Expression Of cIAP2 Research Articles

The effects of NF-κB signal pathway on the process of anastomotic stricture after the radical resection of esophageal carcinoma

The aim of this article is to identify the relationship between the nuclear factor κB (NF-κB) signal pathway expression and the anastomotic stricture. It particularly focuses on the expression of p65, bcl-2, and cIAP-1 in the anastomotic stricture formation after radical resection of esophageal cancer. A total of 82 esophageal squamous carcinoma patients who had undergone esophagectomy by Ivor Lewis procedure were enrolled in the study. Tissues were collected from the patients who developed the anastomotic stricture after the operation, while para anastomotic tissues served as the control. The protein expression of p65, bcl-2, and cIAP-1 was analyzed by immunohistochemistry and western blot analyses, while the messenger RNA (mRNA) levels of p65, bcl-2, and cIAP-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR). The results showed that lower expression of p65, bcl-2, and cIAP-1 proteins was observed in the para anastomotic tissue; in the esophageal stricture’s tissue, the expression of these proteins was significantly higher ( P < 0.05). The mRNA levels of P56, bcl-2, and cIAP-1 in the stricture tissue were remarkably increased ( P < 0.05) compared with the para anastomotic tissues, and the mRNA levels in the sample of grade 3 dysphagia were higher ( P < 0.05) than the levels of grade 1 and 2. In the normal esophageal epithelial cell of stricture patients was upregulated compared with that of no stricture patients. We can confirm that the anastomotic stricture has the relationship with the NF-κB signal pathway. The bcl-2 and cIAP-1, located downstream of NF-κB signal pathway, could be a new preventive and therapeutic target for the anastomotic stricture.

Effect of Circular RNA UBAP2 Silencing on Proliferation and Invasion of Human Lung Cancer A549 Cells and Its Mechanism

背景与目的已有的研究证明环状RNA（circular RNAs, circRNAs）在多种肿瘤发生发展中起重要作用，circUBAP2是一种促癌的circRNA，但是其在肺癌中的作用和机制尚不明确。本研究旨在探讨circular RNA UBAP2（circUBAP2）对人肺癌A549细胞体外增殖和侵袭能力的影响并初步探讨其机制。方法应用CCK-8法检测circUBAP2表达沉默对A549细胞生长的抑制作用，采用流式细胞仪技术检测circUBAP2表达沉默对A549细胞周期和细胞失巢凋亡的影响，Transwell实验检测circUBAP2表达沉默对A549细胞侵袭能力的影响，Western blot和Real-time PCR检测CDK6、cyclin D1、p27、c-IAP1、Bcl-2、Survivin、Bax、FAK、Rac1、MMP2的表达变化以及JNK和ERK1/2的活性，荧光素酶报告技术检测circUBAP2直接靶基因。结果CCK-8实验结果显示circUBAP2表达沉默后，A549细胞株增殖能力受抑制；流式细胞结果显示，circUBAP2表达沉默后，细胞周期停滞在G0期/G1期，且细胞凋亡率显著增加；Transwell实验结果显示，细胞体外侵袭能力明显受到抑制。Western blot和Real-time PCR结果显示，circUBAP2表达沉默后，CDK6、cyclin D1、c-IAP1、Bcl-2、Survivin、FAK、Rac1、MMP2等基因的表达水平显著下降，而p27和Bax的表达显著上升，而且JNK、ERK1/2的活性也受到明显的抑制，circUBAP2直接靶基因为miR-339-5p、miR-96-3p和miR-135b-3p。结论circUBAP2在肺癌的体外增殖和侵袭中发挥重要的作用，抑制其表达，有望成为肺癌分子靶向治疗的新靶点。

Synergistic Effects of a Smac Mimetic with Doxorubicin Against Human Osteosarcoma.

Second mitochondria-derived activator of caspase (Smac) is a proapoptogenic mitochondrial protein that antagonizes inhibitors of apoptosis proteins (IAPs), resulting in induction of apoptosis. In the present study we investigated the effects of a Smac mimetic in combination with doxorubicin against osteosarcoma. In vitro effects of the combination of a Smac mimetic AT-406 and doxorubicin on cell proliferation and apoptosis in osteosarcoma cell lines were examined using cell proliferation assays, flow cytometry, and immunoblot analyses. For in vivo experiments, human osteosarcoma xenografts were treated with combination of the two substances, and tumor volume and apoptotic activity in treated tumors were assessed. In vitro studies revealed that combination of the two substances significantly inhibited osteosarcoma proliferation with decreased cIAP1 expression and induced apoptosis in osteosarcoma cells. Combination of the two substances significantly suppressed osteosarcoma growth in vivo. Moreover, decreased cIAP1 expression and increased apoptotic activity were observed in tumors treated by their combination of the substances. The Smac mimetic AT-406 showed an apoptotic effect and a synergistic antitumor effect with doxorubicin on osteosarcoma. The combination of AT-406 and doxorubicin may serve as a novel therapeutic strategy for osteosarcoma treatment.

