Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L–10 μmol/L; 24–72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.

2-(4-Amino-3-methylphenyl)benzothiazole (DF 203; NSC 674495) and its fluorinated analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786; Fig. 1) belong to a series of antitumor agents distinct from all mechanistic classes of chemotherapeutic agents in current clinical use. The antitumor activity provoked by these agents in vitro is highly selective and exquisitely potent in sensitive phenotypes (1–4). In the National Cancer Institute cell line panel, sensitive cell lines (e.g., MCF-7, T47D, and IGROV-1) reveal GI50 values <10 nmol/L; in contrast, GI50 values exceed 10 μmol/L in resistant cell lines (e.g., MDA-MB-435 and CAKI -1). DF 203 and 5F 203 are rapidly depleted from media supporting growth of sensitive cells (3, 5), as selective sequestration of these agents by sensitive cells has been shown to precede cytosolic aryl hydrocarbon receptor (AhR) binding (6). The AhR-benzothiazole complex translocates to the nucleus, dimerizes with the AhR nuclear transporter, and complexes with the xenobiotic response elements on the cyp1a1 promoter thereby activating gene transcription (6, 7). The subsequent induction of CYP1A1-catalyzed metabolism of DF 203 or 5F 203 to reactive electrophilic species results in DNA-adduct formation in sensitive cells only (8). Insensitive cells fail to deplete drug from nutrient media, cyp1a1 transcription is not achieved, and adducts are not generated in the DNA of these cells as a consequence of exposure to DF 203 or 5F 203 (≤10 μmol/L). The key to the inherent resistance displayed by MDA-MB-435 cells seems to be the constitutive nuclear localization of the AhR (6).

Figure 1.

Chemical structures of Phortress, the dihydrochloride lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), and the primary amine 5F 203, and a summary of the mechanism of action of Phortress.

Figure 1.

Chemical structures of Phortress, the dihydrochloride lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), and the primary amine 5F 203, and a summary of the mechanism of action of Phortress.

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DF 203, owing to antagonistic metabolism, was supplanted by 5F 203 as the favored preclinical candidate (3). In vivo 5F 203 has been shown to induce unequivocal selective antitumor activity (9). Thus, MCF-7 and MDA-MB-435 tumors were implanted in opposite flanks of the same mouse. Following daily (×5) treatment of animals with 5F 203 (4 mg/kg), the growth of MCF-7 tumors only was significantly retarded. Lipophilic 5F 203, however, possesses poor aqueous solubility. Pharmaceutically, an i.v. formulation was desired to avoid potential first-pass metabolism by the liver. 5F 203 was therefore conjugated via the exocyclic amine to amino acids alanine, lysine, and serine, and thus converted to a series of prodrugs (10). The dihydrochloride salt of lysylamide 5F 203 (Phortress; NSC 710305; Fig. 1) liberates 5F 203 in the presence of carcinoma cells in vitro, and in plasma in vivo, and maintains selective, potent antitumor activity (4, 9). Moreover, when MCF-7 and MDA-MB-435 xenografts were recovered from opposite flanks, 24 hours after treatment of mice with Phortress (20 mg/kg, i.p.), CYP1A1 protein and DNA adducts were observed only in MCF-7 tumors.

In this study, we report the consequences of adduct generation on DNA integrity and cell cycle progression. We have probed in silico antitumor activity of these apparently DNA-targeted agents. In addition, using our knowledge of the selective nature of antitumor activity in vivo, the possibility that sensitivity of tumors to Phortress can be identified by single cell gel electrophoresis (SCGE) has been explored.

Two human-derived mammary carcinoma models have been utilized in this study: MCF-7 cells (11) show exquisite sensitivity to the growth inhibitory properties of 2-(4-aminophenyl)benzothiazole analogues in vitro and in vivo; in contrast, MDA-MB-435 cells (12) possess inherent resistance to this class of agent.

Preparation of Cells and Slides for SCGE

MCF-7 and MDA-MB-435 cells were seeded in 25 cm2 tissue culture flasks in RPMI 1640 medium supplemented with 10% FCS. Cells were allowed to attach overnight before addition of 5F 203 (10 nmol/L–10 μmol/L) or DMSO (0.1%). Additional control samples received no treatment.

After incubation for 24, 48, and 72 hours, cells were detached with trypsin, diluted with medium, centrifuged, and the pellet washed twice with ice-cold PBS. To obtain positive control samples, untreated cells were treated with 100 μmol/L H2O2 (20 minutes, 4°C) to generate significant oxidative damage.

Slides were essentially processed according to the manufacturer's instructions (Trevigen, MD, USA). Briefly, harvested cell suspensions (10 μL; 1 × 105/mL)were suspended in 90 μL molten 1% low melting point agarose in 1× PBS at 42°C. Mixtures (75 μL) were loaded onto prewarmed (37°C) CometSlides (Trevigen). Slides were placed flat at 4°C in the dark >3 hours to improve adherence.

