Antitumor Activity Of Oridonin Based On Energy Metabolism In Vitro

At present,the incidence and mortality of cancer are increasing,and it has become the main disease endangering human health.Traditional treatment mainly includes surgery,chemotherapy and radiotherapy,but it often has certain toxic and side effects,such as bone marrow suppression,cardiotoxicity,liver and kidney damage,etc.Traditional Chinese medicine（TCM）plays an important role in the treatment of cancer by exerting its anti-tumor effect through multiple ways and multiple targets.Oridonin is a natural carbon-containing compound of kaurene tetracyclic diterpenoids,which has attracted much attention because of its multi-target and various anti-tumor mechanisms.Tumor cells have a special energy metabolism mode-aerobic glycolysis,that is,tumor cells can still provide energy through glycolysis under normal oxygen conditions.It is found that the anti-tumor effect of oridonin is closely related to the energy metabolism pathway PI3K/AKT,glycolytic enzyme regulator hypoxia inducible factor 1α（HIF-1α）,glucose transporter 1（GLUT-1）,etc.Therefore,based on energy metabolism,this study studied the antitumor effect of oridonin,and preliminarily discussed the relationship between its antitumor effect and energy metabolism.In this experiment,the antitumor activity and mechanism of oridonin were studied in vitro from the perspective of energy metabolism.The main experimental methods are as followed:1.The effects of oridonin on the viability of human gastric cancer cell SGC-7901,human breast cancer cell MCF-7 and human umbilical vein endothelial cell HUVEC were detected by MTT method,and the appropriate concentration was selected according to IC50 value;2.The growth curve method was used to detect the effects of oridonin on the proliferation of three kinds of cells;3.The effects of oridonin on three cell morphology were observed by HE staining;4.Flow cytometry（PI single staining）was used to detect the effects of oridonin on the cell cycle of three kinds of cells;5.Flow cytometry（Annexin V-FITC/PI double staining method）was used to detect the effect of oridonin on the apoptosis rate of three kinds of cells;6.The kit was used to detect the effects of oridonin on the activities of energy metabolism related enzymes including hexokinase（HK）,phosphofructokinase（PFK）,pyruvate kinase（PK）,lactate dehydrogenase（LDH）and succinate dehydrogenase（SDH）,the ATP content and glucose consumption of energy metabolites in three kinds of cells.7.Western blot was used to detect the effects of oridonin on the expression levels of HIF-1α,GLUT-1 and monocarboxylic acid transporter 4（MCT-4）in three kinds of cells.The experimental results are as follows:1.MTT results show that oridonin can inhibit the proliferation of SGC-7901,MCF-7 and HUVEC cells in a dose-dependent manner,with IC50 values of 14.240 μmol/L,11.525 μmol/L and 23.132μmol/L,respectively.Combining the IC50 values of three kinds of cells,14 μmol/L oridonin was selected as the dose concentration in the follow-up experiment.2.The results of growth curve show that oridonin can inhibit the proliferation of three kinds of cells,and the inhibition effect is obvious at 48 h.The inhibition effect of oridonin on the proliferation of tumor cells SGC-7901 and MCF-7 is stronger than that of HUVEC.3.HE staining results showed that oridonin significantly changed the morphology of three kinds of cells,and the influence of oridonin on the number and morphology of SGC-7901 and MCF-7 cells was stronger than that of HUVEC.4.The results of cell cycle showed that oridonin could significantly increase the proportion of S phase in SGC-7901 cells（P<0.01）;The ratio of G0/G1 phase and G2/M phase decreased significantly（P<0.01,P<0.05）.The ratio of G0/G1 phase and S phase in MCF-7 cells increased significantly（P<0.01）,and the ratio of G2/M phase decreased significantly（P<0.01）,and the ratio of G0/G1 phase in HUVEC cells increased significantly（P<0.01,P<0.05）.The blocking effect of oridonin on S phase of MCF-7 cells and G0/G1 phase of HUVEC cells was not significantly different from that of 5-fluorouracil（5-FU）and metformin（P>0.05）.5.The results of apoptosis detection showed that oridonin significantly increased the apoptosis rates of the three kinds of cells（P<0.05,P<0.01）.Its apoptosis-promoting effect on SGC-7901 cells is stronger than that of metformin（P<0.01）,while that on MCF-7 cells is not significantly different from that of 5-FU（P>0.05）,that on HUVEC cells is weaker than that of metformin and 5-FU（P<0.01）,and that on two kinds of tumor cells is stronger than that of HUVEC cells.6.The detection results of energy metabolism-related indicators showed that oridonin could significantly reduce the glucose consumption,HK,PFK,PK,LDH,SDH enzyme activities and metabolite ATP content of SGC-7901 and MCF-7 tumor cells（P<0.01,P<0.05）,its effect on reducing the activities of glycolytic enzymes HK,PFK,PK and the content of energy metabolite ATP in SGC-7901 cells was stronger than that of 5-FU and metformin（P<0.05,P<0.01）.Its effect on reducing the activity of PK enzyme and the content of energy metabolite ATP in MCF-7 cells was stronger than that of 5-FU and metformin（P<0.01）.oridonin could significantly reduce PK,LDH,SDH enzyme activities and the content of energy metabolite ATP in HUVEC cells（P<0.05,P<0.01）,and its effect on reducing glucose consumption,HK and PFK enzyme activities was similar to that of the control group.There was no significant difference（P>0.05）.Oridonin has a significantly stronger effect on reducing glucose consumption,glycolytic enzyme activity and ATP content of two tumor cells than HUVEC cells,indicating that it mainly inhibits tumor cell production by affecting certain links in the glycolysis process.7.Western blot showed that oridonin could significantly reduce the expression of HIF-1α,GLUT-1 and MCT-4 protein in SGC-7901 and MCF-7 tumor cells（P<0.01）,and its effect on reducing the expression of MCT-4 protein in SGC-7901 cells was significantly stronger than that of 5-FU and metformin（P<0.01）.The effect of reducing HIF1α and MCT-4 protein expression in MCF-7 cells was significantly stronger than that of 5-FU（P<0.01）.Compared with the control group,oridonin can significantly reduce the expression of HIF-1α and MCT-4 protein in HUVEC cells（P<0.01）,but has no effect on the expression of GLUT-1 protein（P>0.05）,and its effect on reducing the expression of MCT-4 protein is stronger than that of 5-FU and metformin（P<0.01）,which indicates that oridonin can significantly reduce the expression of HIF-la and MCT-4 protein in HUVEC cells.Therefore,it is concluded that oridonin can inhibit the proliferation,induce cell cycle arrest and promote apoptosis of SGC-7901,MCF-7 and HUVEC cells,and has an effect on SGC-7901 and MCF-7 tumor cells.It is stronger than HUVEC cells,and its mechanism of action is related to reducing the expression of HIF-1α,GLUT-1,and MCT-4,reducing the activity of enzymes related to energy metabolism,and reducing ATP content.It inhibits glycolysis in two tumor cells more than HUVEC cells.more obvious.