Gene Summary

Gene:APAF1; apoptotic peptidase activating factor 1
Aliases: CED4, APAF-1
Summary:This gene encodes a cytoplasmic protein that initiates apoptosis. This protein contains several copies of the WD-40 domain, a caspase recruitment domain (CARD), and an ATPase domain (NB-ARC). Upon binding cytochrome c and dATP, this protein forms an oligomeric apoptosome. The apoptosome binds and cleaves caspase 9 preproprotein, releasing its mature, activated form. Activated caspase 9 stimulates the subsequent caspase cascade that commits the cell to apoptosis. Alternative splicing results in several transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:apoptotic protease-activating factor 1
Source:NCBIAccessed: 26 August, 2015


What does this gene/protein do?
Show (24)
Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 26 August 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • DNA Methylation
  • Transfection
  • Cancer Gene Expression Regulation
  • Base Sequence
  • Testicular Cancer
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Colorectal Cancer
  • bcl-2-Associated X Protein
  • Signal Transduction
  • Cancer DNA
  • Apoptosis Regulatory Proteins
  • p53 Protein
  • Caspases
  • Gene Expression Profiling
  • TNF
  • Oligonucleotide Array Sequence Analysis
  • Tumor Suppressor Gene
  • BCL2 protein
  • Up-Regulation
  • bcl-X Protein
  • Chromosome 12
  • Neoplasm Proteins
  • Death-Associated Protein Kinases
  • Caspase 9
  • Tumor Markers
  • Melanoma
  • Urokinase-Type Plasminogen Activator
  • Proteins
  • Mitochondria
  • Promoter Regions
  • Apoptosis
  • Apoptotic Protease-Activating Factor 1
  • Tumor Suppressor Proteins
  • Caspase 3
  • Cytochromes c
  • Transcription
  • Down-Regulation
  • TP53
  • Messenger RNA
  • Mutation
Tag cloud generated 26 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (3)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: APAF1 (cancer-related)

Liu N, Sun YY, Zhang XW, et al.
Oncogenic miR-23a in Pancreatic Ductal Adenocarcinogenesis Via Inhibiting APAF1.
Dig Dis Sci. 2015; 60(7):2000-8 [PubMed] Related Publications
BACKGROUND: miR-23a, which participates in invasion of pancreatic ductal adenocarcinoma cells into the mesothelial barrier, is a critical regulator in many cancers. It, however, is still unknown whether miR-23a regulates pancreatic cell proliferation and apoptosis or not.
AIMS: We sought to investigate the role of miR-23a in regulation of pancreatic cell proliferation and apoptosis.
METHODS: miRNA, mRNA, and protein expressions were determined by qRT-PCR and Western blot, respectively. Dual-luciferase reporter assay was used in detection for binding ability of miR-23a to APAF1. Ectopic miR-23a and APAF 1 were introduced to pancreatic cells, and their roles in proliferation and apoptosis were detected by MTT, colony formation, and apoptosis assays, respectively.
RESULTS: Up-regulation of miR-23a and down-regulation of APAF 1 were found in pancreatic ductal cancer, respectively. miR-23a significantly inhibited the luciferase activity by targeting APAF 1 3'UTR. Ectopic miR-23a significantly suppressed the APAF 1 gene expression in pancreatic cancer cells. Similar to siAPAF1, miR-23a significantly promoted pancreatic cancer cell proliferation and repressed apoptosis. Furthermore, miR-23a inhibitor and exogenous APAF 1 could recover the effects.
CONCLUSIONS: It is suggested that miR-23a, acting as an oncogenic regulator by directly targeting APAF 1 in pancreatic cancer, is a useful potential biomarker in diagnosis and treatment of pancreatic cancer.

Choi MR, Gwak M, Yoo NJ, Lee SH
Regional Bias of Intratumoral Genetic Heterogeneity of Apoptosis-Related Genes BAX, APAF1, and FLASH in Colon Cancers with High Microsatellite Instability.
Dig Dis Sci. 2015; 60(6):1674-9 [PubMed] Related Publications
BACKGROUND: Apoptosis inactivation and intratumoral heterogeneity (ITH) are common features of cancers, including colorectal cancer (CRC). Inactivation of apoptosis prolongs cancer cell survival, and ITH may contribute to CRC progression.
AIM: To examine the presence and extent of mutational ITH in the pro-apoptotic genes APAF1, BAX, and FLASH and the association of mutational ITH with pathologic parameters of CRC.
METHODS: The ITH of mutations in the mononucleotide repeats of APAF1, BAX and FLASH in different tumors were analyzed in 16 cases of CRC with high microsatellite instability (MSI-H) and 41 cases of CRC with stable MSI/low MSI (MSS/MSI-L) by single-strand conformation polymorphism and DNA sequencing analyses.
RESULTS: Frameshift mutations of APAF1, BAX, and FLASH were identified in 19, 31, and 6 % of CRC with MSI-H, respectively, but also in cases of CRC with MSS/MSI-L. All but one CRC with a mutation (8/9) harbored regional ITH of the APAF1, BAX and FLASH frameshift mutations. ITH, however, was not associated with histopathologic features of CRC with MSI-H, suggesting that ITH might not be related to development of the MSI-H phenotype itself, but rather to disease progression.
CONCLUSIONS: Our results indicate that the APAF1, BAX, and FLASH genes not only harbor frameshift mutations but also demonstrate mutational ITH, which together might play a role in the tumorigenesis of CRC with MSI-H by affecting the apoptosis of cancer cells. Our data also suggest that multiregional mutation analysis is needed for a better evaluation of the mutation status in CRC.

