Apollon Expression Research Articles

Impact of Berberine on the Expression of Apollon Gene in Chronic Lymphocytic Leukemia

Background: The incidence of B-chronic lymphocytic leukemia (B-CLL) resulting from the clonal accumulation of apoptosis-resistant malignant B lymphocytes is growing in the adult population of Iran. Inhibitors of apoptosis proteins (IAPs) are considered as factors that can delay the onset of CLL cell apoptosis. Berberine is an isoquinoline alkaloid isolated from Cotridis rhizoma that exhibits anti-tumor activities through various mechanisms. Objectives: In this study, we investigated the impact of berberine on the level of Apollon expression in peripheral blood mononuclear cells (PBMCs) of 12 cases newly diagnosed with CLL and 6 healthy donors. Methods: At first, the level of Apollon expression was assessed in PBMCs of CLL patients compared to the healthy donors. Peripheral blood mononuclear cells were cultured in RPMI-1640 medium with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin for 48 hours, and the effect of berberine (25 µM) on the level of Apollon expression in CLL patients was assessed and compared to that of healthy donors. Results: We found that the expression level of Apollon was not significantly different between CLL patients and healthy donors (P = 0.640). Moreover, berberine induced no significant differences in Apollon expression as compared to the untreated (control) group (P = 0.545 and P = 0.267 in CLL patients and healthy donors, respectively). Conclusions: Overall, our results suggest that berberine has no direct effect on the expression of Apollon gene in CLL patients, and pro-apoptotic impacts of berberine may be exerted through other mechanisms.

Apollon silencing enhanced the sensitivity to cisplatin-induced cytotoxicity of chemoresistant breast cancer cell line MCF-1/cisplatin

Objective To explore the effect of Apollon on cisplatin-induced chemoresistance of breast cancer cell line MCF-1/cisplatin (DDP). Methods Apollon silencing was performed by transient transfection of small interfering RNA (siRNA). The inhibitory effect of different concentrations of cisplatin (10, 20, 40, 80, and 100 μmol/L) on cell viability was measured by methyl thiazol tetrazolium (MTT) assays. The mRNA and protein expression levels were detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting, respectively. Results As compared with MCF-7 cells, Apollon mRNA and protein levels were 1.70±0.21 folds and 2.23±0.33 folds enhanced in cisplatin-resistant breast cancer cell line MCF-1/DDP, respectively (P=0.007, 0.003). Knockdown of Apollon increased the sensitivity to cisplatin-induced cytotoxicity of MCF-1/DDP. As compared with non-transfected control group, the half maximal inhibitory concentration (IC50) value of cisplatin was significantly decreased from (81.39±3.67) μmol/L to (33.24±2.01) μmol/L (P=0.002). Meanwhile, the mRNA and protein expression levels of Apollon and β-catenin were remarkably declined to 0.43±0.16 (P=0.005), 0.54±0.10 (P=0.006) and 0.23±0.08 (P=0.003), 0.44±0.20 (P=0.005) after Apollon silencing, respectively. In addition, the mRNA and protein expression levels of β-catenin were significantly 1.91±0.23 folds (P=0.0044) and 2.21±0.31 (P=0.0023) folds enhanced with little influence on Apollon expression (P=0.890, 0.730) upon the treatment of WAY-262611 in siRNA-transfected cells. Conclusion Apollon silencing enhanced the sensitivity to cisplatin-induced cytotoxicity of chemoresistant breast cancer cell line MCF-1/DDP, which might be partially dependent on the reduced β-catenin expression. Key words: Breast cancer; Apollon; Cisplatin-resistant

With no interaction, knockdown of Apollon and MDR1 reverse the multidrug resistance of human chronic myelogenous leukemia K562/ADM cells.

Chemotherapy is the main treatment method for patients with chronic myeloid leukemia(CML) and has achieved marked results. However, the acquisition of multidrug resistance(MDR) has seriously affected the quality of life and survival rate of patients. The overexpression of the inhibitors of apoptosis proteins(IAPs) and the adenosine triphosphate(ATP)-dependent binding cassette(ABC) transporters are the two main causes of MDR. Apollon and MDR1 are the most important and representative members, respectively, among the IAPs and ABC transporters. In the present study, we investigated the role of Apollon and MDR1 in chemotherapy resistance and their mechanism of interaction. We respectively knocked down the expression of Apollon and MDR1 using short hairpin RNA(shRNA) in adriamycin(ADM) resistant human CML K562 cells and examined the drug sensitivity, the consequences with regard to ADM accumulation and the alterations in the expression of Apollon and MDR1. The expression levels of Apollon and MDR1 mRNA were higher in the K562/ADM cells compared with the parental K562 cells as determined by reverse transcription‑polymerase chain reaction (RT-PCR). The plasmids of Apollon and MDR1 shRNA were respectively stably transfected into K562/ADM cells using Lipofectamine2000. The transfection efficiency was detected by fluorescence microscopy. Cell Counting Kit-8 (CCK-8) assay revealed that Apollon or MDR1 knockdown significantly increased the chemosensitivity of the K562/ADM cells to ADM. Flow cytometric assay revealed that K562/ADM/shMDR1 cells exhibited a significantly increased intracellular accumulation of ADM, and that changes were not found in the K562/ADM/shApollon cells. Compared with the parental K562/ADM cells, a significantly decreased expression of Apollon mRNA and protein was determined in the K562/ADM/shApollon cells without affecting the expression of MDR1 as determined by RT-PCR and western blotting. Likewise, the expression levels of MDR1 mRNA and protein also markedly downregulated in the K562/ADM/shMDR1 cells had no effect on Apollon expression. Collectively, our findings demonstrated, for the first time, that downregulation of Apollon or MDR1 through stable transfection with the Apollon- or MDR1-targeting shRNA induced MDR reversal through respective inhibition of Apollon or MDR1 expression and function. However, the reversal mechanism of Apollon and MDR1 revealed no direct interaction with each other.

