The Mechanism Research Of Oridonin Suppressing Esophageal Epithelial Cell Transformation

ObjectiveEsophageal cancer(EC) is one of the most common upper digestive tract malignancies. China is the highest incidence of EC area in the world, especially in Hebei, Henan, Fujian and Chongqing. Occurrence and development of EC is a lengthy progress, and it is known that there is a process from esophageal squamous cell slight or moderate to severe hyperplasia, till carcinoma in situ or esophageal squamous cell carcinoma. Esophageal squamous cell slight or moderate hyperplasia which is known to us as a stage before severe hyperplasia belongs to a large scale. If medical interventions are used to inhibit the further progression, cancer prevention may be done. Numerous researches have been reported that there are close relations between EC and aberrant activation of certain signal pathways(Wnt 、 Notch 、Ras/Raf/MEK/ERK、PI3K/Akt/m TOR pathway) in cells. These signal molecules may be targets of preventing and treating EC. Inhibition of some targets can be new trends in preventing and treating EC.Rabdosia rubescens(Hemsl.)Hara is a plant of labiatae which was used to soothe sore throats and prevent EC from progression and one of which main anti-tumor constituents is oridonin. Clinical trials have proved that Rabdosia rubescens(Hemsl.) Hara can relief symptoms of cancers, such as esophageal cancer, cardia cancer, primary hepatic cancer and breast cancer and prolong patients’ life. However, the mechanism of anti-tumor is not clear. This thesis is about the mechanism research of oridonin suppressing EGF-induced SHEE cell transformation through cell toxicity assay, inhibition of proliferation assay, anchorage-independent cell growth assay, human phospho-kinase array and western blot assay, aiming for the early level prevention of EC. Materials and Methods1.Research the toxicity of oridonin to SHEE cell through different concentrations(DMSO, 1, 5, 10, 25, 50 and 100 μM) prior to the next assay.2.Research the inhibition of proliferation of SHEE cell at different time(0,24, 48, 72 and 96h) in different concentrations of oridonin(DMSO, 1, 2.5, 5, 10 and 20μM).3.Research the anchorage-independent SHEE cell growth in different concentrations of oridonin(DMSO, EGF, 1μM+EGF, 2.5μM+EGF, 5μM+EGF, 10μM+EGF and 20μM+EGF, CEGF=10ng/ml).4.Screen for the EGF-induced activated protein kinase which can be inhibited by oridonin(DMSO, EGF and 20μM+EGF, CEGF=10ng/ml) with Human Phospho-Kinase Array Kit.5.Validate the results of human phospho-kinase array through western blot.6.Research the inhibition of different concentrations of oridonin(DMSO, EGF, 1μM+EGF, 2.5μM+EGF, 5μM+EGF, 10μM+EGF and 20μM+EGF, CEGF=10ng/ml) compared with the given inhibitor LY294002 through western blot. Results1.The treament of SHEE cell with different concentrations of oridonin(DMSO, 1, 5, 10, 25, 50 and 100 μM) for 24 h or 48 h tested by CCK-8 assay resulted in that 20μM oridonin could lead to 80 precent of cell viability.2.The treament of SHEE cell with different concentrations of oridonin(DMSO, 1, 2.5, 5, 10 and 20μM) for 0h, 24 h, 48 h, 72 h and 96 h tested by CCK-8 assay resulted in that cell viability decreased with the 20μM oridonin at 72 h and 96h(P<0.05).3.The cell colonies of control didn’t form and a signficant decreased(P<0.05) in colony formation in cells with different concentrations of oridonin compared with EGF-treated group.4.Human phospho-kinase array showed that oridonin could inhibit phosphorylation of Akt/m TOR and Akt/GSK3β signal pathway.5.Western blot assay showed the same results with human phospho-kinase array.6.Western blot assay showed that oridonin could inhibit Akt/m TOR and Akt/GSK3β signal pathway phosphorylation with dose-dependent manner(P<0.05) and was the same with the PI3 K inhibitor LY294002(P>0.05). Conclusions1.Oridonin inhibits the proliferation of SHEE cell with dose-dependent manner and suppresses the EGF-induced cell transformation.2.Oridonin inhibits phosphorylation of Akt/GSK3β and Akt/m TOR pathway, but doesn’t inhibit ERK/RSK2 pathway.