In vitro activity: Norcantharidin (also known as NCTD) is a dual inhibitor of c-Met and EGFR. It has anticancer activity and can induce cell cycle arrest at G2/M phase in HCT116 and HT29 cells. NCTD inhibits not only the expression of the total EGFR and the p-EGFR (phosphorylated EGFR) but also the expression of the total c-Met and the pc-Met (phosphorylated c-Met) in colon cancer cells.

Cell Assay: Norcantharidin is also an inhibitor of protein phosphatase types 1, 2A and 2B, and can arrest the cell cycle at G2/M phase in K562 human myeloid leukemia cells, and inhibit DNA synthesis in HL-60 cells. HT29 and HCT116 cells suspended in complete media are seeded in 96-well plates (2000 cells/well). After growing for 24 h in incubator, media are aspirated and 0.2 mL complete media containing serial concentrations of NCTD are added to each well. After incubating the plate for 24, 48, or 72 h, 20 μL of resazurin (2 mg/mL dissolved in water) is added to each well. The fluorescent signal is monitored using 544 nm excitation and 595 nm emission wavelengths by Spectramax M5 plate reader after incubation at 37 °C for 16 h in the incubator. The number of living cells in each well is proportional to the relative fluorescence unit (RFU) measured by the assay.