Hepatitis C Virus Infection Increases c-Jun N-Terminal Kinase (JNK) Phosphorylation and Accentuates Hepatocyte Lipoapoptosis

BackgroundHepatitis C virus (HCV) infection and metabolic diseases including nonalcoholic steatohepatitis (NASH) exhibit a complex interplay. Although free fatty acid-mediated apoptosis is a prominent feature of NASH, the impact of HCV infection on hepatocyte lipotoxicity has remained largely unexplored. The study aimed at identifying whether infection by HCV affected the apoptotic pathway in hepatocytes during fatty acid assault.Material/MethodsOR6 cells, which are derived from human hepatocellular carcinoma Huh-7 cells and harbor a full-length HCV RNA genome replication system, were treated with palmitate. Apoptosis was examined by 4′,6-diamidino-2-phenylindole staining. Activation and expression of JNK, Bim, cIAP-1, and Mcl-1 were examined by immunoblotting. mRNA expression of CHOP, a major player in endoplasmic reticulum stress-mediated apoptosis, was assessed by real-time PCR.ResultsPalmitate-induced hepatocyte apoptosis was significantly enhanced in OR6 cells compared to cured cells, in which the HCV genome had been eradicated by treatment with interferon-α. Although basal expression of CHOP mRNA was enhanced in OR6 cells compared to cured cells, it was similarly upregulated in both cell lines following palmitate treatment. Notably, palmitate-induced JNK phosphorylation was accentuated in OR6 cells compared to cured cells. Inhibition of JNK with SP600125 attenuated palmitate-induced apoptosis. Palmitate-mediated upregulation of BH3-only protein Bim, which acts downstream of JNK, was also enhanced in OR6 cells compared to cured cells. In contrast, Mcl-1 and cIAP-1 were equally reduced in OR6 cells and cured cells following palmitate treatment.ConclusionsThese findings suggest that during lipoapoptosis, HCV infection may enhance hepatocyte toxicity by increasing JNK phosphorylation.

Cellular inhibitor of apoptosis protein 2 promotes the epithelial-mesenchymal transition in triple-negative breast cancer cells through activation of the AKT signaling pathway.

Triple-negative breast cancer (TNBC) represents approximately 10–17% of all breast cancers, and patients with TNBC show a poorer short-term prognosis than patients with other types of breast cancer. TNBCs also have a higher tendency for early distant metastasis and cancer recurrence due to induction of the epithelial-mesenchymal transition (EMT). Several recent reports have suggested that inhibitor of apoptosis (IAP) proteins function as regulators of the EMT. However, the roles of these proteins in TNBC are not clear. Accordingly, we investigated the roles of cIAP2 in TNBC. Among eight IAP genes, only cIAP2 was upregulated in TNBC cells compared with that in other breast cancer subtypes. Analysis of TMAs revealed that expression of cIAP2 was upregulated in TNBCs. In vitro studies showed that cIAP2 was highly expressed in TNBC cells compared with that in other types of breast cancer cells. Furthermore, silencing of cIAP2 in TNBC cells induced mesenchymal-epithelial transition (MET)-like processes and subsequently suppressed the migratory ability and invasion capacity of the cells by regulation of Snail through the AKT signaling pathway. In contrast, ectopic expression of cIAP2 in luminal-type breast cancer cells induced activation of the AKT signaling pathway. These results collectively indicated that cIAP2 regulated the EMT in TNBC via activation of the AKT signaling pathway, contributing to metastasis in TNBC. Our study proposes a novel mechanism through which cIAP2 regulates the EMT involving AKT signaling in TNBC cells. We suggest that cIAP2 may be an attractive candidate molecule for the development of targeted therapeutics in the future.

