| Objective:1.To analyze the expression of APE1 in thyroid papillary carcinoma and the relationship between its the expression level and the clinicopathological characteristics and its clinical significance.2.To explore the synergistic effect of the APE1 redox inhibitor E3330 on vemurafenib in vitro.3.To reveal the mechanism of APE1 in the treatment tolerance of BRAF mutant thyroid papillary carcinoma with vemurafenib.4.To explore the synergistic effect of the APE1 redox inhibitor E3330 on vemurafenib in vivo.Methods:1.Search the TCGA database and collect the fresh specimens of papillary thyroid carcinoma so as to analyze the expression of APE1 protein in normal tissues and thyroid cancer tissues.2.Immunohistochemical staining was used to detect the expression of APE1 protein in thyroid papillary carcinoma tissues.Univariate and multivariate regression analysis was used to analyze the relationship between the expression of APE1 and clinicopathological features of thyroid papillary carcinoma patients and its clinical significance.3.The synergistic effect of E3330 on vemurafenib was tested in vitro.The experiment of CCK-8,plate cloning,PI staining,Brdu staining,Annexin V-PI double staining,JC-1 staining,Transwell test were used to detect the effects of E333 and vemurafenib on the proliferation,cycle,apoptosis,mitochondrial function,migration and invasion of BCPAP and K-1 cells.4.In vitro experiments was used to verified that E3330 combined with vemurafenib could induce cell senescence induced by autophagy overload.Immunofluorescence,Western-blot and galactosidase staining were used to detect the effects of E3330 and vemurafenib on the content of autophagic substrate protein and cell senescence in BCPAP and K-1 cells.5.Western-blot,small interfering RNA knockdown and Co-IP experiments were used to verify the synergistic effect of E3330 on vemurafenib.6.The model of pulmonary metastasis of thyroid papillary carcinoma in BALB/c immunodeficient mice was established by intravenous injection of BCPAP GFP overexpression stable line.Then observe the synergistic effect of E3330 on vemurafenib in vivo.Results:1.The expression level of APE1 protein in 509 cases of papillary thyroid cancer and59 normal persons was retrieved and analyzed by TCGA database.The results showed that APE1 was highly expressed in papillary thyroid cancer,and the expression level of APE1 in fresh papillary thyroid cancer tissues was higher than that in normal tissues.In paraffin specimens,APE1 has a higher protein expression level than normal tissues.2.The results of univariate analysis between APE1 histochemical staining score and clinicopathological features showed that APE1 expression was significantly correlated with BRAF mutation,lymph node metastasis and TNM stage in thyroid papillary carcinoma,but not with sex,age,multifocal tumors and T stage.Multivariate logistic analysis showed that the expression of APE1,age and T stage were independent prognostic factors for lymph node metastasis of thyroid papillary cancer.3.CCK-8 cell viability test showed that compared with DMSO group,the relative cell viability of BCPAP and K-1 vemurafenib group and combined drug group decreased significantly,and the combined drug group had higher survival inhibition on cancer cells.PI staining for cell cycle and Brdu results showed that compared with the DMSO group,both the vemurafenib group and the combined group showed G1 arrest,and the proportion of S phase(DNA replication)cells was significantly reduced,and the combined drug group had more obvious retardation.The results of plate cloning experiments showed that compared with the DMSO group,the cell proliferation ability of the vemurafenib group,the E3330 group and the combined medicated group were significantly decreased,and the combined medicated group had higher proliferation inhibition on the tumor cells;the results of Annexin V-PI double staining assay showed that compared with the DMSO group,the vemurafenib group,the E3330 group and the combination group significantly increased the early and late apoptotic rates of BCPAP and K-1,and the incidence of apoptosis in the drug combination group was significantly higher than that in the drug alone group;The results of JC-1 staining assay for mitochondrial potential showed that vemurafenib and both drugs caused a significant decrease in mitochondrial potential of BCPAP compared with DMSO,and the combination of the two drugs caused a significant decrease in mitochondrial potential of K-1;The results of Western-blot detection of apoptosis-related proteins showed that the mitochondrial apoptosis-related protein Bcl-2 was significantly decreased,Bax and Cleaved-PARP were significantly increased.In addition,other related pathway proteins of apoptosis were detected.The results showed that compared with DMSO group,Cleaved-caspase 3 was significantly increased in vemurafenib group,E3330 group and combined drug group,while Survivin was significantly decreased in combined drug group,and the degree of changes in combined drug group was significantly higher than that in single drug group.The transwell invasion test showed that the number of cells passing through the polycarbonate membrane was significantly reduced in the BCPAP and K-1vemurafenib,E3330 and combination groups compared with the DMSO group.The number of membrane cells was significantly less than that of the drug alone group.4.The results of immunofluorescence showed that compared with the DMSO group,the autophagy increased in different degrees in the vemurafenib group,the E3330 group and the combination group,and the increased level of autophagy in the combination group was more obvious,and the accumulation of LC3 b and P62 appeared.While the detection of LC3 b,P62 protein levels through Western-blot was consistent with the immunofluorescence results.The results of detection of autophagy upstream regulatory proteins showed that p-m TOR was significantly decreased and p-AMPK protein and ATG5 protein levels were significantly increased in the vemurafenib group and the combination group,and the changes in the combination group were more significant.The results of galactosidase staining showed that compared with the control group,the combination group had cell senescence.Setting up three groups by autophagy activator Rapamycin showed that compared with the DMSO group,the 10 u M vemurafenib +50u M E3330 group,the 10 u M Rapamycin group,the combination group of LC3 b and P62 protein levels were significantly different.At the same time,the senescence staining showed that the two groups had significant aging compared with the DMSO group.5.Four groups of experiments were performed for 24 hours after drug addition.The results of Western-blot experiments showed that the levels of p-MEK and p-ERK protein had various degrees of reduction and which in the combination group were significantly lower than those in the vemurafenib group.Si RNA was used to down-regulated the expression of the MAPK pathway.The results showed that p-MEK and p-ERK expression was decreased in protein level after weakening the expression of APE1.The results of endogenous Co-IP assay showed that APE1 and BRAF had a binding effect.6.In vivo results showed that all mice had lung metastases,and 1 case of liver metastasis occurred in the vemurafenib group.Compared with the blank control group,the growth of metastatic tumors in the vemurafenib group and the combination group was markedly slowed down after 12 days of administration.Moreover,the inhibitory effect of the combination group was more obvious than that of the vemurafenib group.The immunohistochemistry results showed that compared with the blank control group,the expression of Ki-67 was significantly reduced in the vemurafenib group and the combination group,the expression of LC3 b and P62 was significantly increased,the expression of Cleaved-caspase3 significantly increased,and the changes were more obvious in the combination group than vemurafenib.Conclusions:1.The expression of APE1 was significantly higher in thyroid papillary cancer than in normal tissues.In thyroid papillary cancer,the expression of APE1 was significantly correlated with BRAF mutation,lymph node metastasis and TNM stage.The expression of APE1,age and T stage were independent prognostic factors for the lymph node metastasis of papillary thyroid cancer.2.E3330 can enhance the inhibitory effect of vemurafenib on thyroid papillary carcinoma cell lines BCPAP and K-1 including the proliferation,viability,invasion,migration,anti-apoptosis and tumor senescence induction.3.APE1 maintains the continuous activation of MAPK pathway under BRAF protein by binding to BRAF.4.E3330 can synergize vemurafenib in suppressing the progression of papillary thyroid cancer in vivo. |
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