| Malignant tumors are serious diseases that seriously threaten human health.The treatment is mainly based on surgical treatment,radiation therapy,chemotherapy and immunotherapy in recent years.Whether it is radiotherapy,chemotherapy or immunotherapy,the low selectivity of drug resistance and precision medication is a difficult problem to control.Although the advantages of immunotherapy are significant,the response rate of patients is not high,so although there are many unsatisfactory chemotherapy,it still invades the treatment of metastatic tumors.The emergence of targeted drugs in recent years has brought new hope to cancer patients.Targeted drug targets are clear,but resistant,and only a few targeted drugs have resistance markers.At present,there is a lack of accurate and effective indicator markers to predict the effect of chemotherapy and targeted therapy.Therefore,new indicators are urgently needed to accurately use drugs to improve patient survival and survival therapy.AGR2 is a member of the disulfide isomerase family.Although it is an endoplasmic reticulum localization protein,AGR2 can still localize to the cytoplasm of cells and secrete to the outside of cells,and has secretory properties.As an oncogene,AGR2 is highly expressed in a variety of tumors:1)can significantly promote tumor proliferation and invasion and metastasis;2)its secretion characteristics suggest that it can be used as a diagnostic marker for tumors;3)can be used as a drug target,already AGR2 Research reports on antibody drugs.As a drug target,it has been found that AGR2 is associated with drug resistance and is resistant to endocrine therapy of breast cancer.There are few studies on AGR2 and tumor resistance.Therefore,this paper firstly determined the association between AGR2 expression and tumor chemotherapy drugs and targeted drugs through drug screening,and found that its expression significantly affected the drug efficacy.Subsequently,the mechanism of drug resistance was studied.It was found that both intracellular and secreted AGR2 participated in drug resistance through different mechanisms.Finally,the AGR2-mediated drug resistance mechanism was used to explore its response,and the secreted AGR2 was identified as a chemotherapy and vascular target.The drug is indicated to the drug,and a strategy for sequential administration based on the expression of AGR2 is proposed.Part I AGR2 expression and sensitivity screening of tumor cells to cytotoxic chemotherapy drugs and targeted drugsAGR2 is believed to be an oncogene that significantly promotes proliferation,invasion and metastasis of cancer cells.And it has been shown in several reports that AGR2 prevents cancer cells from cell death.So far,it has been found that high expression of AGR2 in breast cancer promotes the tolerance of tamoxifen and doxorubicin.However,the expression of AGR2 in docetaxel-resistant prostate cancer cells was significantly down-regulated in our previous data analysis.Therefore,we first performed drug screening to determine the correlation between AGR2 expression and tumor chemotherapy tolerance,providing a basis for further analysis of AGR2 function.I.Comparison of the sensitivity of basal expression of AGR2 to chemotherapeutic drugs in cancer cellsBy analyzing the basal expression of common tumor cell AGR2,the cells with high and low expression of AGR2 were selected for drug sensitivity screening,and the expression level of AGR2 was found to affect the drug efficacy.1.Low-expression of AGR2 in non-small cell lung cancer and colon cancer cells resistant to chemotherapy drugs.The results of WB showed that the expression of AGR2 in A549 cells was significantly lower than that in H460 cells,and the expression of AGR2 in SW620 was significantly lower than that in SW480.The IC50 values were analyzed according to the MTT results.The results showed that A549 and SW620 cells with low expression of AGR2 were more tolerant to docetaxel,doxorubicin and cisplatin,while H460 and SW480 cells were more sensitive to selected drugs.2.Breast cancer cells with high expression of AGR2 are resistant to cytotoxic chemotherapy drugs.WB experiments showed that AGR2 expression in breast cancer cells MDA-MB-453 was significantly higher than that in MDA-MB-468 cells.MTT results showed that MDA-MB-453 cells were more tolerant to three chemotherapeutic drugs,while MDA-MB-468 cells were resistant.The drug is more sensitive.?.Cell silencing or overexpression of AGR2 expression response to chemotherapy drugs and targeted drugs1.The effect of AGR2 expression changes on the sensitivity of chemotherapy drugs.We silenced the expression of AGR2 in AGR2-expressing PC3,H460,SW480 cells,and analyzed the effect of AGR2 on AGR2 low expression of DU145,A549,SW620.MTT assay showed that prostate cancer,non-small cell lung cancer and colon adenocarcinoma cells were resistant to chemotherapeutic drugs docetaxel,doxorubicin and cisplatin compared with