Molecular mechanism of anti-cancerous potential of Morin extracted from mulberry in Hela cells

The flavonoids are of great interest due to their antioxidant and anti-cancerous potential. The present study investigated the Morin extracted from mulberry leaves and its anti-cancerous mechanism via cell inhibition, cell cycle analysis, apoptosis, mRNA expression and antioxidant mechanism through reactive oxygen species generation. Morin inhibited the proliferation of HeLa cells at IC50 of 214.28 μM and led to morphological changes, followed by induction of cell cycle arrest in G2/M-phase and ultimately resulted into apoptosis. Morin-induced G2/M-phase arrest was accompanied by the increase in mRNA expression of p53, p21 and Wee 1 genes and decreased levels of CDK1, Cdc25c, Survivin, cyclin B1and CHK2. Morin-induced apoptosis was regulated through multiple pathways, including intrinsic and extrinsic pathway. The underlying mechanisms consisted of increased mRNA expression of Bax, Bad, cytochrome c, Apaf-1, caspases-9, DR3, DR5, FasL, FADD, caspases-10, PARP, PI3K, AKT, mTOR, P70S6K and Smac genes as well as decreased expression of Bcl-2, Bcl-xL, AMPK, cIAP-1, cIAP-2, PKCε and NF-kβ. In addition, Morin treatment resulted in the generation of intracellular ROS, which play an essential role in the induction of apoptosis. The present study recommends the use of Morin in the development of functional foods with anti-cancerous potential in the future.

Abstract 5185: Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma

Abstract Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a class of targeted drugs that antagonize selected inhibitor of apoptosis protein (IAP) to block pro-survial signalling, and eventually induce cell apoptosis. The present study was designed to investigate the radiosensitizing effect of one SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We analyzed the correlation between apoptotic molecules and HPV status in HNSCC via mRNA expression using The Cancer Genome Atlas (TCGA) database, as well as protein expression in 6 HNSCC cell lines by immunoblotting. We also examined cIAP1 expression on a tissue microarray (TMA) of HNSCC tumors, and assessed its correlation with HPV status and patient outcome. Clonogenic survival and WST-1 assays were carried out to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis, Annexin-V assays and immunoblotting were performed to investigate effects of LCL161 on radiation induced cell apoptosis. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis and immunoblotting from HNSCC cells revealed that expression of cIAP1, Survivin, DIABLO, p65, IK-b, TNF-α, BCL-XL, Caspase-7, and Caspase-8 were elevated in HPV[-] compared to HPV[+] HNSCC tumors. IHC staining showed cIAP1 expression was associated with HPV[-] status (p=0.0239). In addition, cIAP1 expression was significantly higher in HNSCC compared with adjacent normal tissue (p=0.0003). In univariate analysis, cIAP1 was significantly associated with poor overall survival (p=0.0359). LCL161 degraded cIAP1 at nanomolar concentrations but has minimal single-agent cytotoxicity with IC50 range of 32 μM – 95 μM. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-], but not in HPV[+] HNSCC cells. Mechanistic studies showed LCL161 mediated radiosensitisation was associated with increased cell apoptosis, with enhanced activation of caspases-3, -7, -8, -9, and PARP. Finally, in vivo data showed that LCL161 degraded cIAP1, activated caspases, and profoundly radiosensitized two human HPV[-] HNSCC xenograft models. Conclusion: Taken together, our results demonstrate for the first time that cIAP1 is a potential prognostic and therapeutic biomarker for HNSCC patients. Through targeting cIAP1 and other IAP family members, LCL161, radiosensitizes HNSCC tumors, particularly HPV [-] tumors, via caspase activation and apoptosis induction. Taken together, LCL161 holds promise for future clinical development as a novel radiosensitizer in the treatment of HPV[-] HNSCC tumors. Citation Format: Linlin Yang, Bhavna Kumar, Tianyun Li, Thenodoros N. Teknos, Arnab Chakravarti, Terence M. Williams. Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2017-5185

Abstract LB-269: PFKP/PFKFB3 modulate metabolic switch and chemoresistance to cisplatin in ovarian cancer