Cells embedded in agarose were lysed for 1 hour in prechilled lysis solution [2.5 mol/L NaCl, 100 mmol/L EDTA (pH 10), 10 mmol/L Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100; 4°C] to remove cellular proteins. For alkaline SCGE, slides were placed in freshly prepared alkaline solution [0.3 mol/L NaOH, 1 mmol/L Na2EDTA (pH >13)] for 20 minutes at room temperature in the dark to allow unwinding of DNA and conversion of alkali-labile sites to single strand breaks (SSB) before electrophoresis.

For detection of double strand breaks (DSB), slides were removed from the lysis solution and washed by immersing in 50 mL of 1× Tris-borate EDTA buffer [45 mmol/L Tris-borate, 1 mmol/L EDTA (pH 8.0)] for 5 minutes before electrophoresis. No alkaline treatment was done.

Electrophoresis

For alkaline SCGE, slides were placed in 0.3 mol/L NaOH, 1 mmol/L Na2EDTA (pH >13). Electrophoresis was conducted for 20 minutes at 16 V (1 V/cm), 300 mA. The alkaline conditions caused DNA to denature and made the assay sensitive to SSB. Slides were then drained, placed on a tray, and washed slowly with two changes, 10 minutes each, of neutralization buffer [0.4 mol/L Tris-HCl (pH 7.5)] followed by washing twice with distilled H2O. Slides were then dehydrated in 70% ethanol for 5 minutes and dried at room temperature. The whole procedure was carried out in dim light to minimize artifactual DNA damage.

For detection of DSB, electrophoresis was carried out in neutral conditions (13). After incubation with lysis buffer, slides were washed twice by immersing in 1× Tris-borate EDTA buffer for 5 minutes. SCGE was conducted for 10 minutes at 16 V at room temperature. Slides were dipped in ethanol for 5 minutes and air-dried overnight.

Imaging and Evaluation of DNA Damage

Slides were stained with 50 μL diluted SYBR Green solution [1:10,000 in TE buffer 10 mmol/L Tris-Cl (pH 7.5), 1 mmol/L EDTA] and analyses done at 250× magnification using a fluorescence microscope (λex = 494 nm and λem = 521 nm). Images of 50 randomly selected cells were captured for each sample. Image analysis was achieved by using Scion Image (Scion Co., Frederick, MD, http://www.scioncorp.com; ref. 14). Tail length, measured from the estimated center of the cell, percentage of DNA in the comet tail (%DNA migrated), also measured from the estimated center of the cell, and tail moment (tail length × % migrated DNA) were quantified for each cell as DNA damage parameters. Statistical analyses were conducted using one-way ANOVA. To determine at which time point significance was achieved, one-way ANOVA with post hoc t tests (Dunnett's t test) procedure was utilized. In addition, the significance of each dose against the untreated control was evaluated by the Dunnett's t test. Computer data was analyzed using SPSS package (SPSS V 11.0, 2000, Chicago, IL). A level of probability P < 0.05 was taken as indicating statistical significance.

Cell Cycle Analyses

MCF-7 and MDA-MB-435 cells were seeded at densities of 5 × 105 to 1.5 × 106 cells per 10 mL RPMI 1640 medium in 25 cm2 tissue culture flasks. After allowing overnight attachment, cells were treated continuously for 24, 48, and 72 hours with 5F 203 (10 nmol/L–10 μmol/L). Adherent cells were trypsinized and collected in ice-cold 1% FCS-PBS buffer. Approximately 5 × 105 to 106 cells were used for each determination. Cells were centrifuged at 1,000 rpm for 5 minutes and washed twice in 1% FCS-PBS buffer. The resulting cell pellet was resuspended and fixed in 500 μL of 70% ice-cold ethanol for 30 minutes on ice, then pelleted by centrifugation. The supernatant was removed and cells washed twice in 1% FCS-PBS. The cell pellet was resuspended in 500 μL of 0.2 μm-filtered PBS. RNase A (5 μL; 2,000 units/mL) plus propidium iodide (50 μL, 500 μg/mL) were added, and the tubes incubated at 37°C for 30 minutes.

For each determination, the DNA content of 10,000 cells was analyzed by Beckman Coulter EPICS XL (Miami, FL) flow cytometry in which an argon laser (488 nm) was used to excite propidium iodide, and emission above 550 nm was collected. The single cell population was gated and DNA histograms were generated using EXPO 32 flow cytometry software (Applied Cytometry System, Sheffield, United Kingdom).

