Apoptosis Protease-activating Factor Research Articles

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.

LncRNA UCA1 epigenetically suppresses APAF1 expression to mediate the protective effect of sevoflurane against myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MI/RI) is a leading cause of death globally. Whereas some long noncoding RNAs (lncRNAs) are known to participate in the progression of MI/RI, the role of urothelial carcinoma associated 1 (UCA1) in conjunction with sevoflurane treatment remains largely unknown. H9C2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to establish an in vitro MI/RI model, and sevoflurane was then added. Cell viability, apoptosis, SOD activity, and MDA levels were measured. Levels of inflammatory cytokines and methylation of apoptosis protease-activating factor 1 (APAF1) were determined. Interactions among lncRNA UCA1, enhancer of zeste homologue 2 (EZH2), DNA methyltransferase-1 (DNMT1), and APAF1 were analyzed. After H/R treatment, the viability of H9C2 cardiomyocytes decreased and apoptosis rate, oxidative stress factor levels, inflammatory cytokine levels, and apoptosis-related protein levels all increased. Sevoflurane treatment reversed these changes. LncRNA UCA1 knockdown attenuated the therapeutic effect of sevoflurane on H/R-treated cardiomyocytes, and silencing of APAF1 reversed this role of UCA1 knockdown. Moreover, lncRNA UCA1 recruited DNMT1 through EZH2, thus promoting methylation of the APAF1 promoter region. LncRNA UCA1 recruits DNMT1 to promote methylation of the APAF1 promoter through EZH2, thus strengthening the protective effect of sevoflurane on H/R-induced cardiomyocyte injury.

Response to Different Oxygen Partial Pressures and Evolution Analysis of Apoptosis-Related Genes in Plateau Zokor (Myospalax baileyi)

The plateau zokor (Myospalax baileyi) is a native species of the Qinghai–Tibet Plateau that spends its entire life underground in sealed burrows with hypoxic conditions. The present study aimed to assess the sequence characteristics of apoptosis-related genes and the response to different oxygen partial pressures (pO2) in plateau zokor and Sprague-Dawley rats. The sequences of the p53-induced protein with a death domain (Pidd), p53-upregulated modulator of apoptosis (Puma), insulin-like growth factor binding protein 3 (Igfbp3), and apoptosis protease-activating factor 1 (Apaf1) were evaluated concerning homology and convergent evolution sites, and their mRNA levels were evaluated in different tissues under 14.13 (3,300 m) and 16.12 kPa (2,260 m) pO2 conditions. Our results showed that, (1) the sequences of the apoptosis-related genes in plateau zokor were highly similar to those of Nannospalax galili, followed by Rattus norvegicus; (2). Pidd, Puma, Igfbp3, and Apaf1 of plateau zokor were found to have five, one, two, and five convergent sites in functional domains with N. galili, respectively. Lastly (3), under low pO2, the expression of Pidd and Puma was downregulated in the lung of plateau zokors. In turn, Igfbp3 and Apaf1 were upregulated in the liver and lung, and Puma was upregulated in the skeletal muscle of plateau zokor under low pO2. In Sprague-Dawley rats, low pO2 downregulated Puma and Apaf1 expression in the liver and downregulated Igfbp3 and Puma in the lung and skeletal muscle separately. In contrast, low pO2 upregulated Pidd expression in the liver and skeletal muscle of Sprague-Dawley rats. Overall, the expression patterns of Apaf1, Igfbp3, and Puma showed the opposite pattern in the liver, lung, and skeletal muscle, respectively, of plateau zokor as compared with Sprague-Dawley rats. In conclusion, for the long-time adaptation to hypoxic environments, Pidd, Puma, Igfbp3, and Apaf1 of plateau zokor underwent convergent evolution, which we believe may have led to upregulation of their levels under low oxygen partial pressures to induce apoptosis, so as to suppress tumorigenesis under hypoxic environments in plateau zokor.

Rapamycin maintains the primordial follicle pool and protects ovarian reserve against cyclophosphamide-induced damage.