Expression and clinical significance of Apollon in renal carcinoma.

Apollon, namely baculoviral inhibitor of apoptosis proteins (IAP) repeat containing 6, is an unusually large member of the IAP family, and may be important in oncogenesis. The aim of the present study was to assess the association between renal carcinoma (RC) and Apollon expression, and to highlight the link between Apollon expression and the occurrence, development and prognosis of RC. Apollon expression was detected by immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction in RC tissues, adjacent non-cancerous tissues and paired normal tissues, respectively, in order to analyze the association between Apollon expression and clinicopathological features of RC. Kaplan-Meier survival estimate was used to assess the prognostic significance. It was observed that Apollon expression was higher in carcinoma tissues than in adjacent non-cancerous tissues and normal control tissues at the protein and messenger RNA level (P<0.001). There was a significant difference in T-stage (P=0.006), nodal involvement (P=0.007) and tumor-node-metastasis-stage (P=0.035) in patients categorized according to different Apollon expression levels. A prognostic significance of Apollon was also identified by the Kaplan-Meier method. The results of the present study indicate that Apollon expression is associated with the biological characteristics of renal cancer, and is potentially a valuable predictor and novel target for RC.

Expression and clinical significance of Apollon in esophageal squamous cell carcinoma.

Apollon, an unusually large member of the inhibitors of apoptosis protein family, may be important for oncogenesis development. The aim of the present study was to assess the association between esophageal squamous cell carcinoma (ESCC) and Apollon expression levels, and to highlight the association between Apollon and the occurrence, development and prognosis of ESCC. Apollon expression was detected by immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction in ESCC tissues, adjacent non-cancerous tissues and paired normal tissues respectively, in order to analyze the association between Apollon expression and the clinicopathological features of ESCC. Survival analysis was used to assess the prognostic significance of Apollon expression. It was determined that the mRNA and protein expression levels of Apollon were significantly higher in the carcinoma tissues compared with the adjacent non-cancerous tissues and normal control tissues (P<0.001). There was a significant difference in lymph node involvement and the tumor, nodes, and metastases stage in patients categorized according to different Apollon expression levels. The prognostic significance of Apollon was also determined using the log-rank method. The overexpression of Apollon was associated with shorter overall survival and disease-free survival rates. The present study indicates that Apollon expression is associated with the biological characteristics of ESCC, and may be a valuable prognostic factor and a novel chemotherapeutic target for ESCC treatment.

Apollon modulates chemosensitivity in human esophageal squamous cell carcinoma.

Patients with esophageal squamous cell carcinoma (ESCC) are often diagnosed with advanced diseases that respond poorly to chemotherapy. Here we reported that Apollon, a membrane-associated inhibitor of apoptosis protein, was overexpressed in ESCC cell lines and clinical ESCC tissues, and Apollon overexpression clinically correlated with poor response to chemotherapy (P = 0.001), and short overall survival (P = 0.021). Apollon knockdown increased cisplatin/docetaxel-induced apoptosis, mitochondrial dysfunction and cytochrome c release in two ESCC cell lines. Apollon knockdown potentiated cisplatin/docetaxel-induced long-term cell growth inhibition, and enhanced chemosensitivity of ESCC cells to cisplatin/docetaxel in xenograft tumor models. Apollon knockdown also enhanced cisplatin/docetaxel-induced activation of caspase-8 (extrinsic pathway) and caspase-9 (intrinsic pathway) in ESCC cells and xenograft tumor models. Mechanism studies revealed that the effect of Apollon on chemosensitivity is mainly mediated by Smac. Apollon expression strongly and negatively correlated with Smac expression in clinical ESCC tissues (P = 0.001). Apollon targeted Smac for degradation in ESCC cells. The effect of Apollon on chemosensitivity was reversed by Smac knockdown in ESCC cells. Taken together, our data show association of Apollon expression with chemotherapeutic response in ESCC, and provide a strong rationale for combining Apollon antagonism with chemotherapy to treat ESCC.