Hollow Fiber Preparation

The method of Hollingshead et al. (15) was essentially adopted for hollow fiber procedures. Polyvinylidene difluoride hollow fibers (type f; Spectrum Laboratories, Inc., Rancho Dominguez, CA; 500 kDa cutoff, 1 mm inner diameter) were flushed through with 70% ethanol using a blunt 21 gauge needle (Tycohealthcare, Gosport, United Kingdom) and 10-mL syringe (Terumo, Liverpool, UK). Fibers were immersed in 70% ethanol for 72 hours and flushed through once more with 70% ethanol then distilled H2O and autoclaved at 131°C. Fibers were flushed through with RPMI 1640 culture medium before dispensation of cell suspensions into fibers. Heated smooth jawed needle holders sealed the fiber ends. Additionally, fibers were laid along a demarcated tray, covered with medium, and heat sealed at 2 cm intervals. Cell-laden fiber segments were incubated at 37°C in 75 cm3 flasks containing culture medium overnight before implantation into pure strain 6- to 8-week-old female NMRI mice. Animals were housed in cages in an air-conditioned room with alternating cycles of light and dark and access to food and water ad libitum. Studies were carried out under a home office license and UKCCCR guidelines for the welfare of animals in experimental neoplasia were adhered to throughout the study (16). To each mouse,an estrogen pellet (60 day release 17β-estradiol, 0.72 mg, Innovative Research, Michigan) was given s.c. at the base of the neck, 24 hours before hollow fiber implantation. Two fibers (one of each cell line) were implanted at both s.c. and i.p. sites. For i.p. implants, a small incision was made through the skin and musculature of the ventral abdominal wall. Fibers were placed into the peritoneal cavity and both incisions closed with histoacryl tissue glue (B/Braun Surgical, Barcelona, Spain) and an additional skin staple. For s.c. implants, a small incision was made dorsally near the base of the tail. Fibers were implanted to the left of the dorsal midline in a cranial direction using a sterile trocar. The small incision was closed with tissue glue and a skin staple.

Assessment of Tumor Cell Growth within Hollow Fibers

On each day of growth assessment, a modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide dye conversion assay was done (15, 17). Mice were sacrificed by cervical dislocation, fibers excised and placed in 2 mL of prewarmed RPMI 1640 (20% FCS) for 30 minutes at 37°C. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (5 mg/mL in distilled water): RPMI 1640 (20% FCS) 1:5 was added (1 mL) and fibers incubated at 37°C with 5% CO2 for 4 hours. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide solution was aspirated, and sterile filtered 2.5% protamine sulfate (0.9 g NaCl, 2.5 g protamine sulfate in 100 mL of distilled water; 2 mL) was added, specimens were stored at 4°C for a minimum of 24 hours in the dark to fix the formazan product. When all samples had been fixed, fresh protamine sulfate (2.5%) was added and fibers stored for 2 to 4 hours at 4°C. Each fiber was transferred to a well in a 24-well plate, cut in half and air-dried overnight, protected from light. DMSO (300 μL) was added to each well and formazan product extracted. Volumes of 190 μL of each well solution were transferred to 96-well plates and absorbance read at 540 nm. Mean absorbance on day of retrieval was compared with mean absorbance on implantation day and % net cell growth was calculated.

Comet Assay of Cells Cultured in Hollow Fibers

Mice were treated with Phortress (20 mg/kg in physiologic saline, i.p., 0.1 mL/10 g body weight) 4 days post fiber implantation. Control animals received vehicle alone. Fibers were recovered from mice 24 hours after Phortress treatment and transferred to culture medium (37°C) for 30 minutes. Following transfer to 6-well plates, fibers were flushed through with accutase solution (in Dulbecco's PBS 0.5 mmol/L EDTA). Plates were incubated for 45 minutes (37°C, 5% CO2) on an orbital shaker at 100 g, thus detaching cells from fiber walls. Cells were expelled using a blunt needle and syringe, pelleted from the resulting suspension, and placed on ice in 150 μL PBS to prevent DNA repair. SCGE procedures were adapted (18) and carried out in a dark room under red light.

Frosted edge slides were placed into normal melting point agarose (1%), removed and the underside wiped, slides were dried overnight. Low melting point agarose (1%, 150 μL, 45°C) was added to cell suspensions. Cell suspensions (150 μL) were added to agarose coated slides, coverslips added, and slides placed on ice for 5 minutes. Coverslips were removed, low melting point agarose (0.5%, 150 μL) added as a third layer, coverslips replaced, and slides again kept on ice for 5 minutes. Coverslips were again removed and slides submerged in cold lysis solution [2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris (pH 10), 1% Triton X-100, 10% DMSO; >1 hours, 4°C]. Slides were submerged with buffer [1 mmol/L EDTA and 300 mmol/L NaOH (pH >13)] in a horizontal electrophoresis tank and incubated for 30 minutes to allow DNA unwinding. Electrophoresis was then conducted at 25 V for 30 minutes. Slides were neutralized [Tris (pH 7.5)], washed (PBS), air-dried overnight, rehydrated (distilled H2O) and incubated for 30 minutes with SYBR gold (×10,000 in distilled H2O, (Invitrogen Ltd., Paisley, UK). Using Comet III software (Perceptive Instruments, Suffolk, United Kingdom) connected to a fluorescence microscope and camera (LEICA DMLB), 50 cells per slide were selected randomly for analysis. Tail moment was used as a primary measure of DNA damage and results presented as tail moment histograms (19, 20). Statistical analysis of comet data was done using SPSS (version 11.0) and application of nonparametric Mann Whitney U tests (21–23).