Any abnormal activation of primordial follicles and subsequent depletion can irreversibly diminish the ovarian reserve, which is one of the major chemotherapy-induced adverse effects in young patients with cancer. Herein, we investigated the effects of rapamycin on the activation and development of ovarian follicles to evaluate its fertility-sparing therapeutic value in a cyclophosphamide (CTX)-treated mouse model. Based on ovarian histomorphological changes and follicle counting in 50 SPF female C57BL/6 mice, daily administration of 5 mg/kg rapamycin for 30 days was deemed an ideal dosage and duration for administration in subsequent experiments. Compared with the control group, rapamycin treatment inhibited the activation of quiescent primordial follicles, with no obvious side effects observed. Finally, 48 mice were randomly divided into four groups: control, rapamycin-treated, cyclophosphamide-treated, and rapamycin intervention. Body weight, ovarian histomorphological changes, number of primordial follicles, DDX4/MVH expression, apoptosis of follicular cells, and expression of apoptosis protease-activating factor (APAF)-1, cleaved caspase 3, and caspase 3 were monitored. Co-administration of rapamycin reduced primordial follicle loss and the development of follicular cell apoptosis, thereby rescuing the ovarian reserve after CTX treatment. On analyzing the mTOR signaling pathway, we observed that rapamycin significantly decreased CTX-mediated overactivation of mTOR and its downstream molecules. These findings suggest that rapamycin exhibits potential as an ovarian-protective agent that could maintain the ovarian primordial follicle pool and preserve fertility in young female patients with cancer undergoing chemotherapy.

Apoptotic effect of Physalis minima Linn ethanol extract on breast cancer cells via p53 wild-type and Apaf-1 protein

Purpose: To produce an anti-cancer agent from Physalis minima ethanol extract as well as prevent the growth of NMU-induced breast cancer and MCF-7 cell line.Methods: This research used an animal model (Wistar-Furth rats), and cell line used in this study was normal breast-cell line MCF-7. The rats were administered with the ethanol extract of Physalis minima Linn (100, 250 and 400 mg/kg/day) by gavage, once a day for 4 weeks. Meanwhile, MCF-7 cell lines were cultured in medium and incubated in 100 μg/mL of ethanol extract of Physalis minima for 48 h. The samples were analyzed using histology and immunohistochemistry techniques for expression of p53 antibody DO-1 and APAF-1.Results: The results of immunohistological analysis in the breast organ showed that Physalis minima Linn extract significantly (p < 0.05) increased the tumor suppressor protein p53 at doses of 100, 250 and 400 mg/kg/day. In addition, the extract also significantly (p < 0.05) increased APAF-1, which is a gene determining cell death, at doses of 100, 200 and 400 mg/day.Conclusion: Ethanol extract of Physalis minima Linn inhibits the cytotoxic activity of NMU-induced breast cancer by increasing the tumor suppressor protein p53 and APAF-1. Thus, Physalis minima Linn extract can potentially be used as a complementary treatment for inhibiting the growth of breast cancer cells in patients.
 Keywords: Apoptosis protease-activating factor-1, Breast cancer, MCF-7 cell line, Physalis minima, p53

Bmapaf-1 is Involved in the Response against BmNPV Infection by the Mitochondrial Apoptosis Pathway

