2,5-dimethylcelecoxib Promotes The Expression Of G6PC In Hepatocellular Carcinoma Cells In Inflammatory Environment Through Autophagy

Objective:Inflammation and metabolism affect each other.Inflammation can lead to abnormal cell metabolism,including glycolysis and gluconeogenesis.Excessive metabolites produce cytotoxicity and aggravate the development of inflammation.Because inflammation is also closely related to the progression of tumors,for example,when hepatocellular carcinoma(HCC)cells are stimulated by various inflammatory mediators and the tumor microenvironment changes,their metabolism,especially sugar metabolism,will also change to facilitate Survival,glycogen and lipid accumulation common in HCC are the result of this adaptation.Therefore,reducing the inflammatory response is beneficial to inhibit the progression of HCC.Glucose-6-phosphatase(glucose-6-phosphatase,G-6-pase,also known as G6PC)is a phosphatase that hydrolyzes phosphate compounds.It can catalyze glucose-6-phosphate(G6P)to generate free glucose,which is to maintain glucose The key enzymes of homeostasis play an important role in gluconeogenesis and glycogen decomposition.If the G6 PC enzyme is lacking,glycogen cannot be metabolized normally,and the patient manifests as glycogen storage diseases(GSD),liver steatosis,cirrhosis and even liver cancer.The research team found in the early stage that2,5-dimethylcelecoxib(2,5-dimethylcelecoxib,DMC)can target to inhibit the activity of m PGES-1 enzyme(the terminal rate-limiting enzyme responsible for the production of inducible PGE2 at the site of inflammation),thereby Effectively down-regulate PGE2,but its effect on HCC cell glucose metabolism is still unclear.Therefore,in this study,we used cytological experiments to explore the effect and mechanism of DMC on the changes in glucose metabolism of liver cells in an inflammatory environment,and provide new ideas and strategies for the treatment of HCC and various liver diseases.Methods:1.Treat liver cancer cell HepG2 with 50 μM DMC and perform whole-genome chip analysis.After screening and analysis,select G6 PC,an enzyme related to glycogen metabolism that is significantly different and statistically significant;2.Use different concentrations(0,10,100 ng/ml)of inflammatory factor IL-1β and different concentrations(0,5,20,35 μM)of DMC to treat liver cancer cells(Huh7and SMMC-7721)for 24 h and 48 h,respectively.Western blot was used to detect the changes in cell G6 PC expression,and to explore the effect of the drug DMC on the expression of G6 PC in liver cancer cells in an inflammatory environment;3.Treat liver cancer cells(Huh7 and SMMC-7721)with 10 ng/ml IL-1 β,20μM DMC and 200 n M Bafilomycin A1(BAF1)for 24 hours,and detect the changes of hepatocyte autophagy-related molecules by western blot,and further explore the influence of DMC on liver cells The mechanism of sugar metabolism;4.Construct a stable liver cancer cell line with ULK1 knockdown(sh-ULK1),and explore the relevant molecular mechanism of the drug DMC to regulate the changes of liver cancer cell G6 PC.Results:1.The results of the whole genome microarray suggested that the expression of G6 PC gene m RNA was up-regulated in hepatoma cells treated with DMC,which was further verified by q PCR technique.2.The results of western blot and glycogen assay showed that IL-1 β inhibited the expression of G6 PC in a time and concentration dependent manner,while DMC could reverse these conditions and enhance the expression of G6 PC.3.Western blot showed that the autophagy flux of hepatoma cells induced by DMC was increased,the expression of LC3B-II,p-ULK1(Ser555)was significantly up-regulated,while the expression of p62 ptalk ULK1(Ser757)was down-regulated,and the autophagy inhibitor BAF1 blocked the autophagy flux and partially reduced the induction of G6 PC by DMC.4.Compared with NC group,DMC could only restore a small part of the suppressed G6 PC of sh-ULK1 hepatoma cells.Conclusions:1.Inflammatory factor IL-1 β significantly down-regulates the expression of liver cancer cells G6 PC and accumulates glycogen,while m PGES-1 inhibitor DMC can reverse and restore the expression of liver cancer cells G6 PC in an inflammatory environment and reduce glycogen load;2.DMC induces increased autophagy flux to promote cell G6 PC expression;blocking autophagy flux weakens the effect of DMC on G6 PC expression;3.ULK1 participates in the regulation of DMC on the glycogenolysis process of liver cancer cells.