control cells,and three tumor cells were overexpressed after AGR2.Increased sensitivity to drugs,consistent with previous data2.The effect of cell AGR2 expression changes on the sensitivity of targeted drugsMTT assay showed that the expression of AGR2 was down-regulated in prostate cancer,non-small cell lung cancer and colon adenocarcinoma.The EGFR-targeted drug gefitinib,CDK4/6 targeting drug pabucilide and angiogenesis target Tolerant to the drug cabozantini,sensitive to the proteasome targeting drug bortezomib.After overexpression of AGR2,the three tumor cells were sensitive to gefitinib,pabutramide and cabozantini,and tolerated to bortezomib.3.Breast cancer cell AGR2 expression and chemotherapeutic drug sensitivity screening.MTT assay showed that AGR2 down-regulated breast cancer cells MDA-MB-453 cytotoxic chemotherapy drugs docetaxel,doxorubicin and CDK4/6 compared with control cells The targeted drug,pabuciflozil,the angiogenesis-targeted drug,cabozantinib,and the proteasome-targeted drug,bortezomib,were consistently sensitive.MDA-MB-468 cells overexpressing AGR2 were consistently tolerant to the five chemotherapeutic drugs screened.Part ? Molecular mechanism and response to the secretion of AGR2 against anti-angiogenic targeted drugsIn the first part of the drug screening,we found that AGR2 highly expressed tumor cells were resistant to the anti-angiogenesis drug cabozantinib,which has aroused our interest.Both tumor proliferation and metastasis require adequate nutrient supply,which promotes angiogenesis of tumor tissue in order to ensure nutrients and oxygen,transport metabolites and provide an important gateway for tumor metastasis.Although AGR2 has been studied most frequently in tumor proliferation and invasion,few reports have reported that AGR2 is associated with angiogenesis.Our research team found that AGR2 could significantly promote the invasion and metastasis of orthotopic prostate cancer,and the blood transfusion of tumor in situ overexpressing AGR2 is especially abundant,suggesting that AGR2 promotes invasion and metastasis of prostate cancer through promoting tumor angiogenesis.Combined with the influence of the first part,AGR2's effect on anti-angiogenic drug sensitivity,this section explores the molecular mechanism and coping strategies of AGR2 promoting angiogenesis.?.Extracellular AGR2 synergizes with VEGFA to promote angiogenesis1.Recombinant human AGR2 protein promotion angiogenesisA His-tagged recombinant human AGR2 fusion protein was obtained using a prokaryotic expression purification system.Molecular sieve results show that the protein is present as a dimeric active form.Cell growth curve data indicate that recombinant human AGR2 protein can promote prostate cancer cell proliferation.Tubul formation experiments indicate that rhAGR2 significantly increases the number of tubule formation in endothelial cell HUVECs.The results of ELISA showed that there was no significant change in VEGFA content in HUVECs culture medium after rhAGR2 stimulation,suggesting that AGR2 does not promote the maturation of VEGFA.2.Recombinant human AGR2 protein synergizes with VEGFA to promote angiogenesisTube formation experiments showed that both rhAGR2 and VEGFA could increase the number of tubule formation in HUVECs,and the number of tubule formation in HUVECs was significantly higher than that in the stimulation alone.Scratch experiments showed that the combined stimulation of rhAGR2 and VEGFA enhanced the ability of HUVECs and prostate cancer cells PC3 to move significantly stronger than the stimulation alone.Q-PCR results showed that rhAGR2 combined with VEGFA stimulated the VEGFR2 signaling pathway.?.Extracellular AGR2 interacts directly with VEGFA1.AGR2 interacts directly with VEGFARecombinant human VEGFA fusion protein with His-tag and GST-tagged AGR2 full-field and truncated protein protein were obtained by prokaryotic expression purification system.SDS-PAGE showed that the protein was correct and the purity was high.His-pulldown experiments confirmed the direct interaction of AGR2 with VEGFA.The phalldown assay by VEGFA and AGR2 truncation showed that AGR2 and VEGFA bind to amino acids 81-101 of AGR2.2.AGR2 forms an intermolecular disulfide bond with VEGFAHis-pulldown experiments confirmed that the AGR2 cysteine mutant could not interact with VEGFA,and the reducing agent blocked the interaction of AGR2 with VEGFA.Tubulin formation experiments showed that both cell secreted and purified AGR2 C81A mutant proteins could not promote HUVECs tubule formation.Thus the PDI domain is a key domain for the formation of intermolecular disulfide bonds between AGR2 and other proteins.3.Reducer glutathione blocks AGR2 promoting angiogenesis and pro-invasive activityCo-immunoprecipitation experiments showed that the physiological reducing agent glutathione can disrupt the interaction of extracellular AGR2 with VEGFA.Tubul:in formation experiments showed that glutathione