A Study On The Inhibition Effect Of Celastrol In Colorectal Cancer

In recent years,Chinese medicine for cancer prevention and treatment attracted widespread attention.Celastrol is a pharmacologically active ingredient initially isolated from the roots of the Tripterygium wilfordii plant.In clinical,Celastrol as a traditional drug,is generally used for the therapy of inflammatory and auto-immune disease,such as allergic asthma,amyotrophic lateral sclerosis and rheumatoid arthritis.An increasing number of studies demonstrated that Celastrol also could inhibit a variety of cancer cell growth,including breast cancer,hepatocellular carcinoma,bladder cancer,gastric cancer and prostate cancer.The purpose of this study is to systematically explore the effect of Celastrol on colorectal cancer and its specific molecular mechanism.The results of MTT and cell colony formation assay demonstrated that Celastrol could significantly inhibit SW480 and HCT116 cell growth.When the C57BL/6J-ApcMin/+(APCMin/+)mice and AOM/DSS mice were intraperitoneally injected with Celastrol,and the results showed that Celastrol exerted an obvious tumor growth inhibition in vivo.Moreover,Western blot and TUNEL assay indicated that Celastrol induced cell apoptosis in vitro and in vivo.We found that Celastrol could induce the degradation of β-catenin through the ubiquitin-proteasome system,which inhibited the activation of Wnt/p-catenin signaling pathway.In clinical,the great majority of colorectal cancers(>80%)have detected over-activation of Wnt/β-catenin signaling pathway.These suggested us that inhibiting the Wnt/β-catenin signaling pathway may be the mechanism of Celastrol-mediated inhibition effect on colorectal cancer.At present,previous reports have proved that the Hippo pathway genetically and functionally interacts with Wnt/β-catenin signalling.The Hippo pathway is a highly conservative signaling pathway involved in regulation of organ size.The main downstream effector,yes-associated protein(YAP),could interact with β-catenin,in order to regulate the intestinal regeneration as well as the tumorigenesis and progression of colorectal cancer.Our results also demonstrated that Celastrol-induced p-catenin degradation depended on YAP and its phosphorylation,and YAP was an indispensable component in the destruction complex of β-catenin.Recently,the liver kinase B1(LKB1)as a tumor suppressor,also known as serine-threonine kinase 11(STK11),has been reported to repress YAP activity via promoting YAP phosphorylation,nuclear exclusion,and proteasomal degradation.In SW480 and HCT116 cells,we used CRISPR-Cas9 system to establish the LKB1-deficient stable cell lines and the intestinal-specific LKB1-KO mice(established by Loxp-Cre system)were treated with AOM/DSS.With the treatment of Celastrol,the deficiency of LKB1 could promote the colorectal cancer cell growth and inhibit the degradation of p-catenin induced by Celastrol in vitro and in vivo,which impaired the anti-tumor effect of Celastrol.Moreover,Celastrol up-regulated the expression of heat shock factor 1(HSF1)and enhanced the transcriptional activity of LKB1.LKB1 activated its downstream factor-AMP-activated protein kinase a(AMPKa),to further phosphorylate YAP,which eventually promoted the degradation of β-catenin.In conclusion,our results demonstrated that Celastrol inhibited colorectal cancer cell growth in vitro and in vivo through the degradation of P-catenin via the ubiquitin-proteasome system.Importantly,we found the potential mechanism of P-catenin degradation induced by Celastrol was that Celastrol increased the expression of HSF1,enhanced the transcriptional activity of LKB1 and activated AMPKa which leaded to the phosphorylation of YAP.Phosphorylated YAP existed in the destruction complex of P-catenin and promoted the degradation of β-catenin.Interestingly,YAP and LKB1 played the essential roles in the progression of colorectal cancer and inhibition effect of Celastrol.All these results provide the important theoretical basis for Celastrol as a new drug in clinical treatement of colorectal cancer.Hippo pathway is a highly conservative signaling pathway related to the development of tissues and organs and maintaining the balance of cell proliferation and apoptosis,which has been demonstrated to be strongly linked to the tumorigenesis and tumor progression.As the major downstream effector of Hippo pathway,yes-associated protein(YAP),is a transcriptional activator of target genes that are involved in cell proliferation and survival.As an oncogene,YAP can promote cell growth and inhibit cell apoptosis.Another major downstream effector of Hippo pathway,transcriptional coactivators with PDZ-binding motif(TAZ),is nearly 60%homologous with YAP.Here,we assume that TAZ probably has the similar function to YAP.To test this issue,we established an inducible and a stable expression system of TAZ in T-Rex-293 and HEK293 cells,respectively.The results of cell growth curves and colony formation assay and tumor xenograft growth showed that overexpression of TAZ could promote cell growth in vitro and in vivo.Meanwhile,we found that up-regulated expression of TAZ could partially restore Celastrol-induced cell apoptosis.Induced overexpression of TAZ could upregulate its target genens including ankyrin repeat domain containing protein(ANKRD),cysteine rich 61(CYR61)and connective tissue growth factor(CTGF),increase the expression of Bcl-2,decrease the expression of Bax and activate the PI3K/Akt pathway,which may be the mechanism underlying anti-apoptosis of TAZ.All these findings indicated that TAZ acts as an oncogene that could be a key regulator of cell proliferation and apoptosis.