER) stress [as evidenced by the increase of intracellular Ca(2+) release, the activation of both, inositol-requiring enzyme 1α (IRE1α) and calpain and cleavage of caspase-4] and mitochondrial dysregulation as evidenced by the loss of mitochondrial membrane potential, Cytochrome c release and cleavage of caspases-9 and -3, and poly adenosine diphosphate ribose polymerase (PARP). Also, the activation of apoptosis signal-regulating kinase (ASK) 1, c-jun-N-terminal kinase (JNK) and the transcription factors AP-1 and p53, and the induction of Bax expression were noted in APR-2-expressing cells. Both immune fluorescence staining and western blotting revealed the localization of APR-2 at ER and Bax protein at both mitochondria and ER. However, data of inhibitory experiments demonstrated that APR-2-induced apoptosis of melanoma cells is mediated by three parallel pathways: one of them IRE1/tumour necrosis factor receptor-associated factor 2/ASK1/JNK/Cyt.c/caspase-9/caspase-3/PARP) seems to be mitochondrial dependent, whereas, the other two pathways namely calpain/caspase-4/caspase-9/caspase-3/PARP and protein kinase RNA-like ER kinase/ATF4/C/EBP homologous protein (CHOP)/Bim seem to be mitochondrial independent. In conclusion, our data provide insight into the molecular mechanism of APR-2-induced apoptosis and suggest APR-2 gene transfer as an alternative approach for the treatment of chemoresistance melanoma metastasis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Proliferation; Cells, Cultured; Cytochromes c; Electrophoretic Mobility Shift Assay; Endoplasmic Reticulum; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Humans; JNK Mitogen-Activated Protein Kinases; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Signal Transduction

Silibinin was reported to have high cyto-toxicity in many malignant cell lines, however, it showed low cyto-toxicity in treatment of human melanoma A375-S2 cells and even protected these cells against certain stress insults. Reactive oxygen species was reported to have controversial effects on cancer chemotherapy. In this study we investigated the mechanism of reactive oxygen species generation and the role of reactive oxygen species in protecting cells against silibinin induced cyto-toxicity in A375-S2 cells. We found that silibinin induced the generation of large amount of superoxide anion (O(2)(∙-)) and small amount of hydrogen peroxide (H(2)O(2)) through down-regulating the activity of mitochondrial complex IV and the protein level of cytochrome c. We also discovered that O(2)(∙-) generation activated insulin like growth factor-1 receptor (IGF-1R) and its down-stream phosphatidylinositol 3-kinases-Akt (PI3K-Akt) and phospholipase C γ-protein kinase C (PLC γ-PKC) signaling pathways, which were augmented by H(2)O(2) scavenger catalase. Scavenging O(2)(∙-) by superoxide dismutase (SOD) or inhibition of IGF-1R-PI3K-Akt and IGF-1R-PLC γ-PKC signaling pathways increased cell apoptosis. Therefore, O(2)(∙-) mediated cell resistance to silibinin via activating IGF-1R-PI3K-Akt and IGF-1R-PLC γ-PKC pathways in silibinin treated A375-S2 cells.

Topics: Antioxidants; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Electron Transport Complex IV; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Melanoma; Phosphatidylinositol 3-Kinases; Phospholipase C gamma; Protein Kinase C; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Silybin; Silymarin; Superoxide Dismutase; Superoxides

In this study, we have investigated the chemotherapeutic potential of a purple violet pigment (PVP), which was isolated from a previously undescribed Antarctic Janthinobacterium sp. (Ant5-2), against murine UV-induced 2237 fibrosarcoma and B16F10 melanoma cells.. The 2237, B16F10, C50, and NIH3T3 cells were treated with PVP at different doses and for different times, and their proliferation and viability were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle arrest induced by PVP in 2237 fibrosarcoma cells was assessed by flow cytometry and expression analysis of cell cycle regulatory proteins were done by Western blot. Apoptosis induced by PVP in 2237 cells was observed by annexin-V/propidium iodide double staining flow cytometry assay and fluorescence microscopy. To further determine the molecular mechanism of apoptosis induced by PVP, the changes in expression of Bcl-2, Bax and cytochrome c were detected by Western blot. The loss of mitochondrial membrane potential in PVP treated 2237 cells was assessed by staining with JC-1 dye following flow cytometry. Caspase-3, Caspase-9 and PARP cleavage were analyzed by Western blot and Caspase-3 and -9 activities were measured by colorimetric assays.. In vitro treatment of murine 2237 cells with the PVP resulted in decreased cell viability (13-79%) in a time (24-72 h) and dose (0.1-1 μM)-dependent manner. The PVP-induced growth inhibition in 2237 cells was associated with both G0/G1 and G2/M phase arrest accompanied with decrease in the expression of cyclin dependent kinases (Cdks) and simultaneous increase in the expression of cyclin dependent kinase inhibitors (Cdki) - Cip1/p21 and Kip1/p27. Further, we observed a significant increase in the apoptosis of the 2237 fibrosarcoma cells which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2, disruption of mitochondrial membrane potential, cytochrome c release, activation of caspase-3, caspase-9 and poly-ADP-ribose-polymerase (PARP) cleavage.. We describe the anti-cancer mechanism of the PVP for the first time from an Antarctic bacterium and suggest that the PVP could be used as a potent chemotherapeutic agent against nonmelanoma skin cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Cytochromes c; Fibrosarcoma; Indoles; Melanoma; Melanoma, Experimental; Membrane Potential, Mitochondrial; Mice; Oxalobacteraceae; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

To study the cytotoxic effects and related signaling pathways of curcumin on human uveal melanoma cells in vitro.. Two human uveal melanoma cell lines (M21 and SP6.5), scleral fibroblasts, and choroidal melanocytes were treated with curcumin. The effects of curcumin on cell viability were assessed by using the MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay. Cytosol cytochrome c levels and the activities of caspase-9 and caspase-3 were measured by using an enzyme-linked immunosorbent assay.. Curcumin induced cell death of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 microM) and time-dependent manner (3-48 hr), with IC50 at 19.05 microM and 22.39 microM in M21 and SP6.5 cell lines, respectively. Curcumin at lower concentrations (10-30 microM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of fibroblasts and choroidal melanocytes. Curcumin significantly increased the level of cytosol cytochrome c (2-fold greater than the controls after 2 hr treatment), caspase-9 and caspase-3 activities (approximately 4.5- and 6-fold greater than the controls after 2-6 hr treatment, respectively) in a dose-dependent manner.. Curcumin has selectively potent cytotoxic effects on cultured human uveal melanoma cells. This effect is associated with the release of cytochrome c from the mitochondria and the activation of caspase-9 and caspase-3 in uveal melanoma cells after treatment with curcumin.

