DNA Interaction and Dual Topoisomerase I and II Inhibition Properties of the Anti-Tumor Drug Prodigiosin
http://www.100md.com
《毒物学科学杂志》
Departament de Biologia Cel·lular i Anatomia Patològica, Cancer Cell Biology Research Group, Universitat de Barcelona, Barcelona, Spain E-08907
Departament de Química Orgànica, Universitat de Barcelona, Barcelona, Spain E-08028
Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Barcelona, Spain E-08028
ABSTRACT
Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin–DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confocal microscopy was performed to determine the possible implication of AIF in the prodigiosin–DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Our results demonstrate prodigiosin–DNA interaction in vitro and in cultured cells. It involves prodigiosin–DNA intercalation, with some preference for the alternating base pairs but with no discrimination between AT or CG sequences, dual abolition of topoisomerase I and II activity and, as consequence, DNA cleavage. Prodigiosin–DNA damage is independent of AIF. Furthermore, we found that copper-mediated cleavage activity is associated with pH (occurring at pH 6.8 rather than pH 7.4) and with the Cu2+ ion concentration. These results indicate DNA a therapeutic target for prodigiosin and could explain the apoptosis mechanism of action induced by this antineoplastic drug.
Key Words: DNA damage; prodigiosin; topoisomerase inhibition.
INTRODUCTION
The identification of novel targets and the development of more specific chemotherapy agents are two of the most important goals of research in cancer therapy. Apoptosis is a form of cell death involved in the action of several (and perhaps all) cancer-chemotherapy agents (Cameron and Feuer, 2000). One of its hallmarks is the degradation and concomitant compaction of chromatin, in which DNA fragmentation is initiated and propagated by single-stranded (ss) breaks that result in double-stranded (ds) fragments, which are then further digested to oligonucleosomal ladders. Chromatin processing may proceed by two redundant parallel pathways. The first is caspase-dependent and involves caspase-activated DNAse (CAD), responsible for oligonucleosomal DNA fragmentation and advanced chromatin condensation. The second is caspase-independent and involves apoptosis-inducing factor (AIF), which leads to large-scale DNA fragmentation and peripheral chromatin condensation (Susin et al., 2000). However, some anti-cancer drugs can directly induce DNA damage, and others act indirectly via molecular targets. In both cases, apoptosis could be a consequence of the injury (Hurley, 2002).
Topoisomerases have been established as effective chemotherapeutic targets. These enzymes modulate DNA superhelicity and act by introducing single (type I) or double (type II) DNA breaks. They are involved in DNA repair, replication, transcription, and chromosome segregation during mitosis. Two general classes of topoisomerase inhibitor mechanisms have been described. These are the classic topoisomerase poisons, such as camptothecin and etoposide, for topoisomerase I and II, respectively, which stabilize the cleavable complexes and stimulate enzyme-linked DNA breaks. They should be differentiated from catalytic inhibitors that act by blocking overall catalytic activity of the enzymes, such as aclarubicin and novobiocin (Froelich-Ammon and Osheroff, 1995). For topoisomerase-directed agents, resulting DNA damage can lead to cell cycle arrest and/or cell death by apoptosis (Kaufmann, 1998).
One of the first cellular responses to the introduction of ds breaks into DNA is the phosphorylation of H2AX, a histone H2A family member that forms foci at break sites. The number of introduced ds breaks is proportional to the number of H2AX molecules phosphorylated (p-H2AX) and the severity of the damage (Rogakou et al., 1999). Phosphorylated H2AX appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments, before the appearance of internucleosomal DNA fragments (Rogakou et al., 2000).
Prodigiosins (2-methyl-3-pentyl-6-methoxyprodigiosene), a family of natural red pigments, are synthesized from different microorganisms such as S. marcescens and Streptomyces. They are characterized by a common pyrrolyl pyrromethene skeleton and possess promising immunosuppressive and apoptotic properties (Perez-Tomas et al., 2003). Their potential immunosuppressive activity when administered at non-toxic concentrations is noteworthy (Songia et al., 1997). Therefore, D'Alessio et al. at Pharmacia & Upjohn have attempted the preparation of synthetic analogues of the natural prodigiosins to be used as immunosuppressive agents (D'Alessio et al., 2000). In terms of their anti-cancer properties, the National Cancer Institute (NCI) has also determined the cytotoxic properties of the prodigiosin-group natural products (Boyd, 1987). These molecules facilitate cell death by apoptosis and exhibit selective activity against breast (Soto-Cerrato et al., 2004; Yamamoto et al., 2000a), haematopoietic (Montaner et al., 2000; Yamamoto et al., 2000b), and colon cancer cell lines (Montaner and Perez-Tomas, 2001). They also were shown to act selectively in hepatocellular carcinoma xenografts (Yamamoto et al., 1999) and in B-cell chronic lymphocytic leukemia in 32 patients (Campas et al., 2003).
Comparison of the cytotoxic properties of prodigiosin (Fig. 1), prodigiosene, and 2-methylprodigiosene in vitro revealed the exceptional cytotoxic potency of prodigiosin (i.e., IC50: 225, 275, and 400 nM in the Jurkat, SW-620, and Ramos cell lines, respectively) (Montaner et al., 2000; Montaner and Perez-Tomas, 2001). This cytotoxic potency may be attributed to the presence of the prodigiosin C-6 methoxy substituent (Boger and Patel, 1988), as well as the A-pyrrole ring, which plays a key role in the cytotoxic potency of prodigiosins (Melvin et al., 2000).
Understanding the mechanism of action of the prodigiosins is essential for drug development and will require the identification and characterization of their cellular target. Their action depends in part on their ability to uncouple vacuolar H+-ATPase (V-ATPase) through promotion of the H+/Cl– symporter, and to induce neutralization of the acid compartment of cells, which leads to acidification of the cytoplasm, cell-cycle arrest, and apoptosis (Yamamoto et al., 1999, 2000a, 2000b). Prodigiosins can also activate either or both the p38-MAPK and the SAPK/JNK pathways, thereby inducing apoptosis (Montaner and Perez-Tomas 2002a, 2002b; Yamamoto et al., 2000b). In addition, Melvin et al. (1999, 2000) showed that tambjamine E and prodigiosin bind DNA. Although these data suggest a third possible mechanism of prodigiosin action, in cultured cells confirmation is still awaited.
During a study of prodigiosin internalization, we observed that this auto-fluorescent drug, with a maximum absorbance at 535 nm, promptly reached the nucleus of treated cells. This led to a sequence of research steps: first, to analyze the mode of prodigiosin interaction with DNA, second, to explore potential topoisomerase inhibition properties of prodigiosin, and finally, to evaluate DNA damage induced by prodigiosin in cultured cells.
Our results confirm DNA as a therapeutic target for prodigiosin and could explain the apoptotic mechanism of action of this natural drug.
MATERIALS AND METHODS
The supercoiled plasmid Blue Script cloning vector (pBS) was from Stratagene (La Jolla, CA), and the supercoiled pRYG was from TopoGEN (Columbus, OH). The enzymes XbaI and EcoRI, were purchased from Promega (Madison, WI). Topoisomerase I was from Sigma (St. Louis, MO), and Topoisomerase II from TopoGEN. Polyclonal antibodies to topoisomerase I and II were from TopoGEN (ref. 2011–1 and ref. 2012–2); those to AIF (ref. H-300), from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal antibody to p-H2AX from Upsdate (NY, ref. 05–636). Secondary antibodies were: horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG from BioRad (Hemel Hempstead,UK, ref. 170-6515), FluorolinkCy3 goat anti-rabbit IgG from Amersham Biosciences (Little Chalfont, UK, ref. PA43004), and Alexa fluor 488 goat anti-mouse IgG from Molecular Probes (Eugene, OR, ref. A-11001). Radioactive nucleotides were from Amersham Biotech (UK). All other reagents were obtained from Sigma. All materials used were molecular biology grade where available; otherwise the purest available material was used. Deionized water further purified with a Millipore Milli-Q system (Bedford, MA) was used.
All DNA was purified by two sequential phenol:chloroform:isoamyl alcohol (25:24:1) extractions and ethanol precipitation. The concentration of DNA was determined by OD260.
Prodigiosin was isolated from a S. marcescens 2170 culture broth as described previously (Montaner et al., 2000). Confirmation of its structure was obtained by 1H-nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Electrospray ionization, m/z 324.4 (M+H)+, (C20H25N3O requires 323.4381, MW average); 1H-NMR (CD3OD, 500 MHz, ppm): 10.71 (m, NH), 8.54 (m, NH), 7.08 (s, 1H), 6.95 (s, 1H), 6.88 (m, 1H), 6.83 (m, 1H), 6.30 (m, 1H), 6.25 (s, 1H), 3.96 (s, 3H), 2.43 (t, 2H), 1.58 (s, 3H), 1.2 ± 1.4 (m, 6H), 0.91 (t, 3H). Stock solutions were prepared in dimethyl sulfoxide (DMSO), and concentrations were determined by UV-Vis in 95% EtOH-HCl.
Cell lines and culture conditions.
Acute human T leukemia cells (Jurkat clone E6-1) and human breast adenocarcinoma cells (MCF7) were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco's modified Eagle medium (DMEM):HAM'S F-12 (1:1) medium, respectively. All media were supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine, at 37°C, 5% CO2 in air. In addition, MCF7 was cultured with 10 μg/ml insulin.
DNA intercalation of prodigiosin.
The supercoiled pBS DNA, prepared from DH5 cells, was purified using Qiagen Plasmid Maxi Kits (Qiagen, Hilden, Germany).
The study was performed on relaxed DNA. Supercoiled phosphate buffered saline (0.4 μg) was incubated with 1 unit of topoisomerase I, in 50 mM Tris-HCl pH 7.5, 1 mM ethylene diamnine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), and 20 % glycerol for 30 min, at 37°C. Relaxed DNA was then purified and incubated with prodigiosin (0.5–2 μM), in TE (10 mM Tris-HCl pH 6.8, 1 mM EDTA), overnight (14 h), at 37°C. Finally, samples were applied to 1.2 % agarose gels containing chloroquine (6 ng/μl), in 1x TAE (40 mM Tris-acetic acid pH 7.7, 2 mM EDTA), at 2 V/cm overnight, stained with 0.5 μg/ml ethidium bromide (EtBr), and photographed under UV light.
Competition dialysis assay.
To determine the preference of prodigiosin for base sequences of alternating and non-alternating C-G (CC and CG, respectively), and alternating and non-alternating A-T (AA and AT, respectively) base pairs, different DNA fragments of the same size were selected and dialyzed against a common solution containing prodigiosin. Each fragment provided different alternating and nonalternating sites (CCCCGGGG, 6 CC or GG and 1 CG; CGCGCGCG, 7CC or GG; AAAATTTT, 6 AA or TT and 1 AT; ATATATAT, 7 AT or TA). This method has been used previously with different ligands (Lisgarten et al., 2002; Ren and Chaires, 1999).