Preparation of Xenografts for SCGE

The experimental group consisted of six female NCR nude mice implanted with estrogen pellets and bearing a tumor in each flank. Tumors were established in mice after 4 to 5 weeks. Animals (4) were treated with Phortress (20 mg/kg, i.p.), two control animals received vehicle alone. After 4 and 24 hours, Phortress-treated mice were anesthetized and tumors excised. Control mice were anesthetized and tumors excised at 24 hours. Tumor fragments were placed in 500 μL cold Hank's balanced salt solution containing 20 mmol/L EDTA/10% DMSO and minced using a scalpel blade. The resulting cell suspension was filtered through a 50-μm nylon mesh and centrifuged at 200 × g for 5 minutes. The pellet was resuspended in PBS and 10 to 15 μL of sample were mixed with low melting point agarose. SCGE was done as described for hollow fiber cultures. Volumes were adjusted such that 10,000 cells per slide were analyzed. Each sample was prepared in triplicate and 50 cells scored per slide. Nonparametric Mann-Whitney U tests were applied to the data to determine statistical significance.

Self-Organizing Map–Based Cluster Analysis

Recently, a new set of computational tools, based on methods of a self-organizing map (SOM) has been developed, and their applications to the National Cancer Institute's antitumor drug-screening data explored (24). This suite of tools seeks to identify compounds with similar activities against National Cancer Institute tumor cell lines to facilitate discoveries of potentially new drug leads, new molecular targets and identification of tumor-selective agents. The SOM cluster analysis, which represents an alternative to the more classic COMPARE analysis introduced in 1997, identified relationships between chemotypes of >100,000 screened agents and their effect on six major classes of cellular activities: mitosis (M), nucleic acid synthesis (S), membrane transport and integrity (N), phosphatase- and kinase-mediated cell cycle regulation (P), and two remaining regions arbitrarily labeled Q and R, which could not be assigned to any specific activity class. Utilizing this newly available tool, data mining has been conducted using aminophenylbenzothiazole compounds DF 203 and 5F 203 as seeds.

Induction of DNA DSB and SSB by 5F 203 In vitro

SCGE, comet assays were done to confirm and quantify DNA damage caused by 5F 203 in vitro. Under alkaline conditions (pH >13), SSB and alkali labile sites were detected; investigation of DSB was conducted at neutral pH.

As illustrated in Fig. 2A, aminophenylbenzothiazole-sensitive MCF-7 cells, treated with 5F 203 (10 nmol/L–10 μmol/L) for 72 hours, and analyzed following alkaline SCGE, revealed a bright comet head and a tail whose fluorescence and length increased with 5F 203 concentration. Calculated tail moments for increasing 5F 203 concentrations and incubation times are illustrated in Fig. 2Bi. A clear and steady dose-dependent increase in DNA SSB was observed 24, 48, and 72 hours after treatment (10 nmol/L–1 μmol/L 5F 203). Detection of SSB became considerable following exposure of cells to 100 nmol/L 5F 203 (all time points examined). At higher doses (1–10 μmol/L 5F 203) however, DNA damage seemed to reach a plateau. Visual inspection revealed that this was due to extreme fragmentation and hence, loss of the comet head, leading to lack of detection by the comet software.

Figure 2.

A, DNA SSB induced by 5F 203. MCF-7 cells were treated for 72 hours with (i) medium alone, (ii) DMSO vehicle, (iiivi) 10, 100 nmol/L, 1, and 10 μmol/L 5F 203, respectively, and subjected to alkaline SCGE. Cells were viewed at ×250 using fluorescent microscopy. B, DNA SSB after treatment of (i) MCF-7 and (ii) MDA-MB-435 cells with 5F 203. DNA damage was analyzed by alkaline comet assay. Control samples represent cells treated with medium alone. Columns, representative of three separate experiments.

Figure 2.

A, DNA SSB induced by 5F 203. MCF-7 cells were treated for 72 hours with (i) medium alone, (ii) DMSO vehicle, (iiivi) 10, 100 nmol/L, 1, and 10 μmol/L 5F 203, respectively, and subjected to alkaline SCGE. Cells were viewed at ×250 using fluorescent microscopy. B, DNA SSB after treatment of (i) MCF-7 and (ii) MDA-MB-435 cells with 5F 203. DNA damage was analyzed by alkaline comet assay. Control samples represent cells treated with medium alone. Columns, representative of three separate experiments.