Safa M, Tavasoli B, Manafi R, et al.
Indole-3-carbinol suppresses NF-κB activity and stimulates the p53 pathway in pre-B acute lymphoblastic leukemia cells.
Tumour Biol. 2015; 36(5):3919-30 [PubMed] Related Publications
B cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common type of cancer in children. Dramatic improvements in primary therapy for childhood ALL have led to an overall cure rate of 80 %, providing opportunities for innovative combined-modality strategies that would increase cure rates while reducing the toxic side effects of current intensive regimens. In this study, we report that indole-3-carbinol (I3C), a natural phytochemical found in cruciferous vegetables, had anti-leukemic properties in BCP-ALL NALM-6 cells. I3C induced cell growth inhibition by G1 cell cycle arrest and triggered apoptosis in a dose- and time-dependent manner. p53, p21, and Bax proteins showed increased expression after I3C treatment. Real-time PCR analysis of pro-apoptotic p53 target genes revealed up-regulation of PUMA, NOXA, and Apaf-1. I3C also suppressed constitutive nuclear factor-κB (NF-κB) activation and inhibited the protein expression of NF-kappa B-regulated antiapoptotic (IAP1, Bcl-xL, Bcl-2, XIAP) and proliferative (c-Myc) gene products. Coadministration of I3C with the topoisomerase II inhibitor, doxorubicin, potentiates cytotoxic effects compared with either agent alone. Apoptosis induction by the drug combination was associated with enhanced caspase-9 activation and PARP cleavage. Furthermore, I3C abolished doxorubicin-induced NF-κB activity as evidenced by decreased nuclear accumulation of p65, inhibition of IκBα phosphorylation and its degradation, and decreased NF-κB DNA-binding activity. Western blot analysis revealed that doxorubicin-induced Bcl-2 protein expression was inhibited by I3C. Overall, our results indicated that using nontoxic agents, such as I3C, in combination with anthracyclines might provide a new insight into the development of novel combination therapies in childhood BCP-ALL.

Hoskins JW, Jia J, Flandez M, et al.
Transcriptome analysis of pancreatic cancer reveals a tumor suppressor function for HNF1A.
Carcinogenesis. 2014; 35(12):2670-8 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Pancreatic ductal adenocarcinoma (PDAC) is driven by the accumulation of somatic mutations, epigenetic modifications and changes in the micro-environment. New approaches to investigating disruptions of gene expression networks promise to uncover key regulators and pathways in carcinogenesis. We performed messenger RNA-sequencing in pancreatic normal (n = 10) and tumor (n = 8) derived tissue samples, as well as in pancreatic cancer cell lines (n = 9), to determine differential gene expression (DE) patterns. Sub-network enrichment analyses identified HNF1A as the regulator of the most significantly and consistently dysregulated expression sub-network in pancreatic tumor tissues and cells (median P = 7.56×10(-7), median rank = 1, range = 1-25). To explore the effects of HNF1A expression in pancreatic tumor-derived cells, we generated stable HNF1A-inducible clones in two pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) and observed growth inhibition (5.3-fold, P = 4.5×10(-5) for MIA PaCa-2 clones; 7.2-fold, P = 2.2×10(-5) for PANC-1 clones), and a G0/G1 cell cycle arrest and apoptosis upon induction. These effects correlated with HNF1A-induced down-regulation of 51 of 84 cell cycle genes (e.g. E2F1, CDK2, CDK4, MCM2/3/4/5, SKP2 and CCND1), decreased expression of anti-apoptotic genes (e.g. BIRC2/5/6 and AKT) and increased expression of pro-apoptotic genes (e.g. CASP4/9/10 and APAF1). In light of the established role of HNF1A in the regulation of pancreatic development and homeostasis, our data suggest that it also functions as an important tumor suppressor in the pancreas.

Zhang X, Ge YL, Zhang SP, et al.
Downregulation of KDR expression induces apoptosis in breast cancer cells.
Cell Mol Biol Lett. 2014; 19(4):527-41 [PubMed] Related Publications
Angiogenesis plays a crucial role in the growth, invasion and metastasis of breast cancer. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the key regulators of tumor angiogenesis. VEGFR-2, known as the kinase insert domain receptor (KDR), is a key receptor involved in malignant angiogenesis. We previously showed that knocking down KDR with short interference RNA (KDR-siRNA) markedly decreased KDR expression and suppressed tumor growth in a xenograft model. However, the mechanisms underlying the anti-cancer effects of KDR-siRNA are not clearly understood. This study aimed to elucidate the molecular mechanisms that induce apoptosis in human breast cancer MCF-7 cells after transfection with KDR-siRNA. We studied the effects of KDR-siRNA on proliferation, apoptosis, antiapoptotic and pro-apoptotic proteins, mitochondrial membrane permeability, cytochrome c release and caspase-3 activity. The results indicated that KDR-siRNA treatment significantly inhibited the proliferation and induced the apoptosis of MCF-7 cells, reduced the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl, and increased the level of the pro-apoptotic protein Bax, resulting in a decreased Bcl-2/Bax ratio. KDR-siRNA also enhanced the mitochondrial membrane permeability, induced cytochrome c release from the mitochondria, upregulated apoptotic protease-activating factor-1 (Apaf-1), cleaved caspase-3, and increased caspase-3 activity in MCF-7 cells. Furthermore, KDR-siRNA-induced apoptosis in MCF-7 cells was blocked by the caspase inhibitor Z-VAD-FMK, suggesting a role of caspase activation in the induction of apoptosis. These results indicate that the Bcl-2 family proteins and caspase-related mitochondrial pathways are primarily involved in KDR-siRNAinduced apoptosis in MCF-7 cells and that KDR might be a potential therapeutic target for human breast cancer treatments.

Yong FL, Wang CW, Roslani AC, Law CW
The involvement of miR-23a/APAF1 regulation axis in colorectal cancer.
Int J Mol Sci. 2014; 15(7):11713-29 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Recent advances in microRNAome have made microRNAs (miRNAs) a compelling novel class of biomarker in cancer biology. In the present study, the role of miR-23a in the carcinogenesis of colorectal cancer (CRC) was investigated. Cell viability, apoptosis, and caspase 3/7 activation analyses were conducted to determine the potentiality of apoptosis resistance function of miR-23a in CRC. Luciferase assay was performed to verify a putative target site of miR-23a in the 3'-UTR of apoptosis protease activating factor 1 (APAF1) mRNA. The expression levels of miR-23a and APAF1 in CRC cell lines (SW480 and SW620) and clinical samples were assessed using reverse transcription-quantitative real-time PCR (RT-qPCR) and Western blot. We found that the inhibition of miR-23a in SW480 and SW620 cell lines resulted in significant reduction of cell viability and promotion of cell apoptosis. Moreover, miR-23a up-regulation was coupled with APAF1 down-regulation in CRC tissue samples. Taken together, miR-23a was identified to regulate apoptosis in CRC. Our study highlights the potential application of miR-23a/APAF1 regulation axis in miRNA-based therapy and prognostication.

