| Objective:Renal cell carcinoma(RCC),characterized by high malignancy and poor prognosis,is one of the most common malignant tumors of the adult urinary system.Approximately 30%of RCC paitients are diagnosed with metastatic or advanced carcinoma at first visit.Furthermore,20 to 40%of individuals with local carcinoma who undergo surgical resection subsequently develop metastatic disease.With the understanding of the mechanisms of cancer development,target therapy in molecular level(TARGET,treatment approaches in renal cancer global evaluation trial)became the new hotspot.The representative of targeted drug,sorafenib,aimed at VEGFR,inhibited tumor angiogenesis,induced cell apoptosis.But with the extension of time,the tumor cell will gradually become resistant to drugs,limiting the application of targeted therapy.This essay will discuss the mechanisms on targeted therapy of renal cell carcinoma,and explore methods to reverse drug resistance.The study chose renal cell line 786-0,ACHN for building drug-resistant strains by gradient sorafenib concentration,explore possible resistant mechanism by the research of apoptosis,autophagy and signal pathway,and methods for reversing drug resistance based on the experiment results.Methods:1.Establishment of resistant strains:Renal cell strains,786-0,ACHN,in 96-well plates with different concentration of sorafenib,were measured of its'IC50 by WST-8.Subsequently,sorafenib were dropped in culture solution began with a concentration slightly lower than IC50.The most dynamic cell strain were chosen for passage,and the sorafenib concentration was added gradually every two passages.When the sorafenib concentration reaches 8 ?mol/L,resistant strains can be considered building successfully after keep the concentration for 10 stable passages.2.Contrast detection of drug-resistant strains of 786-O-R,ACHN-R with the non-resistant strains of 786-0,ACHN of apoptosis,autophagy,investigate the mechanism of drug resistance.3.Based on preliminary research and explore methods of reversing drug-resistance strains of renal cell carcinoma.By contrast with the non-resistant strains,detect cell viability,apoptosis,autophagy level for assessment of drug resistance,study of mechanism behind it,verification of preliminary research.4.According to cells experimental result,make animal experiments validation.Building mice model with tumor with cell strain of renal cell carcinoma ACHN,drug-resistant strain ACHN-R,raising two weeks,mouse were randomly divided into five groups,contrast group,drug-resistant contrast group,drug resistant with sorafenib group,drug resistant with ubenimex group,drug resistant with sorafenib and ubenimex group.Measuring the volume of mouse tumor for evaluating the effectiveness of reversing sorafenib resistance.Results:1.Drug-resistant strains of sorafenib 786-O-R,ACHN-R form of sorafenib resistance,drug-resistant strains had lower rate of apoptosis with the same dose,showed that drug-resistant strains had built successfully.2.Significant increases in levels of drug-resistant strains of autophagy,autophagy as a response mechanism participate to sorafenib resistance.3.Akt signal pathway is activated in the resistant strain,the expression of p-Akt,p-mTOR is increased.Akt signal pathway regulates autophagy and apoptosis,and participated in the drug resistance of sorafenib.4.After application of ubenimex,the expression of Akt signal pathway decreased.Compared with placebo,adding ubenimex recovered sensitivity of resistant strains to sorafenib;compared with adding ubenimex merely,obvious better treatment of combination of ubenimex and sorafenib to single ubenimex,proved is not ubenimex induced apoptosis.Conclusion:Sorafenib resistant renal cancer cell has poor reaction to sorafenib,higher level of autophagy by western blott and electron microscope result,and the inhibition of autophagy through Akt pathway can reverse sorafenib resistance,but problems of resistance of other targeted drugs,the effective time of reversing resistance need follow-up experiments with longer time and larger sample. |
PDF Full Text Request