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[Cancer Research 62, 7219-7229, December 15, 2002]
© 2002 American Association for Cancer Research

Biochemistry and Biophysics

Distribution of Furamidine Analogues in Tumor Cells

Targeting of the Nucleus or Mitochondria Depending on the Amidine Substitution1

Amélie Lansiaux, Farial Tanious, Zohar Mishal, Laurent Dassonneville, Arvind Kumar, Chad E. Stephens, Qiyue Hu, W. David Wilson, David W. Boykin and Christian Bailly2

Institut National de la Santé et de la Recherche Médicale U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, 59045 Lille, France [A. L., L. D., C. B.]; Centre National de la Recherche Scientifique, 94800 Villejuif, France [Z. M.]; and Department of Chemistry and Laboratory for Chemical and Biological Sciences, Georgia State University, Atlanta, Georgia 30303 [F. T., A. K., C. E. S., Q. H., W. D. W., D. W. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diphenylfuran diamidines represent an important class of DNA minor groove binders of high therapeutic interest as antiparasitic or antitumor agents depending on the compounds structures. To exert their cytotoxic action, the compounds must first get into the cell and reach the nuclear compartment where the main target, DNA, is located. The forces that drive the drugs into cell nuclei, as well as the influence of the molecular structures on the cell distribution, are not known. To address these issues, we took advantage of the fluorescence of the molecules to analyze their intracellular distribution profiles in tumor cells of different origins (B16 melanoma, MCF7 mammary adenocarcinoma, A549 lung carcinoma, HT29 colon carcinoma, LNCaP, and PC3 prostatic carcinoma) by epifluorescence and confocal microscopy. A homogeneous series of synthetic bis-substituted alkyl or phenyl amidine and reverse amidine derivatives of furamidine was used to dissect the molecular mechanisms that control the distribution of the drugs into the cytoplasm or the nucleus of the cells. The amidine (DB75) and the various N-alkyl derivatives were found to accumulate selectively in the cell nuclei. This is also the case for a guanidine derivative but not for the phenyl-substituted compound DB569, which essentially localizes in cytoplasmic granules. Similar cytoplasmic patterns were observed with a reverse amidine analogue and a pyridine-substituted compound indicating that the presence of aromatic rings on the terminal side chain is the limiting factor that restricts the uptake of the compounds in the nuclear compartment. The use of different organelle-selective fluorescent probes, such as JC-1 and chloromethyl-X-rosamine, both specific to mitochondria and neutral red considered as a lysosome-selective probe, suggests that DB569 preferentially accumulates in mitochondria. Competition experiments with the antitumor drug daunomycin reveal that the diphenylfurans are attracted into the nuclei by the DNA. The DNA minor groove-drug interactions provide the driving force that permits massive accumulation of the fluorescent molecules in the nuclei. The DNA binding properties of the diphenylfuran derivatives were investigated by DNase I footprinting and surface plasmon resonance biosensor experiments to measure sequence selectivity and binding affinities, respectively. Furamidine and its phenyl-substituted analogue that accumulate in the cell nuclei and mitochondria, respectively, share a common selectivity for AT sites and bind equally tightly to these sites. Therefore, it is possible to modulate the intracellular distribution of the furamidine derivatives without affecting their DNA binding and sequence recognition properties. The introduction of aromatic substituents on diphenylfuran diamidines represents a novel strategy to control the intracellular compartmentalization of these DNA binding agents and directs them to mitochondria. This drug design strategy may prove useful to trigger drug-induced apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Furamidine (DB75; Fig. 1Citation ) is a DNA minor groove binder endowed with a pronounced selectivity for sequences containing consecutive AT bp (1, 2, 3) . In therapeutic terms, DB75 is the lead compound of an important class of antimicrobial and antiparasitic agents. This diphenylfuran derivative has shown potent activities against several pathogen microorganisms such as Cryptosporidium parvum (4) , Pneumocystis carinii (5) , and Trypanosoma sp (6 , 7) . An amidoxime prodrug of furamidine (8) is currently undergoing Phase II clinical trials against human African trypanosomes. The mechanism by which the drug enters the parasites is poorly known. For pentamidine (Fig. 1)Citation , a diamidine largely used for the treatment of African trypanosomiasis, specific transporters have been identified recently, such as the adenosine-sensitive pentamidine transporter-1 and the low-affinity pentamidine transporter-1 (9) . These transporters can be inhibited by other diamidines including propamidine, berenil, and stilbamidine (Fig. 1)Citation , and, therefore, it is reasonable to think that furamidine can use the same system to accumulate in the parasite. At present, no such diamidine transporter has been identified in human cells.



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  		Fig. 1. Chemical structures of a few diamidines and the diphenylfuran derivatives used in this study. Hydroxystilbamidine (OHSA) bears an OH group on the phenyl ring (*).

 
In addition to their antiparasitic properties, certain diphenylfuran dications display a useful spectrum of antitumor activities. For example, the imidazoline analogue furimidazoline (DB60), has shown significant antiproliferative activities against various tumor cell lines, including cells resistant to cisplatinum (10) . But here again, the mechanism by which these diamidine drugs enter the cells is poorly understood. These charged compounds enter cells rapidly, and a pure diffusion process seems unlikely (11) . Normal and malignant cells possess a high affinity transport system that controls the entry of polyamines (putrescine, spermidine, and spermine) and guanidines [e.g., methylglyoxal bis(guanylhydrazone); Ref. 12 ]. This membrane transport system may well be exploited by furamidine and its analogues to cross the plasma membrane.

We have described recently the cell distribution profiles of a series of mono, bis, and tetracationic analogues of furamidine (DB75). Unexpectedly, we found that the presence of two or four positive charges favored nuclear uptake, whereas the loss of one of the two cationic side chains of DB75-type compounds, with a remaining mono-amidine or -imidazoline group, was detrimental to the nuclear binding presumably as a result of a reduced affinity for DNA (11) . This key finding prompted us to screen additional furamidine analogues for nuclear versus cytoplasmic staining. Here we present our data with a series of synthetic bis-substituted alkyl or phenyl amidine and reverse amidine derivatives of DB75. For all of the compounds shown in Fig. 1Citation , the distribution in malignant cells was evaluated by epifluorescence microscopy taking advantage of the intrinsic blue-emitting fluorescence of the compounds. High-resolution images were also collected by confocal microscopy, and the relation to DNA binding and cytotoxicity is discussed. Competition experiments with the intercalating drug daunomycin suggest that DNA binding provides the driving force that attract the compounds in the nucleus.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and Chemicals.
Daunomycin was purchased from Sigma. DiOC6,3 JC-1, NBD-ceramide, neutral red, and PI were from Molecular Probes. The synthesis of 9 of the 10 diphenylfuran derivatives used in this study has been described previously: DB75 (6) ; DB181, DB226, DB244, DB249, DB417 (13) ; DB613, DB667, and DB673 (14) . The procedure for the synthesis of the new molecule DB569 is described below.

Chemistry.
A suspension of 2,5-bis(4-carboxyphenyl)furan (13 ; 0.5 g, 0.0016 mol), dry benzene (30 ml), thionyl chloride (2.0 ml, 0.027 mol), and anhydrous dimethylformamide (2 drops) was refluxed for 4 h. The solvent was removed under vacuum, and the residue was triturated with benzene; the benzene was removed under vacuum and the residue triturated with dry ether. The orange solid was filtered and dried under vacuum to yield diacid chloride (0.5 g, 89%), mp >300°C. 1H NMR (CDCl3): 8.17 (d, 4H, J = 7.6), 7.86 (d, 4H, J = 7.6), and 7.02 (s, 2H).

Aniline (0.37 g, 0.004 mol) was added to the diacid chloride from above (0.35 g, 0.001 mol) in 50 ml of dry CH2Cl2, and the mixture was allowed to stir at room temperature for 2.5 h. The solvent was removed under reduced pressure, and water was added. The solid was filtered and washed with 10% HCl, 10% NaHCO3, and water. After drying, the solid was recrystallized from DMF-H2O to give a yellow solid (0.37 g, 80%), mp 355–356°C. 1H NMR (DMSO-d6) was 10.14 (s, 2H), 8.07 (d, 4H, J = 8.0), 7.98 (d, 4H, J = 8.0), 7.78 (d, 4H, J = 7.5), 7.36 (t, 4H, J = 7.5), 7.26 (s, 2H), and 7.11(t, 2H, J = 7.0). 13C NMR (DMSO-d6) was 160.0, 147.9, 134.2, 128.9, 127.6, 123.6, 123.5, 118.8, 118.5, 115.7, and 105.3. Anal. Calcd for C30H22N2O3: C, 78.61; H, 4.80; N, 6.11. Found: C, 78.21; H, 4.60; N, 6.35.

A mixture of 2,5-bis[4-(N-phenylcarbamoyl)phenyl]furan, from above (0.41 g, 0.00089mol), thionyl chloride (0.27 ml, 0.0036 mol), DMF (3 drops), and CH2Cl2 (50 ml, dry) were allowed to reflux overnight. The solution was distilled under reduced pressure to remove solvent. The residue was triturated with dry ether. The solid was filtered (under nitrogen) and dried under vacuum at room temperature to yield a yellow solid (0.40 g, 90%). The bis-imidoyl chloride was used directly in the next step without additional characterization.

A suspension of the bis-imidoyl chloride (0.40 g, 0.00081mol) in CH2Cl2 (50 ml, dry) was saturated with dry ammonia gas while cooling in an ice bath. The mixture was stirred at room temperature for 24 h, and the solvent was removed under reduced pressure. The residue was treated with ice-water, and the pH of the suspension was adjusted to a value of 10 by adding 30% NaOH. The resulting solid was filtered, washed with H2O, and dried under vacuum to yield the free base (0.34 g, 91%). The product was used directly in the next step without additional characterization. The free base was converted to salt by taking up 0.34 g (0.00075 mol) in dry ethanol (40 ml) saturated with HCl and stirring for 2 h. The volume of solvent was decreased to ~10 ml under reduced pressure. Dry ether was added to the solution to cause precipitation of the salt, which was filtered and recrystallized from Ethanol (dry)-ether(dry) to yield yellow solid (0.28 g, 72%), mp 251–253°C. 1H NMR (DMSO-d6) was 11.88 (s, 2H), 10.01 (s, 2H), 8.99 (s, 2H), 8.12(m, 10H), and 7.53(m, 10H). 13C NMR (DMSO-d6) was 169.2, 162.2, 152.5, 134.8, 134.2, 129.0, 128.2, 126.9, 125.5, 123.7, and 111.7. Anal. Calcd for C30H24N4O,2HCl,0.5H2O: C, 66.69; H, 5.37; N, 10.37. Found: C, 66.36; H, 5.19; N, 10.22.

Cell Cultures and Survival Assay.
The human prostatic carcinoma LNCaP and PC3 cells were kindly provided by Dr. Nicole Pommery (Faculty of Pharmacy, Lille, France). These cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 26 mM NaH2CO3 (pH 7.4), 1% L-glutamine, and antibiotics (1% penicillin-streptomycin) in a 37°C incubator supplied with 5% CO2 until they reached a density corresponding to ~106 cells onto 75 cm2 dishes. The colon adenocarcinoma HT29 cell line was a generous gift of Dr. Daniele Demarquay (Institut H. Beaufour, Les Ulis, France). B16 melanoma cells were obtained from Dr. Marie-Claire De Pauw (University of Liège, Belgium). HT29, B16, MCF7, and A549 cells were maintained as monolayers in 150-cm2 culture flasks using culture medium consisting of DMEM-glutaMAX medium supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Cells were grown in a humidified atmosphere at 37°C under 5% CO2. In the exponential phase, the doubling time of these melanocytes ranges from 15 to 18 h, and the confluence stage was achieved at a cell density of 3 x 105 cells/cm2. B16 cells were harvested by trypsinization and plated 20 h before treatment with the test drug. The cytotoxicity of the drugs was assessed using a cell proliferation assay developed by Promega (CellTiter 96 AQueous one solution cell proliferation assay). Briefly, 2 x 104 exponentially growing cells were seeded in 96-well microculture plates with various drug concentrations in a volume of 100 µl. After 72 h incubation at 37°C, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (15) were added to each well, and the samples were incubated for an additional 3 h at 37°C. Plates were analyzed on a Labsystems Multiskan MS reader at 492 nm.

Fluorescence and Confocal Microscopy.
The cells (20,000 cells/cm2) were incubated at 37°C with the test compound, usually at 2 µM for 18 h unless otherwise stated. The medium was removed, and cells were rinsed with ice-cold PBS (10 min) before the fixation with a 2% paraformaldehyde solution for 20 min at +4°C. After washing, the cells were incubated with fluorescent probes DiOC6 (20 nM) for 5 min at 37°C in the dark, washed again with PBS, and then incubated with a solution of PI (0.2 µg/ml) for 5 min at room temperature. A drop of antifade solution was added, and the treated portion of the slide was covered with a glass coverslip. The fluorescence of the drug was detected and localized by fluorescence microscopy using a Zeiss microscope with a x63 or x100 oil objective. Images were captured using the software Quips Smart Capture (Vysis). Alternatively, the fluorescence was detected by confocal microscopy using a Leica DMIRBE microscope controlled by a Leica TCS-NT workstation (Leica Microsystems, Bensheim, Germany) with a 63 x 1.32 NA oil objective, equipped with a 75 mW argon-krypton and Coherent Innova-90-UV laser lines. The emission signal was observed through a dichroic mirror (DD488/568) followed by a filter set (RSP 580, BF 530/30, BP 600/30, and BP460/30 for UV excited probes). The optical sections were obtained in the Z axis and stored on the computer with a scanning mode. In all of the cases, the operating conditions were such that detectable images could not be obtained for cell samples not treated with drugs.

DNA Thermal Melting.
Thermal melting experiments were conducted with Cary 3 or Cary 4 spectrophotometers interfaced to microcomputers as described previously (16) . MES10 buffer {10 mM [2-(N-morpholino) ethanesulfonic acid] and 1 mM EDTA (pH 6.25) with 0.1 M NaCl} was used in the experiments with poly(deoxyadenylate-deoxythymidylic acid). A thermistor fixed into a reference cuvette was used to monitor the temperature. In Tm experiments DNA was added to buffer in 1-cm path length reduced volume quartz cells, and the concentration was determined by measuring the absorbance at 260 nm. The experiments were generally conducted at a concentration of 5 x 10-5 M DNA bp and a ratio of 0.6 compound:bp of DNA.

DNase I Footprinting.
The experimental procedures for the purification and 32P-labeling of the DNA fragments, and the detailed protocol for the DNase I cleavage experiments have been described previously (17 , 18) .

SPR.
Experiments were performed in HBS-EP buffer (from BIACORE) containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% polysorbate 20 (v/v; pH 7.4). Three different DNA oligomers (high-performance liquid chromatography purified and desalted; Midland Certified Reagent Co.) were used in these studies. The 5'-biotin labeled hairpin duplexes are d(Biotin-CGAAATTTCCTCTGAAATTTCG; A3T3 DNA), d(Biotin-CATATATATCCCCATATATATG; AT DNA), and d(Biotin-CGCGCGCGTTTTCGCGCGCG; GC DNA), and the hairpin loop sequences are italicized. The oligomer concentrations were determined optically using extinction coefficients per mol of strand at 260 nm determined by the nearest neighbor procedure.

SPR measurements were performed with a four-channel BIAcore 2000 system and streptavidin-coated sensor chips (SA from BIACORE as described previously; Refs. 19, 20, 21 ). Briefly, the chips were prepared for use by conditioning with three to five consecutive 1-min injections of 1 M NaCl in 50 mM NaOH followed by extensive washing with buffer. 5'-Biotinylated DNA samples (25 nM) in HBS buffer were immobilized on the flow cell surface by noncovalent capture. Three flow cells were used to immobilize DNA oligomer samples, and the fourth cell was left blank as a control. Samples of the compounds were prepared in filtered and degassed MES 10 buffer by serial dilution from stock solutions. Drug samples were injected from 7-mm plastic vials with pierceable plastic crimp caps at a flow rate of 20 µl/min using the KINJECT command. For the furan-DNA complexes a solution of 10 mM glycine (pH 2.0) was used to dissociate all of the furan from DNA to regenerate the surface. An array of different furan concentrations was used in each experiment, and the results were analyzed as described below. The injection of the compound (association) was followed by injection of running buffer (compound dissociation). To reduce the probability of nonspecific binding to the chip surface 50 µl/liter of surfactant P20 was added to the MES buffer. The amount of DNA immobilized was ~350 response units in each flow cell. SPR experiments were performed at 25°C in MES 10. With the SPR technique the change in refractive index occurring at the surface of the sensor chip is monitored. The change in refractive index in terms of RUs is proportional to the amount of compound bound to the DNA.

To obtain the affinity constants the data generated were fitted to different interaction models using Kaleidagraph for nonlinear least squares optimization of the binding parameters using the following equation:

where K1 and K2 are macroscopic equilibrium constants for two types of binding sites, RU is the SPR response at the steady state level, RUmax is the maximum SPR response for binding one molecule per binding site, and Cfree is the concentration of the compound in solution (22) . Poor fits were obtained with a single-site model (K2 = 0), and no significant improvement in the quality of the fits was observed on going to a three-site model. RUmax can be predicted using the following equation:

Where RUDNA is the amount of DNA immobilized in RUs, MW is molecular weight of compound and DNA, respectively, and RII is the refractive index increment ratio of compound to refractive index of DNA (22 , 23) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intracellular Distribution of the Diphenylfuran Derivatives.
B16 melanoma cells were used in the initial investigation of the nuclear staining by the test compounds because of their flattened morphology. Fig. 2Citation shows fluorescence images of B16 cells exposed to DB75 and symmetric N-alkyl amidine derivatives. The cells were treated with each drug at 2 µM for 18 h at 37°C, washed, fixed with 2% paraformaldehyde, and then labeled with the dyes 20 nM DiOC6 and 0.2 µg/ml PI, which stain principally the cytoplasm and the nucleus in green and red, respectively. PI binds strongly to nucleic acids, whereas DiOC6 binds preferentially to mitochondria in the cytoplasm. The three colors, blue, green, and red, can be easily differentiated by fluorescence microscopy. No difference was observed between the unsubstituted parent compound DB75 and the analogues bearing an isopropyl (DB181), isopentyl (DB226), cyclopentyl (DB244), cyclohexyl (DB249), or a methyl (DB417) group. In all of the cases, the molecules are sequestered in the cell nuclei and colocalize with PI. The massive accumulation of the blue fluorescence in the nuclear compartment contrasts with the extensive green fluorescence detected in the cytoplasm. The distribution of the studied compounds in the nuclear compartment is more uniform than that of PI, which concentrates into a few bright red nucleolar-like bodies. As discussed recently (12) , the different nuclear distribution between DB75 and PI can be attributed to their distinct DNA sequence selectivity. Nucleolar DNA is GC-rich and provides a good substrate for the intercalating drugs like PI but not for the minor groove binder DB75, which exhibits a high preference for AT-rich sequences (1, 2, 3) .



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  		Fig. 2. Fluorescence micrographs of B16 melanoma cells stained with the indicated compound (2 µM each in blue), with PI (in red), or with DiOC6 (in green). Images on the right side of the figure show the overlay of the DBN compound with PI (blue + red) or with DiOC6 (blue + green). The cells were incubated with the test drug for 18 h, washed, fixed with 2% paraformaldehyde, and then labeled with PI (0.2 µg/ml PI) or DiOC6 (20 nM) before the microscopy observation (x63). Results are representative of three independent stainings.

 
The guanidine derivative DB673 also concentrates into cell nuclei (Fig. 2)Citation . This compound is relatively weakly fluorescent compared with DB75 and, therefore, a much longer exposure time is required to detect the compound in the cells. In this case, the images are less well resolved because the background fluorescent is higher. All of the studied diphenylfuran derivatives fluoresce in blue ({lambda}excmax = 360–390 nm; {lambda}emmax = 445–460 nm), but the relative fluorescence intensity varies considerably with the compound structure. The exposure time were adjusted to obtain images of equal intensity (Table 1)Citation .


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  		Table 1 Cytotoxicity and DNA binding

 
DB75, its guanidine analogue DB673, and the five N-alkyl derivatives accumulate selectively in the cell nuclei. The situation is radically different with the phenyl-substituted compound DB569. In this case, the fluorescence is essentially localized in cytoplasmic granules, whereas nuclei appear as dark cores (Fig. 2)Citation . The complete absence of nuclear blue fluorescence in all of the B16 cells treated with DB569 was a particularly striking observation, which prompted us to test additional analogues. We then tested the reverse amidine analogue DB613. The terminal phenyl rings are now connected to the diphenylfuran core via a C(NH)NH connector compared with the NHC(NH) spacer of DB569. The reversal of the orientation of the amidine group does not influence the cell distribution profile; DB613 does not enter the nuclei, just as observed with DB569 (Fig. 2)Citation . Similar cytoplasmic patterns were also observed with a compound designated DB667 containing pyridine terminal groups in place of the end phenyls of DB613 (data not shown). The presence of aromatic rings on the terminal side chain appears as the limiting factor that restricts the uptake of the compounds in the nuclear compartment.

The phenyl-amidine compound DB569 is only weakly fluorescent in solution, just like the guanidine analogue DB673, which was found in the nucleus. But in cells, the fluorescence detection of DB569 can be easily obtained by adjusting the exposure time to about 4–5 s, exactly as with the bis-N-isopropyl derivative DB181, which is highly fluorescent in solution (Table 1)Citation . A direct quantitative analysis of compound uptake is impossible because of their different intrinsic fluorescence properties; however, the facile identification of DB569 in cells suggests that this compound massively concentrates in the cytoplasm. But the possibility that this weakly fluorescent compound is transformed into a more fluorescent metabolite retained in the cytoplasm must be kept in mind.

The distinct distribution profiles observed with DB75 and DB569 likely reflect the intrinsic properties of the compounds, but we also considered the possibility that the differences were because of the biochemical nature of the murine B16 melanoma cells. To answer this question, we repeated the experiments using five other cancer cell lines from human origin. Fig. 3Citation shows typical images obtained with HT29 colon carcinoma cells, MCF7 mammary adenocarcinoma cells, A549 lung carcinoma cells, and two prostate tumor cell lines, PC3 and LNCaP. These latter human malignant cells express androgen receptors and are hormonally responsive but not metastatic. In contrast, the metastatic PC3 cells are androgen-insensitive. In these cells, the loss of androgen receptors is generally associated with the progression of prostate cancer to the more invasive carcinoma. These human cell lines gave identical results to the murine cell line. In each case, DB75 was found almost exclusively in the nuclei of the human cells, whereas DB569 was clearly maintained outside the nuclei, in the cytoplasm. Similar results were also obtained with leukemia cells (P388 and K562; data not shown). Therefore, the different distribution profiles observed with DB75 versus DB569 must reflect the chemical properties of the drugs, not the specific capacities of the cells.



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  		Fig. 3. Intracellular distribution of furamidine (DB75) and its N-phenyl derivative DB569 (2 µM each) in murine B16 melanoma cells, human MCF7 mammary adenocarcinoma cells, human A549 lung carcinoma cells, and human prostatic tumor cells PC3 and LNCaP, visualized by fluorescence microscopy (x63) after 18 h incubation. The green and red images correspond to the subsequent incubation of the same cells with DiOC6 (20 nM) and PI (0.2 µg/ml PI), and the images on the right side correspond to the superimposed blue ± green ± red fluorescence.

 
The optical sectioning capabilities of confocal microscopy was particularly useful to precisely locate the drug in cells. Fig. 4Citation is a single confocal slice showing a cross-section of HT29 cells treated with the diphenylfuran DB75 or DB569, and counterstained with DiOC6 and the nuclear specific dye TOTO-3. This dye is used to stain DNA with far-red fluorescence under red (647 nm) excitation (24) . Obviously, DB75 is not evenly distributed over the cell. The blue fluorescence concentrates into the nucleus with no detectable fluorescence in the cytoplasm. The direct observation is reinforced by the fluorescence intensity profiles in Fig. 4Citation , which show the distribution of DB75, PI, and DiOC6 fluorescence along the XY axis indicated on the photographs. The blue fluorescence colocalizes with the red fluorescence. The reverse situation was observed with DB569. In this case, the blue fluorescence essentially follows the green fluorescence. The use of such a high-resolution imaging system attests unambiguously that the two compounds locate in different cell compartments: DB75 in the nuclei and DB569 in the cytoplasm.



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  		Fig. 4. Confocal laser scanning microscopy of HT29 colon carcinoma cells treated with 2 µM DB75 or DB569. The red and green images correspond to the subsequent incubation of the same cells with TOTO-3 (0.5 µM) and DiOC6 (20 nM), and the images on the right side correspond to the superimposed blue + green and blue + red fluorescence. The scales (20 µm) are indicated. The lower images correspond to the superimposed tricolor images (blue + green + red). The graphs shows the distribution of the diphenylfuran (blue), DiOC6 (green), and PI (red) fluorescence along the XY axis indicated in the photographs for both DB75 and DB569.

 
The confocal images also help to identify the cytoplasmic structures that stain with DB569. The localization of the fluorescence blue spots coincides with that of the green probe. The two images obtained with DB569 and DiOC6 are essentially superimposable and, therefore, support the idea that the DB569 molecules accumulate in the mitochondria organelles. Additional experiments performed with the mitochondria-specific probe JC-1 substantiate this observation. This lipophilic cation, commonly used to measure the variation of the mitochondrial membrane potential ({Delta}{Psi}mt; Refs. 25 , 26 ), gives red-fluorescent J-aggregates locally and spontaneously under high mitochondrial {Delta}{Psi}mt, whereas the monomeric form fluoresces green (27) . Typical images of B16 cells treated with DB569 and counterstained with JC-1 are presented in Fig. 5BCitation . The blue (DB569) and green (JC-1) images superimpose relatively well. All of the blue granules are also colored in green suggesting that DB569 and JC-1 essentially locate in the same organelles. In contrast, the overlay is not so good when the cells were counterstained with neutral red (Fig. 5A)Citation , which is considered as a lysosome-specific probe (28 , 29) . Close inspection of the micrographs reveals that the blue spots corresponding to DB569-containing structures contain little red dye. Lysosomes may contain a limited amount of DB569 but a priori the majority of the drug molecules are internalized in the mitochondria. A good correlation was also observed when the cells were colored with DB569 and the red dye chloromethyl-X-rosamine (Mitotracker red), another mitochondria-specific dye. In contrast, no correlation was observed when using the green dye NDB-ceramide considered as a suitable dye for the Golgi apparatus (data not shown). All of the results together strongly suggest that DB569 essentially accumulates in mitochondria, but the molecular basis of this interaction remains to be determined. The mechanism by which DB569 traverses the mitochondrial membrane and is sequestered inside is unknown at present. The internal pH of mitochondria probably plays a role in this sequestration (see "Discussion"). The three compounds that select the mitochondria, DB569, DB667, and DB613, are likely to have pKs three to four units below that of DB75.



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  		Fig. 5. Fluorescence micrographs of B16 melanoma cells incubated for 18 h with 2 µM DB569 and counterstained (A) with neutral red (50 nM, 15mn, 37°C) or (B) with the green fluorescent dye JC-1 (10 µM, 15mn, 37°C). C, confocal laser scanning microscopy of B16 melanoma cells labeled with DB75 in the absence and presence of daunomycin. Panels 1 and 3 refer to B16 murine cells incubated with 8 µM DB75 and 8 µM daunomycin, respectively. Panel 2 refers to B16 cells treated simultaneously with 8 µM DB75 and 8 µM daunomycin for 18 h. In panel 4, the B16 cells were incubated with 8 µM DB75 for 17.5 h and then with 8 µM daunomycin for 30 min before the observation. The scales (20 µm) are indicated. The cells shown are representative of the entire population.

 
The reasons why DB75, but not DB569, is attracted in the nuclei are not known at present, but we hypothesized that the molecules are attracted by the DNA targets in the nuclear compartment. To examine this aspect, competition experiments were performed with antitumor drug daunomycin, which is a well-known DNA intercalating agent and has the additional advantage to fluoresce in red. The specific accumulation of daunomycin and related anthracyclines in cell nuclei has been reported in many studies (30, 31, 32) . Using the confocal equipment, there is no overlap between the blue fluorescence of DB75 and the red fluorescence of daunomycin. Fig. 5CCitation shows representative confocal micrographs of B16 cells stained in blue with DB75 and/or in red with daunomycin. DB75 alone only stains the nuclei of the murine cells (Fig. 5CCitation , panel 1) whereas in the presence of daunomycin, the majority of DB75 molecules is detected in the cytoplasm (Fig. 5CCitation , panel 2). A short treatment with daunomycin suffices to expel the blue compound from the nuclei. Fig. 5CCitation , panel 4 shows B16 cells that have been incubated for 17.5 h with 8 µM DB75 and then for 30 min with 8 µM daunomycin. In this case, the intercalating drug stains the nuclei to a lower extent, but the short incubation period is sufficient to limit the entrance of the diphenylfuran compound in the nuclei. Now the DB75 molecules are distributed equally in the nucleus and the cytoplasm of the tumor cells. Similar results were obtained with the human HT29 cell line (data not shown). Daunomycin was found exclusively in the nuclei, but surprisingly DB75 was confined to the cytoplasm in the presence of the anthracycline competitor. The high affinity of the intercalating drug for DNA must restrict the access of the minor groove binder. These competition experiments strongly support the hypothesis that DNA is responsible for the staining of the nuclei by DB75.

DNA Binding.
The DNA binding properties of the diphenylfuran derivatives was investigated by a combination of spectroscopic, biosensor, and biochemical methods. Melting temperature (Tm) experiments were performed to evaluate the relative affinities of the compounds for poly(deoxyadenylate-deoxythymidylic acid). The {Delta}Tm values ({Delta}Tm = Tmcomplex - TmDNA) measured with each compound, in MES10 buffer at a ratio of 0.6 compound:bp of DNA, are collected in Table 1Citation . All of the compounds bind strongly to DNA with {Delta}Tm values of ~20°C or more. The diamidine DB75 and its phenyl-amidine analogue stabilize the double helix to the same extent, as does the reverse amidine derivative DB613.

A DNase I footprinting study was performed with the two most representative compounds of the present series, the lead compound DB75 and the phenyl-substituted analogue DB569, which exhibit distinct cell distribution properties, with a marked nuclear accumulation for DB75 as opposed to a cytoplasmic staining with DB569. Three DNA restriction fragments of 117, 160, and 265 bp, each uniquely 3'-end radiolabeled, were prepared and subjected to limited cleavage by the nuclease in the presence of different concentrations of DB75 and DB569. Well-resolved footprints were detected with both compounds. The substitution of the amidine ends with a phenyl group does not perturb the capacity of the drug to recognize specific sequences. The footprints are located at the same positions and are equally intense with both compounds (Fig. 6)Citation . The binding curves obtained with DB75 and DB569 superimpose very well, and the sequences protected from DNase I cleavage all correspond to AT-rich sequences, whereas GC-rich sequences are often cut more readily by the enzyme in the presence of the drugs. There is no doubt that the two compounds share a common selectivity for AT sites and bind equally well to these sites.



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  		Fig. 6. Differential cleavage plots comparing the susceptibility of three DNA fragments to DNase I cutting in the presence of the diphenyl-furan derivatives: ({bullet}) DB75 and ({circ}) DB569 (2 µM each). Negative values correspond to a ligand-protected site, and positive values represent enhanced cleavage. Vertical scales are in units of ln(fa) - ln(fc), where fa is the fractional cleavage at any bond in the presence of the drug and fc is the fractional cleavage of the same bond in the control, given closely similar extents of overall digestion. Each line drawn represents a 3-bond running average of individual data points, calculated by averaging the value of ln(fa) - ln(fc) at any bond with those of its two nearest neighbors.

 
A quantitative analysis of the drug-DNA interaction was performed by SPR biosensor experiments. The compounds were passed through three sensor chip flow cells containing DNAs with an A3T3 minor groove-binding site, an alternating AT DNA sequence, or an alternating GC DNA sequence. The compounds were also passed through a control flow cell with no DNA, and the response in this flow cell was subtracted to generate the response sensorgrams. An example set of sensorgrams (RUs versus time) for DB417 binding to the A3T3 oligomer hairpin are shown in Fig. 7ACitation . With these DNAs and compounds the sensorgrams can be divided into three phases: (a) association of the furan with DNA that results in an increase of RU; (b) a steady state region during the solution injection where the rate of compound binding is balanced by dissociation from the complex and there is no change in RU with time; and (c) dissociation of the furan from surface during buffer flow with a decrease in bound compound and observed RU. As can be seen from Fig. 7Citation , the interaction with DB417 is slow at low concentrations and increases in association rate with an increase in the furan concentrations as expected for a bimolecular interaction. Also as expected for a first order reaction, the dissociation reactions are concentration independent. The sensorgrams have a slow dissociation rate up to ~30 RU, but exhibit a second, faster dissociation for the sensorgram region above ~30 RU. The sensorgrams below 30 RU are at concentrations below 1 µM and are characteristic of specific interactions. The region above 30 RU is only reached at higher concentrations (above 1 µM concentration) and represents a weak secondary binding mode for these compounds.



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  		Fig. 7. SPR binding data. A, SPR sensorgrams for the interaction of DB417 with the hairpin DNA oligonucleotide containing the A3T3 sequence. The concentrations are from 1 nM (lowest) to 5 µM (highest). Data were collected at 25°C in MES 10 buffer with a flow rate of 20 µl/min. B, binding plots (SPR response versus drug concentration) used to determine the affinity constants for DB417 complexed with the three different hairpin duplexes [A3T3], [AT]4, and [CG]4. Above 0.5 µM the RU values remain practically unchanged. RU values were obtained from the steady-state region of the SPR sensorgrams.

 
It was possible to reach a steady-state plateau for all of the furan derivatives in Fig. 1Citation in ~200 s at the lowest concentrations used in these experiments. Average fitting of the sensorgrams at the steady state level was performed over a 30-s time period with the BIAevaluation 3.1 software. The binding constants for all of the compounds to the three different DNA sequences were determined by fitting RU at the steady state as a function of concentration, by using the equations in "Materials and Methods," and results for DB417 are shown in Fig. 7BCitation . All of the compounds required a two-site binding equation for the best fit. As expected from the sensorgrams the compounds had a single strong binding site in the DNAs with AT bp as well as a weaker secondary binding site. The primary binding constants are collected in Table 2Citation . The secondary binding exhibits fast dissociation from DNA and has binding constants that are 10–100 times lower than the strong binding constants. The GC sequence exhibited much weaker binding and lower RUs at the same concentration than the AT containing sequences (Fig. 7Citation ; Table 2Citation ). This is the expected result for AT-specific minor groove complex formation. The GC binding constants are only slightly greater than the nonspecific binding observed for the AT DNA duplexes. However, even with the AT DNAs, the compounds show significant structure dependent variations in K values (Table 2)Citation .


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  		Table 2 DNA binding constants determined by SPR

Experiments were conducted at 25°C in MES10 buffer. All K values are for the strongest binding site and are in M-1 units. The K values are all times 106 and can be directly compared.

 
The rates of binding of the furans are slow enough at low concentration to allow determination of association and dissociation kinetics constants. Curves in the strong binding region were fit by a global analysis procedure and equilibrium constants determine by the ratio of association/dissociation constants. In all of the cases the equilibrium constants determined from the rate constants were in good agreement with those determined by the steady state procedure. The rates for binding and dissociation with the GC DNA sequence are too fast to determine by the SPR-biosensor method.

Cytotoxicity.
The cytotoxic potential of the diphenylfuran derivatives was evaluated using a tetrazolium-based survival assay. The drug concentrations required to inhibit cell growth by 50% after incubation in the culture medium for 72 h are indicated in Table 1Citation . DB75 is weakly cytotoxic to the B16 murine melanoma cells, and the substitution of the amidines with a methyl, an isopropyl, and a cyclopentyl group abolishes the cytotoxic potential. The introduction of cyclohexyl groups or the replacement of the amidine with guanidine groups has no effect on cytotoxicity. The substitution of the amidine with isopentyl or a phenyl group only decreases the IC50 by a factor of 2. The most cytotoxic compound in the series is the phenyl-reverse amidine derivative DB613, but nevertheless these diphenylfuran derivatives must be considered as weak cytotoxic agents, and no direct relationship with the distribution profile can be proposed. DB226, which seems to freely enter the nuclei, is equally toxic as DB569, which apparently remains confined in the cytoplasmic compartment of the cells. however, it should be noted that a priori the absence of nuclear DB569 fluorescence does not exclude the possibility that a low concentration of the drug (which is weakly fluorescent) could exist in the nuclei, with the drug molecules inserted in the minor groove of nuclear DNA. It is important to mention that on binding to the minor groove of DNA, the fluorescence of these bis(amidinophenyl)furan derivatives decreases by about 20–25%, and this effect contributes to underestimate the extent of nuclear uptake. The bright blue fluorescence detected in the nuclei of the cells treated with DB75 likely reflects a massive nuclear accumulation.

No correlation between DNA binding strength and cytotoxicity could be established. The graph in Fig. 8Citation shows the distribution of drug equilibrium binding constants for the A3T3 sequence versus IC50 values. The four compounds with K values >108 M-1 are poorly cytotoxic, whereas those with K values <5 x 107 M-1 are more potent at reducing the growth of the melanoma cells.



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  		Fig. 8. Equilibrium DNA binding constants (K values for the A3T3 sequence) versus cytotoxicity values (IC50) are plotted for all 10 compounds. The compounds can be divided in two groups, ({blacksquare}) with high and ({bullet}) low K values.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides three important findings. First, it confirms that furamidine (DB75) selectively accumulates in the nuclei of tumor cells. This conclusion was already drawn from our recent fluorescence study using the murine B16 cell line (11) . Here the use of different human cell lines validates this finding, and the specific sequestration of DB75 in cell nuclei was monitored by confocal microscopy to produce high resolution images. Second, the results indicate that the extent of nuclear uptake by the diphenylfuran derivatives is directly dependent on the compound structure. Substitution of the amidine groups with an alkyl chain does not markedly change the distribution profiles of the drugs in malignant cells. All of the N-alkyl bis(amidinophenyl)furan derivatives tested here mainly distribute in the nuclear compartment where their DNA substrate is located. In contrast, the three derivatives substituted with aromatic rings, phenyl, or pyridine, exhibit distinct distribution profiles with a specific accumulation in cytoplasmic vesicles, most likely the mitochondria. This is important information for future drug design in this series. The incorporation of aromatic rings on the furamidine side chain may provide a suitable delivery strategy to target drugs specifically to mitochondria rather than in the nuclei. Because of the apparently specific non-nuclear uptake, a compound like DB569 has the potential to be used for targeting mitochondrial DNA. Third, the competition studies with daunomycin suggest that the diphenylfurans are attracted into the nuclei by the DNA. The minor groove binders apparently have no intrinsic properties that drive them into the nuclear compartment, and the energetics for transport against the concentration gradient are driven by binding to the nucleic acid targets. DNA is clearly a magnet for these compounds and the DNA minor groove-drug interactions provide the driving force that permits massive accumulation of the fluorescent molecules in the nuclei. In the absence of the competing intercalating drug, DB75 is exclusively sequestered in the nuclear compartment, whereas incubation of the cells with daunomycin profoundly altered the drug compartmentalization by releasing DB75 from the nuclei and shifting it to the cytoplasm.

On entering the cells, furamidine and analogs are attracted by the nucleus where the main known target, DNA, is located. Apparently, these highly polar biscationic compounds have little or no interaction with cell membranes; no staining of the plasma membrane or nuclear envelope was detected. At first sight, DNA attracts the compounds, thus forming a reservoir of molecules in the nuclear compartment. Cationic furamidine-type molecules can be bound to the nucleic acids in the cell nuclei but they could also be attracted by the relatively high negative electric potential across the mitochondrial membranes. For example, certain rhodamine compounds (rhodamine 123, rhodamine 6G, and rhodamine 3B) that are positively charged at physiological pH are able to stain mitochondria specifically, whereas uncharged fluorescein compounds do not (33) . Moreover, the fact that mitochondria contain a significant amount of nucleic acids could also play a role in directing the drug molecules to this compartment. Therefore, one can adopt the view that there may exist a competition or an exchange between nucleus and mitochondrion for the uptake of furamidine-type compounds. Depending on the structure of the terminal groups and the relative affinity for the target, the compound will be driven to the nucleus (e.g., DB75) or to the mitochondrion (e.g., DB569).

The cytotoxic measurements corroborate our previous study showing that nuclear uptake is not associated with cytotoxicity (11) . The biological activity may rather depend on the capacity of these compounds to recognize specific sequences in DNA and to interfere with the correct expression of some critical genes. Interestingly, the three compounds that seem to accumulate in mitochondria are the most cytotoxic compounds in the series. Mitochondrial targeting with phenyl-substituted diphenylfuran diamidines may be more efficient than nuclear targeting to prevent tumor cell growth

The DNA binding data are important because they show that it is possible to modulate the intracellular distribution of the furamidine derivatives without affecting their DNA binding and sequence recognition properties. The footprinting data agree well with the SPR results to indicate that the diamidine DB75 and its phenyl-substituted analogue DB659 both selectively recognize AT-rich sequences in DNA. Linear dichroism measurements (data not shown) indicated that the two compounds bind to the minor groove of DNA. The introduction of aromatic substituents may thus represent a useful strategy to control the intracellular compartmentalization of these DNA-binding agents.

At present, it is unclear whether DB569 is blocked from the nucleus or selectively attracted to the mitochondrion. Blocking seems likely, because the nucleus contains more DNA binding sites. There may be a specific mechanism (e.g., a transporter or an efflux pump) that restricts the nuclear transport of aromatic amidines. At first sight, the lack of nuclear uptake of a compound like DB569 would seem detrimental to its pharmacological effect. One would like to see a DNA-targeted drug entering freely the nucleus to reach its target. But in fact, the lack of nuclear binding and the preferential entrance into mitochondria may be advantageous. As noted above, mitochondria contain a significant proportion of nucleic acids and play a pivotal role in the propagation of the apoptotic signal by which most anticancer drugs kill cells. Mitochondrion is currently viewed as a valid and exploited target for cancer chemotherapy (34) . Several examples can be cited. The cationic rhodacyanine dye MKT-077 (1-ethyl-2-{[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl}pyridinium chloride) exhibits anticarcinoma activity based on selective mitochondria accumulation (35, 36, 37, 38) . Similarly, the DNA binding drug ditercalinium, a dicationic bisintercalator, induces mitochondrial damages (39) . Several reports suggest that mitochondria alterations in carcinomas by different antitumor drugs could play a role in drug-induced cytotoxicity. DNA-binding drugs like ethidium bromide, novobiocine, ellipticine, and paraquat all induce mitochondrial alterations (40, 41, 42, 43) . Therefore, a new strategy can be developed to target mitochondrial DNA using diphenylfuran analogues of DB569.

At this point it is interesting to refer also to the literature on the cellular uptake and distribution of diamidines in tumor cells. The amidine antibiotic hydroxystilbamidine (OHSA), which is the active constituent of the neuronal tracer Fluoro-Gold (44) , was shown to distribute in the cytoplasm and the nuclei of tumor cells by the early 1950s (45, 46, 47) . OHSA is an AT-specific DNA minor groove binder (48, 49, 50) , exactly like the diphenylfuran derivatives used here, and it is supposed to enter human cells by passive diffusion. It has been proposed that the uncharged form of weakly basic compounds is in equilibrium across cell membranes, and that the charged form does not cross membranes readily (51) . As a result, it is expected that if a weak base became protonated intracellularly (e.g., in an acidic compartment such as a lysosome or a mitochondria) it would accumulate in that compartment (51 , 52) . The maximum extent to which a compound can be accumulated into any compartment is equal to the ratio of hydrogen ion concentrations inside and outside the compartment (51) . Considering that the pH gradient across mitochondria is considerably less than that across lysosome (44) , the mitochondrial accumulation of a compound like DB569 is, at first sight, disfavored, but the high affinity of the compound for DNA may well alter the distribution to favor its sequestration in the mitochondria. The hypothesis that nuclear DNA holds DB75 whereas mitochondrial DNA holds DB569 is currently investigated through the use of specific cells such as the HeLa-Rho tumor cell line containing DNA-depleted mitochondria (53) .


   ACKNOWLEDGMENTS
 
We thank the Service Commun d’Imagerie Cellulaire de l’IMPRT (IFR114) for access to the fluorescence microscope. W. D. W. acknowledges an Institut National de la Santé et de la Recherche Médicale "Poste Orange" fellowship for research in Lille at Institut National de la Santé et de la Recherche Médicale U-524.


   FOOTNOTES
 
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 Supported by research grants from the Ligue Nationale Contre le Cancer (Equipe labellisée LA LIGUE; to C. B.), from NIH Grant GM61587 (to W. D. W. and D. W. B.), the Georgia Research Alliance, and a Gates Foundation Grant. Back

2 To whom requests for reprints should be addressed, at Institut National de la Santé et de la Recherche Médicale U-524, IRCL, Place de Verdun, 59045 Lille, France. Phone: 33-320-16-92-18; Fax: 33-320-16-92-29; E-mail: bailly{at}lille.inserm.fr Back

3 The abbreviations used are: DiOC6, 3,3-dihexyloxacarbocyanine iodide; MES, 4-morpholinepropanesulfonic acid; JC-1, tetrachloro-tetraethylbenzimidazolcarbocyanine iodide; SPR, surface plasmon resonance; PI, propidium iodide; RU, response unit; OHSA, 2-hydroxy-4,4'-diamidino stilbene. Back

Received 6/12/02. Accepted 10/17/02.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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