Mechanisms Of Action Of Triptolide Against Colorectal Cancer: Insights From Proteomic And Phosphoproteomic Analyses

Triptergium wilfordii Hook.f is a traditional Chinese medicine,it has the functions of clearing heat and detoxifying,relaxing muscles and promoting blood circulation,and is mainly used in the treatment of rheumatoid arthritis and lupus erythematosus.Triptolide,the major active component of Triptergium wilfordii Hook.f,which has anti-rheumatic,antibacterial,anti-inflammatory,immunomodulatory and anti-tumor effects,and can inhibit the metastasis and proliferation of colorectal cancer.However,the mechanism of the anti-colorectal cancer effect of triptolide remains unclear.In this study,proteomics was used to compare the expression changes of proteome and phosphorylated proteome in the triptolide treated group and the control group of colon cancer cell line HCT116.The differentially expressed proteins and peptides were screened out,and the key differentially expressed proteins and peptides were analyzed by subcellular localization,domain enrichment and gene enrichment.Through kinase prediction analysis,key kinases can be screened and potential therapeutic targets can be obtained,providing strong evidence for rational development of natural anticancer drugs.In our study,962 differentially expressed proteins were identified in colon cancer cells treated with triptolide.Compared with control cells,403 proteins were significantly increased(> 1.2-fold)and 559 proteins were significantly decreased(<0.83-fold)after treatment with triptolide.624 differentially expressed proteins were mainly distributed in the nucleus.Functional enrichment analysis showed that differentially expressed proteins were mainly concentrated in pathways related to cancer cell proliferation and migration,such as ribosome processing,protein synthesis and transcriptional regulation.Domain enrichment analysis showed that it was mainly concentrated in RNA recognition and protein kinase related domains.Four core modules and seven key proteins including BYSL were selected by MCODE and Cytohubba through the analysis of protein interaction network of differentially expressed proteins.A total of 3110(> 1.2-fold)up-regulated and 3161(<0.83-fold)down-regulated phosphorylated peptides were identified in HCT116 cells treated with triptolide.The proteins of differentially expressed phosphorylated peptides were mainly distributed in the nucleus,with a total of 1926.Functional enrichment analysis showed that differentially expressed phosphorylpeptides were mainly enriched in AMPK signal pathway,MAPK signal pathway,transcription,protein processing and modification related pathways.The domains mainly concentrated in protein kinase related domains.57 motifs,including CAMKK,ERK1/2 and tyrosine kinases,were identified by the analysis of phosphate motifs,and 41 newly identified motifs were discovered.Combined analysis of proteomics and phosphorylated proteomics showed that the differentially expressed proteins play a role mainly by causing kinase phosphorylation.The upstream kinase prediction analysis of phosphorylation sites showed that 27 kinases were activated and 30 kinases were inhibited.PRKAA1 kinase may be the key kinase of triptolide against colorectal cancer,and its corresponding AMPK signaling pathway may be the key mechanism of triptolide against colorectal cancer.Conclusions: In this study,proteomic and phosphorylated proteomic maps of triptolide against colorectal cancer were constructed at the cellular level.Quantitative proteomic screening identified 403 up-regulated protein and 259 down-regulated protein,and BYSL,PDCD11,IMP3 may be potential targets of triptolide against colorectal cancer.Phosphorylated proteomics identified 6271 differential expression of phosphorylated peptides,and obtained 27 activated kinase and 30 inhibited kinase.PRKAA1 may be a potential kinase target for the anti-colorectal cancer effect of triptolide,and its corresponding AMPK signaling pathway may be the key pathway.These findings may help shed light on the molecular mechanisms of triptolide against colorectal cancer and may guide the development of new treatments.