| PurposeThe genesis of esophageal cancer is a multi-stage progressive process and influenced by many factors. With the rapid development and application of molecular biology techniques, researchers learn more about the pathogenesis of tumors at molecular levels.Accumulated evidence has indicated that cell signaling pathways will provide new targests for the diagnosis and treatment for esophageal cancer. It will provide new ideas for the treatment of esophageal cancer to aim at particular target or several targets in the process of esophageal cancer genesis and development.Eupatilin (5,7-dihydroxy-3’4’6-tri-methoxy-flavone) is a pharmacologically active flavone, which derived from Artemisia asiatica. Researchers have found eupatilin has antioxidative activity、anti-inflammatory、anti-tumor activity and protecting cell properties, but the mechanism about eupatilin how to act its biological activity and its effects on esophageal cancer is still unclear. The study discusses the mechanisms of eupatilin inhibiting TE1cell proliferation by cell proliferation in vitro、cell soft agar cloning、flow cytometry and western blotting assays, and the study will provide new experiment basis for early chemical intervention of esophageal cancer.Materials and methods1. We performed cytotoxic assay to check the toxic effects of eupatilin on esophageal cancer with different concentration of eupatilin(0、3.125、6.25、12.5、25、50μM).2. We performed cell proliferation experiment to prove the inhibitory effect of eupatilin with different concentration (0、2.5、5、10、20、40μM) on esophageal cancer cell TE1at different time (0、24、48、72、96h).3. We observed the colony-forming ability through different concentrations (0、2.5、5、10、20、40μM) of eupatilin acting on esophageal cancer cell TE1by soft agar cloning assay.4. We used the flow cytometry assay to detect the influence of eupatilin on cell cyle after TE1cells were treated of eupatilin for24hours.5. To verify whether eupatilin activate AKT-GSK3β signaling pathways and MAPK/ERK signaling pathway, we used western blotting to detect the signaling pathways changes after TE1treated with eupatilin.Results1. The toxicity of eupatilin effecting on TE1We measured the cells OD values for cell survival curve after esophageal cancer cell TE1treated with different concentration for24h and48h. We could conclude the IC50concentration was about40μM.We used the concentration gradient in turn:0、2.5、5、10、20、40μM to do the following experiment.2. Eupatilin could inhibit the proliferation of TE1cellsEupatilin restrained the growth of the esophageal cancer cell TE1(P<0.05) in dose and time dependent, compared with the control group.3. Eupatilin could reduce the colony number of TE1on softagar.Compared with the control group, different concentrations of eupatilin could reduce the number of TE1cell clone (P<0.05), and with the concentration of eupatilin increasing, TE1cell clone were reduced (P<0.05).4. Eupatilin inhibited the proliferation of TE1by causing G1phase arrest Flow cytometry results showed that with the increasing concentration of eupatilin effecting on TE1, the percentage of G1phase was increasing, compared with the control group (P<0.05).5. Eupatilin could inhibit the signal pathway of AKT-GSK3β and MAPK/ERKEupatilin could inhibit the signal pathway of AKT-GSK3β,eupatilin could reduce the phosphorylation level of p-AKT and its downstream GSK3β in the signaling pathway of AKT-GSK3β.The phosphorylation level of AKT and GSK3p tended to a downward trend with the concentration of eupatilin was increasing.Eupatilin also can inhibit MAPK/ERK signaling pathway with the level of p-ERK reduced.Conclusion1. Eupatilin can restrain the growth of the esophageal cancer cells TE1by causing cell cycle arrest at G1phase, and can reduce TE1colony number on softagar.2. Eupatilin inhibits the phosphorylation level of AKT and its downstream molecules in TE1cells, and it also inhibits the level of p-ERK in MAPK/ERK signaling pathway. |
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