Apoptotic Protease Activating Factor-1 Expression Research Articles

Zonisamide Ameliorated the Apoptosis and Inflammation in Cerebellar Tissue of Induced Alcohol Addiction Animal Model.

This study investigated the effects of zonisamide treatment on cerebellar tissues in an experimental alcohol addiction (AA) model and its potential mechanisms of action, particularly regarding apoptotic protease activating factor-1 (APAF-1) and tumor necrosis factor-alpha (TNF-α) expression. Thirty rats were divided into three groups: sham, ethanol (EtOH), and EtOH + zonisamide. AA was induced by administering 6 cc of EtOH orally every 8 h for 4 days. Zonisamide (100 mg/kg) was given to rats once daily before EtOH administration. Motor defects were evaluated using an open field maze. Serum TNF-α levels were measured from blood samples. Cerebellar sections were processed for histological examination and immunostained for APAF-1 and TNF-α. Protein interaction networks were constructed using Cytoscape, and functional annotations were performed with ShinyGO (version 0.80) software. The traveled area in the EtOH group was significantly reduced compared to the sham group (p = 0.0005). Rats in the EtOH + zonisamide group covered a larger area, with zonisamide treatment significantly improving locomotor ability compared to the EtOH group (p = 0.0463). Serum TNF-α levels were significantly elevated in the EtOH group compared to the sham group (p < 0.0001) and were significantly decreased in the EtOH + zonisamide group compared to the EtOH group (p = 0.0309). Regular cerebellar histological layers were observed in the sham group, while EtOH induction caused loss of cerebellar tissue integrity, neuronal degeneration, vascular dilatation and congestion, reduced myelin density, and neuropils in the EtOH group. Zonisamide treatment improved these pathologies, enhancing myelination and neuropil formation. Negative APAF-1 and TNF-α expressions were observed across cerebellar layers in the sham group. Due to EtOH toxicity, APAF-1 and TNF-α expression were upregulated in the EtOH group compared to the sham group (p < 0.001 for both). Zonisamide treatment downregulated these protein expressions in the EtOH + zonisamide group compared to the EtOH group (p < 0.001 and p = 0.0087, respectively). APAF-1 was primarily associated with AA through antifolate resistance, endopeptidases, and the interleukin-1 pathway, while TNF-α was predominantly enriched in infections and choline-binding, indicating zonisamide's impact on immune and inflammatory pathways. In conclusion, zonisamide treatment significantly mitigated ethanol-induced cerebellar damage and inflammation in an AA model. Zonisamide improved locomotor function and reduced serum TNF-α levels, as well as APAF-1 and TNF-α expression in cerebellar tissues. These findings suggest that zonisamide exerts its protective effects by modulating immune and inflammatory pathways, thereby preserving cerebellar integrity and function.

Upregulation of apoptotic protease activating factor-1 expression correlates with anti-tumor effect of taxane drug

Drug resistance is a multifactorial process involving a variety of mechanisms and genes. Taxane drug class like Docetaxel is not effective for all types' breast cancers and presents a huge clinical challenge. To improve cancer treatment outcome, it is important to distinguish which proteins can kill the cancer cells and whether the expression levels of these proteins affect treatment. Cancer cells are wildly known to be protected from apoptosis, due to low level of apoptotic protease activating factor-1 (Apaf-1) compared with normal cells. Apaf-1 is an essential protein that defines whether cytochrome c released form mitochondria remains stable or degrades. According to this hypothesis, increasing of Apaf-1 expression in MCF7 breast cancer cells was performed and Docetaxel efficacy examined. The immunoassay techniques were used to investigate Apaf-1 and cytochrome c levels, and different apoptosis assay methods applied to better understand the effect of Apaf-1 expression levels in cellular response to apoptotic stimuli by Docetaxel. Our results determined that cytoplasmic cytochrome c level elevated along with increasing Apaf-1 and MCF7 cells were sensitised to Docetaxel, suggesting that loss of Apaf-1 may cause Docetaxel-resistance in breast cancer cells through less apoptosome formation. ROS level increased in cells transfected with Apaf-1 and induced mitochondrial permeability for cytochrome c release, which subsequently promoted apoptosome formation, intrinsic apoptosis and ATP depletion.