A buffer consisting of 6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA, and 185 mM NaCl pH 7.0, was used. Volumes of 0.5 ml, at 75 μM monomeric unit, of each of the DNA samples were pipetted into separate 0.5 ml Spectra/Por DispoDialyzer units with membrane pores of 1000 Da. These units were placed into a beaker containing 200 ml of dialysate solution and 1 μM ligand, and the contents were allowed to equilibrate, with continuous stirring for 24 h, at room temperature (RT). Then, DNA samples were removed from the DispoDialyzer and were adjusted to a final concentration of 1% (w/v) sodium dodecyl sulfate (SDS) by addition of 10% (w/v) stock solution. An appropriate correction for the slight dilution of the sample resulting from the addition of the SDS solution was made. The total concentration (Ct) of prodigiosin within each dialysis bag was determined spectrophotometrically using the appropriate wavelength and extinction coefficients. The free ligand concentration (Cf) was also determined spectrophotometrically using an aliquot of the dialysate solution, although its concentration did not vary appreciably from the initial concentration of 1 μM. The amount of prodigiosin bound (Cb) was determined by difference (Cb = Ct – Cf).
Topoisomerase I and topoisomerase II cleavage assays.
To study the effect of prodigiosin on topoisomerase I enzyme activity, 0.4 μg of supercoiled pBS DNA was incubated with 1 unit of topoisomerase I and the indicated final concentration of prodigiosin, in 20 μl of reaction mixture (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, and 20% glycerol) for 30 min at 37°C. Reactions were stopped by adding 1% SDS and 0.5 mg/ml proteinase K (final concentrations) and incubating for an additional 30 min at 37°C. Samples were then applied to 1.2% agarose gels in 1x TAE. Gels were run at 2 V/cm, overnight, stained with 0.5 μg/ml EtBr, and photographed under UV light.
For measuring topoisomerase II catalytic activity, 0.2 μg of supercoiled pRYG DNA was incubated with 1 unit of topoisomerase II, and prodigiosin when indicated, in 20 μl reaction buffer (50 mM Tris-HCl pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM DTT, and 30 μg/ml bovine serum albumin [BSA]), for 30 min, at 37°C. After the addition of 5 μl of a solution of 5% sarcosyl, 0.0025% bromophenol blue, 25% glycerol to stop the reaction, samples were observed in agarose gel electrophoresis as described above.
Analysis of topoisomerase I and II covalent complexes in Jurkat cells.
The complex of topoisomerase I and II were analyzed using the TopoGEN in vivo link kit for both enzymes, which is based upon physically separating the topoisomerase/DNA adducts from free DNA and using antibodies to measure bound topoisomerase I or II. Briefly, 1 x 107 Jurkat cells were untreated or treated with 3 μM prodigiosin, 100 μM camptothecin, or 100 μM etoposide, for 1 h. Cells were then collected, washed twice in phosphate buffered saline (PBS), and lysed in 1% sarcosyl in TE pH. Lysates were loaded on top of a CsCl gradient containing four densities (1.37, 1.50, 1.72, and 1.82 g/ml). The tubes were ultracentrifuged in a SW41 rotor (Beckman Coulter) at 31,000 rpm for 14 h at 25°C, and the fractions (400 μl) were collected from the top of the tubes. DNA was determined in each fraction by reading the absorbance at 260 nm. For topoisomerase detection, 50 μl of each fraction were mixed with 100 μl of 25 mM sodium phosphate buffer (pH 6.5), and applied to a nitrocellulose membrane by using a dot blot vacuum manifold. Detection of the topoisomerases/DNA complexes were performed by Western blot using the corresponding polyclonal topoisomerase I or topoisomerase II antibodies according to standard procedures.
DNA cleavage assay and Southern blot analysis in Jurkat cells.
In these assays, 1 x 106 Jurkat cells per ml were incubated in the absence (control cells) or in the presence of increasing amounts of prodigiosin (from 0.2 to 2 μM) or 100 μM etoposide (positive control). After 1 or 3 h incubation, cells were collected and resuspended in PBS at a concentration of 20 x 106 cells/ml. An equal volume of 1% low-melting-point agarose (SeaPlaque GTG, FMC) at 45°C was added, and pills of approximately 4 x 1 mm (width by height) were made with 20 μl of this mixture (containing approximately 2 x 105 cells). Then, the pills were kept at –4°C for 5 min for agarose solidification. The gel pills were incubated in lysis solution (0.5 M EDTA pH 9.0, 1% sarcosyl, 0.5 mg/ml proteinase K) at 50°C for 48 h. They were then washed five or six times in TE (pH 8.0) RT and electrophoresed in 0.75 % agarose. The pills were loaded on the top extreme of the electrophoresis comb before the agarose was carefully added. Pulsed-field gel electrophoresis (PFGE) was carried out with a CHEF-DR III apparatus (Bio-Rad, Hercules, CA) with a 120° reorientation angle. The gel was run in 0.5x TBE buffer (45 mM Tris-HCl pH 8.0, 45 mM boric acid, and 1 mM EDTA) at 20°C at 2 V/cm with a switch time changing linearly from 5 min to 2.5 h over 72 h. After the run, the gel was stained with EtBr for 30 min. DNA was denatured by soaking the gel in a 0.4 NaOH–1.5 M NaCl solution for 15 min, and then transferred onto a nylon membrane for 40 h in the same denaturing solution. The membrane was hybridized with a 32P-labeled Jurkat genomic DNA probe, digested previously overnight with XbaI. After stringency washes, it was exposed to x-ray film (Hyperfilm-max, Amersham), at –80°C.
Immunocytochemical study.
MCF7 (5 x 104 cells/0.5 ml), cultured in a 24-well plate containing glass coverslips, were incubated with prodigiosin (2 μM) for 3 h. Cupric acetate (2 μM) and DPQ (30 μM) were added 30 min before prodigiosin treatment. Then, cells were washed twice with PBS and fixed with methanol:acetic acid (1:1) for 15 min. After washing with PBS, they were permeabilized with 0.2 % Triton X-100 in PBS, for 5 min. Coverslips were incubated with normal goat serum (1:30), for 1 h at RT and overnight with anti-AIF (1:100) or anti-pH2AX (8 μg/ml), at 4°C. Finally, they were rinsed in PBS and incubated for 1 h with the corresponding secondary antibody (1:1000 and 10 μg/ml, respectively) at RT. Confocal microscopic examinations were performed with a Leica TCS SL inverted microscope.
Plasmid DNA cleavage.
This assay was performed as described previously (Borah et al., 1998). Briefly, supercoiled pBS (34 ng per 10 μl) was incubated with prodigiosin (at 1, 2, 5, 10, and 20 μM) plus cupric acetate (prodigiosin:Cu2+ 1:1, 1:2, 2:1, 4:1, or 10:1) in 10 mM Tris-HCl pH 6.8 or pH 7.4, for 30 min, at 37°C. Samples were run on 1.2 % agarose gel in 1x TAE, for 2 h at 80 V, stained with 0.5 μg/ml EtBr, and photographed under UV light.
DNA bands were quantified with the image analysis software program Phoretix 1-D advanced. Results were presented as percent of control (relative to the densitometry values). All data points shown are mean value ± SD of three independent experiments. Statistical significance of differences was assessed by ANOVA (Fisher's protected least significant difference (PLSD) test, p < 0.05, p < 0.01, or p < 0.001).
RESULTS
Prodigiosin Binding to DNA by Intercalation
We studied the effect of chloroquine, an intercalating agent that unwinds the DNA, in relaxed DNA incubated with prodigiosin. For this purpose, we used agarose gel containing chloroquine. Figure 2 shows how chloroquine alters the relative mobility of the topoisomers in lanes 1 and 2, corresponding to control supercoiled and relaxed pBS samples, respectively. By contrast, in prodigiosin-DNA samples (lanes 3–6), chloroquine is not able to unwind the DNA, suggesting that binding of prodigiosin to DNA occurs through intercalation between the stacked base pairs of native DNA. Furthermore, this assay shows that prodigiosin partially uncoils the ds helix.
DNA Sequence Selectivity of Prodigiosin
To find out the DNA sequence selectivity of prodigiosin for small DNA fragments, a competition dialysis assay was performed. Prodigiosin was dialyzed against different self-complementary oligonucleotides, which had the same number of bases. This ensured that the same number of possible similar sites were available, for ease of comparison. The experiment was carried out under experimental conditions at which is widely accepted that DNA is in B-form. Figure 3 shows some preference of prodigiosin for the alternating base pairs, but with no discrimination between AT or CG sequences.
Effect of Prodigiosin on Topoisomerases: Inhibition of Catalytic Activity
A plasmid cleavage assay was used to investigate the effect of prodigiosin on topoisomerase I and II under cell-free conditions. This topoisomerization assay provides a direct means of determining whether the drug affects the unwinding of closed circular duplex DNA. Both enzymes convert supercoiled DNA to nicked and relaxed DNA. When topoisomerase I and II catalytic activity was assayed after conversion of a supercoiled plasmid to relaxed circular DNA (Fig. 4), prodigiosin inhibited this process in a concentration-dependent manner, with an IC50 (μM): 2.2 ± 0.1 for topoisomerase I (Fig. 4A) and 5.1 ± 0.4 for topoisomerase II (Fig. 4B). These observations suggest that prodigiosin inhibits both topoisomerase I and II catalytic activity. The inhibition could result either from direct interference with the enzyme (i.e., stabilization of topoisomerase–DNA cleavable complexes) or from the intercalation of the drug into DNA, which also prevents the relaxation of the plasmid by the enzyme.
Topoisomerase-Mediated DNA Cleavage in Cultured Cells
To determine whether prodigiosin is active against endogenous topoisomerase I and/or II in culture cells, a link assay for cells (TopoGEN) was performed, where the amount of topoisomerase coincident with the DNA peak is a measure of covalent DNA–topoisomerase complexes. Cells were untreated (negative control), or treated for 1 h with 3 μM prodigiosin, 100 μM camptothecin, or 100 μM etoposide, the two drugs used as positive controls for topoisomerase I and II, respectively, and the cell extracts were fractionated by centrifugation with a CsCl gradient. DNA bands were shown between fractions 17 and 20 (as judged from absorbance measurements at 260 nm) near the bottom of the gradient. In the untreated cells, topoisomerase I was only found at the top of the CsCl gradient as free protein, whereas in prodigiosin-treated and camptothecin-treated cells, topoisomerase I signals were also detected in the DNA fractions (Fig. 5A). In the topoisomerase II study, immunoblotting revealed the presence of the enzyme as free protein and also in the DNA fractions for prodigiosin and etoposide (Fig. 5B). These results indicate that prodigiosin stabilizes topoisomerase I and II–DNA complexes.
Effects of Prodigiosin on DNA Integrity
To demonstrate whether prodigiosin, as topoisomerase I/II poison, was able to transform these enzymes into DNA-damaging agents, its effect in genomic DNA integrity was studied. Fragmented chromosomal DNA was allowed to migrate into agarose gels by PFGE and was visualized by hybridization with a labeled genomic probe (Fig. 6). In Jurkat T cells, after either 1 or 3 h, concentrations of prodigiosin from 0.2 μM to 2 μM gave rise to a detectable amount of chromosome damage (lanes 2–13). Lane 1, untreated cells; lane 14, as positive control cells were treated with etoposide (100 μM, 1h), a known topoisomerase II poison that induces DNA damage. The results obtained indicate that the pulsed field gel electrophoresis (PFGE) assay is sensitive enough to detect a level of DNA damage in prodigiosin-treated cells.
