Anti-tumor Efficacy And Molecular Mechanisms Of Fatty Acid Synthase Inhibitor Orlistat In Esophageal Squamous Cell Carcinoma

Backgrounds and ObjectivesFatty acid synthase,FASN)is the catalytic enzyme of the key step in the de novo synthesis of fatty acids.The expression of FASN in cervical cancer,breast cancer,liver cancer and other cancers is up-regulated,while the expression in normal tissues is low or absent.Therefore,targeted inhibition of FASN expression may become a new strategy for tumor therapy.Many studies have shown that the inhibitors of FASN,such as Orlistat,TVB-3166,C75,Resveratrol,Cerulenin,etc.,have shown strong anti-tumor effects in many tumors.However,the role and mechanism of FASN in esophageal squamous cell carcinoma(ESCC)are still unclear.The purpose of this study is to explore the anti-tumor effect of Orlistat,a fatty acid synthase inhibitor,and its possible molecular mechanism.This study provides a theoretical basis for the treatment of ESCC by targeting FASN.Methods1.after analyzing the expression pattern of FASN by shengxin,the expression of FASN in ESCC was confirmed by qRT-PCR and Western blot.2.Using CCK-8 to detect the effect of Orlistat on ESCC proliferation,EdU experiment to detect the effect of Orlistat on ESCC proliferation,clone formation experiment to detect the effect of Orlistat on ESCC clone,flow cytometry to detect the effect of Orlistat on ESCC apoptosis,Transwell chamber was used to detect the effect of Orlistat on migration and invasion of ESCC,Nile red staining was used to detect the effect of Orlistat on lipid production of ESCC,and Western blot was used to detect the expression of lipid-related proteins in ESCC cells treated with different concentrations of Orlistat for 48 hours.3.To detect the inhibitory effect of Orlistat on KYSE150 transplanted tumor in vivo.The nude mice were inoculated with KYSE150 cells subcutaneously,and the solvent control group and Orlistat treatment group were set up.In the treatment group,Orlistat was injected intraperitoneally every day,and the tumor volume were recorded every two days.After the treatment,the eyes of nude mice were harvested and the blood from the posterior venous plexus was taken.After separating the serum,alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum were detected to evaluate the degree of liver injury in nude mice.The tumor tissue was stripped and weighed.The expression of lipid-related proteins FASN,ACLY,SREBF1,ACSs and ACC1 in the tumor tissue was detected by Western blot.4.Immunoprecipitation(IP)assay was used to detect the acetylation level of FASN in Eca109 and KYSE150 cells after Orlistat treatment for 48 h.Western blot was used to detect the expression of FASN,acetyltransferase and deacetylase in Orlistat treated cells.Co-precipitation method(Co-IP)was used to detect acetyltransferase and deacetylase interacting with FASN.5.Use data and image processing software:GraphPad Prism 9.0,Excel and ImageJ to process data and images.Among them,in one-way ANOVA,the independent samples of two groups of data are tested by parameter T for normal distribution,unpaired T for even variance,Welch’s t for uneven variance,and nonparametric T for non-normal distribution(Mann-Whitney test/KolmogorovSmirnov test).The difference between the two groups of paired data samples obeys the normal distribution using the parameter t test,in which the difference is consistent using the paired t test,the difference is different using the ratio paired t test,and the nonparametric t test(Wilcoxon paired test)is used if the difference is not satisfied with the normal distribution.One-way ANOVA(and Nonparametric)is used for multiple groups of data,ANOVA is used for normal distribution,and non-parametric test(Kruskal-Wallis/Friedman)is not used.Two way ANOVA was used.*P<0.05 indicates statistical difference.Results1.The results of the analysis of the generation of confidence indicate that FASN is highly expressed in ESCA.qRT-PCR and Western blot are used to detect the expression of FASN in ESCC tissues or cells,which shows that some tissues are highly expressed,and all ESCC cells are highly expressed.2.CCK8 and EdU experiments show that Orlistat can inhibit the proliferation of ESCC cells,cloning experiments show that Orlistat can inhibit the cloning ability of ESCC cells,apoptosis results show that Orlistat can promote the apoptosis of ESCC cells,Transwell results show that Orlistat can inhibit the migration and invasion of ESCC cells,Nile red staining results show that,Orlistat can inhibit the lipid production of ESCC cells.Western blot results show that Orlistat can significantly down-regulate the expression of lipid-related proteins such as FASN,ACLY,SREBF-1,ACSs and ACC1 in ESCC cells.3.The nude mice transplanted tumor experiment proved that Orlistat could inhibit the proliferation of ESCC transplanted tumor.Serum alanine aminotransferase(alt)and aspartate aminotransferase(ast)were detected,and it was found that the nude mice in the control group and the treatment group had different degrees of liver injury,and there was no significant difference between the two groups.The results of Western blot showed that the expression of FASN,ACLY,SREBF-1,ACSs and ACC1 protein in tumor tissues decreased significantly.4.The immunoprecipitation(IP)experiment showed that the expression of FASN in ESCC cells decreased after Orlistat treatment,but the acetylation level of FASN increased.Orlistat decreased the expression of deacetylases HDAC3 and SIRT2,but promoted the expression of acetylases MOF and PCAF.5.HDAC3,SIRT2,MOF and PCAF all interact with FASN.HDAC3 inhibitor REFP966 decreased the expression of FASN in ESCC cells and increased the acetylation level of FASN.Conclusion1.Orlistat inhibits the proliferation,migration and invasion of ESCC cells,induces apoptosis,and significantly down-regulates the expression of fatty acid de novo protein,suggesting that targeting FASN may become a n ew therapeutic strategy for ESCC.2.In ESCC,acetylation can lead to degradation of FASN,and promote deacetylation of FASN.