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ANTICANCER RESEARCH 25: 2193-2198 (2005)

Relationship between II-DNA Cleavable Complexes, and Cytotoxic Activity of in Human Cervix Carcinoma Cells

BEATA M. GRUBER1, ELZBIETA L. ANUSZEWSKA1, IRENA BUBKO1, ANETA GOZDZIK1, WALDEMAR PRIEBE2 and IZABELA FOKT2

1National Institute of Public Health, Department of Biochemistry and Biopharmaceuticals, Chelmska 30/34 Str., 00-725 Warsaw, Poland; 2The University of Texas, M. D. Anderson Cancer Center, Department of Bioimmunotherapy, 1515 Holcombe Blvd., Houston, TX 77030, U.S.A.

Abstract. The use of anthracyclines as antitumor dates determinant in the cytotoxic action of these compounds. The back to the 1970s, but the mechanism of the cytotoxicity of efficiency of , interpreted as cytotoxic these compounds has long been a matter of debate. There is action, was dependent on cell type. increasing evidence indicating that -induced cytotoxicity commonly converges on the induction of apoptosis. Many Anthracyclines are potent, broad-spectrum chemotherapeutic authors point to the fact that double-strand breaks, resulting agents, effective against solid tumors and malignant from stabilization of cleavable complexes, are the signal for the hematological disease (1, 2). initiation of the apoptotic cascade. In this work, the possible Although the initial intracellular targets of this group of correlation between stabilization of topoisomerase II (topoII)- anticancer drugs may be heterogenous, there is increasing DNA complexes, apoptosis induction and cytotoxicity was evidence indicating that drug-induced cytotoxicity studied. Parental human cervix carcinoma cells, HeLa, and its commonly converges on the induction of programmed cell subline resistant to , KB-V1, were exposed to death (apoptosis). One of the most important routes (DOX) and the novel anthracyclines annamycin resulting in the apoptotic cascade in cells treated with and WP903, given at the concentrations 0.2 and 2.0 Ìg/ml anthracyclines is the stabilization of the topoisomerase II (DOX and annamycin) or 0.2 and 1.0 Ìg/ml (WP903). It was (topoII) - DNA cleavable complex (3, 4). Cardoso et al. (5) found that annamycin was the strongest topoII poison in HeLa note that topoII· is the target of anthracyclines, and cells at both concentrations used, whereas poly (ADP-ribose) preliminary data suggest its promising role as a predictive polymerase (PARP) cleavage was observed dose-dependently in marker of sensitivity to these drugs. KB-V1 cells treated with annamycin or WP903. Simultaneously, DNA are ubiquitous enzymes that can apoptosis, observed as cell morphology or phosphatidylserine solve the topological problem associated with DNA translocation, was evident in both cell types exposed to the novel generated during the key cellular processes such as anthracyclines, independent of concentration. DOX appeared replication, transcription, recombination, repair and to be the weakest apoptotic inducer. On the basis of these chromatin assembly. TopoII introduce transient breaks in studies, it can be suggested that topoII poisoning is not the key both DNA strands (6). Anthracyclines interrupt the process leading to apoptosis and seems to be cell specific. PARP breakage / reunion reaction of topoII resulting in cleavage is probably not an evident marker of anthracycline- accumulation of a topoII-DNA covalent intermediate, the induced apoptosis which, in turn, does not seem to be the cleavable complex (3, 4). This makes topoisomerases powerful poisons that cut up DNA and damage the genome. This action leads to activation of stress-associated signaling pathways, arrest and activation of the biochemical Correspondence to: Beata M. Gruber, National Institute of Public cascade of apoptosis (3, 7). However, the details of the Health , Department of Biochemistry and Biopharmaceuticals, Chelmska 30/34 Str., 00- 725 Warsaw, Poland. e-mail: b-gruber@ downstream signaling from cleavable complexes to cell il.waw.pl death are poorly understood (3, 8). Many authors point to the fact that double-strand breaks, Key Words: Anthracyclines, topo II poison, apoptosis. resulting from stabilization of cleavable complexes and the

0250-7005/2005 $2.00+.40 2193 ANTICANCER RESEARCH 25: 2193-2198 (2005)