Prodigiosin-Induced DNA Cleavage Is Independent of AIF
An immunohistochemical study of p-H2AX was performed to detect genomic DNA ds breaks in MCF7 cells, which do not express caspase 3 because of a gene deletion. The possible mitochondrial AIF release to the nucleus was also studied by dual staining.
The immunostaining of untreated control cells (Fig. 7A) showed a punctate, cytoplasmic staining pattern for AIF (red), consistent with its localization in mitochondria, but no p-H2AX staining (green) was detected. In prodigiosin-treated cells (Fig. 7B), numerous p-H2AX foci appeared in nuclei, indicating a high level of ds, even though the AIF staining pattern did not change.
Copper-Mediated DNA Cleavage is Dependent on pH
The ability of prodigiosin to induce DNA cleavage in the presence of copper was analyzed by agarose gel electrophoresis with supercoiled plasmid DNA. Prodigiosin was used in the presence of Fe3+ and Mn2+ to cleave supercoiled pBS DNA, with negative results (data not shown). In the presence of Cu2+, prodigiosin cleaved supercoiled pBS DNA, generating a single nick into either strand of the supercoiled DNA (nicked DNA) before converting the plasmid to nicked linear DNA through a double-strand break (Figs. 8 and 9). The influence of the buffer pH on the ability of prodigiosin to cleave DNA is illustrated in Figure 8. Lanes 1 and 2 show that prodigiosin:Cu2+ (1:1), at the concentration of 20 and 10 μM, generated ds breaks at pH 6.8 (Fig. 8A and 8C) and 7.4 (Fig. 8B and 8D), as nicked and linear DNA were visible on the gel after 30 min incubation. Furthermore, at lower prodigiosin:Cu2+ (1:1) concentrations, the ability to cleave supercoiled DNA was lower at pH 7.4. At pH 6.8, 1 μM prodigiosin nicked 47.9 ± 3.5% of DNA (Fig. 8A and 8C, lane 5), whereas at pH 7.4, the same concentration of drug nicked 16.5 ± 2.1% of supercoiled DNA (Fig. 8B and 8D, lane 5). In these studies, the EC50 (μM) at pH 6.8 was 2.2 ± 0.04, and at pH 7.4 it was 2.6 ± 0.04.
The influence of the Cu2+ concentration on the ability of prodigiosin to cleave DNA is shown in Figure 9. At pH 6.8, DNA cleavage was greater in the presence of prodigiosin:Cu2+ (1:2) (lane 1) than with prodigiosin:Cu2+ (1:1) (Fig. 9A and 9C, lane 2) (p < 0.001), whereas at pH 7.4 (Fig. 9B and 9D, lanes 1 and 2), there was no significant effect (p > 0.05). Moreover, at lower concentrations of Cu2+ (lanes 3–5) prodigiosin also nicked DNA, but the efficiency of the cleavage was greater at pH 6.8 (Fig. 9A and 9B) than at pH 7.4 (Fig. 9B and 9D). Lane 5 shows that prodigiosin:Cu2+ (20:2) nicked 37.7 ± 6.5% supercoiled DNA at pH 6.8 (Fig. 9A and 9C), but not at pH 7.4 (Fig. 9B and 9C). In these assays, the EC50 (μM) was 1.9 ± 0.17 at pH 6.8 and 2.7 ± 0.19 at pH 7.4.
In both Figures 8 and 9, lanes 6 and 7 are the negative controls; lane 6 corresponds to the pBS plasmid incubated with the highest concentration of prodigiosin used in the assay (20 μM); lane 7, is the pBS plasmid incubated with the highest concentration of Cu2+ used in the assay (20 μM and 40 μM, respectively).
DISCUSSION
Anticancer agents that target DNA are some of the most effective agents in clinical use (Hurley, 2002). Prodigiosin is a promising new antineoplastic agent that triggers apoptosis in different cancer cell lines while acting rapidly, potently, and with no marked toxicity in nonmalignant cell lines (Perez-Tomas et al., 2003). Data in the present study point to DNA as a target of prodigiosin.
Multiple independent studies support the view that prodigiosin interacts directly with DNA. As described previously (Melvin et al., 1999), absorption spectroscopy measurements indicate that prodigiosin slightly increases the melting temperature of DNA in a way similar to that reported by us for another family of ionizable chromophores (Pons et al., 1991). This suggests that prodigiosin stabilizes the DNA double helix, a common feature of DNA-binding chemotherapeutic drugs (Bible et al., 2000). Also, a concentration-dependent inhibition of chloroquine was obtained with prodigiosin-treated DNA (Fig. 2). Chloroquine is an intercalating agent that unwinds DNA, resulting in a decrease in negative superhelicity and thus in the negative superhelical free energy activity. We have shown that prodigiosin competes with chloroquine for the unwinding of DNA and prevents its activity, suggesting that binding of prodigiosin to DNA occurs through intercalation between the stacked base pairs of native DNA. Furthermore, the chromophore prodigiosin interacts with DNA preferentially with alternating base pairs, but with no discrimination between the AT or GC regions (Fig. 3). This property could be explained by the planar tripyrrolic structure of this alkaloid (Fig. 1). Furthermore, our results also show that prodigiosin is a dual DNA topoisomerase I and II inhibitor (Figs. 4 and 5), trapping the enzyme–DNA cleavage complexes and leading to DNA strand breaks (Fig. 6). Apoptosis inducing factor/p-H2AX dual labeling in MCF7 cells suggested that the induction of DNA damage is independent of the apoptotic process (Fig. 7). Additionally, prodigiosin has the property of chelating copper and inducing oxidative DNA cleavage (Melvin et al., 2000). Using a cell free system, we have also observed that this DNA fragmentation depends on copper concentration and on pH (Figs. 8 and 9).
In a previous study of the DNA binding affinity of prodigiosin, carried on by spectrophotometric methods, a major affinity of prodigiosin versus poly(AT) than poly(GC) was detected (Melvin et al., 1999). In our study, in which we used a competition dialysis experiment, we did not find a preferential binding for either the AT or the GC sites, whereas we did detect some preference for alternating (AT, GC) versus non-alternating (AA, GG) base pairs, which is a general trend for intercalator molecules.
Intercalators are the group of compounds that bind with the bases of DNA, thereby interrupting transcription, replication, and/or topoisomerase activities (Portugal et al., 2001). Planar aromatic molecules are capable of interacting with the double helix and poisoning the action of topoisomerases, transforming these essential enzymes into lethal DNA-damaging agents (Turner and Denny, 1996). This property is considered to be important in the therapeutic actions of many anti-tumour agents, such as camptothecin and its analogues (topoisomerase I inhibitor), doxorubicin (topoisomerase II inhibitor; Hurley, 2002), and XR5000 (DACA (dual topoisomerase inhibitors); Baguley et al., 2003). Levels of topoisomerases I and II are generally elevated in cells that are undergoing rapid proliferation, including in some human cancers. This probably contributes to the increased response of aggressive cancers to topoisomerase-targeted agents (Froelich-Ammon and Osheroff, 1995; Holden, 2001). Because of the mechanism of drug action, the higher the physiological concentration of topoisomerases, the more lethal these poisons become. We reported that prodigiosin induces cell cycle arrest in G1–G5 and then apoptosis in Jurkat cells (Perez-Tomas and Montaner, 2003). This effect on proliferating cells agrees with cell response to DNA damage by activating a complex DNA-damage response pathway that includes cell cycle arrest, DNA repair, and in some circumstances, the triggering of apoptosis (Khanna and Jackson, 2001).
We recently showed that prodigiosin induces apoptosis in MCF7 (IC50: 1.1 μM) (Soto-Cerrato et al., 2004), but not oligonucleosomal DNA fragmentation (200 pb, ladder type). Nor was there a pronounced chromatin condensation in these cells (our unpublished observation). These phenomena are a consequence of caspase-activated DNAse (CAD) activity, the initiation of which depends on caspase 3, but caspase 3 is not expressed in MCF7 cells. Apoptosis inducing factor (AIF), a major player in caspase-independent cell death, is also able to induce large-scale DNA fragmentation in the apoptotic process, through translocation of AIF from mitochondria to nucleus (Susin et al., 2000). Interestingly, we observed numerous p-H2AX foci in nuclei of MCF7-treated cells, indicative of large chromatin domains at the sites of DNA damage, whereas AIF appeared in a mitochondrial staining pattern, and no translocation to the nucleus was detected (Fig. 7). These results support the hypothesis that ds DNA damage is independent of the chromatin processing related to the apoptotic process, possibly as consequence of topoisomerases inhibition. Ds-DNA cleavage creates damage which, for obvious reasons, is more difficult for the cell to repair. This could be important from a therapeutic point of view. The observed ability of prodigiosin to induce ds breaks means that it can be added to the short list of agents, mainly of natural origin, that induce ds cleavage and are used as anti-tumour drugs, such as dilanthadine (Branum and Que, 1999).
Additionally, although the ability of prodigiosin to induce oxidative DNA damage has been described previously (Melvin et al., 2000), we have determined a dependence of pH microenvironment and Cu2+ concentration. Thus, in suitable conditions, prodigiosin also can induce DNA cleavage through a copper–prodigiosin system.
In the present study we demonstrated that acidic pH (6.8) is more favorable to prodigiosin-mediated DNA breakage than weakly basic pH (7.4) (Fig. 8). It should be noted that tumours exhibit considerable spatial and possibly temporal heterogeneity in pH, ion concentrations, etc., which may modulate the delivery and cytotoxicity of chemotherapeutics (Kozin et al., 2001). In the complex tumor microenvironment, the intra–extracellular pH gradient is very important. Measurements of extracellular pH (pHe) obtained using microelectrodes have shown that pHe, in solid tumors, is typically in the range 6.6–7.0, whereas in normal tissue pHe is between 7.1 and 7.6 (Wike-Hooley et al., 1984). Although pHe in solid tumors tends to be acid, intracellular pH (pHi) is usually found to have similar values in solid tumors and normal tissues (Gillies et al., 1994). The difference in pHe between tumors and normal tissues provides an opportunity for tumor-selective therapy through the development of drugs that have increased toxicity at low pHe. In fact, the uptake and the cytotoxic efficacy of mitoxantrone, paclitaxel, and topotecan were reduced at pHe 6.5 as compared with pHe 7.4 (Vukovic and Tannock, 1997). Furthermore, chemotherapeutic agents with an appropriate pKa (6.5) can facilitate intracellular uptake and selectivity for cancer cells (Kozin et al., 2001).
