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Biological Testing Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland (E.B., M.C.A., S.F.S.); Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, Nottingham, United Kingdom (V.T., T.D.B., M.F.G.S.); Screening Technologies Branch, Developmental Therapeutics Program, Science Applications International Corporation, Frederick, Maryland (C.D.H); and Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (E.A.S.)
(Received June 24, 2004; accepted September 3, 2004)
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Abstract |
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). These small molecules display antitumor properties that are modulated by substitutions at specific positions on the benzothiazole pharmacophore (Chua et al., 1999; Kashiyama et al., 1999; Wells et al., 2000; Hutchinson et al., 2001, 2002). 2-(4-Amino-3-methylphenyl)benzothiazole (DF 203) represents the lead compound for a recently explored series, and possesses potent antitumor properties in select breast, ovarian, and renal cancer cell lines of the 60-cell line, disease-oriented NCI Anticancer Drug Screen (Hose et al., 2003). DF 203 displays a unique biphasic dose-response relationship, with more potent activity at concentrations below 1 µM but reduced antiproliferative activity at concentrations ranging from 1 to 100 µM. The precise mechanism by which DF 203 and similar benzothiazoles display their antiproliferative effects has not been fully elucidated. Recent studies, however, suggest that their ability to become metabolized and covalently bind to subcellular targets contributes to their anticancer effects (Kashiyama et al., 1999; Chua et al., 2000; Hutchinson et al., 2001). Metabolism and covalent binding to subcellular macromolecules have been shown to play a pivotal role in the anti-cancer activity of other small molecules (Rivera et al., 1999; Efferth et al., 2001; Kuffel et al., 2002). A prior study demonstrated that coincubation of DF 203 with 
-naphthoflavone, a CYP1A1 inhibitor, in sensitive cells can inhibit anticancer activity, which reveals the critical involvement of CYP1A1 in the anticancer activity of the benzothiazoles (Chua et al., 2000).
The fluorinated benzothiazoles (Fig. 1) represent a novel class of anticancer drug candidates that, similar to DF 203, potently and selectively inhibit the growth of renal, breast, and ovarian cancer cell lines and thus retain a pattern of differentiated growth inhibition (Chua et al., 2000). 5F 203 represents one of a series of fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles synthesized to circumvent the biphasic dose-response relationship thought to thwart the antiproliferative activity of the benzothiazoles (Hutchinson et al., 2001).
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As prior reports have shown, DF 203 covalently binds to DNA, forming adducts exclusively in sensitive, growth-inhibited cells (Kashiyama et al., 1999; Trapani et al., 2003). Other recent studies have shown that sensitive MCF-7 breast and IGROV1 ovarian cancer cells treated with 5F 203 formed DNA adducts, including a dominant adduct distinct from that formed after their treatment with DF 203 (Leong et al., 2003; Trapani et al., 2003). Therefore, we sought to clarify whether 5F 203 binds to subcellular macromolecules in addition to DNA in growth-inhibited sensitive cancer cells.
In this study, we determine the ability of the fluorinated benzothiazoles to induce CYP1A1 mRNA and protein expression in sensitive cells and whether CYP1A1 might play a pivotal role in the metabolism and antiproliferative activity of these drug candidates, corroborating previous investigations (Hose et al., 2003). Radiolabeled 5F 203 was used to reveal the extent that 5F 203 binds to subcellular fractions in several cancer cell lines. Metabolism of the fluorinated benzothiazoles and their binding to subcellular macromolecules may constitute a basis for the mechanism of growth inhibition for these and related compounds.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Stock solutions (10 mM) of the fluorinated benzothiazoles were prepared in DMSO or acetonitrile and stored protected from light at -20°C. [14C]5F 203 (14C at the C-2 position of the thiazole ring, 40.5 mCi/mmol) was synthesized at the Research Triangle Institute (Research Triangle Park, NC). The product was 98% pure as determined by radiochemical detector (thin-layer chromatography) and HPLC. For all experiments, [14C]5F 203 was dissolved in DMSO and stored as a 10 mM stock solution at -70°C. HPLC-grade acetonitrile was purchased from J. T. Baker (Philipsburg, NJ). All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) and were of the highest commercial grade available. NADPH was purchased from Calbiochem (San Diego, CA). Cell culture medium and supplements were purchased from Quality Biological, Inc. (Gaithersburg, MD) except for heat-inactivated fetal bovine serum, which was purchased from Hyclone Laboratories (Logan, UT). Leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and aprotinin were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies specific for CYP1A1 (polyclonal antiserum for human CYP1A1/1A2) and microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) were obtained from BD Gentest (Woburn, MA). ECL kits were purchased from Amersham Biosciences Inc. (Piscataway, NJ).