Qiu R, Ma G, Zheng C, et al.
Antineoplastic effect of calycosin on osteosarcoma through inducing apoptosis showing in vitro and in vivo investigations.
Exp Mol Pathol. 2014; 97(1):17-22 [PubMed] Related Publications
Recently, increasing studies have documented that tumorigenesis closely relates to apoptotic processes. Thus, inducing apoptosis is an anti-cancer strategy against osteosarcoma. Here we investigated the anti-proliferative effect of calycosin on human osteosarcoma cell (143B) in vitro. The results showed that calycosin dose-dependently inhibited 143B cell proliferation as reflected in tetrazolium salt (MTT) assay (P<0.01). In addition, calycosin effectively down-regulated cellular mRNA expressions of IκBα, NF-κB p65 and cyclin D1 through RT-PCR assay (P<0.01). Next, calycosin-mediated inhibitory effect on 143B tumor-bearing nude mice and the underlying mechanism were evaluated and discussed. As a result, calycosin administration significantly blocked solid tumor growth in 143B-harbored nude mice (P<0.01). Furthermore, intracellular Bcl-2 protein expression was effectively reduced in 143B-harbored tumor tissue through western blotting analysis (P<0.01), while intratumoral Apaf-1 and cleaved Caspase-3 protein levels were up-regulated, respectively (P<0.01). Taken together, calycosin possesses the anti-osteosarcoma potential, in which the mechanism involved was associated with activation of apoptotic, thus inducing apoptosis.

Moravcikova E, Krepela E, Prochazka J, et al.
Differential sensitivity to apoptosome apparatus activation in non-small cell lung carcinoma and the lung.
Int J Oncol. 2014; 44(5):1443-54 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
The intrinsic apoptosis pathway represents an important mechanism of stress-induced death of cancer cells. To gain insight into the functional status of the apoptosome apparatus in non-small cell lung carcinoma (NSCLC), we studied its sensitivity to activation, the assembly of apoptosome complexes and stability of their precursors, and the importance of X-linked inhibitor of apoptosis (XIAP) in the regulation of apoptosome activity, using cell-free cytosols from NSCLC cell lines and NSCLC tumours and lungs from 62 surgically treated patients. Treatment of cytosol samples with cytochrome c (cyt-c) and dATP induced proteolytic processing of procaspase-9 to caspase-9, which was followed by procaspase-3 processing to caspase-3, and by generation of caspase-3-like activity in 5 of 7 studied NSCLC cell lines. Further analysis demonstrated formation of high-Mr Apaf-1 complexes associated with cleaved caspase-9 in the (cyt-c + dATP)-responsive COLO-699 and CALU-1 cells. By contrast, in A549 cells, Apaf-1 and procaspase-9 co-eluted in the high-Mr fractions, indicating formation of an apoptosome complex unable of procaspase-9 processing. Thermal pre-treatment of cell-free cytosols in the absence of exogenous cyt-c and dATP lead to formation of Apaf-1 aggregates, unable to recruit and activate procaspase-9 in the presence of cyt-c and dATP, and to generate caspase‑3‑like activity. Further studies showed that the treatment with cyt-c and dATP induced a substantially higher increase of caspase-3-like activity in cytosol samples from NSCLC tumours compared to matched lungs. Tumour histology, grade and stage had no significant impact on the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity. Upon addition into the cytosol, the XIAP-neutralizing peptides AVPIAQK and ATPFQEG only moderately heightened the (cyt-c + dATP)-induced caspase‑3‑like activity in some NSCLC tumours. Taken together, the present study provides evidence that the apoptosome apparatus is functional in the majority of NSCLCs and that its sensitivity to the (cyt-c + dATP)-mediated activation is often enhanced in NSCLCs compared to lungs. They also indicate that XIAP does not frequently and effectively suppress the activity of apoptosome apparatus in NSCLCs.

Shang J, Yang F, Wang Y, et al.
MicroRNA-23a antisense enhances 5-fluorouracil chemosensitivity through APAF-1/caspase-9 apoptotic pathway in colorectal cancer cells.
J Cell Biochem. 2014; 115(4):772-84 [PubMed] Related Publications
Current literature provided information that alteration in microRNA expression impacted sensitivity or resistance of certain tumor types to anticancer treatment, including the possible intracellular pathways. The microRNA-23a (miR-23a)-regulated apoptosis in response to the 5-fluorouracil (5-FU)-induced mitochondria-mediated apoptotic pathway was determined in this study. The miR-23a expression in 5-FU-treated and untreated colon cancer cells and tissues was assessed using real-time PCR analysis. To determine the function of miR-23a in the regulation of 5-FU-induced apoptosis, cell-proliferation, cytotoxicity, and apoptosis analyses were performed. Dual luciferase reporter assay was used to identify the apoptosis-related target gene for miR-23a. The activity of caspases-3, -7, and -9 were also assessed in miR-23a antisense and 5-FU treated tumor cells. A xenograft tumor model was established to evaluate the biological relevance of altered miR-23a expression to the 5-FU-based chemotherapy in vivo. We found that the expression of miR-23a was increased and the level of apoptosis-activating factor-1 (APAF-1) was decreased in 5-FU-treated colon cancer cells compared to untreated cells. The activation of the caspases-3 and 7 was increased in miR-23a antisense and 5-FU-treated colon cancer cells compared to negative control. APAF-1, as a target gene of miR-23a, was identified and miR-23a antisense-induced increase in the activation of caspase-9 was observed. The overexpression of miR-23a antisense up-regulated the 5-FU induced apoptosis in colon cancer cells. However, the miR-23a knockdown did not increase the antitumor effect of 5-FU in xenograft model of colon cancer. This study shows that miR-23a antisense enhanced 5-FU-induced apoptosis in colorectal cancer cells through the APAF-1/caspase-9 apoptotic pathway.

