The Effects Of A20on TRAIL Resistance In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma is one of the most common cancers world wide and theChinese morbidity of hepatocellular carcinoma is the first throughout the world.Hepatocellular carcinoma is a serious threat to the people's health due to low surgicalresection rate, high recurrence rate, high mortality, resistance to radiotherapy andchemotherapy without effective clinical treatments.TRAIL emerged as one of the TNF superfamily members in recent years and can inducesthe majority of tumor cells to apoptosis such as breast cancer, bladder cancer, gliomas throughdeath receptor activation pathway. After radical resection, residual liver cancer cells couldevade immune surveillance with rapid recurrence. One of the reasons is that liver cancer cellsis resistant to TRAIL-induced apoptosis via the death receptor pathway whileas themechanism is not clear.A20was initially identified following stimulation of TNF, IL-1or LPS stimulation inhuman umbilical vein endothelial cells. Growing evidence supports that A20is an ubiquitinediting protein that modulates optimal cellular immune and inflammatory responses. Theimportance of A20in immune homeostasis is underscored by the key linkages betweengenetic polymorphisms and multiple autoimmune diseases, including Crohn's disease,systemic lupus erythematosus, rheumatoid arthritis, and type1diabetes. In addition, A20negatively controls the important immune-modulatory transcription factor, NF-κB and, thus,functions as an anti-inflammatory factor. Furthermore, increasing reports also indicate thatA20functions as a tumor suppressor in several lymphomas. However, A20was shown todecrease TNF-mediated apoptosis and necrosis, and increased A20was associated with aresistance to TRAIL in fibrosarcoma, but the specific role of A20is unclear. In our study, we found that A20is highly expressed in HCC tissues and hepatocellularcarcinoma cell lines and participates in the resistance to TRAIL-induced apoptosis. Thecombination of TRAIL and A20gene silencing inhibits hepatocellular carcinoma cell growthand induces hepatocellular carcinoma cell apoptosis, and thus provides an experimental basisfor the combination of TRAIL and A20gene silencing technology or A20targeting inhibitors.In the experiments, we used cell culture, cell death assay, siRNA, flow cytometry,shRNA, Western blotting and other experiments to study A20expression in humanhepatocellular carcinoma tissues and hepatocellular carcinoma cell lines, the combinationeffects of TRAIL and A20gene silencing technology on cell cycle and the protein expressionchanges in the apoptosis pathway. This study aimed to explore the combination effects ofTRAIL and A20gene silencing on inhibition of cell growth and inducing apoptosis inhepatocellular carcinoma with synergetic molecular mechanism to provide a clear theoreticalbasis and experimental evidences for hepatocellular carcinoma targeting therapy.Test results of A20expression in human hepatocellular carcinoma tissues and relativecancer cell lines:(1) A20is highly expressed in human hepatocellular carcinoma tissues and no expressionor low expression is in normal tissues.(2) A20is not expressed in human glioma cell line LN18. Low A20expression is in coloncancer cell lines HCT-8and HT-29. Moderate A20expression is in colon cancer cell linesHCT-8and HT-29. A20is highly expressed in glioma cell line LN443, hepatocellularcarcinoma cell line HepG2and Hep3B.(3) A20may exhibit tissue or cell line-specific characteristics in different carcinomas andfurther mechanism investigations involving each tumor type are needed for the molecularcancer therapeutics.Following experimental results show targeting A20enhances TRAIL-induced apoptosisin hepatocellular carcinoma cells:(1) Caspase-8cleavage is inhibited in TRAIL resistance and targeting A20enhancesTRAIL-induced apoptosis. (2) The synergetic effects of TRAIL and A20silencing enha nce apoptosis by reversingTRAIL resistance in hepatocellular carcinoma cells.(3) The mechanism involving TRAIL-induced apoptosis is that A20silencing mediatesA20-dependent RIP1ubiquitination. A20knockdown causes the de-ubiquitination of RIP1with RIP1cleavage, allowing its association with caspase-8, activating downstream caspase-3and promoting cell death through death receptor signaling pathway.Following experimental results show A20mediates cell growth in hepatocellularcarcinoma cells:(1) Targeting A20effectively enhances the TRAIL-induced apoptosis in hepatocellularcarcinoma cell lines and the antitumor effect of TRAIL was increased in a dose-dependentmanner.(2) RIP1ubiquitination mediated by A20blocks caspase-8activation in hepatocellularcarcinoma in response to TRAIL, whereas A20knockdown rapidly reverses RIP1ubiquitination to enhance TRAIL-induced apoptosis. It emphasizes the crucial role forA20-dependent RIP1ubiquitination in regulating TRAIL resistance.(3) A20knockout inhibits the cell growth in early passages of hepatocellular carcinomacell lines and promotes significant morphological changes in early passages of HepG2cells.A20may regulate cell growth and morphological changes in hepatocellular carcinoma cells.The main innovations of the study:(1) Verifiy A20expression and apoptosis signalings in hepatocellular carcinoma tissuesand hepatocellular carcinoma cell lines to clarify that A20may exhibit tissue or cellline-specific characteristics in different carcinomas.(2) Clarify the molecular mechanism of TRAIL resistance in hepatocellular carcinomacells and analyze the phenomenon that A20is involved in cell growth and morphologicalchanges.(3) Ilustrate the role of A20in TRAIL resistance and the molecular mechanism of thesynergetic effects by targeting A20combined with TRAIL treatment on cell growth inhibitionand apoptosis in hepatocellular carcinoma. Above all, A20is highly expressed both in human hepatocellular carcinoma tissues andhepatocellular carcinoma cell lines and A20may exhibit tissue or cell line-specificcharacteristics in different carcinomas. Caspase-8cleavage is inhibited in TRAIL resistanceand A20is involved in TRAIL resistance. Targeting A20enhances TRAIL-induced apoptosisand the synergetic effects of TRAIL and A20silencing enhance apoptosis by reversingTRAIL resistance in hepatocellular carcinoma cells while the antitumor effect of TRAIL wasincreased in a dose-dependent manner. The mechanism involving TRAIL-induced apoptosisis that A20silencing mediates A20-dependent RIP1ubiquitination. A20knockdown causesthe de-ubiquitination of RIP1with RIP1cleavage, allowing its association with caspase-8,activating downstream caspase-3and promoting cell death through death receptor signalingpathway. A20may also regulate cell growth and morphological changes in hepatocellularcarcinoma cells. So, the combination of TRAIL and the A20gene silencing or targeting A20inhibitors can inhibit cell growth, activate downstream apoptosis pathway in hepatocellularcarcinoma in a better way and provides new ideas and methods for the treatment ofhepatocellular carcinoma.