Study On Mitochondria Oxidate Damage And Mitochondrial DNA Mutation In Colorectal Cancer

Recently, more and more attentions are paid on the theory of oxidative damages to mitochondria and mtDNA in developments of diseases such as tumor.Mitochondria are one of the enzymatic sources of reactive oxygen species (ROS) and could also be a major target for ROS-mediated damage. ROS induce the functional and structural damage of mitochondria and may play an important role in the pathophysiology of cancer. Recent evidence has suggested an intimate link between an excessive generation of ROS and the development of cancer.It is conceivable to speculate that the mtDNA could be a major target for ROS-mediated damage for several reasons. First, mitochondria do not have a complex chromatin organization consisting of histone proteins, which may serve as a protectivebarrier against ROS. Second, mtDNA has a limited repair activity against DNA damage. Third, a large part of O2-, which is formed inside the mitochondria, cannot pass through the membranes. Hence, ROS damage may be contained largely within the mitochondria.Mitochondria contain closed circular, double-stranded DNA of 16.5 kb. Both strands of the mtDNA are transcribed. The mitochondrial genome encodes 13 polypeptides involved in oxidative phosphorylation, including 7 subunits of rotenone-sensitive NADH-ubiquinone oxidoreductase, 1 subunit of ubiquinol-cytochrome c oxidoreductase, 3 subunits of cytochrome c oxidase, and 2 subunits of complex V along with 22 tRNA and 2 rRNA subunits.In order to explore the relationship among the mtDNA mutation, mitochondrial damage and the development of colorectal cancer, we undertook corresponding experiments as followed:1. To investigate the antioxidant system of human colorectal cancer mitochondria, we used spectrometric method to measure the specific activity of scavenger enzymes of free radical in human colorectal cancer mitochondria, including SOD, CAT, GSH-Px. The content of MDA, one of prodution of ROS decompose, was also measured.2. With labeling of Rhodamine123 (Rh123), MMP wasdetected using FCM method. To discuss the mechanism that mitochondria as a potential selective therapeutic target for lipophilic cationic drugs.3. To evaluate the relationship between DNA damage and colorectal cancer development and progress, HPLC-EC method was used to detect the amount of 8-OH-dG in mtDNA and nDNA.4. In the current study, we investigated the mtDNA mutations of colorectal cancer to address the evidence of a role in tumorigenesis and discuss the physical basis, benefits, and limitations of mtDNA analysis as a diagnostic tool.The results are as follows:1. The activity of three scavenger enzymes of free radical including SOD,CAT and GSH-Px in mitochondria of colorectal cancer were remarkably reduced(P<0.01, 0.01