In Vitro Cell Culture. The human breast (MCF-7 and MDA-MB-435), ovarian (IGROV1 and SKOV3), and renal (TK-10 and CAKI-1) cancer cell lines were obtained from the NCI-Frederick repository. The cell types, sources, stocks, morphological characteristics, and methods of propagation of human cancer cell lines have been previously described (Alley et al., 1988; Stinson et al., 1992). Monolayers of cells were cultured at 37°C in an atmosphere of 5% CO2/95% air in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained continuously in the logarithmic (subconfluent) growth phase with routine subculturing.
Sulforhodamine B Dye Assay. The methods used for the 60-cell line panel have been described elsewhere (Monks et al., 1997). Briefly, compounds were solubilized in dimethyl sulfoxide at 200x. The compounds were diluted into RPMI 1640 containing 10% fetal bovine serum, and serial 1-log dilutions were prepared for a total of five concentrations. Generally, the working range for initial testing of each benzothiazole was 10-4 through 10-8 M. The compounds were added to cultures of each of the 60 cell lines used in the panel. After 48 h of exposure to the benzothiazoles, media were removed; the cells were fixed and stained with sulforhodamine B and the total stain quantitated by optical density determinations. Using time 0 as control, cell growth was determined for each cell line, and the concentrations needed to inhibit growth 50% (GI50), to cause total growth inhibition (TGI), and to produce 50% cytocidal activity (LC50) were calculated.
In Vitro Time Course MTT Assay. The cell lines and concentration ranges of benzothiazoles evaluated in the concentration x time (c x t) assays were chosen on the basis of 60-cell line screening data. As described elsewhere (Hollingshead et al., 2004), cells were exposed to the fluorinated benzothiazoles for increasing periods of time before anticancer activity was determined using each of several exposure durations ranging from 
1 h to 144 h. A "stable endpoint" MTT formazan colorimetric endpoint permitted comparative quantitation of anticancer activity in replicate cell culture plates. From the plot of the composite c x t data, the minimum exposure conditions (both concentration and time) required to achieve antiproliferative (GI50), cytostatic (TGI), and/or cytotoxic (LC50) activity in a given cell line were readily determined. Use of c x t indices of GI50, TGI, and LC50 readily permit numerical characterization and comparison of the sensitivities of multiple cell lines as well as the relative activity of the fluorinated benzothiazoles toward one or more cell lines. In addition, such indices provide a means to determine the concentration ranges of a given agent required to confer growth inhibition, cytostasis, and/or cytotoxicity.
Microsomal Metabolism of 5F 203, 6F 203, and 5,6 di-F 203. The ability of microsomes from human B-lymphoblastoid cells expressing human CYP1A1, CYP1A2, and CYP1B1 to metabolize the fluorinated benzothiazoles was determined by previously described methods (Kashiyama et al., 1999; Chua et al., 2000). A typical reaction contained 100 mM K2PO4, 5 mM MgCl2, the appropriate fluorinated benzothiazole (10 µM), 1 mg/ml microsomal protein, and 10 mM NADPH. Control reactions contained microsomes from lymphoblastoid cells containing empty vector. Mixtures were incubated at 37°C with gentle agitation for 2 h. Aliquots were mixed with a 3-fold volume of cold acetonitrile to terminate the reaction. The mixture was centrifuged and supernatants analyzed by HPLC with concurrent UV and fluorescence detection.
Cellular Uptake and Metabolism of 5F 203, 6F 203, and 5,6 di-F 203. MCF-7, MDA-MB-435, TK-10, CAKI-1, IGROV1, and SKOV3 cells in the logarithmic growth phase were passaged into 25-cm2 flasks and allowed to adhere for 24 to 48 h until they reached 50 to 60% confluency. Cell medium was then replaced with medium containing a 1.0 µM concentration of either 5F 203, 6F 203, or 5,6 di-F 203. Control flasks contained either the respective benzothiazoles without cells or cells treated with vehicle alone (0.1% DMSO). Aliquots of cell medium were removed at 24-h intervals for at least 3 days of exposure. In 5F 203-treated MCF-7 cells, aliquots of cell medium were also removed after 12, 14, and 16 h of incubation. All aliquots were analyzed by HPLC as described previously (Chua et al., 2000; Hutchinson et al., 2001). The percentage of each fluorinated benzothiazole remaining was determined by comparing chromatographic peak areas of the fluorinated benzothiazoles at time 0 with those detected during specific periods of incubation.