In addition, metal ions play an important role in biological systems, and, without their catalytic presence in trace or ultratrace amounts, many biochemical reactions would not take place (Theophanides and Anastassopoulou, 2002). Copper is an essential metal and is distributed in all cellular organelles including mitochondria, lysosomes, endoplasmic reticulum, cytosol, and nucleus. Most "free" copper is undetectable. In dry non-cancerous breast tissue, the mean concentration of copper is 23 μM (1.47 ppm), whereas the mean concentration increases to 80 μM (5.12 ppm) in cancerous tissue. The Cu2+-Zn2+ index is also considerably higher in patients with cancer (Poo et al., 1997). These observations suggest that copper may promote prodigiosin-DNA damage in a concentration-dependent manner, as is demonstrated using an in vitro assay in Figure 9, where DNA damage is clearly detected at 5 μM copper, being more efficient at higher concentrations. This may explain the selective cytotoxicity of prodigiosin observed previously (Perez-Tomas et al., 2003).
Taken together, these results provide evidence that prodigiosin binds directly to DNA by intercalation, with preference for the alternating base pairs, is effective in poisoning both topoisomerase I and II enzymes, and is able to induce ss and ds DNA breaks, both in vitro and in cultured cells. The recognition of DNA as target may help to explain the complex cellular effects of the drug. Prodigiosin may ultimately prove of therapeutic value because of its effects on several cellular targets rather than just one. Our results also suggest the possible use of prodigiosin as a chemotherapeutic drug, given its potential cytotoxic selectivity in combination with copper under acidic conditions.
ACKNOWLEDGMENTS
This work was supported by a grant from the Ministry of Science and Technology and the European Union (SAF2001–3545), and by a TV3 Marató grant (ref.# 001510). M.M. is recipient of a research fellowship from the Generalitat de Catalunya (2000FI00035), and R..
REFERENCES
Baguley, B. C., Wakelin, L. P., Jacintho, J. D., and Kovacic, P. (2003). Mechanisms of action of DNA intercalating acridine-based drugs: how important are contributions from electron transfer and oxidative stress Curr. Med. Chem. 10, 2643–2649.
Bible, K. C., Bible, R. H., Kottke, Jr. T. J., Svingen, P. A., Xu, K., Pang, Y. P., Hadju, E., and Kaufmann, S. H. (2000). Flavopiridol binds to duplex DNA. Cancer Res. 60, 2419–2428.
Boger, D. L., and Patel, M. (1988). Total synthesis of prodigiosin, prodigiosene, and desmethoxyprodigiosin: Diels-Alder reactions of heterocyclic azadienes and development of an effective palladium (II)-promoted 2,2'-bipyrrole coupling procedure. J. Org. Chem. 53, 1405–1415.
Borah, S., Melvin, M. S., Lindquist, N., and Manderville, R. A. (1998). Copper-mediated nuclease activity of a tambjamine alkaloid. J. Am. Chem. Soc. 120, 4557–4562.
Boyd, M. R. (1987). The NCI in vitro anticancer drug discovery screen: concept, implementation, and operation, 1985–1995. In Anticancer drug development guide: preclinical screening, clinical trials, and approval (B. A. Teicher, Ed.), pp. 23–42. Humana Press, Totowa, NY.
Branum, M. E., and Que, L., Jr. (1999). Double-strand DNA hydrolysis by dilenthandine complexes. J. Biol. Inorg. Chem. 4, 593–600.
Cameron, R., and Feuer, G. (2000). Molecular, cellular and tissue reactions of apoptosis and their modulation by drugs. In Handbook of experimental pharmacology (R. D. Cameron, and L. A. Feuer, Eds.), pp. 37–58. Springer, Berlin.
Campas, C., Dalmau, M., Montaner, B., Barragan, M., Bellosillo, B., Colomer, D., Pons, G., Perez-Tomas, R., and Gil, J. (2003). Prodigiosin induces apoptosis of B and T cells from B-cell chronic lymphocytic leukemia. Leukemia 17, 746–750.
D'Alessio, R., Bargiotti, A., Carlini, O., Colotta, F., Ferrari, M., Gnocchi, P., Isseta, A., Mongelli, N., Motta, P., Rossi, A., et al. (2000). Synthesis and immunosuppressive activity of novel prodigiosin derivatives. J. Med. Chem. 43, 2557–2565.
Froelich-Ammon, S.J., and Osheroff, N. (1995). Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J. Biol. Chem. 270, 21429–21432.
Gillies, R. J., Liu, Z., and Bhujwalla, Z. (1994). 31P-MRS measurements of extracellular pH tumors using 3-aminopropylphosphonate. Am. J. Physiol. 267, 195–203.
Holden, J. A. (2001). DNA topoisomerases as anticancer drug targets: from the laboratory to the clinic. Curr. Med. Chem. Anticancer Agents 1, 1–25.
Hurley, L. H. (2002). DNA and its associated processes as targets for cancer therapy. Nat. Rev. Cancer 3, 188–200.
Kaufmann, S. H. (1998). Cell death induced by topoisomerase-targeted drugs: more questions than answers. Biochim. Biophys. Acta 1400, 195–211.
Khanna, K. K., and Jackson, S. P. (2001). DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 2, 247–254.
Kozin, S., Shkarin, P., and Gerweck, L. (2001). The cell transmembrane pH gradient in tumors enhances cytotoxicity of specific weak acid chemotherapeutics. Cancer Res. 61, 4740–4743.
Lisgarten, J., Coll, M., Portugal, J., Wright, C. W., and Aymami, J. (2002). The antimalarial drug cryptolepine intercalates specifically into DNA at cytosine-cytosine sites. Nature Struct. Biol. 9, 57–60.
Melvin, M. S., Ferguson, D. C., Lindquist, N., and Manderville, R. A. (1999). DNA binding by 4-methoxypyrrolic natural products. Preference for intercalation at AT sites by tambjamine E and prodigiosin. J. Org. Chem. 64, 6861–6869.
Melvin, M. S., Tomlinson, J. T., Saluta, G. R., Kucera, G.L., Lindquist, N., and Manderville, R. A. (2000). Double-strand DNA cleavage by copper·prodigiosin. J. Am. Chem. Soc. 122, 6333–6334.
Montaner, B., Navarro, S., Pique, M., Vilaseca, M., Martinell, M., Giralt, E., Gil, J., and Perez-Tomas. R. (2000). Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Br. J. Pharmacol. 131, 585–593.
Montaner, B., and Perez-Tomas, R. (2001). Prodigiosin-induced apoptosis in human colon cancer cells. Life Sci. 68, 2025–2036.
Montaner, B., and Perez-Tomas, R. (2002a). Activation of protein kinase C for protection of cells against apoptosis induced by the immunosuppressor prodigiosin. Biochem. Pharmacol. 63, 463–469.
Montaner, B., and Perez-Tomas, R. (2002b). The cytotoxic prodigiosin induces phosphorylation of p38-MAPK but not of SAPK/JNK. Toxicol. Lett. 129, 93–98.
Perez-Tomas, R., and Montaner, B. (2003). Effects of the proapoptotic drug prodigiosin on cell cycle-related proteins in Jurkat T cells. Histol. Histopathol. 18, 379–385.
Perez-Tomas, R., Montaner, B., Llagostera, E., and Soto-Cerrato, V. (2003). The prodigiosins, proapoptotic drugs with anticancer properties. Biochem. Pharmacol. 66, 1447–1452.
Pons, M., Campayo, L., Martinez-Balbas, M. A., Azorin, F., Navarro, P., and Giralt, E. (1991). A novel ionisable chromophore of 1,4-bis (alkylamino)benzo[g]phthalazine which interacts with DNA by intercalation. J. Med. Chem. 34, 82–86.
Poo, J. L., Romero, R. R., Robles, J. A., Momtemayor, A. C., Isoard, F., Estanes, A., and Uribe, M. (1997). Diagnostic value of the copper/zinc ratio in digestive cancer: a case control study. Arch. Med. Res. 28, 259–263.
Portugal, J., Martin, B., Vaquero, A., Ferrer, N., Villamarin, S., and Priebe, W. (2001). Analysis of the effects of daunorubicin and WP631 on transcription. Curr. Med. Chem. 8, 1–8.
Ren, J., and Chaires, J. B. (1999). Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 38, 16067–16075.
Rogakou, E. P., Boon, C., Redon, C., and Bonner, W. M. (1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell. Biol. 146, 905–916.
Rogakou, E. P., Nieves-Neira, W., Boon, C., Pommier, Y., and Bonner, W. M. (2000). Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J. Biol. Chem. 275, 9390–9395.
Songia, S., Mortellaro, A., Taverna, S., Fornasiero, C., Scheiber, E. A., Erba, E., Colotta, F., Mantovani, A., Isetta, A. M., and Golay, J. (1997). Characterization of the new immunosuppressive drug undecylprodigiosin in human lymphocytes. J. Immunol. 158, 3987–3995.
Soto-Cerrato, V., Llagostera, E., Montaner, B., Scheffer, G. L., and Perez-Tomas, R. (2004). The novel anticancer agent prodigiosin triggers mitochondria-mediated apoptosis operating irrespective of multidrug resistance in breast cancer cells. Biochem. Pharmacol. 68, 1345–1352.
Susin, S. A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler, M., Costantini, P., Ferri, K. F., Irinopoulou, T., Prevost, M. C., et al. (2000). Two distinct pathways leading to nuclear apoptosis. J. Exp. Med. 192, 571–579.
Theophanides, T., and Anastassopoulou, J. (2002). Copper and carcinogenesis. Crit. Rev. Oncol. Hemat. 42, 57–64.
Turner, P. R. and Denny, W. A. (1996). The mutagenic properties of DNA minor-grove binding ligands. Mutat. Res. 355, 141–169.
Vukovic, V., and Tannock, I. F. (1997). Influence of low pH on cytotoxicity of paclitaxel, mitoxantrone and topotecan. Br. J. Cancer 75, 1167–1172.
Wike-Hooley, J. L., Haveman, J., and Reinhold, J. S. (1984). The relevance of tumor pH to the treatment of malignant disease. Radiother. Oncol. 2, 343–366.
Yamamoto, C., Takemoto, H., Kuno, K., Yamamoto, D., Tsubura, A., Kamata, K., Hirata, H., Yamamoto, A., Kano, H., Seki, T., et al. (1999). Cycloprodigiosin hydrochloride, a new H+/Cl– symporter, induces apoptosis in human and rat hepatocellular cancer cell lines in vitro and inhibits the growth of hepatocellular carcinoma xenografts in nude mice. Hepatology 30, 894–902.
Yamamoto, D., Kiyozuka, Y., Uemura, Y., Yamamoto, C., Takemoto, H., Hirata, H., Tanaka, K., Hioki, K., and Tsubura, A. (2000a). Cycloprodigiosin hydrochloride, a H+/Cl– symporter, induces apoptosis in human breast cancer cell lines. J. Cancer Res. Clin. Oncol. 126, 191–197.
