Study On The Role Of Ubenimex Targeting CD13 In Inhibiting Autophagy To Overcome Drug Resistance By Disturbing The EMP3/FAK/NF-κB Pathway In GC Cells

Background: Gastric cancer(GC)is the fourth most common malignancy with mortality in China,and its onset is insidious,most GC patients were diagnosed at the advanced stage.Chemotherapy is the primary means of treating patients with advanced or recurrent GC and improving their survival.5-fluorouracil(5-FU)is one of the most widely used chemotherapeutic agents for the treatment of GC,but the crucial problem of the clinical application of 5-FU is chemoresistance.Therefore,exploring the mechanism of chemoresistance and finding methods to reverse the 5-FU resistance in GC have become a research hotspot to be solved urgently.Previous studies found that Ubenimex,as a CD13 inhibitor,has synergistic effect with 5-FU to enhance the antitumor activity of 5-FU in liver,kidney and breast cancer cells,and can also promote chemotherapy-induced apoptosis by targeting CD13 to reverse the drug resistance of liver cancer cells.however,as the only marketed CD13 inhibitor,Ubenimex is only used as an immunopotentiator for the adjuvant treatment of hematological malignancies,there are no studies about Ubenimex with reversing 5-FU resistance in GC.Therefore,this study investigated whether Ubenimex could reverse 5-FU resistance in GC and its mechanism.Methods: Construction of 5-FU-resistant GC cells SGC-7901/5-FU by low-concentration gradient increasing combined with high-dose sequential pulse in vitro.CCK-8 was used to determine the proliferative activity of GC-resistant cells by 5-FU before or after pretreatment with Ubenimex(0.12 mg/ml).After pretreated with 5-FU,the expression levels of autophagy-related proteins in SGC-7901/5-FU cells treated with different concentrations of Ubenimex(0.06 mg/ml,0.12 mg/ml)were detected by Immunofluorescence and Western blot.And the number of autophagosomes in cells before and after pretreatment with Ubenimex was observed by Transmission electron microscope.Next,the expression levels of apoptotic proteins was detected by Western blot and Immunofluorescence,and the ability of Ubenimex to mediate 5-FU-induced apoptosis in GC drug-resistant cells was determined by Flow cytometry.To further explore the molecular mechanisms underlying the effects of Ubenimex,we analyzed the changes in m RNA expression of MKN-45/X cells before and after treatment with Ubenimex by Affymetrixclariom(?) S microarray sequencing;Using the high content screening(HSC)experiment as well as GO function and KEGG pathway enrichment analysis to screen the key genes involved in reversing GC resistance with Ubenimex.And the protein expression and the up-and down-stream regulatory relationship of key genes were further determined using Western blot.SGC-7901/5-FU cells were transfected with overexpression or silencing vectors,and the changes in cell autophagy and related protein expression,apoptosis and related protein expression,the cell sensitivity to 5-FU were evaluated using Flow cytometry,Immunofluorescence,Western blot and CCK-8 assays,thus further determining the role of key proteins and regulatory axis in the reversal of5-FU resistance by Ubenimex in GC.Results: The results of CCK-8 assay indicated that the sensitivity of SGC-7901/5-FU cells to 5-FU was significantly improved by pretreatment with Ubenimex and exhibited a certain dose-time dependence.The results of Western blot and Immunofluorescence indicated that the expression of Beclin-1,ATG5,and LC3 B decreased and the expression of p62 increased as the increase of Ubenimex concentration in SGC-7901/5-FU cells.And the number of autophagosomes in cells treated with Ubenimex was found to be obviously increased by Transmission electron microscope,indicating that Ubenimex can dose-dependently inhibit the autophagy level of SGC-7901/5-FU cells.Flow cytometry,Western blot and Immunofluorescence showed that Ubenimex could promote the apoptosis of 5-FU-resistant cells.The microarray sequencing showed the expression of264 genes including CD13 was down-regulated,and the expression of 227 genes was up-regulated in the Ubenimex-treated MKN-45/X cells.The regulatory axis of CD13/EMP3/FAK was found by HSC assay which may play an important role in the reversal of drug resistance by Ubenimex,GO function and KEGG pathway enrichment analysis indicated that all the above selected genes were enriched in NF-κB which involved in regulating cell autophagy and apoptosis.The following results of Western blot showed that the expression of CD13,EMP3,FAK,and NF-κB was attenuated by Ubenimex in SGC-7901/5-FU cells with a dose-dependent manner;Overexpression of CD13 can reverse the inhibitory effect of Ubenimex on the series of proteins,when EMP3 was overexpressed,it had no significant effect on the ability of Ubenimex to inhibit the expression of CD13,but it could reverse the expression of other signaling pathway proteins;And compared with the control group,the protein expression levels in the CD13 silenced group and the Ubenimex treated group has the same downregulation trend.So it was inferred that Ubenimex could inhibit the pathway activity of CD13/EMP3/FAK/NF-κB in SGC-7901/5-FU cells.Moreover,the autophagy level of SGC-7901/5-FU cells was decreased and the apoptosis level was increased in Ubenimex-treated group,and crucially,the effect of Ubenimex to cell autophagy and apoptosis was reversed after overexpression of CD13;Immunofluorescence also confirmed that Ubenimex could inhibit the phosphorylation level of FAK,and similarly,this effect was reversed after CD13 overexpressed.Therefore,it was confirmed that Ubenimex regulates cell autophagy and apoptosis,in which CD13/EMP3/FAK/NF-κB pathway activity is indispensable.Finally,CCK-8 assay showed that overexpression of CD13/EMP3 could reverse the effect of Ubenimex,exhibited the reduced sensitivity to5-FU in GC cells.Thus,Ubenimex attenuated cell autophagy and reversed 5-FU resistance may be related to the inhibition of CD13/EMP3/FAK/NF-κB pathway activity.Conclusions: Ubenimex can reverse the resistance of SGC-7901/5-FU cells to 5-FU,which is achieved by inhibiting autophagy and promoting its induced apoptosis,and this effect is inseparable from the inhibition of CD13/EMP3/FAK/NF-κB.The study provides a new direction about the use of Ubenimex in the chemotherapy of GC.Accordingly,the combined application of 5-FU and Ubenimex may become a promising strategy in the treatment of GC.