Abstract Background: Late diagnosis and occurrence of chemoresistance contribute ovarian cancer the most malignant gynaecological cancer. High lactate generation and low glucose oxidation, regardless of oxygen availability, termed as the Warburg effect, are commonly found in cancers. Phosphofructokinase-1 (PFK-1) irreversibly converts fructose-6-phosphate to fructose-1,6-bisphosphate, the first committed step in the Warburg effect. PFK1 can be activated by fructose-2,6-bisphosphate (F2,6BP), which in turn is generated and degraded by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB). The platelet isoform of PFK (PFKP) and inducible PFKFB3 isoform modulate glycolysis and contribute to tumorigenesis in other human cancers, the precise mechanisms have yet to be elucidated. Moreover, PFKP and PFKFB3 in ovarian cancer remain to be characterized. We hypothesize that PFKP, which is regulated by mi106a-5p, along with PFKFB3 modulate cancer metabolic switch and chemoresistance to cisplatin in ovarian cancer. Methods: Expression of PFKP and PFKFB3 in ovarian cancer cell lines was investigated by qPCR and immunoblotting. Expression of PFKP in 123 ovarian cancer tissue samples was evaluated by immunohistochemistry. The correlation between PFKP and clinicopathological parameters was analyzed. Ectopic expression or knockdown of PFKP/PFKFB3/miR106a-5p and treatment with 3PO (a PFKFB3 inhibitor), followed by lactate assay and TUNEL assay or annexin V/propidium iodide staining after treatment with cisplatin were performed in ovarian cancer cell lines to detect the effects on cancer metabolic switch and chemoresistance to cisplatin respectively. Additionally, the related apoptosis signaling pathway was explored by RT2 profiler PCR array and immuoblotting. Results: We found up-regulation of PFKP in ovarian cancer cell lines and patient samples with significantly higher PFKP found in chemoresistant than chemosensitive cell lines and clinical samples. Higher PFKP was correlated to shorter overall and disease-free survival. Higher PFKFB3 was also detected in chemoresistant than chemosensitive cell lines. Functionally, knockdown of PFKP/PFKFB3 in ovarian cancer resistant cells decreased lactate level, sensitized cells to cisplatin treatment, which was accompanied by increased cleavage of PARP. 3PO also reduced lactate level and sensitized A2780CP cells to cisplatin treatment, along with altered c-IAP1, c-IAP2, survivin and CD70 expression, and increased cleavage of caspases-9/7 and PARP. Ectopic expression of miR106a-5p decreased PFKP expression, reduced lactate level and sensitized A2780CP cells to cisplatin treatment. Conclusion: Our findings suggest that PFKP/PFKFB3 may overcome chemoresistance in ovarian cancer cells through apoptosis related pathway. Results also show the potential use of PFKFB3 inhibitor as therapeutic targets either alone or in combination with cisplatin and PFKP as a novel prognostic marker for ovarian cancer. Citation Format: Jingjing Wang, Michelle KY Siu, Yuxin Jiang, Thomas HY Leung, Hextan YS NGAN, Annie NY Cheung, Karen KL Chan. PFKP/PFKFB3 modulate metabolic switch and chemoresistance to cisplatin in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-269. doi:10.1158/1538-7445.AM2017-LB-269

Abstract 2016: Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with a five-year survival of ~50%. HNSCC are linked to human papillomavirus (HPV+) or tobacco carcinogenesis (HPV-). The Cancer Genome Atlas recently analyzed 279 HNSCC, revealing that 30% overexpress FADD (Fas-Associated Death Domain), with or without BIRC2/3 genes encoding cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2), which are critical components of the Tumor Necrosis Factor (TNF) Receptor signaling pathways that determine cell death or survival. The frequency of such mutations provides a window for potential therapeutics, as IAP antagonists have been shown to switch cancer cell TNFα signaling from being pro-survival to pro-apoptotic. We recently showed that birinapant, a second mitochondrial activator of caspases (SMAC) mimetic that promotes IAP degradation, sensitizes HNSCC to cell death by TNFα and eradicates tumors in combination with radiation in HPV- HNSCC models overexpressing FADD+/-BIRC2. ASTX660 (developed by Astex Pharmaceuticals) is a novel dual cIAP1/XIAP antagonist currently in clinical trials for treating advanced solid tumors and lymphomas. The objective of the present study is to determine the therapeutic effects of ASTX660 in HPV+/- HNSCC preclinical models. ASTX660 at nanomolar concentrations was found to potently inhibit cell proliferation (measured using XTT assays) and induce apoptosis (Annexin/7-AAD flow cytometry), and displayed combinatorial activity with TNFα, TRAIL, or cisplatin in multiple human HPV+/- HNSCC cell lines. Western blotting showed near complete degradation of cIAP1 expression at nanomolar concentrations in human HNSCC cell lines. Flow cytometry revealed that ASTX660 in combination with either TNFα or cisplatin reduced MHC-I expression and increased PD-L1 expression, suggesting that potential synergistic efficacy could be observed in combination with immune checkpoint inhibitors. Our results demonstrate that ASTX660 is a potential therapeutic agent for both HPV+/- HNSCC. Experiments evaluating the anti-tumor effects of ASTX660 as a monotherapy and in combination with radiation, cisplatin, and anti-PD-1 therapies are ongoing, using both human HNSCC xenograft and syngeneic mouse oral cancer (MOC) models. Supported by NIDCD intramural projects (ZIA-DC-000016, 73 and 74). Citation Format: Roy Xiao, Yi An, Adeeb Derakhshan, Zhong Chen, Nicole C. Schmitt, Carter Van Waes. Novel dual cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human papillomavirus(+) and (-) head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2017-2016