LNC473 Regulating APAF1 IRES-Dependent Translation via Competitive Sponging miR574 and miR15b: Implications in Colorectal Cancer

A growing number of studies have focused on the involvement of non-coding RNAs (ncRNAs) in the internal ribosome entry site (IRES)-mediated translation in tumorigenesis; however, the underlying mechanisms in colorectal cancer (CRC) remain elusive. In this study, we show that LINC00473 (LNC473) exerted its functions as a tumor suppressor in promoting apoptotic protease-activating factor 1 (APAF1) IRES activity through competitively sponging miR574-5p and miR15b-5p in CRC initiation and pathogenesis. Specifically, LNC473 and its downstream target APAF1 were significantly downregulated accompanied by upregulated miR574-5p and miR15b-5p in CRC cells and tissues, which had a significant prognostic impact on clinical outcomes in our CRC cohort (n = 157). Furthermore, ectopic LNC473 significantly sponged endogenous miR574-5p or miR15b-5p and thereby inhibited cell proliferation and colony formation capacity, and it accelerated cell apoptosis through activating the APAF1-CASP9-CASP3 pathway. Notably, LNC473 overexpression resulted in dramatic promotion of APAF1 IRES activity and translation, whereas rescue experiments confirmed the recovery by the existence of LNC473 and miR574/15b-5p. Mechanistically, LNC473 overexpression promoted IRES binding domain exposure and removed the constraints controlling from miR574-5p and miR15b-5p, and subsequently enhanced IRES-mediated APAF1 expression in vitro and in vivo. Therefore, our results uncover a novel LNC473-miR574/miR15b-APAF1 signaling axis, which provides new targets and crosstalk regulation mechanism for CRC prevention and treatment.

LncRNA MEG3 inhibits cell proliferation and induces apoptosis in laryngeal cancer via miR-23a/APAF-1 axis.

Long non‐coding RNA (LncRNA) MEG3 serves a regulatory role in the progression of several types of cancer, but the role of MEG3 in laryngeal cancer is still unknown. The aim of this study was to explore the regulatory role and mechanism of MEG3 in laryngeal cancer. MEG3 expression in 50 laryngeal cancer tissue samples was detected by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). The effects of MEG3 overexpression on laryngeal cancer cells were investigated in vitro and in vivo. The mechanism of competitive endogenous RNA (ceRNA) was validated through luciferase reporter assay, RT‐qPCR and Western blotting. MEG3 was down‐regulated in laryngeal cancer tissues, and the low MEG3 expression was associated with advanced clinical stage. Additionally, MEG3 overexpression inhibited the proliferation and induced the apoptosis of laryngeal cancer cells in vitro and in vivo. Particularly, MEG3 bound to miR‐23a specifically and a reciprocal negative regulation existed between miR‐23a and MEG3. Moreover, MEG3 up‐regulated apoptotic protease activating factor‐1 (APAF‐1), a known miR‐23a's target, thereby leading to the activation of caspase‐9 and caspase‐3. Meanwhile, these activated effects were rescued by miR‐23a overexpression. In conclusion, the present study demonstrated that MEG3 functions as a novel tumour suppressive LncRNA in laryngeal cancer for the first time. Furthermore, MEG3 may act as a ceRNA to regulate APAF‐1 expression by competitive binding to miR‐23a, thereby regulating the progression of laryngeal cancer.

Circular RNA Cdr1as sensitizes bladder cancer to cisplatin by upregulating APAF1 expression through miR-1270 inhibition.

