The Effect Of TNF-? In Tumor Growth Inhibition Induced By Celastrol In Tumor-Bearing Mice

IntroductionThe report The Progress of Chinese Cancer Prevention And Treatment published by the Chinese Medical Association in February 2018 shows that the mortality of cancar ranks the first of Chinese disease spectrum and is higher than the world's average level.Inflammation is a complex defense response against all kinds of damage factors,and both clinical and epidemiological evidences demonstrate that a closely relationship between inflammation and cancer.Inflammation has been identified as an important biomarker of tumor,epidemiological investigations show that almost 20%-25%of all cancers worldwild are caused by microbial infection.Tumor was also known as a wound that do not heal.Tumor microenvironment is a complex environmental system that is essential for tumor survival,and many inflammatory cells?e.g.,macrophage,myeloid derived suppressor cell,natural killer cell,mast cell?infiltrate in it which produces a variety of cytokines?e.g.,TNF-?,IL-6,IL-8,TGF-?,COX-2?through autocrine and paracrine forms,helps the tumor cells to maintain proliferation,limits death,plays an important role in tumor angiogenesis and metastasis,and also in tumor occurrence,development,metastasis and invasion.Targeting tumor microenvironment may become a new focus of tumor prevention and treatment.Celastrol?Cel?is a three ring terpenoid monomer that was extracted from the root bark of Tripterygium Wilfordii Hook.The structural formula is C₂₉H₂₈O₄.Cel has many biological activities,like immunosuppressive,anti-inflammatory,antioxidation,anti-neurodegenerative diseases,anti rheumatism,recent studys show that Cel also has a potent anti-tumor activity on many tumor cells,which will inhibit the proteasome activity,and thereby induce apoptosis of cancer cells.Cel can also downregulate the activity of NF-?B and STAT3 signal pathway,suppresse tumor proliferation and induce the apoptosis of tumor;or inhibit tumor growth through activation of both mitochondria and FasL mediated pathways.Gautam's study demonstrates that Cel inhibits the phosphorylation of I?B?induced by TNF-?,and further inhibits the nuclear transfer and phosphorylation of NF-?B-P65,which enhances the apoptosis and inhibit the invasion of cancer cells induced by TNF-?.Our previous study also showed that Cel could inhibit the activation of the NF-?B pathway induced by TNF-?and enhance the apoptosis of tumor cells by inhibiting the degradation of I?B?and the nuclear transposition of P65 in vitro,suggesting that Cel can enhance the apoptosis induced by TNF-?through adjusting the balance of the signal pathway transduction of TNF-?in the tumor cells.In order to further verify whether TNF-?participates in the tumor growth inhibiton induced by Cel in vivo and the possible mechanism,we established stable TNF-?gene knockout C57 mice through gene knockout technique,and also obtained wildtype C57 mice,which were all inoculated with mouse liver cancer cell line H22 cells in the right flank to establish solid tumor models,and then inject i.p.the wildtype and TNF-?^-/-C57 mice with vehicle or Cel for treatment.The effect of Cel in wildtype and TNF-?^-/-mouse solid tumor and the molecules changes of TNF-?signal transduction were observed to determine the effect and possible mechanism of TNF-?in the apoptosis of tumor cells induced by Cel.Methods?1?Mice were divided into four groups:wildtype control group?WT group?,Cel treatedwildtype group?Cel+WT group?,TNF-?^-/-group,and Cel treated TNF-?^-/-group(Cel+TNF-?^-/-group).All mice were injected with H22 cells in the right flank to establisha solid tumor model.The effect of Cel treatment was measured by the body weight andtumor volume of each group.?2?Mice were divided into four groups:WT group,Cel+WT group,TNF-?^-/-group,andCel+TNF-?^-/-group.The expression of apoptosis related proteins,caspase-3,caspase-8,caspase-9 and PARP in solid tumor tissues was detected by Western blotting afterhomogenizing the solid tumor tissues.?3?Mice were divided into four groups:WT group,Cel+WT group,TNF-?^-/-group,andCel+TNF-?^-/-group.The expression of NF-?B signaling pathway related proteinNF-?B-P65 and I?B?in the solid tumor tissues was detected by Western blotting,qRT-PCR and immunohistochemistry.?4?Mice were divided into four groups:WT group,Cel+WT group,TNF-?^-/-group,andCel+TNF-?^-/-group.The expression of TNF-?in solid tumor tissues of each group wasdetected by Western blotting and qRT-PCR.?5?Mice were divided into four groups:WT group,Cel+WT group,TNF-?^-/-group,andCel+TNF-?^-/-group.The expression of XIAP and cIAP in the solid tumor tissues of micewas detected by Western blotting and qRT-PCR.Results?1?Cel can significantly reduce the volume of solid tumor and inhibit the growth of solidtumor in mice.?2?Compared with vehicle treated WT mice,both the cleavage of PARP and degradation ofCaspase-3,Caspase-8 and caspase-9 were significantly increased in Cel+WT mice.?3?Compared with vehicle treated WT mice,the inhibition of apoptotic proteins XIAP inCel+WT mice was significantly decreased.?4?Compared with the vehicle treated WT mice,the expression of TNF-?was significantlydecreased in Cel+WT mice.?5?Compared with the vehicle treated WT mice,the activation of NF-?B signaling pathwayin Cel+WT mice was inhibited.Conclusions?1?The presence of TNF-?can enhance the inhibition of Cel on the growth of solid tumorsand promote the apoptosis of tumor cells in vivo.?2?Cel may inhibit the activation of NF-?B signaling pathway and inhibit the expression ofapoptosis inhibition related protein XIAP downstream of this pathway,and ultimatelypromote the apoptosis of tumor cells in vivo.