| Background:Gastric cancer is one of the most common malignant tumors of the digestive tract in China,and its morbidity and mortality are among the highest.Invasion and metastasis is a difficult problem in the treatment of gastric cancer.Therefore,it is particularly important to research and develop therapeutic strategies and anti-invasion and metastasis drugs for gastric cancer invasion and metastasis.It is very important to screen and develop new drugs that can inhibit the process of tumor invasion and metastasis and block the pathway of tumor metastasis from traditional Chinese medicine for controlling tumor invasion and metastasis and improving the prognosis and survival rate of patients.Therefore,it is very meaningful to find and develop anti-tumor traditional Chinese medicine.Screening and isolation of effective anti-tumor traditional Chinese medicine components plays an important role in the development and clinical application of anti-tumor traditional Chinese medicine.Celastrus orbiculatus Thunb,a Chinese herbal medicine of the genus Celastrus,is widely used in the treatment of rheumatoid arthritis,lumbago and leg pain.Our research group first found and confirmed that its ethyl acetate extract had anticancer activity,and then isolated and extracted its active components.Its development and application in clinical anti-cancer therapy can produce great economic and social benefits.However,the specific mechanism and effective components of anti-gastric cancer of Celastrus orbiculatus Thunb are still unclear.In this study,after identifying and extracting the effective parts of anti-gastric cancer of Celastrus orbiculatus Thunb,the effective components of anti-gastric cancer of Celastrus orbiculatus Thunb were isolated and determined in vitro.This study is of great significance for the further development and application of Celastrus orbiculatus Thunb.Objective:To investigate the effects of anti-tumor Chinese medicine Triptonoterpene on the apoptosis,invasion and metastasis of human gastric cancer cells,to clarify the specific active components of Chinese medicine Celastrus orbiculatus Thunb in anti-gastric cancer,and to clarify the specific mechanism of the diterpenoid Celastrus orbiculatus Thunb in promoting gastric cancer cell apoptosis and inhibiting gastric cancer cell invasion and metastasis.To lay the experimental foundation for the development and clinical application of Celastrus orbiculatus Thunb.Methods:Routine culture of human gastric cancer BGC-823 and MKN-28 cell lines is to be used.Cell Count Kit 8(CCK-8)was used to detect the effects of Triptonoterpene on the proliferation activity of human gastric cancer BGC-823 and MKN-28 cells,and calculate the semi-inhibitory concentration(IC50)values of Triptonoterpene on different human gastric cancer cells at 24 and 48 hours.According to the IC50 values,three concentrations,low,medium,and high,were selected for subsequent experiments.Cell morphology was observed under a microscope to detect the effect of Triptonoterpene on the biological morphological changes of human gastric cancer cells;Flow cytometry and Hoechst 33342-pyridine iodide(PI)staining experiments were used to evaluate the effects of Triptonoterpene on the apoptosis of human gastric cancer BGC-823 and MKN-28 cells,respectively.The Mitochondrial Membrane Potential Detection Experiment(JC-1)was used to detect the effect of Triptonoterpene on the mitochondrial membrane potential of human gastric cancer cells BGC-823 and MKN-28.The effect of t Triptonoterpene on the levels of reactive oxygen species(ROS)in human gastric cancer BGC-823 and MKN-28 cells was detected by flow cytometry.Western blot was used to detect the expression of important proteins in the apoptosis related protein B-cell lymphoma 2(Bcl-2)family and the expression of cytochrome c,caspase-3,and cleaved caspase-3 proteins.The cell clone formation experiment was used to detect the effect of Triptonoterpene on the cloning ability of gastric cancer BGC-823 and MKN-28 cells.The cell adhesion experiment was used to detect the effect of Triptonoterpene on the adhesion between gastric cancer BGC-823 and MKN-28 cells and the cell matrix.Wound healing assay and Transwell invasion assay were used to investigate the effect of Triptonoterpene on the invasion and metastasis of gastric cancer BGC-823 and MKN-28 cells.High-content dynamic cell imaging technology was used to dynamically observe and detect the inhibitory effect of Triptonoterpene on the motility of gastric cancer BGC-823 and MKN-28 cells.Western blot was used to detect the expression of Epithelial-Mesenchymal Transition(EMT)-related