A mechanism for survival of prostate cancer cells in an androgen-deprived environment remains elusive. Here, we find that expression of neuronal apoptosis inhibitory protein (NAIP) was significantly increased in vivo and in vitro in response to androgen deprivation therapy (ADT). Increased expression of NAIP corresponded to increased DNA-binding activity of NF-κB that physically associated to previously uncharacterized κB-like sites in the NAIP locus. Importantly, expression of NAIP was significantly increased (p=0.04) in clinical samples of CaP from patients receiving ADT. Expression of NAIP may be associated with enhanced survival of prostate cancer in response to castration.
prostate cancer; androgen deprivation; nuclear factor-κB; inhibitors of apoptosis; neuronal apoptosis inhibitory protein
Inhibitors of Apoptosis (IAP) family play a critical role in apoptosis and inflammatory response. Neuronal Apoptosis Inhibitory Protein (NAIP), as a member of both IAPs and NLR families (NOD-Like Receptor), is a unique IAP harboring NOD (Nucleotide Oligomerization Domain) and LLR (Leucine Rich Repeat) motifs. Considering these motifs in NAIP, it has been suggested that the main function of NAIP is distinct from other members of IAPs. As a member of NLR, NAIP mediates the assembly of 'Inflammasome' for inflammatory caspase activation. Pathologic expression of NAIP has been reported not only in some infectious and inflammatory diseases but also in some malignancies. However, there is no report to elucidate NAIP expression in lymphomatic malignancies.
In this study, we examined NAIP protein expression in 101 Formalin-Fixed Paraffin-Embedded blocks including samples from 39 Hodgkin Lymphoma and 23 Non Hodgkin Lymphoma cases in comparison with 39 control samples (30 normal and 9 Reactive Lymphoid Hyperplasia (RLH) lymph nodes) using semi-quantitative immuno-flourecent Staining.
NAIP expression was not statistically different in lymphoma samples neither in HL nor in NHL cases comparing to normal samples. However, we evaluated NAIP expression in normal and RLH lymph nodes. Surprisingly, we have found a statistically significant-difference between the NAIP expression in RLH (M.R of NAIP/GAPDH expression = 0.6365 ± 0.017) and normal lymph node samples (M.R of NAIP/GAPDH expression = 0.5882 ± 0.047) (P < 0.01).
These findings show that the regulation of apoptosis could not be the main function of NAIP in the cell, so the pathologic expression of NAIP is not involved in lymphoma. But, we concluded that the over expression of NAIP has more effective role in the inflammatory response. Also, this study clarifies the NAIP expression level in lymphoma which is required for IAPs profiling in order to be used in potential translational applications of IAPs.
Inhibitor of apoptosis protein; NAIP/BIRC1; Inflammatory caspases; Inflammasome; Hodgkin lymphoma; Non-Hodgkin lymphoma; Semi-quantitative immuno-flourecent staining
The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene.
Neuronal apoptosis inhibitory protein (NAIP, also known as BIRC1) is a member of the conserved inhibitor of apoptosis protein (IAP) family. Lineage-specific rearrangements and expansions of this locus have yielded different copy numbers among primates and rodents, with human retaining a single functional copy and mouse possessing several copies, depending on the strain. Roles for this gene in disease have been documented, but little is known about transcriptional regulation of NAIP. We show here that NAIP has multiple promoters sharing no similarity between human and rodents. Moreover, we demonstrate that multiple, domesticated long terminal repeats (LTRs) of endogenous retroviral elements provide NAIP promoter function in human, mouse, and rat. In human, an LTR serves as a tissue-specific promoter, active primarily in testis. However, in rodents, our evidence indicates that an ancestral LTR common to all rodent genes is the major, constitutive promoter for these genes, and that a second LTR found in two of the mouse genes is a minor promoter. Thus, independently acquired LTRs have assumed regulatory roles for orthologous genes, a remarkable evolutionary scenario. We also demonstrate that 5′ flanking regions of IAP family genes as a group, in both human and mouse are enriched for LTR insertions compared to average genes. We propose several potential explanations for these findings, including a hypothesis that recruitment of LTRs near NAIP or other IAP genes may represent a host-cell adaptation to modulate apoptotic responses.