Topics: Antineoplastic Agents; Caspase 3; Caspase 9; Cell Death; Cell Survival; Curcumin; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Formazans; Humans; Melanocytes; Melanoma; Tetrazolium Salts; Time Factors; Tumor Cells, Cultured; Uveal Neoplasms

The S100B-p53 protein complex was discovered in C8146A malignant melanoma, but the consequences of this interaction required further study. When S100B expression was inhibited in C8146As by siRNA (siRNA(S100B)), wt p53 mRNA levels were unchanged, but p53 protein, phosphorylated p53, and p53 gene products (i.e. p21 and PIDD) were increased. siRNA(S100B) transfections also restored p53-dependent apoptosis in C8146As as judged by poly(ADP-ribose) polymerase cleavage, DNA ladder formation, caspase 3 and 8 activation, and aggregation of the Fas death receptor (+UV); whereas, siRNA(S100B) had no effect in SK-MEL-28 cells containing elevated S100B and inactive p53 (p53R145L mutant). siRNA(S100B)-mediated apoptosis was independent of the mitochondria, because no changes were observed in mitochondrial membrane potential, cytochrome c release, caspase 9 activation, or ratios of pro- and anti-apoptotic proteins (BAX, Bcl-2, and Bcl-X(L)). As expected, cells lacking S100B (LOX-IM VI) were not affected by siRNA(S100B), and introduction of S100B reduced their UV-induced apoptosis activity by 7-fold, further demonstrating that S100B inhibits apoptosis activities in p53-containing cells. In other wild-type p53 cells (i.e. C8146A, UACC-2571, and UACC-62), S100B was found to contribute to cell survival after UV treatment, and for C8146As, the decrease in survival after siRNA(S100B) transfection (+UV) could be reversed by the p53 inhibitor, pifithrin-alpha. In summary, reducing S100B expression with siRNA was sufficient to activate p53, its transcriptional activation activities, and p53-dependent apoptosis pathway(s) in melanoma involving the Fas death receptor and perhaps PIDD. Thus, a well known marker for malignant melanoma, S100B, likely contributes to cancer progression by down-regulating the tumor suppressor protein, p53.

Topics: Apoptosis; Biomarkers, Tumor; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Down-Regulation; Enzyme Activation; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Melanoma; Membrane Potential, Mitochondrial; Multiprotein Complexes; Mutation; Nerve Growth Factors; Phosphorylation; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Transcription, Genetic; Tumor Suppressor Protein p53; Ultraviolet Rays

Tetracyclines were recently found to induce tumour cell death, but the early processes involved in this cytotoxic effect remain unclear.. Viability of human and mouse melanoma cells was determined by MTT assay and flow cytometry. Kinase/protein/caspase activation was measured by Western blotting and mitochondrial membrane potential (DeltaPsi(m)) was analyzed by fluorescence microscopy and flow cytometry.. Human and mouse melanoma cells were treated with doxycycline or minocycline but only doxycycline was cytotoxic. This cell death (apoptosis) in A2058 cells involved activation of caspase-3, -7 and -9 and contributed to inhibition, by doxycycline, of matrix metalloproteinase (MMP) activity and migration of these cells. Doxycycline induced intra-cellular reactive oxygen species (ROS) production, apoptosis signal-regulated kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation at an early stage of treatment and induced mitochondrial cytochrome c release into cytosol and DeltaPsi(m) change during apoptosis. The JNK inhibitor/small interference RNA inhibited doxycycline-induced JNK activation, DeltaPsi(m) change and apoptosis, but did not affect ASK1 activation, suggesting a role of ASK1 for JNK activation in melanoma cell apoptosis. Two ROS scavengers reduced doxycycline-induced JNK and caspase activation, and apoptosis. Taken together, the results suggest the involvement of a ROS-ASK1-JNK pathway in doxycycline-induced melanoma cell apoptosis.. We have shown a promising cytotoxic effect of doxycycline on melanoma cells, have identified ROS and ASK1 as the possible initiators and have demonstrated that JNK activation is necessary for doxycycline-induced melanoma cell apoptosis.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Cytochromes c; Doxycycline; Drug Screening Assays, Antitumor; Free Radical Scavengers; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 5; Melanoma; Membrane Potential, Mitochondrial; Mice; Minocycline; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Up-Regulation

The natural antioxidant gallic acid (GA) has demonstrated a significant inhibition of cell proliferation and induction of apoptosis in a series of cancer cell lines. However, there is no available information to show whether GA induces apoptosis in human skin cancer cells. In the present study, we report GA-induced apoptosis in A375.S2 human melanoma cells. GA affected morphological changes, decreased the percentage of viable cells and induced apoptosis in A375.S2 cells in a dose- and time-dependent manner. Observation of the molecular mechanism of apoptosis in A375.S2 cells showed that GA up-regulated the proapoptotic proteins such as Bax, and induced caspase cascade activity, but down-regulated antiapoptotic proteins such as Bcl-2. GA induced reactive oxygen species (ROS) and intracellular Ca2+ productions and decreased the level of mitochondrial membrane potential (DeltaPsim) in A375.S2 cells in a time-dependent manner. GA triggered cytosolic release of apoptotic molecules, cytochrome c, promoted activation of caspase-9 and caspase-3, and ultimately apoptotic cell death. In addition, GA also promoted cytosolic release of apoptosis-inducing factor (AIF) and endonuclease G (Endo G). Therefore, GA may also induce apoptosis through a caspase-independent pathway. Our results suggest that GA might be a potential anticancer compound; however, in depth in vivo studies are needed to elucidate the exact mechanism.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Calcium; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Gallic Acid; Humans; Melanoma; Membrane Potential, Mitochondrial; Models, Biological; Protein Transport; Reactive Oxygen Species; Signal Transduction

Uveal melanoma, the most common primary intraocular malignancy in adults, is highly resistant to most chemotherapeutic drugs. Arsenic trioxide (ATO) is known to inhibit ocular melanoma cell growth. However, the effects of ATO on human uveal melanoma cells are poorly understood. Therefore, this study evaluated the mechanisms of ATO and its inhibiting effects on a human uveal melanoma cell line (SP6.5). An MTT assay indicated that, compared to human fibroblasts, ATO had a stronger inhibiting effect on SP6.5 cell proliferation in a dose- and time-dependent manner. The apoptosis ratio in SP6.5 cells, which was indicated by cell DNA fragmentation, was 4.1- to 7.7-fold higher after ATO-treatment. The ATO treatment substantially increased the activities of caspase-3 and caspase-9, but not of caspase-8. These findings were consistent with the protein expression observed by Western blots. ATO also significantly enhanced expression of Bax and cytochrome c proteins but suppressed those of Bcl-2. Therefore, ATO-induced apoptosis in uveal melanoma cells occurs mainly through the mitochondrial pathway rather than through the death receptor pathway. This report is the first to evaluate the complete mitochondria-dependent apoptotic pathway of ATO in uveal melanoma cells. These results can be used to improve the clinical effectiveness of ATO treatment for uveal melanoma.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Eye Neoplasms; Humans; Melanoma; Mitochondria; Oxides; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Malignant melanomas are generally drug resistant and have a very poor prognosis. We have studied the effects of a chemical conjugate of pseudomonas exotoxin A (PE) and the antibody 9.2.27, which recognizes the high molecular weight melanoma associated antigen (HMW-MAA) expressed in most malignant melanomas and melanoma cell lines. We demonstrate that the 9.2.27PE immunotoxin (IT) induces cell death in malignant melanoma cells through protein synthesis inhibition followed by some morphological and biochemical features of apoptosis, like rounding up of cells, chromatin condensation and inactivation of PARP. Unlike previous results with the 425.3PE IT in breast cancer cells, we detected no depolarization of the mitochondrial membrane after 9.2.27PE IT treatment. This is likely due to the lack of strong activation of caspase-8 and caspase-3. The lack of depolarization suggests that cytochrome c, a molecule that triggers activation of caspase-3, was retained within the mitochondria. In addition, the protein level of the antiapoptotic Bcl-2 did not decrease in contrast to other antiapoptotic molecules belonging to the inhibitor of apoptosis and the Bcl-2 family. This suggests that Bcl-2 may play a role in maintaining the mitochondrial membrane integrity in the 9.2.27PE-treated cells. Nevertheless, 9.2.27PE IT efficiently killed malignant melanoma cells that can be ascribed to inhibition of protein synthesis followed by some morphological and biochemical features of apoptosis.