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When MCF-7 cells, exposed to concentrations of 5F 203 between 10 nmol/L and 10 μmol/L for 24 to 72 hours, were analyzed by neutral SCGE assay, again statistically significant (P < 0.05), dose- and time-dependent increases in DNA DSB were observed (Fig. 3A and Bi). Of note, is the steep increase in these double strand lesions 72 hours after exposure to 10 nmol/L 5F 203 and 48 hours after treatment of cells with 10 and 100 nmol/L 5F 203.

Figure 3.

A, DNA DSB induced by 5F 203. MCF-7 cells were treated for 72 hours with (i) medium alone, (ii) DMSO vehicle, (iiivi) 10, 100 nmol/L, 1, and 10 μmol/L 5F 203, respectively, and subjected to neutral SCGE. Cells were viewed at ×250 using fluorescent microscopy. B, DNA DSB in (i) MCF-7 and (ii) MDA-MB-435 cells after treatment with 5F 203. DNA damage was quantified following neutral SCGE. Control samples represent cells treated with medium alone. Columns, representative of three separate experiments.

Figure 3.

A, DNA DSB induced by 5F 203. MCF-7 cells were treated for 72 hours with (i) medium alone, (ii) DMSO vehicle, (iiivi) 10, 100 nmol/L, 1, and 10 μmol/L 5F 203, respectively, and subjected to neutral SCGE. Cells were viewed at ×250 using fluorescent microscopy. B, DNA DSB in (i) MCF-7 and (ii) MDA-MB-435 cells after treatment with 5F 203. DNA damage was quantified following neutral SCGE. Control samples represent cells treated with medium alone. Columns, representative of three separate experiments.

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Significantly, no DNA SSB or DSB were observed in aminophenylbenzothiazole-insensitive MDA-MB-435 cells (Figs. 2Bii and 3Bii).

Cell Cycle Perturbation

In an attempt to elucidate more fully the consequences of treatment, the effects of 5F 203 on MCF-7 and MDA-MB-435 cell cycle progression were examined.

As illustrated in Fig. 4, a modest increase of cells in G1 phase and concomitant decrease in G2-M phase cells were observed following treatment with 1 and 10 μmol/L 5F 203 for 24 hours. When MCF-7 cells were treated with 100 nmol/L to 10 μmol/L 5F 203 for 72 hours, the most prominent effect was the accumulation of cells in S phase accompanied by decreased numbers of cells in the G1 phase of the cell cycle. Furthermore, a subpopulation of smaller cells with increased granularity accumulated in the sub-G1 phase >48 hours exposure to 1 and 10 μmol/L 5F 203, suggesting the presence of apoptotic cells. In contrast, 5F 203-insensitive MDA-MB-435 cells failed to show any notable effect on the cell cycle (results not shown).

Figure 4.

Cell cycle profiles following treatment of MCF-7 cells with 5F 203. Cells were recovered after 24, 48, and 72 hours 5F 203 exposure (10 nmol/L–10 μmol/L). DNA content was assayed following propidium iodide staining by fluorescence-activated cell sorting analysis. Insets, % cells in the different cell phases. Y axis, cell number.

Figure 4.

Cell cycle profiles following treatment of MCF-7 cells with 5F 203. Cells were recovered after 24, 48, and 72 hours 5F 203 exposure (10 nmol/L–10 μmol/L). DNA content was assayed following propidium iodide staining by fluorescence-activated cell sorting analysis. Insets, % cells in the different cell phases. Y axis, cell number.

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Tumor Cell Growth within Hollow Fibers In vivo

The growth of MCF-7 and MDA-MB-435 breast carcinoma cells was characterized in hollow fibers in vivo, before Phortress treatment. Cells of both lineages grew optimally over 5 to 6 days when seeded in hollow fibers at densities of 1 × 106 (MCF-7) and 1.25 × 106 (MDA-MB-435) cells/mL (Fig. 5).

Figure 5.

Net cell growth of (A) MCF-7 and (B) MDA-MB-435 breast carcinoma cells grown in hollow fibers in vivo. Cells were seeded into fibers at densities of 1.25 × 106 and 1 × 106 cells/mL, respectively, incubated overnight before implantation i.p. and s.c. into mice. Fibers were retrieved at specified time points. Columns, mean from nine fibers (3/mouse/site); bars, ±SD. Net cell growth (%) was calculated from absorbance values (540 nm) generated using the MTT assay (, i.p; , s.c).

Figure 5.

Net cell growth of (A) MCF-7 and (B) MDA-MB-435 breast carcinoma cells grown in hollow fibers in vivo. Cells were seeded into fibers at densities of 1.25 × 106 and 1 × 106 cells/mL, respectively, incubated overnight before implantation i.p. and s.c. into mice. Fibers were retrieved at specified time points. Columns, mean from nine fibers (3/mouse/site); bars, ±SD. Net cell growth (%) was calculated from absorbance values (540 nm) generated using the MTT assay (, i.p; , s.c).