Circular RNAs (circRNAs) have recently emerged as essential regulators in carcinogenesis and cancer progression. Previous studies have shown that Cdr1as functions as a microRNA (miRNA) sponge in various cancer types. However, the role of Cdr1as in cisplatin chemosensitivity in bladder cancer remains unclear. Here, we used real‐time PCR to examine miRNA and gene expression in bladder cancer tissues and cell lines. The abilities of Cdr1as and its downstream regulatory molecules to induce apoptosis and promote cisplatin‐induced chemosensitivity of bladder cancer cells were determined by flow cytometry and cell counting kit. Bioinformatic analysis was utilized to predict potential miRNA target sites, and biotin‐coupled miRNA capture, biotin‐coupled probe pull‐down assay, and RNA fluorescent in situ hybridization were used to study the interaction between Cdr1as and target miRNAs. Dual‐luciferase reporter assay was also used to validate the target genes of miRNAs. The expression level of apoptotic protease‐activating factor 1 (APAF1) in bladder cancer cells was identified via western blot. Finally, the sensitivity of Cdr1as to cisplatin chemotherapy in nude mice xenografts was evaluated in terms of the size, volume of tumors, and the survival of mice. We report that Cdr1as induced the apoptosis and enhanced the cisplatin chemosensitivity of bladder cancer cells both in vitro and in vivo. Silencing of APAF1 reduced the sensitivity of bladder cancer cells to cisplatin chemotherapy. Furthermore, Cdr1as could directly sponge miR‐1270 and abolish its effect on APAF1. Our study verified that Cdr1as exerts a cisplatin‐chemosensitization effect on bladder cancer cells through the Cdr1as/miR‐1270/APAF1 axis. This newly identified axis may be a potential therapeutic target for bladder cancer patients.

MiR-186 promotes tumor growth in cutaneous squamous cell carcinoma by inhibiting apoptotic protease activating factor-1.

Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of non-melanoma skin cancer worldwide. MicroRNAs (miRNAs or miRs) are a subtype of non-coding RNA associated with the progression of various types of human cancer. MiR-186 has been demonstrated to act as an oncogene in human tumors. However, the role of miR-186 in cSCC remains unclear. The expression of miR-186 and apoptotic protease activating factor 1 (APAF1) was examined using reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence. The correlation between miR-186 and APAF1 was determined using a dual-luciferase assay. Mimics or inhibitors of miR-186 were transfected into A-431 cells to establish cell lines with overexpressed or knocked-down miR-186, respectively. EdU staining and colony formation assays were performed to detect cell proliferation. Transwell and wound-healing assays were performed to analyze cell invasion and migration, respectively. Hoechst staining and flow cytometry were performed to assess cell apoptosis and cell cycle distribution. MiR-186 expression was significantly increased, while APAF1 expression was significantly decreased in cSCC tissues compared with the controls. An miR-186 binding site was predicted in APAF1 and their expression was negatively correlated in cSCC tissues. Cell proliferation, invasion and migration were significantly enhanced in the miR-186-overexpressed A-431 cells and attenuated in miR-186 knockdown cells compared with the control. APAF1 expression was regulated by miR-186, while APAF1 knockdown significantly promoted cell invasion and inhibited cell apoptosis. In summary, the results of the present study indicate that miR-186 serves as an oncogene in cSCC by inhibiting APAF1.

Upregulation of microRNA-23a regulates proliferation and apoptosis by targeting APAF-1 in laryngeal carcinoma.