Simple SummaryApaf-1 is involved in the apoptosis pathway and Bmapaf-1 showed a significant response to BmNPV infection in our previous transcriptome data. In this study, the underlying mechanism of Bmapaf-1 in response to BmNPV infection was studied. To preliminarily determine the relationship of Bmapaf-1 with BmNPV, the expression pattern of Bmapaf-1 was analyzed in different tissues of differentially resistant silkworm strains following virus infection. To further define the role of Bmapaf-1 in BmNPV infection, the alteration of BmNPV infection in BmN cells and the expression patterns of Bmcas-Nc and Bmcas-1 were analyzed following knockdown and overexpression of Bmapaf-1 using siRNA and the pIZT/V5-His-mCherry insect vector, respectively. Furthermore, to analyze whether Bmapaf-1 is involved in BmNPV infection by apoptosis, the inducer NSC348884 and inhibitor Z-DEVD-FMK were used.Discovery of the anti-BmNPV (Bombyx mori nuclearpolyhedrovirus) silkworm strain suggests that some kind of antiviral molecular mechanism does exist but is still unclear. Apoptosis, as an innate part of the immune system, plays an important role in the response against pathogen infections and may be involved in the anti-BmNPV infection. Several candidate genes involved in the mitochondrial apoptosis pathway were identified from our previous study. Bombyx mori apoptosis protease-activating factor-1 (Bmapaf-1) was one of them, but the antiviral mechanism is still unclear. In this study, sequences of BmApaf-1 were characterized. It was found to contain a unique transposase_1 functional domain and share high CARD and NB-ARC domains with other species. Relatively high expression levels of Bmapaf-1 were found at key moments of embryonic development, metamorphosis, and reproductive development. Further, the significant difference in expression of Bmapaf-1 in different tissues following virus infection indicated its close relationship with BmNPV, which was further validated by RNAi and overexpression in BmN cells. Briefly, infection of budded virus with enhanced green fluorescent protein (BV-EGFP) was significantly inhibited at 72 h after overexpression of Bmapaf-1, which was confirmed after knockdown of Bmapaf-1 with siRNA. Moreover, the downstream genes of Bmapaf-1, including Bmnedd2-like caspase (BmNc) and Bmcaspase-1 (Bmcas-1), were upregulated after overexpression of Bmapaf-1 in BmN cells, which was consistent with the RNAi results. Furthermore, the phenomenon of Bmapaf-1 in response to BmNPV infection was determined to be related to apoptosis using the apoptosis inducer NSC348884 and inhibitor Z-DEVD-FMK. Therefore, Bmapaf-1 is involved in the response against BmNPV infection by the mitochondrial apoptosis pathway. This result provides valuable data for clarifying the anti-BmNPV mechanism of silkworms and breeding of resistant silkworm strains.

Copper Induces Oxidative Stress and Apoptosis in the Mouse Liver

Copper (Cu) is an essential trace element involved in the normal physiological processes of animals. However, excessive exposure to Cu can produce numerous detrimental impacts. The aim of this study was to investigate the effects of Cu on oxidative stress and apoptosis as well as their relationship in the mouse liver. Four-week-old ICR mice (n = 240) were randomly assigned to different Cu (Cu2+-CuSO4) treatment groups (0, 4, 8, and 16 mg/kg) for periods of 21 and 42 days. The high doses of Cu exposure could induce oxidative stress, by increasing the levels of reactive oxygen species (ROS) and protein carbonyls (PC) and decreasing the activities of antisuperoxide anion (ASA) and antihydroxyl radical (AHR) and content of glutathione (GSH), as well as activities and mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Moreover, high doses of Cu exposure induced hepatic apoptosis via the mitochondrial apoptotic pathway, as characterized by the depolarization of mitochondrial membrane potential (MMP); significantly increased mRNA and protein expression levels of cytosolic cytochrome (Cyt c), apoptosis-inducing factor (AIF), endonuclease G (Endo G), apoptosis protease-activating factor-1 (Apaf-1), cleaved caspase-9, cleaved caspase-3, cleaved PARP, Bcl-2 antagonist killer (Bak), Bcl-2-associated X protein (Bax), and Bcl-2-interacting mediator of cell death (Bim); and decreased mRNA and protein expression levels of B-cell lymphoma-2 (Bcl-2) and Bcl-extra-large (Bcl-xL). Furthermore, the activation of the tumor necrosis factor receptor-1 (TNF-R1) signaling pathway was involved in Cu-induced apoptosis, as characterized by the significantly increased mRNA and protein expression levels of TNF-R1, Fas-associated death domain (FADD), TNFR-associated death domain (TRADD), and cleaved caspase-8. These results indicated that exposure to excess Cu could cause oxidative stress triggered by ROS overproduction and diminished antioxidant function, which in turn promoted hepatic apoptosis via mitochondrial apoptosis and that the TNF-R1 signaling pathway was also involved in the Cu-induced apoptosis.

Gastroprotective effect of araloside A on ethanol- and aspirin-induced gastric ulcer in mice: involvement of H+/K+-ATPase and mitochondrial-mediated signaling pathway.