Peng SF, Lee CY, Hour MJ, et al.
Curcumin-loaded nanoparticles enhance apoptotic cell death of U2OS human osteosarcoma cells through the Akt-Bad signaling pathway.
Int J Oncol. 2014; 44(1):238-46 [PubMed] Related Publications
Curcumin has potential anticancer activity and has been shown to be involved in several signaling pathways including differentiation and apoptosis. Our previous study showed that water-soluble PLGA curcumin nanoparticles (Cur-NPs) triggered apoptotic cell death through regulation of the function of MDR1 and the production of reactive oxygen species (ROS) in cisplatin-resistant human oral cancer CAR cells. In this study, we investigated the anti-proliferative effects of Cur-NPs on human osteosarcoma U2OS cells. The morphology of Cur-NPs showed spherical shape by TEM analysis. The encapsulation efficiency of curcumin in Cur-NPs prepared by single emulsion was 90.5 ± 3.0%. Our results demonstrated that the curcumin fragments on the mass spectrum of Cur-NPs and the peaks of curcumin standard could be found on the Cur-NPs spectrum by 1H-NMR spectra analysis. Cur-NPs induced anti-proliferative effects and apoptosis in U2OS cells. Compared to the untreated U2OS cells, more detectable amount of Cur-NPs was found inside the treated U2OS cells. Cur-NPs induced DNA fragmentation and apoptotic bodies in U2OS cells. Both the activity and the expression levels of caspases-3/-7 and caspase-9 were elevated in the treated U2OS cells. Cur-NPs upregulated the protein expression levels of cleaved caspase-3/caspase-9, cytochrome c, Apaf-1 and Bad and downregulated the protein expression level of p-Akt in U2OS cells. These results suggest Cur-NPs are effective in enhancing apoptosis in human osteosarcoma cells and thus could provide potential for cancer therapeutics.

Qin R, Shen H, Cao Y, et al.
Tetrandrine induces mitochondria-mediated apoptosis in human gastric cancer BGC-823 cells.
PLoS One. 2013; 8(10):e76486 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Tetrandrine, a bis-benzylisoquinoline alkaloid isolated from the dried root of Hang-Fang-Chi (Stephaniatetrandra S. Moore), has been reported to possess anti-cancer effects on many tumors. In this study, we investigated tetrandrine-induced apoptosis on human gastric cancer BGC-823 cells in vitro and in vivo. The results showed that tetrandrine significantly inhibited cell viability in a dose- and time-dependent manner and induced apoptosis. It increased the apoptosis; upregulation of Bax, Bak, and Bad; and downregulation of Bcl-2 and Bcl-xl in BGC-823 cells. Moreover, tetrandrine increased the activation of caspase-3 and -9, release of cytochrome c, and upregulation of apaf-1, suggesting that tetrandrine-induced apoptosis was related to the mitochondrial pathway. Meanwhile, pretreatment with the pan-caspase inhibitor z-VAD-fmk in BGC-823 cells reduced tetrandrine-induced apoptosis by blocking activation of caspases. Furthermore, tetrandrine effectively inhibited tumor growth via apoptosis induction, which was verified by immunohistochemical analysis in a nude mouse xenograft model. Taken together, we concluded that tetrandrine significantly inhibited the proliferation of gastric cancer BGC-823 cells through mitochondria-dependent apoptosis, which may play a promising role in gastric cancer therapy.

Sanchez R, St-Cyr J, Lalonde ME, et al.
Impact of promoter polymorphisms in key regulators of the intrinsic apoptosis pathway on the outcome of childhood acute lymphoblastic leukemia.
Haematologica. 2014; 99(2):314-21 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
The introduction of multiagent treatment protocols has led to a remarkable increase in survival rates for children diagnosed with acute lymphoblastic leukemia, yet for a subpopulation of patients, resistance to chemotherapeutics remains an obstacle to successful treatment. Here we investigate the role of the mitochondrial (or intrinsic) apoptosis pathway in modulating the onset and outcomes of childhood acute lymphoblastic leukemia. Cell death is a highly regulated process that plays an essential role in regulating cell homeostasis, particularly in tissues with high intrinsic proliferating capacity such as the hematopoietic system. Following the underlying paradigm that cis-acting genetic variation can influence disease risk and outcomes by modulating gene expression, we performed a systematic analysis of the proximal promoter regions of 21 genes involved in apoptosis. Using gene reporter assays, we show that promoter variations in 11 intrinsic apoptosis genes, including ADPRT, APAF1, BCL2, BAD, BID, MCL1, BIRC4, BCL2L1, ENDOG, YWHAB, and YWHAQ, influence promoter activity in an allele-specific manner. We also show that correlated promoter variation and increased expression of MCL1 is associated with reduced overall survival among high-risk patients receiving higher doses of corticosteroid, suggesting that increased expression of this anti-apoptosis gene could lead to reduced cell death and influence treatment response in a disease- and dose-responsive manner.

Wybranska I, Polus A, Mikolajczyk M, et al.
Apoptosis-related gene expression in glioblastoma (LN-18) and medulloblastoma (Daoy) cell lines.
Hum Cell. 2013; 26(4):137-48 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
The expression of apoptosis genes in a commercial pre-designed low-density array from Applied Biosystems was evaluated in two human brain cancer cell models, LN-18 and Daoy (HTB-186™) in comparison to the reference human primary endothelial cells under basic conditions. Analysis of the gene expression in the cancer cell lines compared to the normal control revealed features reflecting anti-apoptotic and inflammatory characteristics of the former. There was an overall downregulation of apoptosis-stimulating genes in both cancer cell lines, along with an upregulation of certain apoptosis inhibitors. A number of genes demonstrated statistically significant changes in their expressions, including BAX (BCL2-associated X protein); the CARD4/NLR family, CARD domain containing 4; CASP10 (caspase 10, apoptosis-related cysteine peptidase); DAP1 (death-associated protein kinase 1), and BIRC5 (baculoviral IAP repeat-containing 5). Anti-apoptotic potential in both cell lines was demonstrated by changes in the Bax:Bcl-2 ratio and downregulation of the APAF1 gene in LN18 cells. There was also significant downregulation of extrinsic signals and the TNF/FADD/inflammatory cascade, and upregulation of caspase inhibitors (IAPs). These results provided a novel molecular characterization of important human cancer cell lines, which might provide a useful research tool for investigating the experimental model of the CNS cell.