5F 203 Cell Treatment and RNA Extraction. MCF-7, TK-10, IGROV1, MDA-MB-435, CAKI-1, and SKOV3 cells grown to 60 to 80% confluency were treated with 0.01, 0.1, or 1.0 µM 5F 203 for 12 or 24 h. Control flasks received vehicle alone (0.1% DMSO). Cells were harvested with trypsin, and total cellular RNA was extracted from 1.5 to 5.0 x 105 cells according to the RNeasy method (QIAGEN, Valencia, CA). The extracted RNA was stored at -20°C until further evaluation. The concentration and purity of the RNA were determined by measurement of the optical densities at 260 and 280 nm. A ratio of >1.7 for A260/A280 was required for use in these studies.
QRT-PCR Analysis. QRT-PCR analysis was used to evaluate CYP1A1 and CYP1B1 gene expression in 5F 203-treated cells. The methodology including the primers and probes used has been previously described in detail (Loaiza-Perez et al., 2002). PCR efficiencies were validated by means of a standard curve.
Western Immunoblot Analysis of CYP1A1. CYP1A1 expression was determined in MCF-7, TK-10, and MDA-MB-435 cells treated with the fluorinated benzothiazoles (24 h, 1 µM) using a method previously described in detail (Chua et al., 2000). In brief, whole cell lysates were prepared and protein content was estimated by Coomassie Blue assay (Pierce Biotechnology, Rockford, IL) using bovine serum albumin as a standard. Solubilized proteins (50 µg per well) were loaded on 10% Tris-glycine precast gels (Novex, San Diego, CA) and transferred in a submerged transfer unit (Novex Xcell II) to polyvinylidene fluoride membranes, in Tris-glycine transfer buffer (Novex) containing 5% methanol for 90 min at 125 V and 4°C. Membranes were then blocked with blocking buffer [phosphate-buffered saline-0.1% Tween 20 (T) and 10% nonfat dried milk] for 1 h at room temperature and rinsed (three times for 5 min with TBS-0.1% T and two times for 5 min with TBS). Blots were then incubated for 2 h at room temperature with polyclonal goat anti-human primary antibody (1:500 dilution in TBS-0.02% T, 1% milk) and rinsed (three times for 5 min with TBS-0.1% T and two times for 5 min with TBS). Membranes were incubated for 1 h at room temperature with the horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (BD Gentest; 1:80,000 dilution in TBS-0.02% T, 1% dried nonfat milk) and rinsed (three times for 5 min with TBS-0.1% T and two times for 5 min with TBS). The ECL Western Blotting Detection Kit (Amersham Biosciences Inc.) was used as recommended by the manufacturer to detect CYP1A1 expression.
Subcellular Binding Analysis. MCF-7, MDA-MB-435, TK-10, CAKI-1, IGROV1, and SKOV3 cells were grown to 60 to 80% confluency in 560-cm2 tissue culture plates (PGC Scientifics, Frederick, Maryland). Cells were treated with 0.1 µM [14C]5F 203 (40.5 mCi/mmol) for 16 h at 37°C before being harvested. After cells were centrifuged and homogenized, subcellular fractions were isolated by differential centrifugation in a manner similar to that described previously (Jones et al., 1995). Protein concentrations were determined by the Coomassie Blue assay (Pierce Biotechnology) using bovine serum albumin as a standard. The resultant fractions were stored at -70°C until further analysis. Proteins in samples were precipitated with trichloroacetic acid and covalent binding of [14C]5F 203 to subcellular fractions was determined using a previously described method (Rivera et al., 1999).
Statistics. All data were analyzed with a one-way analysis of variance with Tukey's post hoc test performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software Inc., San Diego CA, www.graphpad.com). Data are reported as means ± S.D.