Evaluation of the Expression of the Inhibitor of Apoptosis Protein Family and Human Telomerase Reverse Transcriptase in Patients With Advanced Colorectal Adenoma

BackgroundIt has been reported that the expression of the inhibitor of apoptosis protein (IAP) family increases in patients with colon cancer. We evaluated the expression of the IAP family and human telomerase reverse transcriptase (hTERT) in normal colon mucosa from patients with advanced colorectal adenoma and investigated their features according to characteristics of advanced colorectal adenoma.MethodsWhile resections of polyps were performed in patients (n = 80) diagnosed with advanced colorectal adenoma or carcinoma in situ, additional normal tissues were obtained from the sigmoid colon. In healthy patients (n = 16), blind biopsies were performed on the sigmoid colon. The expression of the IAP family, including survivin, XIAP, cIAP1, and cIAP2, and hTERT, were analyzed by real-time PCR in both groups.ResultsA total of 80 advanced colorectal adenoma patients (71.3% male, mean age of 60.4 years) and 16 control patients were enrolled in this study. The mean ranking of cIAP2 was higher in the control group (68.88 vs. 44.43, P = 0.001). The expression levels of hTERT, survivin, XIAP, and cIAP from both groups showed no differences. The expression of survivin, XIAP, cIAP1, cIAP2, and hTERT depending on certain factors of advanced adenoma, including the number (two or fewer vs. three or more), size (smaller than 1 cm vs. larger than 1 cm), grade of dysplasia (low grade adenoma vs. high grade adenoma), pathology (tubular adenoma vs. villous adenoma), and presence of endometrial intraepithelial neoplasms, showed no significant correlations in the Mann-Whitney U-test.ConclusionsThe expression of the IAP family and hTERT, except cIAP2, in the normal mucosa of patients with advanced colorectal adenoma were not different from those of the control group. There were no differences in the IAP family and hTERT according to the characteristics of advanced adenoma.

Effects of bufalin on the mTOR/p70S6K pathway and apoptosis in esophageal squamous cell carcinoma in nude mice.

The aim of this study was to investigate the effects of bufalin on the mammalian target of rapamycin (mTOR/p70S6 kinase (p70S6K) signaling pathway and cell apoptosis in orthotopically transplanted tumors in nude mice. The mice were inoculated with human esophageal squamous cell carcinoma (ESCC) ECA109 cells in order to establish a model of orthotopicall transplanted ESCC tumors. The mice are administered low, medium and high doses of bufalin (0.5, 1.0 and 1.5 mg/kg) or rapamycin, or a combination of both. After the tumors were removed, the mRNA expression levels of mTOR, p70S6K, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), cellular inhibitor of apoptosis protein 1 (cIAP1) and caspase-3 were detected by RT-PCR. In addition, we performed western blot analysis and immunohistochemical analysis to determine the protein expression of mTOR, p70S6K, 4EBP1, cIAP1, active caspase-3, Bcl-2 and Bad in the tumor tissue. The results revealed that bufalin exerted a significant anti-tumor effect in the nude mice with ESCC orthotopically transplanted tumors. This was shown by the decrease in the expression of mTOR, p70S6K and 4EBP1, which suggested that bufalin may possibly be used to inhibit tumor growth via the inhibition of the activation of p70S6K and 4EBP1. We also found that bufalin decreased the expression of cIAP1 and Bcl-2, and increased that of active caspase-3 and Bad, thus indicating that bufalin promoted apoptosis. Thus, our findings suggest that bufalin promotes tumor cell apoptosis, and this may be one of the important anti-tumor mechanisms of action of bufalin.

MicroRNA-34a inhibits tumor invasion and metastasis in osteosarcoma partly by effecting C-IAP2 and Bcl-2.