Chang PY, Peng SF, Lee CY, et al.
Curcumin-loaded nanoparticles induce apoptotic cell death through regulation of the function of MDR1 and reactive oxygen species in cisplatin-resistant CAR human oral cancer cells.
Int J Oncol. 2013; 43(4):1141-50 [PubMed] Related Publications
Curcumin is a polyphenolic compound which possesses anticancer potential. It has been shown to induce cell death in a variety of cancer cells, however, its effect on CAL27‑cisplatin-resistant human oral cancer cells (CAR cells) has not been elucidated to date. The low water solubility of curcumin which leads to poor bioavailability, however, has been highlighted as a major limiting factor. In this study, we utilized water-soluble PLGA curcumin nanoparticles (Cur-NPs), and investigated the effects of Cur-NPs on CAR cells. The results showed Cur-NPs induced apoptosis in CAR cells but exhibited low cytotoxicity to normal human gingival fibroblasts (HGFs) and normal human oral keratinocytes (OKs). Cur-NPs triggered DNA concentration, fragmentation and subsequent apoptosis. Compared to untreated CAR cells, a more detectable amount of Calcein-AM accumulation was found inside the treated CAR cells. Cur-NPs suppressed the protein and mRNA expression levels of MDR1. Both the activity and the expression levels of caspase-3 and caspase-9 were elevated in the treated CAR cells. The Cur-NP-triggered apoptosis was blocked by specific inhibitors of pan-caspase (z-VAD-fmk), caspase-3 (z-DEVD-fmk), caspase-9 (z-LEHD-fmk) and antioxidant agent (N-acetylcysteine; NAC). Cur-NPs increased reactive oxygen species (ROS) production, upregulated the protein expression levels of cleaved caspase-3/caspase-9, cytochrome c, Apaf-1, AIF, Bax and downregulated the protein levels of Bcl-2. Our results suggest that Cur-NPs triggered the intrinsic apoptotic pathway through regulating the function of multiple drug resistance protein 1 (MDR1) and the production of reactive oxygen species (ROS) in CAR cells. Cur-NPs could be potentially efficacious in the treatment of cisplatin-resistant human oral cancer.

Lian S, Shi R, Bai T, et al.
Anti-miRNA-23a oligonucleotide suppresses glioma cells growth by targeting apoptotic protease activating factor-1.
Curr Pharm Des. 2013; 19(35):6382-9 [PubMed] Related Publications
BACKGROUND: Abnormal expression of microRNAs (miRNAs) is closely related to glioma, which is one of the most common malignant brain tumors. The current study is to identify the key miRNAs involved in the pathogenesis of glioma and to discover novel therapeutic targets for this disease.
MATERIALS AND METHODS: Total RNA was extracted from glioma tissues of 100 patients. The microRNA microarray and the northern blot were used to detect the changes of miRNAs expression in 7 pairs of glioma specimens. Relative expressions of miR-23a were validated by real-time reverse transcription polymerase chain reaction (RT-PCR) with specific Taqman probes. In order to evaluate the role of miR-23a, the miR-23a mimics and anti-miR-23a oligonucleotides were transfected to glioma cell lines; the cell proliferation, apoptosis, cell cycle percentage, cell migration and invasion abilities were evaluated in vitro. The target genes of miR-23a were also investigated using the bioinformatics tools. The expression of the apoptotic protease activating factor-1 (APAF1), which might be one of the direct targets of miR-23a, was also analyzed using the luciferase reporter assay and western blot analysis in 293T cells and glioma cell line, respectively.
RESULTS: The microRNA microarray and the northern blot results showed that the expressions of miR-23a in glioma tissues were significantly upregulated. The miR-23a expression levels identified using real time RT-PCR in tumor tissues of 79 samples were higher than in the matched adjacent tissues. By transfection of anti-miR-23a oligonucleotide, the results showed that the proliferation, migration, and invasion of glioma cell lines were significantly suppressed. The bioinformatics searching results showed that APAF1 might be a direct target gene of miR-23a, and it was supported by the luciferase reporter gene assay and western blot analysis results. Finally, experiments showed that overexpression of APAF1 suppressed glioma cell growth and promoted cell apoptosis.
CONCLUSIONS: Our findings characterized the expression properties of miR-23a, contributed to the function and molecular mechanism of miR-23a in glioma and implied that miR-23a might be employed as novel prognostic markers and therapeutic targets of glioma.

Tang JY, Chang HW, Chang JG
Modulating roles of amiloride in irradiation-induced antiproliferative effects in glioblastoma multiforme cells involving Akt phosphorylation and the alternative splicing of apoptotic genes.
DNA Cell Biol. 2013; 32(9):504-10 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Apoptosis is a key mechanism for enhanced cellular radiosensitivity in radiation therapy. Studies suggest that Akt signaling may play a role in apoptosis and radioresistance. This study evaluates the possible modulating role of amiloride, an antihypertensive agent with a modulating effect to alternative splicing for regulating apoptosis, in the antiproliferative effects induced by ionizing radiation (IR) in glioblastoma multiforme (GBM) 8401 cells. Analysis of cell viability showed that amiloride treatment significantly inhibited cell proliferation in irradiated GBM8401 cells (p<0.05) in a time-dependent manner, especially in cells treated with amiloride with IR post-treatment. In comparison with GBM8401 cells treated with amiloride alone, with GBM8401 cells treated with IR alone, and with human embryonic lung fibroblast control cells (HEL 299), GBM8401 cells treated with IR combined with amiloride showed increased overexpression of phosphorylated Akt, regardless of whether IR treatment was performed before or after amiloride administration. The alternative splicing pattern of apoptotic protease-activating factor-1 (APAF1) in cells treated with amiloride alone, IR alone, and combined amiloride-IR treatments showed more consistent cell proliferation compared to that in other apoptosis-related genes such as baculoviral IAP repeat containing 5 (BIRC5), Bcl-X, and homeodomain interacting protein kinase-3 (HIPK3). In GBM8401 cells treated with amiloride with IR post-treatment, the ratio of prosurvival (-XL,-LC) to proapoptotic (-LN,-S) splice variants of APAF1 was lower than that seen in cells treated with amiloride with IR pretreatment, suggesting that proapoptotic splice variants of APAF1 (APAF1-LN,-S) were higher in the glioblastoma cells treated with amiloride with IR post-treatment, as compared to glioblastoma cells and fibroblast control cells that had received other treatments. Together, these results suggest that amiloride modulates cell radiosensitivity involving the Akt phosphorylation and the alternative splicing of APAF1, especially for the cells treated with amiloride with IR post-treatment. Therefore, amiloride may improve the effectiveness of radiation therapy for GBMs.

