Thus, endocrine therapy may play a role in treating hormone-dependent cancers by decreasing the metastases that are caused by MMP7 activation. To test this hypothesis, selleck we examined the ability of TAM to decrease MMP7 activation in the ERβ-positive colon cancer cell line HT29. Methods

Cell culture and treatment HT-29 cells are highly metastatic colon carcinoma cells that were obtained from the American Type Culture Collection, Rockville, MD, USA. Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2. Drug administration schedules TAM and fluorouracil (5-FU) were purchased from Sigma (St Louis, MO). The drug-exposure www.selleckchem.com/products/carfilzomib-pr-171.html schedules, which are summarized in Table 1, were as follows: (a) no treatment; (b) TAM alone (1 × 10-7, 1 × 10-6, 1 × 10-5, or 1 × 10-4 M) for 48 h; (c) 5-FU alone (6.25, 12.5, 25, or 50 μM) for 72 h; (d) 12.5 μM 5-FU for 24 h followed by 12.5 μM 5-FU plus indicated TAM for 48 h. The experiments were performed in triplicate for each time point, and the means ± SD were calculated. Appropriate amounts of drug solution were added directly to the growth

medium the day after plating. Control cells were plated in growth medium supplemented with 0.1% DMSO. Table 1 Schedule of each group of treatment for three different times Group 24 h 48 h 72 h (a) no treatment     (b) TAM TAM   (c) 5-FU 5-FU 5-FU (d) 5-FU 5-FU+TAM 5-FU+TAM Drug sensitivity, as indicated by the MTT assay To induce cell death, cells were treated with either TAM (Sigma, Cat. No. T-9262) dissolved in DMSO or 5-FU. The final concentrations ranged from 1 × 10-7 to 1 × 10-4 M for P-type ATPase TAM and from 6.25 to 50 μM for 5-FU. To test the cytotoxicity of each drug, HT-29 cells in the exponential growth phase were seeded into 96-well cell plates

in 100 μl of culture medium for 24 h prior to drug exposure and then treated with various concentrations of TAM, 5-FU, or a combination of these drugs. Cytotoxicity was evaluated using a tetrazolium-based semi-automated colorimetric (MTT) assay, with an ELISA reader at OD490. Flow cytometry analysis HT-29 cells were seeded in 6-well plates at a density of 4 × 106 cell/well. Cells were treated with various concentrations of each drug for the appropriate times, incubated at 37°C, fixed in 70% ethanol, and labeled with propidium iodide solution (50 μg/ml; Sigma-Aldrich). The DNA content and cell cycle distribution of approximately 1 × 106 stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson). Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was isolated from 4 × 106 cells by TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was reverse transcribed in a total volume of 20 μl containing 2 μg RNA, 0.5 μg olig (dT)15, and 15 μl DEPC-treated water. Reverse transcription reaction was incubated at 30°C for 10 min, 48°C for 30 min, and 99°C for 5 min.