Topics: ADP Ribose Transferases; Antigens, Neoplasm; Apoptosis; Bacterial Toxins; Blotting, Western; Caspase 3; Caspase 8; Cell Line, Tumor; Chromatin; Cytochromes c; Dose-Response Relationship, Drug; Exotoxins; Flow Cytometry; Humans; Immunotoxins; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pseudomonas aeruginosa Exotoxin A; Time Factors; Virulence Factors

Although diallyl trisulphide (DATS) has been found to induce apoptosis in various tumour cells, its cytotoxicity in melanoma cells has not yet been defined and the molecular pathway by which DATS induces apoptosis is not well understood.. To determine growth inhibition of DATS in human melanoma cells (A375 and M14) by inducing apoptosis, and to investigate the mechanism underlying such effects.. Growth inhibition by DATS was estimated by the tetrazolium assay. Apoptosis induction in DATS-treated cells was assessed by staining with 4',6-diamidino-2-phenylindole (DAPI) and double staining with annexin V and propidium iodide. Expression of Bcl-2, Bax, Bcl-xL/Bcl-xS, cytochrome c release, activation of caspase-9 and poly(ADP-ribose) polymerase (PARP) were determined by western blotting. The activity of caspase-3 was measured using a colorimetric assay.. DATS exerted its cytotoxic effect in a time-dependent and dose-dependent manner by inducing apoptosis in A375 and M14 cells. Expression of Bcl-2 and Bcl-xL was downregulated. Release of cytochrome c and activation of the downstream effectors caspase-3, caspase-9 and PARP were detected after DATS sensitization.. DATS inhibits growth of melanoma cells by inducing apoptosis in association with downregulation of Bcl-2 and Bcl-xL and activation of caspases.

Topics: Allyl Compounds; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cytochromes c; Down-Regulation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Skin Neoplasms; Sulfides

This study was carried out to assess the cytotoxicity of several new synthetic steroidal saponins against the human myeloid leukemia cell lines (HL-60 and U937) and against human melanoma cells (SK-MEL-1). Several diosgenyl glycosides analyzed showed strong cell growth inhibition which was associated with alterations in cell cycle progression and induction of apoptosis. Studies of cytochrome c release and caspase-9 activation suggest a main role of the intrinsic pathway of apoptosis in the mechanism of cytotoxicity caused by this kind of compounds.

Topics: Antineoplastic Agents; Apoptosis; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Melanoma; Saponins; U937 Cells

The extracellular gamma-glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. This study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H(2)O(2). Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Blotting, Western; Catalase; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drug Resistance, Neoplasm; gamma-Glutamyltransferase; Humans; Hydrogen Peroxide; Melanoma; Mitochondria; Oxidative Stress; Reactive Oxygen Species

To test the efficacy of resveratrol, a nontoxic plant product, in the treatment of uveal melanoma.. The effect of oral administration and peritumor injection of resveratrol was tested on tumor growth in two animal models of uveal melanoma. The mechanism of resveratrol action on uveal melanoma cells was studied in vitro in a cell-viability assay: with JC-1 dye, to measure mitochondrial membrane potential; by Western blot analysis, to analyze the cellular redistribution of cytochrome c and Smac/diablo; and in a fluorescence assay with specific substrates, to measure activation of different caspases.. Resveratrol treatment inhibited tumor growth in animal models of uveal melanoma. Since oral administration resulted in relatively low bioavailability of resveratrol, the effect of increased local levels was tested by peritumor injection of the drug. This method resulted in tumor cell death and tumor regression. In vitro experiments with multiple uveal melanoma cell lines demonstrate that resveratrol causes a decrease in cell viability, resulting at least in part from an increase in apoptosis through a mitochondrial pathway. An early event in drug action is the direct targeting of mitochondria by resveratrol, which leads to a decrease in mitochondrial membrane potential and the eventual activation of caspase-3.. These data suggest that resveratrol can inhibit tumor growth and can induce apoptosis via the intrinsic mitochondrial pathway and that by further increasing bioavailability of resveratrol the potency of the drug can be increased, leading to tumor regression. The nontoxic nature of the drug at levels needed for therapy make resveratrol an attractive candidate for the treatment of uveal melanoma.

Topics: Administration, Oral; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Biological Availability; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Mitochondrial Proteins; Resveratrol; Stilbenes; Transplantation, Heterologous; Uveal Neoplasms

Geraniin, a form of tannin separated from geranium, causes cell death through induction of apoptosis; however, cell death characteristics for geraniin have not yet been elucidated. Here, we investigated the mechanism of geraniin-induced apoptosis in human melanoma cells and demonstrated that geraniin was able to induce cell apoptosis in a concentration- and time-dependent manner. We also examined the signaling pathway related to geraniin-induced apoptosis. To clarify the relationship between focal adhesion kinase (FAK) and geraniin-induced apoptosis, we treated human melanoma cells with geraniin and found that this resulted dose- and time-dependent degradation in FAK. However, FAK cleavage was significantly inhibited when cells were pretreated with a selective inhibitor of caspase-3 (Ac-Asp-Glu-Val-Asp-CHO). Here, we demonstrated for the first time that geraniin triggered cell death by caspase-3-mediated cleavage of FAK. There were two possible mechanisms for activating caspase-3, mitochondria-mediated and receptor-mediated apoptosis. To confirm the geraniin-relevant signaling pathway, using immunoblot analysis we found that geraniin-induced apoptosis was associated with the up-regulation of Fas ligand expression, the activation of caspase-8, the cleavage of Bid, and the induction of cytochrome c release from mitochondria to the cytosol. Treatment with geraniin caused induction of caspase-3 activity in a dose- and time-dependent manner followed by proteolytic cleavage of poly-(ADP-ribose) polymerase, and DNA fragmentation factor 45. The geraniin-induced apoptosis may provide a pivotal mechanism for its cancer-chemopreventive action.

