benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Melanoma

NY-ESO-1-specific CD4(+) T cells are of interest for immune therapy against tumors, because it has been shown that their transfer into a patient with melanoma resulted in tumor regression. Therefore, we investigated how NY-ESO-1 is processed onto MHC class II molecules for direct CD4(+) T cell recognition of melanoma cells. We could rule out proteasome and autophagy-dependent endogenous Ag processing for MHC class II presentation. In contrast, intercellular Ag transfer, followed by classical MHC class II Ag processing via endocytosis, sensitized neighboring melanoma cells for CD4(+) T cell recognition. However, macroautophagy targeting of NY-ESO-1 enhanced MHC class II presentation. Therefore, both elevated NY-ESO-1 release and macroautophagy targeting could improve melanoma cell recognition by CD4(+) T cells and should be explored during immunotherapy of melanoma.

Topics: Acetylcysteine; Antigen Presentation; Antigens, Neoplasm; Autophagy; Autophagy-Related Protein 12; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Chloroquine; Dendritic Cells; Endocytosis; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class II; Humans; Immunotherapy, Adoptive; Leupeptins; Lymphocyte Activation; Lysosomal-Associated Membrane Protein 2; Melanoma; Membrane Proteins; RNA Interference; RNA, Small Interfering; Small Ubiquitin-Related Modifier Proteins

The proteasome is a promising target in cancer therapy. However, it is ubiquitous and its inhibitors cause side effects. To target melanoma cells we synthesized new peptide aldehyde and vinylsulfone inhibitors of the proteasome conjugated to the melanin-targeting ligand (MTL) derived from radiotracer [(123)I]-N-(2-diethylaminoethyl)benzamide ([(123)I]BZA) or [(125)I]-N-(4-dipropylaminobutyl)-4-iodobenzamide ([(125)I]BZ18). Influence on the cytotoxicity of the benzamide alkyl side chain length and the composition of the amino acid sequence was assessed. Among the conjugates evaluated, compound 16 and 22 presented the highest cytotoxicity (IC(50), 0.71 and 0.64 μM respectively), which persisted in the presence of an MTL derived from N-(dialkylaminoalkylenyl)benzamide residue.

Topics: Aldehydes; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Design; Humans; Inhibitory Concentration 50; Ligands; Melanins; Melanoma; Molecular Targeted Therapy; Protease Inhibitors; Proteasome Inhibitors; Sulfones

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor invitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas. Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.

Topics: Animals; Cell Line, Tumor; Etoposide; G1 Phase; Gene Deletion; Genes, p16; Half-Life; Humans; Lactones; Leupeptins; Melanocytes; Melanoma; Mice; Mice, Transgenic; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Tumor Suppressor Protein p53; Ubiquitin

SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood.. In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β.. Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

Topics: Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Proteasome Inhibitors; Proto-Oncogene Proteins; RNA Interference; Skin Neoplasms; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

Most melanoma cells are characterized by the V600E mutation in B-Raf kinase. This mutation leads to increased expression of interleukin (CXCL8), which plays a key role in cell growth and angiogenesis. Thus CXCL8 appears to be an interesting therapeutic target. Hence, we performed vaccination of mice with GST-CXCL8, which results in a reduced incidence of syngenic B16 melanoma cell xenograft tumors. We next addressed the molecular mechanisms responsible for aberrant CXCL8 expression in melanoma. The CXCL8 mRNA contains multiples AU-rich sequences (AREs) that modulate mRNA stability through the binding of tristetraprolin (TTP). Melanoma cell lines express very low TTP levels. We therefore hypothesized that the very low endogenous levels of TTP present in different melanoma cell lines might be responsible for the relative stability of CXCL8 mRNAs. We show that TTP is actively degraded by the proteasome and that extracellular-regulated kinase inhibition results in TTP accumulation. Conditional expression of TTP in A375 melanoma cells leads to CXCL8 mRNA destabilization via its 3' untranslated regions (3'-UTR), and TTP overexpression reduces its production. In contrast, downregulation of TTP by short hairpin RNA results in upregulation of CXCL8 mRNA. Maintaining high TTP levels in melanoma cells decreases cell proliferation and autophagy and induces apoptosis. Sorafenib, a therapeutic agent targeting Raf kinases, decreases CXCL8 expression in melanoma cells through reexpression of TTP. We conclude that loss of TTP represents a key event in the establishment of melanomas through constitutive expression of CXCL8, which constitutes a potent therapeutic target.

