5-aminolevulinic Acid-based Sonodynamic Therapy Induces The Apoptosis Of Osteosarcoma In Vitro And In Vivo

Osteosarcoma is a common malignant bone tumor which occurs mostly in young adults.Its morbidity is in the first place of primary bone tumors,threatening young people worldwide.Although the clinical diagnoses and treatment technologies have been continuously developped,malignant bone tumor still has very high mortality and disability rate,resulting about 60% 5-year survival rate.In this thesis,we performed an in-depth study of its molecular mechanisms and treatments,aiming to provide new ideas and theoretical basis for its cl inical treatment,as well as to improve patients' quality of life.Sonodynamic therapy(SDT)is a new treatment for tumors based on similar principle as photodynamic therapy.It overcomes the disadvantages of weak penetrability and thermal effect in photodynamic therapy,and has some additional advantages,such as tumor specific aggregation of sonosensitizers,good targeting capability,noninvasiveness,and repeatable ultrasound therapy with little toxic side effects,providing a broad application prospect for the clinical treatment of malignant tumors.In this study,low-intensity ultrasound combined with sonosensitizer was adopted to investigate the molecular mechanisms of rat osteosarcoma cells(UMR-106)apoptosis in vitro and in vivo.First,the optimal concentration of 5-ALA in osteosarcoma cells,the peak time of Protoporphyrin IX(Pp IX)transformation and the optimal time for ultrasound sonication were screened by the MTT method;the metabolic distribution of 5-ALA in UMR-106 cells was detected by a fluorescence microscopy;the apoptosis was detected by Hochest 33342 staining;the cell necrosis was examined by Propidine iodide(PI)staining;the cell apoptotic rate and necrotic rate were compared among different experimental groups;the morphologies of apoptotic cells were detected by a laser scanning confocal microscope after Annexin V-FITC / PI staining;the cell survival rate was detected by a flow cytometry;and changes of cell ultrastructure were observed by a transmission electron microscopy.It was founded that the peak time of Pp IX in cells was 6 h,2.0 m M was the best concentration of 5-ALA,the optimal time for ultrasonic irradiation was 7 min,and the best sound intensity of low-intensity ultrasound was 2.0 W/cm2.Incubation with 2.0 m M 5-ALA for 6 h and exposure to 2.0 W/cm2 low-intensity ultrasound for 7 min could significantly provoke cell apoptosis.In terms of analyzing the mechanism of cell apoptosis,this study examined the generation of reactive oxygen species(ROS)by a fluorescence spectrophotometer.We determined the changes of intracellular Calcium ion level using fluorescent probe staining.The changes of mitochondrial membrane potential(MMP)were analyzed by JC-1 staining.We found that the sonodynamic treatment for tumors mainly uses ultrasound penetration to activate Pp IX in tissues.This results in the production of ROS and Calcium overload,which induce destruction of the cell membrane and mitochondria,and the release of lysosome,causing the destruction of the molecular structure of nucleic acids in tumor cells.Expressions of apoptosis related proteins were detected by Western blotting,and it was found that expressions of Bax(Bcl-2 associated X protein),Cytochrome C(Cyt-C),Caspase-3 and Caspase-9 were up-regulated and the expression of B cell lymphoma-2(Bcl-2)was down-regulated in SDT group.DCFH-DA(2',7'-dichlorofluorescin diacetate)stainging showed that large amount of ROS was produced in the SDT group.A decrease in mitochondrial membrane potential of SDT group was detected by JC-1.The above results showed that endogenous mitochondrial apoptosis was the main pathway for SDT-induced apoptosis of osteosarcoma cell.Built on the above results,this study further investigated the effect of SDT on tumor growth of transplantation tumors in nude mice.Results showed that the growth of transplanted tumors in the SDT group was much slower.TUNEL(Terminal deoxynucleotidyl transferase-mediated d UTP nick end-labeling)test showed an increase in the apoptosis of tumor cells.PCNA(Proliferation cell nuclear antigen)staining showed that the cell proliferation was inhibited.On the other hand,the apoptosis rates in the Ultrasound group and the 5-ALA group had no significant difference compared with that of the Control group.The normal surrounding tissue was not greatly influenced by the SDT treatment.Immunohistochemical staining results showed that the positive expressions of Bax,Caspase-3,p53(Tumor protein p53)and other genes in the SDT group were significantly increased and the expression of Bcl-2 was down-regulated;transmission electron microscopy detected large amount of apoptotic tumo r cells in the SDT group,which further proved that ultrasound activates the sonosensitizer and then induces apoptosis of tumor cells,thereby inhibits the growth of tumors.In conclusion,this thesis study confirmed that SDT can induce apoptosis of osteosarcoma cells and inhibit the growth of osteosarcoma cells.Our results suggest that SDT may produce ROS and induce apoptosis of osteosarcoma tumor.possibly by mitochondria apoptosis pathway.Considering sonodynamic therapy is highly targeting and noninvasive,it shows a great potentail in the field of oncotherapy.