Topics: Anticarcinogenic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; Focal Adhesion Protein-Tyrosine Kinases; Glucosides; Humans; Hydrolyzable Tannins; Melanoma; Up-Regulation

We recently demonstrated that two new prenylflavanones, propolin A and propolin B, isolated and characterized from Taiwanese propolis, induced cytotoxicity effect in human melanoma A2058 cells and shows a strong capability to scavenge free radicals. In this study, propolin A effectively induced a cytotoxic effect on five different cancer cell lines. Similar results were obtained for propolin B. DNA flow cytometric analysis and DNA fragmentation ladder indicated that propolin A and propolin B actively induced apoptosis in A2058 cells. To address the mechanism of the apoptosis effect of propolin A and propolin B, we evaluated the apoptosis-related proteins in A2058 cells. The levels of procaspase-8, Bid, procaspase-3, DFF45, and PARP were decreased in dose- and time course-dependent manners. Furthermore, also found propolin A and propolin B was capable of releasing cytochrome c from mitochondria to cytosol. The findings suggest that propolin A and propolin B may activate a mitochondria-mediated apoptosis pathway. On the other hand, our data show that propolin B inhibitied xanthine oxidase activity more efficiently than propolin A or CAPE. However, CAPE suppressed ROS-induced DNA strand breakage more efficiently than propolin A or propolin B. All these results indicated that propolin A and propolin B may trigger apoptosis of A2058 cells through mitochondria-dependent pathways and also shown that propolin A and propolin B were strong antioxidants.

Topics: Apoptosis; Blotting, Western; Caffeic Acids; Caspase 3; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Breaks; DNA Fragmentation; DNA, Circular; Dose-Response Relationship, Drug; Flavonoids; Flow Cytometry; HL-60 Cells; Humans; Melanoma; Mitochondria; Molecular Structure; Phenylethyl Alcohol; Propolis; Reactive Oxygen Species; Signal Transduction; Xanthine Oxidase

Isocostunolide is a sesquiterpene lactone isolated from the roots of Inula helenium. Its chemical structure was determined by NMR and FAB-MS spectra. No biological activities of this compound have yet been reported. In this study, we found isocostunolide could effectively induce cytotoxicity in three cancer cell lines (A2058, HT-29, and HepG2), with an IC(50) of 3.2, 5.0, and 2.0 micro g/mL, respectively. DNA flow cytometric analysis indicated that isocostunolide actively induced apoptosis of cancer cells accompanied by a marked loss of G0/G1 phase cells. To address the mechanism of the apoptotic effect of isocostunolide, we analyzed the induction of apoptosis-related proteins in A2058. The levels of pro-caspase-8, Bid, pro-caspase-3, and poly(ADP-ribose) polymerase (PARP) decreased. However, the level of Fas was increased markedly in a dose-dependent manner. Furthermore, this compound markedly induced a depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol. The findings suggest that isocostunolide may activate a mitochondria-mediated apoptosis pathway. To address this, we found that isocostunolide-induced loss of mitochondrial membrane potential occurred via modulation of the Bcl-2 family proteins. The production of intracellular reactive oxygen species (ROS) in A2058 was not elicited. In summary, for the first time, we have isolated and characterized isocostunolide from I. helenium. This compound induces apoptosis through a mitochondria-dependent pathway in A2058 cells.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; HT29 Cells; Humans; Lactones; Melanoma; Mitochondria; Mitochondrial Membranes; Molecular Structure; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction; Time Factors

In this work we tried to estimate the role of mitochondria in the ability of cells of two: melanotic and amelanotic transplantable melanoma lines to undergo spontaneous and camptothecin-induced apoptosis. We measured mitochondrial transmembrane potential (DeltaPsi) changes during culture without (spontaneous apoptosis) and with camptothecin (induced apoptosis) by using JC-1 staining and flow cytometry analysis. Cytochrome c release and PARP cleavage as the biological effects of DeltaPsim changes belonging to the phenomena observed during apoptosis were estimated by Western blotting. The results of our investigations showed in both transplantable melanoma cells the features indicating apoptosis: DeltaPsi changes, cytochrome c release and PARP cleavage, but the degree of observed changes depended on the phenotype of melanoma cells examined. After camptothecin treatment the changes were more pronounced in the amelanotic melanoma cells- the more aggressive line.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Camptothecin; Cricetinae; Cytochromes c; Cytosol; Flow Cytometry; Male; Melanoma; Membrane Potential, Mitochondrial; Mesocricetus; Poly(ADP-ribose) Polymerases; Time Factors; Tumor Cells, Cultured

TR3 was originally recognized for its role in the regulation of cell survival and differentiation, however, it was recently found to be a potent pro-apoptotic protein. In order to characterize the role of TR3 in melanoma cell apoptosis, we studied expression of TR3 in melanoma cell lines and tissues, its subcellular distribution and function during apoptosis using various expression and RNA interference vectors. We found that TR3 was constitutively expressed in both cultured melanoma cells and melanoma tissues. TR3 expression was significantly decreased in advanced melanomas comparing to benign nevi. Over-expression of wild type TR3 or mutant TR3 lacking the DNA binding domain resulted in massive apoptosis in melanoma cells, whereas stable knockdown of TR3 using RNA interference resulted in melanoma cell resistance to apoptosis induced by chemotherapeutic agents ATRA and cisplatin. We further demonstrated that apoptosis in melanoma cells was mediated, at least partially, through TR3 mitochondrial translocation but not alteration in TR3 expression levels. Our results suggest that TR3 is an important apoptosis inducing factor in melanoma cells. Decreased expression of TR3 in metastatic melanoma cells may contribute to their reduced apoptotic potential and increased resistance to chemotherapy.

Topics: Apoptosis; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA-Binding Proteins; Humans; Melanoma; Mitochondria; Nuclear Receptor Subfamily 4, Group A, Member 1; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Skin Neoplasms; Transcription Factors; Tretinoin

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient of hot chili peppers, has been reported to possess substantial anticarcinogenic and antimutagenic activities. In the present study, we investigated the effect of capsaicin on induction of apoptosis in highly metastatic B16-F10 murine melanoma cells. Capsaicin inhibited growth of B16-F10 cells in a concentration-dependent manner. Proapoptotic effect of capsaicin was evidenced by nuclear condensation, internucleosomal DNA fragmentation, in situ terminal nick-end labeling of fragmented DNA (TUNEL), and an increased sub G1 fraction. Treatment of B16-F10 cells with capsaicin caused release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Furthermore, Bcl-2 expression in the B16-F10 cells was slightly down-regulated by capsaicin treatment. In contrast, there were no alterations in the levels of Bax in capsaicin-treated cells. Collectively, these findings indicate that capsaicin-induces apoptosis of B16-F10 melanoma cells via down-regulation the Bcl-2.