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Results |
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The fluorinated benzothiazoles were synthesized in an attempt to overcome the biphasic dose-response curve in MCF-7 and TK-10 cells treated with DF 203; yet, a distinct biphasic dose-response curve resulted when these cells were treated with 6F 203. This biphasic response was present, albeit less pronounced, when MCF-7 and TK-10 cells were treated with 5,6 di-F 203 (Fig. 2, A and B). In contrast, 5F 203 potently inhibited the growth of MCF-7, IGROV1, and TK-10 cells without this characteristic biphasic dose-response curve (Fig. 2, A-C). Interestingly, we did not observe a biphasic dose-response curve in IGROV1 cells treated with either of the fluorinated benzothiazoles or with DF 203 (Fig. 2C).
In addition to antiproliferative activity (which is defined as the ability of these drug candidates to inhibit cancer cell growth), the fluorinated benzothiazoles at submicromolar concentrations conferred cytostasis (which is defined as that concentration needed to cause total growth inhibition) after brief exposures in multiple cell lines. In the in vitro c x t assays, as summarized in Table 2, each of the fluorinated benzothiazoles inhibited the growth of MCF-7 cells with GI50 values <0.03 µM and TGI values <0.5 µM. However, none of these derivatives displayed substantial cytotoxicity in MCF-7 cells even with 48- or 144-h exposures to 100 µM (Tables 1 and 2). LC50 activity was achieved in TK-10 after <1 h x 6 µM, whereas IGROV1 required >24 h x 0.74 µM. By contrast, 5F 203, 6F 203, and 5,6 di-F 203 failed to confer LC50 activity in MCF-7 cells, even after 144 h x 100 µM (Table 2; Fig. 3C).
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Sensitive Ovarian, Breast, and Renal Cancer Cells Take Up and Metabolize the Fluorinated Derivatives. Previous studies demonstrated that DF 203 uptake and biotransformation occurs exclusively in growth-inhibited, sensitive cells (Chua et al., 1999, 2000; Kashiyama et al., 1999). To determine whether sensitive cells also take up and biotransform the fluorinated benzothiazoles, aliquots of cell culture medium from both sensitive cells and resistant cells treated with 1 µM 5F 203, 6F 203 or 5,6 di-F 203 for at least 3 days were analyzed using HPLC. The concentrations of fluorinated benzothiazoles in nutrient medium from MCF-7, IGROV1, and TK-10 sensitive cell lines decreased with increasing exposure times (Fig. 4, A-C). In MCF-7 and IGROV1 cells, the decline in peak areas corresponding to 5F 203, 6F 203, and 5,6 di-F 203 was accompanied by the concurrent emergence of chromatographic peaks of as yet uncharacterized metabolites (data not shown). When incubated with TK-10 cells, 5F 203 and 5,6 di-F 203 completely disappeared from culture medium within 2 to 3 days, but 40% of 6F 203 remained after the 3-day incubation (Fig. 4C). Complete disappearance of 6F 203 from the culture medium of TK-10 cells required more than 6 days of incubation (data not shown). After a 12-h incubation, 5F 203 peak areas were reduced by 40% and 50% (10 µM and 5 µM, respectively) in MCF-7 cells, resulting in the emergence of three uncharacterized, minor metabolites (Fig. 5A). The concentration levels of these metabolites in the culture medium increased with incubation time (Fig. 5, B and C).
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In contrast, the concentrations of all three fluorinated benzothiazoles remained largely unchanged even after 3 days of incubation in resistant MDA-MB-435, CAKI-1, and SKOV3 cells (Fig. 4, D-F). Only 70% of the original amount of 6F 203 remained in medium after 2 days of incubation in CAKI-1 cells; however, we detected no additional 6F 203 disappearance, even after 6 days of incubation (data not shown). No metabolite peaks were observed in aliquots of media from any of the insensitive cells incubated with the fluorinated benzothiazoles.