When retroviruses infect cells, the viral DNA inserts into the cellular genome. If this happens in gametes (egg or sperm), the viral DNA will be transmitted from parent to offspring, like all chromosomal DNA. Through evolutionary time, such infections of gametes have been so prevalent that 8%–10% of the normal human and mouse genomes are now composed of ancient viral DNA, termed endogenous retroviruses (ERVs). In human, these ERVs are mutated or “dead” but it has been shown that ERV regulatory regions can be employed by the host to help control expression of cellular genes. Here, we report on a remarkable example of this phenomenon. We demonstrate that both the human and rodent neuronal apoptosis inhibitory protein (NAIP) genes, involved in preventing cell death, use different ERV sequences to drive gene expression. Moreover, in each of the primate and rodent lineages, two separate ERVs contribute to NAIP gene expression. This repeated ERV recruitment by NAIP genes throughout evolution is very unlikely to have occurred by chance. We offer a number of potential explanations, including the intriguing possibility that it may be advantageous for anti-cell death genes like NAIP to use ERVs to control their expression. These results support the view that not all retroviral remnants in our genome are simply junk DNA.
Neuronal degeneration linked to apoptosis can be inhibited by a family of proteins known as inhibitors of apoptosis proteins (IAPs). We examined three members of the IAP family that are implicated in the regulation of neuronal death. We assessed NAIP, XIAP, and cIAP-2 protein levels in the entorhinal cortex of non-demented, cognitively impaired and Alzheimer's disease cases. Levels of paired helical filament-1 (PHF-1), a marker of neurofibrillary tangles, were assessed to determine their relationship to IAP levels. NAIP was decreased in AD cases compared to mildly-impaired and unimpaired cases, and this decrease was associated with increased PHF-1 levels. Low NAIP levels were associated with higher Braak and Braak tangle stage and cognitive dysfunction. XIAP levels were higher in AD cases and cIAP-2 levels did not vary with clinical status. Our data suggest that decreased NAIP may place neurons at risk for the development of tangles and apoptosis.
inhibitor of apoptosis; neurodegeneration; Alzheimer's disease; NAIP; XIAP; cIAP-2; caspase activation; tau; neurofibrillary tangles; MMSE
Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuronopathies characterized by selective degeneration of anterior horn cells. The causative genes to be reported are survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes. The deletion of telomeric copy of SMN (SMN(T)) gene was observed in over 95% of SMAs. The deletion rate of NAIP gene is 20-50% according to disease severity. The objective of this article is to genetically characterize the childhood-onset spinal muscular atrophy in Koreans. Five Korean families (14 constituents containing 5 probands) with SMA were included in this study. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used for the deletion analysis of SMN(T). Multiplex PCR method was used for NAIP analysis. Four probands showed deletion of SMNT gene. Deletion of SMN(C) (centromeric SMN) gene was found in one proband who did not show the deletion of SMN(T) gene and in the father of one proband who showed the deletion of SMN(T) gene. The deletion of NAIP gene was not found among all the studied individuals. The extent of deletion in Koreans was smaller than that in other studied population. PCR-RFLP deletion analysis can be applied to diagnose SMA and make a prenatal diagnosis.
The frequency of deletions within the survival motor neurone
(SMN) and neuronal apoptosis inhibitory protein (NAIP) genes in
patients with spinal muscular atrophy (SMA), and the impact of this on
the diagnosis and prenatal diagnosis of SMA, were investigated by
molecular analysis of stored DNA and retrospective review of case
notes. In type I SMA, 16 of 17 cases were homozygously deleted for
exons 7 and 8 of SMN, 14 of 17 were homozygously deleted for exon 5 of
NAIP, and 13 of 17 were deleted for both. In types II and III SMA,
seven of nine cases were deleted for exons 7 and 8 of SMN. Deletions of
SMN and NAIP occurred in four of nine cases. With one exception, the
deletion genotypes of probands, affected siblings, and terminated
fetuses were identical. Molecular studies are replacing conventional
investigations for SMA and have a high uptake prenatally.
Neuronal apoptosis inhibitor proteins (NAIPs) are members of Nod-like receptor (NLR) protein family. Recent research demostrated that some NAIP genes were strongly associated with both innate immunity and many inflammatory diseases in humans. However, no similar phenomena have been reported in other mammals. Furthermore, some NAIP genes have undergone pseudogenization or have been lost during the evolution of some higher mammals. We therefore aimed to determine if functional divergence had occurred, and if natural selection had played an important role in the evolution of these genes. The results showed that NAIP genes have undergone pseudogenization and functional divergence, driven by positive selection. Positive selection has also influenced NAIP protein structure, resulting in further functional divergence.
Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMA1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.
Spinal muscular atrophy (SMA); Survival motor neuron (SMN) gene; Neuronal apoptosis inhibitory protein (NAIP) gene; Mutation
Oxymatrine, an isolated extract from traditional Chinese herb Sophora Flavescens Ait, has been traditionally used for therapy of anti-hepatitis B virus, anti-inflammation and anti-anaphylaxis. The present study was to investigate the anti-cancer effect of oxymatrine on human pancreatic cancer PANC-1 cells, and its possible molecular mechanism.
The effect of oxymatrine on the viability and apoptosis was examined by methyl thiazolyl tetrazolium and flow cytometry analysis. The expression of Bax, Bcl-2, Bcl-x (L/S), Bid, Bad, HIAP-1, HIAP-2, XIAP, NAIP, Livin and Survivin genes was accessed by RT-PCR. The levels of cytochrome c and caspase 3 protein were assessed by Western blotting.
Oxymatrine inhibited cell viability and induced apoptosis of PANC-1 cells in a time- and dose-dependent manner. This was accompanied by down-regulated expression of Livin and Survivin genes while the Bax/Bcl-2 ratio was upregulated. Furthermore, oxymatrine treatment led to the release of cytochrome c and activation of caspase-3 proteins.
Oxymatrine can induce apoptotic cell death of human pancreatic cancer, which might be attributed to the regulation of Bcl-2 and IAP families, release of mitochondrial cytochrome c and activation of caspase-3.
Spinal muscular atrophy (SMA) is characterised by degeneration of anterior horn cells of the spinal cord and represents the second most common, lethal, autosomal recessive disorder after cystic fibrosis. Based on the criteria of the Internatinal SMA Consortium, childhood SMAs are classified into type I (Werdnig-Hoffmann disease), type II (intermediate form), and type III (Kugelberg-Welander disease). Recently, two genes have been found to be associated with SMA. The survival motor neurone gene (SMN) is an SMA determining gene as it is absent in 98.6% of patients. A second gene, XS2G3, or the highly homologous neuronal apoptosis inhibitory protein gene (NAIP) have been found to be more frequently deleted in type I than in the milder forms (types II and III). We investigated the correlation between the clinical phenotype and the genotype at this loci. A total of 106 patients were classified into type I (44), type II (31), and type III (31) and analysed using SMN, markers C212 and C272, and NAIP mapping upstream and downstream from SMN respectively. The combined analysis of all markers showed a large proportion of type I patients (43%) carried deletions of both SMN and its flanking markers (C212/272) and NAIP exon 5), as compared with none of the patients with type II or III SMA. The presence of large scale deletions involving these loci is specific to Werdnig-Hoffman disease (type I) and allows one to predict the severity of the disease in our series.
A gene involved in the development of spinal muscular atrophy (SMA) has been found on human chromosome 5 after a 4-year search. Named the neuronal apoptosis inhibitor protein (NAIP) gene, it is believed to inhibit the normal process of apoptosis--the disintegration of single cells that results from programmed cell death--in motor neurons. The researchers who found the NAIP gene also discovered that healthy people carry one complete copy of the gene along with many other partial copies. Many children with SMA have the partial copies but not the complete gene. This discovery facilitates the accurate genetic diagnosis of SMA. But gene therapy for SMA will not be possible until researchers find a suitable vector to stably introduce activated and intact copies of the gene into the motor neurons of children with SMA in time to stop motor neuron loss.
DNA methylation-dependent epigenetic mechanisms underlie the development and function of the mammalian brain. MeCP2 expresses highly in neurons, and functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Previous in vitro studies showed neuronal activity-induced phosphorylation (NAIP) of MeCP2 precedes its release from the Bdnf promoter and the ensuing Bdnf transcription. However, the in vivo function of this phosphorylation event remains elusive. We generated knockin mice that lack NAIP of MeCP2, and show here the Mecp2 phospho-mutant mice perform better in hippocampus-dependent memory tests, present enhanced LTP at two synapses in the hippocampus, and show increased excitatory synaptogenesis. At the molecular level, the phospho-mutant MeCP2 protein binds more tightly to several MeCP2 target gene promoters and alters the expression of these genes. Our results supply the first genetic evidence that NAIP of MeCP2 is required in modulating dynamic functions of the adult mouse brain.
