RESUMO
Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Dioxanos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glucosiltransferases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Pirrolidinas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dioxanos/administração & dosagem , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Pirrolidinas/administração & dosagem , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.
Assuntos
Ciclo Celular/genética , Senescência Celular/genética , Complexo de Endopeptidases do Proteassoma/deficiência , Transativadores/deficiência , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , DNA/análise , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G1/genética , Expressão Gênica/genética , Células HeLa , Humanos , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Fase de Repouso do Ciclo Celular/genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Sulfotransferases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transfecção , Proteínas Ubiquitinadas/metabolismo , Regulação para Cima/genética , beta-Galactosidase/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
BACKGROUND: A major focus of prostate cancer research has been to identify genes that are deregulated during tumor progression, potentially providing diagnostic markers and therapeutic targets. METHODS: We have employed serial analysis of gene expression (SAGE) and microarray hybridization to identify alterations that occur during malignant transformation in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Many of these alterations were validated by real-time PCR (rtPCR). RESULTS: We identified several hundred mRNAs that were deregulated. Cluster analysis of microarray profiles with samples from various stages of the disease demonstrated that androgen-independent (AI) primary tumors are similar to metastases; 180 transcripts have expression patterns suggesting an involvement in the genesis of late-stage tumors, and our data support a role for phospholipase A2 group IIA in the acquisition of their highly aggressive characteristics. CONCLUSIONS: Our analyses identified well-characterized genes that were previously known to be involved in prostate cancer, validating our study, and also uncovered transcripts that had not previously been implicated in prostate cancer progression.