Osteosarcoma is a common primary malignant bone tumor that occurs mainly in children and adolescents. Recent evidence has demonstrated that miR-34a is involved in the invasion and metastasis of osteosarcoma. This study aims to explore the effect of biological behavior of miR-34a on osteosarcoma. First, we collect osteosarcoma and adjacent specimens, and the relative expression of miR-34a and C-IAP2 messenger RNA was quantitated by real-time polymerase chain reaction. Furthermore, miR-34a stimulant is synthesized and transfected onto osteosarcoma MG-63 cells. The effect of overexpression of miR-34a on osteosarcoma was detected by colony-forming assay, Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit I, Transwell assay, and animal experiment in vivo. Finally, the relative levels of C-IAP2 and Bcl-2 protein were checked by western blot, and the activity of caspase-3 and caspase-9 was tested by spectrophotometry assay. In conclusion, miR-34a was downregulated in osteosarcoma cells. And the expression of C-IAP2 and Bcl-2 protein was drastically inhibited, and the activities of caspase-3 and caspase-9 were significantly increased after transfecting miR-34a onto osteosarcoma MG-63 cells. And the overexpression of miR-34a can inhibit cell invasion and metastasis, promote cell apoptosis, and arrest cells in G0/G1 period. And the animal experiment in vivo demonstrated that the overexpression of miR-34a could significantly inhibit the growth of osteosarcoma in animal skin. Taken together, we indicated that miR-34a can inhibit tumor invasion and metastasis in osteosarcoma, and its mechanism may be partly related to downregulating the expression of C-IAP2 and Bcl-2 protein directly or indirectly.

CIAP2 promotes gallbladder cancer invasion and lymphangiogenesis by activating the NF-κB pathway.

Several studies have produced contradictory findings about the prognostic implications for inhibitor of apoptosis proteins (IAP) in different types of cancer. Cellular inhibitor of apoptosis 2 (cIAP2/BIRC) is one of the most extensively characterized human IAP. To date, no studies have focused on the expression level of cIAP2 in human gallbladder cancer (GBC), and the mechanism of cIAP2 in GBC invasion and lymphangiogenesis remains unclear. Therefore, in the present study, cIAP2 expression in GBC was detected using quantitative real‐time polymerase chain reaction and immunohistochemistry, and the relationship between cIAP2 levels in cancer tissues and the clinicopathological characteristics of patients was analyzed. The biological effect of cIAP2 in GBC cells was tested using the Cell Counting Kit‐8 Assay, Transwell assays and the ability of human dermal lymphatic endothelial cells (HDLEC) to undergo tube formation. The role of cIAP2 in activating the NF‐κB pathway was determined using a dual‐luciferase reporter assay, immunofluorescence staining, western blotting and ELISA. Finally, an animal model was used to further confirm the role of cIAP2 in lymphangiogenesis. We showed that cIAP2 expression was elevated in human GBC tissues and correlated with a negative prognosis for patients. Moreover, cIAP2 was identified as a lymphangiogenic factor of GBC cells and, thus, promoted lymph node metastasis in GBC cells. Our study is the first to suggest that cIAP2 can promote GBC invasion and lymphangiogenesis by activating the NF‐κB pathway.

The SMAC mimetic birinapant attenuates lipopolysaccharide-induced liver injury by inhibiting the tumor necrosis factor receptor–associated factor 3 degradation in Kupffer cells

It was demonstrated that second mitochondria-derived activator of caspases (SMAC) mimetic inhibites tumor necrosis factor receptor-associated factor 3 (TRAF3) degradation and the mitogen-activated protein kinase (MAPK) signaling pathway activation induced by lipopolysaccharide (LPS) in vitro. However, the effect of Smac mimetic in vivo is not clear. The present study was to investigate the role of Smac mimetic in LPS-induced liver injury in mice and its possible mechanism. An animal model of LPS-induced liver injury was established by intraperitoneally injecting mice with 10mg/kg LPS pretreatment with or without Smac mimetic birinapant (30mg/kg body weight). Birinapant significantly improved the survival rate of endotoxemic mice (P<0.05) and attenuated LPS-induced liver pathologic damage and inflammatory response. IL-1 and TNF-α levels in the serum were markedly decreased in birinapant pretreatment mice compared with control mice (P<0.05).The cellular inhibitor of apoptosis protein 1 (cIAP1) expression in liver resident macrophage (Kupffer cells, KCs) was significantly decreased in the Birinapant group compared to the Vehicle group (P<0.05). At the same time, total TRAF3 protein abundance in KCs rapidly declined after LPS stimulation in the Vehicle group. However, it remained constant in the Birinapant group. Moreover, K48-linked polyubiquitination of TRAF3 in KCs was markedly impressed in the birinapant group compared with the control group. At last, the JNK and p38 MAPK activation in KCs was significantly inhibited by birinapant pretreatment (P<0.05). These results suggested that birinapant attenuated liver injury and improved survival rates in endotoxemic mice by inhibited the expression of cIAP1, degradation of TRAF3 and aviation of MAPK signaling pathway.

