Altered conformational dynamics contribute to species-specific effects of cytochrome c mutations on caspase activation.

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.

Activation of pregnane X receptor protects against cholestatic liver injury by inhibiting hepatocyte pyroptosis.

Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg-1·d-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

Impairment of apoptosis pathway via Apaf1 downregulation during chlorpyrifos and/or cypermethrin induced lung damage.

Chlorpyrifos is an organophosphate and the cypermethrin is type 2 pyrethroid insecticide that are used for indoor and outdoor pest control. The present study aimed to investigate differential transcriptional profiling to identify the candidate gene associated with lung injury following exposure to chlorpyrifos and/or cypermethrin in a mouse model system. Swiss male albino mice (n = 24) were divided into three treatment groups (n = 6 each) that were given chlorpyrifos (2.76 mg kg-1 body weight), cypermethrin (2 mg kg-1 body weight) and the combination of both pesticides orally dissolved in corn oil and one control group (n = 6) that received corn oil for 90 days. The pulmonary expression of the Apaf1 was observed using RT2 Profiler PCR Array. The results showed that chronic exposure to chlorpyrifos, cypermethrin and their combination downregulated (67, 63 and 66 genes) and upregulated (4, 2 and 2 genes), respectively. The pulmonary expression of Apaf1 that plays important role in apoptosis was found to be downregulated. The immunohistochemistry depicted reduced expression of Apaf1 in both airway epithelium and alveolar septa following exposure to chlorpyrifos and/or cypermethrin. In conclusion, results demonstrated that exposure to chlorpyrifos, cypermethrin and their combination cause lung damage by the dysregulation of Apaf1 gene expression.

Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Upregulation of APAF1 and CSF1R in Peripheral Blood Mononuclear Cells of Parkinson's Disease.

Increased oxidative stress and neuroinflammation play a crucial role in the pathogenesis of Parkinson's disease (PD). In this study, the expression levels of 52 genes related to oxidative stress and inflammation were measured in peripheral blood mononuclear cells of the discovery cohort including 48 PD patients and 25 healthy controls. Four genes, including ALDH1A, APAF1, CR1, and CSF1R, were found to be upregulated in PD patients. The expression patterns of these genes were validated in a second cohort of 101 PD patients and 61 healthy controls. The results confirmed the upregulation of APAF1 (PD: 0.34 ± 0.18, control: 0.26 ± 0.11, p < 0.001) and CSF1R (PD: 0.38 ± 0.12, control: 0.33 ± 0.10, p = 0.005) in PD patients. The expression level of APAF1 was correlated with the scores of the Unified Parkinson's Disease Rating Scale (UPDRS, r = 0.235, p = 0.018) and 39-item PD questionnaire (PDQ-39, r = 0.250, p = 0.012). The expression level of CSF1R was negatively correlated with the scores of the mini-mental status examination (MMSE, r = -0.200, p = 0.047) and Montréal Cognitive Assessment (MoCA, r = -0.226, p = 0.023). These results highly suggest that oxidative stress biomarkers in peripheral blood may be useful in monitoring the progression of motor disabilities and cognitive decline in PD patients.

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

Intracellular leucine-rich alpha-2-glycoprotein-1 competes with Apaf-1 for binding cytochrome c in protecting MCF-7 breast cancer cells from apoptosis.

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

Apaf1 nanoLuc biosensors identified lentinan as a potent synergizer of cisplatin in targeting hepatocellular carcinoma cells.

