* 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
1 To whom correspondence should be addressed at Departament de Biologia Cel·lular i Anatomia Patològica. CCBR Group, Pavelló del Govern, 5a planta. LR 5101 C. Feixa Llarga s/n, E-08907 L'Hospitalet (Barcelona), Spain. Fax: +34 93 402 9082. E-mail: rperez{at}ub.edu.
Received November 30, 2004; accepted March 15, 2005
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ABSTRACT |
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Key Words: DNA damage; prodigiosin; topoisomerase inhibition.
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INTRODUCTION |
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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
).
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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.
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MATERIALS AND METHODS |
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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 (0.52 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 NaOH1.5 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-ßmax, 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).
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RESULTS |
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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.
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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).
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DISCUSSION |
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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 enzymeDNA 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 G1G5 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 copperprodigiosin 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 intraextracellular 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 6.67.0, 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.
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ACKNOWLEDGMENTS |
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