The Antitumor Mechanism Of A Novel Diterpene Jaridon 6 From Isodon Rubescens On Gastric Cancer Resistant Cell MGC803/5-Fu

During treatment of malignant tumors,drug resistance due to chemotherapy has an important effect on the efficacy and prognosis of cancer patients.Rabdosia rubescens is a natural plant with anti-tumor efficacy.Jaridon 6 is a novel diterpene compound extracted and isolated from rubescens,its anti-tumor mechanism has been studied and conformed in esophageal cancer cells,but its anti-tumor activity and molecular mechanism in drug-resistant cells are not yet clear,so this study mainly researched the anti-tumor activity and mechanism of Jaridon 6 on gastric cancer-resistant cells MGC803/5-Fu to provide some ideas for the research on new anti-tumor compounds.Firstly,we studied the effect of Jaridon 6 through the Akt pathway on the proliferation of resistant cells MGC803/5-Fu.DAPI staining and Annexin V/PI staining were used to observe the effect of compound Jaridon 6 on the apoptosis of MGC803/5-Fu apoptosis.At the same time,Annexin V/PI staining was used to detect the effect of Jaridon 6 on the cell cycle of MGC803/5-Fu.Western blot assay detected the expression of protein p-PI3 K,PI3K,p-Akt,Akt,p-GSK-3β,GSK-3β,cleaved-Caspase-9,cleaved-Caspase-3,cleaved-Caspase-7 and PARP-1.The results showed that Jaridon 6 had a good inhibitory effect on the proliferation of MGC803/5-Fu cells,and it could inhibit the formation of MGC803/5-Fu cell colony.The cell MGC803/5-Fu was observed under microscope after DAPI staining,cellular rounding and nuclear shrinkage;Annexin V/PI double staining method detected that compound Jaridon 6 promoted early apoptosis of MGC803/5-Fu;simultaneously blocked MGC803/5-Fu in G0/G1 phase;Western blot experiments showed that the expression of proteins p-PI3 K,p-Akt and p-GSK-3β in MGC803/5-Fu cells were decreased,the expression of proteins PI3 K,Akt and GSK-3β remained unchanged,while the expression of proteins cleaved-Caspase-9,cleaved-Caspase-3,cleaved-Caspase-7,and cleaved-PARP-1 were increased,which activated the intracellular Caspase pathway and promoted cell apoptosis.Then,we researched the effect of Jaridon 6 on autophagy of MGC803/5-Fu cells.Immunofluorescence technique was used to detect the autophagy and localization of the autophagic marker protein LC3 B in MGC803/5-Fu cells.Transmission electron microscopy showed that the ultrastructure of intracellular autophagosome after compound Jaridon 6 acted on MGC803/5-Fu.Western blot was used to detect the expression of proteins LC3B-Ⅱ and p62,and the expression of LC3B-Ⅱ and p62 were further detected by adding autophagy inhibitor 3-MA.The results showed that after Jaridon 6 acted on MGC803/5-Fu,the LC3 B was mainly localized in the cytoplasm by immunofluorescence,and the fluorescence intensity increased with the increase of the compound concentration;transmission electron microscopy was used to observe the autophagosome structure surrounded by bilayer membrane or multilayer membrane and the autophagic lysosome structure surrounded by monolayer membrane.The compound Jaridon 6 can upregulate protein LC3 B –Ⅱand downregulate the expression of p62 protein in MGC803/5-Fu cells by Western blot.After adding autophagy inhibitor 3-MA,comparing with negative control group,protein LC3B-Ⅱ was downregulated,the expression of protein LC3B-Ⅱ was increased in compound and 3-MA group,and slightly lower than the compound group,but the expression of protein p62 in the group of compound and 3-MA was decreased compared to the negative control group and increased compared to the compound group.Finally,we explored the effect and mechanism of compound Jaridon 6 on the cell metastasis of MGC803/5-Fu.The effect of compound Jaridon 6 on the metastasis of MGC803/5-Fu cells was detected by wound healing assay and transwell assay at the cell level;Western blot was used to detect the expression of E-cadherin,ZO-1,N-cadherin,Vimentin,Snail,Slug and other proteins at the protein level.The results showed that compound Jaridon 6 can inhibit the metastasis of MGC803/5-Fu cells;Western blot showed that the expression of E-cadherin and ZO-1 proteins were increased,the expression of N-cadherin and Vimentin proteins were decreased,the protein β-Catenin was upregulated in cytoplasm and downregulated in nucleus,and the expression of Snail and Slug proteins were also decreased.In summary,we studied the effect and mechanism of the novel compoundJaridon 6 on PI3K/Akt pathway,autophagy and metastasis of gastric cancer-resistant cell MGC803/5-Fu.The main innovations are:1.We proposed and demonstrated that Jaridon 6 activates the Caspase pathway through the PI3K/Akt pathway and promotes the apoptosis of drug-resistant MGC803/5-Fu cells;2.Jaridon 6 can induce autophagy in MGC803/5-Fu cells.3.Jaridon 6 affected the cell metastatic process of MGC803/5-Fu by inhibiting the development of EMT.This study clarified the anti-tumor mechanism of Jaridon 6 and laid the foundation for the discovery of other novel antitumor drug-resistant compounds.