Topics: Animals; Antibodies; Antineoplastic Agents; Apoptosis; Autophagy; Benzamides; Benzenesulfonates; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL1; Chemokine CXCL5; Dichlororibofuranosylbenzimidazole; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Genes, Reporter; Half-Life; Humans; Immunotherapy, Active; Interleukin-8; Leupeptins; MAP Kinase Kinase Kinases; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Niacinamide; Phenylurea Compounds; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins; Pyridines; Receptors, Interleukin-8B; RNA Stability; RNA, Messenger; RNA, Small Interfering; Sorafenib; Transfection; Tristetraprolin; Tumor Cells, Cultured; Vaccination

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, alphaB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that alphaB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate alphaB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. alphaB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of alphaB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that alphaB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, alphaB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.

Topics: alpha-Crystallin B Chain; Bleomycin; Butadienes; Cells, Cultured; Cyclin D1; Cycloheximide; DNA Damage; Etoposide; Humans; Leupeptins; Melanocytes; Melanoma; Mutant Proteins; Mutation; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Tetracycline

In the present study, we determined whether or not imidazole affects B16 murine melanoma cell migration to prevent melanoma metastasis.. To determine the effects of imidazole on melanoma cell migration, B16 cells were treated with imidazole at various concentrations, and the migration was measured using a scratch migration assay.. Imidazole did not exhibit cytotoxic effects on B16 cells at a concentration below 100 microm. The anti-migratory activity of imidazole was determined by the scratch migration assay. Our results showed that imidazole significantly inhibits B16 cell migration. It is known that the Wnt/beta-catenin signalling pathway regulates the progression of melanocytic tumours and determines the prognosis in cutaneous melanomas. Western blot analysis demonstrated that imidazole increases phosphorylation of beta-catenin and subsequent degradation of beta-catenin. Moreover, inhibition of melanoma cell migration by imidazole was restored by MG132, a proteasome inhibitor, via inhibition of beta-catenin degradation.. Imidazole inhibits B16 cell migration through beta-catenin degradation, suggesting that imidazole is a potential candidate for the treatment of metastatic melanoma.

Topics: Animals; Antineoplastic Agents; beta Catenin; Blotting, Western; Cell Line, Tumor; Cell Movement; Imidazoles; Leupeptins; Melanoma; Mice; Phosphorylation; Signal Transduction

The Bcl-2 family is important in modulating sensitivity to anticancer drugs in many cancers, including melanomas. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-X(L)/Bcl-w. In this report, we examined whether ABT-737 is effective in killing melanoma cells in combination with the proteasome inhibitor MG-132, and further evaluated the mechanisms of action. Viability, morphological, and Annexin V apoptosis assays showed that ABT-737 alone exhibited little cytotoxicity, yet it displayed strong synergistic lethality when combined with MG-132. In addition, the detection of Bax/Bak activation indicated that the combination treatment synergistically induced mitochondria-mediated apoptosis. Furthermore, mechanistic analysis revealed that this combination treatment induced expression of the pro-apoptotic protein Noxa- and caspase-dependent degradation of the anti-apoptotic protein, Mcl-1. Finally, siRNA-mediated inhibition of Mcl-1 expression significantly increased sensitivity to ABT-737 in these cells, and knocking down Noxa expression protected the cells from cytotoxicity induced by the combination treatment. These findings demonstrate that ABT-737 combined with MG-132 synergistically induced Noxa-dependent mitochondrial-mediated apoptosis. In summary, this study indicates promising therapeutic potential of targeting anti-apoptotic Bcl-2 family members in treating melanoma, and it validates rational molecular approaches that target anti-apoptotic defenses when developing cancer treatments.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Drug Synergism; Humans; Leupeptins; Melanoma; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052-mediated sensitization may result from NF-kappaB inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-kappaB and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-kappaB inhibition in sensitization was corroborated with the NF-kappaB inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052-mediated sensitization. These findings show that NPI-0052 modifies the NF-kappaB-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cisplatin; Humans; Lactones; Leupeptins; Male; Melanoma; Membrane Potential, Mitochondrial; NF-kappa B; Phosphatidylethanolamine Binding Protein; Prostatic Neoplasms; Proteasome Inhibitors; Proto-Oncogene Proteins c-raf; Pyrazines; Pyrroles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Snail Family Transcription Factors; TNF-Related Apoptosis-Inducing Ligand; Transcription Factors; Transfection; Tumor Cells, Cultured

