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Interactions of Antitumor Triazoloacridinones with DNA

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Interactions of Antitumor Triazoloacridinones with DNA Vol. 54 No. 2/2007, 297–306 on-line at: www.actabp.pl Regular paper Interactions of antitumor triazoloacridinones with DNA * Marcin Koba and Jerzy Konopa Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Gdańsk, Poland Received: 12 February, 2007; revised: 20 March, 2007; accepted: 21 March, 2007 available on-line: 19 April, 2007 Triazoloacridinones (TA) are a new group of potent antitumor compounds, from which the most active derivative, C-1305, has been selected for extended preclinical trials. This study investi- gated the mechanism of TA binding to DNA. Initially, for selected six TA derivatives differing in chemical structures as well as cytotoxicity and antitumor activity, the capability of noncova- lent DNA binding was analyzed. We showed that all triazoloacridinones studied stabilized the DNA duplex at a low-concentration buffer but not at a salt concentration corresponding to that in cells. DNA viscometric studies suggested that intercalation to DNA did not play a major role in the mechanism of the cytotoxic action of TA. Studies involving cultured cells revealed that triazoloacridinone C-1305 after previous metabolic activation induced the formation of interstrand crosslinks in DNA of some tumor and fibroblast cells in a dose dependent manner. However, the detection of crosslink formation was possible only when the activity of topoisomerase II in cells was lowered. Furthermore, it was impossible to validate the relevance of the ability to crosslink DNA to biological activity of TA derivatives. Keywords: triazoloacridinones, DNA intercalation, DNA interstrand crosslinks, structure–activity relationship, biological activity, metabolic activation INTRODUCTION Previous investigations demonstrated that triazoloacridinones inhibited cleavable complexes of Triazoloacridinones (TA) (see Table 1, for topoisomerase II–DNA and also the catalytic activity structures) are a class of antitumor agents synthe- of this enzyme (Skladanowski et al., 1999; Lemke et sized in our laboratory (Cholody et al., 1990). TA al., 2004), as well as induced G2 block of the cell cy- exhibited high cytotoxic activity against a broad cle followed by apoptosis (Augustin & Konopa, 1999; spectrum of tumor cell lines in vitro and high anti- Augustin et al., 2000; 2006). These effects suggested tumor activity against transplantable tumors such as that cellular DNA might be an important target for leukemia P388, melanoma B16, ascites colon 26 ad- the TA drugs. Triazoloacridinone C-1305 strongly enocarcinoma and colon 38 adenocarcinoma in mice inhibited proliferation of cells lacking poly(ADP-ri- (Cholody et al., 1990; Kusnierczyk et al., 1994). The bose) polymerase 1 (PARP-1), which was a surpris- most active and promising derivate from the TA ing observation because such cells are generally re- group, C-1305, was selected for extended preclinical sistant to other topoisomerase II poisons (Węsierska- trials. Gądek et al., 2004). Moreover, recent data obtained Corresponding author: J. Konopa, Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, G. Narutowicza 11/12, 80-952 Gdańsk, Poland; phone/fax: (+48 58) 347 1516; e-mail: [email protected] *Present address: Department of Drug Chemistry, Faculty of Pharmacy, Collegium Medicum of Nicolaus Copernicus University, Bydgoszcz, Poland. Abbreviations: ATA, aurintricarboxylic acid; bp, base pairs; ctDNA, calf thymus DNA; DMEM, Dulbecco’s modified Ea- gle’s medium; FCR, fraction of crosslinked DNA; FCS, fetal calf serum; MEM, minimal essential medium; PBS, phos- phate-buffered saline; POPOP, 1,4-bis-(5-phenyl-2-oxazolyl)-benzene; PPO, 2,5-diphenyl-oxazole; SDS, sodium dodecylsul- fate; SRB, sulforhodamine; topo II, topoisomerase II; TA, triazoloacridinones. 298 M. Koba and J. Konopa 2007 Table 1. Chemical structures and biological activities of triazoloacridinones studied. Values are means ± S.D. from at least three independent experiments. R1 O R4 N (CH2)n N R3 R2 8 5 N N N Chemical structure Biological activity aCells in vitro bP388 leukemia in mice Compound n R1 R2 R3 R4 HeLa S3 OD ILS –1 EC50 [µM] (mg kg ) (%) C-1233 2 CH3 CH3 H H 37.6 ± 0.55 100 77.0 C-1296 3 CH3 CH3 CH3 H 7.6 ± 0.32 100 18.0 C-1303 2 CH3 CH3 OH H 2.8 ± 0.78 25 102.0 C-1305 3 CH3 CH3 OH H 8.6 ± 0.32 25 165.0 C-1533 2 CH3 CH3 OH CH3 37.4 ± 0.23 25 9.5 c C-1567 2 CH3 CH3 C(CH3)3 H 27.2 ± 0.93 N 0.0 a b EC50, the concentration of the compound inhibiting growth of cells by 50% after 72 h incubation. Data taken from (Cholody et al., 1990). OD, the optimal dose and ILS is the increase in survival time of treated to control animals at optimal dose. cN, means that the compound was not active. in our department indicated that triazoloacridinone gents were from Sigma-Aldrich (Poznań, Poland). C-1305 preferentially intercalated to DNA in gua- All reagents used were at least of the analytical nine