Effects of Apollon antisense oligonucleotide on proliferation and apoptosis of K562 cells

Objective To investigate the effects of Apollon antisense oligonucleotide (ASODN) on proliferation, apoptosis and drug resistance of human leukemia(K562) cells. Methods Specific phosphorothioate ASODN and missense oligonucleotide(MSODN) of Apollon mRNA were synthesized and transfected into K562 cells following cationic liposome.The proliferation inhibition of K562 cells was assessed by MTT.The apoptosis rate was detected by Annexin V-FITC.The sensitivity of K562 cells to etoposide and vincristine was detected by MTT. Results Apollon antisense oligonucleotide inhibition of K562 cells with the concentration and time increased.ASODN at a final concentration of 600 nmol/L could significantly inhibit the K562 cell proliferation.The apoptosis rate was apparently increased (P<0.01). Conclusions Apollon ASODN may decrease Apollon gene expression, suppress K562 cells proliferation effectively, and induce significant apoptosis of K562 cells.Apollon ASODN is able to reverse the drug resistance via inhibition of Apollon expression and inducement of apoptosis. Key words: Apollon; Antisense oligonucleotide; K562; Cell proliferation; Apoptosis; Drug resistance

Role of Apollon in Human Melanoma Resistance to Antitumor Agents That Activate the Intrinsic or the Extrinsic Apoptosis Pathways

To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression. Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal-regulated kinase (MEK)-, BRAF(V600E)-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents. Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAF(V600E)-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAF(V600E)-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAF(V600E)-specific inhibitors. Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways.

Apollon/RNF41 Myocardial Messenger RNA Diagnoses Cardiac Allograft Apoptosis in Rejection

Endomyocardial biopsy (EMB) remains the gold standard for acute cellular rejection (ACR) diagnosis in cardiac transplantation yet is subject to interobserver variability. A method that could avoid discordant EMB analysis would be desirable. The apoptosis rate in EMB correlates with ACR severity. Apollon inhibits apoptosis, and RNF41 catalyzes its degradation. Whether tissue Apollon/RNF41 could diagnose ACR is not known. This study addressed this issue. Apollon/RNF41 messenger RNA (mRNA) was measured by real time reverse-transcriptase polymerase chain reaction and apoptosis was quantified with TUNEL assays in EMBs of 268 transplant recipients. EMBs were obtained at 1, 2, 3, 4, 7, 12, 24, and 52 posttransplant weeks. At all time points posttransplant, Apollon mRNA decreased significantly in EMBs with ACR grades 2R/3R combined (P<or=0.0010) compared with 0/1R combined, although RNF41 mRNA significantly increased in EMBs with ACR grade 1R (P<0.0001) or 2R/3R combined (P<0.0001) compared with 0. At the identified cut-off level of less than or equal to 168.2 arbitrary units, Apollon mRNA identified ACR grades 2R/3R with 100% sensitivity and 84% specificity, whereas RNF41 mRNA at the cut-off level of more than or equal to 51.8 identified ACR grades 1R-3R with 99% sensitivity and 95% specificity. Increased RNF41 (rs, 0.728; P<0.0001) and decreased Apollon (rs, -0.562; P<0.0001) expression correlated significantly with the degree of apoptosis in EMBs. Combined Apollon/RNF41 mRNA quantitatively and specifically identifies ACR associated with apoptosis in cardiac allografts and could validate ACR grading variability associated with histologic EMB analysis.

Overexpression of Apollon, an Antiapoptotic Protein, Is Associated with Poor Prognosis in Childhood De novo Acute Myeloid Leukemia

The genes that encode inhibitor of apoptosis proteins are frequently overexpressed in human cancers and can be associated with resistance to therapy. The overexpression of Apollon, a member of inhibitor of apoptosis proteins, is intuitively expected to be associated with unfavorable clinical features in malignant diseases; however, there have been no clinical studies reporting the prognostic relevance of Apollon expression in human malignancies. This study was done to investigate the clinical relevance of the expression of Apollon in childhood de novo acute myeloid leukemia. In 55 pediatric patients with de novo acute myeloid leukemia, the level of Apollon expression was determined by using quantitative reverse transcriptase-PCR and was analyzed with respect to the patients' clinical features and treatment outcomes. Apollon expression was found to be higher in patients with a leukocyte number of >or=10,000/microL, patients with extramedullary disease, and patients with the French-American-British classification subtype M7. In addition, Apollon overexpression (>or=median expression) was associated with an unfavorable day 7 response to induction chemotherapy and also associated with a poorer 3-year relapse-free survival rate (48.3 +/- 11.2% versus 78.7 +/- 8.5%, P = 0.040). This is the first study demonstrating the prognostic implication of the Apollon expression in human cancers, indicating that Apollon overexpression may be used as a poor prognostic marker in childhood acute myeloid leukemia through validation by further studies.