Experimental Study Of AhR Function In Endometrial Carcinoma Proliferation And Invasion

Endometrial cancer (EC) is the most prevalent gynecologic malignancy amongwomen worldwide. EC has been traditionally divided into two types based on thehistopathology and clinical behavior: type I and type II. Women with these cancersoften have risk factors such as long-term unopposed estrogen therapy, obesity,tamoxifen therapy, polycystic ovarian syndrome (PCOS), obesity, diabetes mellitus, andhypertension. Over the past decades, great advances in our understanding ofendometrial cancer biology, the patients still have an extremely poor prognosis.Undoubtedly, a better understanding of molecular pathogenesis of EC will assist thedevelopment of new therapeutic methods for this deadly disease.The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor of thePER-ARNT-SIM (PAS) family, mediates multiple biological processes includingimmune system homeostasis, cell growth and differentiation, vasular remodeling.Unliganded AHR exists in the cytoplasm as part of a multimeric, upon ligand binding,AhR translocates to the nucleus where it associates with ARNT to form a functionaltranscription factor complex, binds to specific enhancer sequences adjacent to targetpromoters termed dioxin responsive elements and subsequently regulates the expression of Cytochrome P4501A1(CYP1A1) and other xenobiotic target genes to combat theeffects of environmental contaminants such as3-methylcholanthrene (3MC).For the last decades, AhR has been widely studied as a receptor for environmentaltoxicants. Recently, increasing evidence has disclosed the potential role of AhR in thetumor development. The important role of AhR in carcinogenic pathways is evidencedin AhR knockout mice, which are refractory to polycyclic aromatic hydrocarbons(PAHs)-induced carcinogenesis. Increased expression of AhR has been observed inbreast, skin, and lung cancers in humans. Meanwhile, several studies have clearlydemonstrated that AhR-mediated responses are closely correlated with proliferation indifferent types of cancer cells, suggesting that AhR might be as a potential target forcancer treatment. However, the precise role of AhR in EC tumorigenesis is still lacking.In this study, we evaluated AhR expression in EC tissues as well as cell lines, andinvestigated the effects of AhR protein levels and3-MC on endometrial cancer cellgrowth using Ishikawa and ECC-1as cell models. Our data clearly showed thatknockdown of AhR did not alter EC cells proliferation and invasion. However,3-MCdose-dependently inhibited EC cells proliferation via AhR-mediated pathway. Thecontents of present study are as follows:(1) distribution and clinical significance ofAhR expression in endometrial carcinoma;(2) the function of AhR in proliferation andinvasion of endometrial carcinoma cell line;(3) the effect of3-MC on endometrialcarcinoma cell proliferation and invasion.1. Distribution and clinical significance of AhR expression in endometrialcarcinoma. Human endometrial cancer tissue microarray was used for immunolocalization ofAhR in endometrial cancer tissues. the expression and location of AhR protein wasexamined in normal endometrium， proliferative endometrium and endometrialcarcinoma. Furthermore RT-PCR and Western-blot techniques were used to evaluatethe mRNA and protein expression of AhR in fresh tissues of endometrial cancerobtained from5patients compared with their adjacent normal ones. It was demonstratedthat the positive rate of AhR expression was4％,12％,20%and52%in normalendometrium， proliferative endometrium, atypical proliferative endometrium andendometrial carcinoma．The positive rate of AhR expression in endometrial carcinomawas higher than that of the other groups，with a statistical significance(P<0.05)．Inendometrial carcinoma，the expression intensity of AhR was related to surgical stage，the degree of differentiation and metastasis of lymph nodes， with a statisticalsignificance(P<0.05)， but it was not obviously related to pathological type andmyometrial invasion(P>0.05)．The over-expression of AhR was well related with thegeneration，development and metastasis of endometrial carcinoma，and AhR may serveas an clinical diagnosis indicator and a target for chemotherapy of endometrialcarcinoma．2. The function of AhR in proliferation and invasion of endometrial carcinoma cellline.In this part of study, we investigated the function of AhR in proliferation andinvasion of endometrial carcinoma using Ishikawa and ECC-1cells lines. Both mRNAand protein levels of AhR in human endometrial cancer tissues and cell lines were determined by RT-PCR and Western blot analysis. Our data showed that the AhRantibody detected a band approximately at95kDa. After normalized to GAPDH, AhRprotein levels in cancer tissues were2.0fold higher (p <0.05) than those in normaltissue. Also, we detected high protein levels of AhR in both Ishikawa and ECC-1cells.Given AhR is highly expressed in the human endometrial cancer tissues and cell lines,we investigate whether knockdown of AhR would alter the malignant features of ECcells. As shown in Western blot results, the transfection of AhR siRNA reduced theAhR protein levels in both Ishikawa and ECC-1cells in comparison with the cellstreated with the scrambled siRNA. And this inhibitory effect maintained at least72hrs.To gain further insight into the potential role of AhR in the proliferation and invasion ofEC cells, we performed cell proliferation and invasion assays after tranfected with AhRsiRNA. The transfection of AhR siRNA did not change cell proliferation and invasionin EC cells in comparison with the cells treated with the scrambled siRNA. Theseresults suggested that AhR protein levels may not be involved in the features ofmalignant in endometrial cancer.3. The effect of3-MC on endometrial carcinoma cell proliferation and invasion.In this part of study, we investigated the effect of3-MC on endometrial carcinomacell proliferation and invasion. Cells were cultured in the complete growth media withdifferent concentrations of3-MC for48hrs. Additional cells were transfected withscrambled and AhR siRNA for determining their proliferation responses to3-MC.Meanwhile, cells were cultured in complete growth media in the absence or presence of3-MC for24hours. Cells were subjected to the invasion assay. After16hours, the numbers of invaded cells were stained and counted. Results demonstrated that3-MCdose-dependently inhibited EC cell proliferation, but not invasion. Importantly,knockdown of AhR using AhR specific siRNA completely blocked3-MC-nihibited ECcell proliferation, indicating that this proliferative suppression is mediated by AhRpathway.