Tacrolimus (FK506, Fujimycin) is a macrolide immunosuppressant that binds to FKBP12 to form a high-affinity complex (Ki = 0.2 nM), which inhibits calcineurin phosphatase activity, thereby suppressing T lymphocyte signal transduction and the transcription and release of IL-2. It exerts potent immunosuppressive effects and can be used to induce immunosuppression models.

SN-38 glucuronidation (human liver microsomes):3.58 μM, Number of circulating helper-T cells:6.7 ng/mL, FKBP12:0.2 nM (Kd), SN-38 glucuronidation (rat liver microsomes):10.33 μM, Lymphocyte proliferation:0.47 nM

Tumor cell proliferation was determined by the MTT. Briefly, after MH3924A cells had reached the logarithmic growth phase, a 0.2-ml cell suspension at 1×10^4 cells/ml was added into each well of a 96-well plate and cultured in DMEM with 10% FBS, 10 μg/l vascular endothelial growth factor and 0.1 g/l heparin for 24 h. When adherent growth was established, different concentrations of FK506 (10, 100 and 1,000 μg/l), AMD3100 (10, 50 and 100 μg/l) and FK506 (0 and 100 μg/l) + AMD3100 (0, 10, 50 and 100 μg/l) were added into the plates. Untreated cells cultured in medium alone were used as controls. After culturing for 48 h, 10 μl MTT (5 g/l) was added, and each well was incubated for 6 h; next, 150 μl/well dimethyl sulfoxide was added, followed by measurements of the absorbance at 570 mm on a spectrophotometer reader. Each well was measured three times, and each sample was assayed in triplicate [2].

Experiments were performed in 16 healthy August Copenhagen Irish rats (male, 16–20 weeks, weighing 240–300 g). The rat model of liver tumor was established as follows: First, MH3924A cells were collected and injected into the alar skin of rats. The tumors were removed from alar skin when grown to 2×1×1 mm3, and intrahepatic tumor implantation of rats was performed under aseptic conditions as described previously (25,26). Five days later, rats were randomly divided into two groups: one group was subcutaneously injected with normal saline for 14 days (NS group, n=8, 3 mg/kg/day), and the second group was subcutaneously injected with FK506 for 14 days (FK506 group, n=8, 0.3 mg/kg/day). Forty days following implantation, rats were sacrificed, and the weight of tumor, the volume of the fluid in the ascites, the incidence of lymphatic metastasis in the abdominal cavity and of abdominal wall metastasis were measured. In addition, the lungs were irrigated with 15% Indian ink, followed by counting of the number of metastatic nodules in the lung. The tumor and adjacent tissues, as well as healthy liver tissues, were harvested and preserved in 4% formalin for later use [2].