CYP1A1, CYP1A2, and CYP1B1 metabolize 5F 203, 6F 203, and 5,6 di-F 203. DF 203 has been previously shown to mediate its anticancer activity by activating the aryl hydrocarbon receptor (AhR) signaling pathway (Loaiza-Perez et al., 2002). The AhR signaling pathway modulates the transcription of genes encoding CYP1A1, CYP1B1, and CYP1A2. Since these microsomal proteins are able to metabolize DF 203, we suspected that these P450s might also play a role in fluorinated benzothiazole metabolism. The metabolism of 5F 203, 6F 203, and 5,6 di-F 203 was investigated using microsomes from human B-lymphoblastoid cells expressing human recombinant CYP1A1, CYP1A2, or CYP1B1. After a 2-h incubation, peak areas of the fluorinated benzothiazoles in the chromatograms declined (Fig. 6), whereas peak areas corresponding to uncharacterized metabolites emerged. These metabolites were initially detected after 30 min of incubation (data not shown). All three CYP1 isoforms extensively metabolized fluorinated benzothiazoles after the 2-h incubation, but CYP1A1 exhibited the greatest capacity to metabolize the benzothiazoles (10 µM; Fig. 6A). Isoteric replacement of hydrogen with fluorine at the 5' position successfully blocked the formation of two metabolites, which likely included the 6-OH 203 metabolite. CYP1A1 microsomes produced metabolites with greater peak areas compared with those produced from CYP1A2 and CYP1B1 microsomes. No peaks corresponding to metabolites resulted from reaction mixtures containing microsomes derived from cells with empty vector.
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5F 203 Induces CYP1A1 and CYP1B1 mRNA Expression in Sensitive Cancer Cell Lines. Previous studies have demonstrated that benzothiazoles inhibit the growth of sensitive cancer cells concurrent with induction of CYP1A1 and, to a lesser extent, CYP1B1 expression (Chua et al., 2000; Loaiza-Perez et al., 2002; Trapani et al., 2003). We used quantitative real-time RT-PCR (QRT-PCR) to determine whether 5F 203, which lacks a multiphasic quality to the dose-response curve for growth inhibition, induced CYP1A1 and CYP1B1 mRNA expression in sensitive (MCF-7, TK-10, and IGROV1) cells or resistant (MDA-MB-435, CAKI-1, and SKOV3) cells. In general, induction of CYP1A1 expression was greater than that seen for CYP1B1 expression relative to controls. CYP1A1 expression increased 2.5-fold when the concentration increased from 0.01 µM to 0.1 µM and 8-fold when the concentration increased from 0.1 to 1.0 µM after 12 h of 5F 203 treatment of MCF-7 cells. We detected a 4-fold and 2.5-fold increase in CYP1A1 expression corresponding to increases in concentration from 0.01 µM to 0.1 µM and from 0.1 µM to 1.0 µM, respectively, after 24 h of 5F 203 treatment of these cells (Fig. 7A). CYP1A1 expression increased in a time- and dose-dependent manner (1.5- to 2-fold) in TK-10 cells treated with 5F 203 compared with untreated controls (Fig. 7B). A 10-fold increase in CYP1A1 expression occurred when IGROV1 cells were treated with 5F 203, corresponding to an increase in concentration from 0.1 µM to 1.0 µM (24 h; Fig. 7C). However, lower concentrations and shorter exposure times resulted in no increases in CYP1A1 expression relative to untreated controls. CYP1B1 expression increased 8-fold in MCF-7 cells treated with 1.0 µM compared with 0.1 µM 5F 203 (12 h; Fig. 8A). CYP1B1 expression increased approximately 5-fold in MCF-7 cells treated with 0.1 µM compared with 0.01 µM 5F 203 (24 h; Fig. 8A). Similar to induction of CYP1A1 expression, CYP1B1 expression increased in a time- and dose-dependent manner (1.5- to 2-fold) in TK-10 cells treated with 5F 203 (Fig. 8B). On the other hand, none of the resistant cells demonstrated increases in either CYP1A1 or CYP1B1 mRNA expression in comparison with untreated controls (Figs. 7 and 8). CYP1B1 mRNA induction observed under all exposure conditions in IGROV1 cells resembled that of untreated controls (Fig. 8C).
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Fluorinated Benzothiazoles Induce CYP1A1 Expression in Sensitive MCF-7 and TK-10 cells. Previous studies indicated that DF 203 induced CYP1A1 expression in MCF-7 cells but not in MDAMB-435 cells (Chua et al., 2000). To determine whether the fluorinated benzothiazoles induced CYP1A1 protein expression differentially in sensitive cells compared with resistant cells, we used immunoblot analysis to detect CYP1A1 protein expression in MCF-7, TK-10, and MDA-MB-435 cells that were treated with several fluorinated benzothiazoles including the lysylamide prodrug of 5F 203 (Phortress). Western blot analysis revealed that 4F 203, 5F 203, 6F 203, 5,6 di-F 203, and Phortress induced CYP1A1 expression in sensitive MCF-7 cells, whereas 7F 203 caused only minimal CYP1A1 induction (1.0 µM, 24 h; Fig. 9A). CYP1A1 induction was less pronounced in sensitive TK-10 cells, similar to what has been previously shown for DF 203 (Chua et al., 2000), even though TK-10 and MCF-7 cells showed sensitivities comparable to those of the fluorinated benzothiazoles (1.0 µM, 24 h; Figs. 2, 3, and 9B). CYP1A1 induction was prominent after TK-10 cells were exposed to 5F 203, 6F 203, 5,6 di-F 203 and Phortress, and, to a much lesser extent, when these cells were treated with 4F 203 and 7F 203. Figure 9C shows that, similar to DF 203, the fluorinated benzothiazoles were unable to induce CYP1A1 protein expression in resistant MDA-MB-435 cells (Chua et al., 2000; Bradshaw et al., 2002a; Loaiza-Perez et al., 2002).
