| Mitochondrial dysfunction of tumor cells is closely related to tumor cell survival,proliferation and metastasis,and plays an important role in maintaining the normal physiology of tumor cells.Mutations in hepatocellular carcinoma mitochondrial DNA at conserved sites such as cytochrome c oxidase and NADH dehydrogenase may inhibit oxidative phosphorylation of hepatocellular carcinoma cells and promote the occurrence and development of hepatocellular carcinoma.Studies have shown that exogenous mitochondrial transplantation can inhibit the proliferation of tumor cells and change their respiratory status,but its mechanism is not clear.It is also reported that there are differences between M-Mito and F-Mito in respiration capacity and mitochondrial activity.This project aims to explore the inhibitory effect and mechanism of exogenous mitochondria on H22 hepatocellular carcinoma,and compare the difference between the inhibitory effect of M-Mito and F-Mito on hepatocellular carcinoma,so as to provide a new strategy for the treatment of hepatocellular carcinoma.Firstly,we measured the mitochondrial membrane potential,SDH and Mito-ICDH activities of M-Mito and F-Mito in vitro.The results showed that the mitochondrial membrane potential,SDH and Mito-ICDH activities of F-Mito are slightly higher than those of M-Mito.According to the experimental results,we believe that the vitality of FMito is higher than that of M-Mito.Secondly,immunofluorescence was used to verify that H22 cells can take up exogenous mitochondria.Thirdly,the method of CCK-8 was used to determine the changes in the proliferation of H22 cells after treatment with 0～400 μg/m L M-Mito and F-Mito for 8 hours.The results showed that when the concentration of mitochondria was in 100～400 μg/m L,exogenous mitochondria could significantly inhibit the proliferation of H22 cells,and the inhibitory effect of F-Mito on the proliferation of H22 cells was better than that of M-Mito.Finally,flow cytometry was used to determine the changes in H22 cell cycle and apoptosis after H22 cells were treated with 200 μg/m L M-Mito and F-Mito for 4 hours.The results showed that exogenous mitochondria could arrest the H22 cell cycle and promote cell apoptosis.In addition,the ability of F-Mito to arrest cell cycle and promote cell apoptosis of H22 cells was better than M-Mito.The above results indicated that exogenous mitochondria might inhibit the proliferation of H22 cells by arresting the H22 cell cycle and promoting apoptosis in vitro,and that F-Mito is better than M-Mito in inhibiting tumor cell proliferating,which provide a foundation for experimental in vivo.Firstly,the H22 hepatocellular carcinoma subcutaneous transplantation tumor model was used to evaluate the inhibitory effects of M-Mito and F-Mito on the growth of H22 subcutaneous tumors in vivo.After 6 days of treatment,the tumor volume and weight of the exogenous mitochondria treatment group were significantly reduced compared with the model group,and the tumor volume and weight of the F-Mito treatment group decreased more compared with M-mito treatment group.Then,it was found that exogenous mitochondria could increase the necrotic area of tumor tissue,down-regulate Ki-67 expression in tumor tissue and induce tumor cell apoptosis in tumor tissue by HE staining,Ki-67 immunohistochemistry and TUNEL staining of tumor tissue.And the corresponding results of the F-Mito treatment group are better than those of the M-Mito treatment group.The above results indicated that exogenous mitochondria could inhibit the growth of H22 subcutaneous tumors in vivo,and the inhibitory effect of F-Mito on tumors is stronger than that of M-Mito.In order to explore the mechanism of exogenous mitochondria inhibiting the growth of H22 subcutaneous tumors,we first detected the change of redox,glycolysis and TCA cycle related indicators in tumor tissue.The results showed that SOD activity,CAT activity,GSH content and T-AOC in the exogenous mitochondrial treatment group were significantly increased,on the contrary,the content of ROS was significantly reduced.It showed that exogenous mitochondria could increase tumor tissue T-AOC by increasing SOD activity,CAT activity and GSH content,thereby reducing the high ROS content of tumor tissue itself,bringing it close to the normal level,so as to eliminate the protection of endogenous ROS on tumors.In addition,in the mitochondrial treatment group,HK activity,lactic acid content and ATP content related to aerobic glycolysis of tumor cells were significantly reduced.On the contrary,in the mitochondrial treatment group,the activities of PDH,SDH and Mito-ICDH which are related to the cell TCA cycle were increased significantly.These showed that the exogenous mitochondria not only inhibited the glycolysis of tumor cells,but also promoted the occurrence of the TCA cycle,which proved that the exogenous mitochondria could promote the transformation of tumor cell metabolism from aerobic glycolysis to oxidative phosphorylation in vivo.The results of flow cytometry and staining of histopathological sections both suggested that the molecular mechanism of exogenous mitochondria inhibiting tumor growth might be related to cell cycle and cell apoptosis.Therefore,we immediately used Western Blot(WB)to detect the expression of cell cycle-related proteins,apoptosis-related proteins and related regulatory factors.The results showed that exogenous mitochondria could significantly down-regulate the expression of cycle-related proteins,such as Fox M1,Cyclin D1,Cyclin A2,and CDK1,and the expression of HIF-1a was also significantly down-regulated,which is the regulatory factor of Fox M1.The stability of HIF-1a in the cytoplasm is regulated by ROS and SDH that regulate the stability of HIF-1a by PHD2.Results of WB showed that exogenous mitochondria can up-regulate the expression of PHD2.Combined with the effect of exogenous mitochondria on tumor cell ROS and SDH,the results showed that exogenous mitochondria can promote the degradation of HIF-1 in the cytoplasm,down-regulate the expression of Fox M1,and then down-regulate the expression of Cyclin D1,Cyclin A2 and CDK1 to arrest the cell cycle by increasing the activity of SDH and reducing he content of ROS.In addition,exogenous mitochondria could also down-regulate the expression of p-Bad and Bcl-2,and up-regulate the expression of Bax,Caspase-9 and Caspase-3.The phosphorylation level of p-Bad is regulated by glycolysis.Combined with the effect of exogenous mitochondria on glycolytic of tumor cells,the results showed that exogenous mitochondria could inhibit glycolysis to promote the dephosphorylation of p-Bad,down-regulate the expression of Bcl-2,and promote the accumulation of Bax,thereby up-regulating the expression of Casepase-9 and Caspase-3 to promote cell apoptosis.In the corresponding results,the data of the F-Mito treatment group was better than that of the M-Mito treatment group.In summary,exogenous mitochondria could inhibit the proliferation of H22 cells in vitro and in vivo.The mechanism might be that exogenous mitochondria could increase tumor cell SDH activity and decrease ROS content to promote the degradation of HIF-1a in the cytoplasm,down-regulate the expression of the nuclear gene Fox M1,then down-regulate the expression of genes which are related to cell cycle to arrest tumor cells cycle.In addition,the mechanism of exogenous mitochondria inhibiting tumor growth might also be that exogenous mitochondria inhibited glycolysis of tumor cell to promote tumor cell apoptosis through the p-Bad/Bcl-2/Bax/Caspase-9/Caspase-3 signaling pathway.In addition,the inhibitory effect of F-Mito on tumor growth was better than that of M-Mito,which may be related to the fact that the activity of F-Mito was higher than that of M-Mito. |
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