Topics: Adenosine Diphosphate Ribose; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Capsaicin; Capsicum; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Genes, bcl-2; Humans; In Situ Nick-End Labeling; Melanoma; Mice; Proto-Oncogene Proteins c-bcl-2

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

DC-81, an antitumor antibiotic produced by the Streptomyces species, belongs to pyrrolo[2,1-c] [1,4]benzodiazepine (PBD), which are potent inhibitors of nucleic acid synthesis. We previously reported an efficient synthesis of PBD hybrids linked with indole carboxylates. This is the first demonstration on the mechanism of the anticancer effect of PBD hybrid (IN6CPBD) agent on human melanoma A375 cells. IN6CPBD-treated cells exhibited higher cytotoxicity than DC-81 and displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, a degradation of caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage. Because degradative changes associated with apoptosis are often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in IN6CPBD-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with IN6CPBD resulted in the loss of mitochondrial membrane potential (DeltaPsimt), a decrease in intracellular pH (pHi), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and cytochrome c release. Collectively, our studies indicate that IN6CPBD induces apoptosis in A375 cells through a mitochondrial dysfunction pathway, leading to caspase-3 substrate PARP cleavage and subsequent apoptotic cell death.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Azepines; Benzodiazepines; Blotting, Western; Caspase 3; Catalase; Cell Line, Tumor; Cell Survival; Collagen Type XI; Cytochromes c; Dioxins; Dose-Response Relationship, Drug; Flow Cytometry; G1 Phase; Humans; Hydrogen-Ion Concentration; Indoles; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Nucleic Acids; Oligomycins; Organophosphorus Compounds; Reactive Oxygen Species; Superoxide Dismutase

The concept of 'vasculogenic mimicry' (VM) was introduced to describe the unique ability of highly invasive tumor cells to form capillary-like structures (CLS) and matrix-rich patterned network in three-dimensional culture that mimic embryonic vasculogenic network. Recently, we have shown that CLS formation requires apoptotic cell death through activation of caspase-3-dependent mechanism. In this study, to identify some molecular determinants driving aggressive melanoma cells to express a latent 'angiogenic program' that recapitulates the early events of CLS formation, we focused on the involvement of antioxidants (AOs) in the process of melanoma VM. We have studied the effects of resveratrol, (-)-epigallocathechin gallate, N-acetyl-cysteine (NAC) and Trolox on the ability of melanoma cells to form/destroy CLS. We observed that the formation of CLS was strongly related to reactive oxygen species level. In vivo animal experiments confirmed the involvement of reactive oxygen species level in melanoma VM. To understand the molecular mechanisms of this phenomenon, we specifically looked for induction of apoptosis and vascular endothelial growth factor (VEGF) release. Western blot analysis revealed that the level of VEGF, VEGF receptors (VEGF-Rs) and active caspase-3 dramatically decreased in cells treated with AOs. Here, we also report further experiments designed to determine whether the crosstalk between AOs and apoptosis exists in melanoma VM.

Topics: Angiogenesis Inhibitors; Animals; Antioxidants; Apoptosis; Capillaries; Caspase 3; Cell Line, Tumor; Cytochromes c; Female; Humans; Melanoma; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Reactive Oxygen Species; Resveratrol; Stilbenes; Vascular Endothelial Growth Factor A

Mitogen-activated protein kinase (MAPK) is activated in the majority of melanomas, and its activity is essential for cell survival. In this report, we examined the effects of a novel raf inhibitor BAY 43-9006 on melanoma cell viability and intracellular signaling and found that it induces apoptosis through a caspase-independent mechanism. At concentrations that suppress extracellular signal-regulated kinase (ERK) phosphorylation, BAY 43-9006 dephosphorylates Bad on Ser(75) and Ser(99), activates Bak and Bax, and reduces the mitochondrial transmembrane potential. BAY 43-9006 (sorafenib) down-modulates the levels of Bcl-2 and Bcl-X(L) in a MAPK-independent manner in A2058 and SKMEL5 melanoma cells but not in the more resistant A375 cells. Of the three lines tested, only A375 cells were rescued from BAY 43-9006-induced apoptosis by knocking down Bad. BAY 43-9006 induced poly(ADP-ribose) polymerase cleavage and the mitochondrial release of cytochrome c and SMAC. However, the pan-caspase inhibitor Z-VAD-fmk had only a modest protective effect against the drug, suggesting that BAY 43-9006-induced apoptosis is largely caspase independent. BAY 43-9006 but not the MAP/ERK kinase inhibitors PD98059 or U0126 induced the nuclear translocation of apoptosis-inducing factor (AIF) in A2058 and SKMEL5 cells, and the introduction of a small interfering RNA (siRNA) for AIF partially protected these cells from BAY 43-9006-induced apoptosis. The AIF siRNA had little effect in A375 cells, in which drug-induced AIF release was negligible. These data indicate that in sensitive cell lines, BAY 43-9006-induced apoptosis is independent of Bad dephosphorylation and caspase activation and largely mediated through the nuclear translocation of AIF.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; Benzenesulfonates; Caspases; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Humans; Intracellular Signaling Peptides and Proteins; Melanoma; Mitochondria; Mitochondrial Proteins; Mitogen-Activated Protein Kinases; Niacinamide; Phenylurea Compounds; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyridines; raf Kinases; Sorafenib

We investigated the possible mechanisms by which petrotetrayndiol A, a polyacetylene from the sponge Petrosia sp., exerts its anti-proliferative activity in cultured SK-MEL-2 human melanoma cells. Petrotetrayndiol A-treated SK-MEL-2 cells showed growth inhibition and induction of apoptosis in a dose-dependent manner as measured by MTT assay, fluorescent microscopy and flow cytometric analysis. Flow cytometric analysis revealed that petrotetrayndiol A resulted in G2/M arrest in the cell cycle progression which was associated with a marked decrease in the protein expression of cyclin B1 and its activating partner Cdc2 with concomitant inductions of p21WAF1/CIP1. The increase in apoptosis was associated with a dose-dependent up-regulation of cytosolic factor, such as Bax and release of cytochrome c, and down-regulation of Bcl-2. We also observed activation of caspase-9 and caspase-3, DNA ladder formation, proteolytic degradation of poly(ADP-ribose)-polymerase (PARP), and selective down-regulation of cIAP-1. The apoptotic manifestations, such as PARP cleavage and DNA fragmentation, were abolished in the presence of the tripeptide caspase inhibitor z-VAD-fmk and a caspase-3-specific inhibitor Ac-DEVD-cho. Our data thus demonstrate that petrotetrayndiol A-induced apoptosis and growth inhibition of SK-MEL-2 cells is dependent on caspase activation.