Table I. IC50 values (Ìg/ml) determined by the MTT test. MTT test. The suspensions of cells were diluted in MEM to 105 cells/ml and 0.05 ml of each suspension was placed into individual Anthracycline HeLa cells KB-V1 cells wells on a 96-well multiplate. Then, 0.05 ml of each drug solution in MEM were added at double strength dilution for 48 h. The final Dox 0.52±0.08 0.61±0.09 concentrations of DOX, annamycin and WP903 ranged from 0.058 Annamycin 1.45±0.03* 1.38±0.08* to 5.8 Ìg/ml. The cells treated as controls were kept in drug-free WP903 0.57±0.04 0.54±0.02 MEM. The MTT test was performed, as previously described (15). The IC values were determined as the concentrations of the Cells were exposed to the drugs, each in the range: 0.058-5.8 Ìg/ml for 50 drugs required for 50% cell growth inhibition. 48 h, then the MTT test was done as described in Materials and Methods. Results represent the mean ± SEM (n=8-16). Morphological evaluation. The 24-h cell cultures were exposed to *IC values statistically different from IC determined for DOX and 50 50 the following concentrations of the drugs tested for 24 h: DOX and WP903 independently (p<0.05). annamycin; 0.2 and 2.0 Ìg/ml; WP903, 0.2 and 1.0 Ìg/ml. The drug doses were defined as lower or higher than the respective IC50 values. The exposure time was defined on the basis of the earlier experiments (16). Cell morphology was evaluated inability of the cells to repair DNA lesions, are the signal using a light fluorescence microscope Nikon 100 Eclipse TS, after for apoptosis (9-11). However, the role of topoII and staining the cells with Hoechst 33342 (5 Ìg/ml). cleavable complexes in anthracycline cytotoxicity is still TopoII protein and apoptosis detection ( PARP cleavage). Treatment controversial (12, 13). protocol. Twenty-four-h cell cultures were exposed to the drugs used at Another doubt discussed in many papers is the role of concentrations as given above (Morphological evaluation) for 24 h. apoptotic processes in the death of tumor cells exposed to anthracyclines. Some authors have shown that anthracyclines Nuclear extract preparation. Cells were washed with cold PBS, induce apoptosis, but that this event is not directly related to scraped very gently, spun and washed repeatedly at 15,000 rpm for cell death. Fedier et al. (14) found that DOX was a strong 2 min. The cell pellet was resuspended in lysis buffer A (1 M Hepes, pH = 7.9/ 2 M KCl/ 0.5 M EDTA, pH = 8.0/ 0.1 M EGTA, pH = inducer of apoptotic processes in mouse lung fibroblasts, 7.0) with addition of 0.1 M DTT, 100 mM PMSF, leupeptin 1 mg/ml, regardless of their susceptibility to that agent. aprotinin 1 mg/ml and benzamidine 250 mg. During incubation, the Due to the serious problem of multidrug resistance and cells were disrupted and nuclei were released, visible as the high cardiotoxicity in and, in particular, transparent pellet. Nuclear extracts were obtained by incubation of anthracycline therapy, new anthracycline derivatives with that pellet on ice lysis buffer B (1 M Hepes, pH = 7.9/ 5 M NaCl/ better therapeutic indices are being sought. 0.5 M EDTA, pH = 8.0/ 0.1 M EGTA, pH = 7.0) with addition of As a part of a collaboration with the Priebe group, at the 0.1 M DTT, 100 mM PMSF, leupeptin 1 mg/ml, aprotinin 1 mg/ml, benzamidine 250 mg and frequent intermittent vortexing. MD Anderson Cancer Center, Houston, Texas, USA, for the screening studies of a number of novel anthracyclines, Western blot. The extracted nuclear proteins were resolved by WP903 and annamycin were chosen as the most promising 12% SDS PAGE electrophoresis (each well was loaded with 50 drugs in anticancer therapy. DOX, as a known agent, was Ìg of protein) and electroblotted onto a nitrocellulose membrane, treated as the reference drug. 0.45 nm. After blocking with 3% non-fat milk, topoII· protein The aim of this work was to define a possible correlation was detected with rabbit polyclonal antibody, 200 Ìg/ml IgG between the topoII poison activity of the compounds, (1:100), Santa Cruz Biotechnology Inc., USA (corresponding to apoptosis intensity and cytotoxic activity. the fragment at the carboxy terminus of DNA topoII· of human origin), and PARP protein was detected with rabbit polyclonal Apoptotic events were morphologically evaluated using antibody, 200 Ìg/ml IgG (1:100), Santa Cruz Biotechnology Inc. the fluorescence microscope and in PARP cleavage assay. (corresponding to amino acids 764 - 1014 of the carboxy terminus Stabilization of cleavable complexes as well as PARP of PARP). The secondary antibody was polyclonal goat anti- cleavage were studied using the Western blot technique. rabbit IgG-HRP conjugated (1:500), Dako, Denmark. Proteins were visualized with HRP Color Development Reagent (4 CN Materials and Methods (4 – chloro-1- naphthol)).