The proteasome inhibitor bortezomib is emerging as a potent anti-cancer agent. Still, recent clinical trials have revealed a significant secondary toxicity of bortezomib. Consequently, there is much interest in dissecting the mechanism of action of this compound to rationally improve its therapeutic index. The cytotoxic effect of bortezomib is frequently characterized by interfering with downstream events derived from the accumulation of proteasomal targets. Here we identify the first chemical agent able to act upstream of the proteasome to prevent cell killing by bortezomib. Specifically, we show that the polyhydroxyl compound Tiron can function as a competitive inhibitor of bortezomib. This effect of Tiron was surprising, since it is a classical radical spin trap and was expected to scavenge reactive oxygen species produced as a consequence of bortezomib action. The inhibitory effect of Tiron against bortezomib was selective, since it was not shared by other antioxidants, such as vitamin E, MnTBAP, L-N-acetyl-cysteine, and FK-506. Comparative analyses with nonboronated proteasome inhibitors (i.e. MG132) revealed a specificity of Tiron for bortezomib. We exploited this novel feature of Tiron to define the "point of no return" of proteasome inhibition in melanoma cells and to block cell death in a three-dimensional model of human skin. Cells from T-cell lymphoma, breast carcinoma, and non-small cell lung cancer were also responsive to Tiron, suggesting a broad impact of this agent as a bortezomib blocker. These results may have important implications for the analysis of bortezomib in vivo and for the design of drug mixtures containing proteasome inhibitors.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Antineoplastic Agents; Antioxidants; Binding, Competitive; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Death; Cell Line; Cell Line, Tumor; Cell Survival; Drug Antagonism; Fibroblasts; Free Radicals; Humans; Immunoblotting; Kinetics; Leupeptins; Melanocytes; Melanoma; Membrane Potentials; Models, Biological; Protease Inhibitors; Proteasome Inhibitors; Pyrazines; Reactive Oxygen Species; Skin; Time Factors

Low levels of p27Kip1 expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27Kip1 for degradation, was overexpressed and was inversely related to p27Kip1 levels in malignant melanoma with poor prognosis.. We investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27Kip1 down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo.. We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo.. Skp2 protein was decreased and the p27Kip1 protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo.. Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27Kip1 degradation in malignant melanoma.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Melanoma; Melanoma, Experimental; Mice; Mice, Nude; RNA Interference; RNA, Small Interfering; S-Phase Kinase-Associated Proteins

Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 micromol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the

Topics: Benzamides; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Dose-Response Relationship, Radiation; Enzyme Inhibitors; Humans; Leupeptins; MAP Kinase Signaling System; Melanoma; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Radiation-Sensitizing Agents; Time Factors; Transfection

CCAAT/enhancer-binding protein (C/EBP) family transcription factors are critical for transcription of several genes involved in tissue development and cellular function, proliferation, and differentiation. Here we show that inhibitory/regulatory C/EBP family proteins, Ig/EBP (C/EBPgamma) and CHOP (C/EBPzeta), but not positively functioning NF-IL6 (C/EBPbeta), are constitutively multiubiquitinated and subsequently degraded by the proteasome. In addition, ubiquitination and degradation of these proteins are suppressed by forming dimer through their leucine zipper domains. Deletion of leucine zipper domain in NF-IL6 caused the loss of its homodimerization activity and the degradation of protein by the ubiquitin-proteasome system. In addition, Ig/EBP with its leucine zipper domain substituted for that of NF-IL6 formed homodimer and was stabilized. These observations suggest that mammalian cells equip a novel regulatory system abrogating the excess C/EBP family transcription factors bereft of dimerizing partner.

Topics: Carcinoma, Squamous Cell; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Cell Line; Cycloheximide; Cysteine Endopeptidases; Dimerization; Humans; Kidney; Leucine Zippers; Leupeptins; Melanoma; Methyl Methanesulfonate; Multienzyme Complexes; Neoplasm Proteins; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Recombinant Fusion Proteins; Sequence Deletion; Transcription Factor CHOP; Transcription Factors; Tumor Cells, Cultured; Ubiquitin