Yamamoto, D., Uemura, Y., Tanaka, K., Nakai, K., Yamamoto, C., Takemoto, H., Kamata, K., Hirata, H., and Hioki, K. (2000b). Cycloprodigiosin hydrochloride, H+/Cl– symporter, induces apoptosis and differentiation in HL-60 cells. Int. J. Cancer 88, 121–128.(Beatriz Montaner, Wilmar )
Departament de Química Orgànica, Universitat de Barcelona, Barcelona, Spain E-08028
Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Barcelona, Spain E-08028
ABSTRACT
Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin–DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confocal microscopy was performed to determine the possible implication of AIF in the prodigiosin–DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Our results demonstrate prodigiosin–DNA interaction in vitro and in cultured cells. It involves prodigiosin–DNA intercalation, with some preference for the alternating base pairs but with no discrimination between AT or CG sequences, dual abolition of topoisomerase I and II activity and, as consequence, DNA cleavage. Prodigiosin–DNA damage is independent of AIF. Furthermore, we found that copper-mediated cleavage activity is associated with pH (occurring at pH 6.8 rather than pH 7.4) and with the Cu2+ ion concentration. These results indicate DNA a therapeutic target for prodigiosin and could explain the apoptosis mechanism of action induced by this antineoplastic drug.
Key Words: DNA damage; prodigiosin; topoisomerase inhibition.
INTRODUCTION
The identification of novel targets and the development of more specific chemotherapy agents are two of the most important goals of research in cancer therapy. Apoptosis is a form of cell death involved in the action of several (and perhaps all) cancer-chemotherapy agents (Cameron and Feuer, 2000). One of its hallmarks is the degradation and concomitant compaction of chromatin, in which DNA fragmentation is initiated and propagated by single-stranded (ss) breaks that result in double-stranded (ds) fragments, which are then further digested to oligonucleosomal ladders. Chromatin processing may proceed by two redundant parallel pathways. The first is caspase-dependent and involves caspase-activated DNAse (CAD), responsible for oligonucleosomal DNA fragmentation and advanced chromatin condensation. The second is caspase-independent and involves apoptosis-inducing factor (AIF), which leads to large-scale DNA fragmentation and peripheral chromatin condensation (Susin et al., 2000). However, some anti-cancer drugs can directly induce DNA damage, and others act indirectly via molecular targets. In both cases, apoptosis could be a consequence of the injury (Hurley, 2002).
Topoisomerases have been established as effective chemotherapeutic targets. These enzymes modulate DNA superhelicity and act by introducing single (type I) or double (type II) DNA breaks. They are involved in DNA repair, replication, transcription, and chromosome segregation during mitosis. Two general classes of topoisomerase inhibitor mechanisms have been described. These are the classic topoisomerase poisons, such as camptothecin and etoposide, for topoisomerase I and II, respectively, which stabilize the cleavable complexes and stimulate enzyme-linked DNA breaks. They should be differentiated from catalytic inhibitors that act by blocking overall catalytic activity of the enzymes, such as aclarubicin and novobiocin (Froelich-Ammon and Osheroff, 1995). For topoisomerase-directed agents, resulting DNA damage can lead to cell cycle arrest and/or cell death by apoptosis (Kaufmann, 1998).
One of the first cellular responses to the introduction of ds breaks into DNA is the phosphorylation of H2AX, a histone H2A family member that forms foci at break sites. The number of introduced ds breaks is proportional to the number of H2AX molecules phosphorylated (p-H2AX) and the severity of the damage (Rogakou et al., 1999). Phosphorylated H2AX appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments, before the appearance of internucleosomal DNA fragments (Rogakou et al., 2000).
Prodigiosins (2-methyl-3-pentyl-6-methoxyprodigiosene), a family of natural red pigments, are synthesized from different microorganisms such as S. marcescens and Streptomyces. They are characterized by a common pyrrolyl pyrromethene skeleton and possess promising immunosuppressive and apoptotic properties (Perez-Tomas et al., 2003). Their potential immunosuppressive activity when administered at non-toxic concentrations is noteworthy (Songia et al., 1997). Therefore, D'Alessio et al. at Pharmacia & Upjohn have attempted the preparation of synthetic analogues of the natural prodigiosins to be used as immunosuppressive agents (D'Alessio et al., 2000). In terms of their anti-cancer properties, the National Cancer Institute (NCI) has also determined the cytotoxic properties of the prodigiosin-group natural products (Boyd, 1987). These molecules facilitate cell death by apoptosis and exhibit selective activity against breast (Soto-Cerrato et al., 2004; Yamamoto et al., 2000a), haematopoietic (Montaner et al., 2000; Yamamoto et al., 2000b), and colon cancer cell lines (Montaner and Perez-Tomas, 2001). They also were shown to act selectively in hepatocellular carcinoma xenografts (Yamamoto et al., 1999) and in B-cell chronic lymphocytic leukemia in 32 patients (Campas et al., 2003).
Comparison of the cytotoxic properties of prodigiosin (Fig. 1), prodigiosene, and 2-methylprodigiosene in vitro revealed the exceptional cytotoxic potency of prodigiosin (i.e., IC50: 225, 275, and 400 nM in the Jurkat, SW-620, and Ramos cell lines, respectively) (Montaner et al., 2000; Montaner and Perez-Tomas, 2001). This cytotoxic potency may be attributed to the presence of the prodigiosin C-6 methoxy substituent (Boger and Patel, 1988), as well as the A-pyrrole ring, which plays a key role in the cytotoxic potency of prodigiosins (Melvin et al., 2000).
Understanding the mechanism of action of the prodigiosins is essential for drug development and will require the identification and characterization of their cellular target. Their action depends in part on their ability to uncouple vacuolar H+-ATPase (V-ATPase) through promotion of the H+/Cl– symporter, and to induce neutralization of the acid compartment of cells, which leads to acidification of the cytoplasm, cell-cycle arrest, and apoptosis (Yamamoto et al., 1999, 2000a, 2000b). Prodigiosins can also activate either or both the p38-MAPK and the SAPK/JNK pathways, thereby inducing apoptosis (Montaner and Perez-Tomas 2002a, 2002b; Yamamoto et al., 2000b). In addition, Melvin et al. (1999, 2000) showed that tambjamine E and prodigiosin bind DNA. Although these data suggest a third possible mechanism of prodigiosin action, in cultured cells confirmation is still awaited.
During a study of prodigiosin internalization, we observed that this auto-fluorescent drug, with a maximum absorbance at 535 nm, promptly reached the nucleus of treated cells. This led to a sequence of research steps: first, to analyze the mode of prodigiosin interaction with DNA, second, to explore potential topoisomerase inhibition properties of prodigiosin, and finally, to evaluate DNA damage induced by prodigiosin in cultured cells.
Our results confirm DNA as a therapeutic target for prodigiosin and could explain the apoptotic mechanism of action of this natural drug.
MATERIALS AND METHODS
The supercoiled plasmid Blue Script cloning vector (pBS) was from Stratagene (La Jolla, CA), and the supercoiled pRYG was from TopoGEN (Columbus, OH). The enzymes XbaI and EcoRI, were purchased from Promega (Madison, WI). Topoisomerase I was from Sigma (St. Louis, MO), and Topoisomerase II from TopoGEN. Polyclonal antibodies to topoisomerase I and II were from TopoGEN (ref. 2011–1 and ref. 2012–2); those to AIF (ref. H-300), from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal antibody to p-H2AX from Upsdate (NY, ref. 05–636). Secondary antibodies were: horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG from BioRad (Hemel Hempstead,UK, ref. 170-6515), FluorolinkCy3 goat anti-rabbit IgG from Amersham Biosciences (Little Chalfont, UK, ref. PA43004), and Alexa fluor 488 goat anti-mouse IgG from Molecular Probes (Eugene, OR, ref. A-11001). Radioactive nucleotides were from Amersham Biotech (UK). All other reagents were obtained from Sigma. All materials used were molecular biology grade where available; otherwise the purest available material was used. Deionized water further purified with a Millipore Milli-Q system (Bedford, MA) was used.
All DNA was purified by two sequential phenol:chloroform:isoamyl alcohol (25:24:1) extractions and ethanol precipitation. The concentration of DNA was determined by OD260.
Prodigiosin was isolated from a S. marcescens 2170 culture broth as described previously (Montaner et al., 2000). Confirmation of its structure was obtained by 1H-nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Electrospray ionization, m/z 324.4 (M+H)+, (C20H25N3O requires 323.4381, MW average); 1H-NMR (CD3OD, 500 MHz, ppm): 10.71 (m, NH), 8.54 (m, NH), 7.08 (s, 1H), 6.95 (s, 1H), 6.88 (m, 1H), 6.83 (m, 1H), 6.30 (m, 1H), 6.25 (s, 1H), 3.96 (s, 3H), 2.43 (t, 2H), 1.58 (s, 3H), 1.2 ± 1.4 (m, 6H), 0.91 (t, 3H). Stock solutions were prepared in dimethyl sulfoxide (DMSO), and concentrations were determined by UV-Vis in 95% EtOH-HCl.
Cell lines and culture conditions.
Acute human T leukemia cells (Jurkat clone E6-1) and human breast adenocarcinoma cells (MCF7) were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco's modified Eagle medium (DMEM):HAM'S F-12 (1:1) medium, respectively. All media were supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine, at 37°C, 5% CO2 in air. In addition, MCF7 was cultured with 10 μg/ml insulin.
DNA intercalation of prodigiosin.
The supercoiled pBS DNA, prepared from DH5 cells, was purified using Qiagen Plasmid Maxi Kits (Qiagen, Hilden, Germany).
The study was performed on relaxed DNA. Supercoiled phosphate buffered saline (0.4 μg) was incubated with 1 unit of topoisomerase I, in 50 mM Tris-HCl pH 7.5, 1 mM ethylene diamnine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), and 20 % glycerol for 30 min, at 37°C. Relaxed DNA was then purified and incubated with prodigiosin (0.5–2 μM), in TE (10 mM Tris-HCl pH 6.8, 1 mM EDTA), overnight (14 h), at 37°C. Finally, samples were applied to 1.2 % agarose gels containing chloroquine (6 ng/μl), in 1x TAE (40 mM Tris-acetic acid pH 7.7, 2 mM EDTA), at 2 V/cm overnight, stained with 0.5 μg/ml ethidium bromide (EtBr), and photographed under UV light.
Competition dialysis assay.
To determine the preference of prodigiosin for base sequences of alternating and non-alternating C-G (CC and CG, respectively), and alternating and non-alternating A-T (AA and AT, respectively) base pairs, different DNA fragments of the same size were selected and dialyzed against a common solution containing prodigiosin. Each fragment provided different alternating and nonalternating sites (CCCCGGGG, 6 CC or GG and 1 CG; CGCGCGCG, 7CC or GG; AAAATTTT, 6 AA or TT and 1 AT; ATATATAT, 7 AT or TA). This method has been used previously with different ligands (Lisgarten et al., 2002; Ren and Chaires, 1999).
