Effects of estradiol, progesterone and their antagonists (modulators) on both Methotrexate and Myotrexate induced apoptosis on endometrial stromal cell line in vitro

Uvod: Endometrijalne hiperplazije odlikuju se prekomernim rast žlezdanog i stromalnog dela endometrijuma sa ili bez atipije koje su udružene sa iregularnim krvarenjima van perioda menstruacionog ciklusa. Najčešći uzrok za razvoj endometrijalne hiperplazije predstavljaju povišeni nivo i produženo delovanje estrogena sa izostankom progesteronskog dejstva u uterusu. Cilj: Cilj ovog istraživanja bilo je utvrđivanje pojedinačnog i kombinovanog efekta ispitivanih lekova (Raloksifen, Mifepriston) i ženskih polnih hormona (Estrogen i Progesteron) na citotoksični i apoptotični efekat Metotreksata i Miotreksata kao i utvrđivanje mehanizama apoptoze u endometrijalnoj stromalnoj ćelijskoj liniji. Metode: Ćelijska linija koja je korišćena za ispitivanje predstavlja humane endometrijalne stromalne ćelije nalik na fibroblaste ThESC ćelijska linija. Ćelije su bile podeljene na dve grupe- eksperimentalna grupa ćelija (ćelije tretirane ispitivanim supstancama) i kontrolna grupa ćelija (ćelije bez tretmana). Ćelije su tretirane rastvorima različitih koncentracija ispitivanih supstanci Raloksifen, Mifepriston, Estrogen, Progesteron, Metotreksat i Miotreksat u periodu od 24 časa. Nakon tretmana ispitivanim supstancama (pojedinačno i u kombinacijama) od 24 časa koristeći MTT test određivan je citotoksični efekat ispitivanih supstanci, potom korišćenjem metode Annexin V FITC određivan je procenat apoptotičnih ćelija eksperimentalne grupe i metodom imunofluoroscencije određivane su ekspresija i lokalizacija ključnih proteina za apoptozu. Upoređivanje dobijenih rezultata sprovedenih metoda vršeno je između eksperimetalne i kontrolne grupe ćelija. Metodom imunofluoroscencije utvrđen je i mehanizam apoptotičnog dejstva ispitivanih supstanci što do sada u istraživanjima nije pokazano.Introduction: Endometrial uterine hyperplasias are characterized with both glandular and stromal endometrial over proliferation in presence or absence of the cell atypia followed with irregular bleeding outside menstrual cycle. The most common cause for the development of endometrial hyperplasia represents both elevated level of estrogen and prolonged estrogen effect in the absence of progesterone effect in uterus. Aim: The aim of this study was to investigate single and combined effects of Raloxifene, Mifepristone, female sex hormones (Estrogen and Progesterone) on both cytotoxic and apoptotic effect of Methotrexate and Myotrexate and to the determinate their apoptotic mechanism in the endometrial stromal cell line. Method: The cell line used for the experiments represents fibroblast like, human endometrial stromal cell line, ThESC cell line. The cells were divided into two groups- experimental group (cells treated with investigated substances) and control group of the cells (untreated cells). Treatment of the cells was performed with solutions of investigated substances (Raloxifene, Mifepristone, Estrogen, Progesterone, Methotrexate and Myotrexate) with different concentrations for 24 hour period. After 24 hour of single and combined treatment with investigated substances MTT assay was used in order to determine cytotoxic effect, followed by Annexin V FITC assay for determination of the percentage of apoptotic cells and by using immunofluoroscence method both expression and localization of key regulatory apoptotic proteins were determined. Results obtained using all methods were compared between experimental and control group of the cells. Using immunofluoroscence method the precise apoptotic mechanism was determined which by now had not been showed in ongoing experiments