| Background:Colon cancer is a common malignant disease,which mostly occurs at the junction of the rectum and the sigmoid colon,ranks third among malignant tumors.Due to unhealthy life style and other reasons,its incidence is still increasing.At present,the main therapeutic method for colon cancer is surgery.Although various comprehensive treatments and personalized treatments,such as chemotherapy and immunotherapy,can be provided according to the patient’s condition,chemotherapy is often accompanied by adverse reactions and drug resistance.Therefore,the development and search of new anti-tumor drugs is extremely important for improving the treatment of patients with colorectal cancer.Tanshinone ⅡA(Tan ⅡA)is one of the main fat-soluble compositions in the roots of Salvia miltiorrhiza Bge.It is the most abundant diterpene quinone in Salvia miltiorrhiza and is a natural inhibitor of monoacylglycerol lipase.Tan ⅡA has a variety of pharmacological effects,including anti-inflammatory,anti-cancer,anti-atherosclerotic activities,and cardiovascular protection.At present,the anti-tumor activity of Tan ⅡA has been reported in a variety of cancer cells,such as cell cycle arrest,apoptosis,autophagy and cell migration inhibition.Recently,studies have shown that Tan ⅡA can suppressed colon cancer development.However,the mechanism is till unclear.Therefore,this study intends to explore the mechanism of Tan ⅡA in colorectal cancer progression.Firstly,we evaluated the effect of Tan ⅡA on the proliferation,invasion and metastasis of colorectal cancer cells.Secondly,we studied the protein changes affected by Tan ⅡA,and analyzed related signal pathways,through quantitative proteomics analysis.Thirdly,the signal pathways of the specific effects of Tan ⅡA were studied to clarify the specific mechanism of Tan ⅡA and provide an experimental basis for the clinical application of Tan ⅡA.Methods:1.Evaluate the concentration dependent and time dependent effects of Tan ⅡA on colon cancer cells by CCK-8 experiment.2.Through CCK-8,cell count,clony formation and nude mouse xenograft experiments,the effects of selected concentrations of Tan ⅡA on the proliferation of colon cancer cells were studied.3.Study the effect of Tan ⅡA on the migration and invasion of colon cancer cells,through transwell experiment.4.Detect the effect of Tan ⅡA on the apoptosis of colon cancer cells by flow cytometry.5.Detect the level of GSH,NADPH and ATP in the cells,to explore the effect of Tan ⅡA on the intracellular redox homeostasis and ROS level.6.Exploring the changes in the proteome of colon cancer cells by Tan ⅡA treatment through label-free proteomics strategy,and studying the influence of Tan ⅡA on intracellular signal pathways through bioinformatics analysis.7.Western blot,real-time PCR,luciferase reporter assay,and chromatin immunoprecipitation were used to detect the effect of Tan ⅡA on the Nrf2/ARE signaling pathway.8.Detect the effect of Tan ⅡA on the subcellular localization of Nrf2 by nuclear plasma separation experiment.9.Through the Nrf2 replenishment experiment,combined with flow cytometry and RT-PCR to detect the effect of Nrf2 on the inhibitory effect of Tan ⅡA on colon cancer cells.Results:1.Tan ⅡA inhibits colon cancer cells proliferation and tumor growth.By treating colon cancer cell lines HCT-116 and LoVo with different concentrations of Tan ⅡA for different time,it was found that Tan ⅡA had a time and dose dependent on the proliferation of colon cancer cells.The results of CCK-8,clony formation and cell proliferation showed that Tan ⅡA significantly inhibited the proliferation of colon cancer cells.The results of mice xenografts experiment also indicated that Tan ⅡA inhibited tumor growth.2.Tan ⅡA inhibits the invasion and migration of colon cancer cells.Through cell wound healing and transwell migration and invasion experiments,it was found that Tan ⅡA significantly inhibited the healing of scratches and reduced the number of cells passing through the transwell chamber.3.Tan ⅡA increases the level of ROS in colon cancer cells and promotes apoptosis.Through flow cytometry,it was found that Tan ⅡA significantly promoted the occurrence of colon cancer cell apoptosis.Further analysis found that Tan ⅡA can increase the level of intracellular ROS,while reducing intracellular GSH,NAPDH and ATP levels,and antioxidants treatment can inhibit the cell death and ROS increment caused by Tan ⅡA.Therefore,Tan ⅡA participates in the regulation of intracellular oxidation homeostasis.4.Tan ⅡA treatment affects the Nrf2/ARE signal pathway in cells.Proteomic study of cells in the control group and the Tan ⅡA treatment group,through the bioinformatics analysis of 122 differential expressed proteins,the results suggest that Tan ⅡA may affect the Nrf2/ARE signaling pathway,especially the expression of its downstream classic antioxidant genes,including NQO1,HO1,and GCLC.5.Tan ⅡA can cause cell death by inhibiting the Nrf2/ARE signaling pathway.Through RT-PCR and Western blot analysis,the inhibitory effect of Tan ⅡA on the expression of downstream genes of Nrf2 was confirmed.Reporter gene and chromatin immunoprecipitation experiments showed that Tan ⅡA inhibited the transcriptional activity of Nrf2.Since Nrf2 needs to be localized in the nucleus to perform its function,nucleus isolation experiment confirmed that Tan ⅡA affects the nuclear localization of Nrf2.Further research found that Nrf2 expressed in the nucleus can inhibit the cell death and the increase of ROS caused by Tan ⅡA,and it can also inhibit the down-regulation of antioxidant gene expression caused by Tan ⅡA.Conclusion:The results of the study show that Tan ⅡA as a traditional Chinese medicine extract has anti-cancer effects.It can induce apoptosis by increasing the level of ROS in cells and inhibit the metastasis and invasion ability of colon cancer cells.Hence,Tan ⅡA inhibits the expression of antioxidant genes by inhibiting the Nrf2/ARE signaling pathway,thereby breaking the mechanism of cell apoptosis caused by the oxidation homeostasis in the cell.Therefore,the elucidation of this mechanism provides a theoretical and experimental basis for the treatment of Tan ⅡA for colorectal cancer,which is of great significance. |
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