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Determination of Phortress-Induced SSB in the Hollow Fiber Model

The two cell lines were grown simultaneously at i.p. and s.c. sites in NMRI mice. On day 4, mice were treated with Phortress or vehicle alone. Following 24 hours exposure to Phortress, fibers were recovered, tumor cells retrieved, and comet assays done. The results revealed selective formation of SSB in the DNA of MCF-7 cells recovered from hollow fibers implanted i.p. or s.c. following treatment of mice with Phortress (Fig. 6A and C). Tail moment histograms of MDA-MB-435 comets following retrieval of cells from mice treated with Phortress were indistinguishable from those of untreated control animals (Fig. 6B and D). MCF-7 cells retrieved from Phortress and vehicle-treated mice at i.p. sites (Fig. 6A) had mean tail moment values of 12.88 ± 16.51 and 2.07 ± 4.91, respectively (P ≤ 0.001). MCF-7 cells retrieved from s.c. sites (Fig. 6C) of Phortress-treated mice also had mean tail moments greater than those from vehicle-treated control animals ( P = 0.031).

Figure 6.

Tail moment histograms of comets derived from cells cultured in hollow fibers invivo. Hollow fibers were seeded with either MCF-7 (A and C; 1 × 10 6 cells/mL) or MDA-MB 435 (B and D; 1.25 × 10 6 cells/mL) cells. Two fibers (one of each cell line) were implanted into NMRI mice at both i.p. ( A and B) and s.c. ( C and D)sites. On day 4, mice were treated with either Phortress (, 20 mg/kg) or physiological saline (□) by a single i.p. injection. After 24 hours, fibers were excised and cells retrieved and analyzed for DNA SSB using the alkaline comet assay. Columns, analyses of 150 cells (3 mice × 1 fiber × 50 comets).

Figure 6.

Tail moment histograms of comets derived from cells cultured in hollow fibers invivo. Hollow fibers were seeded with either MCF-7 (A and C; 1 × 10 6 cells/mL) or MDA-MB 435 (B and D; 1.25 × 10 6 cells/mL) cells. Two fibers (one of each cell line) were implanted into NMRI mice at both i.p. ( A and B) and s.c. ( C and D)sites. On day 4, mice were treated with either Phortress (, 20 mg/kg) or physiological saline (□) by a single i.p. injection. After 24 hours, fibers were excised and cells retrieved and analyzed for DNA SSB using the alkaline comet assay. Columns, analyses of 150 cells (3 mice × 1 fiber × 50 comets).

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In contrast, there was no significant difference between mean tail moment values of MDA-MB-435 cells retrieved from hollow fibers of Phortress and vehicle-treated mice: values obtained at i.p. sites were 1.14 ± 2.32 and 1.44 ± 3.06, respectively; at s.c. sites, values were 1.28 ± 2.17 and 1.38 ± 2.84, respectively. From the tail moment histograms representative of these mean data, Phortress-sensitive and -resistant phenotypes can clearly be distinguished.

Induction of SSB by Phortress in the Xenograft Model

Samples of MCF-7 tumors recovered from untreated mice contained cell nuclei that were predominantly rounded, or with low-level tail moment values (0–5). However, there were considerable numbers of cells with tail moments between 5 and 10. Evidence also revealed a population of nuclei with extensive tail moments, thought to be consistent with apoptotic cells. After 4 hours Phortress treatment (20 mg/kg) tail moments of nuclei were greater than those of controls, and the frequency profile of this treatment group was significantly different from the tail moment frequency of cell nuclei extracted from untreated mice ( Fig.7;P = 0.029). After 24 hours treatment (20 mg/kg Phortress), samples showed a reduction in comets with low tail moment values (0–5), and generation of more comets with intermediate (10–20) and high (20–35) tail moment values, which proved to be highly significant (P < 0.0001). Comets with extensive tail moments were also evident, as observed in control samples. DNA SSB inflicted upon MCF-7 tumors as a direct consequence of Phortress administration to tumor-bearing animals has clearly been detected.

Figure 7.

Tail moment histograms of comets derived from MCF-7 xenografts 4 h () and 24 h () after treatment of NMRI mice (i.p.) with 20 mg/kg Phortress or saline vehicle (24 h; □).

Figure 7.

Tail moment histograms of comets derived from MCF-7 xenografts 4 h () and 24 h () after treatment of NMRI mice (i.p.) with 20 mg/kg Phortress or saline vehicle (24 h; □).

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Self-Organizing Map-Based Cluster Analysis

Previously, by Computer Pattern Recognition (COMPARE),the growth inhibitory profiles of antitumor 2-(4-aminophenyl)benzothiazoles were analyzed (2). It was concluded that these agents, which possess an activity fingerprint highly distinctive within the class, as shown by high Pearson correlation coefficients, were COMPARE negative to all other classes of clinical antitumor agent. When the GI50 data of agents DF 203 and 5F 203 were used as seeds, both compounds projected on the SOM in region P (k7.13 and k7.15, respectively), suggesting that these molecules may be modulating kinases and phosphatases associated with cell cycle regulation and apoptosis (Fig. 8).