Statistical analysis. The statistical evaluation of the results was Drugs. Doxorubicin (DOX ) was purchased from Fluka, Germany; performed using Student’s t-test and Cochrane’s Cox test for WP903 and annamycin were from the MD Anderson Cancer unrelated samples. Center, Houston, TX, USA.

Cells. The HeLa human cervix carcinoma cell line and its subline Results resistant to vinblastine, KB-V1, were purchased from the American Tissue Culture Collection. Cells were grown in MEM Cytotoxic activity. The cytotoxic activity of the drugs tested supplemented with 10% fetal calf serum and antibiotics. KB-V1 was determined by the MTT test. As shown in Table I, both cells were maintained in MEM with vinblastine, 0.5 Ìg/ml. cell types exhibited comparable sensitivity to all drugs tested.

2194 Gruber et al: Topo II and Apoptosis in Anthracycline Cytotoxicity

Figure 1. Stabilization of cleavable complexes topo II·-DNA by DOX (a); annamycin (b); WP903 (c). Cell cultures were treated according to the procedure given in Materials and Methods. Results represent the mean ± SEM.

However, it can be observed that annamycin is about 3-fold concentrations, i.e., 0.2 and 2.0 Ìg/ml. Slight quenching of less cytotoxic than DOX or WP903. The cytotoxic activities the topoII· band was observed in KB-V1 cells exposed to of DOX and WP903 were comparable. annamycin at the 0.2 Ìg/ml concentration (Figure 1b). Neither DOX nor WP903 was noted as having any influence Stabilization of cleavable complexes. The effect of stabilization on topoII·-DNA complexes (Figure 1a, c). of cleavable complexes topoII-DNA was observed using the Western blot technique. Anthracyclines can block topoII· in Apoptosis induction. Cell morphology was evaluated in the cell complexes with DNA, resulting in lower intensity quenching cultures treated with the drugs studied in comparison with the of the topoII· bands. As shown in Figure 1b, the strongest control cell cultures (untreated). The use of Hoechst 33342 effect of topoII· blocking in cleavable complexes occurred allows for the monitoring of any structural changes in genetic in the case of annamycin given to HeLa cells at both material, due to intercalation into DNA, which results in a

2195 ANTICANCER RESEARCH 25: 2193-2198 (2005)

Figure 2. PARP cleavage induced by DOX (a); annamycin (b); WP903 (c). Light fields stand for PARP, 116 kDa; dark fields stand for PARP fragments, 86 kDa. Cell cultures were treated according to the procedure given in Materials and Methods. Results represent the mean±SEM.

blue fluorescence visible by fluorescence microscope. Typical activation of caspase-3 and -7, is one of the apoptotic effects late apoptotic symptoms appeared in HeLa and KB-V1 cells and results in the formation of fragments such as 86 kDa, exposed only to new anthracyclines, i.e., annamycin or WP903, which can be detected with specific antibodies. independently of the concentrations used. The treatment As presented in Figure 2b, the strongest effects defined resulted in rings of condensed chromatin around the nuclear as PARP cleavage were noted in KB-V1 cells treated with membranes and apoptotic body formation (data not annamycin, 2 Ìg/ml or with WP903, 0.2 or 1.0 Ìg/ml (Figure shown).The same significant difference in apoptotic induction 2b, c). A slight PARP cleavage effect was observed in HeLa between DOX and WP903 was also confirmed in annexin V cells treated with: DOX, 2.0 Ìg/ml; annamycin, 2.0 Ìg/ml; or assay by flow cytometry (Gruber, unpublished). with WP903, 0.2 or 1.0 Ìg/ml (Figure 2a, b, c).

PARP cleavage. Apoptosis observed from morphological Discussion features was confirmed with the PARP cleavage assay employing the Western blot technique. PARP, 116 kDa, is a The cytotoxic effect of anthracycline antibiotics is nuclear enzyme . Its proteolytic cleavage, linked to the multidirectional. One of the mechanisms consists of blocking