proteins and Matrix metalloproteinases(MMPs)-related proteins in gastric cancer cells.Result:Compared with the control group,the proliferation rate of BGC-823 cells treated with Triptonoterpene(20,40,80,160 μM)for 24 h and 48 h was significantly decreased(P<0.01).The 24 h and 48 h IC50 of thymol terpene on human gastric cancer cells were 59.07 μM and 54.18 μM,respectively.Compared with the control group,the morphology of the cells in the Triptonoterpene groups(20,40,80 μM)changed significantly,and a large number of vacuoles appeared in the cells in the high concentration group,showing a honeycomb shape.Compared with the control group,the apoptosis rate of human gastric cancer cells in the Triptonoterpene group was significantly increased(P<0.05,P<0.01).Compared with the control group,the red fluorescence indicating JC-1 aggregation was weakened,and the green fluorescence indicating more monomeric JC-1 was enhanced in the cells of the Triptonoterpene group.Compared with the control group,the fluorescence intensity of ROS in the Triptonoterpene group was significantly increased(P<0.01).Compared with the control group,the number of human gastric cancer cells stained by PI in the Triptonoterpene group was significantly increased(P<0.01).Compared with the control group,the expression of Cytochrome c in cytoplasm was increased,the expression of Bcl-2 and B cell lymphoma-xL(Bcl-xL)protein was decreased,and the expression of Bcl-2 associated X protein(Bax)was increased after the intervention of Triptonoterpene.The expression of Caspase-3 and cleaved Caspase-3 protein was also increased(P<0.05,P<0.01).The results of CCK-8 assay and colony formation assay showed that compared with the control group,the proliferation activity of gastric cancer BGC-823 and MKN-28 cells treated with Triptonoterpene(20,40,80,160 μM)for 24 h and 48 h was significantly decreased(P<0.05,P<0.01).Cell adhesion assay showed that the adhesion between BGC-823 and MKN-28 cells and cell matrix was significantly inhibited after treated with Triptonoterpene(20,40,80μM)for 24 h(P<0.001).In addition,cell migration assay showed that the wound-healing ability of gastric cancer BGC-823 and MKN-28 cells was significantly inhibited after treated with Triptonoterpene(20,40,and 80μM)for 24 h(P<0.001).The results of Transwell assay showed that after the BGC-823 and MKN-28 cells were treated with Triptonoterpene(20,40,80 μM)for 24 h,the number of transmembrane cells was significantly reduced(P<0.001).The results of high-content cell dynamic tracking experiment showed that the range of the movement path of BGC-823 cells was reduced after the treatment of Triptonoterpene(20,40,80 μM).At the same time,the total migration distance and displacement of gastric cancer cells were also shortened(P<0.001).In addition,the changes of gastric cancer cell motility were also tracked by high-content imaging technology.We found that the average migration speed of gastric cancer cells was decreased in the Triptonoterpene group(P<0.001).The results of Western blot showed that the expression levels of EMT-related classical proteins in gastric cancer BGC-823 and MKN-28 cells were changed after the treatment of Triptonoterpene(20,40,80μM).The expression level of E-cadherin in gastric cancer BGC-823 and MKN-28 cells was increased,while the expression levels of lower N-cadherin,Vimentin and Slug were decreased(P<0.05).Moreover,the expressions of MMP-9,MMP-2 and TIMP-1 in gastric cancer BGC-823 and MKN-28 cells were also significantly decreased(P<0.05).Conclusion:As a monomeric compound isolated from the traditional Chinese medicine Celastrus orbiculatus Thunb,Triptonoterpene has significant proliferation inhibition and pro-apoptosis effects on human gastric cancer cells.Moreover,the migration and invasion of gastric cancer cells were significantly inhibited by Triptonoterpene.As a natural product of Celastrus orbiculatus Thunb,Triptonoterpene has a significant anti-gastric cancer effect,and it is likely to be one of the important components of the anti-tumor effective components in Celastrus orbiculatus Thunb.This study has clarified the material basis of Celastrus orbiculatus Thunb inhibiting gastric cancer,elucidated the effect and mechanism of Celastrus orbiculatus Thunb in inhibiting various malignant phenotypes of gastric cancer,and laid a certain experimental foundation for elucidating the anti-tumor molecular mechanism of Celastrus orbiculatus Thunb,and also laid an important foundation for the clinical application of Celastrus orbiculatus Thunb and the development of anti-gastric cancer traditional Chinese medicine. |
PDF Full Text Request