ABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/physiology , Apoptosis/drug effects , Apoptosis/physiology , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/physiology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/physiology , Peptides/isolation & purification , Peptides/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Apoptosis Regulatory Proteins , Cell Line , HeLa Cells , Humans , In Vitro Techniques , Inhibitor of Apoptosis Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Neoplasm Proteins/genetics , Peptide Library , Peptides/genetics , Protein Binding , Protein Interaction Mapping , RNA Interference , Tumor Stem Cell Assay , Two-Hybrid System TechniquesABSTRACT
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 (
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Biomarkers, Tumor/metabolism , Carcinoma, Renal Cell/pathology , Inhibitor of Apoptosis Proteins/metabolism , Kidney Neoplasms/pathology , Neoplasm Proteins/metabolism , Nephrectomy/methods , Aged , Carcinoma, Renal Cell/mortality , Carcinoma, Renal Cell/surgery , Cohort Studies , Female , Humans , Kidney Neoplasms/mortality , Kidney Neoplasms/surgery , Male , Middle Aged , Multivariate Analysis , Prognosis , Prospective Studies , Risk Factors , Survival Analysis , Treatment OutcomeABSTRACT
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.
Subject(s)
Apoptosis/genetics , Carcinoma, Renal Cell/pathology , Cell Proliferation , DNA-Binding Proteins/genetics , Kidney Neoplasms/pathology , Transcription Factors/genetics , Base Sequence , Carcinoma, Renal Cell/genetics , DNA Primers , Enhancer of Zeste Homolog 2 Protein , Gene Silencing , Humans , Kidney Neoplasms/genetics , Polycomb Repressive Complex 2 , RNA Interference , RNA, Messenger/genetics , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Apoptosis/genetics , Carcinoma, Non-Small-Cell Lung/physiopathology , Inhibitor of Apoptosis Proteins/physiology , Lung Neoplasms/physiopathology , Neoplasm Proteins/physiology , Adaptor Proteins, Signal Transducing/genetics , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Line, Tumor , Etoposide/pharmacology , Humans , Inhibitor of Apoptosis Proteins/genetics , Neoplasm Proteins/genetics , RNA Interference , RNA, Small Interfering , Transfection , Tumor Stem Cell Assay , Ultraviolet RaysABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/physiology , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Apoptosis/drug effects , Apoptosis/radiation effects , Apoptosis Regulatory Proteins , Caspase 3 , Caspases/metabolism , Colony-Forming Units Assay , Etoposide/pharmacology , Gene Silencing , HeLa Cells/pathology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Isoforms , RNA Interference , Tumor Necrosis Factor-alpha/pharmacology , Ultraviolet RaysABSTRACT
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.
Subject(s)
Cell Physiological Phenomena , Hepatitis B virus/physiology , Oligopeptides/metabolism , Papillomaviridae/physiology , Signal Transduction/physiology , Animals , Drug Design , Humans , Oligopeptides/genetics , Oligopeptides/therapeutic use , Protein Array Analysis , Protein Binding/genetics , Two-Hybrid System Techniques , Virus Replication/drug effects , Virus Replication/physiologySubject(s)
Betaherpesvirinae/genetics , Chromosomes, Artificial, Bacterial/genetics , Genome, Viral , Cell Line , Cloning, Molecular , Cytomegalovirus/genetics , DNA, Viral/genetics , DNA, Viral/isolation & purification , Electroporation , Escherichia coli/genetics , Humans , Mutagenesis , Plasmids/genetics , Plasmids/isolation & purification , Recombination, Genetic , TransfectionABSTRACT
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.