DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells

British Journal of Cancer (2003) 88, 599 – 605 & 2003 Cancer Research UK All rights reserved 0007 – 0920/03 $25.00 www.bjcancer.com DNA damage and cell cycle arrest induced by 2-(4-amino-3methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells V Trapani1, V Patel2, C-O Leong1, HP Ciolino3, GC Yeh3, C Hose4, JB Trepel5, MFG Stevens1, EA Sausville6 and AI Loaiza-Pérez*,6 1 School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK; 2Oral and Pharyngeal Cancer Branch, National Institutes of Dental and Craniofacial Research, Bethesda, MD, USA; 3Cellular Defense and Carcinogenesis Section, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702-1201, USA; 4Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Frederick, MD 21702-1201, USA; 5Medical Oncology Clinical Research Unit, Medicine Branch, National Cancer Institute, National Institutes of Health, MD 20892, USA; 6Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 6N115, Bethesda, MD 20892, USA The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G1 and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AHR100 variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptasepolymerase chain reaction (RT – PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AHR100 cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AHR100 cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203. British Journal of Cancer (2003) 88, 599 – 605. doi:10.1038/sj.bjc.6600722 www.bjcancer.com & 2003 Cancer Research UK 2-(4-Amino-3-methylphenyl)benzothiazoles are novel compounds with potent and unique antitumour properties (Shi et al, 1996; Bradshaw et al, 1998a, b; Kashiyama et al, 1999). It was demonstrated that selective metabolism in vitro of the parent agent DF 203 (NSC 674495) correlated very highly with its antiproliferative activity, with uptake and biotransformation observed only in those cell lines that are sensitive to the compound, such as MCF-7 and T47D breast carcinoma cells (Kashiyama et al, 1999). CYP1A1, whose expression and activity are induced only in sensitive cells, appears to be responsible for the metabolic hydroxylation of DF 203 in position 6, which produces an inactive and antagonistic molecule (Chua et al, 2000). CYP1A1 is also postulated to have a crucial role in mediating the antitumour activity of DF 203, possibly generating an electrophilic *Correspondence: Dr AI Loaiza-Pérez; E-mail: Received 20 May 2002; revised 17 October 2002; accepted 23 October 2002 intermediate responsible for the formation of DNA adducts in sensitive cells (Stevens et al, 2001). Fluorinated derivatives of the parent drug DF 203 (see Figure 1 for structures) were synthesised in order to prevent deactivation resulting from metabolic ring hydroxylation (Hutchinson et al, 2001). Fluoro-analogues retain potency and selectivity, and successfully reduce or abolish the production of inactive and antagonistic metabolites and the consequent biphasic dose – response phenomenon. Drug-induced induction of CYP1A1, a crucial event in determining the antitumour specificity of this series of benzothiazoles (Chua et al, 2000), was not compromised by fluorination (Hutchinson et al, 2001). 2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) has emerged as the most potent of the new generation of antitumour benzothiazoles both in vitro and in vivo and is currently the focus of pharmaceutical and preclinical development, as it can be converted to a readily soluble prodrug with appropriate pharmaceutical properties (Bradshaw et al, 2002). As for the parent drug DF 203, depletion of 5F 203 from culture media and subsequent induction of CYP1A1 correlate with cell Experimental Therapeutics Keywords: 2-(4-aminophenyl)benzothiazoles; aryl hydrocarbon receptor; CYP1A1; DNA damage; S-phase arrest; MCF-7 5F 203-induced cytotoxicity V Trapani et al 600 CH 3 R N CH 3 F N NH 2 S NH 2 NH (CH 2 )4 .2HCl O NH 2 S DF 203 (NSC 674495): R = H Phortress (NSC 710305) 5F 203 (NSC 703786): R = F Figure 1 Chemical structures of antitumour 2-(4-amino-3-methylphenyl)benzothiazoles. responsiveness. Only in sensitive cells does drug uptake occur and CYP1A1 enzyme expression increase to detectable levels; naturally occurring drug-resistant cells do not show significant changes in either drug levels in medium or CYP1A1 expression (Brantley et al, 2001). We have previously reported that the parent compound DF 203 induces activation of the AhR in sensitive cells such as human breast epithelial cancer MCF-7 cells. In contrast, nonresponsive cells, for example, breast carcinoma MDA-MB-435 or prostate carcinoma PC-3 cells, showed no activation of the AhR and no induction of CYP1A1 after drug treatment (Loaiza-Pérez et al, 2002). We also have reported a suitable model to investigate the role of the AhR in mediating drug cytotoxicity (Loaiza-Pérez et al, 2001), consisting of AhR-deficient AHR100 cells, derived from MCF-7 human breast epithelial cancer cells by continuous exposure to escalating concentrations of benzo[a]pyrene (Yeh et al, 2001). AHR100 cells display relative resistance to the cytotoxic effects of several polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene and dimethylbenz[a]anthracene (DM (...truncated)