combined with rhAGR2 stimulated the number of tubule formation in HUVECs significantly decreased.Scratch experiments showed that glutathione combined with rhAGR2 stimulated the ability of PC3 cells to metastasize significantly.In vivo tumor invasion and metastasis model found that glutathione can reduce the number of lung metastases in rhAGR2 mice,but the anti-angiogenic drug bevacizumab can not inhibit the number of lung metastases in rhAGR2 mice.?.Secreted AGR2 antagonistic angiogenesis targeted drugs and drug selection studiesTube formation experiments showed that both cabozantinib and bevacizumab could effectively inhibit the number of tubule formation in HUVECs,but rhAGR2 could not reverse the effect of beboluzumab and inhibited the number of tubule formation in HUVECs.Tumor-bearing experiments in nude mice showed that intraperitoneal injection of rhAGR2 attenuated the antitumor activity of bevacizumab,but had no effect on the antitumor activity of cabozantinib.It is suggested that AGR2 indicates anti-angiogenesis drug administration.Part ?:Molecular mechanism of AGR2 low expression-mediated multidrug resistance in tumor chemotherapyIn section one,we found that low expression of AGR2 mediated chemotherapy resistance in hormone-independent prostate cancer cells,non-small cell lung cancer cells,and colon adenocarcinoma cells.The screening results revealed that down-regulation of AGR2 mediated the tolerance of cancer cells to various chemotherapeutic drugs with different structures and different targets,suggesting that AGR2 mediated multidrug resistance of tumor chemotherapy.The mechanism of AGR2 promoting angiogenesis cannot explain this phenomenon,so we explored the novel molecular mechanism of AGR2-mediated tumor chemotherapy tolerance.I.AGR2 negatively regulates MRP2 expression and mediates multidrug resistance of tumor chemotherapySince low levels of AGR2 can mediate chemotherapeutic drug resistance in a variety of different structures and mechanisms of action,and drug-pump proteins are an important mechanism mediating multidrug resistance,the first analysis of drug pump proteins in AGR2 low expression-mediated resistance The role of medicine.Flow cytometry results showed that PC3 cells down-regulated AGR2,and the accumulation level of intracellular doxorubicin decreased significantly,indicating that the drug pump protein participated in its drug resistance process.MTT results showed that AGR2 expression in prostate cancer,non-small cell lung cancer and colon adenocarcinoma cells was negatively correlated with P-gp non-affinity drug sensitivity.The expression of AGR2 was silenced in PC3 and 1-H460 cells.The level of MRP2 protein was increased,and immunofluorescence results showed a decrease in MRP2 on the membrane of overexpressed AGR2 cells.It is suggested that AGR2 mediates MRP2-dependent multidrug resistance.?.ARRB1 mediates MRP2 protein degradationFlow cytometry detected silencing of ARRB1 expression,and intracellular accumulation of doxorubicin was significantly reduced.WB results showed that silencing ARRB1 expression,MRP2 protein expression increased,while overexpression of ARRB1,cell MRP2 protein expression decreased.Co-mmunoprecipitation was used to detect the interaction of MRP2 with the same family of proteins MRP1 and ARRB1.Protein ubiquitination assay revealed overexpression of ARRB1,MRP2 ubiquitination increased,and ARRB1 S412D inactivation mutation did not increase MRP2 ubiquitination.The final co-immunoprecipitation results showed that MRP2 interacts with the E3 ubiquitin ligase MDM2.?.AGR2 up-regulates ARRB1 is essential in multidrug resistance in tumor chemotherapy1.Down-regulation of ARRB1 mediates multidrug resistance of tumor chemotherapyMTT assay showed that AR3? was silenced in PC3 cells and H460 cells.The cells were resistant to three cytotoxic chemotherapeutic drugs such as docetaxel,doxorubicin and cisplatin,while ARRB1 was overexpressed in DU 145 and A459 cells.Increased drug sensitivity.Animal experiments have shown that ARRB1 tumors are down-regulated to docetaxel.2.AGR2 up-regulates ARRB1 protein expression mediating drug toleranceWB results showed that H460 cells down-regulated AGR2 expression and ARRB1 protein expression decreased.The results of co-immunoprecipitation showed that AGR2 interacted with ARRB1.MTT results indicated that overexpression of ARRB1 reversed down-regulation of AGR2-mediated tumor chemotherapy tolerance,whereas silencing of ARRB1 expression reversed overexpression of AGR2-mediated drug sensitivity.3.PDI domain participates in AGR2-mediated drug toleranceWB results showed that the AGR2 C81A mutant with a PDI domain mutation could not upregulate ARRB1 expression.MTT results showed that the AGR2 C81A mutant could not affect the sensitivity of tumor cells to docetaxel and doxorubicin.4.AGR2 Up-regulates MRP2 protein expression mediates drug toleranceMTT results indicate that silencing of MRP2 expression