In vivo assessment of anti-tumor activity

The transplantable murine tumor cell line, namely Ehrlich ascites carcinoma cells, was obtained from National Cancer Institute, Cairo, Egypt. Ehrlich ascites carcinoma cells were maintained in vivo in female Swiss albino mice by intraperitoneal transplantation of 2 × 10⁶ cells/mouse every 10 days. Ascitic fluid was drawn from tumor-bearing mice at the log phase of the tumor cells (7–8th day of tumor bearing). The freshly drawn fluid was diluted with ice-cold sterile isotonic saline and each animal received 200 μl of tumor cell suspension containing 2 × 10⁶ tumor cells subcutaneously.

Female Swiss albino mice weighing 20–25 g were divided into four groups (ten animals per group). When mice developed palpable mass (tumor volume range: 80–120 mm³), treatment was started (day 0). Group 1 was given the vehicle daily and considered as untreated control. Group 2 was given the vehicle once daily for 10 days and on day 7, cisplatin was injected at a single dose of 5 mg/kg i.p. Group 3 was given indole-3-carbinol at a dose of 20 mg/kg orally once daily for 10 days and on day 7, cisplatin was injected at a single dose of 5 mg/kg i.p. Group 4 was given 20 mg/kg indole-3-carbinol orally once daily for 10 days. The dose of cisplatin was selected according to previous studies^36,37. Longest and shortest diameters of the tumor were measured using a digital Vernier caliper. Tumor volume of each animal was calculated using the following formula:

On day 11, animals were sacrificed and tumor specimens were excised and fixed in 10% formalin for histopathological examination using light microscopy. Also, Kidney specimens were taken for the assessment of histopathological alterations induced in the different treatment groups according to the method previously described.