2196 Gruber et al: Topo II and Apoptosis in Anthracycline Cytotoxicity the cleavable DNA-topoII complexes governing the super- As shown by many authors, apoptosis induced by helicity of DNA strands. It is well established that DNA anthracyclines results in caspase activation, especially double-strand breaks can trigger apoptotic cell death (9, 17, that of caspase-3, which cleaves PARP (26, 27). Faderl et 18). It is suggested that the phenomenon of blocking cleavable al. (28) have noted that apoptosis induced by WP744, a complexes leads to cell death by a mechanism not entirely novel anthracycline, in human acute myeloid elucidated (13). A very important target site for anthracycline cell lines K562, KBM-3 and OCIM-2 appeared to be antibiotics are cell proliferation systems including apoptotic mediated by caspase-3. WP744 induced cleavage of processes. The effect of this group of compounds as apoptotic PARP. Caspase activation, in association with PARP stimulators on tumor cells is indirect, i.e., they influence the cleavage and protein kinase C delta, has been confirmed activity of specific genes and their products, regulatory by Huigsloot et al. (29) in rat mammary adenocarcinoma proteins (19). As given by Li and Liu (9), the blocking of MTLn3 cells exposed to DOX. cleavable topoII-DNA complexes may be involved in the Our results point to the fact that PARP cleavage is not commitment step of apoptotic cell death. such an evident marker of anthracycline-induced apoptosis, The results obtained in this study do not confirm any and that such a route of apoptosis may depend on the cell correlation between stabilization of topoII-DNA complexes and type or time of exposure to the drug. Yeung et al. (30) have apoptosis induction observed from specific morphology (data noted that 18-h-treatment with DOX did not induce PARP not shown) and PARP cleavage, confirmed with membrane cleavage in human anaplastic thyroid cell lines. Holleman phosphatidylserine translocation assay (Gruber, unpublished). et al. (31) did not observe the correlation between caspase- In addition, the cytotoxic activity of the drugs tested does not 3 activation or PARP cleavage and resistance to seem to be dependent on these apoptotic events. These results in human acute lymphoblastic leukemia cell do not confirm the observations made by some authors that lines. According to the authors, it seems that caspase-3 reduced levels of topoII proteins or gene expression are activation and PARP inactivation are not essential for correlated with cell sensitivity to anthracyclines. Such a daunorubicin-induced apoptosis. Cell specificity of that conclusion was drawn by Evans et al. (20), testing small-cell lung apoptotic event has also been shown by Hopkins- cancer cell lines H69AR resistant to DOX and its parental H69 Donaldson et al. (32) in neuroblastoma cells, invasive cell line. Park et al. (21) have noted that the response of breast (N-type cells) and non-invasive (S-type cells), treated with cancers to DOX chemotherapy was increased in the cases with DOX. DOX was shown to induce caspase-3, -7, -8 and -9 topoII amplification, whereas the response rate was significantly activation in S-type cells whereas N-type cells were killed decreased in the case without topoII amplification. De Jong et by a caspase-independent mechanism. al. (22) have suggested that resistance of the human small cell The different cell responses and cytotoxic efficiencies of lung carcinoma cell line, GLC4, to DOX was, in part, due to the drugs reported here suggest that the mechanisms the reduced drug-induced formation of the cleavage complexes. responsible for the cytotoxic activity of anthracyclines are However, Den Boer et al. (10) have noted that neither topoII· cell specific. This observation was confirmed for expression, nor activity correlated with the anthracycline annamycin, which was 3-fold less cytotoxic than DOX for cytotoxicity in leukemic cells. According to the authors , topoII· HeLa and KB-V1 cells. Ling et al. (24) have reported may be an indicator of the proliferative status of cells without a annamycin as 50-fold more cytotoxic than DOX against direct relationship with anthracycline resistance in acute P388 cells resistant to DOX. leukemia. Some authors point to the fact that the cytotoxicity The results obtained here allow for the following of lesions generated by stabilization of cleavable complexes conclusions to be drawn: depends on the locus of the interactions: drug-topoII-DNA (7). 1. Stabilization of cleavable topoII-DNA complexes by Apoptosis, a carefully regulated process of cell death, anthracyclines seems to be a cell-specific process. may proceed through mechanisms varying according to cell 2. Stabilization of cleavable complexes is not condition sine type (23). The results obtained by Ling et al. (24) have qua non for apoptosis induction by anthracyclines. demontrated that the cell-killing effect observed in murine 3. PARP cleavage is not an evident marker of anthracycline- leukemia cells, P388, is mediated, at least in part, by the induced apoptosis, and this event depends on the cell type. induction of apoptosis. Such correlation was not exhibited in 4. Apoptotic events do not seem to be the determinants in the the current study with human cervix carcinoma cells, HeLa cytotoxic action of anthracyclines. and KB-V1, and is in agreement with our earlier work on 5. The cytotoxic activity of anthracyclines depends on their human melanoma cells treated with DOX (16), as well as that chemical structure. of Gariboldi et al. (25) who studied non-small cell lung Acknowledgements carcinoma cells exposed to DOX and found no relationship between drug response and expression and/or subcellular This work was presented at the 34th Annual Meeting of EEMS in localization of apoptosis-related proteins. Maastricht, Holland, 4-8 September, 2004.

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