The Mechanism Of Senescence Induced By TNF-α In Mouse Melanoma Cells

ObiectiveTumor necrosis factor alpha (TNF-a) is one of the strongest bioactive factor that can directly kill tumor cells and has no obvious toxicity to normal cells.Previous studies found that TNF-a can induce mouse melanoma B16cells autophagy,but whether it can induce senescence of mouse melanoma cells is not clear.Therefore, this study aims to investigate whether TNF-a can induce mouse melanoma cells senescence,and explore the mechanism of senescence.Methods1. The mouse melanoma B16and B16F10cells were cultured in vitro,they were divided into three groups according to different treating methods:the control group,TNF-a treated group (12h,24h,48h after treated with20ng/ml TNF-a),3-MA+TNF-a treated group (pretreat with lOmM3-MA for2hours,then treat with20ng/ml TNF-a for12h,24h,48h).2. Observe the change of lipid droplets’number after oil red O staining in B16cells via microscope.3. Detect the G1/GO phase percentage of B16and B16F10cells after treated with TNF-a at different time using Flow cytometry (FCM).4. Collect B16and B16F10cells of different treated groups and extract total RNA.The mRNA expression level of senescence-releated gene pl6,p21,p53, CDK2,CDK4,CyclinD, CyclinE,Rb,E2F-1were detected by real-time polymerase chain reaction (RT-PCR).5. Collect B16cells of different treated and extract total protein by RIPA.The expression level of senescence-releated protein p53,pRb,E2F-l,and K-Ras,pErk, NF-κB in Ras-Raf-MAPK signaling pathway,autophagy-related marker protein LC3and Beclinl were detected by western blot.Results1. Compared with control group,there is a little lipid droplets at24h and a large number of lipid droplets at48h in B16cells after treated with TNF-a,and the number of lipid droplets was increased in time dependent manner after treatment of TNF-a;2. Compared with control group,the G1/G0phase percentage of B16cells increased significantly after treated with TNF-a at48h, and it is lower than3-MA+TNF-a treated group in B16cells;Compared with control group,the G1/G0phase percentage of B16F10cells increased significantly after treated with TNF-a,and the G1/G0phase percentage was increased in time dependent manner after treatment of TNF-a;3. p16gene could be detected in Mela cells but not in mouse melanoma B16and B16F10cells;4. Compared with control group,the change of mRNA expression of senescence-related gene p21was not obvious after treated with TNF-a at12h,but the mRNA expression of p53was up-regulated significantly after treated with TNF-a at12h.The mRNA expression of p21and p53in3MA+TNF-a treated group were higher than TNF-a treated group in B16cells;5. Compared with control group,the mRNA expression of downstream key genes CyclinE and E2F-1of p21and p53were down-regulated significantly,the mRNA expression of Rb was up-regulated significantly in B16cells;6. Compared with control group,the mRNA expression of p21was not obvious,the mRNA expression of p53was up-regulate significantly at24h and down-regulated to initial level at48h in B16F10cells;7. Compared with control group,the mRNA expression of downstream key genes CDK2,CDK4,CyclinE and E2F-1of p21and p53were down-regulated significantly.the mRNA expression of CyclinD was up-regulate significantly at24h,but down-regulated at48h;the change of mRNA expression of Rb was not obvious in B16F10cells;8. Compared with control group,the expression of senescence marker protein p53was up-regulated significantly after treated with3-MA+TNF-a,and higher than TNF-a treated group in B16cells;9. Compared with control group,the phosphorylation level of Rb was down-regulated significantly both in TNF-a and3-MA+TNF-a treated group in B16cells; 10. Compared with control group,the expression of E2F-1was down-regulated at12h after treated with TNF-a but up-regulated at24h in B16cells;11. Compared with control group,the expression of autophagy marker protein LC3and Beclinl were up-regulated and higher than3-MA+TNF-a treated group in B16F10cells;12. Compared with control group,the expression of K-Ras and pErk were up-regulated significantly after treated with TNF-α,but it is lower than3-MA+TNF-a treated group in B16cells;13. Compared with control group,the expression of NF-κB was up-regulated significantly after treated with TNF-α,and it is lower than3-MA+TNF-a treated group.Conclusion1. TNF-a could induce B16F10cells autophagy and could also induce mouse melanoma cells senescence.2. TNF-a may via autophagy induced senescence in B16cells,and moderate autophagy could delay the senescence of B16cells.3. TNF-a could induce B16cells senescence by Ras-Raf-MAPK and P53-P21-Rb signaling pathways.4. NF-κB may plays a major role in regulating senescence of B16cells.5. p16gene may be deficiented in mouse melanoma cells.