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5F 203 Binds to Subcellular Macromolecules in Sensitive Cancer Cells. DF 203 covalently binds to DNA exclusively in sensitive cancer cells (Kashiyama et al., 1999). We measured subcellular localization of [14C]5F 203 within sensitive and resistant cancer cells to determine whether a correlation exists between the ability of 5F 203 to bind to subcellular macromolecules within cells and the sensitivity of cancer cells to 5F 203. We found that [14C]5F 203, or one or more metabolites, bound to macromolecules in nuclear, mitochondrial, microsomal, and cytosolic fractions of the sensitive cell lines (Fig. 10). In MCF-7 cells, the majority of bound radioactivity was localized in the nuclear and microsomal fractions along with a considerable amount in the mitochondrial fraction (Fig. 10A). A similar pattern was observed when ZR-75-1 cells (another estrogen receptor-positive breast cancer cell line) were treated with [14C]5F 203 (data not shown). The subcellular fractions of IGROV1 cells showed relatively indistinct differences in their capacities to bind to 5F 203 (Fig. 10B). The extent of binding to subcellular fractions in this cell line was less pronounced than in MCF-7 and TK-10 cells, possibly because CYP1A1 expression is not induced to as great an extent in IGROV1 cells as in MCF-7 and TK-10 cells after 5F 203 treatment. Substantial [14C]5F 203 binding occurred in the microsomal fraction of TK-10 cells, and the mitochondrial and nuclear fractions also showed considerable binding (Fig. 10C). A minimal amount of bound radioactivity in subcellular fractions of resistant cell lines MDA-MB-435 and SKOV3 was detected (Fig. 10, A and B). Nuclear binding of [14C]5F 203 was minimal yet more apparent in nuclear fractions of CAKI-1 cells (Fig. 10C). This was anticipated since CAKI-1 cells were less resistant to 5F 203 than either MDA-MB-435 or SKOV3 cells.
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; Chua et al., 2000; Hose et al., 2003; Monks et al., 2003). Our data clearly demonstrate that sensitive cells and cytochromes P450 (1A1, 1A2, and 1B1) readily metabolized the fluorinated benzothiazoles. In addition, we show that [14C]5F 203 or its metabolite(s) bound to macromolecules in cells proportionate to the cells' sensitivity to the growth-inhibitory effects of 5F 203. Interestingly, we demonstrate that these metabolites bound not only to the nuclear fraction, as would be expected from a prior demonstration of DNA adduct formation (Leong et al., 2003; Cain, 2003), but also to mitochondrial, microsomal, and cytosolic targets.
The fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles were originally synthesized to circumvent the formation of the inactive metabolite 6-OH 203, which likely contributes to the biphasic dose-response relationship of DF 203 (Bradshaw et al., 1998b, 2001; Hutchinson et al., 2001). In this study, fluorination of the benzothiazole in the 5-position resulted in a monophasic dose-response curve as opposed to a biphasic dose-response curve for growth inhibition. This finding therefore obviates the concern that dose escalation in clinical trials would need to account for the possibility that higher doses may actually compromise potential efficacy.