The aim of this study was to elucidate the gastroprotective activity and possible mechanism of involvement of araloside A (ARA) against ethanol- and aspirin-induced gastric ulcer in mice. The experimental mice were randomly divided into control, model, omeprazole (20mg/kg, orally) and ARA (10, 20 and 40mg/kg, orally). Gastric ulcer in mice was induced by intragastric administration of 80% ethanol (10mL/kg) containing 15mg/mL aspirin 4h after drug administration on day 7. The results indicated that ARA could significantly raise gastric juice volume and acidity; ameliorate gastric mucosal blood flow, gastric binding mucus volume, ulcer index and ulcer inhibition rate; suppress H+/K+-ATPase activity, which was confirmed by computer-aided docking simulations; inhibit the release of mitochondrial cytochrome c into the cytoplasm; inhibit caspase-9 and caspase-3 activities and down-regulate mRNA expression levels; down-regulate the mRNA and protein expressions of apoptosis protease-activating factor-1 and protein expression of cleaved poly(ADP ribose) polymerase-1; and up-regulate Bcl-2 mRNA and protein expressions and down-regulate Bax mRNA and protein expressions, thus elevating the Bcl-2/Bax ratio in a dose-dependent manner. Histopathological observations further provided supportive evidence for the aforementioned results. The results demonstrated that ARA exerted beneficial gastroprotective effects on alcohol- and aspirin-induced gastric ulcer in mice, which was related to suppressing H+/K+-ATPase activity as well as pro-apoptotic protein expression, and promoting anti-apoptotic protein expression, thus alleviating gastric mucosal injury and cell death.

Propofol attenuates cytokine-mediated upregulation of expression of inducible nitric oxide synthase and apoptosis during regeneration post-partial hepatectomy.

To determine the effects of propofol and ketamine anesthesia on liver regeneration in rats after partial hepatectomy (PHT). Male Wistar albino rats were assigned randomly to four groups of 10. Anesthesia was induced and maintained with propofol in groups 1 and 2, and with ketamine in groups 3 and 4. PHT was undertaken in groups 1 and 3. Rats in groups 2 and 4 (control groups) underwent an identical surgical procedure, but without PHT. At postoperative day-5, rats were killed. Regenerated liver was removed, weighed, and evaluated (by immunohistochemical means) for expression of inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), apoptosis protease-activating factor (APAF)-1, and proliferating cell nuclear antigen (PCNA). Also, blood samples were collected for measurement of levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Between groups 2 and 4, there were no differences in tissue levels of iNOS, eNOS, and APAF-1 or plasma levels of TNF-α and IL-6. eNOS expression was similar in group 1 and group 3. Expression of iNOS and APAF-1 was mild-to-moderate in group 1, but significantly higher in group 3. Groups 1 and 3 showed an increase in PCNA expression, but expression in both groups was comparable. Plasma levels of TNF-α and IL-6 increased to a lesser degree in group 1 than in group 3. Propofol, as an anesthetic agent, may attenuate cytokine-mediated upregulation of iNOS expression and apoptosis in an animal model of liver regeneration after partial hepatectomy.

Overexpression of miRNA-221 promotes cell proliferation by targeting the apoptotic protease activating factor-1 and indicates a poor prognosis in ovarian cancer.

MicroRNAs are a class of small non-coding, endogenous RNAs involved in cancer development and progression. MicroRNA-221 (mir-221) has been reported to have both an oncogenic and tumor-suppressive role in human tumors, but the role of miR-221 in ovarian cancer is poorly understood. In the present study, the expression levels of miR-221 and the apoptosis protease activating factor 1 (APAF1) protein in 63 samples of ovarian cancer tissues and the cell lines, IOSE25, A2780, OVCAR3, SKOV3 and 3AO were detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Cell proliferation was measured using Cell Counting kit-8 (CCK-8); cell migration and invasion were detected using a Transwell assay; cell apoptosis was evaluated by flow cytometry and Hoechst staining, and a luciferase assay was performed to verify a putative target site of miR-221 in the 3′-UTR of APAF1 mRNA. Expression of miR-221 was upregulated in ovarian cancer tissues. Patients with increased miR-221 expression levels had a reduced disease-free survival (P=0.0014) and overall survival (P=0.0058) compared with those with low miR-221 expression. Transfection of SKOV3 and A2780 cell lines with miR-221 inhibitor induced APAF1 protein expression, suppressed cell proliferation and migration and promoted tumor cell apoptosis. In conclusion, the APAF1 gene was confirmed as a direct target of miR-221 and overexpression of APAF1 suppressed ovarian cancer cell proliferation and induced cell apoptosis in vitro. These findings indicate that miR-221-APAF1 should be studied further as a potential new diagnostic or prognostic biomarker for ovarian cancer.

