Eugenol Acid Antagonizes Pancreatic Cancer By Inhibiting NF-?B-induced Anterior Gradient 2 Expression

Background and objectiveThe survival rate of pancreatic cancer is one of the worst among all malignant tumors.Although technologies such as radiotherapy and chemotherapy have developed rapidly,surgical resection is still the only possible cure.The survival rate of pancreatic cancer is still low.The mechanism of occurrence and development is of great significance to the development of new pancreatic cancer treatment drugs and to improve the survival rate of pancreatic cancer.Eugenol is a phenylpropene isolated from essential oils extracted from clove,nutmeg,cinnamon,or bay leaf,and is commonly used in the production of flavors or perfumes.It is also used clinically as a local anesthetic and preservative.An increasing number of studies show that the anti-cancer ability of eugenol acid has been shown in many tumors.However,the clinical effect of eugenol acid in the treatment of pancreatic cancer has not been reported.Therefore,this study aims to explore the effect of eugenol acid on pancreatic cancer cells,and to explore its biological function and mechanism of action,in order to find new drugs for the treatment of pancreatic cancer.MethodPanc-1 was cultured in ordinary medium and medium containing eugenol solution,and colony formation experiments were performed to observe the effect of eugenol on the growth of PANC-1 cells in vitroThe parental cells of the PANC-1 cell line were subcutaneously inoculated into the lower flank of mice to establish a xenograft model.The tumor growth was regularly monitored and recorded.The xenograft tumor mice were equally divided into two groups,one group was given eugenol treatment,each The drug was administered 3 times a week until the end of the experiment.The other group was used as a blank control.Twelve pairs of pancreatic cancer patients and their adjacent tissue samples were collected,and qRT-PCR was used to detect the expression of subunit RelA of AGR2 and NF-?B.The human pancreatic cancer cell line PANC-1 was futher selected.Pancreatic cell line H6c7,qRT-PCR method was used to detect the expression level of AGR2 and NF-?B subunit RelA,and Western blot was performed simultaneously Method to detect protein expression of AGR2 and RelA.The expression levels of AGR2 and rela were analyzed by TCGA database.RNA and protein were extracted from xenograft tumors immediately after eugenol-treated xenograft mice and mice without eugenol-treated xenograft tumors.qRT-PCR was used to detect mRNA expression of AGR2 and RelA,and Western blot was performed simultaneously Method to detect protein expression of AGR2 and RelA.Chromatin immunoprecipitation experiments verified direct binding between RelA and the AGR2 promoter.The sequences containing RelA and AGR2 binding sites were cloned into the pMIR-Report Luciferase vector,and the WT-luf vector(wt)and MUT-Luf base mutation vector(mut)were constructed and treated 'with eugenol.Luciferase reporter gene experiment It was verified that eugenol further regulates AGR2 by regulating NF-?B.Construction of a PANC-1 cell line that overexpresses/silences AGR2.A silent AGR2 pancreatic cancer xenograft mouse model was constructed and compared with the empty vector pancreatic cancer xenograft tumor mice.The survival analysis was performed by Log-Rank method.Construct a mouse xenograft model of PANC-1 cell line overexpressing AGR2 and a mouse xenograft model of PANC-1 cell line with an empty vector AGR2,respectively,given or not given eugenol treatment,divided into AGR2+EA group,Empty vector Three groups of mice in the+EA group and Empty vector+Vehicle group were studied to compare the survival time,the size of the transplanted tumor,the weight of the tumor in the three groups of pancreatic cancer xenograft mice,and the mice were sacrificed on the 21st day.The tumors were subjected to western blot to detect the protein levels of AGR2 and NF-?B subunit RelA.ResultThe number of colony-forming colonies of PANC-1 cells cultured in eugenol-containing medium was significantly reduced compared with ordinary medium.The weight of xenograft tumors in mice treated with eugenol was significantly reduced compared with the blank control group.Using Kaplan-Meier analysis,we found that the eugenol group significantly prolonged the overall survival of pancreatic tumor xenograft mice compared with the blank group rate.Compared with adjacent cancer tissues,the expression of AGR2 and RelA mRNA in human pancreatic tumor tissues was significantly increased;the expression of AGR2 and RelA mRNA in pancreatic cancer cell line PANC-1 was increased compared with normal pancreatic cells H6c7.TCGA database analysis showed that the expression of AGR2 and RelA were significantly increased in human pancreatic tumor tissues.Detection of eugenol-trealed xenograft tumor mice and non-engenol-treated xenograft tumor mice by qRT-PCR showed that the expression of AGR2 and RelA in xenograft tumors of eugenol-treated mice The expression of AGR2 and RelA in xenograft tumors of mice not treated with eugenol was significantly down-regulated.Chromatin immunoprecipitation experiments show that there is a large amount of AGR2 transcript enrichment in the RelA immunoprecipitation complex,confirming that RelA and AGR2 directly bind to play a role.The dual luciferase report experiment showed that eugenol can inhibit the luciferase activity of WT-luf compared with the control group,and the inhibitory effect of eugenol on luciferase can be offset by the interference mutation of MUT-Luf.The survival time of mice in the Empty vector+EA group was longer than that in the AGR2+EA group and Empty vector+EA group,and the mass and volume of xenograft tumors were larger in the other two groups of mice.Western blot analysis showed that in the Empty vector+Vehicle group,the expressions of RelA and AGR2 both increased,and the expression of RelA decreased in the AGR2+EA group,while the expression of AGR2 increased,and the expressions of RelA and AGR2 decreased in the Empty vector+EA group.ConclusionThe increased expression of AGR2 and rela is an important factor in the development of pancreatic cancer.AGR2 is target gene of NF-?B.Eugenol acid is a potential drug for the treatment of pancreatic cancer by inhibiting NF-?B,thereby inhibiting AGR2,and reducing the ability of pancreatic cancer cells to grow.