RESUMO
IRE1 is a conserved dual endoribonuclease/protein kinase that is indispensable for directing the endoplasmic reticulum (ER) stress response in yeast, flies, and worms. In mammalian systems, however, the precise biological activities carried out by IRE1alpha are unclear. Here, molecular and chemical genetic approaches were used to control IRE1 activity in a number of prostate cancer cell lines and the resulting impact on gene transcription, cell survival, and proliferation was examined. Modulating IRE1alpha activity had no transcriptional effect on the induction of genes classically associated with the ER stress response (Grp78 and CHOP) or cell survival when confronted with ER stress agents. Rather, IRE1alpha activity was positively correlated to proliferation. Since Xbp-1 mRNA is the sole known substrate for IRE1 endoribonuclease activity, the role of this transcription factor in mediating proliferation was examined. Repressing total Xbp-1 levels by siRNA techniques effectively slowed proliferation. In an effort to identify IRE1/XBP-1 targets responsible for the cell cycle response, genome-wide differential mRNA expression analysis was performed. Consistent with its ability to sense ER stress, IRE1alpha induction led to an enrichment of ER-Golgi, plasma membrane, and secretory gene products. An increase in cyclin A1 expression was the only differentially expressed cell cycle regulatory gene found. Greater cyclin A protein levels were consistently observed in cells with active IRE1alpha and were dependent on XBP-1. We conclude that IRE1alpha activity controls a subset of the ER stress response and mediates proliferation through tight control of Xbp-1 splicing.
Assuntos
Ciclina A1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Ciclina A1/genética , Proteínas de Ligação a DNA/genética , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/genética , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
Inducing apoptosis via the extrinsic death receptor pathway is an attractive anti-cancer treatment strategy, however, numerous cancer cells exhibit significant resistance to death ligand stimuli. Here, we investigated the anti-neoplastic capability of proteasome inhibition, through the administration of Velcade, to synergize with a death receptor agonist in vivo. The death ligand-resistant LNCaP prostate xenograft model was utilized. Tumors were established and mice were treated with Velcade, TRAIL (TNF-Related Apoptosis Inducing Ligand) or the combined regimen. Only mice treated with a combination of Velcade and TRAIL was tumor growth inhibited with a corresponding loss of the hemorrhagic phenotype, decreased tumor cell proliferation and increased tumor cell apoptosis. Next, to determine if the extrinsic pathway is critical for mediating the anti-tumor efficacy that can be achieved in some cell types with Velcade treatment alone, the death receptor sensitive PC-3 xenograft model was used. PC-3 tumors exhibited a 54% decrease in tumor volume in response to Velcade, while c-FLIP overexpressing PC-3 xenografts were resistant to the treatment. These findings suggest that the extrinsic apoptotic pathway can mediate the anti-tumor effects of Velcade and support the therapeutic use of proteasome inhibition in combination with a death receptor stimulus in the treatment of prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Divisão Celular/efeitos dos fármacos , Neoplasias da Próstata/patologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Ácidos Borônicos/uso terapêutico , Bortezomib , Linhagem Celular Tumoral , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Pirazinas/uso terapêutico , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêuticoRESUMO
BACKGROUND: Proteasome inhibition through the administration of Velcade is a viable chemotherapeutic strategy that is approved to treat multiple myeloma and is being evaluated for efficacy against prostate cancer. Currently, the apoptotic pathways that contribute to this anticancer response are poorly understood. Our goal is to test the extent to which proteasome inhibition modulates apoptosis through death receptor pathways. METHODS: Several prostate cancer cell lines and primary prostate epithelial cells (PrECs) were used as models. The death receptor pathway was activated by the expression of Fas ligand (FasL) or addition of TNF-related apoptosis-inducing ligand (TRAIL) in the presence or absence of proteasome inhibitors. The apoptotic response was quantified by annexin V, TUNEL and nuclear condensation assays. Western blot analysis was conducted to quantify protein levels and enzyme assays were used to measure caspase activity. RESULTS: Proteasome inhibition markedly sensitized prostate cancer cells to apoptosis initiated by Fas ligand (FasL) or TRAIL. In the presence of either death ligand, procaspase-8 processing occurred, but led to minimal amounts of active caspase-8. The addition of Velcade, however, led to robust active caspase-8 protein abundance and activity. In the presence of Velcade the caspase-8 p18 subunit half-life increased from 22 min to over 2 hr. CONCLUSIONS: These findings demonstrate that proteasome inhibition can sensitize cells to apoptosis elicited by tumor necrosis factor ligands and retarding caspase-8 degradation provides one explanation for this activity. This study suggests that the clinical efficacy of Velcade may result, at least in part, from the activity of death ligands.
Assuntos
Apoptose/fisiologia , Caspase 8/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteassoma , Receptores de Morte Celular/metabolismo , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteína Ligante Fas/metabolismo , Meia-Vida , Humanos , Masculino , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells; however, in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation.
Assuntos
Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Inibidores Enzimáticos/farmacologia , Humanos , Interleucina-8/química , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ressonância Magnética Nuclear Biomolecular , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Impaired tau catabolism may contribute to tau accumulation and aggregation in Alzheimer's disease and neurofibrillary tangle formation. This study examined the effects of proteasome and calpain inhibition on tau levels and turnover in primary rat hippocampal neurons and differentiated SH-SY5Y human neuroblastoma cells. Administration of proteasome (MG-115, lactacystin) or calpain (MDL28170) inhibitors for up to 24 hours did not alter tau levels in differentiated SH-SY5Y cells or rat hippocampal neurons. Addition of 1 microM and 10 microM MG-115 did not change total tau levels, but did result in increased reactivity of phosphorylation-dependent tau antibodies (PHF-1, CP-13) and decreased Tau-1 immunoreactivity. Administration of cycloheximide to inhibit de novo protein synthesis also did not alter tau levels in the presence or absence of lactacystin. These results demonstrate that although the proteasome and calpain protease systems are capable of degrading tau in cell-free assays, their inhibition does not alter cellular tau levels in primary neurons or differentiated neuroblastoma cells.