A buffer consisting of 6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA, and 185 mM NaCl pH 7.0, was used. Volumes of 0.5 ml, at 75 μM monomeric unit, of each of the DNA samples were pipetted into separate 0.5 ml Spectra/Por DispoDialyzer units with membrane pores of 1000 Da. These units were placed into a beaker containing 200 ml of dialysate solution and 1 μM ligand, and the contents were allowed to equilibrate, with continuous stirring for 24 h, at room temperature (RT). Then, DNA samples were removed from the DispoDialyzer and were adjusted to a final concentration of 1% (w/v) sodium dodecyl sulfate (SDS) by addition of 10% (w/v) stock solution. An appropriate correction for the slight dilution of the sample resulting from the addition of the SDS solution was made. The total concentration (Ct) of prodigiosin within each dialysis bag was determined spectrophotometrically using the appropriate wavelength and extinction coefficients. The free ligand concentration (Cf) was also determined spectrophotometrically using an aliquot of the dialysate solution, although its concentration did not vary appreciably from the initial concentration of 1 μM. The amount of prodigiosin bound (Cb) was determined by difference (Cb = Ct – Cf).
Topoisomerase I and topoisomerase II cleavage assays.
To study the effect of prodigiosin on topoisomerase I enzyme activity, 0.4 μg of supercoiled pBS DNA was incubated with 1 unit of topoisomerase I and the indicated final concentration of prodigiosin, in 20 μl of reaction mixture (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, and 20% glycerol) for 30 min at 37°C. Reactions were stopped by adding 1% SDS and 0.5 mg/ml proteinase K (final concentrations) and incubating for an additional 30 min at 37°C. Samples were then applied to 1.2% agarose gels in 1x TAE. Gels were run at 2 V/cm, overnight, stained with 0.5 μg/ml EtBr, and photographed under UV light.
For measuring topoisomerase II catalytic activity, 0.2 μg of supercoiled pRYG DNA was incubated with 1 unit of topoisomerase II, and prodigiosin when indicated, in 20 μl reaction buffer (50 mM Tris-HCl pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM DTT, and 30 μg/ml bovine serum albumin [BSA]), for 30 min, at 37°C. After the addition of 5 μl of a solution of 5% sarcosyl, 0.0025% bromophenol blue, 25% glycerol to stop the reaction, samples were observed in agarose gel electrophoresis as described above.
Analysis of topoisomerase I and II covalent complexes in Jurkat cells.
The complex of topoisomerase I and II were analyzed using the TopoGEN in vivo link kit for both enzymes, which is based upon physically separating the topoisomerase/DNA adducts from free DNA and using antibodies to measure bound topoisomerase I or II. Briefly, 1 x 107 Jurkat cells were untreated or treated with 3 μM prodigiosin, 100 μM camptothecin, or 100 μM etoposide, for 1 h. Cells were then collected, washed twice in phosphate buffered saline (PBS), and lysed in 1% sarcosyl in TE pH. Lysates were loaded on top of a CsCl gradient containing four densities (1.37, 1.50, 1.72, and 1.82 g/ml). The tubes were ultracentrifuged in a SW41 rotor (Beckman Coulter) at 31,000 rpm for 14 h at 25°C, and the fractions (400 μl) were collected from the top of the tubes. DNA was determined in each fraction by reading the absorbance at 260 nm. For topoisomerase detection, 50 μl of each fraction were mixed with 100 μl of 25 mM sodium phosphate buffer (pH 6.5), and applied to a nitrocellulose membrane by using a dot blot vacuum manifold. Detection of the topoisomerases/DNA complexes were performed by Western blot using the corresponding polyclonal topoisomerase I or topoisomerase II antibodies according to standard procedures.
DNA cleavage assay and Southern blot analysis in Jurkat cells.
In these assays, 1 x 106 Jurkat cells per ml were incubated in the absence (control cells) or in the presence of increasing amounts of prodigiosin (from 0.2 to 2 μM) or 100 μM etoposide (positive control). After 1 or 3 h incubation, cells were collected and resuspended in PBS at a concentration of 20 x 106 cells/ml. An equal volume of 1% low-melting-point agarose (SeaPlaque GTG, FMC) at 45°C was added, and pills of approximately 4 x 1 mm (width by height) were made with 20 μl of this mixture (containing approximately 2 x 105 cells). Then, the pills were kept at –4°C for 5 min for agarose solidification. The gel pills were incubated in lysis solution (0.5 M EDTA pH 9.0, 1% sarcosyl, 0.5 mg/ml proteinase K) at 50°C for 48 h. They were then washed five or six times in TE (pH 8.0) RT and electrophoresed in 0.75 % agarose. The pills were loaded on the top extreme of the electrophoresis comb before the agarose was carefully added. Pulsed-field gel electrophoresis (PFGE) was carried out with a CHEF-DR III apparatus (Bio-Rad, Hercules, CA) with a 120° reorientation angle. The gel was run in 0.5x TBE buffer (45 mM Tris-HCl pH 8.0, 45 mM boric acid, and 1 mM EDTA) at 20°C at 2 V/cm with a switch time changing linearly from 5 min to 2.5 h over 72 h. After the run, the gel was stained with EtBr for 30 min. DNA was denatured by soaking the gel in a 0.4 NaOH–1.5 M NaCl solution for 15 min, and then transferred onto a nylon membrane for 40 h in the same denaturing solution. The membrane was hybridized with a 32P-labeled Jurkat genomic DNA probe, digested previously overnight with XbaI. After stringency washes, it was exposed to x-ray film (Hyperfilm-max, Amersham), at –80°C.
Immunocytochemical study.
MCF7 (5 x 104 cells/0.5 ml), cultured in a 24-well plate containing glass coverslips, were incubated with prodigiosin (2 μM) for 3 h. Cupric acetate (2 μM) and DPQ (30 μM) were added 30 min before prodigiosin treatment. Then, cells were washed twice with PBS and fixed with methanol:acetic acid (1:1) for 15 min. After washing with PBS, they were permeabilized with 0.2 % Triton X-100 in PBS, for 5 min. Coverslips were incubated with normal goat serum (1:30), for 1 h at RT and overnight with anti-AIF (1:100) or anti-pH2AX (8 μg/ml), at 4°C. Finally, they were rinsed in PBS and incubated for 1 h with the corresponding secondary antibody (1:1000 and 10 μg/ml, respectively) at RT. Confocal microscopic examinations were performed with a Leica TCS SL inverted microscope.
Plasmid DNA cleavage.
This assay was performed as described previously (Borah et al., 1998). Briefly, supercoiled pBS (34 ng per 10 μl) was incubated with prodigiosin (at 1, 2, 5, 10, and 20 μM) plus cupric acetate (prodigiosin:Cu2+ 1:1, 1:2, 2:1, 4:1, or 10:1) in 10 mM Tris-HCl pH 6.8 or pH 7.4, for 30 min, at 37°C. Samples were run on 1.2 % agarose gel in 1x TAE, for 2 h at 80 V, stained with 0.5 μg/ml EtBr, and photographed under UV light.
DNA bands were quantified with the image analysis software program Phoretix 1-D advanced. Results were presented as percent of control (relative to the densitometry values). All data points shown are mean value ± SD of three independent experiments. Statistical significance of differences was assessed by ANOVA (Fisher's protected least significant difference (PLSD) test, p < 0.05, p < 0.01, or p < 0.001).
RESULTS
Prodigiosin Binding to DNA by Intercalation
We studied the effect of chloroquine, an intercalating agent that unwinds the DNA, in relaxed DNA incubated with prodigiosin. For this purpose, we used agarose gel containing chloroquine. Figure 2 shows how chloroquine alters the relative mobility of the topoisomers in lanes 1 and 2, corresponding to control supercoiled and relaxed pBS samples, respectively. By contrast, in prodigiosin-DNA samples (lanes 3–6), chloroquine is not able to unwind the DNA, suggesting that binding of prodigiosin to DNA occurs through intercalation between the stacked base pairs of native DNA. Furthermore, this assay shows that prodigiosin partially uncoils the ds helix.
DNA Sequence Selectivity of Prodigiosin
To find out the DNA sequence selectivity of prodigiosin for small DNA fragments, a competition dialysis assay was performed. Prodigiosin was dialyzed against different self-complementary oligonucleotides, which had the same number of bases. This ensured that the same number of possible similar sites were available, for ease of comparison. The experiment was carried out under experimental conditions at which is widely accepted that DNA is in B-form. Figure 3 shows some preference of prodigiosin for the alternating base pairs, but with no discrimination between AT or CG sequences.
Effect of Prodigiosin on Topoisomerases: Inhibition of Catalytic Activity
A plasmid cleavage assay was used to investigate the effect of prodigiosin on topoisomerase I and II under cell-free conditions. This topoisomerization assay provides a direct means of determining whether the drug affects the unwinding of closed circular duplex DNA. Both enzymes convert supercoiled DNA to nicked and relaxed DNA. When topoisomerase I and II catalytic activity was assayed after conversion of a supercoiled plasmid to relaxed circular DNA (Fig. 4), prodigiosin inhibited this process in a concentration-dependent manner, with an IC50 (μM): 2.2 ± 0.1 for topoisomerase I (Fig. 4A) and 5.1 ± 0.4 for topoisomerase II (Fig. 4B). These observations suggest that prodigiosin inhibits both topoisomerase I and II catalytic activity. The inhibition could result either from direct interference with the enzyme (i.e., stabilization of topoisomerase–DNA cleavable complexes) or from the intercalation of the drug into DNA, which also prevents the relaxation of the plasmid by the enzyme.
Topoisomerase-Mediated DNA Cleavage in Cultured Cells
To determine whether prodigiosin is active against endogenous topoisomerase I and/or II in culture cells, a link assay for cells (TopoGEN) was performed, where the amount of topoisomerase coincident with the DNA peak is a measure of covalent DNA–topoisomerase complexes. Cells were untreated (negative control), or treated for 1 h with 3 μM prodigiosin, 100 μM camptothecin, or 100 μM etoposide, the two drugs used as positive controls for topoisomerase I and II, respectively, and the cell extracts were fractionated by centrifugation with a CsCl gradient. DNA bands were shown between fractions 17 and 20 (as judged from absorbance measurements at 260 nm) near the bottom of the gradient. In the untreated cells, topoisomerase I was only found at the top of the CsCl gradient as free protein, whereas in prodigiosin-treated and camptothecin-treated cells, topoisomerase I signals were also detected in the DNA fractions (Fig. 5A). In the topoisomerase II study, immunoblotting revealed the presence of the enzyme as free protein and also in the DNA fractions for prodigiosin and etoposide (Fig. 5B). These results indicate that prodigiosin stabilizes topoisomerase I and II–DNA complexes.
Effects of Prodigiosin on DNA Integrity
To demonstrate whether prodigiosin, as topoisomerase I/II poison, was able to transform these enzymes into DNA-damaging agents, its effect in genomic DNA integrity was studied. Fragmented chromosomal DNA was allowed to migrate into agarose gels by PFGE and was visualized by hybridization with a labeled genomic probe (Fig. 6). In Jurkat T cells, after either 1 or 3 h, concentrations of prodigiosin from 0.2 μM to 2 μM gave rise to a detectable amount of chromosome damage (lanes 2–13). Lane 1, untreated cells; lane 14, as positive control cells were treated with etoposide (100 μM, 1h), a known topoisomerase II poison that induces DNA damage. The results obtained indicate that the pulsed field gel electrophoresis (PFGE) assay is sensitive enough to detect a level of DNA damage in prodigiosin-treated cells.
