Inhibitory Effect Of Exogenous Mitochondria On B16 Tumors In Mice And Its Mechanism

Malignant melanoma is one of the most malignant tumors in human skin tumors,and its mortality accounts for 80% of skin tumor mortality.The malignant degree of melanoma is related to the degree of mitochondrial abnormality.Experiments have shown that the delivery of exogenous healthy mitochondria to melanoma mice can significantly inhibit the proliferation of melanoma,and the mitochondria extracted from the liver of young mice have better tumor suppression effects than old mice.The mechanism of action is not clear.In addition,according to reports in the literature,there is a clear difference between female mitochondria and male mitochondria.Therefore,in this study,when exploring the mechanism of exogenous mitochondria to inhibit tumor proliferation,the mitochondria were divided into female mitochondria and male mitochondria for further research.In the in vitro experiment,first use cellular immunofluorescence to prove that exogenous mitochondria can enter B16 cells.Secondly,the difference between female mitochondria and male mitochondria was explored.The protein concentration,membrane potential and pyruvate dehydrogenase activity of the two mitochondria were measured.It was found that female mitochondria had higher pyruvate dehydrogenase activity.Finally,the Alma blue method and flow cytometry were used to explore the changes of B16 cell proliferation,apoptosis and cycle after the intervention of exogenous mitochondria.The results showed that when the mitochondrial protein concentration was 0-0.5 ?g/?L,it inhibited cell proliferation It was dose-dependent,and at 0.4 ?g/?L,the concentration was low and the inhibitory effect was good;the apoptosis rate increased and the cycle was blocked,and the female mitochondrial group had more significant results than the male mitochondrial group.The above results indicate that exogenous mitochondria can inhibit the proliferation of tumor cells,promote the increase of apoptosis and block the cycle,and that female mitochondria have a stronger effect of inhibiting tumor proliferation than male mitochondria.In the in vivo experiments,two tumor models were established—in situ subcutaneous tumors and metastatic lung tumors.In the subcutaneous tumor model,exogenous mitochondria are injected into the subcutaneous tumor model mice through the tail vein,and the effect of exogenous mitochondria on subcutaneous tumors is explored from the tumor volume and tumor weight.The results show that mitochondria can significantly inhibit the rapid growth of melanoma,and the female mitochondrial inhibitory effect is significantly stronger than male mitochondria.In order to explore the mechanism of action of exogenous mitochondria,this experiment examined substances related to glycolysis,including pyruvate kinase activity,lactic acid content,and ATP content.The results showed that all three were significantly reduced,indicating that exogenous mitochondria can inhibit glycolysis.An increase in apoptosis and autophagy was observed from electron microscopic observations.Therefore,the proteins associated with apoptosis and autophagy were detected by immunoblotting,and the expression of pro-apoptotic proteins Bax,Caspase-9,and Caspase-3 were significantly increased.And Mfn2,LC3?/LC3?,Parkin,PINK1 also have an increasing trend.The above results indicate that exogenous mitochondria can promote the apoptosis of tumor cells and increase the production of autophagy.In the mouse metastatic lung tumor model,it was found that exogenous mitochondria have the same effect on lung metastases and subcutaneous tumors.In order to further explore the mechanism of exogenous mitochondria to suppress tumors,using transcriptomics to find differential genes,it was found that exogenous mitochondria can significantly inhibit the expression of genes related to cell cycle.Among them,the nuclear gene FoxM1 is extremely down-regulated,which can be regulated by the mitochondrialnuclear reverse regulatory signal ROS via HIF-1?,thus speculating that exogenous mitochondria regulate the cell cycle through ROS/HIF-1?/FoxM1.In the differential gene function classification,multiple metabolic-related pathways were also found to be related to the effect of mitochondrial therapy.Therefore,the high-resolution mass spectrometry(HRMS)detection technology was used to screen the differential metabolites of mice with metastatic melanoma after injection of exogenous mitochondria.The results proved that some of the differential metabolite content related to lipid metabolism and sugar metabolism decreased.Lipid metabolism and sugar metabolism can provide tumors with the necessary substances for proliferation.It shows that exogenous mitochondria can prevent the formation of metabolites necessary for tumor cell proliferation in terms of inhibiting lipid metabolism and sugar metabolism;and by comparing the differential metabolites of female mitochondria and male mitochondria,it was found that male mitochondrial group and female mitochondrial group There is a clear difference in onecarbon metabolism.Based on the results of biochemical detection,transcriptomics and metabolomics,this study revealed that the possible mechanism of exogenous mitochondria inhibiting metastatic lung tumors is that after exogenous mitochondria enter tumor cells,HIF-1? degradation is promoted by reducing ROS content,Causes FoxM1 gene expression to be down-regulated,thereby blocking the cell cycle and inhibiting tumor cell metastasis.And it can also inhibit the metabolism of cancer cells and lipid metabolism,resulting in insufficient energy and fatty acids required for tumor cell proliferation,which leads to the inhibition of cell proliferation and apoptosis.The female mitochondria are superior to male mitochondria in inhibiting tumors because female mitochondria have a better effect on inhibiting one-carbon metabolism,and thus have a better effect on inhibiting tumor proliferation.