Yamaguchi M
The anti-apoptotic effect of regucalcin is mediated through multisignaling pathways.
Apoptosis. 2013; 18(10):1145-53 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Regucalcin (RGN/SMP30) was originally discovered in 1978 as a calcium-binding protein that does not contain the EF-hand motif of as a calcium-binding domain. The name, regucalcin, was proposed for this calcium-binding protein, which can regulate various Ca(2+)-dependent enzymes activation in liver cells. The regucalcin gene is localized on the X chromosome, and its expression is mediated through many signaling factors. Regucalcin plays a pivotal role in regulation of intracellular calcium homeostasis in various cell types. Regucalcin also has a suppressive effect on various signaling pathways from the cytoplasm to nucleus in proliferating cells and regulates nuclear function in including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis. Overexpression of endogenous regucalcin was found to suppress apoptosis in modeled rat hepatoma cells and normal rat kidney proximal epithelial NRK52 cells induced by various signaling factors. Suppressive effect of regucalcin on apoptosis is related to inhibition of nuclear Ca(2+)-activated DNA fragmentation, Ca(2+)/calmodulin-dependent nitric oxide synthase, caspase-3, Bax, cytochrome C, protein tyrosine kinase, protein tyrosine phosphatase in the cytoplasm and nucleus. Moreover, regucalcin stimulates Bcl-2 mRNA expression and depresses enhancement of caspase-3, Apaf-1 and Akt-1 mRNAs expression. This review discusses that regucalcin plays a pivotal role in rescue of apoptotic cell death, which is mediated through various signaling factors.

Barione DF, Lizarte FS, Novais PC, et al.
Gene expression study related with the intrinsic pathway of apoptosis in bladder cancer by real-time PCR technique.
Genet Mol Res. 2013; 12(2):878-86 [PubMed] Related Publications
We examined the expression of anti-apoptotic genes (XIAP and Bcl-2) and apoptotic genes (cytochrome c, caspase-9, Apaf-1) in tissue samples of patients with superficial bladder cancer. Thirty-two bladder cancer tissue samples (8 papillary urothelial neoplasm of low malignant potential, 10 low-grade, and 14 high-grade) and 8 normal bladder tissue samples from necropsy were used for the study of gene expression by real-time PCR analysis. Analysis of the expression of apoptotic gene constituents of an apoptosome demonstrated an increase in Apaf-1 expression in the three tumor grades when compared with the control (P < 0.01, P < 0.05, and P < 0.01), low expression of caspase-9 in all groups (P < 0.05), and an increase in cytochrome c expression in all tumor grades in relation to the control, although without statistically significant difference. The expression of anti-apoptotic genes revealed an increase in XIAP expression in all tumor grades in relation to the control, although without statistically significant difference, and low expression of Bcl-2 in all tumor grades and the control (P < 0.05). The results proved that there is low evidence of apoptotic activity by the intrinsic pathway, demonstrated by the low expression of caspase-9 and considerable increase in XIAP expression, which may render these genes potential therapeutic targets in bladder cancer treatment.

Hsu TH, Chu CC, Hung MW, et al.
Caffeic acid phenethyl ester induces E2F-1-mediated growth inhibition and cell-cycle arrest in human cervical cancer cells.
FEBS J. 2013; 280(11):2581-93 [PubMed] Related Publications
Caffeic acid phenyl ester (CAPE) has been identified as an active component of propolis, a substance that confers diverse activities in cells of various origins. However, the molecular basis of CAPE-mediated cellular activity remains to be clarified. Here, we show that CAPE preferentially induced S- and G2 /M-phase cell-cycle arrests and initiated apoptosis in human cervical cancer lines. The effect was found to be associated with increased expression of E2F-1, as there is no CAPE-mediated induction of E2F-1 in the pre-cancerous cervical Z172 cells. CAPE also up-regulated the E2F-1 target genes cyclin A, cyclin E and apoptotic protease activating of factor 1 (Apaf-1) but down-regulated cyclin B and induced myeloid leukemia cell differentiation protein (Mcl-1). These results suggest the involvement of E2F-1 in CAPE-mediated growth inhibition and cell-cycle arrest. Transient transfection studies with luciferase reporters revealed that CAPE altered the transcriptional activity of the apaf-1 and mcl-1 promoters. Further studies using chromatin immunoprecipitation assays demonstrated that E2F-1 binding to the apaf-1 and cyclin B promoters was increased and decreased, respectively, in CAPE-treated cells. Furthermore, E2F-1 silencing abolished CAPE-mediated effects on cell-cycle arrest, apoptosis and related gene expression. Taken together, these results indicate a crucial role for E2F-1 in CAPE-mediated cellular activities in cervical cancer cells.

Yao TT, Mo SM, Liu LY, et al.
5-Aza-2'-deoxycytidine may influence the proliferation and apoptosis of cervical cancer cells via demethylation in a dose- and time-dependent manner.
Genet Mol Res. 2013; 12(1):312-8 [PubMed] Related Publications
The methylation of tumor suppressor genes has been shown to be involved in many human cancers. 5-Aza-2'-deoxycytidine (5-Aza-CdR) can reactivate the expression of methylated tumor suppressor genes. In our study, 2 human cervical cancer cell lines, HeLa and SiHa, were treated with different concentrations (20, 10, 5, and 2.5 μM) of 5-Aza-CdR for 24, 48, and 72 h. After incubation, cells were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry. The expression of RASSF1A and APAF-1 was detected by RT-PCR. 5-Aza-CdR inhibited the growth of HeLa and SiHa cells at different concentrations. The strongest inhibition and apoptosis rates were obtained after incubation for 72 h (5.63 ± 1.38 and 8.24 ± 2.40%, respectively). No significant difference in the expression of RASSF1A was found upon drug treatment, while APAF-1 expression increased in HeLa cells after treatment (0.790 ± 0.056%). Our results suggest that the tumor-suppressive effect of 5-Aza-CdR may result from the reactivation of silenced APAF-1 through demethylation.