Isobaric tags for relative and absolute quantitation analysis on serum markers of pancreatic cancer based on mass spectrometry

Objective To determine the expression of serum proteins in pancreatic cancer patients based on mass spectrometry to screen differential proteins and to find potential molecular biomarkers to prediagnosis of pancreatic cancer. Methods The technique of isobaric tags for relative and absolute quantitation (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) were used to analyze the serum proteins in 15 pancreatic cancer patients and 10 healthy controls. Screening for serum differential proteins was performed via retrieving Panther, Mascot and Scaffold databases. Ingenuity Pathway Analysis was used to detect the correlation between proteins. Results The expression level of 442 proteins was quantified, and 76 proteins were found to be differentially expressed which changed corresponding biological process. The up-regulation of DNA repair protein 50 (RAD50) and down-regulation of transforming growth factor β1 (TGFβ1) and apoptosis protease-activating factor 1 (APAF1) which were correlated with cell growth and apoptosis were found in pancreatic cancer. A closely interacted network was formed between these three differential proteins and other molecules, which could effect metabolism in pancreatic cancer patients. Conclusion Differential expression of serum proteins screened by iTRAQ in pancreatic cancer patients may be the potential markers of pancreatic cancer, which can provide molecular basis for early diagnosis of pancreatic cancer. Key words: Pancreatic neoplasms; Tumor markers, biological; Isobaric tags for relative and absolute quantitation; Proteomics; Serum

Promoter Methylation and mRNA Expression of APAF-1 Gene in Breast Cancer

Background: Apoptosis protease-activating factor-1 (APAF-1) is an important tumor suppressor gene, which plays a central function in DNA damage-induced apoptosis. The present study aimed to examine the epigenetic regulation of APAF-1 in breast cancer to gain a better understanding of tumor biology. Methods: The promoter methylation of APAF-1 gene was determined by methylation-specific polymerase chain reaction (MS-PCR) method, using 63 paraffin-embedded breast tumors and their corresponding normal tissues as the control tissue samples. Expression analysis was performed on all tissue samples, using quantitative PCR (qPCR) method. Results: Significant down-regulation was observed for APAF-1 expression among tumor tissues, compared to the control group (P = 0.011). APAF-1 promoter hypermethylation was more frequent in breast tumors in comparison with normal tissues (57% vs. 21%; P = 0.001) and was correlated with gene mRNA level (P < 0.05). Methylation level increased from the primary to the advanced stage of the disease (P = 0.001), which suggests the possible role of APAF-1 methylation in disease progression. Conclusions: Methylation is an important epigenetic factor which might contribute to the down-regulation of APAF-1 gene in breast tumor tissues.

Decreased expression of long non-coding RNA GAS5 promotes cell proliferation, migration and invasion, and indicates a poor prognosis in ovarian cancer.

Long non-coding RNA growth arrest-specific 5 (GAS5) was reported to be aberrantly expressed in various types of cancers. However, the role of GAS5 in the evolution and progression of ovarian cancer remains elusive. In the present study, we aimed to investigate the cellular function and clinical significance of GAS5 in ovarian cancer. GAS5 expression levels in 63ovarian cancer tissues were detected by quantitative real-time PCR. Cell Counting Kit-8 (CCK-8) assay was performed to analyze the effect of GAS5 on cell proliferation. The effect of GAS5 on cell migration and invasion was detected using Transwell assay. Cell apoptosis was evaluated by flow cytometry and Hoechst staining. SKOV3 cells with stable expression of GAS5 were injected into nude mice to study the effect of GAS5 on tumorigenesis invivo. Western blotting was used to determine the protein levels of GAS5 potential targets. The results showed that GAS5 was markedly decreased in tumor tissues and a lower expression of GAS5 was detected in tumors with larger size, deeper invasive depth and higher tumor stage. Patients with low GAS5 expression level had poorer disease-free (P<0.0001) and overall survival (P=0.0016) than those with high GAS5 expression. Moreover, overexpression of GAS5 was demonstrated to suppress ovarian cancer cell proliferation invitro and invivo. Finally, we found that GAS5 influenced ovarian cancer cell proliferation, partly via regulating cyclinD1, p21 and apoptosis protease activating factor1 (APAF1) expression. Our findings suggest that lncRNA GAS5 may represent a novel indicator of poor prognosis in ovarian cancer and may be a potential therapeutic target for diagnosis and therapy.

