Characterization of cannabinoid receptors in breast cancer cell linesKrstanović, Fran (Avtor) Debeljak, Nataša (Mentor) Lah Turnšek, Tamara (Komentor) Breast cancercannabinoid receptorsCB1CB2cannabidiolBreast cancer is the most common malignancy in women across the globe, with over 1.5 million new cases reported yearly. Apart from invasive methods, phenotype-specific system therapy is used in suppression of disease development and recurrence. Recent research revealed that the endocannabinoid system (ECS) is involved in many diseases, including cancer. The endocannabinoid receptors are receptors associated with G-proteins, which are found in the central nervous system (Type 1 receptor, CB1) and the immune system (Type 2 receptor, CB2). While some cannabinoids (such as tetrahydrocannabinol, THC) are associated with psychotropic effects when bound to (CB1), others (such as cannabinol, CBD) have a plethora of biological effects, indicating their potential therapeutic value. In this Master Thesis we aimed to reveal (1) the expression levels of cannabinoid receptors and (2) the effects of cannabidiol (CBD) on cell viability alone, or in combination with commonly used phenotype-specific therapeutic on selected breast cancer cells. We hypothesized that the selected breast cancer cell lines express CBs on a protein level and that receptor CB1 and CB2 levels would correspond to the biological effects of CBD on cell viability. We evaluated the expression of CB1 and CB2 in breast cancer cell lines of different subtypes and receptor status (ER+/PR+/HER2-, ER-/PR-/HER2+ and triple-negative). The expression levels of CB1 and CB2 receptor proteins were analyzed using western blot (WB) and immunocytochemistry (ICC) method, comparing two different sets of primary antibodies. We tested one of the most used commercial polyclonal antibodies (manufactured by Abcam) against both CBs, and were among the first to test the new monoclonal antibody (manufactured by Santa Cruz Biotechnology) against both CBs. Both sets of antibodies successfully detected CBs in selected cell lines with the WB method, with rather different specificity. No constant pattern of expression was observed with the ICC method, as receptor staining highly varied amongst tested cell lines and antibodies. With the cell viability assay, we also tested the therapeutic potential of cannabidiol (CBD) alone, or in combination with common phenotype-specific therapeutics. Tamoxifen was used for hormone receptor-positive (ER+, PR+) T-47D, Herceptin (HER) for human epidermal growth factor 2 receptor positive (HER+) SK-BR-3 and cisplatin (CIS) for triple-negative (ER-, PR-, HER-) MDA-MB-231 cell line. CBD treatment alone was shown to decrease cell viability in all tested cell lines, in either a concentration-dependent (T-47D) or threshold response (SK-BR-3, MDA-MB-231) manner. To our knowledge, we were the first to test and show CBD effects in combination with the above listed therapeutics. Possible synergism between CBD and therapeutics was observed in all tested cell lines, which holds a promising potential for further exploitation of ECS in treatment of breast cancer.20192019-09-12 16:50:08Magistrsko delo/naloga110200VisID: 6581COBISS_ID: 1538448579sl