7 12 dimethylbenzanthracene dmba

All experiments were carried out in accordance with the Ethical Guidelines from the Institutional Animal Care and Use Committee (CEUA) and the NIH Guidelines for the Care and Use of Laboratory Animals (http://oacu.od.nih.gov/regs/index.htm. 8th Edition; 2011). The protocol used was approved by the CEUA of the West Zone State University (UEZO) with protocol code 008/2019. The animals were housed in polyethylene cages at the animal facility and were maintained under a 12-h light/dark cycle with a controlled temperature (25 °C) and free access to water and food.
Thirty female Wistar rats (150–200 g and 8-week-old) were used for the experimental induction of breast cancer using the method described by Deepalakshmi and Mirunalini [35 (link)] and our group [21 (link), 36 (link)]. In summary, the rats were anesthetized (ketamine and xylazine) and the breast tumour was induced by subcutaneous injection in the mammary region with one single dose of 25 mg/kg 7,12-dimethylbenzanthracene (DMBA) (Sigma-Aldrich, St. Louis, MO) dissolved in 0.5 mL of physiological saline and 0.5 mL of sunflower oil. The DMBA was used according to the manufacturer’s instructions. Rats were palpated in the mammary gland once a week to detect the presence of breast tumours for 15 weeks.

MCF-7 and T47D human breast cancer cell lines were purchased from the America Type Culture Collection (Manassas, VA, USA). DMEM (UFC-Biotech, Riyadh, Saudi Arabia) supplemented with 15% (v/v) foetal calf serum (FCS, Biowhitaker, Lonza, Belgium), 2 mM glutamine, penicillin (100 IU/mL), and streptomycin (Sigma St. Louis, MO, USA) (100 g/mL) was used to maintain the cell lines. Conditions of 37 °C, 5% CO_2, and 95% relative humidity were maintained on all cell lines in the incubator. A 10 mM solution of TQ (Sigma-Aldrich, Louis, MO, USA) in 10% Dimethyl sulfoxide (DMSO) (Millipore, Molsheim, France) was used for all treatments and for preparing the requisite working concentrations with cell culture medium. In both controls, the overall DMSO concentration remained less than 0.1%. 7,12-dimethylbenzanthracene (DMBA) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Corn oil was used to prepare the stock solutions of thymoquinone.