Liver cancer is one of the most common malignancies that is difficult to treat due to late diagnosis and chemo-resistance. In the present study, we developed and validated a cell based split nanoLuc biosensor to monitor the Apaf1-Apaf1 interactions in response to apoptosis-inducing drugs such as cisplatin. We showed that the activity of split nanoLuc is reconstituted only in response to apoptotic inducer, cisplatin and in a dose-dependent manner. Apaf1 mutants which were unable to oligomerize failed to recover nanoLuc activity while constitutively active variant increased the nanoLuc activity. Generation of Apaf1 knockout HepG2 and treatment with cisplatin showed dramatic reduction in cell death suggesting that cisplatin mainly targets liver cancer cells through apoptosis. As the natural products are potent sources of compounds for adjuvant therapy, we screened a collection of natural products and identified lentinan as an inducer of apoptosome formation, a key step for induction of apoptosis. Lentinan is a polysaccharide with antitumor, pro-apoptotic properties that functions with poorly understood mechanisms. Lentinan was shown to have cytotoxic effects with the IC50 of 650 µM. Sub-lethal lentinan concentration doubled the nanoLuc activity when co-treated with cisplatin. We also showed that lentinan hugely reduced the dose of cisplatin to induce certain amount of death and that lentinan co-treatment with cisplatin enhanced the Apaf1 transcription in HepG2 cells while lentinan or cisplatin alone failed to alter the transcription. In addition, lentinan and cisplatin co-treatment induced mitochondrial depolarization. This suggested that lentinan combinatorial therapy with cisplatin engaged a different signalling pathway to kill the liver cancer cells and that adjuvant therapy with lentinan can reduce the dose of cisplatin and thus reduce the possibility of chemo-resistance.

Over Fifty Years of Life, Death, and Cannibalism: A Historical Recollection of Apoptosis and Autophagy.

Research in biomedical sciences has changed dramatically over the past fifty years. There is no doubt that the discovery of apoptosis and autophagy as two highly synchronized and regulated mechanisms in cellular homeostasis are among the most important discoveries in these decades. Along with the advancement in molecular biology, identifying the genetic players in apoptosis and autophagy has shed light on our understanding of their function in physiological and pathological conditions. In this review, we first describe the history of key discoveries in apoptosis with a molecular insight and continue with apoptosis pathways and their regulation. We touch upon the role of apoptosis in human health and its malfunction in several diseases. We discuss the path to the morphological and molecular discovery of autophagy. Moreover, we dive deep into the precise regulation of autophagy and recent findings from basic research to clinical applications of autophagy modulation in human health and illnesses and the available therapies for many diseases caused by impaired autophagy. We conclude with the exciting crosstalk between apoptosis and autophagy, from the early discoveries to recent findings.

Solitary Fibrous Tumor: Integration of Clinical, Morphologic, Immunohistochemical and Molecular Findings in Risk Stratification and Classification May Better Predict Patient outcome.

Although solitary fibrous tumors (SFTs) have an unpredictable evolution, some specific clinicopathologic factors have been associated with the final outcome. We retrieved clinical, pathological and molecular data of 97 patients with a histological diagnosis of SFT and Signal transducer and activator of transcription 6 (STAT6) positivity. We retrospectively studied the pathological factors predictive of recurrence/metastasis and compared them with the clinical outcome. A wide immunohistochemical study and molecular analysis to detect NAB2/STAT6 gene fusion, tumor protein-53 (TP53) and/or (telomerase reverse transcriptase) TERT promotor mutation were performed. The risk of metastasis was calculated using the Demicco risk stratification system (RSS). The results were combined and examined to assess the accuracy of risk stratification and classification. The most common location was in non-extremities; 66% were located in soft tissue or subcutaneous areas and 92.8% in deep locations. On microscopic analysis, 38.1% of tumors revealed hypercellularity with a predominant patternless and/or hemangiopericytic growth pattern; 13.4% had ≥4 mitoses/10HPF; 16.5% showed necrosis, and almost half the tumors showed at least focal myxoid areas. Dedifferentiation was observed in three tumors. Immunomarker expression in SFTs was as follows: CD34 92.9%, CD99 57.1%, Bcl2 67.9%, neuroendocrine markers (at least 1) 25.7%, Desmin 14.3%, CK(AE1/AE3) 3%, Apoptotic Protease Activating Factor (APAF-1) 87% and finally Ki-67 ≥ 10% in 14.4%. The NAB2/STAT6 gene fusion was detected in 50 tumors. After a median follow-up of 90 months, 9.3% recurred, 11.3% metastasized, 10.3% died of disease and 76.2% were free of disease. TERT mutations were detected in 40.6% of the SFTs; the TP53 mutation was detected in 17%, and only 9.3% showed both mutations. According to the Demicco RSS, 6.1%, 11.3% and 82.4% of the tumors were classified as high, intermediate or low-risk of metastasis, respectively. All high-risk tumors had ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10, HTER and/or TP53 mutation and poor evolution. The intermediate risk SFTs with worse evolution displayed the HTER mutation. Almost all low-risk tumors had a favorable evolution, although four showed at least one adverse factor (Ki-67 ≥ 10, ≥4 mitoses/10HPF or high tumor size) and had a worse evolution. An integration of clinical, morphologic, immunohistochemical and molecular findings may improve risk stratification and classification and better predict patient outcome. The unfavorable course seems to be more frequent in high-risk SFTs, although it is not exceptional in low-risk SFTs either; hence, a long-term follow-up is required independently of the assigned risk stratification score. The inclusion of molecular findings in risk stratification systems could improve the precision in the classification of SFTs, especially those of intermediate risk. Future studies will be required to determine the most effective way to incorporate molecular analyses into RSS on SFTs. The coexistence of several adverse factors such as ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10%, mutations in HTER and/or p53 may suggest a closer clinical follow-up regardless of the histological appearance of the tumor.