CIAP-2 Expression Increases in Elderly Patients with Squamous Cell Carcinoma of the Head and Neck

Summary Background Evidence indicates apoptosis deregulation is associated with aging. We correlated expression of cIAP-2, a member of the inhibitor of apoptosis protein family, with survival and age in patients with resected squamous cell carcinoma of head and neck (SCCHN). Methods Immunohistochemical (IHC) staining for cIAP-2 and other markers were performed using tissue microarrays from 103 resected SCCHN patients. Expression was grouped according to IHC scores (0–3) and statistical association with clinical–pathological factors was determined. Results High expression of cIAP-2 was found to be increased in patients aged 60 or greater. High expression of cIAP-2 and an age ≥60 were significantly associated with worse survival in univariate analysis, but only an age ≥60, and not cIAP-2 expression, was prognostic in multivariate analysis. Conclusion High expression of cIAP-2 was found more frequently in elderly SCCHN patients but was not independently associated with survival after adjusting other clinical–pathologic factors.

The Role of cIAP1 and XIAP in Apoptosis Induced by Tumor Necrosis Factor Alpha in Esophageal Squamous Cell Carcinoma Cells

The inhibitor of apoptosis protein (IAP) family are reported to play important roles in cancer cells evading apoptosis. However, the significance of their expression in human esophageal squamous cell carcinoma (ESCC) cells remains uncertain. The present study aimed to investigate the role of the IAP family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human ESCC cells. Five human ESCC cell lines were pretreated with TNF-α, cycloheximide (CHX, protein synthesis inhibitor), epoxomicin (proteasome inhibitor). Apoptosis assay and protein study with Western blot testing were conducted. Knockdown experiments with IAP siRNA were conducted, and the effect on cell apoptosis was analyzed. Significant apoptosis was induced in five ESCC cell lines by TNF-α plus CHX stimulation, but not when treated with TNF-α or CHX alone. The protein expression levels of cIAP1 and XIAP were decreased by treatment with TNF-α in the presence of CHX, and the degree of cIAP1 and XIAP expression decrease was correlated with sensitivity to TNF-α plus CHX-induced apoptosis. Epoxomicin suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP, in addition to apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but did not suppress degradation of survivin, cIAP1, and XIAP. Furthermore, cIAP1 or XIAP siRNA transfected cells underwent apoptosis in response to treatment with TNF-α alone. Double knockdown of both genes resulted in further increased apoptosis. cIAP1 and XIAP play an essential role in the resistance of ESCC cells against apoptosis.

P2.03a-059 LCL161 Increases Paclitaxel-Induced Apoptosis by Degrading cIAP1 and cIAP2 in NSCLC: Topic: Clinical Trials

LCL161, a novel Smac mimetic, is known to have anti-tumor activity and improve chemosensitivity in various cancers. However, the function and mechanisms of the combination of LCL161 and paclitaxel in non-small cell lung cancer (NSCLC) remain unknown. Cellular inhibitor of apoptotic protein 1 and 2 (cIAP1 and cIAP2) expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry. The correlations between cIAP1,2 expression and clinicopathological characteristics, prognosis were analyzed. Cell viability and apoptosis were measured by MTT assays and Flow cytometry. Western blot and co-immunoprecipitation assay were performed to measure the protein expression and interaction in NF-κB pathway. siRNA-mediated gene silencing and caspases activity assays were applied to demonstrate the role and mechanisms of cIAP1,2 and RIP1 in lung cancer cell apoptosis. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of LCL161 alone or in combination with paclitaxel. The expression of cIAP1 and cIAP2 in Non-small cell lung cancer (NSCLC) tumors was significantly higher than that in adjacent normal tissues. cIAP1 was highly expressed in patients with late TNM stage NSCLC and a poor prognosis. Positivity for cIAP1 and cIAP2 was an independent prognostic factor that indicated a poorer prognosis in NSCLC patients. LCL161, an IAP inhibitor, cooperated with paclitaxel to reduce cell viability and induce apoptosis in NSCLC cells. Molecular studies revealed that paclitaxel increased TNFα expression, thereby leading to the recruitment of various factors and the formation of the TRADD-TRAF2-RIP1-cIAP complex. LCL161 degraded cIAP1 and cIAP2, releasing RIP1 from the complex. Subsequently, RIP1 was stabilized and bound to caspase-8 and FADD, thereby forming the caspase-8/RIP1/FADD complex, which activated caspase-8, caspase-3 and ultimately lead to apoptosis. In nude mouse xenograft experiments, the combination of LCL161 and paclitaxel degraded cIAP1,2, activated caspase-3 and inhibited tumor growth with few toxic effects. Thus, LCL161 could be a useful agent for the treatment of NSCLC in combination with paclitaxel.

Defective expression of apoptosis-related molecules in multiple sclerosis patients is normalized early after autologous haematopoietic stem cell transplantation.

Defective apoptosis might be involved in the pathogenesis of multiple sclerosis (MS). We evaluated apoptosis-related molecules in MS patients before and after autologous haematopoietic stem cell transplantation (AHSCT) using BCNU, Etoposide, AraC and Melphalan (BEAM) or cyclophosphamide (CY)-based conditioning regimens. Patients were followed for clinical and immunological parameters for 2 years after AHSCT. At baseline, MS patients had decreased proapoptotic BAD, BAX and FASL and increased A1 gene expression when compared with healthy counterparts. In the BEAM group, BAK, BIK, BIMEL , FAS, FASL, A1, BCL2, BCLXL , CFLIPL and CIAP2 genes were up-regulated after AHSCT. With the exception of BIK, BIMEL and A1, all genes reached levels similar to controls at day+720 post-transplantation. Furthermore, in these patients, we observed increased CD8+ Fas+ T cell frequencies after AHSCT when compared to baseline. In the CY group, we observed increased BAX, BCLW, CFLIPL and CIAP1 and decreased BIK and BID gene expressions after transplantation. At day +720 post-AHSCT, the expression of BAX, FAS, FASL, BCL2, BCLXL and CIAP1 was similar to that of controls. Protein analyses showed increased Bcl-2 expression before transplantation. At 1 year post-AHSCT, expression of Bak, Bim, Bcl-2, Bcl-xL and cFlip-L was decreased when compared to baseline values. In summary, our findings suggest that normalization of apoptosis-related molecules is associated with the early therapeutic effects of AHSCT in MS patients. These mechanisms may be involved in the re-establishment of immune tolerance during the first 2 years post-transplantation.

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein.

Growth Hormone Mediates Its Protective Effect in Hepatic Apoptosis through Hnf6.

Background and AimsGrowth hormone (GH) not only supports hepatic metabolism but also protects against hepatocyte cell death. Hnf6 (or Oc1) belonging to the Onecut family of hepatocyte transcription factors known to regulate differentiated hepatic function, is a GH-responsive gene. We evaluate if GH mediates Hnf6 activity to attenuate hepatic apoptotic injury.MethodsWe used an animal model of hepatic apoptosis by bile duct ligation (BDL) with Hnf6 -/- (KO) mice in which hepatic Hnf6 was conditionally inactivated. GH was administered to adult wild type WT and KO mice for the 7 days of BDL to enhance Hnf6 expression. In vitro, primary hepatocytes derived from KO and WT liver were treated with LPS and hepatocyte apoptosis was assessed with and without GH treatment.ResultsIn WT mice, GH treatment enhanced Hnf6 expression during BDL, inhibited Caspase -3, -8 and -9 responses and diminished hepatic apoptotic and fibrotic injury. GH-mediated upregulation of Hnf6 expression and parallel suppression of apoptosis and fibrosis in WT BDL liver were abrogated in KO mice. LPS activated apoptosis and suppressed Hnf6 expression in primary hepatocytes. GH/LPS co-treatment enhanced Hnf6 expression with corresponding attenuation of apoptosis in WT-derived hepatocytes, but not in KO hepatocytes. ChiP-on-ChiP and electromobility shift assays of KO and WT liver nuclear extracts identified Ciap1 (or Birc2) as an Hnf6-bound target gene. Ciap1 expression patterns closely follow Hnf6 expression in the liver and in hepatocytes.ConclusionGH broad protective actions on hepatocytes during liver injury are effected through Hnf6, with Hnf6 transcriptional activation of Ciap1 as an underlying molecular mediator.