Ultrasound Combined With Protoporphyrin ¢ù Damage Effects And Mechanisms Of Human Breast Cancer Cell Line Mcf-7

Sonodynamic therapy (SDT) is based on the photodynamic therapy (PDT), which firstly put forwarded by Japanese researcher Umemura. The ultrasound with different intensity and frequency can activate cytotoxicity of the sonosensitizers. Sonosensitizers specialy retent in tumor combined with the ultrasound penetrate deeply into tissues can generate irreversible anti-tumor effect. Hematoporphyrin (Hp) and their derivatives(HpD) is the earliest sonosensitizers. ProtoporphyrinIX (PpⅨ) as an compound of hematoporphyrin derivatives, which has obvious anti-tumor effect when combine with ultrasound. Previously, the anticancer effect of PpⅨ-SDT mainly focus on the induction of cell apoptosis, the role of autophagy in the anticancer effect become the study spotlight in recent years following the development of the research in autophagy. Our research group early observed morphology of autophagy in cell apoptosis induction by SDT, therefrom it can be supposed that SDT can caused diverse death mode of cells. The purpose of this thesis aim to discuss the SDT damageeffect on the human breast cancer MCF-7 cells and the induction mechanism of autophagy, which can complement the mechanism of action in SDT and provide new thinking or settlement for clinical treatment.This paper is supported by the Foundation of Doctorialtutor. Based on the previous work, cell killing effects of PpⅨ-SDT on MCF-7 cells in vitro and the induction mechanism of autophagy were studied by diverse means of molecular biology technology, be involved with different microscopic structure and molecular levels.The present exprimental results are as follows:1. The results indicated that linear relation between concentration of PpⅨin the 0-400 nmol/L and fluorescence intensity were good. The intracellular content of PpⅨreached a maximum at 2.5 h with different initial concentrations such as 0.5μM,1μM and 2.5μM.2. By Laser scanning confocal microscope, the subcellular localization of PpⅨwas evaluated, which showed that the subcellular localization of PpⅨcoincidence with MitoTracker Green as the probe of mitochondrion subcellular localization.The PpⅨsubcellular localization is mitochondrion.3. The relative viability of MCF-7 cells treated with PpⅨ, ultrasound or ultrasound combined with PpⅨrespectively in order to screen out the optimum intensity of ultrasound and concentration of PpⅨin the PpⅨ-SDT. The result indicated that the damage of PpⅨon MCF-7 cells in a dose-dependent manner. When the concentration of PpIX is less than 2.5μM, it has no growth inhibition. The concentration of PpIX was 2.5μM showed slight cytotoxicity on MCF-7 cells. The concentration of PpIX was 5μM showed obvious cytotoxicity on MCF-7. The damage of ultrasound on MCF-7 cells in a intensity-dependent manner, the cell growth inhibition effects increase with the different intensity of ultrasound such as 1 W/cm2,2 W/cm2,3 W/cm2,4 W/cm2. PpⅨ-SDT compared to the PpⅨor ultrasound alone has obviously synergetic effect. So the concentration of 2.5μM PpⅨand 3 W/cm2 of ultrasound were choosed as the parameters for the future studies.4. Optical microscope observed the morphology of MCF-7 cells treated by PpⅨ-SDT, which showed that cell treated with PpⅨalone compared control has slight impair on MCF-7 cells. PpⅨ-SDT compared to the PpⅨor ultrasound alone has obviously serious effect on MCF-7 cells, it shows that the number and the morphology changed obviously, accompany with the incressed cell debris.5. Mitochondrial membrane potential of cell were analyzed using flow cytometry. The result indicated that mitochondrial membrane potential of MCF-7 cells were most affected by PpⅨ-SDT group. PpⅨ-SDT group shows more significant effect on mitochondrial membrane potential than PpⅨalone or ultrasound alone.6. The induction of autophagy was analysed by Acrine Orange(AO) fluorescent staining, which showed that it had more acidic vesicle organelles in PpⅨ-SDT group compared control. The PpⅨ-SDT group appeared autophagy.7. The expression of autophagy-related proteins was assessed by Western Blotting. The result showed that the phenomenon of LC3I conversion to LC3II appear in PpⅨ-SDT group. Furthermore the protein expression of Vps34, Cathepsin B and UVRAG increased in PpⅨ-SDT treated cells. These results indicated the MCF-7 cells treated by ultrasound combined PpⅨcan activate autophagy.