Long Noncoding RNA Hypoxia-Inducible Factor-1 Alpha-Antisense RNA 1 Regulates Vascular Smooth Muscle Cells to Promote the Development of Thoracic Aortic Aneurysm by Modulating Apoptotic Protease-Activating Factor 1 and Targeting let-7g.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a severe threat that is characterized by the increased aortic diameter. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of TAA. Previous research indicated that long noncoding RNAs (lncRNAs) were involved in the development of TAA. This article aimed to explore the role of lncRNA hypoxia-inducible factor-1 alpha-antisense RNA 1 (HIF1A-AS1) and potential action mechanisms in VSMCs. METHODS: The expression of HIF1A-AS1, collagen I, collagen III, microRNA let-7g (let-7g) and apoptotic protease-activating factor 1 (APAF1) was detected by quantitative real-time polymerase chain reaction. Cell proliferation and cell apoptosis were assessed by Cell Counting Kit-8 and flow cytometry assays, respectively. The protein levels of proliferating cell nuclear antigen, Cleaved caspase-3 (Cleaved-cas3), B cell lymphoma/leukemia-2 (Bcl-2), Collagen I, Collagen III, and APAF1 were quantified by Western blot. The relationship between let-7g and HIF1A-AS1 or APAF1 was predicted by the online bioinformatics tool and verified by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: HIF1A-AS1 was upregulated in TAA tissues and was a valuable diagnostic marker of TAA. HIF1A-AS1 overexpression suppressed proliferation, induced apoptosis, and reduced the expression of extracellular matrix proteins in VSMCs. let-7 g was a target of HIF1A-AS1, and its inhibition functioned the same role as HIF1A-AS1 overexpression. APAF1 was a target of let-7g, and its knockdown played the opposite role with HIF1A-AS1 overexpression. The reintroduction of let-7g or APAF1 knockdown reversed the effects of HIF1A-AS1 overexpression in VSMCs. CONCLUSIONS: HIF1A-AS1 regulated the proliferation, apoptosis ,and the activity of extracellular matrix proteins in VSMCs through modulating APAF1 by targeting let-7g, leading to the development of TAA.

Molecular mechanism of C-phycocyanin induced apoptosis in LNCaP cells.

The usefulness of Marine-derived products as the source of anticancer agents has been explored for many decades. The objective of our study was to investigate the molecular mechanism by which C-PC induces apoptosis in monotherapy as well as in combination treatment with a known chemotherapeutic drug named Topotecan (TPT) using prostate cancer cells (LNCaP). To determine the intracellular mechanism of action, we analyzed the gene expression profile of C-PC treated cells using human apoptosis RT2 profiler PCR array, which indicated that C-PC was able to regulate both anti- and pro-apoptotic genes significantly. Detailed analysis revealed increases in the levels of Bax, Apaf-1 (pro-apoptotic proteins) along with the activation of the key apoptotic proteases such as caspase-8, caspase-9, and caspase-3. Similarly, analysis of anti-apoptotic proteins demonstrated a decrease in the expression of Bcl-2, Mcl-1, and survivin. Results from the whole-cell incubation studies indicated that C-PC was only binding to the plasma membrane-associated receptor proteins. LNCaP cells treated with C-PC alone and in combination with TPT showed increased expression of the death receptor FAS (also known as FAS or CD95) along with cleaved PARP, confirming its importance. Our study is significant since it is providing greater insight into the apoptotic mechanisms triggered by C-PC as well as emphasizing the involvement of FAS in mediating its effects. Furthermore, our results with combination treatments suggest that-PC could improve the anticancer effects of drugs such as TPT that are currently used for cancer treatments. In addition, use of C-PC in combination can also diminish the side effects resulting from conventional chemotherapeutic agents such as TPT.