MicroRNA-23a (miR-23a) is a potential biomarker for laryngeal cancer. Apoptotic protease activating factor 1 (APAF-1) was recently demonstrated to be a target of miR-23a. However, whether miR-23a exerts its effects via APAF-1 in laryngeal cancer, remains unknown. In the present study, miR-23a expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). APAF-1 mRNA and protein expression levels were assayed by RT-qPCR and western blotting, respectively. Binding of miR-23a to APAF-1 was monitored by a luciferase reporter assay. Gain-of-function and loss-of-function studies were performed in order to investigate the roles of miR-23a and APAF-1 in Hep2 cell proliferation and apoptosis. miR-23a and APAF-1 were found to be significantly upregulated and downregulated, respectively, in laryngeal cancer tissues, and there was a significant negative correlation between APAF-1 and miR-23a expression. The results of the luciferase reporter assay demonstrated that miR-23a bound directly to the APAF-1 mRNA 3'-untranslated region. Ectopic expression of miR-23a and knockdown of APAF-1 significantly promoted cell proliferation and colony formation, and inhibited early apoptosis in Hep2 cells. In conclusion, miR-23a acts as an oncogenic regulator in laryngeal carcinoma by directly targeting APAF-1, and may be a useful biomarker in the diagnosis and treatment of laryngeal carcinoma.

High expression of APAF-1 elevates erythroid apoptosis in iron overload myelodysplastic syndrome

Apoptotic protease-activating factor 1 (APAF-1) is a central component of the intrinsic pathway of apoptosis. Our study aims at searching the role of APAF-1 in iron overload myelodysplastic syndrome (MDS). Erythroid apoptosis rate, mRNA expression levels of APAF-1, and caspase-9 activity were determined by flow cytometry, quantitative real-time PCR, and colorimetric assay in MDS patients, respectively. In addition, K562 and MDS-L cell lines were incubated with different concentrations of ferric ammonium citrate (FAC) or ferric ammonium citrate + desferrioxamine (FAC + DFO) in vitro to observe the alteration in erythrocyte apoptosis rate, APAF-1 mRNA, and protein expression levels. Moreover, as control, erythroid apoptosis rate and APAF-1 mRNA expression were detected after silencing APAF-1 expression by endoribonuclease-prepared small interfering RNAs (esiRNAs) in K562 and MDS-L cell lines. Both erythroid apoptosis rate and APAF-1 mRNA expression of the iron overload (IO) group were significantly higher than those of the non-IO group (P < 0.001 and P < 0.001). There is a significant difference of caspase-9 activity between the IO group and the non-IO group (P < 0.001). Erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines significantly elevated after FAC incubation in different concentrations (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L), while erythroid apoptosis rate and APAF-1 mRNA expression in the FAC + DFO group declined (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). After silencing of APAF-1 expression with specific esiRNAs, erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines markedly decreased (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). APAF-1 plays an important role in iron-induced erythroid apoptosis increase in MDS.

