The Role Of Mitochondrial DNA Content Reduction In The Initiation And Development Of Colorectal Cancer

Part Ⅰ Detection of the copy number of mitochondrial DNA in colorectal cancerObjective Changes of mitochondrial DNA(mtDNA)copy number were widely reported in colorectal cancers.Both an increase and a decrease in mtDNA copy number had been reported.In order to detect the copy number of mitochondrial DNA in colorectal cancer,we designed the experiment.Methods The expression of genes encoded by mitochondrial DNA in colorectalcancer and normal tissues was analyzed by GEPIA and immunohistochemistry.Results The GEPIA showed that MT-ND1,MT-ND2,MT-ND3,MT-CO1,MT-CO2,MT-CO3,MT-ATP6 and MT-ATP8 were lower expression in colorectal cancer and there were statistically significant differences between colorectal cancer and nomal tissue.Compared with benign tissues,the expression of COX I is lower in colorectal cancer tissues.Conclusion The above results both suggested that the copy number of mtDNA was decreased in colorectal cancer tissues.Part Ⅱ Establishment of mitochondrial DNA-reduced NCM460 cell model and detection of changes in biological behavior of this cell modelObjective Changes of mitochondrial DNA(mtDNA)copy number were widely reported in colorectal cancers.However,the role of mtDNA content in malignant transformation of human colonic intestinal epithelial cells remains largely obscure.Methods Cell exposure to low concentration of EB for a long time selectively reduces the mtDNA content,which is reversible after EB removal from the culture medium.NCM460 cells partially depleted of mtDNA were established as mtDNA-reduced cell model and the effects of mtDNA reduction in malignant transformation of human colonic intestinal epithelial cells were investigated.Results The growth rate of the mtDNA-reduced NCM460 cells was markedly slower compared with the parent and reverted cells.We found that the mtDNA-reduced NCM460 cells enhanced glucose uptake and generated markedly higher level of lactate,accompanied with slightly reduced ATP production compared with the parent and reverted cells.The activity of glycolytic enzymes,such as hexokinase(HK)and phosphofructokinase(PFK),was increased in mtDNA-reduced cells.These results suggested a switch to aerobic glycolysis in mtDNA-reduced cells,which helped the cells to gain a survival advantage.When mtDNA content was restored in these cells,metabolism returned to normal.What’s more,we demonstrated that mtDNA reduction in NCM460 promoted anoikis resistance and cell invasion.When the mtDNA content was restored,anoikis resistance and cell invasion were reversible.In addition,mtDNA reduction in NCM460 cells induced the activation of PI3K/AKT/mTOR pathway.LY294002,the PI3K inhibitor,was sufficient to restore sensitivity to detachment-induced apoptosis and curtail cell invasion.Conclusion MtDNA reduction might confer survival and invasive advantage to NCM460 cells through PI3K/AKT/mTOR signaling.Part Ⅲ Establishment of mitochondrial DNA-reduced SW480 cell model and detection of changes in biological behavior of this cell modelObjective Mutations and reductions in mitochondrial DNA(mtDNA),often detected in many tumors,including colorectal cancer,may lead to mitochondrial dysfunction and further contribute to enhancing their malignant phenotypes.However,the roles of mtDNA abnormalities in colorectal cancer remain unknown.Methods Cell exposure to low concentration of EB for a long time selectively reduces the mtDNA content,which is reversible after EB removal from the culture medium.MtDNA-reduced cell model was established by partial depletion of mtDNA in SW480 cells and the effects of mtDNA reduction in colorectal cancer cells were investigated.Results We found that mitochondria of parent SW480 cells were elongated with complete membranc structures and transversely arranged ridges that were tidy and compact.Whereas the shape of mitochondria of mtDNA-reduced cells were changed,the membranes were damaged,ridge structures were disorganized or even invisible.Upon removal of EB,mitochondrial morphology was restored and the transversely arranged ridges reappeared.The ROS production and mitochondrial membrane potential was decreased in mtDNA-reduced cells.We found that mtDNA-reduced cells had enhanced glucose uptake and generated markedly higher level of lactate.These changes were accompanied by only a slight reduction in ATP production compared with the parent cells.Furthermore,the activity of the glycolytic enzymes,hexokinase(HK)and phosphofructokinase(PFK),was increased in mtDNA-reduced cells.These results suggested a switch to aerobic glycolysis in mtDNA-reduced cells,which helped the cells to gain a survival advantage.Notably,when mtDNA content was restored,metabolism returned to normal.In addition,the mtDNA-reduced cells were highly resistant to 5-fluorouracil-and oxaliplatin-induced apoptosis and this drug resistance was reversible following recovery of the mtDNA content.The mtDNA-reduced cells also had an enhanced invasion ability.We also found that the PI3K/Akt/mTOR pathway was activated in the mtDNA-reduced cells.This pathway might play a significant role in increased glucose uptake,drug resistance,enhanced invasion ability in the mtDNA-reduced cells.Conclusion Our results supported the notion that mtDNA reduction induced aerobic glycolysis,an enhanced invasion ability and a reversible apoptosis-resistant phenotype in SW480 cells,and that the PI3K/Akt/mTOR pathway might be involved in these phenotypes.