Moringa oleifera leaves ethanolic extract influences DNA damage signaling pathways to protect liver tissue from cobalt -triggered apoptosis in rats.

This study assessed the potential of Moringa oleifera leaves ethanol extract (MLEE) in attenuating the detrimental effects of cobalt dichloride (CoCl2) on rat liver. Forty rats were assigned to five equal groups: control group, MLEE-treated group, CoCl2-treated group, prophylaxis co-treated group, and therapeutic co-treated group. The levels of Co, hepatic injury markers, total antioxidant capacity (TAC), and oxidative stress biomarkers (reactive oxygen species [ROS] and protein carbonyl [PC]) were evaluated. Comet assay was used to evaluate the extent of DNA damage. Further, the expression profile of DNA-damage effector genes was assayed by real-time quantitative polymerase chain reaction (qRT-PCR) analysis. Immunohistochemical analysis of heat shock protein (HSP-70) in hepatocytes was conducted. The results showed that the exposure of CoCl2 to rats resulted in declined TAC, elevated oxidative injury, and induced DNA damage markers. Upregulation of mRNA expression of tumor suppressor protein (P53), apoptosis inducing factor (AIF), and apoptotic peptidase activating factor 1 (Apaf-1) was observed. The immunostaining density of HSP-70 expression was found to be elevated. Thus, MLEE reduced the CoCl2-induced genotoxicity by preventing CoCl2-induced generation of ROS, and protected against ROS mediated-oxidative injury and DNA damage. Moreover, the expression of DNA damage effector genes was affected. Based on these results, we conclude that MLEE is more effective when administered as a prophylactic regimen with the exposure to CoCl2.

TRIAP1 Inhibition Activates the Cytochrome c/Apaf-1/Caspase-9 Signaling Pathway to Enhance Human Ovarian Cancer Sensitivity to Cisplatin.

OBJECTIVE: To investigate whether TRIAP1inhibition affects the ovarian cancer cell resistance to cisplatin (DDP) via the Cyt c/Apaf-1/caspase-9 pathway by in vitro and in vivo experiments. METHODS: CCK8 assay was performed to find out how treatment with both TRIAP1 siRNA and DDP affects the cell viability of SKOV3 cells and DDP-resistant human ovarian carcinoma cell line SKOV3/DDP. SKOV3/DDP cells were transfected with control siRNA or TRIAP1 siRNA before 24 h of treatment with DDP (5 µg/mL). Flow cytometry was employed to detect cell apoptosis and Western blot to examine the expressions of Cyt c/Apaf-1/caspase-9 pathway-related proteins. SKOV3/DDP cells transfected with control siRNA or TRIAP1 siRNA were subcutaneously injected into BALB/c-nu/nu nude mice followed by the intraperitoneal injection of DDP (4 mg/kg). Cyt c/Apaf-1/caspase-9 pathway in transplanted tumors was detected by immunohistochemistry. RESULTS: TRIAP1 expression declined in SKOV3 cells when compared with SKOV3/DDP cells. The proliferation rate was lower in SKOV3/DDP cells transfected with TRIAP1 siRNA combined with treatment of DDP (1, 2, 4, 6, 8, 16, 32 µg/mL) than in those transfected with control siRNA. Moreover, the TRIAP1 siRNA group had an increased SKOV3/DDP cell apoptosis rate with the activation of the Cyt c/Apaf-1/caspase-9 pathway. During DDP treatment, nude mice in TRIAP1 siRNA group had slower growth and smaller size of transplanted tumor than those in control siRNA group, with increased expression of Cyt c, Apaf-1, and caspase-9. CONCLUSION: TRIAP1 inhibition may enhance the sensitivity of SKOV3/DDP cells to cisplatin via activation of the Cyt c/Apaf-1/caspase-9 pathway.

Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome.

Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions.IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.

Apoptosome formation upon overexpression of native and truncated Apaf-1 in cell-free and cell-based systems.

Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network. When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1 molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay was used to compare apoptosome formation in cell-free and cell-based systems. This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal fragment of P. pyralis luciferase. In cell based-system, the apoptosome formation is induced inside the cells which express Apaf-1 tagged with complementary fragments of luciferase while in cell-free system, the apoptosome formation is induced in extracts of the cells. In cell-free system, cytochrome c dependent luciferase activity was observed with full length Apaf-1. However, luciferase activity due to apoptosome formation was much higher in cell based system compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats (ΔApaf-1) interacted with endogenous Apaf-1 in a different fashion compared to native form as confirmed by different retention time of eluate in gel filtration and binding to affinity column. The interactions between endogenous Apaf-1 and ΔApaf-1 is stronger than its interaction with native exogenous Apaf-1 as indicated by dominant negative effect of ΔApaf-1 on caspase-3 processing.

Prognostic impact of Apaf-1, Cyclin D1, and AQP-5 in serous ovarian carcinoma treated with the first-line chemotherapy.

Chemoresistance is the major obstacle to effective treatment in patients with serous ovarian carcinoma (SOC), which frequently related to the failure of chemotherapeutic agents to induce apoptosis. In this study, the immunohistochemical expression of Apaf-1, Cyclin D1, and Aquaporin-5 (AQP-5) was studied in 50 paraffin blocks of SOC. Data on overall survival (OS), disease-free survival (DFS) and response to the first-line chemotherapy were collected and then statistically analyzed. Apaf-1 expression was observed in 84% of the SOC cases with a significant down-regulation with higher tumor grade, lymph node metastasis, and advanced FIGO stage. Cyclin D1 expression was found in 70% of the cases with a significant up-regulation with higher tumor grade, lymph node metastasis, and advanced FIGO stage. Positive AQP-5 expression was noted in 84% of the cases with a significant positive association with higher tumor grade, lymph node metastasis, and advanced FIGO stage. During the follow-up period, the Apaf-1 expression had a significant negative association with OS and DFS (p < 0.001 for each), while both Cyclin D1 and AQP-5 expression had a significant positive association with unfavorable OS and DFS. The cases of SOC treated with suboptimal surgery revealed a significant association of low Apaf-1, high Cyclin D1, and strong AQPs with the poor response to the first-line chemotherapy (p = 0.047, p < 0.001, and 0.006 respectively). CONCLUSIONS: Down-regulation of Apaf-1 protein and the overexpression of Cyclin D1 and AQP-5 proteins possibly contribute to an aggressive SOC with a high risk of recurrence and poor response to the first-line chemotherapy.

A multi-target protein of hTERTR-FAM96A presents significant anticancer potent in the treatment of hepatocellular carcinoma.