Fluorination of small molecules frequently blocks metabolic hydroxylation, which can modulate their anticancer activity (Akama et al., 1997). 6F 203-treated MCF-7 and TK-10 cells displayed a biphasic dose-response curve, corroborating findings from a previous investigation (Hutchinson et al., 2001). The biphasic dose-response detected, albeit in a less pronounced fashion, in 5,6-di F 203-treated MCF-7 and TK-10 cells was unanticipated, since a previous investigation predicted that the difluorinated benzothiazoles, including 5F 203, would produce neither exportable metabolites nor a biphasic dose-response curve (O'Brien et al., 2003). Using HPLC with an exceptionally low limit of detection, we were able to detect minor metabolites resulting from both 5,6 di-F 203-treated cells and 5F 203-treated cells to a comparable extent (data not shown). It is possible that "low level" metabolites were produced which antagonized the antiproliferative activity of 5,6 di-F 203. A biphasic dose-response curve was previously observed when IGROV1 cells were treated with DF 203 containing 3' halogen substitution and a pronounced biphasic dose-response curve was detected in OVCAR-5 ovarian cells treated with nonhalogenated DF 203 (Bradshaw et al., 1998a). Thus, the tendency of a benzothiazole to produce a biphasic dose-response curve may depend on the cancer cell type, the presence or absence of functional groups on the benzothiazole pharmacophore, or the extent the benzothiazole metabolizes into products that might antagonize its antiproliferative activity.
This study demonstrated that the fluorinated benzothiazoles were capable of producing antiproliferative, cytostatic, and, in some instances, cytotoxic effects exclusively in sensitive cancer cell lines. It was unanticipated that the fluorinated benzothiazoles would not produce cytotoxic activity even after extensive exposures (>6 days) in MCF-7 cells, since previous studies indicate that the most potent fluorinated benzothiazole, 5F 203, produces apoptosis and DNA damage in MCF-7 cells, which is characteristic of cytotoxic activity (Loaiza-Perez et al., 2002; Trapani et al., 2003). Although the endpoints of protein mass measured in sulforhodamine B assays and the viable cell mass measured in MTT assays used in this study were unable to reveal substantial cytotoxicity (LC50 activity) in MCF-7 cells with 6-day exposure to high drug concentration, such activity was readily detectible in IGROV-1 and TK-10 cells under the same in vitro assay conditions.
Cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1) have been shown to metabolize anticancer drugs (Kashiyama et al., 1999; Rochat et al., 2001) and prodrugs (Sladek, 1994). CYP1A1 preferentially metabolizes DF 203 to an inactive hydroxyl metabolite as well as a reactive electrophilic species, which frontier molecular orbital studies predict as being a nitrenium ion (O'Brien et al., 2003). In this study, CYP1A1 preferentially metabolized 5F 203, 6F 203, and 5,6 di-F 203. This is consistent with observations that a few anticancer agents induce the expression of P450s in human cancer cells, subsequently resulting in their metabolism to display their cytotoxic effects (Chase et al., 1998; Jounaidi et al., 1998). 5F 203 treatment caused a stronger induction of CYP1A1 mRNA and protein expression in sensitive MCF-7 cells than in sensitive TK-10 cells, supporting results from previous investigations (Bradshaw et al., 2002a; Hose et al., 2003; Monks et al., 2003).
The AhR signaling pathway activates the transcription of CYP1A1, CYP1B1, and CYP1A2 genes (Whitlock, 1999) and mediates the sensitivity of MCF-7 cells to the anticancer effects of DF 203 and 5F 203 (Loaiza-Perez et al., 2002; Trapani et al., 2003). These candidate anticancer agents activate the AhR signaling pathway in sensitive but not in resistant cells. Once this pathway is activated, AhR heterodimerizes with the AhR nuclear translocator protein (Jain et al., 1994; Safe and Krishnan, 1995). This heterodimer complex translocates from the cytosol into the nucleus where it binds to the xenobiotic response element located within the 5'-flanking region of responsive genes to initiate transcription, leading to the synthesis of their protein products (Denison et al., 1998; Lampen et al., 1998).
We and others show that cell sensitivity to a particular anticancer agent can depend upon a cell's ability to take up and metabolize this agent (Kashiyama et al., 1999; Hutchinson et al., 2001; Bradshaw et al., 2002a,b). Previous reports indicate that a few drug candidates, including DF 203, become metabolically activated subsequent to inducing CYP1A expression in sensitive cancer cells (Chua et al., 2000; Kuffel et al., 2002; Pichard-Garcia et al., 2004). In this study, metabolism occurred exclusively in sensitive cancer cells, suggesting that the fluorinated benzothiazoles are metabolically activated.
