In vivo tumor xenograft model

Athymic male mice were acquired from the San Diego Institute of NxGen Biosciences were kept under a contamination-free environment (12 h day/12 h night schedule), provided with a sterilized food ad libitum. CWR22Rν1 cells were selected for evaluating the in vivo impact of MEM as they generate fast tumors in mice. The implantation of CWR22Rν1 cells is also responsible for the secretion of marked quantities of PSA in the bloodstream of the mice. Cells were analyzed, suspended in complete RPMI medium 1640. Tumor xenografts CWR22Rν1 cells in mice were established by injecting cells (1 × 106) subcutaneously near tail mixed with RPMI plus (Collaborative Biomedical Products, Bedford, MA) Matrigel in a ratio of 1:1. Twelve mice were randomly selected into two groups containing six animals each. Mice first group served as control were fed with normal drinking water. Mice of groups two were injected with an intraperitoneal injection of MEM 2.5 mg. Throughout the study weight of the mice's body, food, and water used were noted two times a week. Tumor volume was calculated by the formula 0.5238 × L1 × L2 × H (L1 = long diameter, L2 = short diameter, and H = height of the tumor) and tumor sizes were measured twice weekly. Animals were euthanized using the CO2 inhalation method following ARAC guidelines when tumor volumes reached ~ 1200mm³. Blood samples were collected by the mandibular bleed and serum separated from the whole blood was stored at − 20 °C. Serum PSA levels were assayed by the PSA ELISA kit described above. H&E stained slides of lung, kidney, liver, heart, and brain were prepared to examine the possible toxic effects of MEM treatment.