triplet regions and induced specific changes in grade. Ultra pure water (18 MΩ) was used in all ex- DNA structures (Lemke et al., 2005). The structural periments. resemblance of triazoloacridinones to other anti- Cell culture and media. HL60 (human my- cancer drugs, such as imidazoacridinones (Cholody eloblastic leukemia), HeLa S3 (human cervix carci- et al., 1992; Kusnierczyk et al., 1994; Dziegielewski noma) and NIH 3T3 (mouse fibroblasts) cell lines o et al., 2002) and anthracenediones (Kapuscinski & were cultivated at 37 C in 5% CO2/air atmosphere Darzynkiewicz, 1985; Skladanowski et al., 2000) (flat in appropriate culture medium (HL60: RPMI-1640, structure of aromatic acridine ring (Arjunan et al., 5% FCS; HeLa S3: MEM, 5% FCS; NIH 3T3: DMEM, 1993) and diamino alkylamino group in the side 10% FCS, respectively) supplemented with 100 µg/ chain) indicated that DNA binding, including inter- ml streptomycin, 100 U/ml penicillin. The RPMI- strand crosslinking of DNA, may play a role in the 1640 medium, minimal essential medium (MEM) TA mechanism of action. and Dulbecco’s modified Eagle’s medium (DMEM) In this study, we examined the TA capabil- supplemented with 1000 mg/l glucose and fetal calf ity of intercalation to DNA and formation of in- serum (FCS) were from Gibco Europe Ltd (Paisley, terstrand crosslinks in cellular DNA, and tried to UK); antibiotics were from Sigma-Aldrich (Poznań, evaluate the importance of these phenomena for Poland); proteinase K was from Merck (Darmstadt, the biological activity of these compounds by cor- Germany); nuclease S1 was from Fermentas (Vilnius, relation analysis. Lithuania); sulforhodamine (SRB) and calf thymus DNA (ctDNA, type II) were from Sigma-Aldrich (Poznań, Poland). MATERIALS AND METHODS Growth inhibition assay. Growth inhibition assays were carried out with exponentially grow- Chemicals. Triazoloacridinones were resyn- ing cells. The cells were seeded into 24-well plates thesized and purified as described earlier (Cholody (20 000/2 ml per well) about 24 h before drug treat- et al., 1990) by Dr. Barbara Horowska from this de- ment and exposed to drugs for 6 h followed by 72 h partment and were dissolved in water. Aurintricar- postincubation in drug-free medium or for 72 h of boxylic acid (ATA) and sodium dodecylsulfate (SDS) continuous incubation. Cytotoxic activity was de- were from Serva (Heidelberg, Germany); NaClO4 termined by cell counting (Bhuyan et al., 1992) (cell was from Fluka (Sigma-Aldrich, Poland); 2,5-diphe- number was determined with ZBI Coulter Counter) nyl-oxazole (PPO) and 1,4-bis-(5-phenyl-2-oxazolyl)- or by sulforhodamine assay (Skehan et al., 1990). benzene (POPOP) were from Merck (Darmstadt, DNA thermal denaturation studies. Stabiliza- Germany); methyl-[14C]thymidine was from Amer- tion of secondary structure of DNA by TA was stud- sham International (Amersham, UK) and other rea- ied by determining melting temperatures of calf thy- Vol. 54 Interactions of triazoloacridinones with DNA 299 mus DNA in the presence of 10 µM triazoloacridi- were seeded 48 h prior to treatment and concomi- nones in phosphate buffer (7.5 mM NaH2PO4, 1 mM tantly during this time DNA was radiolabeled. After EDTA, pH 7.0). Compounds tested were mixed with changing the medium, the cells were treated with DNA at a 1 : 4 drug/bp (base pair) molar ratio. Sam- TA for indicated times. In all cases, after treatment ples were heated over the range 45–95oC (using a with the compounds the cells were transferred to heating rate of 1oC/min) and absorbance at 260 nm test tubes and washed with PBS twice. After wash- was monitored in a Varian Cary 300 Bio UV-Vis ing the cells were mixed with 0.2 ml of lysing solu- spectrophotometer equipped with a programmable tion (10 mM Tris/HCl, 10 mM EDTA, 10 mM NaCl, heating unit. The ‘melting’ temperature (tm) was 0.5% SDS, pH 8.0) supplemented with proteinase K read from the plot of relative hyperchromicity versus (1 mg/ml). After 1 h at 37oC, 1.8 ml of denaturating the temperature as the midpoint of the hyperchro- solution (5.25 M sodium perchlorate, 1 mM EDTA, mic transition (Pawlak et al., 1983). 0.2% N-lauryl sarcosine, 20% methanol, pH 7.0) was DNA viscometry studies. Viscometric meas- added and incubation was continued for additional urements were performed as described previously 1 h. DNA was denatured by heating at 50oC for (Reinert, 1991; Suh & Chaires, 1995) in a Micro-Ub- 30 min, then renatured by rapid dilution with 10 ml belohde microviscometer with a 0.53 mm capillary of ice-cold zinc buffer (1 mM zinc acetate, 5 mM so- (Schott) at 25oC. Titration of ligands was performed dium acetate, 10 mM NaCl, pH 4.4) and cooling at by the addition of small volumes (20 µl) of concen- –18oC. The fraction of crosslinked DNA (FCR) was trated stock solution (2 mM) in TE buffer (10 mM calculated according to the formula: Tris/HCl, 1 mM EDTA, pH 7.4) to the viscometer chamber containing calf thymus DNA (2 mM in bp) − FCR[%] = FDS treat FDS contr x100 1− sonicated to approx.
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