The Inhibitory Effect And Mechanism Of Sonodynamic Therapy With HMME On UMR-106 Cells

PARTⅠAcute toxicity in UMR-106 cells induced by Sonodynamic Therapy with Hematoporphyrin Monomethyl Ether (HMME-SDT)Objective: The aims of the present study were to observe HMME as a sonosensitizer and investigate the acute toxicity in UMR 106 cells induced by HMME-SDT.Methods: The effects of various ultrasound irradiation time (0 s,10 s,30 s,60 s,90 s)and various concentrations (0μg/ml,10μg/ml,20μg/ml,40μg/ml,50μg/ml ) of HMME on the survival of UMR 106 cells were determined by MTT. To definite irradiation time and concentration of HMME.The cells were divided four groups, which are control (C), HMME, ultrasound (U) and SDT groups. The Acute toxicity was observed by MTT. The death modality was investigated by flowcytometry and the ultramicrostructure was observed by transmission electron microscope.Results: There was a significant difference(P<0.05) when the irradiation time were 30 s,60 s,90 s. However, there were no significant differences(P>0.05) for HMME. The irradiation time:10 s, HMME: 20ug/ml, the survival of the HMME-SDT group was significantly lower than the C,HMME and U groups (P<0.05). Necrosis and apoptosis be observed by flowcytometry and electron microscope.Conclusion: HMME is an effective sonosensitizer. HMME-SDT can significantly kill UMR-106 osteogenic sarcoma cells , there were necrosis and apoptosis .PARTⅡInhibition of osteogenic sarcoma UMR-106 cells induced by sonodynamic therapy with hematoporphyrin Monomethyl Ether (HMME-SDT)Objective: Inhibition of UMR-106 osteogenic sarcoma cells induced by SDT with HMME was studied to provide preliminary foundation for osteogenic sarcoma treatment by sonodynamic therapy (SDT).Methods: UMR-106 cells were divided into four groups including control (C), HMME, ultrasound (U) and SDT groups. The parameters of SDT were ultrasonic frequency: 10.50 MHz, intensity:0.5W/cm2, and irradiation time:10s,the concentration of HMME was 20μg/ml, respectively. The survival of the UMR-106 cells cells were determined by MTT assay method at various time (12 h, 24 h, 36 h, and 48 h). The mitochondrial membrane potential (reagent: Rhodamine 123), cellular reactive oxygen (reagent: DCFH-DA), the concentration changes of cellular calcium ion(reagent: Fluo-3-Am) were also investigated by flow cytometry correspondingly. Nucleolar morphous was observed by 33258 nucleoli stain.Results: The survival of HMME-SDT group were 69.42%(12 h), 50.10%(24 h), 44.37%(36 h), and 43.26%(48 h). There was a significant difference between the SDT group and C, HMME and U groups at same time (p< 0.01). There were significant differences between the HMME-SDT group and HMME,U and C groups on cellular reactive oxygen,△Ψm and cellular calcium ion (p<0.01). The results of karyotin stain demonstrated necrosis and apoptosis,nucelus concentration and floccuent change as well as nuclear margination.Conclusions: HMME-SDT can inhibit the proliferation of UMR-106 cells significantly, which may be associated with the elevated cellular reactive oxygen and cellular calcium ion, the damage of cell membrane.PARTⅢInhibition effect of SDT with HMME on inoculated UMR-106 transplantation tumorObjective: To investigate the distribution of HMME in the SD rats with inoculated UMR-106 transplantation tumor and the inhibition effect of SDT with HMME on the transplantation tumor. Methods: HMME was injected into SD rats intravenously and then the concentrations of HMME accumulated in the livers, muscle and tumors of inoculated rats at various time points were measured by HPLC. The optimal ultrasound time was decided by the distribution results. The rats with inoculated UMR-106 cells were divided into four groups , including contro(l C); HMME; ultrasonic( U); SDT groups. The antitumor effects were reflected by the changes of tumors on volumes and weights. The morphology changes of tumor tissues were observed using HE stain. The expression of PCNA and TUNNEL was analyzed by immunohistochemistry stain, and IPP image analysis.Results: After HMME intravenous injection through tail veins, the concentration of HMME in tumors achieved peak value at 3 h. Compared with other three groups, the volumes and weights of tumors treated by SDT were reduced and showed significant difference (p<0.01). HE staining demonstrated tumor tissues necrosis,nuclear condense,nuclear decomposition。The results of immunohistochemistry stain and IPP image analysis demonstrated the reduction of PCNA expression and increase of TUNNEL expression.Conclusion: HMME SDT can show effective inhibition on the proliferation of inoculated UMR-106 transplantation tumor, which may be related with its inhibitotory effect on the proliferation and induced apoptosis of the inoculated UMR-106 transplantation tumor.