Increased Effects Of 2,5-dimethylcelecoxib On Sensitivity Of Hepatocellular Carcinoma Cells To Sorafenib Via CYP3A5 Expression And Activation Of AMPK

Objective: Sorafenib is a first-line drug approved for systemic treatment of advanced hepatocellular carcinoma(HCC),but its clinical efficacy is limited by drug resistance and adverse reactions.Since the occurrence and development of HCC is often accompanied by inflammation,the combination of sorafenib and other therapeutic drugs,especially anti-inflammatory drugs,is one of the directions of current exploration.2,5-dimethylcelecoxib(DMC)is a derivative of celecoxib,which targets to inhibit membrane-bound PGE2 synthetase 1(mPGES-1).DMC does not have the ability of COX-2 inhibition and does not affect the coagulation function while inhibiting the synthesis of PGE2.We have been committed to the study of DMC in the early stage.In this study,we will explore the effect of DMC combined with sorafenib on enhancing the inhibition of HCC.Methods:1.HepG2 cells were treated with DMC at the optimal concentration,and the untreated HepG2 cells were used as negative control.The effects of DMC on HepG2 cells were analyzed by microarray.The results of microarray were analyzed comprehensively,and the differentially expressed genes with statistical significance were selected as target genes.The effect of DMC on the expression of target genes was verified by Western blotting assay in several hepatoma cell lines.Using online public database gepia website(www.gepia.com)To explore the differential expression of target genes in HCC tissues and adjacent tissues and the evaluation of survival analysis.2.Western blotting assay was used to detect the changes of CYP3A5 expression and related signaling pathways in hepatoma cells treated with sorafenib.In order to detect the effects of sorafenib and / or DMC on the migration ability of hepatoma cells,we used wond healing assay and migration Transwell assay to detect the changes of cell migration ability after sorafenib and / or DMC treatment.Western blotting assay,wild healing assay and migration Transwell assay were used to detect the effects of upregulation of CYP3A5 on related signaling pathways and migration ability of hepatoma cells.3.CCK8 assay was used to detect the effects of different concentrations of sorafenib and / or DMC on cell viability,and compusyn software was used to create the equivalent line diagram and calculate the combination index(CI).CCK8 assay,colony formation assay and flow cytometry were used to detect the effects of sorafenib and/or DMC on cell proliferation and apoptosis,and Western blotting assay was used to detect the expression of apoptotic protein in cells.4.Through the drug target prediction website Pharma mapper to obtain the potential protein targets related to DMC,and use go and KEGG analysis to screen the signal pathways that may be affected by DMC,and verify them in hepatoma cells by Western blotting assay.The effect of the combination of drugs on intracellular signal pathway was restored by pretreatment of hepatoma cells with pathway inhibitors.The changes of cytotoxic and proliferative inhibition ability of drugs were detected by CCK8 assay.Results:1.The microarray results showed that compared with the control group without DMC treatment,the expression of CYP3A5 in HepG2 cells increased after DMC treatment.KEGG analysis showed that the expression of CYP3A5 was related to multiple pathways,such as metabolic pathway,drug metabolism cytochrome P450,the metabolism of cytochrome P450 to heterobiotics,steroid biosynthesis,retinol metabolism,etc.Western blotting assay also verified that the expression of CYP3A5 in hepatoma cells induced by DMC was time-dependent and concentration dependent.Bioinformatics analysis showed that the expression of CYP3A5 in HCC was significantly lower than that in adjacent tissues,and its high expression was associated with better overall survival(OS).2.Western blotting assay showed that sorafenib inhibited the expression of CYP3A5 protein and activated Akt phosphorylation.Compared with the control group,sorafenib had a limited ability to inhibit the migration of hepatoma cells,but the ability to inhibit migration was significantly enhanced when combined with DMC.Compared with the control group,the phosphorylation level of Akt in sorafenib treated cells was decreased,and the migration ability was significantly decreased.3.According to the calculation results of compusyn},the combination of DMC and sorafenib can synergistically enhance the drug sensitivity(CI<1).At the same time,the combination of drugs improved the inhibition effect of single drug treatment on the proliferation of hepatoma cells and the promotion effect of apoptosis,and increased the expression of apoptotic protein.4.The AMPK pathway and PI3K/Akt pathway were selected to verify by microarray results and pharma mapper prediction results.The activation of AMPK pathway and the inhibition of PI3K/Akt pathway were observed in DMC cells treated with sorafenib,which could be reversed by AMPK pathway inhibitors.At the same time,after using AMPK pathway inhibitor,the proliferation inhibition and drug killing effect of combination therapy were weakened.Conclusions:1.DMC treatment increased the expression of CYP3A5 in HCC cells.The survival analysis of database showed that the expression of CYP3A5 was associated with better overall survival rate.2.Sorafenib inhibited the expression of CYP3A5 and promoted Akt phosphorylation,and up-regulated the expression of CYP3A5 or combined with DMC could enhance the inhibitory effect of sorafenib on the migration of hepatoma cells.3.DMC combined with sorafenib has synergistic effect and enhances the ability of sorafenib to inhibit the proliferation and promote apoptosis of hepatoma cells.4.The mechanism of DMC combined with sorafenib to enhance the anti-tumor ability includes activating AMPK and inhibiting Akt.