The antiapoptotic properties of the inhibitor of apoptosis (IAP) family of proteins have been linked to caspase inhibition. We have previously described an alternative mechanism of XIAP inhibition of apoptosis that depends on the selective activation of JNK1. Here we report that two other members of the IAP family, NAIP and ML-IAP, both activate JNK1. Expression of catalytically inactive JNK1 blocks NAIP and ML-IAP protection against ICE- and TNF-α-induced apoptosis, indicating that JNK1 activation is necessary for the antiapoptotic effect of these proteins. The MAP3 kinase, TAK1, appears to be an essential component of this antiapoptotic pathway since IAP-mediated activation of JNK1, as well as protection against TNF-α- and ICE-induced apoptosis, is inhibited when catalytically inactive TAK1 is expressed. In addition, XIAP, NAIP, and JNK1 bind to TAK1. Importantly, expression of catalytically inactive TAK1 did not affect XIAP inhibition of caspase activity. These data suggest that XIAP's antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.
In this study was investigate IAPs in normal human prostate (NP), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma (PC), and their involvement in apoptosis/proliferation via NF-kB (TNF-α, IL-1) stimulation.
Immunohistochemical and Western blot analyses were performed in 10 samples of normal prostates, 35 samples of BPH, 27 samples diagnosis of PIN (with low-grade PIN or high-grade PIN) and 95 samples of PC (with low, medium or high Gleason grades).
In NP, cytoplasm of epithelial cells were positive to c-IAP1/2 (80% of samples), c-IAP-2 (60%), ILP (20%), XIAP (20%); negative to NAIP and survivin. In BPH, epithelial cells were immunostained to c-IAP1/2 (57.57%), c-IAP-2 (57.57%), ILP (66.6%), NAIP (60.6%), XIAP (27.27%), survivin (9.1%). Whereas low-grade PIN showed intermediate results between NP and BPH; results in high-grade PIN were similar to those found in PC. In PC, epithelial cells were immunostained to c-IAP1/2, c-IAP-2, ILP, NAIP, XIAP (no Gleason variation) and survivin (increasing with Gleason).
IAPs could be involved in prostate disorder (BPH, PIN and PC) development since might be provoke inhibition of apoptosis and subsequently cell proliferation. At the same time, different transduction pathway such as IL-1/NIK/NF-kB or TNF/NF-kB (NIK or p38) also promotes proliferation. Inhibitions of IAPs, IL-1α and TNFα might be a possible target for PC treatment since IAPs are the proteins that inhibited apoptosis (favour proliferation) and IL-1α and TNFα would affect all the transduction pathway involucrate in the activation of transcription factors related to survival or proliferation (NF-kB, Elk-1 or ATF-2).
Spinal muscular atrophy (SMA), a clinically and genetically heterogeneous group of neuromuscular diseases, is a disorder of motor neurones characterised by degeneration of spinal cord anterior horn cells and muscular atrophy.
SMA is an autosomal recessive disorder with a carrier frequency of about 1/50. Three candidate genes, the survival motor neurone (SMN) gene, the neuronal inhibitory protein (NAIP) gene, and the p44 (subunit of basal transcription factor TFIIH) gene, have been considered as genes involved in this condition. The region spanning these genes has a complex organisation including duplications, repetitive sequences, truncated genes, and pseudogenes, which makes molecular analysis of this condition difficult. Although deletions have been found in the majority of SMA patients, a few microrearrangements (like duplications, missense mutations, microdeletions, and gene conversions) localised in the telomeric form of the SMN gene have also been reported.
The function of the protein encoded by the SMN gene is still not fully understood but recent studies have indicated that it is found intracellularly in gems, novel nuclear structures. Its interaction with other proteins suggests a role in mRNA processing and metabolism. Whether the NAIP gene protein and other apoptosis associated proteins are directly involved in the initial stages of neurone degeneration and apoptosis, or acting downstream on the pathological pathway, has been difficult to determine. Further studies will be required to elucidate possible functional interactions between these proteins.
Keywords: SMA; SMN; NAIP
The crystal structures of the human NAIP BIR2 and cIAP2 BIR3 domains have been determined. Both BIR domains harbors an amino-terminal tetrapeptide in its peptide-binding groove.
