A Preliminary Study Of USF1 Mediates The Drug Resistance Mechanism Of Bladder Cancer Cells To Oxaliplatin

Objective: Bladder caner（BC）ranks the tenth malignant tumor incidence rate in the world,and is also the second most common malignancy in the genitourinary system.Muscle-invasive bladder cancer（MIBC）is aggressive and has poor prognosis.Chemotherapy is still one of the most important and effective treatments for MIBC.Since the late 1980 s,cisplatin（CIS）based combination chemotherapy has been the standard treatment.However,up to50% of the patients were screened as unsuitable for cisplatin based chemotherapy,especially those with poor renal function and poor functional score,and the problem of extensive drug resistance in the application of CIS is serious.Oxaliplatin（oxa）,as the third generation of platinum drugs,has been proved to be effective for BC in a number of phase II clinical trials,and has the potential to be a substitute for CIS.At the same time,many studies show that oxa inevitably produces drug resistance in the process of anti-tumor.P38 MAPK pathway is one of the key pathways of oxa induced apoptosis in tumor cells.It can activate p38 MAPK pathway and induce apoptosis by promoting the phosphorylation of p38 MAPK.Upstream transcription factor 1（USF1）is one of the downstream transcription factors of p38 MAPK pathway,which has been confirmed to be associated with tumor drug resistance.However,the drug resistance mechanism of USF1 in the treatment of bladder cancer with oxa remains unclear.In this study,we constructed oxa resistant bladder cancer cell lines to explore the role of USF1 and p38 MAPK pathway in the mechanism of oxa resistance in bladder cancer.Methods: Bladder cancer cell lines（T24,253J）were used as experimental sensitive strains,and oxa resistant cell lines（T24-oxa,253J-oxa）were induced gradually by concentration gradient method.CCK8 method was used to detect the IC50 value of the drug-resistant cell lines.The expression of p38 MAPK and p-p38 MAPK in sensitive and drug-resistant cell lines were detected by immunofluorescence technique and Western blot（WB）.Annexin V/PI double staining kit was used to detect the apoptosis of T24-oxa and 253J-oxa.The difference of USF1 expression between sensitive and resistant strains was verified by fluorescence immunoassay and WB.After construction of lentivirus Lv-sh USF1 and control lentivirus Lv-sh Control which can specifically inhibit the expression of USF1,the infection of oxa resistant cell lines T24-oxa and253J-oxa was down regulated CCK8 kit was used to determine the IC50 value of oxa to the above cell lines,and to compare the sensitivity of drug-resistant cell lines to oxa after downregulation of USF1.The adenovirus Ad-USF1 overexpressing USF1 and the control virus ad Ad-EGFP were used to infect the oxa sensitive cell lines T24 and 253 J with low expression of USF1.The IC50 value of oxa to the above cell lines was determined by CCK8,and the drug resistance of the oxa sensitive cell lines after overexpression of USF1 was compared.Results: The IC₅₀ values of oxa to sensitive and resistant cell lines were determined by CCK8 method as follows（unit:μM）,T24（0.802 ± 0.175）,T24-oxa（9.763 ± 1.457）,253J（0.739 ± 0.043）and 253J-oxa（12.47 ± 2.480）.The m RNA expression levels of MDR1,MRP1 and GSTP1 and Bcl-2,Bcl-w,XIAP and Survivin were significantly increased in T24-oxa and 253J-oxa.The phosphorylation of p38 MAPK（p-p38 MAPK）was detected by immunofluorescence and WB in T24,T24-oxa,253 J and 253J-oxa cells treated with oxa.However,PARP was not cleaved in T24-oxa and 253J-oxa cells.Annexin V/PI double staining kit was used to detect T24-oxa and 253J-oxa.Under the action of oxa,apoptotic cells could hardly be detected;WB and immunofluorescence further confirmed that the expression of USF1 in drug-resistant cell lines was significantly up-regulated.The IC50 of T24-oxa and253J-oxa cell lines infected with lentivirus Lv-sh USF1 and control lentivirus Lv-sh Control were（9.114 ± 0.873）,（14.562 ± 1.176）,（2.894 ± 0.361）and（6.046 ± 0.522）μ M,respectively.The IC50 of T24 and 253 J cell lines infected with Ad-USF1 and Ad-EGFP were（3.872 ± 0.552）,（0.804 ± 0.111）,（1.675 ±0.154）and（0.743 ± 0.108）μ M,respectively.Conclusion: After Oxa treated T24 and drug-resistant cell lines T24-Oxa and 253 J and drug-resistant cell lines 253J-Oxa,the p38 MAPK pathway was activated,but the p38 MAPK pathway of drug-resistant cell lines was activated but did not undergo apoptosis.USF1 is significantly increased in Oxa resistant strains.Silencing the expression of USF1 can effectively increase the sensitivity of T24-Oxa and 253J-Oxa cells to Oxa.Increasing the expression of USF1 can effectively reduce the sensitivity of T24 and 253 J cells to Oxa.The abnormal increase of USF1 is manifested through the p38 MAPK pathway,which is closely related to the resistance of bladder cancer cells to Oxa.