The Synthesis Of Mitochondrial Targeting Coumarin And Their Antitumor Effects And Radiation Sensitization

Objective:Coumarin derivatives have good antitumor potential,but mitochondria targeted-coumarin has not been reported.In this paper,mitochondria targeted-coumarin with various targeting groups were synthesized and their antitumor and radio sensitizing effects were measuredMethods:We obtained mitochondrial-targeted coumarin through brominating of 6-methyl coumarin,followed by coupling the brominated product with a targeting group.MTT assay was used to detect the inhibitory effect of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups on tumor cells and the toxicity to normal cells A31 cells.Clone formation assay was used to detect the proliferation effect of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups on tumor cells.With multi-target single-hit model,the radiosensitization effect of 6-(4-(dimethylamino)pyridine)methyl coumarin was investigated through sensitizing enhancement ratio(SER).DCFH-DA method was used to detect the influence on tumor cells ROS levels of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups.Rh123 method was used to detect the influence on tumor cells mitochondria membrane potential of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups.Rh-2-AM method was used to detect the influence on tumor cells mitochondria Ca2+ concentration of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups.Hoechst 33258 was used to detect the influence on tumor cells apoptosis of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups.NAO method was used to detect the influence on tumor cells mitochondria mass of untargeted coumarin and mitochondrial targeted-coumarin with various targeting groups.Results:We confirmed the structure of the targeted compounds through mass spectrometry and nuclear magnetic resonance spectroscopy.The MTT results showed that mitochondrial targeted-coumarin with various targeting groups can effectively reduce the survival rate of tumor cells and they have no apparently toxicity to normal cells(A31 cells)in the dose range from 50 ?mol/L to 500 ?mol/L,while untargeted coumarin has no effect on the survival rate of.tumor cels.The clone formation results showed that mitochondrial targeted-coumarin with various targeting groups could effectively inhibit the proliferation of tumor cells,while untargeted coumarin has no effect on the proliferation of tumor cells.The results also showed that the radiosensitization ratio of the compound at the dose of IC20 was 1.58.The ROS results showed that 6-(4-(dimethylamine)pyridine)methyl coumarin significantly increased the ROS level in A549 cells compared with the irradiation group.While compared with the control group,both 6-(nicotinamide)methyl coumarin and 6-(triphenylphosphine)methyl coumarin significantly increased the cancer cells within the ROS levels,however untargeted coumarin has no significantly influence on the ROS levels of tumor cells.The Rh123 experiment results demonstrated that both 6-(nicotinamide)methyl coumarin and 6-(triphenylphosphine)methyl coumarin were effective in reducing the mitochondrial membrane potential of tumor cells compared with the control group,while untargeted coumarin has no significantly influence on the mitochondrial membrane potential of tumor cells.The results of detecting mitochondrial Ca2+ concentration showed that 6-(triphenylphosphine)methyl coumarin significantly increased the mitochondrial Ca2+concentration in HeLa cells,while untargeted coumarin has no significantly influence on mitochondrial Ca2+ concentration.The results of Hoechst 33258 staining experiment demonstrated that 6-(nicotinamide)methyl coumarin and 6-(triphenylphosphine)methyl coumarin could induce tumor cells apoptosis,while untargeted coumarin is not able to induce HeLa cells apoptosis effectively.The result of NAO staining experiment showed that 6-(triphenylphosphine)methyl coumarin effectively reduced the mitochondrial mass of HeLa cells,while untargeted coumarin has no influence on the mitochondrial mass of HeLa cells.Conclusion:untargeted coumarin cannot inhibit the survival rate,proliferation and apoptosis of HeLa cells.6-(4-(dimethylamine)pyridine)methyl coumarin significantly increased the sensitivity of A549 cells to radiation.By promoting the level of ROS and reduced tumor cell mitochondrial membrane potential,6-(nicotinamide)methyl coumarin promoted cancer cell apoptosis.6-(triphenylphosphine)methyl coumarin can induce tumor cell apoptosis through reducing mitochondrial membrane potential of cancer cells,increasing the level of ROS in cancer cells,increasing mitochondrial Ca2+ concentration in cancer cells and decreasing mitochondria mass in cancer cells.Therefore,through designing untargeted coumarin to mitochondrial targeting drugs improved its antitumor activity.