Abstract A27: MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1

Abstract Over the past 30 years, there has been a dramatic increase in the incidence of obesity worldwide. As of 2010, approximately one-third of U.S. adults are obese, while another one-third of adults are overweight. Obesity is one of the risk factors for ovarian cancer. Greater than 75% of ovarian cancer patients present with advanced stage disease. Tumors preferentially spread to the omentum, a major site of abdominal fat, where ovarian cancer cells infiltrate omental adipose tissue. Omental adipocytes may interact directly with cancer cells to create a microenvironment niche that supports cancer growth. Recent studies have shown that cells can communicate through the exchange of bioactive molecules via microvesicles (exosomes and ectosomes). Microvesicles fuse with target cell membranes and transfer proteins, lipids, mRNAs and non-coding RNAs from donor to recipient cells. We hypothesize that adipocytes secrete microvesicles containing tumor-supporting factors that promote ovarian cancer progression. Ion Torrent RNA sequencing and subsequent qRT-PCR analyses were used to identify non-coding microvesicle RNAs secreted by primary cultures of cancer associated adipocytes (CAA), cancer associated fibroblasts (CAF), normal adipocytes, normal fibroblasts and ovarian cancer cells. Of the non-coding microvesicle RNAs identified, microRNA 21 (miR21) showed significantly higher expression in microvesicles secreted from CAA when compared to those from CAF and normal omental adipocytes, fibroblasts, and ovarian cancer cells. Using qRT-PCR of RNA isolated from microdissected frozen tissue and In-situ hybridization on FFPE tissue sections, CAA demonstrated significant higher miR21 expression levels compared to the adjacent cancer cells and normal adipocytes. MiR21 was up-regulated in metastatic ovarian cancer cells in the omentum as compared to those in the primary tumor site. Moreover, strong miR21 staining was observed in omental cancer cells particularly at the invasion front, suggesting that miR21 may be transferred from CAA to their neighboring ovarian cancer cells. To test this hypothesis, SKOV3 ovarian cancer cells were co-cultured with adipocytes transfected with miR21-FAM or incubated with fluorescently labeled adipocyte-derived microvesicles. Confocal microscopy confirmed the delivery of miR21 by adipocyte-derived microvesicles to cancer cells. Since miR21 expression in tumor cells has been linked with resistance to a variety of chemotherapeutic agents, we further delineated the direct effect of miR21 on the chemoresistance of ovarian cancer cell. The ovarian cancer cell lines, OVCA432 and SKOV3, transfected with miR21 precursor showed an increase in cell survival and a decrease in apoptosis in the presence of taxol. Furthermore, miR21 was significantly up-regulated in taxol-resistant HeyA8-MDR and SKOV3-TR cells as compared to the parental cell lines, HeyA8 and SKOV3, respectively. These data strongly suggest that miR21 may confer taxol resistance to ovarian cancer cells. Using microarray analysis of RNA isolated from SKOV3 cells transfected with the miR21 precursor, we identified a set of chemoresistance-related genes associated with miR21 expression. One of the most significantly down-regulated genes is Apoptotic Protease Activating Factor 1 (APAF1). By qRT-PCR, we confirmed that miR21 over-expression decreased APAF1 mRNA expression levels in OVCA432 and SKOV3 cells. A strong miR21 binding site is predicted on the 3′-UTR of APAF1, suggesting APAF1 is the direct target of miR21. In summary, up-regulation of miR21 in omental ovarian cancer cells is mediated through transport of miR21 in adipocyte-derived microvesicles from CAA in the omentum. Further, miR21 confers taxol-resistance in ovarian cancer cells by down-regulating APAF1 expression in ovarian cancer cells to decrease taxol-induced apoptosis. Citation Format: Ngai Na Co, Rosemarie Schmandt, Karen Lu, Samuel Mok. MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1 [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A27.

Lack of Apoptotic Protease Activating Factor-1 Expression and Resistance to Hypoxia-Induced Apoptosis in Cervical Cancer

Clinical observations suggest that intratumoral hypoxia increases the aggressiveness of tumors through clonal selection of cancer cells that have lost their apoptotic potential. The aim of this study, therefore, was to investigate the expression of the proapoptotic protein apoptotic protease activating factor-1 (Apaf-1) in cervical cancers and to analyze its relation to intratumoral hypoxia and apoptosis. Furthermore, the effect of hypoxia and apoptosis on survival was examined. In 56 patients, intratumoral oxygenation measurements and subsequent needle biopsies were done. The obtained tissue was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays and by immunohistochemistry with an Apaf-1 antibody. Apaf-1 was expressed in 86% of cancers. The median apoptosis rate was 1.0%. There was no correlation between Apaf-1 expression and intratumoral hypoxia. However, Apaf-1 expression was negative in 37.5% of hypoxic cervical cancers (pO(2) <or= 10 mmHg) with low apoptosis rates (<or=1.0%) compared with only 5.0% in nonhypoxic cancers and hypoxic cancers with high apoptosis (P = 0.005; Fisher's exact test). With a median follow-up period of 44 months, there was a nonsignificant trend toward worse prognosis in the hypoxic low-apoptotic group (P = 0.08). Although Apaf-1 is expressed in the vast majority of cervical cancers, a significant proportion of tumors with low apoptosis rates despite intratumoral hypoxia showed a lack of Apaf-1 expression. This finding suggests that loss of Apaf-1 expression is a mechanism by which hypoxic cervical cancers acquire resistance to apoptosis. Thus, low Apaf-1 expression in hypoxic tumors may be an unfavorable prognostic factor.