Prodigiosin-Induced DNA Cleavage Is Independent of AIF
An immunohistochemical study of p-H2AX was performed to detect genomic DNA ds breaks in MCF7 cells, which do not express caspase 3 because of a gene deletion. The possible mitochondrial AIF release to the nucleus was also studied by dual staining.
The immunostaining of untreated control cells (Fig. 7A) showed a punctate, cytoplasmic staining pattern for AIF (red), consistent with its localization in mitochondria, but no p-H2AX staining (green) was detected. In prodigiosin-treated cells (Fig. 7B), numerous p-H2AX foci appeared in nuclei, indicating a high level of ds, even though the AIF staining pattern did not change.
Copper-Mediated DNA Cleavage is Dependent on pH
The ability of prodigiosin to induce DNA cleavage in the presence of copper was analyzed by agarose gel electrophoresis with supercoiled plasmid DNA. Prodigiosin was used in the presence of Fe3+ and Mn2+ to cleave supercoiled pBS DNA, with negative results (data not shown). In the presence of Cu2+, prodigiosin cleaved supercoiled pBS DNA, generating a single nick into either strand of the supercoiled DNA (nicked DNA) before converting the plasmid to nicked linear DNA through a double-strand break (Figs. 8 and 9). The influence of the buffer pH on the ability of prodigiosin to cleave DNA is illustrated in Figure 8. Lanes 1 and 2 show that prodigiosin:Cu2+ (1:1), at the concentration of 20 and 10 μM, generated ds breaks at pH 6.8 (Fig. 8A and 8C) and 7.4 (Fig. 8B and 8D), as nicked and linear DNA were visible on the gel after 30 min incubation. Furthermore, at lower prodigiosin:Cu2+ (1:1) concentrations, the ability to cleave supercoiled DNA was lower at pH 7.4. At pH 6.8, 1 μM prodigiosin nicked 47.9 ± 3.5% of DNA (Fig. 8A and 8C, lane 5), whereas at pH 7.4, the same concentration of drug nicked 16.5 ± 2.1% of supercoiled DNA (Fig. 8B and 8D, lane 5). In these studies, the EC50 (μM) at pH 6.8 was 2.2 ± 0.04, and at pH 7.4 it was 2.6 ± 0.04.
The influence of the Cu2+ concentration on the ability of prodigiosin to cleave DNA is shown in Figure 9. At pH 6.8, DNA cleavage was greater in the presence of prodigiosin:Cu2+ (1:2) (lane 1) than with prodigiosin:Cu2+ (1:1) (Fig. 9A and 9C, lane 2) (p < 0.001), whereas at pH 7.4 (Fig. 9B and 9D, lanes 1 and 2), there was no significant effect (p > 0.05). Moreover, at lower concentrations of Cu2+ (lanes 3–5) prodigiosin also nicked DNA, but the efficiency of the cleavage was greater at pH 6.8 (Fig. 9A and 9B) than at pH 7.4 (Fig. 9B and 9D). Lane 5 shows that prodigiosin:Cu2+ (20:2) nicked 37.7 ± 6.5% supercoiled DNA at pH 6.8 (Fig. 9A and 9C), but not at pH 7.4 (Fig. 9B and 9C). In these assays, the EC50 (μM) was 1.9 ± 0.17 at pH 6.8 and 2.7 ± 0.19 at pH 7.4.
In both Figures 8 and 9, lanes 6 and 7 are the negative controls; lane 6 corresponds to the pBS plasmid incubated with the highest concentration of prodigiosin used in the assay (20 μM); lane 7, is the pBS plasmid incubated with the highest concentration of Cu2+ used in the assay (20 μM and 40 μM, respectively).
DISCUSSION
Anticancer agents that target DNA are some of the most effective agents in clinical use (Hurley, 2002). Prodigiosin is a promising new antineoplastic agent that triggers apoptosis in different cancer cell lines while acting rapidly, potently, and with no marked toxicity in nonmalignant cell lines (Perez-Tomas et al., 2003). Data in the present study point to DNA as a target of prodigiosin.
Multiple independent studies support the view that prodigiosin interacts directly with DNA. As described previously (Melvin et al., 1999), absorption spectroscopy measurements indicate that prodigiosin slightly increases the melting temperature of DNA in a way similar to that reported by us for another family of ionizable chromophores (Pons et al., 1991). This suggests that prodigiosin stabilizes the DNA double helix, a common feature of DNA-binding chemotherapeutic drugs (Bible et al., 2000). Also, a concentration-dependent inhibition of chloroquine was obtained with prodigiosin-treated DNA (Fig. 2). Chloroquine is an intercalating agent that unwinds DNA, resulting in a decrease in negative superhelicity and thus in the negative superhelical free energy activity. We have shown that prodigiosin competes with chloroquine for the unwinding of DNA and prevents its activity, suggesting that binding of prodigiosin to DNA occurs through intercalation between the stacked base pairs of native DNA. Furthermore, the chromophore prodigiosin interacts with DNA preferentially with alternating base pairs, but with no discrimination between the AT or GC regions (Fig. 3). This property could be explained by the planar tripyrrolic structure of this alkaloid (Fig. 1). Furthermore, our results also show that prodigiosin is a dual DNA topoisomerase I and II inhibitor (Figs. 4 and 5), trapping the enzyme–DNA cleavage complexes and leading to DNA strand breaks (Fig. 6). Apoptosis inducing factor/p-H2AX dual labeling in MCF7 cells suggested that the induction of DNA damage is independent of the apoptotic process (Fig. 7). Additionally, prodigiosin has the property of chelating copper and inducing oxidative DNA cleavage (Melvin et al., 2000). Using a cell free system, we have also observed that this DNA fragmentation depends on copper concentration and on pH (Figs. 8 and 9).
In a previous study of the DNA binding affinity of prodigiosin, carried on by spectrophotometric methods, a major affinity of prodigiosin versus poly(AT) than poly(GC) was detected (Melvin et al., 1999). In our study, in which we used a competition dialysis experiment, we did not find a preferential binding for either the AT or the GC sites, whereas we did detect some preference for alternating (AT, GC) versus non-alternating (AA, GG) base pairs, which is a general trend for intercalator molecules.
Intercalators are the group of compounds that bind with the bases of DNA, thereby interrupting transcription, replication, and/or topoisomerase activities (Portugal et al., 2001). Planar aromatic molecules are capable of interacting with the double helix and poisoning the action of topoisomerases, transforming these essential enzymes into lethal DNA-damaging agents (Turner and Denny, 1996). This property is considered to be important in the therapeutic actions of many anti-tumour agents, such as camptothecin and its analogues (topoisomerase I inhibitor), doxorubicin (topoisomerase II inhibitor; Hurley, 2002), and XR5000 (DACA (dual topoisomerase inhibitors); Baguley et al., 2003). Levels of topoisomerases I and II are generally elevated in cells that are undergoing rapid proliferation, including in some human cancers. This probably contributes to the increased response of aggressive cancers to topoisomerase-targeted agents (Froelich-Ammon and Osheroff, 1995; Holden, 2001). Because of the mechanism of drug action, the higher the physiological concentration of topoisomerases, the more lethal these poisons become. We reported that prodigiosin induces cell cycle arrest in G1–G5 and then apoptosis in Jurkat cells (Perez-Tomas and Montaner, 2003). This effect on proliferating cells agrees with cell response to DNA damage by activating a complex DNA-damage response pathway that includes cell cycle arrest, DNA repair, and in some circumstances, the triggering of apoptosis (Khanna and Jackson, 2001).
We recently showed that prodigiosin induces apoptosis in MCF7 (IC50: 1.1 μM) (Soto-Cerrato et al., 2004), but not oligonucleosomal DNA fragmentation (200 pb, ladder type). Nor was there a pronounced chromatin condensation in these cells (our unpublished observation). These phenomena are a consequence of caspase-activated DNAse (CAD) activity, the initiation of which depends on caspase 3, but caspase 3 is not expressed in MCF7 cells. Apoptosis inducing factor (AIF), a major player in caspase-independent cell death, is also able to induce large-scale DNA fragmentation in the apoptotic process, through translocation of AIF from mitochondria to nucleus (Susin et al., 2000). Interestingly, we observed numerous p-H2AX foci in nuclei of MCF7-treated cells, indicative of large chromatin domains at the sites of DNA damage, whereas AIF appeared in a mitochondrial staining pattern, and no translocation to the nucleus was detected (Fig. 7). These results support the hypothesis that ds DNA damage is independent of the chromatin processing related to the apoptotic process, possibly as consequence of topoisomerases inhibition. Ds-DNA cleavage creates damage which, for obvious reasons, is more difficult for the cell to repair. This could be important from a therapeutic point of view. The observed ability of prodigiosin to induce ds breaks means that it can be added to the short list of agents, mainly of natural origin, that induce ds cleavage and are used as anti-tumour drugs, such as dilanthadine (Branum and Que, 1999).
Additionally, although the ability of prodigiosin to induce oxidative DNA damage has been described previously (Melvin et al., 2000), we have determined a dependence of pH microenvironment and Cu2+ concentration. Thus, in suitable conditions, prodigiosin also can induce DNA cleavage through a copper–prodigiosin system.
In the present study we demonstrated that acidic pH (6.8) is more favorable to prodigiosin-mediated DNA breakage than weakly basic pH (7.4) (Fig. 8). It should be noted that tumours exhibit considerable spatial and possibly temporal heterogeneity in pH, ion concentrations, etc., which may modulate the delivery and cytotoxicity of chemotherapeutics (Kozin et al., 2001). In the complex tumor microenvironment, the intra–extracellular pH gradient is very important. Measurements of extracellular pH (pHe) obtained using microelectrodes have shown that pHe, in solid tumors, is typically in the range 6.6–7.0, whereas in normal tissue pHe is between 7.1 and 7.6 (Wike-Hooley et al., 1984). Although pHe in solid tumors tends to be acid, intracellular pH (pHi) is usually found to have similar values in solid tumors and normal tissues (Gillies et al., 1994). The difference in pHe between tumors and normal tissues provides an opportunity for tumor-selective therapy through the development of drugs that have increased toxicity at low pHe. In fact, the uptake and the cytotoxic efficacy of mitoxantrone, paclitaxel, and topotecan were reduced at pHe 6.5 as compared with pHe 7.4 (Vukovic and Tannock, 1997). Furthermore, chemotherapeutic agents with an appropriate pKa (6.5) can facilitate intracellular uptake and selectivity for cancer cells (Kozin et al., 2001).
