Metabolism of estrogens and cancer of the tongue Free

B19

Background. Squamous cell carcinoma (SCC) of the tongue is the most common cancer of the oral cavity. Recently, the incidence of tongue cancer has increased dramatically in young adults, particularly in women of premenopausal age. While tobacco smoking is a major risk factor for developing tongue cancer, the potential contribution of hormonal factors to the increased susceptibility of females must be considered. We hypothesize that estrogens and products of their oxidative metabolism participate in carcinogenesis in the tongue. The goal of the present study was to determine if estrogen metabolism genes are expressed in the tongue epithelium and if their expression can be modulated by 17β-estradiol (E₂), the most potent estrogen.
 >Methods. Immunohistochemical (IHC) analyses of murine tongue tissue and tissue microarrays of human head and neck specimens (31 non-neoplastic and 111 SCCs, in duplicate) were performed using antibodies against cytochrome P450 (CYP) 1B1, estrogen receptor (ER) β and E₂. Squamous epithelial cells were isolated from frozen sections of tongue tissue by laser capture microdissection. SCC9 cells (80% confluent) were plated and treated with either vehicle (0.1% ethanol) or 10 nM E₂ for 24 hours. RNA, extracted from both LCM samples and cultured cells, was subjected to quantitative real-time PCR, performed in triplicate, using primers specific for CYP1B1, CYP1A1, CYP3A4, catechol-O-methyltransferase (COMT), sulfotransferase(SULT) 1A1, UDP-glucuronosyltransferase (UGT) 1A, and glutathione S-transferase (GST) P1. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) served as a reference gene.
 >Results: IHC analysis of the tongues of intact female A/J mice revealed the presence of CYP1B1 and ERβ within the nuclei of squamous epithelial cells. Staining of E₂ was localized to both the cytoplasm and nucleus. Evaluation of the mRNA levels of estrogen metabolism genes in the microdissected tongue epithelium of ovariectomized A/J mice revealed the expression of CYP1B1, CYP1A1, COMT, SULT1A1, UGT1A, and GSTP1. Consistent with these mouse data, staining for E₂, ERβ and CYP1B1 was detected in both non-neoplastic and neoplastic human head and neck epithelium. Comparison of the intensity of staining of each antibody in non-neoplastic and neoplastic tissue revealed a significant increase in CYP1B1 levels in SCCs as compared to non-neoplastic epithelium (p=0.001). Expression of the estrogen metabolism genes CYP1B1, CYP1A1, CYP3A4, COMT, SULT1A1, UGT1A and GSTP1 was observed in human tongue carcinoma (SCC9) cells. Treatment of SCC9 cells with E₂ resulted in induction of the expression of the CYP1B1 gene, which encodes the protein that oxidizes estrogens to mutagenic metabolites, but not COMT, a gene that encodes the major conjugating enzyme, as compared to untreated control cells. In summary, estrogen metabolism genes are expressed in both mouse and human tongue tissue. Both the increased levels of CYP1B1 in head and neck SCCs and the ability of E₂ to modulate its expression suggest that estrogen metabolism may contribute to tongue carcinogenesis.