Potential role of microRNA-10b down-regulation in cardiomyocyte apoptosis in aortic stenosis patients.

MicroRNAs have been associated with cardiomyocyte apoptosis, a process involved in myocardial remodelling in aortic valve (Av) stenosis (AS). Our aim was to analyse whether the dysregulation of myocardial microRNAs was related to cardiomyocyte apoptosis in AS patients. Endomyocardial biopsies were obtained from 28 patients with severe AS (based on pressure gradients and Av area) referred for Av replacement and from necropsies of 10 cardiovascular disease-free control subjects. AS patients showed an increased (P<0.001) cardiomyocyte apoptotic index (CMAI) compared with controls. Two clusters of patients were identified according to the CMAI: group 1 (CMAI ≤ 0.08%; n=16) and group 2 (CMAI > 0.08%; n=12). Group 2 patients presented lower cardiomyocyte density (P<0.001) and ejection fraction (P<0.05), and higher troponin T levels (P<0.05), prevalence of heart failure (HF; P<0.05) and NT-proBNP levels (P<0.05) than those from group 1. miRNA expression profile analysed in 5 patients randomly selected from each group showed 64 microRNAs down-regulated and 6 up-regulated (P<0.05) in group 2 compared with group 1. Those microRNAs with the highest fold-change were validated in the full two groups corroborating that miR-10b, miR-125b-2* and miR-338-3p were down-regulated (P<0.05) in group 2 compared with group 1 and control subjects. These three microRNAs were inversely correlated (P<0.05) with the CMAI. Inhibition of miR-10b induced an increase (P<0.05) of apoptosis and increased expression (P<0.05) of apoptosis protease-activating factor-1 (Apaf-1) in HL-1 cardiomyocytes. In conclusion, myocardial down-regulation of miR-10b may be involved in increased cardiomyocyte apoptosis in AS patients, probably through Apaf-1 up-regulation, contributing to cardiomyocyte damage and to the development of HF.

Comparison of combined antioxidants and thymoquinone in the prevention of testis ischemia - reperfusion injury.

We aimed to compare the preventive effects of combined antioxidants (CA1, 2) with a single antioxidant drug (thymoquinone; TQ) on experimental testis Ischemia/Reperfusion (I/R) injury. Thirty-five adult male Wistar rats were divided into five groups of seven rats each: control, testis I/R, testis I/R+CA1, testis I/R+CA2, and testis I/R+TQ. After 1h of testicular ischemia, reperfusion was achieved by detorsion for 4h. Antioxidants were intraperitoneally administered for 30min prior to reperfusion. All rats were sacrificed 4h after reperfusion to evaluate the tissue levels of malondialdehyde (MDA) and total antioxidant status (TAS) and the immunohistochemical evaluation of tissue inducible and endothelial nitric acid synthase (iNOS, eNOS) and apoptosis protease-activating factor 1 (APAF-1). MDA levels were lower and TAS values were higher in the I/R+antioxidant groups than in the I/R group (p<0.05). iNOS and eNOS levels in the I/R+antioxidant groups were also lower than those in the I/R group (p<0.05). There were no significant differences between the CA groups and the TQ group according to aforementioned parameters. In addition, tissue APAF-1 values were significantly higher in the I/R group than in the other groups. However, there was a significant difference between the TQ and CA groups in APAF-1 levels, which were highest in the TQ group (p<0.05). Although TQ alone increased TAS values and reduced tissue iNOS and eNOS levels, combined antioxidant treatment may more effectively reduce apoptosis and increase preventive effects in testis I/R injury.

The effects of tadalafil and pentoxifylline on apoptosis and nitric oxide synthase in liver ischemia/reperfusion injury