In addition, metal ions play an important role in biological systems, and, without their catalytic presence in trace or ultratrace amounts, many biochemical reactions would not take place (Theophanides and Anastassopoulou, 2002). Copper is an essential metal and is distributed in all cellular organelles including mitochondria, lysosomes, endoplasmic reticulum, cytosol, and nucleus. Most "free" copper is undetectable. In dry non-cancerous breast tissue, the mean concentration of copper is 23 μM (1.47 ppm), whereas the mean concentration increases to 80 μM (5.12 ppm) in cancerous tissue. The Cu2+-Zn2+ index is also considerably higher in patients with cancer (Poo et al., 1997). These observations suggest that copper may promote prodigiosin-DNA damage in a concentration-dependent manner, as is demonstrated using an in vitro assay in Figure 9, where DNA damage is clearly detected at 5 μM copper, being more efficient at higher concentrations. This may explain the selective cytotoxicity of prodigiosin observed previously (Perez-Tomas et al., 2003).
Taken together, these results provide evidence that prodigiosin binds directly to DNA by intercalation, with preference for the alternating base pairs, is effective in poisoning both topoisomerase I and II enzymes, and is able to induce ss and ds DNA breaks, both in vitro and in cultured cells. The recognition of DNA as target may help to explain the complex cellular effects of the drug. Prodigiosin may ultimately prove of therapeutic value because of its effects on several cellular targets rather than just one. Our results also suggest the possible use of prodigiosin as a chemotherapeutic drug, given its potential cytotoxic selectivity in combination with copper under acidic conditions.
ACKNOWLEDGMENTS
This work was supported by a grant from the Ministry of Science and Technology and the European Union (SAF2001–3545), and by a TV3 Marató grant (ref.# 001510). M.M. is recipient of a research fellowship from the Generalitat de Catalunya (2000FI00035), and R..
REFERENCES
Baguley, B. C., Wakelin, L. P., Jacintho, J. D., and Kovacic, P. (2003). Mechanisms of action of DNA intercalating acridine-based drugs: how important are contributions from electron transfer and oxidative stress Curr. Med. Chem. 10, 2643–2649.
Bible, K. C., Bible, R. H., Kottke, Jr. T. J., Svingen, P. A., Xu, K., Pang, Y. P., Hadju, E., and Kaufmann, S. H. (2000). Flavopiridol binds to duplex DNA. Cancer Res. 60, 2419–2428.
Boger, D. L., and Patel, M. (1988). Total synthesis of prodigiosin, prodigiosene, and desmethoxyprodigiosin: Diels-Alder reactions of heterocyclic azadienes and development of an effective palladium (II)-promoted 2,2'-bipyrrole coupling procedure. J. Org. Chem. 53, 1405–1415.
Borah, S., Melvin, M. S., Lindquist, N., and Manderville, R. A. (1998). Copper-mediated nuclease activity of a tambjamine alkaloid. J. Am. Chem. Soc. 120, 4557–4562.
Boyd, M. R. (1987). The NCI in vitro anticancer drug discovery screen: concept, implementation, and operation, 1985–1995. In Anticancer drug development guide: preclinical screening, clinical trials, and approval (B. A. Teicher, Ed.), pp. 23–42. Humana Press, Totowa, NY.
Branum, M. E., and Que, L., Jr. (1999). Double-strand DNA hydrolysis by dilenthandine complexes. J. Biol. Inorg. Chem. 4, 593–600.
Cameron, R., and Feuer, G. (2000). Molecular, cellular and tissue reactions of apoptosis and their modulation by drugs. In Handbook of experimental pharmacology (R. D. Cameron, and L. A. Feuer, Eds.), pp. 37–58. Springer, Berlin.
Campas, C., Dalmau, M., Montaner, B., Barragan, M., Bellosillo, B., Colomer, D., Pons, G., Perez-Tomas, R., and Gil, J. (2003). Prodigiosin induces apoptosis of B and T cells from B-cell chronic lymphocytic leukemia. Leukemia 17, 746–750.
D'Alessio, R., Bargiotti, A., Carlini, O., Colotta, F., Ferrari, M., Gnocchi, P., Isseta, A., Mongelli, N., Motta, P., Rossi, A., et al. (2000). Synthesis and immunosuppressive activity of novel prodigiosin derivatives. J. Med. Chem. 43, 2557–2565.
Froelich-Ammon, S.J., and Osheroff, N. (1995). Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J. Biol. Chem. 270, 21429–21432.
Gillies, R. J., Liu, Z., and Bhujwalla, Z. (1994). 31P-MRS measurements of extracellular pH tumors using 3-aminopropylphosphonate. Am. J. Physiol. 267, 195–203.
Holden, J. A. (2001). DNA topoisomerases as anticancer drug targets: from the laboratory to the clinic. Curr. Med. Chem. Anticancer Agents 1, 1–25.
Hurley, L. H. (2002). DNA and its associated processes as targets for cancer therapy. Nat. Rev. Cancer 3, 188–200.
Kaufmann, S. H. (1998). Cell death induced by topoisomerase-targeted drugs: more questions than answers. Biochim. Biophys. Acta 1400, 195–211.
Khanna, K. K., and Jackson, S. P. (2001). DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 2, 247–254.
Kozin, S., Shkarin, P., and Gerweck, L. (2001). The cell transmembrane pH gradient in tumors enhances cytotoxicity of specific weak acid chemotherapeutics. Cancer Res. 61, 4740–4743.
Lisgarten, J., Coll, M., Portugal, J., Wright, C. W., and Aymami, J. (2002). The antimalarial drug cryptolepine intercalates specifically into DNA at cytosine-cytosine sites. Nature Struct. Biol. 9, 57–60.
Melvin, M. S., Ferguson, D. C., Lindquist, N., and Manderville, R. A. (1999). DNA binding by 4-methoxypyrrolic natural products. Preference for intercalation at AT sites by tambjamine E and prodigiosin. J. Org. Chem. 64, 6861–6869.
Melvin, M. S., Tomlinson, J. T., Saluta, G. R., Kucera, G.L., Lindquist, N., and Manderville, R. A. (2000). Double-strand DNA cleavage by copper·prodigiosin. J. Am. Chem. Soc. 122, 6333–6334.
Montaner, B., Navarro, S., Pique, M., Vilaseca, M., Martinell, M., Giralt, E., Gil, J., and Perez-Tomas. R. (2000). Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Br. J. Pharmacol. 131, 585–593.
Montaner, B., and Perez-Tomas, R. (2001). Prodigiosin-induced apoptosis in human colon cancer cells. Life Sci. 68, 2025–2036.
Montaner, B., and Perez-Tomas, R. (2002a). Activation of protein kinase C for protection of cells against apoptosis induced by the immunosuppressor prodigiosin. Biochem. Pharmacol. 63, 463–469.
Montaner, B., and Perez-Tomas, R. (2002b). The cytotoxic prodigiosin induces phosphorylation of p38-MAPK but not of SAPK/JNK. Toxicol. Lett. 129, 93–98.
Perez-Tomas, R., and Montaner, B. (2003). Effects of the proapoptotic drug prodigiosin on cell cycle-related proteins in Jurkat T cells. Histol. Histopathol. 18, 379–385.
Perez-Tomas, R., Montaner, B., Llagostera, E., and Soto-Cerrato, V. (2003). The prodigiosins, proapoptotic drugs with anticancer properties. Biochem. Pharmacol. 66, 1447–1452.
Pons, M., Campayo, L., Martinez-Balbas, M. A., Azorin, F., Navarro, P., and Giralt, E. (1991). A novel ionisable chromophore of 1,4-bis (alkylamino)benzo[g]phthalazine which interacts with DNA by intercalation. J. Med. Chem. 34, 82–86.
Poo, J. L., Romero, R. R., Robles, J. A., Momtemayor, A. C., Isoard, F., Estanes, A., and Uribe, M. (1997). Diagnostic value of the copper/zinc ratio in digestive cancer: a case control study. Arch. Med. Res. 28, 259–263.
Portugal, J., Martin, B., Vaquero, A., Ferrer, N., Villamarin, S., and Priebe, W. (2001). Analysis of the effects of daunorubicin and WP631 on transcription. Curr. Med. Chem. 8, 1–8.
Ren, J., and Chaires, J. B. (1999). Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 38, 16067–16075.
Rogakou, E. P., Boon, C., Redon, C., and Bonner, W. M. (1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell. Biol. 146, 905–916.
Rogakou, E. P., Nieves-Neira, W., Boon, C., Pommier, Y., and Bonner, W. M. (2000). Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J. Biol. Chem. 275, 9390–9395.
Songia, S., Mortellaro, A., Taverna, S., Fornasiero, C., Scheiber, E. A., Erba, E., Colotta, F., Mantovani, A., Isetta, A. M., and Golay, J. (1997). Characterization of the new immunosuppressive drug undecylprodigiosin in human lymphocytes. J. Immunol. 158, 3987–3995.
Soto-Cerrato, V., Llagostera, E., Montaner, B., Scheffer, G. L., and Perez-Tomas, R. (2004). The novel anticancer agent prodigiosin triggers mitochondria-mediated apoptosis operating irrespective of multidrug resistance in breast cancer cells. Biochem. Pharmacol. 68, 1345–1352.
Susin, S. A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler, M., Costantini, P., Ferri, K. F., Irinopoulou, T., Prevost, M. C., et al. (2000). Two distinct pathways leading to nuclear apoptosis. J. Exp. Med. 192, 571–579.
Theophanides, T., and Anastassopoulou, J. (2002). Copper and carcinogenesis. Crit. Rev. Oncol. Hemat. 42, 57–64.
Turner, P. R. and Denny, W. A. (1996). The mutagenic properties of DNA minor-grove binding ligands. Mutat. Res. 355, 141–169.
Vukovic, V., and Tannock, I. F. (1997). Influence of low pH on cytotoxicity of paclitaxel, mitoxantrone and topotecan. Br. J. Cancer 75, 1167–1172.
Wike-Hooley, J. L., Haveman, J., and Reinhold, J. S. (1984). The relevance of tumor pH to the treatment of malignant disease. Radiother. Oncol. 2, 343–366.
Yamamoto, C., Takemoto, H., Kuno, K., Yamamoto, D., Tsubura, A., Kamata, K., Hirata, H., Yamamoto, A., Kano, H., Seki, T., et al. (1999). Cycloprodigiosin hydrochloride, a new H+/Cl– symporter, induces apoptosis in human and rat hepatocellular cancer cell lines in vitro and inhibits the growth of hepatocellular carcinoma xenografts in nude mice. Hepatology 30, 894–902.
Yamamoto, D., Kiyozuka, Y., Uemura, Y., Yamamoto, C., Takemoto, H., Hirata, H., Tanaka, K., Hioki, K., and Tsubura, A. (2000a). Cycloprodigiosin hydrochloride, a H+/Cl– symporter, induces apoptosis in human breast cancer cell lines. J. Cancer Res. Clin. Oncol. 126, 191–197.
Yamamoto, D., Uemura, Y., Tanaka, K., Nakai, K., Yamamoto, C., Takemoto, H., Kamata, K., Hirata, H., and Hioki, K. (2000b). Cycloprodigiosin hydrochloride, H+/Cl– symporter, induces apoptosis and differentiation in HL-60 cells. Int. J. Cancer 88, 121–128.(Beatriz Montaner, Wilmar )