Anti-Pancreatic Cancer Activity And Mechanism Of Natural Sesquiterpenoids Carabrone And Nardoguaianone L In Vitro

Pancreatic cancer is the seventh leading cause of cancer-related death in human,and is called as the“king of cancer”.It is difficult to diagnose and has a very poor prognosis.Currently,there is no effective targeted therapy drug in clinical practice.Therefore,it is important to search for and discover therapeutic reagent related to pancreatic cancer.Active molecules from natural sources usually have the characteristics of multi-target,wide range of action,and easy access,and are an important source of drug discovery.Carabrone is a carabane-type sesquiterpene compound extracted from Carpesium cernuum,which has anti-liver cancer,anti-breast cancer and anti-oral cancer activities.As a potential anti-tumor molecule,there are few research reports on Carabrone’s anti-tumor activity in pancreatic cancer.Nardoguaianone L（G-6）is a novel guaiane-type sesquiterpenoid from Nardostachys jatamansi.In this paper,the evaluation of the anti-pancreatic cancer activity and the preliminary mechanism research were carried out for the single-drug use of Carabrone,G-6 and G-6 combined with gemcitabine in vitro.First,the effects of drugs on cell viability,cell migration,intracellular MMP2 and MMP9proteins,cell morphology,cell proliferation effects and population dependence,mitochondrial membrane potential,ROS,and cell death.In addition,PI single staining and Annexin V/PI double staining were used to detect cell cycle and apoptosis by flow cytometry.Finally,according to the analysis of proteomics and combined with JC-1 single staining and DCFH-DA single staining,the mechanism of action of the drug to inhibit the growth of pancreatic cancer cells was preliminarily clarified.The research results are as follows:Firstly,Carabrone inhibited the proliferation of pancreatic cancer Capan-2,PANC-1,SW1990,and CFPAC-1 cells in a dose-dependent manner,and its IC₅₀ values were47.62±1.72μM,7.78±2.62μM,5.53±1.19μM and 48.72±2.90μM.Carabrone anti-pancreatic cancer SW1990 cell activity results in vitro showed that,compared with the control group,Carabrone could reduce the colony formation rate of SW1990 cells by 7fold,the 2-4 fold reduction in cell migration rate,and the expressions of migration-related proteins MMP2 and MMP9 were inhibited.Carabrone could induce apoptosis and G2phase cycle arrest in SW1990 cells,increase the level of ROS in SW1990 cells,and reduce the mitochondrial membrane potential.Carabrone-treated SW1990 cells regulated the Hippo pathway by up-regulating CSNK1E and down-regulating WWTR1,and induced ferroptosis by up-regulating HMOX1,SLC7A11 and MAPKAP1.Secondly,G-6 inhibited the proliferation of pancreatic cancer cells Capan-2,PANC-1,SW1990,and CFPAC-1 in a dose-dependent manner.At 72 h,the IC₅₀ value of SW1990 cells was 30.14±16.11μM,and the IC₅₀ values of the other three types of cells were all greater than 50μM,and inhibited the migration of SW1990 cells in a concentration-dependent manner.Compared with the GEM treatment group,G-6combined with GEM decreased the survival rate of SW1990,Capan-2 and PANC-1 cells by 1.35-3.39 fold,1.12-2.39 fold and 1.32-2.83 fold respectively,but had no significant inhibitory effect on CFPAC-1 cells.After G-6 and GEM were treated respectively,the cell morphology was changed,and after combined treatment,the cells became brighter and the gaps became dirtier,and nuclear agglutination and nucleolus shrinkage appeared.G-6 combined with GEM could significantly inhibit the proliferation of SW1990 cells,induce apoptosis and G1 phase cycle arrest in SW1990 cells,and the effect was better than that of single drug treatment group.G-6 combined with GEM induced the increase of ROS and the decrease of mitochondrial membrane potential in SW1990 cells,and regulated the AGE-RAGE signaling pathway by down-regulating PI3K,TGFβ1 and PLCD1.In summary,Carabrone exhibited cytotoxicity to pancreatic cancer SW1990 cells through the inhibition of proliferation and migration mediated by the Hippo signaling pathway,and arrested the G2 phase of the cell cycle,and eventually induced ferroptosis in cells.Compared with single administration,G-6 combined with GEM increased the anti-tumor activity of SW1990 cells,which mainly regulated the AGE-RAGE signaling pathway by down-regulating PI3K,TGFβ1 and PLCD1 to induce the increase of ROS and destroy mitochondrial membrane potential,induction of apoptosis.The results of this study provide experimental data and theoretical support for the sources of pancreatic cancer drugs and their molecular mechanisms.