Catecholestrogens induce oxidative stress and malignant transformation in human endometrial glandular cells: Protective effect of catechol-O-methyltransferase Salama A. Salama 1 * , Marwa Kamel 1,2 , Mohamed Awad 1 , Abdel-Hakim Ben Nasser 1 , Ayman Al-Hendy 1 , Shaleen Botting 1 and Concepcion Arrastia 1 1 Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX 2 Department of Cancer Biology, Unit of Pharmacology, National Cancer Institute, Cairo University, Egypt Prolonged exposure to unopposed estrogens is a major risk factor for the development of endometrial cancer. Oxidative metabolism of estradiol (E 2 ) into the catecholestrogens (CEs), 4-hydroxyestra- diol (4-OHE 2 ) and 2-hydroxyestradiol (2-OHE 2 ), may play an im- portant role in estrogen carcinogenicity. CEs can be oxidized to the corresponding ortho-quinone derivatives with concomitant forma- tion of the reactive oxygen species (ROS). Catechol-O-methyltrans- ferase (COMT) is the major enzyme involved in the detoxification of CEs in extrahepatic tissues. We investigated the potential of E 2 , 2-OHE 2 and 4-OHE 2 to induce microsatellite instability (MSI) and neoplastic transformation of immortalized human endometrial glandular (EM) cells. We also investigated the functional signifi- cance of COMT gene expression on modulating the effects of E 2 and CEs in EM cells. Our data indicated that E 2 and 4-OHE 2 induce MSI, ROS and neoplastic transformation in EM cells. The capacity of E 2 and its catechol metabolites to induce MSI, ROS and neoplastic transformation in EM cells is ranked as follows: 4- OHE 2 > E 2 > 2-OHE 2 . Knockdown of COMT expression in EM cells resulted in increased estrogenic milieu and increased estro- gen-induced cell proliferation. More importantly, knockdown of COMT increased the propensity of E 2 or CEs to induce ROS, MSI and neoplastic transformation of EM cells. In contrast, overexpres- sion of COMT in EM cells significantly reduced the cellular estro- genic milieu and protected against E 2 - or CEs-induced, ROS, MSI and neoplastic transformation. The capacity of E 2 or CEs to induce neoplastic transformation of human endometrial glandular cells in vitro may suggest that E 2 -induced endometrial cancer is mediated by its metabolism into CEs. Our study clearly indicates that COMT gene expression plays a critical role in modulating the hormonal and carcinogenic effects of E 2 and CEs and, consequently, modifies the risk for E 2 -induced endometrial cancer. To the best of our knowledge, this is the first study to (i) demonstrate the potential capacity of estrogen and its catechol metabolites to induce neoplas- tic transformation of immortalized human endometrial glandular cells; and (ii) illustrate the important role of COMT gene expres- sion in protecting against E 2 -induced endometrial cancer. ' 2008 Wiley-Liss, Inc. Key words: catecholestrogens; COMT; genomic instability; endometrial cancer Endometrial cancer is the most common cancer of the genital tract in American women and ranks first in incidence and second in mortality among gynecological malignancies. 1 It is estimated that the rates of endometrial cancer in American women are among the highest in the world. 2 The risk factors for endometrial cancer include nulliparity, early age at menarche, late age at men- opause, obesity and long-term use of estrogen replacement ther- apy (ERT). These risk factors seem to relate, either directly or indirectly, prolonged exposure to an unopposed estrogen. 3 Most endometrial carcinomas are Type I estrogen-associated endome- trioid adenocarcinomas, 4–6 and it is evident that long-term expo- sure to synthetic and endogenous estrogens is unequivocally involved in the pathobiology of endometrial cancer. However, efforts to identify the precise mechanisms of estrogen-induced carcinogenesis have not led to a consensus, and the mechanisms through which estrogens cause endometrial cancer remain elu- sive. 7 Two distinctive, but complementary, mechanisms have been proposed for estrogen-induced cancers. One commonly held theory is that estrogens can induce cell proliferation, increase cell division and, thus, cause random errors during DNA replication. 