The aim of this study was to investigate the effects of tadalafil (TDF) and pentoxifylline (PTX) on hepatic apoptosis and the expressions of endothelial and inducible nitric oxide synthases (eNOS and iNOS) after liver ischemia/reperfusion (IR). Forty Wistar albino rats were randomly divided into five groups (n=8) as follows: sham group; IR group with ischemia/reperfusion alone; low-dose and high-dose TDF groups received 2.5 mg/kg and 10 mg/kg TDF, respectively; and PTX group received 40 mg/kg PTX. Blood was collected for the analysis of serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, uric acid, malondialdehyde (MDA), and total antioxidant capacity (TAC). MDA and TAC also were measured in liver tissue. Histopathological examination was performed to assess the severity of hepatic injury. Apoptosis was evaluated using the apoptosis protease-activating factor 1 (APAF-1) antibody; the expressions of eNOS and iNOS were also assessed by immunohistochemistry in all groups. Serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, uric acid, MDA, and TAC, tissue MDA and TAC levels, hepatic injury, and score for extent and for intensity of eNOS, iNOS, and apoptosis protease-activating factor 1 were significantly different in TDF and PTX groups compared to the IR group. High dose-TDF and PTX have the best protective effect on IR-induced liver tissue damage. This study showed that TDF and PTX supplementation may be helpful in preventing free oxygen radical damage, lipid peroxidation, hepatocyte necrosis, and apoptosis in liver IR injury and minimizing liver damage.

Studies on tumor suppressor gene apoptosis protease-activating factor1, ARF tumor suppressor , RUNT-related transcription factor 3 methylation in tissue of colorectal cancer

Objective To investigate abnormal promoter methylation status of apoptotic protease activating factor-1 (APAF1), ARF tumor suppressor (p14ARF) and RUNT-related transcription factor 3 (RUNX3) genes and analyze the value of these genes in the diagnosis of colorectal cancer. Methods Methylation specific polymerease chain reaction (MSP) was used to detect the methylation status of APAF1, p14ARF and RUNX3 genes in 70 cases of colorectal cancer tissues. Results The methylation rate of APAF1, p14ARF and RUNX3 in tumor tissue of colorectal cancer was 78.5%, 54.2% and 47.1% respectively. RUNX3 and APAF1 genes were highly expressed in pathological stage Ⅱ, and p14ARF and APAF1 in pathological stage Ⅲ. Aberrant methylation rate of APAF1, p14ARF and RUNX3 genes in colorectal cancer was more significantly different than the polyp of colon (P<0.05). Conclusion Aberrant methylation status of APAF1, p14ARF and RUNX3 is closely related with incidence of colorectal cancer. Key words: Colorectal cancer; Tumor suppressor gene; Methylation specific polymerease chain reaction

Expression Pattern and Regulatory Role of microRNA-23a in Conjugated Linoleic Acids-Induced Apoptosis of Adipocytes

Background/Aims: Conjugated linoleic acids (CLAs) are known to induce apoptosis in adipocytes; however, the cellular mechanisms involved remained illdefined. We explored the different apoptotic induction effects of two CLA isomers on adipocytes and then investigated the expression and function of microRNAs (miRNAs) related to the apoptosis. Methods: TUNEL and FCM assays were used to detect CLAs-induced adipocyte apoptosis. Microarrays were used to compare the differential expression of miRNAs. MiR-23a, a miRNA that showed significant changes in expression in the CLA-treated cells, was selected for the subsequent functional studies via over-expression and knock down in in vivo and in vitro experiments. Results: C9, t11-CLA exhibited a stronger induction of apoptosis in the differentiated 3T3-L1 adipocytes than t10, c12-CLA. However, t10, c12-CLA could rapidly activate NF-κB, which may have caused their different apoptotic effects. MiR-23a was markedly down-regulated by the CLAs treatment and miR-23a over-expression attenuated CLA-induced apoptosis. Apoptosis protease-activating factor 1 (APAF1) was identified as a target gene of miR-23a. In an in vivo experiment endogenous miR-23a was down-regulated in mice fed with a mixture of both CLAs. The mice also exhibited less fat deposition and more apoptotic fat cells in adipose tissue. Moreover, endogenous miR-23a was suppressed in mice via intravenous injection with an antagomir which resulted in decreased body weight, increased number of apoptotic fat cells and increased APAF1 expression in adipose tissue. Conclusion: Taken together, our results suggest that miR-23a plays a critical role in CLA-induced apoptosis in adipocytes via controlling APAF1 expression.