Topics: Acetylene; Animals; Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Enzyme Activation; Humans; Inhibitor of Apoptosis Proteins; Melanoma; Polyynes; Porifera; Proto-Oncogene Proteins c-bcl-2

G3139, an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in 518A2 and other melanoma cells. G3139-mediated apoptosis appears to be independent of its ability to down-regulate the expression of Bcl-2 protein, because the release of mitochondrial cytochrome c precedes in time the down-regulation of Bcl-2 protein expression. In this study, we demonstrate the ability of G3139 and other phosphorothioate oligonucleotides to bind directly to mitochondria isolated from 518A2 cells. Furthermore, we show that this interaction leads to the release of cytochrome c in the absence of a mitochondrial membrane permeability transition. Our data further demonstrate that there is an interaction between G3139 and VDAC, a protein that can facilitate the physiologic exchange of ATP and ADP across the outer mitochondrial membrane. Evidence from the electrophysiologic evaluation of VDAC channels reconstituted into phospholipid membranes demonstrates that G3139 is capable of producing greatly diminished channel conductance, indicating a closed state of the VDAC. This effect is oligomer length-dependent, and the ability of phosphorothioate homopolymers of thymidine of variable lengths to cause the release of cytochrome c from isolated mitochondria of 518A2 melanoma cells can be correlated with their ability to interact with VDAC. Because it has been suggested that the closure of VDAC leads to the opening of another outer mitochondrial membrane channel through which cytochrome c can transit, thus initiating apoptosis, it appears that VDAC may be an important pharmacologic target of G3139.

Topics: Base Sequence; Cell Line, Tumor; Cytochromes c; Humans; Ion Channel Gating; Melanoma; Mitochondria; Mitochondrial Membranes; Oligonucleotides; Phosphates; Thionucleotides; Voltage-Dependent Anion Channels

Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo.. 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured.. In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors.. The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.

Topics: Animals; Apoptosis; Base Sequence; Cytochromes c; DNA, Antisense; Down-Regulation; Gene Silencing; Humans; Melanoma; Mice; Mice, Inbred ICR; Mice, SCID; Mitochondria; Molecular Sequence Data; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Small Interfering; Thionucleotides; Xenograft Model Antitumor Assays

Cell-cell adhesion is considered to be important in the development and maintenance of organ tissue. The spatial association between melanocytes and keratinocytes within human epidermis is achieved by homophilic interaction of E-cadherin molecules located on adjacent cells. In contrast, downregulation of E-cadherin expression in melanoma cells is considered as a key event in metastasis. Besides the adhesive properties, E-cadherin serves as a signal receptor linking to the cadherin-catenin signaling complex. As cadherins act as negative regulators of beta-catenin, a contribution to tumor formation seems likely. In the present study, it was tested whether ectopic expression of E-cadherin triggers apoptosis in human melanoma cell lines (G-361, JPC-298, SK-Mel-13). It was found that restoration of E-cadherin caused sensitization against drug-induced apoptosis. Particularly, the release of mitochondrial cytochrome c was increased in response to staurosporine. Moreover, activation of caspase-3 and caspase-8 was elevated. Similarly, DNA fragmentation, serving as a marker for advanced apoptosis, was amplified in cells transduced with E-cadherin. Interestingly, transduction with an E-cadherin construct lacking the extracellular domain showed no modified apoptosis. In conclusion, our findings suggest therapeutic strategies that enable expression of E-cadherin in order to sensitize human melanoma cells towards apoptosis.

Topics: Apoptosis; Cadherins; Caspase 3; Caspase 8; Catenins; Cell Adhesion; Cell Line, Tumor; Cytochromes c; Cytosol; DNA Fragmentation; Humans; Melanoma; Neoplasm Metastasis; Protein Structure, Tertiary; Staurosporine

Apoptotic deficiency is one of the mechanisms leading to chemoresistance due to the potential of many chemotherapeutic drugs to induce apoptosis. We have examined drug-induced apoptosis in the chemosensitive human melanoma cell line MeWo, as well as in its resistant sublines, which were selected by continuous exposure to etoposide (MeWo(Eto1)) and cisplatin (MeWo(Cis1)). In former studies, activation of the mitochondrial pro-apoptotic pathway could not be demonstrated in etoposide-resistant cells after exposure to etoposide. A significant reduction of PARP [poly (ADP-ribose) polymerase] cleavage and caspase activation, but unimpaired DNA fragmentation, was seen in cisplatin-resistant cells after treatment with cisplatin. In the current study, we investigated effects of chemotherapeutic drugs different from the selecting agents cisplatin and etoposide on the observed modulations of the mitochondrial apoptotic pathway. We analysed dose-dependent release of cytochrome c, caspase-9 activation, cleavage of PARP and activation of effector caspases in etoposide and cisplatin-resistant cells after exposure to etoposide, teniposide, cisplatin or fotemustine. In analogy to etoposide exposure, we could not demonstrate any activation of the apoptotic pathway in etoposide-resistant cells after exposure to teniposide, another topoisomerase-II inhibitor. In contrast, exposure to cisplatin and fotemustine led to apoptotic cell death in these cells. This suggests that the deficiency of apoptosis in etoposide-resistant cells is dependent on the trigger by topoisomerase-II inhibitors. Analysis of cisplatin-resistant cells after etoposide and fotemustine exposure revealed an increased activity of the apoptotic pathway when compared with cisplatin exposure at corresponding survival rates in these cells. These results suggest that the observed modulations of the apoptotic pathway in resistant melanoma cell lines are specific for an anti-neoplastic drug and are not fixed at the molecular level, as different chemotherapeutic drugs are capable of overcoming these alterations.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspases; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; Etoposide; Humans; Melanoma; Poly(ADP-ribose) Polymerases; Signal Transduction; Tumor Cells, Cultured

In a previous study, we showed that G3139, an antisense phosphorothioate oligonucleotide that down-regulates the expression of Bcl-2 protein, did not cause chemosensitization of 518A2 melanoma cells. In this work, we show that G3139, and the 2-base mismatch, G4126, can initiate apoptosis in this and other melanoma cell lines as shown by increased cell surface Annexin V expression, typical nuclear phenotypic changes as assessed by 4',6-diamidino-2-phenylindole staining, activation of caspase-3 (but not caspase-8) and Bid, appearance of DEVDase (but not IETDase) activity, and cleavage of poly(ADP-ribose)-polymerase 1. Depolarization of the mitochondrial membrane occurs as a relatively late event. All of these processes seem to be substantially, but perhaps not totally, Bcl-2 independent as shown by experiments employing an anti-Bcl-2 small interfering RNA, which as shown previously down-regulated Bcl-2 protein expression but did not produce apoptosis or chemosensitization in melanoma cells. In fact, these G3139-induced molecular events were not dramatically altered in cells that forcibly overexpressed high levels of Bcl-2 protein. Addition of irreversible caspase inhibitors (e.g., the pan-caspase inhibitor zVAD-fmk) to G3139-treated cells almost completely blocked cytotoxicity. Examination of the time course of the appearance of caspase-3 and cleaved poly(ADP-ribose)-polymerase 1 showed that this could be correlated with the release of cytochrome c from the mitochondria, an event that begins only approximately 4 hours after the end of the oligonucleotide/LipofectAMINE 2000 5-hour transfection period. Thus, both G3139 and cytotoxic chemotherapy activate the intrinsic pathway of apoptosis in these cells, although Bcl-2 expression does not seem to contribute strongly to chemoresistance. These findings suggest that the attainment of G3139-induced chemosensitization in these cells will be difficult.

