The Anti-proliferation Mechanism Of Triptolide On Colon Cancer Cells

Background: Triptolide is a triepoxide diterpenoid lactone compound, extractedfrom the plant Tripterygium wilfordii Hook F., is one of the main activeingredients of Tripterygium. Triptolide have anti-inflammatory, anti-fertility, andimmune suppression activity. Rencent studies indicated that triptolide also hassatisfactory anti-tumor activity, and it can inhibit a variety of tumor cellsproliferation and induce tumor cells apoptosis. Our previous study demonstratedthat triptolide inhibited colon cancer cells proliferation and migration. However,the mechanisms of its anti-proliferation effect remain unclear. This study was tofurther investigate the mechanism of triptolide inhibiting colon cancer cellsproliferation, and to provide a theoretical reference for the application oftriptolide.Objective: To assess the anti-proliferation effects of triptolide on colon cancer HT29and SW480cells and the molecular mechanisms.Methods:Part I: To investigate the effects of triptolide on proliferation, cell cycle andapoptosis in colon cancer cells HT29and SW480. MTT assay and BrdUincorporation experiment were used to observe the effects of triptolide on coloncancer cells’ proliferation. Flow cytometry analysis was employed to observe theinfluence of triptolde on colon cancer cell cycle and apoptosis after incubationwith triptolide for24h.Part II: Molecular mechanism. The microarray was used to detect the genesexperssion and provide clues to study the mechanisms; We used western blot toanalyse the phosphorylation level of proliferation-related protein ERK1/2, MEK1/2, MEK1, AKT, and then used qPCR to detect the effect of triptolide on ERKdownstream gene GABPα mRNA expression to explore the mechanism oftriptolide on cell proliferation; Western blot was used to determine the effects oftriptolide on the expression of cell cycle related proteins p21, p53, CDK2,p-CDK2, CDK4, CDK6, cyclin D1, and cyclin A1, and co-immunoprecipitationto observe the effect of triptolide on the interaction of p21and CDK4/CDK6inSW480cells, for the purpose of investigating the mechanism oftriptolide-induced G1arrest. To explore the effects of triptolide on the Wntpathway, SW480cells were incubated with or without triptolde for24h,cytoplasmic proteins and nuclear proteins were extracted from SW480cellsrespectively, and then the expression of β-catenin in the cytoplasm and nucleuswas detected by Western blot. QPCR was used to assay the β-catenin geneCTNNB1and its downstream target genes LEF1and TCF7mRNA expression.Results: 1Results of MTT showed that triptolide inhibited HT29and SW480cellsproliferation in a dose and time dependent manner, and it was more potent than5-FU and doxorubicin. The IC50was13.4nM and15.1nM respectively afterHT29or SW480cells were incubated with triptolide for72h. BrdU incorporationexperiments further confirmed that the anti-proliferation effects of150nMtriptolide on HT29and SW480cells. Incubating HT29or SW480cells withtriptolide for24h, the BrdU-positive rate was significantly decreased in bothcells (p <0.01). Flow cytometry results showed that triptolide induced HT29andSW480cells cell cycle G1phase arrest in a dose-dependent manner and had noobvious effects on cell apoptosis.2Microarray results suggest that incubation with triptolide for24h,196geneswere upregulated and706genes were downregulated in SW480cells, andpathway analysis revealed that down-regulated genes were mainly involved in theMAPK pathway, the cycle pathway, the Wnt pathway and the Ubiquitin mediatedproteolysis pathway. Triptolide significantly inhibited the phosphorylation ofERK and AKT and the expression of ERK downstream gene GABPα mRNA.However, we did not detect alteration of phosphorylation level of MEK1/2andMEK1. Those results suggested that the triptolide inhibited the cell proliferationby affecting the ERK and its downstream molecules. We did not detect the effectof triptolide on the expression of CDK4and CDK6, but observed that triptolideupregulated the expression of p21, cyclin D1, p53and CDK2, and downregulatedthe expression of cylinA1, inhibited the phosphorylation of CDK2.Co-immunoprecipitation results showed that triptolide enhanced p21binding toCDK4/CDK6, and blocked the function of CDK4/CDK6. So we hypothesizedthat triptolide blocked the function of CDK4/CDK6through upregulation of p21 expression and downregulation of cyclin A1to inhibit the cell through G1/Scheck point entry into S phase, which led to the cell cycle G1phase arrest. TheqPCR results showed that triptolide inhibited the β-catenin gene CTNNB1and itsdownstream target genes LEF1and TCF7mRNA expression. We did not observethe alteration of β-catenin expression in the total protein. But we found that theβ-catenin in nucleus was decreased. Therefore, we hypothesized that triptolideinhibited the expression of the LEF1/TCF7through inhibition of β-cateninentering into the nucleus, thereby inhibited many genes’ transcription andtranslation and then inhibited cell proliferation.Conclusion:1Triptolide significantly inhibited the proliferation of colon cancer cells.2Triptolide inhibited cell proliferation through affecting ERK and thedownstream molecules，induced cell cycle G1arrest through upregulating p21and downregulating cyclin A1expression, and affected the Wnt pathway throughblocking β-catenin into the nucleus.