The kinase reaction is performed in 50 μL of 50 mM HEPES (pH 7.3), containing 125 mM NaCl, 24 mM MgCl₂, 0.1 mM Na₃VO₄, 20 μM ATP, 1.6 μg/mL EGF, and 15 ng of EGFR, affinity purified from A431 cell membranes. The compound in DMSO is added to give a final DMSO concentration of 2.5%. Phosphorylation is initiated by addition of ATP and proceeded for 8 mm at room temperature, with constant shaking. The kinase reaction is terminated by aspiration of the reaction mixture and is washed 4 times with wash buffer. Phosphorylated PGT is measured by 25 mim of incubation with 50 μL per well HRP-conjugated PY54 antiphosphotyrosine antibody, diluted to 0.2 μg/mL in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). Antibody is removed by aspiration, and the plate is washed 4 times with wash buffer. The colonmetric signal is developed by addition of TMB Microwell Peroxidase Substrate, 50 μL per well, and stopped by the addition of 0.09 M sulfuric acid, 50μL per well. Phosphotyrosine is estimated by measurement of absorbance at 450 nm. The signal for controls is typically 0.6-1.2 absorbance units, with essentially no back ground in wells without AlP, EGFR, or POT and is proportional to the time of incubation for 10 mm^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

To test the viability of cells treated with B-DIM, Erlotinib, or the combination, BxPC-3 and MIAPaCa cells are plated (3,000-5,000 per well) in a 96-well plate and incubated overnight at 37°C. A range of concentrations for both B-DIM (10-50 µM) and Erlotinib (1-5 µM) is initially tested. Based on the initial results, the concentration of B-DIM (20 µM) and Erlotinib (2 µM) are chosen for all assays. The effects of B-DIM (20 µM), Erlotinib (2 µM), and the combination on BxPC-3 and MIAPaCa cells are determined by the standard MTT assay after 72 h and is repeated three times. The color intensity is measured by a Tecan microplate fluorometer at 595 nm. DMSO-treated cells are considered to be the untreated control and assigned a value of 100%. In addition to the above assay, we have also done clonogenic assay for assessing the effects of treatment^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Female ICR-SCID (6-7 weeks old) mice are randomized into the following treatment groups (n=7): (a) untreated control; (b) only B-DIM (50 mg/kg body weight), intragastric once every day; (c) Erlotinib (50 mg/kg body weight), everyday i.p. for 15 days; and (d) B-DIM and Erlotinib, following schedule as for individual treatments. All mice are killed on day 3 following last dose of treatment, and their body weight is determined. One part of the tissue is rapidly frozen in liquid nitrogen and stored at −70°C for future use and the other part is fixed in formalin and processed for paraffin block. H&E staining of fixed tissue section is used to confirm the presence of tumor(s) in each pancreas.
Rats^[3]
Six-week-old male Sprague-Dawley (SD) rats weighing 180 to 210 g are used. Cisplatin (CP) is freshly prepared in saline at a concentration of 1 mg/mL and then injected intraperitoneally in SD rats (n=28) at a dose of 7 mg/kg on day 0. To investigate the effect of Erlotinib, 28 CP-N rats are divided into two groups. Separate groups (n=14) each of animals are administered with either Erlotinib (20 mg/kg) (CP+E, n=14) or vehicle (CP+V, n=14) daily by oral gavage from day -1 (24 hours prior to the CP injection) to day 3. Vehicle-treated groups receive an equivalent volume of saline. Five male SD rats at the age of 6 weeks are used as a normal control group (NC, n=5). The NC rats are given an equivalent volume of saline daily by oral gavage from day -1 to day 3. At day 4 (96 hours after CP injection), each rat is anesthetized and sacrificed by exsanguination after the cardiac puncture; blood is collected by cardiac puncture and kidneys are collected. Renal tissue is divided; separate portions are snap-frozen in liquid nitrogen or fixed in 2% paraformaldehyde/phosphate-buffered saline (PBS) for later use. All surgery is performed under diethyl ether gas anesthesia, and all efforts are made to minimize suffering.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Moyer JD, et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res. 1997, 57(21), 4838-4848.  [Content Brief]

  [2]. Ali S, et al. Apoptosis-inducing effect of erlotinib is potentiated by 3,3'-diindolylmethane in vitro and in vivo using an orthotopic model of pancreatic cancer. Mol Cancer Ther, 2008, 7(6), 1708-1719.  [Content Brief]

  [3]. Wada Y, et al. Epidermal growth factor receptor inhibition with erlotinib partially prevents cisplatin-induced nephrotoxicity in rats. PLoS One. 2014 Nov 12;9(11):e111728.  [Content Brief]