Topics: Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; Cytoplasm; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Lipids; Melanoma; Membrane Potentials; Mitochondria; Oligodeoxyribonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Thionucleotides; Transfection

The roles of p33ING2 as a tumor suppressor candidate have been shown through regulation of gene transcription, induction of cell cycle arrest, and apoptosis. As p33ING2 shares 58.9% homology with p33ING1b, we hypothesized that p33ING2 shares functional similarities with p33ING1b. We previously found that p33ING1b cooperates with p53 to enhance UVB-induced apoptosis. Here, we report that overexpression of p33ING2 enhanced apoptosis in UVB-irradiated and non-irradiated melanoma MMRU cells. We demonstrate that enhancement of apoptosis by p33ING2 requires the presence of functional p53. Furthermore, we found that overexpression of p33ING2 significantly downregulated the expression of Bcl-2 after UVB irradiation, resulting in an increased Bax/Bcl-2 ratio. Moreover, we found that p33ING2 promoted Bax translocation to mitochondria, altered the mitochondrial membrane potential, and induced cytochrome c release and thus the activation of caspases 9 and 3. In addition, we showed that under non-stress conditions p33ING2 upregulates Fas expression and activates caspase 8. Taken together, we demonstrate that p33ING2 cooperates with p53 to regulate apoptosis via activation of both the mitochondrial/intrinsic and death-receptor/extrinsic apoptotic pathways.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspases; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; fas Receptor; Homeodomain Proteins; Humans; Melanoma; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ultraviolet Rays; Up-Regulation

Solid tumors are often placed under stress conditions, such as glucose starvation which may result in topoisomerase II drug resistance. In this study, we investigated whether glucose deprivation or substitution by fructose regulates tumor cell apoptosis induced by 2-acetyl furanonaphthoquinone (FNQ). We now show that FNQ exerts much greater antitumor activity than either 7-methoxy 2-ethyl FNQ or 2-ethyl FNQ. Whereas 0.8 microM FNQ induces apoptosis after 16 hours in glucose-supplemented conditions irrespective of bcl-2 overexpression in K1735 melanoma, 0.5 microM FNQ is also effective within 12 hours in low glucose or in fructose-supplemented medium. Under the latter conditions, apoptosis-associated PARP cleavage and cytosolic cytochrome C are increased, together with induction and partial translocation to mitochondria of phosphorylated Jun-N-terminal kinase and massive upregulation of mitochondrial Mn superoxide dismutase. We propose that mitochondrial colocalization of these activities is important in this synergistic anti-tumor effect of FNQ and glucose depletion. Since glucose limitation slows proliferation and decreases efficacy of some genotoxic drugs that trigger apoptosis in rapidly dividing cells, we propose evaluating FNQ as a novel therapeutic anti-cancer adjuvant against slowly proliferating tumors.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cytochromes c; Furans; Glucose; Glycolysis; Humans; MAP Kinase Kinase 4; Melanoma; Mitochondria; Naphthoquinones; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms; Superoxide Dismutase; Transcriptional Activation; Tumor Cells, Cultured

The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk/Bik) has been described to inhibit Bcl-2 and Bcl-xL, thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk/Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk/Bik only weakly at the mRNA and protein level. Conditional expression of Nbk/Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk/Bik in vivo, stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk/Bik expression. By investigating the mechanism of Nbk/Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk/Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk/Bik.

Topics: Animals; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspases; Chromatin; Cytochromes c; Diphosphonates; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; fas Receptor; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Injections, Subcutaneous; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Pamidronate; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Skin; Tetracycline; Tumor Cells, Cultured

Melanoma is the most aggressive form of skin cancer and advanced stages are invariably resistant to conventional therapeutic agents. Using bortezomib as a prototypic proteasome inhibitor, we have identified a novel and critical role of the proteasome in the maintenance of the malignant phenotype of melanoma cells that could have direct translational implications. Thus, melanoma cells from early, intermediate, and late stages of the disease could not sustain proteasome inhibition and underwent an effective activation of caspase-dependent and -independent death programs. This effect was tumor cell selective, because under similar conditions, normal melanocytes remained viable. Intriguingly, and despite of interfering with a cellular machinery in charge of controlling the half-life of the vast majority of cellular proteins, bortezomib did not promote a generalized disruption of melanoma-associated survival factors (including NF-kappaB, Bcl-2, Bcl-x(L), XIAP, TRAF-2, or FLIP). Instead, we identified a dramatic induction in vitro and in vivo of the BH3-only protein Noxa in melanoma cells (but not in normal melanocytes) in response to proteasome inhibition. RNA interference validated a critical role of Noxa for the cytotoxic effect of bortezomib. Notably, the proteasome-dependent regulation of Noxa was found to extend to other tumor types, and it could not be recapitulated by standard chemotherapeutic drugs. In summary, our results revealed Noxa as a new biomarker to gauge the efficacy of bortezomib specifically in tumor cells, and provide a new strategy to overcome tumor chemoresistance.

Topics: Animals; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Caspases; Cisplatin; Cytochromes c; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Activation; Female; Humans; Melanocytes; Melanoma; Mice; Mice, Nude; NF-kappa B; Protease Inhibitors; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrazines; RNA Interference; Up-Regulation; Xenograft Model Antitumor Assays

p53, a commonly mutated gene in human cancers, participates in cell cycle arrest, DNA repair and apoptosis. A small pharmacological compound, CP-31398, was found to have the ability to promote proper p53 protein folding, activate p53 transcription of downstream targets, and slow tumour growth in mice. Additionally, CP-31398 was found to be able to convert mutant p53 to wild-type conformation in several cell lines.. To examine if CP-31398 can revert all mutant p53 proteins to wild-type function.. We studied a series of apoptotic responses to CP-31398 in three melanoma cell lines varying in p53 mutation status.. Upon a moderate dose of CP-31398 treatment (15 microg mL(-1)), only the wild-type p53 MMRU and the single p53 point mutation MeWo cells exhibited apoptosis. Another melanoma cell line, Sk-mel-110, containing multiple p53 mutations, did not exhibit apoptosis. Although CP-31398 enhanced overall p53 protein level, its ability to promote proper folding of p53 protein was limited to CP-31398-sensitive MMRU and MeWo cells. These sensitive cells showed an increased Bax and PUMA transcription, altered mitochondrial membrane potential, followed by the release of cytochrome c, and cleaved caspase-9 and caspase-3. We also demonstrated that Apaf-1 was not involved in CP-31398-mediated apoptosis.. Our results suggest that the ability of CP-31398 to revert mutant p53 proteins to wild-type conformation may be correlated to p53 mutational status. More studies are necessary, to further investigate the effect of CP-31398 on mutant p53 and its potential applications as an anticancer agent.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Caspases; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Genes, p53; Humans; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Potentials; Mitochondria; Proteins; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We had demonstrated that two prenylflavanones, propolin A and propolin B, isolated and characterized from Taiwanese propolis, induced apoptosis in human melanoma cells and significantly inhibited xanthine oxidase activity. Here, we have isolated a third compound called propolin C. The chemical structure of propolin C has been characterized by NMR and HRMS spectra, and was identical to nymphaeol-A. However, no biological activities of this compound have ever been reported. In the present study, propolin C effectively induced a cytotoxic effect on human melanoma cells, with an IC(50) of about 8.5 microM. DNA flow cytometric analysis indicated that propolin C actively induced apoptosis in human melanoma cells and there is a marked loss of cells from the G2/M phase of the cell cycle. To address the mechanism of the apoptosis effect of propolin C, we evaluated the effect of propolin C on induction of apoptosis-related proteins in human melanoma cells. The levels of procaspase-8, Bid, procaspase-3, and poly(ADP-ribose) polymerase were decreased in dose- or time course-dependent manners. Moreover, propolin C was capable of releasing cytochrome c from mitochondria to cytosol. The findings suggest that propolin C may activate a mitochondria-mediated apoptosis pathway. On other hand, propolin C is a potential antioxidant agent and shows a strong capability to scavenge free radicals and inhibit on xanthine oxidase activity with IC(50) of about 17.0microM. In conclusion, the isolation and characterization of propolin C from bee propolis are described for the first time, and this compound is a powerful inducer of apoptosis in human melanoma cells.

