All cell culture works were carried out in RPMI 1640 medium containing 10% FBS +1X Pen/Strep/Ampho [5] , [11] . Cell growth was quantitated by MTT proliferation assay.

BLM cells were suspended in growth medium (GM) and plated at a density of 1 × 10⁴ cells/well in a 96 well plate. Cells were left overnight at 37˚C to attach to the plate. Following day, growth medium was replaced by GM containing hormones at different concentrations and incubated for 48 hrs. After 48 hrs, medium was replaced by 100 μl of 1 in 10 diluted (in GM) MTT solution and incubated for another 4 hrs at 37˚C. After 4 hrs, MTT solution was removed. MTT was reduced by metabolically viable cells to a colored (purple) water insoluble formazan salt. The purple color precipitate was solubilized by adding 100 μl of isopropanol and shaken for 20 - 30 min at room temperature. Intensity of resultant purple color was measured at 570 nm in a SLT spectra plate reader.

Trypan blue is a dye that does not interact with cell unless the cell membrane is damaged. Healthy, undamaged cells exclude the dye, but it is readily absorbed by damaged cells and renders them clearly visible under microscope. After 48 hrs of incubation of BLM cells with RU-486, 100 μl of 0.4% trypan blue in PBS was added to the chamber slide containing 1 × 10⁴ cells and incubated for 5 min at room temperature. Cells were washed with PBS to remove excess trypan blue dye and were examined under microscope. Only dead cells would take up the dye and appear as dark stained cells under microscope.

DAPI is a blue fluorescent probe. DAPI stains nucleus specifically, with little or no cytoplasmic labeling. After 48 hrs of incubation of BLM cells with RU-486, cells in the chamber slide were fixed with 2% paraformaldehyde for 20 min. Cells were washed with PBS to remove paraformaldehyde. One hundred μl of 1 in 1000 diluted DAPI was added to the cells and was incubated in the dark for 5 min. Excess DAPI was removed by washing with PBS and was viewed under bright and fluorescent light.

TUNEL assay was performed to check DNA fragments arising out of apoptosis. Procedure was carried out as per manufacturer’s protocol. Briefly, cells grown on slide were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Labeled with TdT reaction mix and stained with 100 μl of DAB and visualized under a light microscope.

Agarose gel (1.2%) was made by mixing agarose in 1X TAE buffer. After cooling the solution, ethidium bromide was added to the solution and poured into the gel apparatus. Fifteen microgram of DNA was loaded on the well and ran at 100 volts till the bromophenol dye moved of the gel. The gel was photographed under UV light.

Caspase-3/7 is a critical executioner of apoptosis, as it is responsible for the proteolytic cleavage of many key proteins. Assays were carried out as per the manufacturer’s protocol. Briefly, cells (25000 cell/well) were lysed with lysis buffer. Two X (2X) reaction buffer was added, followed by 2X substrate working solution was added. The plate was covered and incubated at room temperature for 30 min. The plate was read at excitation wavelength of 499 nm and emission wavelength of 521 nm.

As there were conflicting reports on the physical presence of progesterone and estrogen receptors in various melanoma cell lines, the mechanism of RU-486 action was determined by a bioassay. A bioassay, which involved pre-incubation of melanoma cells with progesterone followed by RU-486 treatment, was carried out. In the pre-incubation experiment, melanoma cells were pre-treated with various concentrations of (1, 2.5, 5, 10 μM) of progesterone for 60 - 90 min. Afterwards, RU-486 at various concentrations (10, 50, 100 μM) were added and incubated for 48 hrs. At the end of 48 hrs, MTT assay was carried out.

Initially dose response curve of dexamethasone (Dex) was carried out on BLM cells for 48 hrs. Then 1 μM dexamethasone was co-incubated with various concentrations of RU-486. Dose response curve of straight RU-486 treated cells was compared with dose response curve of Dex and RU-486 co-incubated BLM cells.

All experimental points were carried out in triplicate (3 wells). Each experiment was repeated a minimum of three times to check for consistency in results. Each experimental point was expressed as Mean ± SEM. Significance between any two experimental conditions was decided using student’s “t” test. Unpaired t-test was used to determine the p-values and a p-value of 0.05 or less was considered significant.

Initially dose response curve was carried out with RU-486 starting from 100 nM to 200 μM to find out RU-486 effect on human melanoma (BLM) cell growth. There were qualitative and quantitative changes. Qualitatively cell morphology was changed as shown in Figure 1(a). Quantitatively, there was a dose-dependent (sigmoidal) inhibition of human melanoma cell growth as shown in Figure 1(b). A linear decrease in cell growth was seen between 10 μM and 100 μM concentrations. Secondly, to optimize the length of incubation time, percentage of cell growth with a single concentration of RU-486 (50 μM) was monitored at 6, 12, 24, 48 and 72 hrs time points. A significant decrease in cell growth (43.7%) was observed at 48 hrs as shown in Figure 1(c). So, it was decided to incubate melanoma cells with RU-486 for 48 hrs.

There was a significant decrease in melanoma cell growth with increasing concentrations of RU-486. There were also cells floating in the medium, suggesting cell growth inhibition was due to cell death. So, the mechanism of cell death was investigated.

Cell death by Necrosis occurs when the cell membrane is ruptured. Necrosis is a process of cellular lysis with a corresponding release of cytoplasmic components into the surrounding tissue. Trypan blue dye could get into the cell and stain the dead cell. Viable cells would exclude trypan blue from inside. Trypan blue dye exclusion test was used to check if necrosis had taken place after RU-486 treatment. Trypan blue staining did not show any significant staining in the number of dead cells between untreated control and RU-486 treated cells (Figure 3), indicating necrosis was not the mechanism of inhibition of cell growth.

Apoptosis, also known as programed cell death proceeds systematically. Chromatin condensation takes place followed by change in nuclear shape. So DAPI was used to stain the nucleus to check for change in nuclear shape.

RU-486 is a synthetic steroid hormone and was shown to act through progesterone receptor [7] as well as glucocorticoid receptor [8] . Hence, it was important to study the mechanism of RU-486 action.

In the previous papers [5] [11] co-incubation study showed that the action of progesterone was not mediated through progesterone receptor. Yet another way to check the involvement of progesterone receptor was to pre-incubate BLM cells with progesterone at different concentrations (1, 2.5, 5, 10 μM) for 60 - 90 min, followed by RU-486 treatment (Figure 5(a)). Progesterone would bind to progesterone receptor during the initial 60 - 90 min pre-incubation time and when RU-486 was added later, only very limited number of progesterone receptors would be available for RU-486 to act, resulting in an increased cell growth compared to straight RU-486 treated cells. However, the growth of progesterone pretreated cells followed by RU-486 treatment showed statistically significant decrease in cell growth compared to straight RU-486 treated cells (Figure 5(b)). This experiment not only suggested that both progesterone and RU-486 might be acting through different mechanism, but also indicated that RU-486 action was not mediated through progesterone receptor.

Before carrying out co-incubation with RU-486, effect of Dex alone on BLM cell growth was determined by carrying out dose-response curve (Figure 6(a)), which did not show any dose-dependent effect on cell growth. In fact, it was a straight line, suggesting Dex had no effect on BLM cell growth. Then co-incubation of 1 μM of Dex with varying concentrations of RU-486 was carried out. When the dose-response curve of straight RU-486 treated cells was compared with the dose-response curve of co-incubated cells (Figure 6(b)), there was no difference at all in their response, suggesting the action of RU-486 was not mediated through glucocorticoid receptor also.