Study On The Mechanism Of Triptolide Against Renal Cancer

Background:Renal cell carcinoma(RCC)is one of the most common malignant tumors in humans.The incidence and mortality of RCC in China are still increasing.However,while the treatment regimens for RCC have progressed from the early era of cytokine therapy,to the modern era of targeted therapy and immunotherapy,these progresses have not yet translated into significant improvement in survival.Importantly,advanced RCC is still an incurable disease while the overall prognosis for RCC is still quite poor.Therefore,the development of new and more effective treatments for RCC remains an unmet urgent need.Celastrol is a compound extracted from Tripterygium wilfordii,which has been extensively studied due to its unique biological activities.Celastrol has been reported to exhibit significant preclinical anti-tumor effects on a variety of tumors,including lung cancer,breast cancer,liver cancer,identifying it as one of the promising agents for development new treatments for cancers.The current study is intended to investigate the potential of celastrol as an agent for treating RCC,either in the form of celastrol-based single agent therapies or in certain combinatorial therapies.Objective:This study intends to evaluate the killing effects of celastrol both in vitro on cultured renal cancer cells of various RCC cell lines and in the standard preclinical setting,i.e.the mouse subcutaneous RCC xenograft models;and to explore the underlying mechanism of these effects.Methods:(1)The growth or survival effects of celastrol on SN-12C,ACHN,TK-10,A498 and Renca renal carcinoma cell lines were accessed by a standard proliferation assay using lncucyte-ZOOM platform.(2)The mechanisms for the growth inhibitory effects of celastrol against RCC cells were interrogated by transcriptome analysis.(3)Annexin V/PI double staining was used to assess whether celastrol exhibit any significant apoptosis effects on the various types of RCC cell lines.(4)Western blotting was used to assess the expression levels of various proteins of intertests.(5)A subcutaneous RCC model based on the mouse Renca RCC cell line was used to evaluate the in vivo anti-RCC effect of celastrol.Specifically,in vivo,the seizes of tumors in control and celastrol-treated mice were monitored.In addition,samples were also taken at various time points for Tunel staining to assess the apoptotic-inducing effects of celastrol.(6)Flow cytometry was used to detect the expression of surface PD-L1 of subcutaneous renal cancer cells.(7)A subcutaneous RCC model based on the mouse Renca RCC cell line was used to evaluate the in vivo anti-RCC effect of celastrol combined with PD-L1 blockade.(8)Immunohistochemical technique(IHC)was used to detect the expression of a number of proteins of interest,including Ki67,p-ERK1/2,p-p90RSK,p-pGSK-3β,GSK-3β,PD-L1 on the RCC model.(9)The injury of celastrol combined with PD-L1 blockade on the liver and kidney of mice was detected by H&E staining,PAS staining and Tunel technique.(10)Biochemical analysis of celastrol combined with PD-L1 blockade on liver and kidney function in mice.DBIL,ALT,AST,ALP for liver function test and CREA,BUN for kidney function test.Results:(1)At celastrol 750 nM dose had a significant inhibitory effect on the proliferation or survival of all RCC cell line examined(p<0.001 for SN-12C;p<0.05 for ACHN;p<0.001 TK-10;p<0.05;for A498;and p<0.01 for Renca,respectively).(2)Treatment of SN-12C and TK-10 with celastrol resulted in a total of 399 overlapping differential expression genes(DEGs),including 255 up-regulated genes and 144 down-regulated genes.The results of cluster analysis of DEGs showed that the expression patterns of celastrol-treated group and control group could be completely separated.A GO enrichment analysis revealed that the DEGs were mainly enriched in the biological processes related to cell death or apoptosis.DEGs on the KEGG database were mainly enriched in signal pathways including TNF,NF-κB,MAPK,NOD-like receptors,chemokine.interaction between cytokines and cytokine receptors,antigen processing and presentation.In addition,DEGs were enriched in rheumatoid arthritis,bladder cancer disease.(3)Celastrol promoted apoptosis in all renal cancer cell lines examined.In addition,celatrrol significantly promoted the expression of Pro-caspase3 and increased the levels of cleaved PARP.(4)Celastrol inhibited the expression of a number of proteins related to TNF and NF-κB signaling pathway,including TNF,IL-1β,p-NF-kappa B and p-IκBalpha.(5)Celastrol significantly inhibited tumor growth in a mouse model for RCC model(p<0.001).(6)Celastrol resulted in a significantly elevation of the levels of apoptosis in mouse subcutaneous RCC model.(7)At celastrol 3 mg/kg dose which is effective for RCC model,however,it led to a significant reduction in body weights and the living condition was poor with adverse reactions such as sweating,hair removal,hematochezia and so on.At celastrol 1 mg/kg dose did not cause any significant adverse effects,but with poor anti-tumor effect.(8)Western blotting and IHC results showed that the expression of p-Raf,p-MEK,p-ERK1/2,p-p90RSK,p-GSK-3β and PD-L1 in RCC treated with celastrol increased significantly,while the expression of GSK-3β decreased.Flow cytometry showed that the expression of PD-L1 on the surface of mouse subcutaneous RCC model treated with celastrol was significantly higher than that of the control group(P<0.001).(9)Celastrol combined with PD-L1 blockade enhanced inhibitory effect on mouse RCC model:the tumor growth rate treated with 1 mg/kg celastrol combined with PD-Ll blockade group was significantly slower than that of control group,1 mg/kg celastrol group and PD-L1 blockade group(P<0.01).(10)Toxicity analysis of celastrol combined with PD-L1 blockade in mice:the body weight changes of mice during the period of treatment showed that there was no significant difference compare with control group,the living condition of mice was well and no obvious adverse reaction was found.The liver and kidney injury was not found in each group.(11)Compared with the control group,there were no difference in liver function indexes ALT,AST,DBIL and renal function indexes CREA and BUN with the combination therapy.However,the serum AST and DBIL in the 3 mg/kg celastrol group increased significantly(P<0.05).Conclusion:(1)Celastrol could promote the apoptosis of renal cancer cells by inhibiting NF-kappa B signaling pathway.(2)3mg/kg celastrol could significantly inhibit the growth of RCC tumors in mice,but with causing a significant reduction in body weight and some adverse reactions including depilation,sweating,hematochezia,liver function impairment.(3)Celastrol could activate MAPK/p90RSK/GSK-3β pathway and up-regulate the expression of PD-L1 in RCCs.(4)The combination of celastrol and PD-L1 blockade could lead to a significantly the growth inhibitory effect on RCC tumors without causing significant adverse reactions.In summary,celastrol could promote the apoptosis of renal cancer cells by inhibiting NF-κB pathway to achieve anti-tumor effect.Celastrol could inactivate GSK-3β by activating MAPK/p90RSK pathway,thus promoting the expression of PD-L1.Celastrol could increase the anti-RCC effect of a PD-L1 blockade-based treatment.Thus,overall,the results have suggested that celastrol represents a promising new agent for developing effective treatments for RCC.