in PC3 and H460 cells reverses down-regulation of AGR2-mediated drug sensitivity.Part ?.Proteasome inhibitors overcome the role of AGR2 low expression-mediated multidrug resistance in tumor chemotherapy and its molecular mechanismIn the pre-screening process,we found that the drug sensitivity of the proteasome inhibitor bortezomib was negatively correlated with AGR2 expression,suggesting that our targeted proteasome has the potential to be a low-expression of AGR2-mediated multidrug resistance in tumor chemotherapy coping strategies.This section analyzes the mechanism of AGR2 regulating proteasome activity and provides theoretical support for the reversal strategy of tumor chemotherapy tolerance.?.AGR2 negatively regulates the proteasome activity of tumor cells1.AGR2 is associated with the ubiquitin-proteasome degradation pathwayThe BioGRID database analyzes the AGR2 protein interaction network,and clustering results show that AGR2 interacting proteins are associated with protein ubiquitination.WB results showed that the total ubiquitinated protein in silencing AGR2 expression cells decreased,while the accumulation of ubiquitinated proteins in overexpressed AGR2 cells increased.2.AGR2 negatively regulates proteasome activityThe results of proteasome activity analysis showed that the expression of AGR2 was silenced,trypsin-like activity,chymotrypsin-like activity and peptidyl-glutamyl hydrolase-like activity were increased in the proteasome,while overexpression ofAGR2,chymotrypsin-like activity and Peptidyl-glutamyl hydrolase-like activity is reduced,and trypsin-like activity is substantially unchanged.?.AGR2 regulates the binding of the 19s regulatory particle of the proteasome to the 20s core particle1.AGR2 binds to the 19s proteasome regulatory subunit via the PDI domainProteasome activity assay results showed that the purified AGR2 protein did not inhibit proteasome activity compared to the control BSA protein.Co-immunoprecipitation showed that AGR2 interacted with the 19s regulatory subunit protein PSMD2,and AGR2 C81A with PDI domain mutation could not interact with PSMD2.Proteasome activity assay indicated that AGR2 C81Acould not inhibit protein media activity.MTT assay showed that AGR2 C81A could not reduce the sensitivity of tumor cells to the proteasome inhibitor bortezomib.2.AGR2 inhibits the binding of the 19s regulatory particle to the 20s core particleThe results of co-immunoprecipitation showed that overexpression of AGR2 increased the binding of PSMD2 to AGR2 and decreased to the core particle protein PSMA7.It is suggested that high expression of AGR2 inhibits the binding of the 19s regulatory particle to the 20s core particle and decreases the proteasome activity.?.Docetaxel and Bortezomib Sequential Overcomes Chemotherapy Acquired ToleranceAnimal experimental data showed that downregulation of AGR2 mediates tumor cell tolerance to docetaxel and is sensitive to proteasome inhibitors.Docetaxel increases proteasome activity by down-regulating AGR2.The MTT results showed that the treatment of dobuticol for 12 h and the replacement of bortezomib for 12 h resulted in significantly lower tumor cell survival rate than docetaxel alone and bortezomib alone or in combination.Animal experiments have shown that the effect of treatment with bortezomib after docetaxel treatment is better than the combination of docetaxel and bortezomib alone or in combination.Conclusion1.Low level of AGR2 expression is associated with multidrug resistance in tumor cells.2.AGR2 synergizes with VEGFA to promote tumor angiogenesis and bevacizumab tolerance,while glutathione and cabozantini can be used as coping strategies.3.Low expression of AGR2 mediates tumor chemotherapy tolerance by down-regulating ARRB1 expression.4.AGR2 negatively regulates the proteasome by inhibiting the binding of the proteasome core particle to the regulatory particle.5.Bortezomib overcomes low expression of AGR2-mediated tumor multidrug resistance,and docetaxel and bortezomib sequential overcomes clinical chemotherapy acquired tolerance.Innovation1.We identify that VEGFA is a direct interacting protein of extracellular AGR22.It is first reported that ARRB1 mediates the initiation of degradation of MRP2,and E3 ubiquitin ligase MDM2 mediates the ubiquitination of MRP2.3.We first identify that PSMD2 is an interactor of AGR2,and it inhibits the association of proteasome core particle to regulatory particle.4.The propensity regimen of docetaxel and bortezomib sequential is proposed for the first time,and AGR2 is proposed as an indicator to guide the chemotherapy of clinical tumors.Inadequacies1.Failure to analyze the molecular mechanism of AGR2 up-regulation in multidrug resistance in breast cancer cells2.The mechanism of AGR2 promoting MRP2 degradation is not complete clear,and the role of AGR2 in the degradation process of MRP2 should be further analyzed. |
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