Methods
Human breast cancer cells (MC-F7) were grown in culture and a variety of experimental assays were performed to determine the growth characteristics of these cells alone or in the presence of the aforementioned drugs.
Cell Culture:
MCF-7 cells were maintained in regular growth media (DMEM + 10% fetal bovine serum) in 7 mL tissue culture flasks. The cells were grown to confluency, removed from the flasks and plated onto either glass cover slips or 35mm plastic culture dishes. The cells were then treated with the test drugs (pimozide, mibefradil, or tamoxifin) at various doses (100nM to 100uM) for 24 hours before being subjected to a variety of cells assays to determined cell growth (propidium iodide proliferation assay) and the specific pathway of cell death (Ethidium Bromide/Acridine Orange fluorescent experiment).
Morphological Assessment of MCF-7 cells through Propidium Iodide Assay
To visualize and quantify the number of cells surviving drug treatment as well as cell morphology. Cells treated with drugs for 24 or 48 hr (see above) will be washed with HBSS and stained with the dye propidium iodide that stains only cells that have lost their membrane integrity (dying cells) and observed using differential interference microscopy (DIC) on a micoscope at 200X magnification. High power micoscopy fields will be captured using a CCD camera connected to a personal computer and images will be stored on computer disc. Numbers of viable and dying cells will be counted using a commercial image analysis program (ImagePRO) and cell appearance observed e.g number of cells and cell colonies, cell swelling, multinucleated cells, size of cells, cell spreading.
The cells (from the culture wells) were washed with fresh MEM, and incubated in growth media minus NGF / serum together with appropriate doses of the drugs. The wells were rinsed were gently with PBS. One ml of ethidium bromide / acridine orange solution was added to cover the cells. They were left to incubate for five minutes in the dark at room temperature. The cells were then rinsed gently with a solution of PBS / glycerol (1:1). The cells were then viewed under flourence. Viable cells had intact nuclei and stained green / yellow. Apoptotic cells had very visible piknotic nuclei with DNA condensation. Necrotic cells fluoresced orange. Cells were counted and the numbers of viable, apoptotic and necrotic cells were determined per group.
Cell Proliferation Measurement:
Cell proliferation was quantified using the Cell Titer 96AQ kit. Cells were grown in 96 well plates and treated with varying concentrations of drug. The quantity of formazin product formed (directly proportional to the number of viable cells) was measured on a plate reader and converted to cell number.