In vitro activity: Oridonin is a cell-permeable diterpenoid compound that possesses anti NF-κB activity and displays antiproliferative (ED50 ~ 2.7 μg/ml in lymphoid malignant cells) and antiangiogenic properties (significantly inhibits network formation of HMEC-1 cells at 2.5 μg/ml). Reported to affect DNA synthesis, induce apoptosis and initiate cell cycle arrest. Shown to efficiently block both TNF-α and LPS-induced NF-κB activity in Jurkat and in RAW264.7 murine macrophages, and inhibit p65 NF-κB transcriptional activity (IC50 ~ 5 μg/ml in MT-1 cells) by disrupting NF-κB DNA-binding activity without interfering with its nuclear translocation.

Kinase Assay: or the AKT kinase assay, the ADP-Glo(TM) Kinase Assay Kit is used. Active AKT1 or AKT2 kinase and inactive GSK-3β as substrate are mixed by 1× reaction buffer and then added to a white 96-well plate. Pure ATP provided in the kit is serially diluted obtain a final concentration of 0, 1, 10, 50, and 100 μM. GSK-3β is added to reach a final concentration of 2.5, 5, 10 or 20 μM and DMSO is used as a control. The mixed solution is incubated at room temperature and luciferase activity is measured using the Luminoskan Ascent plate reader

Cell Assay: Cells are seeded (6×103 cells/well for KYSE70; 2.5×103 cells/well for KYSE410; 2×103 cells/well for KYSE450) in 96-well plates and incubated for 24 h and then treated with different amounts of Oridonin or vehicle. After incubation for 24, 48 or 72 h, cell proliferation is measured by the MTT assay. For anchorage-independent cell growth assessment, cells (2.5, 5 or 10 μM Oridonin) suspended in complete medium are added to 0.3% agar with vehicle, 2.5, 5 or 10 μM Oridonin in a top layer over a base layer of 0.5% agar with vehicle, 2.5, 5 or 10 μM Oridonin. The cultures are maintained at 37°C in a 5% CO2 incubator for 3 weeks and then colonies are visualized under a microscope and counted using the Image-Pro Plus software program.