Rogalińska M, Franiak-Pietryga I, Błoński JZ, et al.
Toward personalized therapy for chronic lymphocytic leukemia: DSC and cDNA microarray assessment of two cases.
Cancer Biol Ther. 2013; 14(1):6-12 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
The differences in clinical course of chronic lymphocytic leukemia could have an impact on variations in a patient's response to therapy. Our published results revealed that thermal transition (95 ± 5°C) in differential scanning calorimetry profiles appear to be characteristic for the advanced stage of CLL. Moreover, a decrease/loss of this transition in nuclei from leukemic cells exposed to drugs ex vivo could indicate their diverse efficacy. It seems that the lack of changes in thermal profile could predict patient's drug resistance. In this study, we demonstrate the results obtained after drug treatment of leukemic cells by calorimetry, apoptosis-related parameters involved in expression of genes using cDNA microarray and western blot. These data were compared with the patients' clinical parameters before and after RCC therapy (rituximab + cladribine + cyclophosphamide). The complementary analysis of studied cases with opposite clinical response (CR or NR) revealed a strong relationship between clinical data, differences in thermal scans and apoptosis-related gene expression. We quantified expression of eight of apoptosis-related 89 genes, i.e., NOXA, PUMA, APAF1, ESRRBL1, CASP3, BCL2, BCL2A1 and MCL1. Particular differences in NOXA and BCL2 expression were revealed. NOXA expression in cells of patients who achieved a complete response to RCC therapy was 0.44 times higher in comparison to control ones. Interestingly, in the case of patients who did not respond to immunotherapy, NOXA expression was highly downregulated (RQ = 4.39) as compared with untreated cells. These results were confirmed by distinct cell viability, protein expression as well as by differences in calorimetry profiles.

Ottosson-Wadlund A, Ceder R, Preta G, et al.
Requirement of apoptotic protease-activating factor-1 for bortezomib-induced apoptosis but not for Fas-mediated apoptosis in human leukemic cells.
Mol Pharmacol. 2013; 83(1):245-55 [PubMed] Related Publications
Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

Lo Muzio L, Sartini D, Santarelli A, et al.
Expression and prognostic significance of apoptotic genes in oral squamous cell carcinoma.
Mol Carcinog. 2014; 53(4):264-71 [PubMed] Related Publications
Oral squamous cell carcinoma (OSCC) is the most common malignancy of oral cavity. Human cancers are characterized by an imbalance of regulatory mechanisms controlling different cellular pathways, including apoptosis. Apoptosis occurs in a wide variety of physiological processes, such as embryonic development, tissue homeostasis or immune defense, and its role is to remove harmful, damaged, or unwanted cells. Defective apoptosis represents an important causative factor in the development/progression of cancer, and the ability of tumor cells to evade apoptosis can play a significant role in their resistance to conventional anticancer treatment. We investigated the expression profile of genes involved in the apoptotic mechanism in 21 paired tissue samples (OSCC and adjacent normal oral mucosa) by cDNA macroarray, in order to identify differentially expressed genes in oral cancer compared to normal tissue. To validate the results obtained by cDNA macroarray, quantitative real-time PCR, Western blot, and immunohistochemical analyses were performed. Results obtained by cDNA macroarray analysis showed different expression levels of CRADD, FADD, ATM, APAF1, and TP63 genes in OSCC compared to normal mucosa. Differential gene expression measurements (tumor vs. normal tissue) performed by real-time PCR showed an overexpression of FADD and a downregulation of ATM. Moreover, Western blot analysis confirmed that both CRADD and APAF-1 were decreased in OSCC compared to normal oral mucosa. As showed by immunohistochemistry, OSCC exhibited increased expression of p63 compared to normal tissue. Interestingly, a statistically significant positive correlation was found between p63 expression and the histological grade.

Zang YS, Zhong YF, Fang Z, et al.
MiR-155 inhibits the sensitivity of lung cancer cells to cisplatin via negative regulation of Apaf-1 expression.
Cancer Gene Ther. 2012; 19(11):773-8 [PubMed] Related Publications
MicroRNA-155 (miR-155) overexpression is often found in malignancies including lung cancer. The objective of this study is to verify the hypothesis, based on the results of bioinformatics analysis, that miR-155 modulates cellular apoptosis and DNA damage through the regulation of Apaf-1 and is thus involved in the development and progression of lung cancer. First, we measured the expression of miR-155 and the Apaf-1 protein in lung cancer tissues. The results showed that expression of miR-155 was significantly higher in lung cancer tissues than in paracancerous and normal tissues; whereas Apaf-1 expression was lower in the lung cancerous tissues. We then established miR-155-silenced and Apaf-1-overexpressed A549 cell lines by transfection with pMAGic2.0-BIC-siRNA and pcDNA3.1-Apaf-1, respectively. These cell lines were then treated with cisplatin, and apoptosis and DNA damage were assessed, with non-transfected A549 cells used as negative controls. The results showed that, relative to controls, the silencing of miR-155 resulted in elevated expression of the Apaf-1 protein, whereas Apaf-1 mRNA levels remained unchanged. Both the silencing of miR-155 and the overexpression Apaf-1 greatly increased the sensitivity of A549 cells to cisplatin treatment, as evidenced by elevated rates of apoptosis and DNA damage. Furthermore, dual-transfection of A549 cells with miR-155 siRNA and Apaf-1 siRNA resulted in the attenuation of apoptosis and DNA damage. In conclusion, the inhibition of miR-155 can enhance the sensitivity of A549 cells to cisplatin treatment by modulation of cellular apoptosis and DNA damage through an Apaf-1-mediated pathway.

Sabirzhanov B, Stoica BA, Hanscom M, et al.
Over-expression of HSP70 attenuates caspase-dependent and caspase-independent pathways and inhibits neuronal apoptosis.
J Neurochem. 2012; 123(4):542-54 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
HSP70 is a member of the family of heat-shock proteins that are known to be up-regulated in neurons following injury and/or stress. HSP70 over-expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over-expression by transfection with HSP70-expression plasmids in primary cortical neurons and the SH-SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2-ceramide, and β-Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease-activating factor 1, as well as apoptosis-inducing factor, key molecules involved in development of caspase-dependent and caspase-independent PCD, respectively. Markers of caspase-dependent PCD, including active caspase-3, caspase-9, and cleaved PARP were attenuated in neurons over-expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase-dependent and caspase-independent PCD pathways.

Eißmann M, Melzer IM, Fernández SB, et al.
Overexpression of the anti-apoptotic protein AVEN contributes to increased malignancy in hematopoietic neoplasms.
Oncogene. 2013; 32(20):2586-91 [PubMed] Related Publications
AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms.

