Abstract 3679: Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells.

Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferases (GST) via the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear Factor- E2-related factor (Nrf2) signaling pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-quinone (CE-3,4-Q), while GSTs detoxify it through nucleophilic addition. CE-3,4-Q can bind with DNA to form depurinating DNA adducts. Thus, SFN, a isothiocyanate found in broccoli, may alter estrogen metabolism to protect against estrogen-mediated carcinogenesis. MCF10A cells were treated with either vehicle or SFN (10 μM) and either estradiol (E 2 ) or 4-OHE 2 (10 μM). NQO1 activity increased 3.2-fold by SFN treatment compared to vehicle. Estrogen metabolites and depurinating DNA adducts in the cell culture medium were partially purified by solid phase extraction and then analyzed by UHPLC-MS/MS. Following E 2 treatment, 4-OHE 1/2 -1-N3Ade and 4-OHE 1/2 -1-N7Gua adducts were reduced by 60% in SFN treatment; 4-OHE 1/2 - glutathione conjugates increased 1.9 while 4-OCH 3 E 1/2 increased 3.0 fold with SFN. Following treatment with the proximate metabolite 4-OHE 2 , 4-OHE 1/2 -1-N3Ade and 4-OHE 1/2 -1-N7Gua adducts also decreased 60% in SFN treated cells compared to vehicle; while 4-OHE 1/2 -glutathione-conjugates increased 5.0-fold and 4-OCH 3 E 1/2 levels were 3.4-fold higher. To constitutively enhance the expression of Nrf2-regulated genes including NQO1, cells were treated with either scrambled or siKEAP1 RNA and E 2 or 4-OHE 2 . NQO1 activity increased 2.2-fold with siKEAP1 treatment. Following E 2 treatment, 4-OHE 1/2 -1-N3Ade and 4-OHE 1/2 -1-N7Gua adducts dropped 70% in siKEAP1 treated cells compared to scrambled; 4-OHE 1/2 -glutathione conjugates increased 1.3-fold; however, 4-OCH 3 E 1/2 decreased 50% with siKEAP1 treatment. Following 4-OHE 2 treatment, the 4-OHE 1/2 -1-N3Ade and 4-OHE 1/2 -1-N7Gua adducts declined 60% in siKEAP1 treated cells compared to scrambled; 4-OHE 1/2 -glutathione conjugates increased 1.4-fold while 4-OCH 3 E 1/2 declined 60% with siKEAP1 treatment. SFN or siKEAP1 have similar effects on up-regulating NQO1 transcripts, protein expression and activity levels and on diminution of depurinating estrogen DNA adducts following E 2 or 4-OHE 2 challenge. However, these pharmacologic and genetic approaches have different effects on COMT. siKEAP1 down-regulates COMT expression, which inhibits 4-OCH 3 E 1 but not 4-CH 3 E 2 formation whilst SFN elevates COMT expression and subsequently levels of 4-OCH 3 E 1/2 . Thus, activation of the Nrf2 pathway may account for some but not all of the protective effects of SFN against estrogen-mediated DNA damage. Supported by DOD BCRP Postdoctoral Fellowship103928. Citation Format: Li Yang, Muhammad Zahid, Eleanor G. Rogan, Ercole L. Cavalieri, James D, Yager, Kala Visvanathan, John Groopman, Nancy E. Davidson, Thomas W. Kensler. Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2013-3679