8 However, the fact that ER-a knockout mice that express the Wnt- 1 oncogene (ERKO/Wnt-1) develop mammary tumors provides direct evidence that estrogens may cause cancer through a geno- toxic, non-ER-a-mediated mechanism. 9,10 Furthermore, ovariec- tomized mice treated with estradiol (E 2 ) develop breast cancer, even in the presence of pure antiestrogen (ICI-182,780). 11 Alter- natively, there is a growing consensus that the oxidative metabo- lism of estrogens into catecholestrogens (CEs) could play a detri- mental role in the initiation and/or evolution of estrogen-related cancerous processes. 11–14 In extrahepatic tissues, estrogens undergo oxidative metabolism by cytochrome P450 1A1 and 1B1 to produce 2-hydroxyestradiol (2-OHE 2 ) and 4-hydroxyestradiol (4-OHE 2 ), respectively. 15 CEs, particularly 4-OHE 2 , are sus- pected carcinogens and their carcinogenic potential is attributed to their rapid oxidization (even by autooxidation) to their corre- sponding ortho-quinone derivatives (2,3-OHE 2 -o-quinone and 3,4-OHE 2 -o-quinone), which have the potential to both initiate and promote carcinogenesis. These CEs-derived ortho-quinones are electrophilic compounds that can react with DNA to form covalent adducts and induce mutagenesis and, eventually, carci- nogenesis. 16,17 In addition, CEs-derived ortho-quinones are potent redox cycling agents capable of generating the reactive oxygen species (ROS), which is implicated in carcinogenesis. 18–20 Recently, it has been reported that in human breast epithelial cells (MCF-10F), an estrogen receptor-negative cell line, treat- ment with E 2 or CEs at low concentrations cause a loss of hetero- zygosity and mutation in many genes in different chromosomes, which demonstrates that E 2 , independent of receptor function, can induce mutation and neoplastic transformation. 21 Similarly, con- siderable evidence indicates that metabolic activation of E 2 into CEs is involved in the development of endometrial cancer. Indeed, CEs have been shown to induce endometrial adenocarcinoma in mice 22–24 ; it is noteworthy that the 4-OHE 2 -treated mice had a 9- fold higher incidence of endometrial cancer compared with the E 2 -treated group. 24 This notion supports the hypothesis that the formation of 4-OHE 2 represents the metabolic activation of E 2 to a reactive intermediate capable of causing genomic instabilities and mutations, and, eventually, cancer in the endometrium. In extrahepatic tissues, CEs are primarily inactivated by O- methylation catalyzed by catechol-O-methyltransferase (COMT). 25–27 Thus, the level of CEs in target tissues is determined by the Abbreviations: CEs, catecholestrogens; CE-Q, quinone; CE-SQ, chemi- cally reactive estrogen semiquinone; COMT, catechol-O-methyltransfer- ase; DCF, dichlorofluorescein; DCFDA, dichlorofluorescein diacetate; DMEM, Dulbecco’s minimal essential medium; DMSO, dimethylsulf- oxide; E 2 , estrogen; EM, endometrial glandular cells; FCS, fetal calf serum; 2-OHE 2 , 2-hydroxycatecholestrogen; 4-OHE 2 , 4-hydroxycatecho- lestrogen; MMR, mismatched repair gene; MSI, microsatellite instability; OD, optical density; ROS, reactive oxygen species; SEM, standard error of the mean; O2*-, superoxide; SHE, Syrian hamster embryo. Grant sponsor: M.D. Anderson SPORE Career Development award; Grant number: CA098258; Grant sponsor: UTMB NIEHS Center Pilot Project; Grant number: E506676. *Correspondence to: Department of Obstetrics and Gynecology, Uni- versity of Texas Medical Branch, Galveston, TX 77555-0587, USA. Fax: 1409-747-0492. E-mail: sasalama@utmb.edu Received 25 October 2007; Accepted after revision 27 February 2008 DOI 10.1002/ijc.23653 Published online 19 June 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 1246–1254 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer