To monitor the subcellular distribution of mixed epidermal growth factor (EGF) receptor (EGFR)–DNA targeting drugs termed combi-molecules, we designed AL237, a fluorescent prototype, to degrade into a green fluorescent DNA damaging species and FD105, a blue fluorescent EGFR inhibitor. Here we showed that AL237 damaged DNA in the 12.5 to 50 μmol/L range. Despite its size, it blocked EGFR phosphorylation in an enzyme assay (IC50 = 0.27 μmol/L) and in MDA-MB468 breast cancer cells in the same concentration range as for DNA damage. This translated into inhibition of extracellular signal-regulated kinase 1/2 or BAD phosphorylation and downregulation of DNA repair proteins (XRCC1, ERCC1). Having shown that AL237 was a balanced EGFR-DNA targeting molecule, it was used as an imaging probe to show that (a) green and blue colors were primarily colocalized in the perinuclear and partially in the nucleus in EGFR- or ErbB2-expressing cells, (b) the blue fluorescence associated with FD105, but not the green, was colocalized with anti-EGFR red-labeled antibody, (c) the green fluorescence of nuclei was significantly more intense in NIH 3T3 cells expressing EGFR or ErbB2 than in their wild-type counterparts (P < 0.05). Similarly, the growth inhibitory potency of AL237 was selectively stronger in the transfectants. In summary, the results suggest that AL237 diffuses into the cells and localizes abundantly in the perinuclear region and partially in the nucleus where it degrades into EGFR and DNA targeting species. This bystander-like effect translates into high levels of DNA damage in the nucleus. Sufficient quinazoline levels are released in the cells to block EGF-induced activation of downstream signaling. Mol Cancer Ther; 9(4); 869–82. ©2010 AACR.

The epidermal growth factor (EGF) receptor (EGFR) and its closest family member p185neu, the product of the HER2 gene, are transmembrane receptor tyrosine kinases (TK), which transduce signals associated with tumor cell proliferation (14). The variety of approaches currently used to target EGFR includes small monoclonal antibody strategy to block ligand binding and TK inhibitors (57). Over the past 10 years, molecules that inhibit receptor autophosphorylation and downstream intracellular signaling have been developed and have shown significant antitumor activity in vitro (812). Several of them including gefitinib (Iressa), erlotinib (Tarceva), and lapatinib (Tykerb/Tyverb) have been approved for clinical use (13, 14). However, none of these drugs are used in the clinic as single agents in the therapy of advanced cancers (15, 16). Despite their ability to block growth signaling associated with EGFR or p185neu, cancer cells have the ability to evade the growth inhibitory effect of these drugs by activating alternative signaling pathways. Moreover, these drugs are reversible inhibitors, indicating that the tumor cells may resume proliferation following drug clearance. Therefore, for an effective therapy, combination with a drug capable of killing the cell by a different mechanism (e.g., DNA damage or inhibition of DNA synthesis) is required. However, despite this overwhelming reality of cancer therapy, the development of mono-targeted drugs through high throughput screening or rational drug design remains the most generally adopted strategy. Over the past several years, we have developed a paradigm shifting strategy that seeks to design a single molecule with multiple functions termed combi-molecule (1722). These molecules, despite their combination-based design, were not developed with the purpose of eventually replacing the traditional chemotherapy but rather complementing it. Albeit, we showed that many prototypes (e.g., SMA41, FD137) showed stronger antiproliferative activity than classic combinations of drugs with the same mechanism of action (20, 2326).

As outlined in Fig. 1A, the combi-molecules (see I-Alk) were designed to bind to EGFR on their own and to decompose into another EGFR inhibitor (I) plus a DNA alkylating species (Alk). Previous studies from our laboratory have shown that indeed the combi-molecules (e.g., SMA41; Fig. 1A) could directly block EGFR in short exposure assay in vitro at room temperature in serum-containing media (18). Additionally, we showed that they are capable of blocking EGFR phosphorylation and significantly damaging DNA in human tumor cells in vitro and in vivo, indicating that our combi-molecules induce a bifunctional activity in whole cells (22, 27). Using the fluorescence of the generated inhibitor I (excitation, 294 nm; emission, 451 nm) and 14C-radiolabeled alkyl moiety of SMA41, we previously confirmed that the combi-molecule could indeed decompose in the intracellular compartment into an EGFR inhibitor (I) and a methyldiazonium species (Alk) that damages DNA (27, 28). Whereas the fluorescence property of the aminoquinazoline (I) permitted the observation of its subcellular distribution or localization, that of the short-lived [14C]methyldiazonium could not be imaged (28). Here we designed a novel probe, AL237, in which the fluorescent dansyl tag is attached to a 3-alkyl triazene moiety (Fig. 1B; I-Alk-Dansyl), which when hydrolysed will release a fluorescent alkylating agent (see Alk-Dansyl). Alkylation of DNA by this fluorescent alkylating molecule (see Fig. 1B) would lead to green nuclei (excitation, 340 nm; emission, ∼525 nm), and the release of the aminoquinazoline (I, FD105; excitation, 294 nm; emission, 451 nm) would generate blue areas in the cells. Thus, fluorescence microscopy would allow us to image the complete fragmentation of the combi-molecule and its colocalization with one of its targets, EGFR, using immunofluorescence. Here, we test these hypotheses with AL237 and correlate its biodistribution profile with its dual mechanism of action. For purpose of comparison, a nonconjugated dansylated alkyl agent, N-dansylaziridine, was used (see structure in Fig. 5C). The latter, as previously reported, can only alkylate nucleic acids (29). It is to be noted that this study does not seek to establish the growth inhibitory potency of AL237 but rather to show its binary EGFR-DNA targeting property and to be used as a probe to image the subcellular localization of the two bioactive degradation products responsible for its EGFR-DNA binary targeting mechanism.

Figure 1.

A, schematic representation of the combi-targeting concept. B, stepwise decomposition of AL237. I-Alk-Dansyl, to generate EGFR inhibitor (I, FD105) and dansylated alkylating DNA species (Alk-Dansyl). The entire AL237 molecule and the dansylated DNA damaging species both emit at 525 nm (green).

Figure 1.

A, schematic representation of the combi-targeting concept. B, stepwise decomposition of AL237. I-Alk-Dansyl, to generate EGFR inhibitor (I, FD105) and dansylated alkylating DNA species (Alk-Dansyl). The entire AL237 molecule and the dansylated DNA damaging species both emit at 525 nm (green).

Close modal

Cell culture

MDA-MB-468 human breast carcinomas were obtained from American Type Culture Collection. Mouse fibroblast cells NIH 3T3 used as control or NIH 3T3her14 (transfected with erbB1/EGFR gene) and NIH 3T3neu (transfected with erbB2 gene) were provided by Dr. Moulay Aloui-Jamali (Montreal Jewish General Hospital). All cells were maintained in DMEM supplemented with 10% fetal bovine serum, 10 mmol/L HEPES, 2 mmol/L l-gutamine, and antibiotics (all reagents were purchased from Wisent, Inc.) as previously described (18). Cells were maintained at exponential growth at 37°C in a humidified environment with 5% CO2. In all assays cells were plated 24 h before drug administration.

Drug treatment

AL237 and JDA41 were synthesized in our laboratory. The methods used for AL237 and JDA41 complete synthesis were described elsewhere (22, 30). N-dansylaziridine was purchased from Biomol, and Temozolomide and Iressa were purchased from the hospital pharmacy and extracted from pills in our laboratory. EGF was obtained from Roche Molecular Diagnostics. In all assays, drugs were dissolved in DMSO and subsequently diluted in phenol red/fetal bovine serum–free DMEM before added to cells. The concentration of DMSO never exceeded 0.2% (v/v) during treatment.

Growth inhibition assay

Cells were plated in 96-well flat-bottomed microtiter plates at 5,000 cells per well (NIH 3T3her14, NIH 3T3neu, MDA-MB-468) or 10,000 cells per well (NIH 3T3). After 24 h cells were exposed to different drug concentrations for 4 d. Briefly, following drug treatment, cells were fixed with 10% ice-cold trichloroacetic acid for 60 min at 4°C, stained with sulforhodamine B (0.4%) for 4 h at room temperature, rinsed with 1% acetic acid, and allowed to dry overnight (31). The sulforhodamine B absorbance was recorded at 492 nm using a Bio-Rad microplate reader. The results were analyzed by GraphPad Prism (GraphPad Software, Inc.), and the sigmoidal dose response curve was used to determine IC50. Each point represents the average of at least three independent experiments run in triplicate.

In vitro enzyme assay

The EGFR and src kinase assays are similar to one described previously (27). Briefly, the kinase reaction was done in 96-well plates using 4.5 ng/well EGFR or src (Biomol). Following drug addition (range, 0.0001–10 μmol/L), phosphorylation of the EGFR was initiated by supplementing the reaction with ATP. The phosphorylated substrate was detected using a horseradish peroxidase–conjugated anti-phospho-tyrosine antibody (Santa Cruz Biotechnology), and the colorimetric reaction was monitored at 450 nm using a Bio-Rad reader. The results were analyzed by GraphPad Prism, and IC50 was calculated.

Western blot analysis

Cells were grown to 80% confluence in six-well plates and serum starved for 24 h (serum-free DMEM), followed by a 2-h incubation with AL237 at the indicated concentrations. Cells were washed from the drug with PBS, and then cells were stimulated with EGF (50 ng/mL) for 15 min. Cells were collected and lysed in ice-cold protein extraction buffer for 30 min [20 mmol/L Tris-HCl (pH 7.5), 1% NP40, 10 mmol/L EDTA, 150 mmol/L NaCl, 20 mmol/L NaF, 1 mmol/L Na vanadate, complete protease inhibitor cocktail (Roche Molecular Diagnostics)]. Equal amounts of proteins were separated on 10% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were blocked with 5% milk in TBST (20 mmol/L Tris-HCl, 137 mmol/L NaCl, 0.1% Tween 20) for 3 h. Primary antibodies used for immunodetection were dissolved in antibody buffer [5 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% (v/v) Tween 20, 0.05% (w/v) Na azide, 0.25% (w/v) gelatin] or TBST buffer as follows: anti-phospho-tyrosine (clone 4G10, Upstate; 1:1,000), anti-EGFR (sc-03, Santa Cruz; 1:1,000), anti-phospho-EGFR (Tyr1068, 1:1,000), anti-XRCC1 (33-2-5, ThermoFisher Scientific; 1:1,000), anti-ERCC1 (clone 3H11, ThermoFisher Scientific; 1:1,000), and anti-phospho-γH2AX (1:1,000, Abcam). Anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2; Thr202/Tyr204; 1:4,000), anti-ERK1/2 (p44/p42 mitogen-activated protein kinase; 1:2,500), anti-phospho-BAD (Ser112; 1:1,000), anti-BAD (1:250) were obtained from Cell Signaling Technology. Anti-tubulin-α (clone DM1A, NeoMarkers; 1:2,000) was used as a loading control. Secondary horseradish peroxidase–conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories. The bands were visualized using enhanced chemiluminescence (Amersham Bioscience). Western blot experiments were done at least twice from two independent cell treatments.

Alkaline comet assay

Cells were exposed to AL237 or N-dansylaziridine (0, 6.25, 12.5, 25, 50, 100 μmol/L) for 2 h, and the alkaline comet assay was done as previously described (27, 32). During this procedure, cells were protected from direct light to minimize DNA damage. Comets were visualized at 10× magnification using Leica microscope after staining with SYBR Gold (1:10,000, Molecular Probes). DNA damage was quantified using Comet Assay IV software (Perceptive Instruments), and the degree of DNA damage was expressed as tail moments. A At minimum, 50 comets were analyzed for each cell treatment and the mean tail moments were calculated from three independent experiments.

Neutral comet assay

Cells were treated and collected as in the alkaline comet assay, embedded in agarose at the same cell density, and lysed with a neutral buffer for 2 h at room temperature [154 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.8), 10 mmol/L EDTA, 0.5% (v/v) N-lauroyl-sarcosine (pH 8.0)]. Gels were soaked for 30 min in neutral Tris-borate EDTA buffer [90 mmol/L Tris-HCl (pH 7.8), 90 mmol/L boric acid, 2 mmol/L EDTA] and electrophorezed for 20 min at 20 V (33, 34). Modification of the assay permitted to retain the fluorescent dansyl tag to the sites of DNA adducts and allowed to observe nuclei without using a specific DNA staining dye.

Intracellular fluorescence by UV flow cytometry

NIH 3T3, NIH 3T3her14, and NIH 3T3neu were plated at 0.5 × 106 cells per well in six-well plates, allowed to adhere overnight, and treated with AL237 (0, 6.25, 12.5, 25, 50, 100 μmol/L) for 45 min. Cells were collected, washed, and resuspended in 300 μL PBS supplemented with 1% fetal bovine serum to minimize cell clumping. Cellular fluorescence levels were measured using a Becton Dickinson LSR flow cytometer (BD Biosciences). Cells were excited with 340-nm light emitting laser, and the AL237 hydrolyzed fragments were detected as follows: the aminoquinazoline emitted at 451 nm (blue) and the dansylated DNA damaging species emitted at 525 nm (green). Fluorescence levels determined by fluorescence-activated cell sorting were analyzed with GraphPad Prism software, and the accumulated fluorescence for each cell line was expressed as percentage of mean fluorescence over control. Four independent experiments were done in duplicate.

Nuclear fluorescence UV flow cytometry

NIH 3T3, NIH 3T3her14, and NIH 3T3neu were plated at 0.5 × 106 cells per well in six-well plates, incubated overnight, and exposed for 45 min to AL237 or N-dansylaziridine (0, 6.25, 12.5, 25, 50, 100 μmol/L). After washing with PBS, cells were incubated in 300 μL of Vindelov solution for 15 min at 37°C [10 mmol/L Tris-HCl (pH 7.5); 10 mmol/L NaCl, 0.1% Nonidet P40 (v/v); 100 μg/mL; 50 units/mL RnaseA; ref. 35]. Nuclei were analyzed on a BD LSR flow cytometer as described earlier. A minimum of 10,000 cell nuclei were acquired per sample, and each drug concentration was done in duplicate. The fluorescence levels were quantified with CellQuest Pro software (Becton Dickinson), and results are reported as percentage fluorescence over control from three independent experiments.

Immunofluorescence of EGFR and phospho-tyrosine

MDA-MB-468 cells were plated at 60% confluence on a microcover glass (VWR) placed in a 24-well plate. Cells were starved overnight, followed by treatment with 25 μmol/L AL237 for 2 h, and then stimulated with 50 ng/mL EGF for 15 min. Subsequently, cells were washed twice with PBS, fixed with 100% ice-cold methanol at −20°C for 5 min, followed by 1 h blocking with 5% normal goat serum. Double immunostaining was done using directly-coupled mouse anti-phospho-tyrosine-FITC (1:100), mouse anti-EGFR-PE (1:100), or the appropriate IgG-PE– or IgG-FITC–conjugated controls (1:100, purchased from Santa Cruz Biotechnology, Inc.). Thereafter, cells were washed twice with PBS, stained with 5 ng/mL 4′,6-diamidino-2-phenylindole solution (Sigma), and mounted with a gel mounting media (Fisher Scientific). Immunofluorescence images were captured with Leica microscope (Leica) using the appropriate filters and analyzed with Leica application suite software.

Live cell fluorescence imaging of AL237 in NIH 3T3 cells

NIH 3T3, NIH 3T3her14, and NIH 3T3neu were plated at 70% confluence in six-well plates, allowed to adhere overnight, and treated with 25 μmol/L AL237 for 2 h. At the indicated time points, cells were washed with PBS twice and images were saved with Leica DFC300FX camera with the appropriate filters.

Immunofluorescence of phospho-γH2AX

MDA-MB-468 cells were plated on slides and incubated overnight, followed by a treatment with 25 and 50 μmol/L of AL237 for 2 h. Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 10 min, followed by 1 h blocking with 5% normal goat serum. Slides were incubated with mouse anti-phospho-γH2AX antibody (Abcam) for 3 h and antimouse FITC-labeled secondary antibody (Sigma) for 1 h. Typical for double-strand DNA breaks, phospho-γH2AX foci were observed on a fluorescent Leica microscope at 40×.

Annexin V/propidium iodide binding assay

MDA-MB-468 cells were plated in a six-well plate and treated with a dose range of each drug for 48 h. Thereafter, cells were harvested and incubated with Annexin V–FITC and propidium iodide (PI) using the apoptosis detection kit (BenderMedSystems, Inc.) following the protocol provided by the supplier. Annexin V–FITC and PI binding were analyzed with a Becton Dickinson FACScan. Data were collected using logarithmic amplification of both the FL1 (FITC) and FL2 (PI) channels. Quadrant analysis of coordinate dot plots was done with CellQuestPro software.

Analysis of binary EGFR-DNA targeting potentials

Inhibition of EGFR phosphorylation

For AL237, being a bulky molecule, we first determined whether the long spacer attached to the 6-position of the quinazoline ring affects its ability to inhibit receptor phosphorylation in purified EGFR enzyme assay. Our results showed that AL237 was capable of inhibiting EGFR phosphorylation with an IC50 of 0.27 μmol/L. Moreover, we have addressed AL237 selective EGFR binding by measuring inhibition of tyrosine phosphorylation on a src kinase. The result indicates that, despite its significant bulkiness, AL237 binds strongly to the ATP-binding site of EGFR and is a selective EGFR inhibitor (Fig. 2A). Indeed, varieties of similar compounds with bulky side chains have been synthesized in our laboratory and have been shown to retain strong EGFR binding affinity (23, 36, 37). It is also known that most anilinoquinazolines show unspecific binding to the ErbB2 gene product, which is the closest family member of EGFR (13). More importantly, to verify the ability of AL237 to block EGFR phosphorylation in whole cells, we analyzed phospho-EGFR levels (Tyr1068) in the two panels of EGFR-expressing cells. AL237 induced ∼100% inhibition of EGFR phosphorylation at 6.25 μmol/L in NIH 3T3 EGFR transfectant (Fig. 2B) and at 12.5 μmol/L in the MDA-MB-468 cells (Fig. 2C).

Figure 2.

AL237 inhibits EGFR phosphorylation and downstream signaling. A, inhibition of EGFR and src tyrosine phosphorylation by AL237 were measured by in vitro binding assay. Points, mean of three independent experiments run in duplicate; bars, SD. B, inhibition of EGFR phosphorylation by AL237 was analyzed in NIH 3T3 cells transfected with EGFR (NIH 3T3her14) by Western blot. Cellular proteins were isolated after exposure to increasing AL237 concentrations for 2 h, followed by EGF stimulation (50 ng/mL). C, MDA-MB-468 cellular proteins (50 μg) were analyzed for phospho-EGFR inhibition after treatment with the indicated doses of AL237 for 2 h, followed by EGF stimulation. Membranes were incubated with anti-phospho-EGFR (Tyr1068) antibody. D, MDA-MB-468 cells were starved overnight, treated with the indicated concentrations of AL237 for 2 h, and thereafter stimulated or not stimulated with EGF (50 ng/mL) for 15 min. Afterwards, cellular proteins were analyzed by Western blot for the effect of AL237 on phospho-ERK1/2 and phospho-BAD (Ser112) protein levels followed by incubation with anti-ERK1/2 and anti-BAD antibodies. Blots were also incubated with antibodies against ERCC1 and XRCC1 to detect DNA repair proteins, and anti-tubulin antibody was used to control for equal loading. Western blots were repeated twice, and similar results from two independent treatments were obtained.

Figure 2.

AL237 inhibits EGFR phosphorylation and downstream signaling. A, inhibition of EGFR and src tyrosine phosphorylation by AL237 were measured by in vitro binding assay. Points, mean of three independent experiments run in duplicate; bars, SD. B, inhibition of EGFR phosphorylation by AL237 was analyzed in NIH 3T3 cells transfected with EGFR (NIH 3T3her14) by Western blot. Cellular proteins were isolated after exposure to increasing AL237 concentrations for 2 h, followed by EGF stimulation (50 ng/mL). C, MDA-MB-468 cellular proteins (50 μg) were analyzed for phospho-EGFR inhibition after treatment with the indicated doses of AL237 for 2 h, followed by EGF stimulation. Membranes were incubated with anti-phospho-EGFR (Tyr1068) antibody. D, MDA-MB-468 cells were starved overnight, treated with the indicated concentrations of AL237 for 2 h, and thereafter stimulated or not stimulated with EGF (50 ng/mL) for 15 min. Afterwards, cellular proteins were analyzed by Western blot for the effect of AL237 on phospho-ERK1/2 and phospho-BAD (Ser112) protein levels followed by incubation with anti-ERK1/2 and anti-BAD antibodies. Blots were also incubated with antibodies against ERCC1 and XRCC1 to detect DNA repair proteins, and anti-tubulin antibody was used to control for equal loading. Western blots were repeated twice, and similar results from two independent treatments were obtained.

Close modal

Inhibition of downstream signaling

After analyzing the effect of AL237 on EGFR phosphorylation, we further studied its role on downstream signaling in MDA-MB-468 cells by Western blotting. Cells were treated with and without EGF to determine EGF-dependent EGFR inhibition by AL237 on downstream signaling. Inhibition of EGF-induced phosphorylation of EGFR was accompanied by reduced p44/p42 mitogen-activated protein kinases (ERK1/2) and BAD (Ser112) phosphorylation (Fig. 2D). EGF-induced signaling was also accompanied by a slight downregulation of XRCC1 and ERCC1, two DNA repair proteins involved in the repair of AL237-induced DNA damage (Fig. 2D).

Induction of DNA damage in MDA-MB-468 breast cancer cells

To test the DNA damaging potency of our combi-molecule and the ability to alkylate DNA, cells were treated with AL237 for 2 hours. DNA damage was assessed by the alkaline comet assay and quantified by measuring comet tail moment using Comet Assay IV software (Fig. 3A). Strong dose-dependent DNA damage was measured with increasing AL237 concentrations, and SYBR gold-stained nuclei with typical comet tail formation were imaged (Fig. 3A). To verify if the DNA-alkylated adducts formed by AL237 resulted in the formation of double-strand DNA breaks and the typical γH2AX foci indicative of the assembly of DNA repair protein complexes, we analyzed phospho-γH2AX activation by Western blotting and immunofluorescence. We observed a dose-dependent increase in γH2AX protein phosphorylation and the formation of 5 to 10 phospho-γH2AX foci per nucleus compared with untreated cells (Fig. 3B). This moderate increase in γH2AX phosphorylation reflects the inability of alkylating lesions to induce high levels of γH2AX accumulation or double-strand DNA breaks (38, 39). Overall, the observed strong DNA damaging potency of AL237 and its ability to block cell signaling associated with EGF stimulation confirmed that it behaved as a true combi-molecule. Having shown its dual targeting, the higher cytotoxicity induced by the combi-molecule was addressed by quantitating the levels of apoptosis after 48 hours.

Figure 3.

DNA damage induced by AL237 in MDA-MB-468. A, cells were exposed to AL237 for 2 h, followed by assessment of drug-induced DNA damage using an alkaline comet assay. Comet tail moments were quantitated by comet IV software (Perceptive Instruments). Columns, average comet tail moment calculated from 50 comets based on three independent experiments for each concentration (0, 6.25, 12.5, 25, 50, 100 mmol/L). Representative images of DNA comets stained with SYBR Gold dye and visualized by fluorescence microscopy at 10× were shown for each dose. B, extracts from cells treated for 2 h with increasing concentrations of AL237 were analyzed by Western blot with anti-phospho-γH2AX antibody. Membrane was probed with anti-tubulin antibody as a loading control. MDA-MB-468 cells were preincubated on a slide, treated with 0, 25, or 50 μmol/L of AL237, and analyzed for phospho-γH2AX foci. Cells were observed at Leica fluorescent microscope (40×) by indirect immunofluorescence using primary mouse anti-phospho-γH2AX antibodies, followed by FITC-labeled antimouse antibody. C, Annexin V/PI-stained MDA-MB-468 cells were analyzed by fluorescence-activated cell sorting 48 h after treatment with AL237 and N-dansylaziridine. D, fluorescence distribution of AL237-EGFR-binding aminoquinazoline species at 25 μmol/L was imaged over time in MDA-MB-468 cells (top). Fluorescence distribution of AL237 hydrolyzed degradation products were observed either alone (bottom left) or in the presence of equimolar concentrations (25 μmol/L) with a competitive EGFR binding nonfluorescent molecule JDA41 (bottom right) at 20 min.

Figure 3.

DNA damage induced by AL237 in MDA-MB-468. A, cells were exposed to AL237 for 2 h, followed by assessment of drug-induced DNA damage using an alkaline comet assay. Comet tail moments were quantitated by comet IV software (Perceptive Instruments). Columns, average comet tail moment calculated from 50 comets based on three independent experiments for each concentration (0, 6.25, 12.5, 25, 50, 100 mmol/L). Representative images of DNA comets stained with SYBR Gold dye and visualized by fluorescence microscopy at 10× were shown for each dose. B, extracts from cells treated for 2 h with increasing concentrations of AL237 were analyzed by Western blot with anti-phospho-γH2AX antibody. Membrane was probed with anti-tubulin antibody as a loading control. MDA-MB-468 cells were preincubated on a slide, treated with 0, 25, or 50 μmol/L of AL237, and analyzed for phospho-γH2AX foci. Cells were observed at Leica fluorescent microscope (40×) by indirect immunofluorescence using primary mouse anti-phospho-γH2AX antibodies, followed by FITC-labeled antimouse antibody. C, Annexin V/PI-stained MDA-MB-468 cells were analyzed by fluorescence-activated cell sorting 48 h after treatment with AL237 and N-dansylaziridine. D, fluorescence distribution of AL237-EGFR-binding aminoquinazoline species at 25 μmol/L was imaged over time in MDA-MB-468 cells (top). Fluorescence distribution of AL237 hydrolyzed degradation products were observed either alone (bottom left) or in the presence of equimolar concentrations (25 μmol/L) with a competitive EGFR binding nonfluorescent molecule JDA41 (bottom right) at 20 min.

Close modal

Induction of apoptosis in MDA-MB-468 breast cancer cells

Annexin V–FITC and PI staining were used to distinguish viable (PI−/FITC−), early apoptotic (PI−/FITC+), late apoptotic (PI+/FITC+), and necrotic (PI+/FITC−) cells after 48 hours of exposure to the combi-molecule. We observed a strong and a dose-dependent increase in apoptosis by AL237 at IC50 range reaching as high as 60% apoptotic cells 48 hours posttreatment (Fig. 3C). In contrast, much lower levels were observed when cells were exposed to 100 μmol/L Temozolomide (9%), 25 μmol/L N-dansylaziridine (12.5%), or combinations of 25 μmol/L Temozolomide and 25 μmol/L Iressa (35%; see Supplementary Fig. S1). Thus, the combined EGFR TK inhibition and DNA damaging properties of AL237 were sufficient to confer high levels of apoptosis in MDA-MB-468 cells.

Next, we used AL237 to determine its selective growth inhibition in EGFR/ErbB2-expressing cells and to image the release of its bioactive species in the intracellular compartment.

Imaging of AL237 in MDA-MB-468 human breast cancer cells and NIH 3T3 transfectants

Imaging of MDA-MB-468 cells treated with AL237

Fluorescence emission released by AL237 was analyzed in MDA-MB-468 cells at different time points using 25 μmol/L of the drug. The two hydrolyzed degradation products were detectable in the cells as early as 5 minutes after addition, reaching a maximum 20 minutes later (Fig. 3D, top). We observed the blue fluorescence associated with the aminoquinazoline FD105 (451 nm) and the green fluorescence with the DNA damaging dansylated moiety (525 nm) in the cytoplasm and in the vicinity of the nucleus. Whereas the entire molecule can still fluoresce in green, the detection of blue FD105 fragment is indicative of a degradation of the molecule, which as reported elsewhere has a half-life of 22 minutes (30). Whether the combi-molecule was partially or completely decomposed, the green fluorescence was consistently localized in the perinuclear area (Fig. 3D, bottom left). To challenge the EGFR-directed localization of the combi-molecule in the perinucalear region, we used JDA41 (IC50 = 0.081 mmol/L), a nonfluorescent EGFR TK inhibitor (22). The results showed that competitive exposure of AL237 (IC50 = 0.27 mmol/L) with JDA41 delocalized the latter into the cytoplasm, indicating that the perinuclear colocalization may be directed by EGFR binding (Fig. 3D, bottom right).

Qualitative analyses of isogenic NIH 3T3 EGFR/ErbB2 transfectants

AL237-released green and blue fluorescence were also observed in an isogenic context using NIH 3T3, NIH 3T3her14 (transfected with EGFR), and NIH 3T3neu (transfected with erbB2). As we have shown on the merged images in MDA-MB-468 cells (Fig. 4A, top right), in NIH 3T3 cells, the blue and green fluorescence produced by AL237 or its decomposition products were similarly colocalized in the perinuclear area or in the vicinity of the nucleus (Fig. 4B). Interestingly, in the isogenic cells, AL237 or its degradation products were colocalized, with a more pronounced perinuclear distribution in EGFR- and ErbB2-expressing cells than in their wild-type counterpart. Moreover, quantitative flow cytometric analysis of NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells further confirmed at a single-cell level that AL237 decomposed in the cells and released its two fluorescent degradation products: blue fluorescence corresponding to the aminoquinazoline moiety (Fig. 4C, top graph) and green fluorescence due to the entire molecule and/or the released dansylated DNA damaging species (Fig. 4C, bottom graph).

Figure 4.

Cellular fluorescence of AL237 and EGFR in MDA-MB-468, NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells. A, AL237 was hydrolyzed to an aminoquinazoline fragment that emitted at 451 nm (blue), whereas the entire AL237 molecule and the dansylated DNA damaging species both emitted at 525 nm (green). Direct drug fluorescence was analyzed in MDA-MB-468 cells after 2 h of drug treatment with 25 μmol/L AL237 (top). Each fluorescent fragment was detected using individual filters on Leica fluorescent microscope (40× magnification), and the overlaid image was generated to observe colocalization of the two hydrolyzed AL237 fragments (top right). Following drug treatment, MDA-MB-468 cells were immunostained with EGFR PE-labeled antibody (red, bottom left). Merged images of AL237 (blue, EGFR inhibitor fragment) with EGFR (red) were assembled to outline the area of colocalization (magenta), and an enlarged cell is represented (bottom right). The DNA-targeting fragment of AL237 (green) was also overlaid with EGFR to observe the level of colocalization at the perinuclear and nuclear regions. B, NIH 3T3 and NIH 3T3her14 live cells were imaged to observe biodistribution of the molecule in EGFR-expressing and non–EGFR-expressing cells. This qualitative analysis was complemented by a quantitative fluorescence measurement of each AL237 species at a single-cell level. C, NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells were exposed to the drug for 45 min and washed with PBS, and thereafter, each fragment fluorescence was measured by flow cytometry: The accumulated drug-associated fluorescence was measured from 10,000 cells and then normalized over the background cell fluorescence in the control. Blue fluorescence of the aminoquinazoline species (top) and green fluorescence of the dansylated alkyldiazonium fragment (bottom) were quantified for each cell type. Each point is the average value of three independent experiments done for each cell line in duplicate at each drug concentration. D, MDA-MB-468 cells were treated with 25 μmol/L AL237 for 2 h, followed by EGF stimulation, methanol fixed, and directly stained with anti-EGFR-PE and anti-phospho-tyrosine-FITC antibodies. 4′,6-Diamidino-2-phenylindole stain was used to define nuclei (top and middle), but it was omitted from the samples that were drug treated because 4′,6-diamidino-2-phenylindole–stained nuclei also emitted strong green fluorescence, which interfered with AL237 fluorescence (bottom).

Figure 4.

Cellular fluorescence of AL237 and EGFR in MDA-MB-468, NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells. A, AL237 was hydrolyzed to an aminoquinazoline fragment that emitted at 451 nm (blue), whereas the entire AL237 molecule and the dansylated DNA damaging species both emitted at 525 nm (green). Direct drug fluorescence was analyzed in MDA-MB-468 cells after 2 h of drug treatment with 25 μmol/L AL237 (top). Each fluorescent fragment was detected using individual filters on Leica fluorescent microscope (40× magnification), and the overlaid image was generated to observe colocalization of the two hydrolyzed AL237 fragments (top right). Following drug treatment, MDA-MB-468 cells were immunostained with EGFR PE-labeled antibody (red, bottom left). Merged images of AL237 (blue, EGFR inhibitor fragment) with EGFR (red) were assembled to outline the area of colocalization (magenta), and an enlarged cell is represented (bottom right). The DNA-targeting fragment of AL237 (green) was also overlaid with EGFR to observe the level of colocalization at the perinuclear and nuclear regions. B, NIH 3T3 and NIH 3T3her14 live cells were imaged to observe biodistribution of the molecule in EGFR-expressing and non–EGFR-expressing cells. This qualitative analysis was complemented by a quantitative fluorescence measurement of each AL237 species at a single-cell level. C, NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells were exposed to the drug for 45 min and washed with PBS, and thereafter, each fragment fluorescence was measured by flow cytometry: The accumulated drug-associated fluorescence was measured from 10,000 cells and then normalized over the background cell fluorescence in the control. Blue fluorescence of the aminoquinazoline species (top) and green fluorescence of the dansylated alkyldiazonium fragment (bottom) were quantified for each cell type. Each point is the average value of three independent experiments done for each cell line in duplicate at each drug concentration. D, MDA-MB-468 cells were treated with 25 μmol/L AL237 for 2 h, followed by EGF stimulation, methanol fixed, and directly stained with anti-EGFR-PE and anti-phospho-tyrosine-FITC antibodies. 4′,6-Diamidino-2-phenylindole stain was used to define nuclei (top and middle), but it was omitted from the samples that were drug treated because 4′,6-diamidino-2-phenylindole–stained nuclei also emitted strong green fluorescence, which interfered with AL237 fluorescence (bottom).

Close modal

Colocalization of EGFR and AL237 in MDA-MB-468 cells

To verify whether AL237 binds to EGFR, we used direct immunofluorescence by staining MDA-MB-468 cells with PE-labeled EGFR and FITC-labeled phospho-tyrosine antibodies. After EGF stimulation, we observed activated EGFR at the plasma membrane (Fig. 4D, middle), which was strongly inhibited after the cells were exposed for 2 hours to 25 μmol/L of AL237, a dose at which EGFR phosphorylation was also shown to be significantly depleted using Western blot analysis (see Fig. 2B and C). Whereas in EGF-stimulated cells, the EGFR showed a more membrane localization, in AL237-treated cells, it was redistributed in the cytoplasm in endosome-like structures primarily concentrated around the perinuclear region (Fig. 4A and D, bottom right). The localization of the fluorescence is in agreement with the ability of the released blue aminoquinazoline (see Fig. 1) to bind to EGFR TK and also with that of the green dansylated alkylating species to alkylate DNA.

Selective nuclear localization and growth inhibition

Quantitative and qualitative nuclear analysis

When total subcellular fluorescence was analyzed in the isogenic cells, although the trend was toward greater fluorescence in cells transfected with EGFR and its closest homologue ErbB2, the differences in fluorescence intensity were not statistically significant when compared with the wild type (P > 0.05; Fig. 4C). However, we believed that if the primary localization of AL237 in the perinuclear region was partially due to its binding to EGFR or related proteins, concomitantly released dansylated alkylating species that covalently bind to DNA might induce high levels of green fluorescence in the nuclei of these cells. Hence, we attempted to detect the levels of green fluorescence directly bound to DNA using a neutral comet assay and flow cytometric analysis of nuclei isolated by the Vindelov method (35). Under neutral conditions, we expected the alkylated dansyl species to remain bound to nuclei, thereby allowing direct imaging of the adducted DNA. For purpose of comparison, N-dansylaziridine, a dansylated alkylating agent deprived of the quinazoline moiety required for binding to EGFR, was used. As depicted in Fig. 5, strong green fluorescence intensity was observed from nuclei of cells treated with AL237 (Fig. 5A, left), with higher intensity in EGFR and ErbB2-transfected cells. Intensities were lower with N-dansylaziridine and not selectively stronger in the transfectants (Fig. 5A, right). Quantitative flow cytometric analysis confirmed that AL237 released significantly higher levels of fluorescence in the NIH 3T3her14 and NIH 3T3neu nuclei than in their NIH 3T3 wild-type counterpart (Fig. 5B). Two- to 3-fold differences in green fluorescence intensity were observed between NIH 3T3 and ErbB2 or EGFR. Statistical analysis was done with a two-tailed unpaired t test between NIH 3T3 and ErbB2 (at 50 μmol/L, P = 0.0256; 100 μmol/L, P = 0.0175) and between NIH 3T3 and EGFR (at 50 μmol/L, P = 0.0281; 100 μmol/L, P = 0.0121). Similarly, higher blue fluorescence intensity in the nuclei of the transfectants was observed and the difference when compared with the wild-type cells was statistically significant at the highest dose (100 μmol/L, P = 0.0287 and P = 0.0234). Importantly, no selective green fluorescence distribution was observed in nuclei from isogenic cells treated with N-dansylaziridine (Fig. 5C) that does not contain an EGFR targeting moiety. This is an indirect evidence supporting the implication of EGFR in the selective nuclear accumulation of AL237.

Figure 5.

Nuclear accumulation of AL237 and N-dansylaziridine in NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells. A, cells were treated with 100 μmol/L AL237 or N-dansylaziridine for 2 h. After cells were lysed and electrophoresed under neutral conditions, the nuclei of all three cell lines were analyzed by fluorescence microscopy (10× magnification) for the accumulation of either the dansylated fragment of AL237 (left) or N-dansylaziridine control (right). B, flow cytometry analysis of NIH 3T3 cells and the transfectants treated with five doses of AL237 for 45 min. Cells were washed twice with PBS and then lysed with Vindelov's solution for single nuclei isolation. C, schematic of N-dansylaziridine used as a control and flow cytometry analysis for its nuclear accumulation in NIH 3T3 cells and the transfectants. A minimum of 10,000–cell nuclei fluorescence was measured and represented as mean fluorescence normalized over the control. AL237 green fluorescence and quinazoline-derived blue fluorescence (B) and N-dansylaziridine fluorescence (C) were calculated as mean ± SD determined from four independent experiments done in duplicates for each drug concentration. Statistical analysis was done with a two-tailed unpaired t test; statistical significance at P < 0.05 [between NIH 3T3 and NIH 3T3neu at 50 μmol/L (P = 0.0256), 100 μmol/L (P = 0.0175); between NIH 3T3 and NIH 3T3her14 at 50 μmol/L (P = 0.0281), 100 μmol/L (P = 0.0121)] for green fluorescence. Statistical analysis for blue fluorescence intensity was significant at 100 μmol/L dose (P = 0.0287 and P = 0.0234).

Figure 5.

Nuclear accumulation of AL237 and N-dansylaziridine in NIH 3T3, NIH 3T3her14, and NIH 3T3neu cells. A, cells were treated with 100 μmol/L AL237 or N-dansylaziridine for 2 h. After cells were lysed and electrophoresed under neutral conditions, the nuclei of all three cell lines were analyzed by fluorescence microscopy (10× magnification) for the accumulation of either the dansylated fragment of AL237 (left) or N-dansylaziridine control (right). B, flow cytometry analysis of NIH 3T3 cells and the transfectants treated with five doses of AL237 for 45 min. Cells were washed twice with PBS and then lysed with Vindelov's solution for single nuclei isolation. C, schematic of N-dansylaziridine used as a control and flow cytometry analysis for its nuclear accumulation in NIH 3T3 cells and the transfectants. A minimum of 10,000–cell nuclei fluorescence was measured and represented as mean fluorescence normalized over the control. AL237 green fluorescence and quinazoline-derived blue fluorescence (B) and N-dansylaziridine fluorescence (C) were calculated as mean ± SD determined from four independent experiments done in duplicates for each drug concentration. Statistical analysis was done with a two-tailed unpaired t test; statistical significance at P < 0.05 [between NIH 3T3 and NIH 3T3neu at 50 μmol/L (P = 0.0256), 100 μmol/L (P = 0.0175); between NIH 3T3 and NIH 3T3her14 at 50 μmol/L (P = 0.0281), 100 μmol/L (P = 0.0121)] for green fluorescence. Statistical analysis for blue fluorescence intensity was significant at 100 μmol/L dose (P = 0.0287 and P = 0.0234).

Close modal

Selective growth inhibition of EGFR-expressing MDA-MB-468 and NIH 3T3 cells

To determine whether the binary EGFR/DNA targeting property of AL237 that showed selective biodistribution in EGFR/ErbB2 transfectants would translate into increased growth inhibitory potency on EGFR-expressing cells, we tested its growth inhibitory effect on MDA-MB-468 and NIH 3T3 transfectants (Fig. 6). AL237 showed 5-fold stronger inhibition on EGFR- and ErbB2-expressing cells than in control NIH 3T3 cells (P < 0.05, unpaired t test; Fig. 6A). AL237 also induced strong growth inhibition in the MDA-MB-468 cells that overexpress EGFR (Fig. 6B).

Figure 6.

Selective growth inhibition by AL237 in NIH 3T3 and MDA-MB-468 cells. IC50 values of AL237 in NIH 3T3 wild type and transfectants (A) and in MDA-MB-468 cells (B) were determined by sulforhodamine B assay, and values were averaged from three independent experiments ran in triplicates.

Figure 6.

Selective growth inhibition by AL237 in NIH 3T3 and MDA-MB-468 cells. IC50 values of AL237 in NIH 3T3 wild type and transfectants (A) and in MDA-MB-468 cells (B) were determined by sulforhodamine B assay, and values were averaged from three independent experiments ran in triplicates.

Close modal

The combi-molecules are a novel type of structures designed to block divergent targets in tumor cells. The growth of refractory tumors is driven by multiple signaling disorders that often cannot be blocked by the use of a single drug. The combi-molecule approach is the first that seeks to create molecules capable of blocking at least two divergent targets in the cells by allowing the intact molecules to block one target on their own and to degrade into other species directed at the same or different targets (18, 20, 22, 23, 26, 40, 41). To gain insight into the subcellular distribution and degradation of combi-molecules, we designed a new prototype termed AL237 to (a) be fluorescent on its own and (b) degrade under physiologic conditions to FD105 (an EGFR inhibitor) that fluoresces in the blue and a DNA alkylating fragment that fluoresces in the green (see Fig. 1). Thus, this was designed to not only allow us to visualize the release of the EGFR inhibitor in the cells but also to image the concomitantly generated DNA damaging species. Because the green fluorescence released from the dansyl moiety attached to the intact combi-molecule is undistinguishable from that emitted by the alkylating dansyl species, the colocalization of blue and green fluorescence suggests that both the intact molecule and its dissociated DNA alkylating dansylated species may be released concomitantly and primarily in the same locations. The high intensity of the green fluorescence in the perinuclear area indicates that the combi-molecules might be primarily localized and decompose to release both the aminoquinazoline inhibitor of EGFR FD105 and the DNA damaging species therein. A fraction of the combi-molecule might also decompose in the nucleus, because blue fluorescence was also detected therein. The fact that good colocalization was seen between the blue fluorescence of FD105 and red fluorescence associated with anti-EGFR antibody lends support to the ability of the released blue FD105 to bind to EGFR. Correspondingly, the poor colocalization observed for the green fluorescence associated with the dansylated alkylating DNA damaging agent with EGFR was in agreement with the inability of the latter species to bind to EGFR but rather to DNA in the nuclei.

Previous work with the 14C-methyl labeled combi-molecules showed preferential perinuclear distribution of SMA42, an analogue of FD105, released from the model combi-molecule SMA41 (24). The corresponding 14C-labeled alkyl group was bound to all three major macromolecules of the cells (DNA, RNA, and protein; ref. 27). Here, the fluorescence-labeled alkyl group presents the advantage of being observable by fluorescence microscopy and being quantified by fluorescence intensity per cell or nucleus by flow cytometry. The results obtained from immunofluorescence and flow cytometric analyses indicate that the combi-molecule and its derived species, despite being abundantly distributed in the perinuclear or partially in the nuclear region, is available at high enough concentrations to block EGFR TK activity at the level of the plasma membrane, downregulate the mitogen-activated protein kinase pathway, and prevent downstream induction of DNA repair genes, such as the XRCC1 and ERCC1. Thus, it suggests that the combi-molecules simultaneously damages DNA and impairs its repair mechanism and also the phosphorylation of the proapoptotic protein BAD. Unfortunately, we were not able to image the membrane localization of AL237, perhaps due to insufficiently high population of bound combi-molecules in the latter area. However, the fact that phosphorylation of EGFR was strongly inhibited by AL237 is an indirect evidence of the presence of a fraction of the intact molecule or its derived quinazoline at the level of the membrane. It should be noted that, whereas the data suggest that EGFR overexpression is associated with elevated nucleus binding of the dansylated DNA moiety, the compound through its DNA damaging moiety is capable of damaging tumor cells that do not express EGFR although to a lesser extent as exemplified by the NIH 3T3 wild type. This suggests that subpopulation of non–EGFR-expressing cells present in heterogeneous tumors may also be killed by the combi-moecule through its DNA damaging arm.

Importantly, we showed that the dansyl moiety is strongly bound to the nucleus, which is in agreement with the high levels of DNA damage observed by the comet assay and a slight increase in γH2AX phosphorylation and foci formation. The rather moderate increase in γH2AX phosphorylation is due to the inability of alkylating lesions to induce significant levels of double-strand DNA breaks. In term of nuclear staining, the most striking observation was the significant difference in fluorescence intensity observed between the NIH 3T3 transfectants and their wild-type counterpart. This is consistent with our previous observation of selectively high levels of DNA damage induced by the combi-molecule SMA41 in NIH 3T3 cells transfected with EGFR when compared with its wild type (42). A similar observation was made in MDA-MB-435 cells transfected with EGFR or ErbB2 (43). In this study, nuclei of cells transfected with EGFR or its closest homologue ErbB2 emitted higher green and blue fluorescence intensity than those of NIH 3T3 wild type. This can be rationalized in light of the high levels of EGFR observed in the perinuclear region. Perhaps EGFR and related proteins localized in the perinuclear region serve as anchorage from which the free dansyl alkyldiazonium moiety (see Fig. 1) can diffuse toward genomic DNA. Indeed, many reports not only described perinuclear distribution of EGFR but also its nuclear translocation (4448). Recent studies by Dittmann et al. (48) showed that EGFR translocates to the nucleus in response to radiation-induced DNA lesions, and more importantly, the nuclear EGFR is shown to be involved in DNA repair. Our observations that N-dansylaziridine, which does not contain a quinazoline EGFR targeting moiety, does not emit higher fluorescence intensity in the transfectants is an indirect evidence of the implication of EGFR and related proteins in the selectivity of nuclear staining by AL237.

This study conclusively showed that the combi-molecule AL237 is indeed an agent that (a) penetrates the cells and primarily localizes in the perinuclear region, (b) releases species that block signaling associated with EGFR activation, (c) damages DNA, and (d) significantly inhibits tumor cell growth. Thus, it behaved as a valid agent to image the distribution of not only the EGFR inhibitory but also the DNA binding species.

No potential conflicts of interest were disclosed.

We thank National Cancer Institute of Canada and Canadian Institutes of Cancer Research for financial support and MUHC Research Institute for an equipment grant that supported the acquisition of our Leica fluorescence microscope.

Grant Support: National Cancer Institute of Canada grant 018475, Canadian Institutes of Cancer Research grant FRN 49440, and Fonds de la Recherche en Santé du Québec doctoral award (M. Todorova). M. Todorova is supported by Fonds de la Recherche en Santé du Québec doctoral training award.

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.

1
Slamon
DJ
,
Clark
GM
,
Wong
SG
, et al
. 
Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene
.
Science (Washington, DC)
1987
;
235
:
177
82
.
2
Aaronson
SA
. 
Growth factors and cancer
.
Science
1991
;
254
:
1146
53
.
3
Modjtahedi
H
,
Dean
C
. 
The receptor for EGF and its ligands: expression, prognostic value and target for therapy in cancer (review)
.
Int J Oncol
1994
;
4
:
277
96
.
4
Normanno
N
,
De Luca
A
,
Bianco
C
, et al
. 
Epidermal growth factor receptor (EGFR) signaling in cancer
.
Gene
2006
;
366
:
2
16
.
5
Mendelsohn
J
. 
Epidermal growth factor receptor inhibition by a monoclonal antibody as anticancer therapy
.
Clin Cancer Res
1997
;
3
:
2703
7
.
6
Ciardiello
F
,
Tortora
G
. 
A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor
.
Clin Cancer Res
2001
;
7
:
2958
70
.
7
Albanell
J
,
Gascon
P
. 
Small molecules with EGFR-TK inhibitor activity
.
Current Drug Targets
2005
;
6
:
259
74
.
8
Bos
M
,
Mendelsohn
J
,
Kim
YM
, et al
. 
PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number-dependent manner
.
Clin Cancer Res
1997
;
3
:
2099
106
.
9
Moyer
JD
,
Barbacci
EG
,
Iwata
KK
, et al
. 
Induction of apoptosis and cell cycle arrest by CP-358774, an inhibitor of epidermal growth factor receptor tyrosine kinase
.
Cancer Res
1997
;
57
:
4838
48
.
10
Barker
AJ
,
Gibson
KH
,
Grundy
W
, et al
. 
Studies leading to the identification of ZD1839 (Iressa): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer
.
Bioorg Med Chem Lett
2001
;
11
:
1911
4
.
11
Rusnak
DW
,
Lackey
K
,
Affleck
K
, et al
. 
The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo
.
Mol Cancer Ther
2001
;
1
:
85
94
.
12
Normanno
N
,
Bianco
C
,
De Luca
A
,
Maiello
MR
,
Salomon
DS
. 
Target-based agents against ErbB receptors and their ligands: a novel approach to cancer treatment
.
Endocr Relat Cancer
2003
;
10
:
1
21
.
13
Wissner
A
,
Brawner Floyd
M
,
Rabindran
SK
, et al
. 
Syntheses and EGFR and HER-2 kinase inhibitory activities of 4-anilinoquinoline-3-carbonitriles: analogues of three important 4-anilinoquinazolines currently undergoing clinical evaluation as therapeutic antitumor agents
.
Bioorg Med Chem Lett
2002
;
12
:
2893
7
.
14
Moy
B
,
Goss
PE
. 
Lapatinib: current status and future directions in breast cancer
.
Oncologist
2006
;
11
:
1047
57
.
15
Huang
S
,
Armstrong
EA
,
Benavente
S
,
Chinnaiyan
P
,
Harari
PM
. 
Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor
.
Cancer Res
2004
;
64
:
5355
62
.
16
Baumann
M
,
Krause
M
,
Dikomey
E
, et al
. 
EGFR-targeted anti-cancer drugs in radiotherapy: preclinical evaluation of mechanisms
.
Radiother Oncol
2007
;
83
:
238
48
.
17
Katsoulas
A
,
Rachid
Z
,
McNamee
JP
,
Williams
C
,
Jean-Claude
BJ
. 
Combi-targeting concept: an optimized single-molecule dual-targeting model for the treatment of chronic myelogenous leukemia
.
Mol Cancer Ther
2008
;
7
:
1033
43
.
18
Matheson
SL
,
McNamee
J
,
Jean-Claude
BJ
. 
Design of a chimeric 3-methyl-1,2,3-triazene with mixed receptor tyrosine kinase and DNA damaging properties: a novel tumor targeting strategy
.
J Pharmacol Exp Ther
2001
;
296
:
832
40
.
19
Brahimi
F
,
Matheson
SL
,
Dudouit
F
, et al
. 
Inhibition of epidermal growth factor receptor-mediated signaling by “combi-triazene” BJ2000, a new probe for combi-targeting postulates
.
J Pharmacol Exp Ther
2002
;
303
:
238
46
.
20
Qiu
Q
,
Dudouit
F
,
Matheson
SL
, et al
. 
The combi-targeting concept: a novel 3,3-disubstituted nitrosourea with EGFR tyrosine kinase inhibitory properties
.
Cancer Chemother Pharmacol
2003
;
51
:
1
10
.
21
Qiu
Q
,
Domarkas
J
,
Banerjee
R
, et al
. 
Type II combi-molecules: design and binary targeting properties of the novel triazolinium-containing molecules JDD36 and JDE05
.
Anticancer Drugs
2007
;
18
:
171
7
.
22
Qiu
Q
,
Domarkas
J
,
Banerjee
R
, et al
. 
The combi-targeting concept: in vitro and in vivo fragmentation of a stable combi-nitrosourea engineered to interact with the epidermal growth factor receptor while remaining DNA reactive
.
Clin Cancer Res
2007
;
13
:
331
40
.
23
Banerjee
R
,
Rachid
Z
,
McNamee
J
,
Jean-Claude
BJ
. 
Synthesis of a prodrug designed to release multiple inhibitors of the epidermal growth factor receptor tyrosine kinase and an alkylating agent: a novel tumor targeting concept
.
J Med Chem
2003
;
46
:
5546
51
.
24
Matheson
SL
,
Brahimi
F
,
Jean-Claude
BJ
. 
The combi-targeting concept: intracellular fragmentation of the binary epidermal growth factor (EGFR)/DNA targeting “combi-triazene” SMA41
.
Biochem Pharmacol
2004
;
67
:
1131
8
.
25
Qiu
Q
,
Dudouit
F
,
Banerjee
R
,
McNamee
JP
,
Jean-Claude
BJ
. 
Inhibition of cell signaling by the combi-nitrosourea FD137 in the androgen independent DU145 prostate cancer cell line
.
Prostate
2004
;
59
:
13
21
.
26
Domarkas
J
,
Dudouit
F
,
Williams
C
, et al
. 
The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR)
.
J Med Chem
2006
;
49
:
3544
52
.
27
Matheson
SL
,
McNamee
JP
,
Wang
T
, et al
. 
The combi-targeting concept: dissection of the binary mechanism of action of the combi-triazene SMA41 in vitro and antitumor activity in vivo
.
J Pharmacol Exp Ther
2004
;
311
:
1163
70
.
28
Matheson
SL
,
Mzengeza
S
,
Jean-Claude
BJ
. 
Synthesis of 1-[4-(m-tolylamino)-6-quinazolinyl]-3-[14C]methyltriazene: a radiolabeled probe for the combi-targeting concept
.
J Labelled Comp Rad
2003
;
46
:
729
35
.
29
Broo
K
,
Wei
J
,
Marshall
D
, et al
. 
Viral capsid mobility: a dynamic conduit for inactivation
.
Proc Natl Acad Sci U S A
2001
;
98
:
2274
7
.
30
Larroque-Lombard
AL
,
Todorova
M
,
Golabi
N
,
Williams
C
,
Jean-Claude
B
. 
Synthesis and differential uptake of fluorescence-labeled combi-molecule bu P-gp-preficient and defficient uterine sarcoma cells MES-SA and MES-SA/DX5
.
J Med Chem
2009
,
submitted
.
31
Skehan
P
,
Storeng
R
,
Scudiero
D
, et al
. 
New colorimetric cytotoxicity assay for anticancer-drug screening
.
J Natl Cancer Inst
1990
;
82
:
1107
12
.
32
McNamee
JP
,
McLean
JR
,
Ferrarotto
CL
,
Bellier
PV
. 
Comet assay: rapid processing of multiple samples
.
Mutat Res
2000
;
466
:
63
9
.
33
Olive
PL
,
Banath
JP
. 
The comet assay: a method to measure DNA damage in individual cells
.
Nat Protocols
2006
;
1
:
23
9
.
34
Wojewodzka
M
,
Buraczewska
I
,
Kruszewski
M
. 
A modified neutral comet assay: elimination of lysis at high temperature and validation of the assay with anti-single-stranded DNA antibody
.
Mutat Res
2002
;
518
:
9
20
.
35
Vindelov
LL
. 
Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei
.
Virchows Arch B Cell Pathol
1977
;
24
:
227
42
.
36
Rachid
Z
,
Brahimi
F
,
Qiu
Q
, et al
. 
Novel nitrogen mustard-armed combi-molecules for the selective targeting of epidermal growth factor receptor overexperessing solid tumors: discovery of an unusual structure-activity relationship
.
J Med Chem
2007
;
50
:
2605
8
.
37
Larroque
A-L
,
Peori
B
,
Williams
C
, et al
. 
Synthesis of water soluble bis-triazenoquinazolines: an unusual predicted mode of binding to the epidermal growth factor receptor tyrosine kinase
.
Chem Biol Drug Des
2008
;
71
:
374
9
.
38
Huang
X
,
Halicka
HD
,
Darzynkiewicz
Z
. 
Detection of histone H2AX phosphorylation on Ser-139 as an indicator of DNA damage (DNA double-strand breaks)
.
Curr Protoc Cytom
2004
;
Chapter 7
:
Unit 7 27
.
39
Kinner
A
,
Wu
W
,
Staudt
C
,
Iliakis
G
. 
γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin
.
Nucleic Acids Res
2008
;
36
:
5678
94
.
40
Rachid
Z
,
Brahimi
F
,
Katsoulas
A
,
Teoh
N
,
Jean-Claude
BJ
. 
The combi-targeting concept: chemical dissection of the dual targeting properties of a series of “combi-triazenes”
.
J Med Chem
2003
;
46
:
4313
21
.
41
Brahimi
F
,
Rachid
Z
,
Qiu
Q
, et al
. 
Multiple mechanisms of action of ZR2002 in human breast cancer cells: a novel combi-molecule designed to block signaling mediated by the ERB family of oncogenes and to damage genomic DNA
.
Int J Cancer
2004
;
112
:
484
91
.
42
Matheson
SL
,
McNamee
JP
,
Jean-Claude
BJ
. 
Differential responses of EGFR-/AGT-expressing cells to the “combi-triazene” SMA41
.
Cancer Chemoth Pharm
2003
;
51
:
11
20
.
43
Banerjee
R
,
McNamee
JP
,
Jean-Claude
BJ
. 
The combi-targeting concept: selective targeting of the epidermal growth factor receptor (EGFR)- and Her2-expressing cancer cells by the complex combi-molecule RB24 (NSC 741279)
.
J Pharmacol Exp Ther
2009
,
Acceptable pending revision
.
44
Lin
S-Y
,
Makino
K
,
Xia
W
, et al
. 
Nuclear localization of EGF receptor and its potential new role as a transcription factor
.
Nature Cell Biology
2001
;
3
:
802
8
.
45
Lo
HW
,
Xia
W
,
Wei
Y
, et al
. 
Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer
.
Cancer Res
2005
;
65
:
338
48
.
46
Lo
HW
,
Hsu
SC
,
Hung
MC
. 
EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization
.
Breast Cancer Res Treat
2006
;
95
:
211
8
.
47
Kim
J
,
Jahng
WJ
,
Di Vizio
D
, et al
. 
The phosphoinositide kinase PIKfyve mediates epidermal growth factor receptor trafficking to the nucleus
.
Cancer Res
2007
;
67
:
9229
37
.
48
Dittmann
K
,
Mayer
C
,
Kehlbach
R
,
Rothmund
MC
,
Peter Rodemann
H
. 
Radiation-induced lipid peroxidation activates src kinase and triggers nuclear EGFR transport
.
Radiother Oncol
2009
;
92
:
379
82
.

Supplementary data