SAT-305 - Involvement of Integrin in Progesterone and RU-486 Mediated Action on Adhesion Function of Human Melanoma (BLM) Cell Line

Our previous in-vitro studies with progesterone (P) and RU-486 (RU) on human melanoma cell line (BLM) demonstrated anti-cancer action in terms of decreased cell growth, adhesion and migration functions. Cell growth and adhesion functions recovered significantly following 96 hrs recovery period after treatment with P or RU. However, migration function did not show any significant recovery following 96 hrs recovery period. Suppression and recovery of adhesion function following P or RU treatment prompted us to check for the involvement of integrin in the process. Previous studies by others identified the presence of integrin (v3) on the membrane of BLM cell, but did not show any involvement of integrin in adhesion function after treatment with P or RU. So, we designed a study involving treatment of BLM cells with progesterone (50 M) alone or progesterone + 3-Methyladenine (3-MA) 2-mM (rescued experiment). Similarly RU-486 50 M treatment alone or RU-486 + pan caspase inhibitor (CI) 20 M (rescued experiment)). Following forty-eight hours of treatment, cells were harvested and plated on a 96 well plate, coated with BSA or FN (1 g/ml concentration). 30 x103 cells were added to each well (BSA and FN coated) and cells in the uncoated wells served as control. After 60 min of incubation at 370C, wells were washed with PBS to remove unattached cells and the cells adhered to the wells were stained with crystal violet, dried and solubilized with isopropanol and quantitated. Untreated cells served as control with 100% adhesion. With respect to P treatment in the uncoated well, adhesion was 80%, whereas in the FN coated well, adhesion was 89.5%. With respect to rescued cells P+3-MA, adhesion in the uncoated well was 89% and in the FN coated well was 109%. With respect to RU-486 treatment, adhesion in the uncoated well was 68%, whereas in the FN coated wells, adhesion was 78%. With respect to rescued RU-486 + CI cells, adhesion in the uncoated well was 66% and in the FN coated well, adhesion was 77%, suggesting FN coating enhanced adhesion to the well indicating the involvement of integrin in the adhesion process. However, further work is needed to find out the type of integrin involved in the adhesion process by iummunohistochemical staining.

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