The abilities to escape apoptosis induced by anticancer drugs are an essential factor of carcinogenesis and a hallmark of resistance to cancer therapy. In this study, we identified hTERTR-FAM96A (human telomerase reverse transcriptase-family with sequence similarity 96 member A) as a new efficient agent for apoptosome-activating and anti-tumor protein and investigated the potential tumor suppressor function in hepatocellular carcinoma. The hTERTR-FAM96A fusion protein was constructed by genetic engineering and its anticancer function of hTERTR-FAM96A was explored in vitro and in vivo by investigating the possible preclinical outcomes. Effects of hTERTR-FAM96A on improvement of apoptotic sensitivity and inhibition of migration and invasion were examined in cancer cells and tumors. Our results showed that the therapeutic effects of hTERTR-FAM96A were highly effective for inhibiting tumor growth and inducing apoptosis of hepatocellular carcinoma cells in H22-bearing nude mice. The hTERTR-FAM96A fusion protein could specifically bind with Apaf-1 and hTERT, which further induced apoptosis of hepatocellular carcinoma cells and improved apoptosis sensitivity. Our results indicated that hTERTR-FAM96A treatment enhanced cytotoxic effects by upregulation of cytotoxic T lymphocyte responses, interferon-γ release, and T lymphocyte infiltration. In addition, hTERTR-FAM96A led to tumor-specific immunologic cytotoxicity through increasing apoptotic body on hepatocellular tumors. Furthermore, hTERTR-FAM96A dramatically inhibited tumor growth, reduced death rate, and prolonged mice survival in hepatocellular carcinoma mice derived from three independent hepatocellular carcinoma mice cohorts compared to control groups. In summary, our data suggest that hTERTR-FAM96A may serve as an efficient anti-tumor agent for the treatment of hepatocellular carcinoma.

The anti-cancer effects of Tualang honey in modulating breast carcinogenesis: an experimental animal study.

BACKGROUND: Honey has been shown to have anti-cancer effects, but the mechanism behind these effects is not fully understood. We investigated the role of Malaysian jungle Tualang honey (TH) in modulating the hematological parameters, estrogen, estrogen receptors (ER1) and pro and anti-apoptotic proteins expression in induced breast cancer in rats. METHODS: Fifty nulliparous female Sprague-Dawley rats were used and grouped as follows: Group 0 (healthy normal rats control), Group 1 (negative control; untreated rats), Groups 2, 3 and 4 received daily doses of 0.2, 1.0 and 2.0 g/kg body weight of TH, respectively. The rats in groups 1, 2, 3, 4 were induced with 80 mg/kg of 1-methyl-1-nitrosourea (MNU). TH treatment in groups 2, 3 and 4 was started one week prior to tumor induction and continued for 120 days. RESULTS: The TH-treated rats had tumors of different physical attributes compared to untreated negative control rats; the tumor progression (mean 75.3 days versus 51.5 days); the incidence (mean 76.6% versus 100%); the multiplicity (mean 2.5 versus 4 tumor masses per rat); the size of tumor mass (mean 0.41 cm versus 1.47 cm [p < 0.05]) and the weight of the tumor mass (mean 1.22 g versus 3.23 g; [p < 0.05]). Histological examinations revealed that cancers treated with TH were mainly of grades I and II compared with the non-treated control, in which the majority were of grade III (p < 0.05). TH treatment was found to modulate hematological parameters such as Hb, RBCs, PCV, MCV, RDW, MCHC, polymorphs and lymphocytes values. TH treatment groups were found to have a lower anti-apoptotic proteins (E2, ESR1 and Bcl-xL) expression and a higher pro-apoptotic proteins (Apaf-1 and Caspase-9) expression at serum and on cancer tissue level (p < 0.05). CONCLUSION: Tualang Honey alleviates breast carcinogenesis through modulation of hematologic, estrogenic and apoptotic activities in this experimental breast cancer animal model. Tualang Honey may be used as a natural 'cancer-alleviating' agent or as a supplement to chemotherapeutic agents.

Role of Methylation in the Regulation of Apoptosis Genes APAF1, DAPK1, and BCL2 in Breast Cancer.

Changes in the levels of expression of proapoptotic genes APAF1 and DAPK1 and antiapoptotic gene BCL2 were studied by real time PCR in specimens of tumors and histologically intact tissue from 28 patients with breast cancer. The expression of APAF1 and DAPK1 was below the normal in the majority of tumor samples (p<0.05), while the level of BCL2 mRNA more often surpassed the normal (p<0.1). Study of the same sample of specimens by methylspecific PCR showed predominance of APAF1 and DAPK1 hypermethylation (p<0.05 and p<0.1, respectively) and more frequent hypomethylation of BCL2. A significant correlation between changes in the levels of expression and methylation (r=0.40-0.49; p<0.05) was detected for all three genes (APAF1, DAPK1, and BCL2). The results suggest that methylation play an important role in the regulation of these apoptosis system genes in breast cancer.