RESUMO
Livin (ML-IAP) is a cancer-associated member of the inhibitor of apoptosis protein (IAP) family. By yeast two-hybrid screening of a randomized peptide expression library, we isolated short linear peptides that specifically bind to Livin, but not to other IAPs. Intracellular expression of the peptides sensitized livin-expressing cancer cells toward different pro-apoptotic stimuli. The bioactive peptides neither showed sequence homologies to Smac-derived IAP inhibitors, nor did they interfere with the binding of Livin to Smac. Intracellular expression of the peptides did not affect the levels or the subcellular distribution of Livin. Growth of livin-expressing tumor cells was inhibited in colony formation assays by the Livin-targeting peptides. These findings provide evidence that the targeted inhibition of Livin by peptides represents a viable approach for the apoptotic sensitization and growth inhibition of tumor cells. The inhibitory peptides isolated here could form a novel basis for the development of therapeutically useful Livin inhibitors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/fisiologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Linhagem Celular , Células HeLa , Humanos , Técnicas In Vitro , Proteínas Inibidoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Biblioteca de Peptídeos , Peptídeos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Interferência de RNA , Ensaio Tumoral de Célula-Tronco , Técnicas do Sistema de Duplo-HíbridoRESUMO
The enhancer of zeste homolog 2 (EZH2) gene has been recently linked to human malignancies where it may serve as a new target for cancer therapy. Here, we analyzed EZH2 expression in primary renal cell carcinoma (RCC) specimens and in nontumorous tissue samples from adult kidney. EZH2 transcripts were detectable in all RCC specimens examined. Expression levels were significantly higher in tumor tissue (p < or = 0.0001) than in samples from normal adult kidney. Moreover, inhibition of endogenous EZH2 expression in RCC cell lines by RNA interference (RNAi) led to reduced proliferation and increased apoptosis in RCC cells. These data show that EZH2 is overexpressed in RCC. Furthermore, they indicate that the EZH2 gene plays a role for both the proliferation and the apoptosis resistance of RCC cells. Targeted inhibition of EZH2 could therefore represent a novel strategy to improve the therapeutic response of RCC.
Assuntos
Apoptose/genética , Carcinoma de Células Renais/patologia , Proliferação de Células , Proteínas de Ligação a DNA/genética , Neoplasias Renais/patologia , Fatores de Transcrição/genética , Sequência de Bases , Carcinoma de Células Renais/genética , Primers do DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Inativação Gênica , Humanos , Neoplasias Renais/genética , Complexo Repressor Polycomb 2 , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
OBJECTIVES: To assess the protein expression of Livin, an apoptosis inhibitor, in renal cell carcinoma (RCC) and to determine its prognostic relevance. PATIENTS AND METHODS: Immunohistochemical staining for Livin was performed in tissue microarrays (TMAs), including tumour tissue cores, from patients with RCC who had undergone renal surgery. In 682 TMAs cytoplasmatic staining intensity and nuclear staining quantity were evaluated, and the association of Livin expression with progression-free survival (PFS) and cancer-specific survival (CSS) was analysed with a multivariate Cox regression model. RESULTS: Over a median (range) follow-up of 5.2 (0-16.1) years, 204 patients (28%) had died from their disease. The CSS rates at 1 and 5 years for the entire cohort was 88% and 71%. Cytoplasmatic Livin staining was absent in 516 (76%) specimens; staining was positive in 166 (24%) specimens. Weak nuclear Livin staining (
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Renais/patologia , Proteínas de Neoplasias/metabolismo , Nefrectomia/métodos , Idoso , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/cirurgia , Estudos de Coortes , Feminino , Humanos , Neoplasias Renais/mortalidade , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Estudos Prospectivos , Fatores de Risco , Análise de Sobrevida , Resultado do TratamentoRESUMO