The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1′–P4′ side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3′ position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2′ and P4′ pockets make similar interactions to those seen in other BIR domain–peptide complexes. The structures also reveal how a serine in the P1′ position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.
apoptosis; cancer; inflammation; IAP; NAIP; cIAP; BIR
Detection of microbial products by host inflammasomes is critical for innate immune surveillance. Inflammasomes activate the CASPASE-1 (CASP1) protease, which processes the cytokines interleukin(IL)-1β and -18, and initiates a lytic host cell death called pyroptosis1. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD domain containing 4) inflammasome2–4. Here we show that systemic inflammasome activation by flagellin leads to loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (< 30 minutes) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4, and CASP1, but is independent of IL-1β/-18 production. Instead, inflammasome activation results, within minutes, in an ‘eicosanoid storm’ – a pathological release of signaling lipids that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1 (COX-1), a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by activation of cPLA2 (cytosolic phospholipase A2) in resident peritoneal macrophages, which are specifically primed for production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Thus, our results identify eicosanoids as a novel cell type-specific signaling output of the inflammasome with dramatic physiological consequences in vivo.
The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR–mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.
Two candidate genes (NAIP and SMN) have recently been reported for childhood onset spinal muscular atrophy (SMA). Although affected subjects show deletions of these genes, these deletions can lead to either a very mild or a severe phenotype. We have analysed a large number of clinically well defined patients, carriers, and normal controls to assess the frequency and extent of deletions encompassing both of these genes. A genotype analysis indicates that more extensive deletions are seen in the severe form of SMA than in the milder forms. In addition, 1 center dot 9% of phenotypically normal carriers are deleted for the NAIP gene; no carriers were deleted for the SMN gene. Our data suggest that deletions in both of these genes, using the currently available assays, are associated with both a severe and very mild phenotype.
This study was designed to determine the levels of survivin expression and identify its clinical significance as a prognostic factor for stage III non-small cell lung cancer (NSCLC). A total of 210 cases of stage III NSCLC were collected and the expression levels of survivin and vascular endothelial growth factor A (VEGF-A) in tumor tissues were investigated using immunohistochemistry (IHC). The medical records of the patients were reviewed to determine the association with clinical course. Of the 210 NSCLC tissues, 112 (53.3%) cases demonstrated positive expression of survivin protein. Coexpression of survivin and VEGF-A was identified. The 5-year survival rate of patients with positive survivin expression was significantly lower compared with the survivin-negative cancer patients (P<0.05). The expression of survivin in NSCLC correlated with tumor size. Survivin and VEGF-A were independent prognostic factors of stage III NSCLC. Survivin protein is a valuable marker of prognosis in stage III NSCLC patients.
survivin; VEGF-A; immunohistochemistry; prognostic marker
Survivin, a member of the inhibitor of apoptosis-protein family suppresses apoptosis and regulates cell division. It is strongly overexpressed in the vast majority of cancers. We were interested if survivin detected by immunohistochemistry has prognostic relevance especially for patients of the two soft tissue sarcoma entities leiomyosarcoma and synovial sarcoma.
Tumors of leiomyosarcoma (n = 24) and synovial sarcoma patients (n = 26) were investigated for their expression of survivin by immunohistochemistry. Survivin expression was assessed in the cytoplasm and the nucleus of tumor cells using an immunoreactive scoring system (IRS).
We detected a survivin expression (IRS > 2) in the cytoplasm of 20 leiomyosarcomas and 22 synovial sarcomas and in the nucleus of 12 leiomyosarcomas and 9 synovial sarcomas, respectively. There was no significant difference between leiomyosarcoma and synovial sarcoma samples in their cytoplasmic or nuclear expression of survivin. Next, all sarcoma patients were separated in four groups according to their survivin expression in the cytoplasm and in the nucleus: group 1: negative (IRS 0 to 2); group 2: weak (IRS 3 to 4); group 3: moderate (IRS 6 to 8); group 4: strong (IRS 9 to 12). In a multivariate Cox's regression hazard analysis survivin expression detected in the cytoplasm or in the nucleus was significantly associated with overall survival of patients in group 3 (RR = 5.7; P = 0.004 and RR = 5.7; P = 0.022, respectively) compared to group 2 (reference). Patients whose tumors showed both a moderate/strong expression of survivin in the cytoplasm and a moderate expression of survivin in the nucleus (in both compartments IRS ≥ 6) possessed a 24.8-fold increased risk of tumor-related death (P = 0.003) compared to patients with a weak expression of survivin both in the cytoplasm and in the nucleus.
