Sulfation of 4-Hydroxytamoxifen is a Novel Pathway of Tamoxifen-Induced Apoptosis in Breast Cancer Cell Lines

Proc Amer Assoc Cancer Res, Volume 46, 2005 5323 For the past thirty years, tamoxifen therapy for estrogen receptor positive breast cancer has been the gold standard of therapy and has been a major factor in increasing overall survival from this disease. The primary mechanism of action of tamoxifen’s effect has been thought to be mediated by binding of the active metabolite, 4-hydroxytamoxifen, to the estrogen receptor, thus blocking estrogen-stimulated cellular proliferation. However, it is known that tamoxifen exerts many effects on tumor cells, including the induction of apoptosis. 4-Hydroxytamoxifen is sulfated to form 4-sulfoxytamoxifen (4-SO4TAM) in a reaction catalyzed by human sulfotransferase 1A1 (SULT 1A1). SULT1A1 is a member of the sulfotransferase superfamily of Phase II detoxification enzymes. The net result of catalysis by SULT1A1 is generally to produce a metabolite that is more polar, and thus, more easily excretable from the organism. A single nucleotide polymorphism in exon 7 of the SULT 1A1 ( SULT 1A1*2 ) gene produces a less thermostable enzyme with approximately two-fold lower activity than that of the common allele SULT 1A1*1 . We have shown previously that the SULT1A1*2 low activity allele is associated with significantly poorer overall survival in woman treated with tamoxifen for breast cancer. Since this finding was converse to what one would expect if sulfation were an elimination step for this active metabolite, we investigated the hypothesis that 4-SO4TAM plays an important role in the anti-carcinogenic mechanism of tamoxifen through a pathway independent of detoxification. We synthesized 4-SO4TAM and compared the potential of TAM, 4-OHTAM and 4-SO4TAM to initiate DNA damage and apoptosis in various breast cancer cell lines. Comet assays revealed that at equimolar concentrations, significantly more DNA damage was evident in ZR75-1 cells exposed to 4-SO4TAM compared to the other compounds. Average olive tail moments (a measure of DNA breakage) were 3.3, 2.5 and 2.3 for 4-SO4TAM, 4-OHTAM and TAM, respectively. Cytoxicity assays showed that 4-SO4TAM had no cytotoxic properties. Subsequent flow cytometry analysis demonstrated the presence of significant numbers of apoptotic cells when ZR75-1 (ER positive) and MDA-MB231 (ER negative) cells were exposed to 4-SO4TAM. This is the first study to show that 4-SO4TAM can cause apoptosis and therefore it may play a significant role in the efficacy of tamoxifen as an anti-carcinogenic treatment.