Necrosis Was Induced By Fas Signal Pathway In Human Lung Cancer A549/H460Cell Lines With Up-regulated Gene Expression Of CYLD

Objective: To investigate the expression level of tumor-suppressing gene CYLD inhuman lung carcinoma and the effect of programmed cell death (PCD) induced by Fas/FasLpathway in human lung cancer NCI-A549/H460cell lines complemented with exogenousFLAG-CYLD. Methods: The expression level of endogenous CYLD gene with canceroustissue of five operative specimens in lung adenocarcinoma cancer was assessed by real-timePCR, and adjacent normal lung tissue as control.The recombinate vectorpcDNA3.0-FLAG-CYLD was transfected into NCI-A549/H460cell lines. FLAG-CYLD/A549/H460cells were selected by culture medium containing G418and continuedto perform culturing. Compare the express level of CYLD gene after transfection tountransfected NCI-A549/H460by real-time PCR. Mem-FasL was added and incubatedrespectively with FLAG-CYLD/and FLAG-CYLD/A549/H460for24h. In addition toabove design, Caspase inhibitor zVAD-fmk was mixed in cell culture fluid for30min priorto induction by Mem-FasL. The PCD rates were assessed by flow cytometry (FCM). It wasanalyzed that whether CYLD could change Ub-RIP1into de-ubiquitinated RIP1whichwould be found in secondary complexes associated with caspase-8, and whether all thatcould promote A549cell to death by the methods of WB and Co-IP. Results: EndogenousCYLD gene expression was higher in adjacent normal lung tissue than cancerous tissue by2.53timesï¼›The differences were statistically significant (P<0.05). After exogenousFLAG-CYLD transfection, the value of CYLD expression was higher than before by1.40times and1.83times each in A549and H460cell lines. The rate of necrosis induced by Fassignal was raised after transfecting FLAG-CYLD into A549and H460cell lines. In thelevel of necrosis in A549cells, the WB and Co-IP results showed that the interactionexisted between in RIP1, CYLD and caspase-8. Apart from this, RIP1could combine withRIP3especially in pretreatment with caspase inhibitor zVAD-fmk. Conclusions: The gene expression of CYLD with adjacent cancerous tissue of the operative specimens in lungcancer was down-regulated. The change in gene expression was distinctly up-regulatedafter that FLAG-CYLD plasmid was transfected into A549and H460cell line. The necrosisis observed obviously and enhanced induced by Fas signal with mem-FasL inFLAG-CYLD/A549and H460cell lines. In the level of necrosis in A549cells, theanalysis showed that CYLD protein could change Ub-RIP1into de-ubiquitinated RIP1which would be found in secondary complexes associated with caspase-8andde-ubiquitinated RIP1could further recruit another homologous protein RIP3to compositenecrosis complexes which could lead to necrosis as a form of cell death.