The fluorinated benzothiazoles retain potent and selective anticancer activity and the capacity to become metabolized in vivo (Chua et al., 1999; Bradshaw et al., 2002a,b). Observed concentrations of 5F 203 and 6F 203 in mouse plasma during a previous study exceed the "c x t" parameters of cell exposure required to achieve cytostasis in MCF-7 and IGROV-1 cells, and cytocidal activity in TK-10 cells observed in this current study (Alley et al., 2000). Pharmacological evaluations of L-lysylamide prodrugs of 5F 203 and 6F 203 following single i.v. bolus administrations to mice at well tolerated dosages demonstrate efficient in vivo conversion of each prodrug to 5F 203 and 6F 203 (Alley et al., 2000).
We demonstrate that fluorinated benzothiazoles disappeared from nutrient media, correlating with their capacity to be metabolized and bind covalently to macromolecules within "sensitive" cells but not "resistant" cancer cells. Prior studies have demonstrated the ability of benzothiazoles or their metabolites to bind covalently to DNA in vitro to mediate their anticancer effects (Leong et al., 2003; Trapani et al., 2003). A 5F 203 derivative has previously been shown to covalently bind to DNA in IGROV-1 and MCF-7 cells in vivo in proportion to its ability to inhibit tumor growth (Bradshaw et al., 2002a). Although previous studies demonstrated the ability of the benzothiazoles to bind to nuclear and microsomal macromolecules (Chua et al., 2000; Loaiza-Perez et al., 2002), ours is the first to demonstrate the affinity of 5F 203 for mitochondrial and cytosolic macromolecules in sensitive cells. Since 5F 203 has been previously shown to induce apoptosis (Trapani et al., 2003), its affinity for the mitochondria raises the possibility that this drug candidate facilitates cytochrome C release to initiate the caspase cascade essential for executing apoptosis (Cain, 2003). Further experiments will seek to explore the consequences of 5F 203 interaction with the functional properties of mitochondria.
This work demonstrates that the fluorinated benzothiazoles, and in particular, 5F 203, exhibit promising anticancer activity that warrants their continued development. Efforts are under way to characterize any 5F 203 metabolites that may possess anticancer activity, thereby yielding additional clues regarding the mechanism of antiproliferative activity for 5F 203. In the United Kingdom, Phortress (L-lysylamide prodrug of 5F 203) will enter clinical trials under the auspices of the Cancer Research Campaign.
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Disclaimer: The content of this article does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. government.
This work was previously presented in abstract form at the 91st Annual American Association for Cancer Research meeting in San Francisco, California, April 1-5, 2000; the 92nd Annual American Association for Cancer Research meeting in New Orleans, Louisiana, March 24-28, 2001, and the 14th World Congress of Pharmacology meeting in San Francisco, California, July 7-12, 2002.
This is part 25 of the series "Antitumor 2-(4-aminophenyl)benzothiazoles."
ABBREVIATIONS: DF 203, 2-(4-amino-3-methylphenyl)benzothiazole (NSC 674495); 5F 203, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (NSC 703786); 6F 203, 2-(4-amino-3-methylphenyl)-6-fluorobenzothiazole (NSC 702156); 5,6 di-F 203, 2-(4-amino-3-methylphenyl)-5,6-difluorobenzothiazole (711671); 4F 203, 2-(4-amino-3-methylphenyl)-4-fluorobenzothiazole (NSC 706705); 7F 203, 2-(4-amino-3-methylphenyl)-7-fluorobenzothiazole (NSC 711670); 6-OH 203, 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole (NSC 703785); Phortress, lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; AhR, aryl hydrocarbon receptor; QRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; TBS, Tween-buffered solution; GI50, concentration of compound needed to cause 50% growth inhibition; TGI, total growth inhibition; LC50, concentration of compound needed to cause 50% cell death (cytocidal activity); NCI, National Cancer Institute.
1 Current address: Consorzio Mario Negri Sud, Departimento di Biologia Cellulare e Oncologia via Nazionale, 66030 S. Maria Imbaro (CH). 
2 Current address: University of Maryland Greenebaum Cancer Center, 22 S. Greene Street, Baltimore, MD.
Address correspondence to: Dr. Sherman F. Stinson, P.O. Box B, Bldg. 1047, Room 6, BTB, DTP, DCTD, National Cancer Institute at Frederick, Frederick, MD 21701. E-mail: stinson{at}dtpax2.ncifcrf.gov
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) or IGROV1 cells (¶) at a given time point (P < 0.05). Bars shown to reach 100 µM actually exceed this value but by no more than 10%.