Figure 8.

A, Complete SOM according to the cellular activities of anticancer agents screened by the National Cancer Institute http://spheroid.ncifcrf.gov). B, location of DF 203 and 5F 203 on the SOM, projecting on subregion P11 (k7.13 and k9.15, respectively).

Figure 8.

A, Complete SOM according to the cellular activities of anticancer agents screened by the National Cancer Institute http://spheroid.ncifcrf.gov). B, location of DF 203 and 5F 203 on the SOM, projecting on subregion P11 (k7.13 and k9.15, respectively).

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Previously, we have shown that in vitro and in vivo antitumor activities of 5F 203 and its lysylamide prodrug, Phortress, are mediated through the formation of DNA adducts in sensitive tumor cells (8). Herein, we have investigated the biological consequences of DNA lesions induced by 5F 203 to comprehend more clearly the mechanism of action of this class of agent.

SCGE conducted under alkaline and neutral conditions to detect SSB and DSB, respectively, showed significant and selective DNA damage in MCF-7 cells only, following exposure to 5F 203. Thus, previous reports illustrating selective formation of benzothiazole-derived adducts in DNA of sensitive MCF-7 mammary carcinoma cells only are corroborated (8). Moreover, alkaline SCGE revealed DNA damage inflicted upon MCF-7 cells is not compromised (a) following prodrug modification and (b) when cells are grown in vivo. Phortress-induced generation of SSB in MCF-7 cells in vivo is significant whether the compared group represents inherently resistant MDA-MB-435 tumor cells cultured in hollow fibers transplanted in animals that receive subsequent Phortress treatment, or MCF-7 xenografts recovered from untreated mice.

The selective formation of SSB and DSB in MCF-7 cells was both dose and time dependent. SSB in DNA may result from a number of different reaction types including base and nucleotide excision repair, direct scission of the DNA backbone by chemical or radical attack, strand breakage following binding of intercalating agents, alkali-labile DNA adducts, endonuclease or topoisomerase action and DNA hydrolase release from lysosomes (25–27). As with most of the monofunctional alkylating agents, induction of SSB in MCF-7 by 5F 203 could directly result from the presence of bulky and helix-distorting DNA adducts which weaken the N-glycosylic bond. This reaction would lead to depurination/depyrimidination of the adducted bases and the appearance of alkali-labile abasic sites (27–29). Abasic sites are removed by abasic endonucleases, which cleave DNA adjacent to these sites, creating SSB in DNA. Bulky DNA lesions are normally repaired rapidly and efficiently by NER (30, 31), using the undamaged DNA as template (32).

The most notable cellular response to DNA damage is the temporary arrest of cell cycle progression at the G1-S phase by cell cycle checkpoint mechanisms (32, 33). Indeed, when MCF-7 cells were exposed to 5F 203 (10 nmol/L–10 μmol/L, 24–72 h), significant numbers of cells did accumulate in the G1-S and S phase of the cell cycle (Fig. 4). Transcription of DNA-damage response genes, p53-induced gene 3p21/Cip1 and Wip1, was activated by DF 203 and 5F 203 in sensitive MCF-7 cells (34). In addition, expression of p53 protein, a transcription factor which becomes stabilized and active upon DNA damage, was highly induced in MCF-7 cells following treatment with DF 203 (≥30 nmol/L; ref. 35). P21Waf1/Cip1 cyclin-dependent kinase inhibitor (also known as CDKN1A) is capable of silencing the cyclin-dependent kinases, which are essential for cell cycle progression through G1-S phase (36, 37). Human wild-type p53-induced phosphatase 1 (Wip1) encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner with a pattern similar to that of p21 (38–40). The major role of Wip1 is in regulation of the choice of response to p53. When highly expressed, Wip1 attenuates UV-induced p38-MAPK (mitogen-activated protein kinase) mediated phosphorylation of Ser33 and Ser46 in p53, thereby inhibiting p53-mediated apoptosis in cells sustaining repairable damage(41).

The role of S phase arrest in 5F 203 treated cells remains obscure. The intra-S phase checkpoint is known to operate in a p53/p21-independent manner (36). The majority of lesions evoked by DNA-reactive agents occur during DNA synthesis, therefore, 5F 203-induced adducts (SSB and DSB) may directly disrupt DNA synthesis, causing delay within S phase.

A unifying theme of cell cycle arrest is that, functionally, it provides protection, allowing additional time for DNA repair at critical junctures in cell cycle progression (37). Up-regulation of the DNA damage recognition gene DDB2 has been confirmed by microarray analyses of MCF-7 cells exposed for 24 hours to 5F 203 and DF 203 (1 μmol/L; ref.34), corroborating a role for NER in benzothiazole-DNA adduct repair.

