In-Vitro Anti-Cancer Actions of Progesterone on Human Melanoma Cells Based on Rescue and Recovery studies of Cell Growth, Adhesion and Migration Functions Pandurangan Ramaraj Dept. of Biochemistry Kirksville College of Osteopathic Medicine A.T.Still University of Health Sciences Kirksville, MO 63501 Presentation Progesterone and its receptor antagonist RU-486 effects on Melanoma cells - inhibition of cell growth Mechanism of cell death induced by Progesterone Effect of Progesterone on melanoma cell growth, Rescue with 3-methyl adenine (MA) and recovery studies Effect of Progesterone on melanoma cell adhesion, Rescue with 3-methyl adenine (MA) and recovery studies Effect of Progesterone on melanoma cell migration, Rescue with 3-methyl adenine (MA) and recovery studies A, B, C, D E of Melanoma Skin - Organized into outer epidermal and inner dermal layers Embedded in the epidermis a) Keratinocytes b) Melanocytes and c) Langerhans cells Melanocytes Melanin pigment brown in color Melanoma cancer of the melanocyte Fatal form of skin cancer - metastasize to other parts of the body Melanoma - Skin Cancer Skin Structure Malignant Melanoma (C43): European Age-Standardised Mortality Rates per 100,000 Population, UK Difference in Mortality rate between Males and Females Clinical Studies: Overall survival outcome for young women (45 years of age and under) was far superior to older women (55 years of age and older) and men of any age group Involvement of hormones in the improved survival of young women with melanoma A 22% survival advantage and 17% 5 year disease-free interval advantage for females women were found to survive longer than men after the development of stage III disease Instances of melanoma diagnosed in premenopausal women metastasizing many years later in the post-menopausal period - loss of some protective effect existing in the younger patient Female hormones may exert a protective effect against melanoma metastases No correlation with sex steroid hormones in these studies Involvement of Female Sex Hormones in Melanoma Steroidogenesis Sex steroids such as Androgens, Estrogen and Progesterone are needed for a healthy skin The major androgens in the skin are testosterone (T), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA) and androstenedione (AD) Androgens affect sebaceous gland growth and differentiation, hair growth and wound healing Literature search for estrogen in melanoma. Neither estrogen nor its receptor antagonist (TAM) showed any effect on melanoma Literature survey showed that information on the role of progesterone in melanoma was inconclusive There was no correlation with progesterone and protection on melanoma Literature Survey Neat ControlAlcohol ControlDHEA 100 uM T-100 uMP-100 uM AD 100 uM Effect of various sex steroids on B16 melanoma cell growth Dose-dependent effect of Progesterone on mouse melanoma cell growth P-1 M P-100 M B16F10 BLM cell line Comparison of progesterone dose curves between B16 and BLM cell lines control Spermidine 100 M 3-MA 2 mM Spermidine + 3-MA 2 mM AUTOPHAGY 3-Methyl Adenine Rescue Prog 10 M Prog 1 M Prog-10 M + 3-MA 2 mMProg-1 M + 3-MA 2 mM 3- Methyl Adenine (2 mM) rescue of progesterone treated cells Control P-10 M R-10 M R-50 MR-100 M P-10 M + R-10 M P-10 M + R-50 M P-10 M+ R-100 M Progesterone + RU-486 co-incubation Control P-10 M (Treated) P10 M (Recovered) Recovery cell growth assay after progesterone Treatment 96 well plate P-0.06 P-0.13 Trial Cell growth rescue assay with 3-Methyladenine P-0.06 P-50 M column P-50 M + 3-MA column Treated row First 48 hrs Recovery row Second 48 hrs Recovery row Control column Cell Recovery Growth - Pictures P P-0.18 P P