Survivin protein expression in the cytoplasma and in the nucleus detected by immunohistochemistry is significantly associated with prognosis of leiomyosarcoma and synovial sarcoma patients.
Alternative splicing of survivin mRNA gives rise to multiple isoforms, that is, survivin and 3 splice variants, survivin-2B, survivin-3B and survivin-ΔEx3. The aim of this study was to compare the expression of survivin, survivin-2B and survivin-ΔEx3 in normal breast tissue, fibroadenomas, primary breast cancer and axillary nodal metastases. Survivin, survivin-2B and survivin-ΔEx3 mRNA were measured using semiquantitative RT–PCR. In the primary carcinomas, we related mRNA for each form of survivin to both survivin protein and apoptosis. For each type of breast tissue, survivin was the predominant form detected, being present in 146 out of 156 (93.6%) primary breast carcinomas, 11 out of 11 (100%) axillary nodal metastases, 21 out of 31 (67.7%) fibroadenomas and five out of 22 (22.7%) specimens of normal breast tissue. Levels of the three forms of survivin were significantly higher in the carcinomas compared to normal breast tissue (P<0.0001). Levels of both survivin-2B and survivin-ΔEx3 but not survivin were significantly higher in nodal metastases than primary carcinomas. Survivin mRNA levels correlated significantly with survivin protein. Finally, both survivin and survivin-ΔEx3 but not survivin-2B correlated positively with apoptosis. Although survivin, survivin-2B and survivin-ΔEx3 were all detected in both malignant and nonmalignant breast tissue, the predominant form was survivin. Our results suggest that the different forms of survivin may have different roles in apoptosis in breast cancer.
inhibitor of apoptosis; apoptosis; programmed cell death; breast carcinoma
Although it was observed that inhibition of the antiapoptotic protein survivin expression in lung cancer cells induces apoptosis, the expression and role of survivin variants (survivin-2B and survivin-ΔEx3) in lung cancer have not yet been characterized. We analyzed 24 non-small-cell lung cancer (NSCLC) samples by semi-quantitative RT-PCR. Surprisingly, our results revealed that high-level expression of survivin-2B is significantly associated with the patient category of “no relapse and alive” (p-value < 0.0001). In contrast, high-level expression of survivin-ΔEx3 is highly associated with the patient category of “relapse and dead” (p-value < 0.0001). Consistent with this observation, exogenous expression of survivin-2B in A549 lung cancer cells inhibited cell growth, disrupted the mitochondria potential, and induced apoptotic cell death, while expression of survivin-ΔEx3 protected the mitochondria potential and facilitated cell survival. These findings provide evidence that survivin-2B and survivin-ΔEx3 play opposite roles in disease relapse and NSCLC cell survival, which is likely through the differential modulation of mitochondrial potential. Thus, controlling the differential expression of survivin-2B and survivin-ΔEx3 may represent novel approaches for cancer therapeutics in NSCLC.
Survivin-2B; Survivin-ΔEx3; Non-small-cell lung cancer
The identification of differentially regulated apoptotic signals in normal and tumor cells allows the development of cancer cell-selective therapies. Increasing evidence shows that the inhibitor of apoptosis (IAP) proteins survivin and XIAP are highly expressed in tumor cells but are absent or have very low levels of expression in normal adult tissues. We found that inhibiting AKT activity with 10 to 100 nM deguelin, a small molecule derived from natural products, markedly reduced the levels of both survivin and XIAP, inducing apoptosis in human breast cancer cells but not in normal cells. It is noteworthy that we detected an elevated level of cleaved poly(ADP-ribose) polymerase, a signature of caspase activation, without a significant increase in caspase activity in deguelin-treated cancer cells. Our results suggest that severe down-regulation of the IAPs by deguelin releases their inhibitory activity over pre-existing active caspases present in cancer cells, inducing apoptosis without the need for further caspase activation. Because normal cells have very low levels of p-AKT, XIAP, survivin, and preexisting caspase activity, deguelin had little effect on those cells. In addition, we found that combining deguelin with chemotherapy drugs enhanced drug-induced apoptosis selectively in human tumor cells, which suggests that deguelin has great potential for chemosensitization and could represent a new therapeutic agent for treatment of breast cancer.