Protoporphyrin ? On S180 And L1210 Tumor Cell DNA Damage And Its Mechanism

The porphyrin componds have been used as photosensitizer in clinical diagnosis and treatment of malignant tumar over past 20 years, and hematoporphyrin derivatives (HpD) is the most typical among them. ProtoporphyrinⅨ(PpⅨ)is an efficient component of HpD. The biosynthesis inside the body originates from 8-aminolevulinic acid (5-ALA) through the effect of a series of enzymes, and finally generates endogenous PpⅨin mitochondria. Previois research has shown that exogenous PpⅨcan specifically concentrate on tumor cell result in killing effect, which involves different cell structures, such as cell membrane damage, the change of organelles structure and DNA damage. As an important genetic material of cell, the structure and function of DNA changes under the action of exogenous PpⅨ,and further influences the gene expression and regulation, inhibits and kills the tumor cell.This thesis was supported by the National Natural Science Foundation of China (Grant No. 30270383 and No.10904087).Based on the previous work, we studied the damage of DNA by applying exogenous PpⅨto tumor cells S180 and L1210 in vitro culture. Our present exprimental results were as follows.1.By using fluorescence microscope, it was observed that when the concentration of PpⅨwas 0.1,1,and 10μg/ml, respectively, both tumor cells S180 and L1210 showed different levels of damages, such as the emergence of apoptotic body, and the nuclear damage becomes more serious with the increasing of the concentration of PpⅨ.2. Mitochondrial membrane potential of tumor cells S180 and L1210 was detected by Rhodamine 123 with the concentration of PpⅨin 0.1μg/ml,1μg/ml and 10μg/ml.The result indicated that mitochondrial membrane potential of treatment groups declined sharply compared wih the control group.3.Different concentrations of PpⅨwere incubated with tumor cells S180 and L1210 for 1 h,3 h, 5 h and 9 h, respectively. By using single cell gel electrophoresis, it was observed that the DNA damage was the most obvious at 1 h and 3 h incubations, and that 5 h later, for the low concentration PpⅨgroup, the damage of cell nucleus has been repaired to some extent, and that 9h later, the DNA damage for all groups have been repaired gradually.4. S180 and L1210 tumor cells treated with exogenous PpⅨwas detected by kinetic measurement, which showed that endogenous PpⅨcontent were rising gradually with the increasing of PpIX concentration, content of PpⅨin each group cells reached a maximum at 3 h,then it decreased, content of PpⅨin cells reached a mininum at 5 h. The results suggested that the fact that the degree of DNA damage decreases at 5 h was related with the decrease of endogenous PpⅨcontent.5.In the presence of ciclosporin A, the mitochondria PT hole specificity inhibitor,when the PpⅨconcentrate was 10μg/ml and incubated with S180 and L1210 tumor cells at 1h, the degree of DNA damage significantly decreased. This result revealed that DNA damage was related with mitochondria damage.6. After different concentration of PpⅨtreated with S180 and L1210, using immunofluorescence staining, confocal laser scanning microscope,we observed that AIF which translocated from mitochondria to nucleus increased siginificantly when compared with the control group. When ciclosporin A was added, the releasing of AIF decreased. DNA damage also decreased, which was detected by single cell gel eletrophoresis. This indicated that the fact that AIF translocated to nucleus may be one of the reasons of the DNA fragmentation.