Treatment Of Osteosarcoma With Mild Microwave Ablation Combined With HSP90 Inhibitor And TGF-β1 Inhibitor

Part Ⅰ:Microwave ablation combined with HSP90 inhibitor(PF-04929113),TGF-β1 inhibitor(SB-525334)in the treatment of osteosarcoma in vitro.Objective To explore the combination of microwave ablation combined with TGF-β1 inhibitor(SB-525334)and HSP90 inhibitor(PF-04929113)at the lower ablation temperature,and to vernfy its tumor killing effection.Methods Osteosarcoma cells(VX2)were divided into 4 groups according to the temperature range:37 ℃ group,41 ℃ group,48 ℃ group,60 ℃ group.Each temperature group was set up in the Control group(Control group),HSP90 inhibitor(PF-04929113)treatment group(p group),TGF-pl inhibitor(SB-525334)treatment group(s group),HSP90 inhibitor(PF-04929113)and TGF-pl inhibitor(SB-525334)combination group(p+s group).The survival rate of each group was determined by MTT method,and the best combination of inhibitor combined with low temperature ablation was found.Calcein was used to detect cell death;flow cytometry was used to detect the proportion of apoptosis in each group.Cellμlar immunofluorescence was used to detect changes in target proteins:TGF-β1,Smad4,HSP90,Cytochrome C(Cyto-C),casepase3,and casepase9.Results The MTT results showed that the survival rate of VX2 cells in the 48℃group was 86.08%in the Control group,64.81%in the p group,63.18%in the s group,and 37.18%in the p+s group.There was no significant difference in survival rate among the other groups.The M(48 ℃)+p+s reached a similar rate to the 60 ℃M group(13.01%),and the apoptosis/necrosis rate was significantly higher than that of the microwave or microwave combined with an inhibitor group.Both calcein detection and flow cytometry showed consistent results with MTT.When microwave ablation(48oC)combined with TGF-β1 inhibitor(SB-525334)and HSP90 inhibitor(PF-04929113),the expression of HSP90,TGF-pl and Smad4 was not significantly enhanced,while the expression of Cyto-C,caspase 3 and caspase 9 in the apoptotic signaling pathway was enhanced.Conclusion Microwave ablation combined with TGF-β1 inhibitor(SB-525334)and HSP90 inhibitor(PF-04929113)has good tumor killing effection at 48 ℃,and can protect normal tissues around the tumor while killing tumor tissue.Part Ⅱ:The initial in vivo verification and mechanism study of mild microwave ablation combined with HSP90 inhibitor(PF-04929113)and TGF-β1 inhibitor(SB-525334)in the treatment of osteosarcoma.Objective To further demonstrate the efficacy of mild microwave ablation combined with TGF-β1 inhibitor(SB-525334)and HSP90 inhibitor(PF-04929113)in the treatment of osteosarcoma in vivo,and to study the molecular mechanism of this killing effect.Methods Animal experiments were performed using New Zealand white rabbits to establish a bone tumor model,which was divided into Control group,microwave ablation group(M group),microwave ablation + HSP90 inhibitor(PF-04929113)group(M+p group),microwave ablation + TGF-β1 inhibitor(SB-525334)group(M+s group),microwave ablation + HSP90 inhibitor(PF-04929113)+ TGF-β1 inhibitor(SB-525334)group(M+p+s group)When the tumor size was about 3x3 cm,the mice were treated with microwave,and the tumor size and body weight were recorded every other day.On the 21st day,the tumor tissues were taken for HE staining and target protein detection(Immunohistochemistry detection of TGF-β1,Smad 4,HSP90,Cyto-C,casepase3,casepase9).Results The tumor volume of each group increased within 0-21 days after microwave ablation.There was no significant difference in tumor volume between the groups before ablation,but the M+p+s group had the smallest volume and the Control group had the largest volume at 21 days after surgery.The weight of each group of tumor rabbits increased within 0-21 days after operation,but there was no significant difference in body weight between the groups at each time point.The target protein immunohistochemistry of tumor tissues showed that the expression of tumor growth-promoting factors TGF-β1,SMAD4 and HSP90 was decreased in M+p+s group,and the expression of pro-apoptotic proteases Cyto-C,caspase 3 and caspase 9 were increased.HE staining showed that the proportion of tumor tissue in the M+p+s group was significantly lower than that in the other groups.Conclusion Microwave ablation combined with TGF-β1 inhibitor(SB-525334)and HSP90 inhibitor(PF-04929113)has good tumor killing effect at 48 ℃,and can protect normal tissues around the tumor while killing tumor cells.Blocking TGF-β1 promotes tumor cell proliferation signaling pathway and HSP90 tumor cell protection pathway,and blocks the synergistic effect of these two signaling pathways,and promotes tumor cell apoptosis.