Piotrowska H, Myszkowski K, Ziółkowska A, et al.
Resveratrol analogue 3,4,4',5-tetramethoxystilbene inhibits growth, arrests cell cycle and induces apoptosis in ovarian SKOV-3 and A-2780 cancer cells.
Toxicol Appl Pharmacol. 2012; 263(1):53-60 [PubMed] Related Publications
In the screening studies, cytotoxicity of 12 methylated resveratrol analogues on 11 human cancer cell lines was examined. The most active compound 3,4,4'5-tetramethoxystilbene (DMU-212) and two ovarian cancer cell lines A-2780 (IC(50)=0.71 μM) and SKOV-3 (IC(50)=11.51 μM) were selected for further investigation. To determine the mechanism of DMU-212 cytotoxicity, its ability to induce apoptosis was examined. DMU-212 arrested cell cycle in the G2/M or G0/G1 phase which resulted in apoptosis of both cell lines. The expression level of 84 apoptosis-related genes was investigated. In SKOV-3 cells DMU-212 caused up-regulation of pro-apoptotic Bax, Apaf-1 and p53 genes, specific to intrinsic pathway of apoptosis, and a decrease in Bcl-2 and Bcl 2110 mRNA expressions. Conversely, in A-2780 cells an increased expression of pro-apoptotic genes Fas, FasL, TNF, TNFRSF10A, TNFRSF21, TNFRSF16 specific to extracellular mechanism of apoptosis was observed. There are no data published so far regarding the receptor mediated apoptosis induced by DMU-212. The activation of caspase-3/7 was correlated with decreased TRAF-1 and BIRC-2 expression level in A-2780 cells exposed to DMU-212. DMU-212 caused a decrease in CYP1A1 and CYP1B1 mRNA levels in A-2780 by 50% and 75%, and in SKOV-3 cells by 15% and 45%, respectively. The protein expression was also reduced in both cell lines. It is noteworthy that the expression of CYP1B1 protein was entirely inhibited in A-2780 cells treated with DMU-212. It can be suggested that different CYP1B1 expression patterns in either ovarian cell line may affect their sensitivity to cytotoxic activity of DMU-212.

Saha A, Lu J, Morizur L, et al.
E2F1 mediated apoptosis induced by the DNA damage response is blocked by EBV nuclear antigen 3C in lymphoblastoid cells.
PLoS Pathog. 2012; 8(3):e1002573 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
EBV latent antigen EBNA3C is indispensible for in vitro B-cell immortalization resulting in continuously proliferating lymphoblastoid cell lines (LCLs). EBNA3C was previously shown to target pRb for ubiquitin-proteasome mediated degradation, which facilitates G1 to S transition controlled by the major transcriptional activator E2F1. E2F1 also plays a pivotal role in regulating DNA damage induced apoptosis through both p53-dependent and -independent pathways. In this study, we demonstrate that in response to DNA damage LCLs knocked down for EBNA3C undergo a drastic induction of apoptosis, as a possible consequence of both p53- and E2F1-mediated activities. Importantly, EBNA3C was previously shown to suppress p53-induced apoptosis. Now, we also show that EBNA3C efficiently blocks E2F1-mediated apoptosis, as well as its anti-proliferative effects in a p53-independent manner, in response to DNA damage. The N- and C-terminal domains of EBNA3C form a stable pRb independent complex with the N-terminal DNA-binding region of E2F1 responsible for inducing apoptosis. Mechanistically, we show that EBNA3C represses E2F1 transcriptional activity via blocking its DNA-binding activity at the responsive promoters of p73 and Apaf-1 apoptosis induced genes, and also facilitates E2F1 degradation in an ubiquitin-proteasome dependent fashion. Moreover, in response to DNA damage, E2F1 knockdown LCLs exhibited a significant reduction in apoptosis with higher cell-viability. In the presence of normal mitogenic stimuli the growth rate of LCLs knockdown for E2F1 was markedly impaired; indicating that E2F1 plays a dual role in EBV positive cells and that active engagement of the EBNA3C-E2F1 complex is crucial for inhibition of DNA damage induced E2F1-mediated apoptosis. This study offers novel insights into our current understanding of EBV biology and enhances the potential for development of effective therapies against EBV associated B-cell lymphomas.

Wen X, Lin ZQ, Liu B, Wei YQ
Caspase-mediated programmed cell death pathways as potential therapeutic targets in cancer.
Cell Prolif. 2012; 45(3):217-24 [PubMed] Related Publications
The caspase family is well characterized as playing a crucial role in modulation of programmed cell death (PCD), which is a genetically regulated, evolutionarily conserved process with numerous links to many human diseases, most notably cancer. In this review, we focus on summarizing the intricate relationships between some members of the caspase family and their key apoptotic mediators, involving tumour necrosis factor receptors, the Bcl-2 family, cytochrome c, Apaf-1 and IAPs in cancer initiation and progression. We elucidate new emerging types of cross-talk between several caspases and autophagy-related genes (Atgs) in cancer. Moreover, we focus on presenting several PCD-modulating agents that may target caspases-3, -8 and -9, and their substrates PARP-1 and Beclin-1, which may help us harness caspase-modulated PCD pathways for future drug discovery.

Moravcikova E, Krepela E, Prochazka J, et al.
Down-regulated expression of apoptosis-associated genes APIP and UACA in non-small cell lung carcinoma.
Int J Oncol. 2012; 40(6):2111-21 [PubMed] Related Publications
The Apaf-1 interacting protein (APIP) and the uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) belong to endogenous regulators of the apoptosome apparatus, but their role in tumourigenesis and progression of non-small cell lung carcinoma (NSCLC) is not known. Previous studies demonstrated that APIP inhibits the apoptosome-mediated procaspase-9 activation while UACA induces translocation of Apaf-1 from the cytoplasm into the nucleus. Here, we report for the first time that the expression of APIP and UACA genes is down-regulated on the level of both mRNA and protein in NSCLC cells and tumours. In particular, the expression of APIP protein was strikingly decreased and the expression of UACA mRNA and protein was frequently down-regulated in NSCLC tumours of different histopathological types. Moreover, stage IA NSCLC tumours showed significantly lower expression of UACA mRNA compared to higher stage tumours. The weak increase of both APIP and UACA mRNA levels in the 5-aza-2'-deoxycytidine-treated NSCLC cells indicates that mechanisms other than DNA methylation are involved in the regulation of APIP and UACA gene expression in these cancer cells. Taken together, the down-regulation of APIP and UACA expression suggests that the threshold to activate the apoptosome apparatus may be decreased in NSCLC cells due to the lack of APIP-mediated suppression and UACA-assisted Apaf-1 nuclear entry. Moreover, the loss of UACA-assisted Apaf-1 nuclear translocation may underlie the failure of DNA damage checkpoint activation in NSCLC cells leading to their genomic instability.

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