Livin (alternatively called ML-IAP or KIAP) is a cancer-associated member of the antiapoptotic inhibitor of apoptosis protein family. Two splicing variants of Livin, designated Livin alpha and Livin beta, have been identified. The significance of these isoforms for Livin-mediated apoptosis inhibition is largely unclear. Using an isoform-specific RNA interference (RNAi) strategy, we silenced endogenous Livin expression in HeLa cells. We found that the targeted inhibition of Livin beta, but not of Livin alpha, blocked the growth of HeLa cells in clonogenic survival assays. In addition, silencing of Livin beta, but not of Livin alpha, sensitized HeLa cells to different proapoptotic stimuli such as UV irradiation, tumor necrosis factor alpha, or etoposide. These events were linked to activation of caspase-3 and increased poly(ADP-ribose) polymerase cleavage, specifically upon silencing of Livin beta. The proapoptotic sensitization of HeLa cells upon RNAi-mediated silencing of the endogenous livin gene was specifically reverted by ectopic expression of Livin beta but not of Livin alpha. We conclude that the Livin beta isoform plays the key role for the antiapoptotic protection of HeLa cells by the livin gene. Our results show that the Livin isoforms can strongly differ in their functional significance for the antiapoptotic resistance of tumor cells. Studies evaluating Livin as a novel diagnostic and prognostic tumor marker should benefit from isoform-specific expression analyses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspases/metabolismo , Ensaio de Unidades Formadoras de Colônias , Etoposídeo/farmacologia , Inativação Gênica , Células HeLa/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Isoformas de Proteínas , Interferência de RNA , Fator de Necrose Tumoral alfa/farmacologia , Raios UltravioletaRESUMO
Cancer cells are typically characterized by increased resistance towards apoptosis. Livin (alternatively called ML-IAP or KIAP) is an anti-apoptotic protein which is expressed in several cancer forms. Using real-time reverse transcription-polymerase chain reaction (RT-PCR), we confirmed livin expression in a significant portion of non-small cell lung cancer (NSCLC) tissue samples and, in addition, detected livin expression in a number of NSCLC cell lines. In order to elucidate whether livin contributes to the apoptotic resistance of lung cancer cells, we silenced endogenous livin expression in a panel of cancer-derived NSCLC cell lines by RNA interference (RNAi). We observed that the targeted inhibition of livin strongly sensitized NSCLC cells to different pro-apoptotic stimuli, such as UV-irradiation or the chemotherapeutic drug etoposide. In addition, long-term silencing of livin blocked the outgrowth of NSCLC cells in colony formation assays. These effects of small interfering (si)RNA were specific for livin-expressing tumor cells. Our results indicate that Livin is an important contributor to the apoptosis resistance of NSCLC cells and may serve as a novel molecular target for therapeutic inhibition in NSCLC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Proteínas Inibidoras de Apoptose/fisiologia , Neoplasias Pulmonares/fisiopatologia , Proteínas de Neoplasias/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno , Transfecção , Ensaio Tumoral de Célula-Tronco , Raios UltravioletaRESUMO
Increased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific antiapoptotic factors may provide a rational basis for the development of novel therapeutic strategies. We investigated here whether the RNA interference (RNAi) technology could be used to increase the apoptotic susceptibility of cancer cells. As a molecular target, we chose the antiapoptotic livin (ML-IAP, KIAP) gene, which is expressed in a subset of human tumors. We identified vector-borne small interfering (si)RNAs, which could efficiently block endogenous livin gene expression. Silencing of livin was associated with caspase-3 activation and a strongly increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin, UV-irradiation, or TNFalpha. The effects were specific for Livin-expressing tumor cells. Our results (i) provide direct evidence that the intracellular interference with livin gene expression resensitizes human tumor cells to apoptosis, (ii) define the livin gene as a promising molecular target for therapeutic inhibition, and (iii) show that the livin gene is susceptible to efficient and specific silencing by the siRNA technology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/genética , Inativação Gênica , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas Inibidoras de ApoptoseRESUMO
In recent years, peptide aptamers have emerged as novel molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to and inactivate a given target protein at the intracellular level, they provide a new experimental strategy for functional protein analyses, both in vitro and in vivo. In addition, by using peptide aptamers as "pertubagens", they can be employed for genetic analyses, in order to identify biochemical pathways, and their components, that are associated with the induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be developed into diagnostic tools for the detection of a given target protein or for the generation of high-throughput protein arrays. Finally, the peptide aptamer technology has direct therapeutic implications. Peptide aptamers can be used in order to validate therapeutic targets at the intracellular level. Moreover, the peptide aptamer molecules themselves should possess therapeutic potential, both as lead structures for drug design and as a basis for the development of protein drugs.