Topics: Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caffeic Acids; Carrier Proteins; Caspases; Cell Cycle; Cell Division; Cell Survival; Cytochromes c; DNA Fragmentation; Enzyme Activation; Flavonoids; Free Radicals; Humans; Melanoma; Mitochondria; Phenylethyl Alcohol; Propolis; Xanthine Oxidase

Apoptosis protease-activating factor-1 (Apaf-1), the central element in the mitochondrial pathway of apoptosis, is frequently absent or poorly expressed in metastatic melanomas, a tumor type showing a low degree of spontaneous apoptosis and a poor response to conventional therapies. In the present study, we used the Apaf-1-positive Me665/2/21 melanoma cell line to investigate the fate of Apaf-1 during cisplatin-induced apoptosis. As novel findings described for the first time in melanoma cells, we observed that Apaf-1 was markedly decreased during apoptosis, already at early stages of cell damage; concurrently, an immunoreactive N-terminal fragment of congruent with 26 kDa was evident. In spite of the remarkable decrease of Apaf-1 in apoptotic cells, caspase-9 was found to be processed and enzymatically active. Both Apaf-1 depletion and its proteolytic cleavage were markedly prevented in presence of the caspase-3/-7 inhibitor ac-DEVD-CHO. In presence of ac-DEVD-CHO, caspase-9 activity was also inhibited, along with a partially different pattern of caspase-9 processing forms. Unexpectedly, the inhibition afforded by ac-DEVD-CHO on several components, that is, caspase-3/-7 and caspase-9 activities, and Apaf-1 proteolytic degradation, did not abrogate the apoptotic morphology and cell detachment, nor the proteolytic degradation of crucial targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. Together, our results suggest that caspase-3 and -7, proved to be dispensable for the above apoptosis-associated events, play a role on Apaf-1 handling and possibly on apoptosome function.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Line, Tumor; Cisplatin; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Lamin Type B; Macromolecular Substances; Melanoma; Mitochondria; Peptide Fragments; Peptide Hydrolases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Signal Transduction

Staurosporine has long been used in vitro as an initiator of apoptosis in many different cell types, but the mechanism involved remains poorly understood. In the present study, we have examined the apoptosis-inducing potential of staurosporine in cultured melanoma cell lines and dissected the staurosporine-induced apoptotic signaling pathway. We report that although staurosporine activated Bax and the mitochondrial caspase-dependent apoptotic pathway, it also induced apoptosis of melanoma by caspase-independent pathways. The caspase-dependent apoptotic pathway was activated relatively soon after exposure to staurosporine and was associated with release of cytochrome c and Smac/DIABLO from mitochondria and cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNase. This pathway was inhibitable by broad caspase inhibitors. A second apoptotic pathway that appeared to be involved in late apoptotic events was caspase independent in that inhibitors of caspases did not prevent the late onset of apoptosis. Overexpression of Bcl-2 inhibited the early onset of apoptosis but not the later, caspase-independent pathway. Apoptosis-inducing factor may be responsible for the late apoptotic execution in that its translocation from mitochondria into the nucleus coincided with the late onset of apoptosis and could not be inhibited by either a pan-caspase inhibitor or overexpression of Bcl-2. Our results indicate that staurosporine is able to bypass resistance of melanoma cells to mitochondrial caspase-dependent apoptotic pathways; hence, derivatives of staurosporine may warrant further evaluation either alone or with other apoptosis-inducing agents.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Carrier Proteins; Caspases; Cell Line, Tumor; Cytochromes c; Cytosol; Enzyme Activation; Flavoproteins; Humans; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Protein Conformation; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Staurosporine; Time Factors

Aroylhydrazone and thiosemicarbazone iron (Fe) chelators have potent antitumor activity. The aim of the current study was to examine the antitumor effects and mechanisms of action of a novel series of Fe chelators, the di-2-pyridyl thiosemicarbazones. Of 7 new chelators synthesized, 4 showed pronounced antiproliferative effects. The most active chelator was Dp44mT, which had marked and selective antitumor activity-for example, an IC(50) of 0.03 microM in neuroepithelioma cells compared with more than 25 microM in mortal fibroblasts. Indeed, this antiproliferative activity was the greatest yet observed for an Fe chelator. Efficacy was greater than it was for the cytotoxic ligand 311 and comparable to that of the antitumor agent doxorubicin. Strikingly, Dp44mT significantly (P <.01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining demonstrated apoptosis in tumors taken from mice treated with Dp44mT. This chelator caused a marked increase of caspase-3 activity in murine Madison-109 (M109) cells. Caspase activation was at least partially mediated by the release of mitochondrial holo-cytochrome c (h-cytc) after incubation with Dp44mT. In conclusion, Dp44mT is a novel, highly effective antitumor agent in vitro and in vivo that induces apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Division; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Iron; Iron Chelating Agents; Lung Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mitochondria; Neoplasm Transplantation; Neuroectodermal Tumors, Primitive, Peripheral; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Reactive Oxygen Species; Thiosemicarbazones; Transferrin; Tumor Cells, Cultured

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.

Topics: Antigens, Neoplasm; Blotting, Western; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Lysosomes; Mass Spectrometry; Melanoma; Microscopy, Electron; Mitochondria; Nuclear Proteins; Proteomics

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that beta-Carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents beta-Carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. beta-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-kB activation is involved in beta-Carotene-induced caspase cascade. These results support a pharmacological role for beta-Carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells.

Topics: Adenocarcinoma; Apoptosis; beta Carotene; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; HL-60 Cells; Humans; Melanoma; Membrane Potentials; Mitochondria; Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured