| Objective:1.To investigate the effect of the VEGFR2 inhibitor apatinib on the growth of human papillary thyroid cancer cells.2.In vitro experiments and BALB/c immunodeficient mouse papillary thyroid carcinoma xenograft model in vivo study of apatinib treatment of thyroid papillary carcinoma and its mechanism.3.To explore the role and mechanism of autophagy in the treatment of thyroid cancer cells with apatinib.The effect of combined use of autophagy inhibitor and apatinib on the growth of thyroid papillary carcinoma and its mechanism.Methods:1.The expression of VEGFR2 in tissues of patients with papillary thyroid carcinoma was detected by immunohistochemical staining and clinically relevant.Q-PCR and Western-blot were used to detect the expression of VEGFR2 mRNA and protein in tumor tissue and normal tissues of thyroid papillary carcinoma,and the expression of VEGFR mRNA and protein in different thyroid cell lines.2.The effect of apatinib on the proliferation of thyroid papillary carcinoma cell line K-1 and KTC-1 was detected by CCK-8 method at different concentrations and different times.The effects of apatinib on the migration and invasion of tumor cells were examined using scratch test and Transwell assay.The effect of apatinib on the apoptosis of thyroid papillary carcinoma cells was detected by Annexin V/PI double staining method.The effect of apatinib on the cell cycle was detected by PI staining.Protein level was detected by Western-blot method.The levels of Akt phosphorylation and the changes of apoptosis-related protein levels in K-1 and KTC-1 cells after treatment.3.The in vivo effect of apatinib on thyroid papillary carcinoma was established by establishing a tumor model of papillary thyroid carcinoma in BALB/c immunodeficient mice.4.The expression of autophagy marker protein LC3B in thyroid cancer cells after treatment with apatinib was detected by immunofluorescence.The papillary carcinoma cell lines K-1 and KTC-1 were examined by transmission electron microscopy after apatinib treatment.The formation of autophagosomes in the cytoplasm;the treatment of cells with apatinib combined with autophagy inhibitor chloroquine(HCQ)followed by Western-blot method to detect the expression level of autophagic marker protein LC3B.5.The autophagy inhibitor 3-MA was combined with apatinib to treat cells.The expression of LC3B in the cytoplasm was detected by immunofluorescence,and the formation of autophagosomes was observed by transmission electron microscopy.Thyroid papillary cancer cells were identified.Level of phagocytosis,and the effect of apatinib combined with 3-MA on the apoptosis of papillary thyroid carcinoma cells was detected by Annexin V/PI double staining;the expression of autophagy-related and apoptotic proteins was detected by Western-blot method.6.The expression of autophagy-related gene ATG5 was down-regulated by transfection of small interfering RNA ATG5,DMSO and apatinib were used again to treat the two groups of cells,and the expression of autophagy-related and apoptotic proteins was detected by Western-blot.Results:1.The expression level of VEGFR2 in tumor tissues of papillary thyroid carcinoma is significantly higher than that in adjacent normal tissues,and the expression level of VEGFR2 is related to tumor size,number of tumors,and lymph node metastasis.The mRNA and protein expression levels of papillary thyroid cancer cell line K-1 and KTC-1 VEGFR2 were significantly higher than those of normal thyroid follicular epithelial cells Nthy Ori3-1.2.The thyroid papillary carcinoma cell line K-1 and KTC-1 were studied.The results of CCK-8 showed that apatinib inhibited the proliferation of K-1 and KTC-1,and showed concentration and time dependence.Sex.The scratch test and Transwell experiment results show that apatinib can inhibit the migration and invasion of K-1 and KTC-1.Clone formation experiments showed that colony formation of apatinib cells was concentration-dependent.The assay by Annexin V/PI double staining assay showed that apatinib induced apoptosis and G0/G1 phase arrest in a concentration-dependent manner.Western-blot showed that apatinib can increase the phosphorylation level of VEGFR2,increase the expression of cleaved-PARP and Bax,decrease the expression of apoptosis-inhibiting protein Bcl-2,and down-regulate CyclinD 1 and P21.Increased.3.BALB/c immune-deficient mice were used as the research object.The thyroid papillary carcinoma cell line K-1 was used to establish the transplanted tumor model.The subcutaneous tumors were grown to 3*3*3mm3,oral apatinib 100mg/kg/day.After 4 weeks of continuous medication and 2 weeks of observation after discontinuation,the results showed that the transplant tumors of the apatinib group were significantly smaller than those of the control group.Immunohistochemical staining showed that the expression of VEGFR2,Ki-67,Bcl-2 was down-regulated in the apatinib group,and cleaved-PARP was up-regulated.4.Thyroid papillary carcinoma cell line K-1 and KTC-1 were studied.Immunofluorescence staining showed that the expression of autophagy marker protein LC3B in cytoplasm was significantly increased after apatinib treatment.Cells were observed under transmission electron microscope.The autophagic corpuscles in the cytoplasm increased significantly.Western-blot results showed that the expression of LC3B in apoptin and HCQ treated autophagy inhibitors decreased,the expression of autophagic substrate P62 was up-regulated,and the expression of apoptotic protein cleaved-PARP was up-regulated.5.The thyroid papillary carcinoma cell line K-1 was used as the research object.After the cells were treated with apatinib,the autophagy inhibitor 3-MA was used to inhibit autophagy,and Annexin V/PI double staining was used to detect cell apoptosis.The results show that 3-MA significantly enhances the ability of apatinib to induce K-1 apoptosis,and administration alone does not result in apoptosis of K-1.The results of Western-blot showed that 3-MA reduced the expression of LC3B.The combination of apatinib and 3-MA increased the expression of cleaved-PARP and down-regulated the expression of Bcl-2.6.ATG5 specifically interferes with ATG5 expression by inhibiting autophagy by transfecting the small interfering RNA ATG5.The cells were then treated with apatinib.The results of Western-blot showed that ATG5 expression was inhibited by the apoptotic cells.After treatment,the expression of apoptotic protein cleaved-PARP was significantly up-regulated.Conclustion:1.The expression level of VEGFR2 in papillary thyroid carcinoma is higher than that in normal thyroid tissue,and it is related to tumor size,multifocal and lymph node metastasis.2.In vivo and in vitro experiments confirmed that apatinib can inhibit the growth of thyroid papillary carcinoma and promote apoptosis through phosphorylation of VEGFR2 pathway.3.Apatinib can induce cytoprotective autophagy in thyroid cancer cells,autophagy inhibitor autophagy inhibitor in combination with apatinib can further inhibit the proliferation of thyroid papillary cancer cells,induce PARP/Bcl-2-mediated apoptosis. |
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