Abstract 1017: Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines

Abstract Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, providing a selective advantage for tumor cell survival, due in part, to its ability to inhibit cell death via APAF1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis. Methods: The expression of HSP70 and TRIAP1 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1) - three independent transductions, in triplicate. Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry with annexin V and propidium iodide (PI) staining. We also evaluated APAF1 and Caspase9 genes expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results: The cell lines RPMI8226 (without deletion of TP53 by FISH) and U266 (deletion of one allele of TP53 by FISH) were chosen for the transduction experiments because they showed significant levels of expression of HSP70 and TRIAP1. The efficiency of transduction, with GFP reporter gene, was 70% for both cell lines. Silencing of both genes was confirmed by qPCR and Western blotting. Inhibition of TRIAP1 increased the percentage of cells in late apoptosis (p&amp;lt;0.001 for RPMI8226 and P&amp;lt;0.01 for U266) and was accompanied by increased expression of Caspase9 in both MM cell lines (p&amp;lt;0.001 for RPMI8226 and p&amp;lt;0.05 for U266). Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines (p&amp;lt;0.01 for RPMI8226 and p&amp;lt;0.05 for U266) and these results were confirmed by increased activity of Caspase3/7 (p&amp;lt;0.01 for RPMI8226 and p&amp;lt;0.05 for U266). Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact of this method on the induction of late apoptosis, through APAF1/Caspase9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever FISH status for TP53 (Support by FAPESP 2010/17668-6). Citation Format: Veruska Lia Fook Alves, Daniela B. Zanatta, mariana bleker oliveira, angela isabel pereira eugenio, rodrigo carlini fernando, Bryan E. Strauss, Gisele Wally Braga Colleoni. Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2015-1017

Silencing of HSP70 and TRIAP1 Genes in Multiple Myeloma Cell Lines Induces Apoptosis through Apaf-1/Caspase 9 Pathway: New Potential Targets on the Way?

Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, and that high expression of this chaperone is linked with increasing tumor grade and/or poor prognosis. The overexpression of HSP70 may provide a selective advantage for tumor cell survival, due in part, to its ability in inhibiting cell death via APAF-1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Some studies have shown that HSP70 gene silencing using antisense RNA, abundantly induced cell death in breast cancer lines and it was not toxic to normal breast epithelial cells or human fibroblasts. This cell death was not dependent on P53status or inhibited by Bcl2 pathway. Other studies using HSP70 antisense RNA have also indicated apoptosis of tumor cells in lung cancer, oral cavity, colon, prostate, liver, and brain cell lines. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM).Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis.Methods: The expression of TRIAP1 and HSP70 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1). Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry after annexin V and propidium iodide (PI) staining. We also evaluated APAF-1 and Caspase 9 gene expression by qPCR and Caspase 9 and Caspase 3/7 protein activity.Results: The cell lines RPMI8226 (without deletion of P53 by FISH) and U266 (deletion of one allele of P53 by FISH) were chosen for the transduction experiments because they showed relevant expression of TRIAP1 and HSP70. The efficiency of transduction, as measured in both cell lines transduced with the pLKO vector containing the GFP reporter gene, was 70%, demonstrating that there would be no technical restriction to perform this experiment. RPMI8226 and U266 were submitted to three independent transductions, in triplicate, with the lentiviral vector containing the constructs pLKO shRNATRIAP1, shRNAHSP70 and shRNAscramble. We obtained the silencing of TRIAP1 and HSP70 genes in both MM cell lines when the transduced cell lines were compared with shRNAscramble. Silencing was confirmed by relative qPCR and Western blotting (for HSP70 only). Inhibition of TRIAP1 expression significantly increased the percentage of cells in late apoptosis (annexin V+/Propidium Iodide+) analysis (p<0.001 for RPMI8226 and P<0.01 for U266) one week after transduction and it was accompanied by increased expression of Caspase 9 in both MM cell lines (p<0.001 for RPMI8226 and p<0.05 for U266 cell lines) (One-Way ANOVA with Tukey´s multiple comparison test) when transduced cells were compared with the respective wild type cell lines. Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines when the inhibited cells were compared with cells transduced with shRNAscramble one week after transduction (p<0.01 for RPMI8226 and p<0.05 for U266 cell lines) and the results were confirmed by increased activity of Caspase 3/7 (p<0.01 for RPMI8226 and p<0.05 for U266). We observed significantly increase of Caspase 9 activity when RPMI8226 was transduced with shRNAHSP70 (p<0.001).Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact on the induction of late apoptosis, through APAF-1/Caspase 9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever P53 status assessed by FISH, as suggested by previous studies in other types of cancer (Support by FAPESP 2010/17668-6). DisclosuresNo relevant conflicts of interest to declare.