Human DDB1and DDB2 genes encode the 127 kDa (p127) and 48 kDa (p48) subunits, respectively, of the damage specific DNA-binding protein (DDB; ref. 42). Mutations in the DDB2 gene inactivate DDB in individuals from complementation group E of xeroderma pigmentosum, an autosomal recessive disease characterized by extreme sensitivity to sunlight, severe risk of skin cancer, and defective NER (42, 43). Human DDB protein has been implicated in global genomic repair of UV-damaged DNA and has also been shown to bind to a broad range of other forms of damaged DNA, including DNA damaged by cisplatin, nitrogen mustard, psoralen, abasic sites, and ssDNA (44). Alternative roles of DDB2 (p48) protein include remodeling chromatin at sites of DNA damage to facilitate global genomic repair (43) and regulation of cell cycle arrest at G1-S checkpoint (42).

However, when the burden of genomic insult overwhelms the DNA repair mechanism, cells initiate programmed cell death (apoptosis; ref. 32). DSB are considered the most important type of lesion for the cytotoxic effects of DNA reactive agents; these lesions are difficult to repair, and levels correlate closely with cell killing (24). Figures. 2B and 3B, representing detection of 5F 203–dependent SSBs and DSBs, respectively, illustrate distinct dose- and time-dependent profiles. The appearance of sub-G1, apoptotic MCF-7 populations (1 and 10 μmol/L, >48 hours; Fig. 4) correlates with formation of DSB. Microarray analyses (34), demonstrating induction of the apoptosis-initiating receptor CD95/FAS(TNFRSF6), and simultaneous repression of Myc and Bcl-2 (35) in MCF-7 cells corroborate mediation of cell killing via induction of apoptosis.

Thus, the biological consequences of DNA adduct formation are closely dependent on the severity of the damage in relation with their reparability (45). DSB sever the chromosomes and are highly lethal to cells unless repaired (32). DNA DSB can be formed directly by oxygen free radicals and ionizing radiation, or indirectly by topoisomerase II inhibitors (46, 47). The formation of DSB induced by 5F 203 may be a direct consequence of SSB conversion to DSB, which occurs if the replication machinery leaves an interrupted daughter strand when it encounters a SSB (48). In this case, the replication process will be interrupted and a rapid, vigorous cellular response would be evoked.

Our knowledge of the mechanism of action of Phortress is summarized in Fig. 1. In view of the CYP1A1-mediated generation of DNA damage, it may be argued that Phortress represents a P450-activated cytotoxic class of agent comparable to oxazaphosphorine anticancer agents. However, these prodrugs, which include ifosfamide, are activated by liver P450 enzymes and produce circulating toxic metabolites. Indeed, the appearance of 4-hydroxyifosfamide correlated with DNA damage determined by comet assays in peripheral blood lymphocytes 24 hours after the start of chemotherapy (49). In contrast, metabolic activation of 5F 203 occurs within specific tumors following benzothiazole-induced transcription of cyp1a1, resulting in the stark selectivity associated with the aminophenylbenzothiazole class of agent. DNA damage and perturbation of cell cycle occur in sensitive cells only. From this distinction, one may hope to encounter fewer systemic adverse effects, as the case against a pan cytotoxic is argued. Indeed, in silico SOM cluster analyses of the mechanisms of action,according to their cellular antitumor activities, mapped 5F 203 and DF 203 within the region for kinase/phosphatase regulation of cell cycle machinery (Fig. 8). Microarray data (34) consolidate this view, as 5F 203 and DF 203 perturb transcription of genes associated with regulation of the cell cycle and apoptosis, specifically in sensitive cells. Previously, the GI50 antitumor profile computer pattern recognition algorithm identified these agents as COMPARE negative (2). Once again, the unique mechanism of action of antitumor aminophenylbenzothiazoles is substantiated as 5F 203 and its nonfluorinated congener DF 203 were not classified with any class of agents currently available in the anticancer armory.

In the present study, it has been confirmed that exposure to benzothiazole compounds in vitro and in vivo leads to formation of DNA SSB and DSB in sensitive tumor cells. These DNA lesions, in turn, evoke manifold biological/cellular sequelae, including DNA damage recognition, cell cycle arrest, and apoptosis. That treatment-induced DNA damage reveals clinical relevance has been shown. Comet assays determined fewer strand breaks in response to ifosfamide and doxorubicin treatment in fine needle aspirates of breast cancer patients who subsequently relapsed (49).

We believe that Phortress represents a novel class of antitumor agent, which has the potential to exact selective antitumor activity in a significant minority of human cancers. Moreover, we conclude from work presented herein that by SCGE, Phortress-sensitive and -inherently resistant phenotypes may be distinguished.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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