| Objective:Hepatocellular carcinoma(HCC)is the sixth most common malignant tumor globally,ranking as the third cancer-related deaths,which posing a great threat to people’s health[1].As a minimally invasive interventional treatment technology,Radiofrequency ablation(RFA)has a radical effect on HCC with diameter less than 3 cm,and the overall efficacy is equivalent to surgical resection,and the incidence of complications is lower[3].Unfortunately,due to the uneven distribution of ablation energy in the tumor,the efficiency of killing distal tumor cells is not sufficient,which may eventually lead to local recurrence,even enhance the invasive phenotype of cancer cells,and finally lead to poor prognosis[4].In this study,we constructed incomplete thermal ablation models of HCC cells in vivo and in vitro,then we explored the mechanism of residual HCC cells after incomplete RFA(iRFA),and evaluated the efficiency of a newly oral HSP90 inhibitor XL888 on HCC cells under incomplete RFA conditions,Our findings provide a new insight into the mechanism of the recurrence HCC after iRFA,and evaluate the efficiency of small molecule inhibitor XL888 combined with RFA on HCC cells.Methods:1.CCK8 was used to detect the cell activity after HT and XL888 treatment;2.Colony formation assay was used to detect the inhibitory effect of HT and drug on cell proliferation;3.Hoechst 33258 was used to detect the cell apoptosis after HT and drug treatment;4.Western blotting was used to detect the cell apoptosis;5.Construction of incomplete RFA model in vivo;6.Detection of mRNA level of target gene by RT qPCR;7.Detection of intracellular expression and localization of target protein by confocal immunoassay;8.Overexpression of target protein by cDNA plasmid;9.Analysis of protein complex formation by immunoprecipitation;10.The expression of heterologous protein were detected of by immunohistochemistry in tumor tissue;Results:1.Firstly,we evaluated the correlation between HSP90 and STAT3 in the tissues of liver cancer.The analysis of clinical HCC mRNA data in TCGA database showed that the correlation coefficient between Hsp90 and STAT3 was 0.32.2.The co-expression of HSP90 and STAT3 was increased in tumor.The expression of STAT3 was up-regulated in the samples with high expression of HSP90,and they were closely related to tumor stage and high AFP;3.The model of sublethal HT in vitro was constructed,and the cell activity was detected by CCK8 after different temperature and time gradient treatment.The cell activity was significantly inhibited at 48℃ and above,but decreased at 46℃.The results showed that the cell activity was almost unaffected under the condition of low temperature.Similarly,the treatment time of 10 min was selected as the condition of subsequent sublethal treatment.4.The interaction between HSP90 and STAT3 was enhanced under sublethal HT.IP results confirmed that there was interaction between HSP90 and STAT3 under physiological conditions,and HT increased the interaction between HSP90 and STAT3.The results showed that the expression of STAT3 protein was increased,and the expression of Mcl-1 and Bcl-xL,which were important downstream anti-apoptotic proteins,were also increased,and their expression was also confirmed at the mRNA level;5.The activation of STAT3 was increased and p-STAT3 was promoted into the nucleus under HT,the nuclear protein was extracted for Western blotting.The results of blotting showed that HT induced p-STAT3 into the nucleus,and enhanced transcription factor activity;6.Under the condition of HT,the combination of HSP90 and STAT3 showed anti-apoptotic activity in vivo.We successfully transplanted hepatoma cell lines in nude mice by subcutaneous tumorigenesis.When the maximum diameter of tumor reached 0.8 cm,the radiofrequency needle with effective radiofrequency diameter of 1 cm was used to puncture into mice.IRFA was performed under the condition of 5W and 60s,and then the tumor was dissected to see the transition zone around coagulation necrosis in the tumor,and the mice survived well without infection and other complications,indicating the successful construction of inRFA model in vivo;7.IRFA stimulated the transformation of tumor to malignant phenotype.The maximum diameter and vertical transverse diameter of tumor were measured every week.The results showed that the tumor volume in iRFA group was significantly smaller than that in the control group in the early stage,but the growth rate of tumor was significantly faster with the prolongation of feeding time.Although the volume was still smaller than that in the control group at the end of feeding,the growth rate had increased significantly.Ki-67 was detected by immunohistochemistry in iRFA.The results showed that with the increase of XL888 concentration and time,the inhibition of cell activity was gradually obvious;9.Heat treatment combined with XL888 promoted the apoptosis of hepatoma cells.After selecting 100nm as the treatment concentration,CCK8 was used to detect the cell activity after combined treatment.The results showed that the cell activity of combined treatment group was significantly lower than that of single drug and HT group,and with the increase of XL888 concentration,the killing effect of combined treatment group was more obvious.The results of 33258 staining showed that the combined treatment could induce nuclear pyknosis,suggesting that XL888 could enhance the heat sensitivity of HCC cells;10.XL888 combined with HT could promote the apoptosis of HCC cells by activating Bcl-2 pathway and inducing caspase 3 activation;Western blotting results showed that the changes of Mcl-1 and Bcl-xL,the important members of Bcl-2 family,were significant,and caspase 3 and PARP were also activated;11.The results of IP showed that the binding of HSP90 and STAT3 was significantly decreased after XL888 treatment,and the activities of STAT3 and p-STAT3 were significantly decreased after XL888 treatment;12.Combined treatment inhibited the entry of p-STAT3 into the nucleus,and the expression of downstream target proteins Mcl-1 and Bcl-xL was decreased;13.Combined treatment of XL888 and heat promoted apoptosis through STAT3.After overexpression of STAT3 combined treated HCC cells,CCK8 detection showed that the cell activity was restored and the expression of apoptosis related proteins was reduced compared with the combined treatment group;14.XL888 enhanced the killing effect of iRFA in vivo.Constructed iRFA model in vitro,and treated with XL888 by gavage at the same time.The tumor diameter of mice was measured regularly and the mice were killed 21 days later.The results showed that the tumor volume and weight of mice were higher than those of control group and single group 15.X1888 has no obvious damage to the normal liver and kidney tissues of mice.In terms of safety,the liver and kidney tissues of mice were isolated for immunohistochemistry.HE staining results showed that there was no obvious tissue damage and necrosis in all the treated groups.Conclusion:HSP90 and STAT3 were co-related in HCC tissues,and there is a direct binding effect between them in HCC cell lines.In addition,heat treatment can enhance the direct binding effect of HSP90 and STAT3,and the formation of HSP90-STAT3 complex could induce STAT3 activation and promote the translocation of p-STAT3 into nucleus,and increase the expression of STAT3 downstream anti-apoptotic proteins—Mcl-1 and Bcl-xL,finally inhibit the proliferation of HCC cells under heat shock.The combination of XL888 and HT can destroy the formation of HSP90-STAT3 complex,therefore enhance the expression of Bcl-2 family proteins,activated caspase 3 and PARP protein,and increase the heat sensitivity of HCC cells. |
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