Mechanisms of induction of apoptosis by ... - Springer Link

Apoptosis 2005; 10: 1497–1514


C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands. DOI: 10.1007/s10495-005-1540-9

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation ´ zwiak A. Koceva-Chyła, M. Je¸drzejczak, J. Skierski, K. Kania and Z. Jo´ Department of Thermobiology, University of Łod´ ´ z, Łod´ ´ z, Poland (A. Koceva-Chyła, M. Je¸drzejczak, K. Kania, Z. Jo´ ´ zwiak); Flow Cytometry Laboratory, National Institute of Public Health, Warsaw, Poland (J. Skierski)

Published online: 3 October 2005 We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.

Keywords: anthraquinones; apoptosis; caspases; cytotoxicity; immortalized cells; necrosis.

Introduction Anthraquinone compounds, anthracyclines and anthracenediones, have long been used as effective anticancer drugs against a broad spectrum of tumours.1–3 Depending on their chemical structure, anthrachinone Correspondence to: A. Koceva-Chyła, Department of Thermobiology, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland. Tel.: +48 42 635-4477; Fax: +48 42 635-4473; e-mail: [email protected]

drugs can kill tumor cells by diverse mechanisms, involving different initial intracellular targets that normally contribute to drug-induced toxicity and the induction of apoptosis. Caspases, mainly caspase-3, the chief executioner of apoptosis, has been shown to play an important role in the apoptotic cell death induced by anticancer drugs. A blockage of apoptosis has been found in cells deficient in the caspase-3 gene.4–8 Conversely, a caspase-independent pathway has been well documented in vitro as well.9–11 Moreover, caspase-3 activation has been proposed as a factor determining the apoptotic or necrotic mode of cell death.12 Although the direct effects of anthracycline drugs such as doxorubicin (DOX) on damage to DNA have been intensively studied, the sequence of morphological and biochemical events that mediate cell death in response to other anticancer drugs is still unclear. Different courses and pathways of apoptosis induced by the same drug in different cell types have been reported.13–15 There is also much controversy regarding the role of apoptosis and necrosis in the cytotoxic action of anthracyclines. Results obtained for the same drug with different cell lines also show inconsistency e.g. some authors suggested a direct connection between the extent of apoptosis and the cytotoxicity of anthracyclines, whereas others did not observe a positive correlation between the induction of apoptosis and cell sensitivity to these drugs.16–21 Moreover, evidence for the necrotic mode of death induced by the anthracycline drugs doxorubicin and daunorubicin in tumor cells has been provided.22 Little is known about the induction of apoptosis by aclarubicin (ACL) and mitoxantrone (MTX). Thus, the objective of our study was to compare the mode of cell death induced by aclarubicin and mitoxantrone with the most widely explored doxorubicin as well as to investigate the possible involvement of the caspase-3 activation in the development of the apoptotic events and their significance for drug cytotoxicity. Apoptosis · Vol 10 · No 6 · 2005

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In the experiments we used two spontaneously immortalized rodent cell lines as a model for cells with neoplastic phenotype: mouse embryo fibroblasts (NIH 3T3 cell line) and Chinese hamster peritoneal fibroblasts (B14 cell line). These cell lines display noticeably different sensitivity towards aclarubicin, doxorubicin and mitoxantrone and thus seem particularly suited for investigating the relation between apoptosis and drug cytotoxicity. Using a flow cytometry method, agarose gel electrophoresis, fluorescence microscopy analysis and caspase-3 activity assay we compared the investigated drugs with respect to their ability to induce apoptotic/necrotic morphological changes at the single cell level, caspase-3 activation and DNA fragmentation. We investigated molecular events and morphological features associated with apoptosis induced in cells exposed to these drugs by estimation: the time-course of apoptotic events, the dependence of the induction of apoptosis on the drug dose, the mode of cell death induced in the presence or absence of caspase-3 inhibitor Ac-DEVD-CHO. Another aim of the study was to examine the possible relation between the induction of apoptosis and drug cytotoxicity.

Materials and methods Chemicals Cell culture media, supplements and calf neonatal serum were obtained from Gibco (Edinburgh, Scotland). Doxorubicin was purchased from Pharmacia-Upjohn, Upsala, Sweden; aclarubicin—from Behring Institut GmBH, Vienna, Austria; mitoxantrone—from Polfa, Zielona G´ora, Poland. Caspase Z-DEVD-AMC Assay Kit #1 was provided by Molecular Probes, Eugene, OR, USA. All other reagents were supplied by Sigma, St. Louis, USA.

Cell culture NIH 3T3 cells were grown in monolayer with Dulbecco’s Modified Eagle Medium (DMEM), containing glucose (4.5 g/L) and supplemented with 10% heat inactivated neonatal calf serum, 1 mM L-glutamine and 1 mM sodium pyruvate. B14 cells were cultured in monolayer with MEM Eagle medium, containing 10% heat-inactivated neonatal calf serum. The cells were kept in a watersaturated incubator at 37◦ C, in the atmosphere containing 5% CO2 and maintained in the exponential growth phase by routine subculturing every 2–3 days. The cells were monitored periodically to ensure they were mycoplasma free.

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Drug treatment Drugs (ACL, DOX and MTX) at the final concentration of 2 mg/ml were aliquoted in small portions (50–100 µl) each and stored, protected from light, frozen at –20◦ C. Concentrated drug solutions were thawed immediately before use, and after dilution with PBS added directly to the cell medium at the desired final concentration. Cells were subcultured for 16 h prior to drug treatment to ensure they were in the exponential growth phase and incubated with the appropriate drug concentrations for 2 h under culture conditions. After a drug treatment, the medium was removed, and the cell monolayer was washed twice with pre-warmed (37◦ C) PBS and permitted to grow in drug-free medium for different time periods of between 6 and 72 h. At the defined time points cells were harvested, pelleted by centrifugation at 200 × g, washed with cold (4◦ C) PBS, resuspended in PBS and used for further analysis. Cytotoxicity assay The cytotoxic effect of the investigated drugs was determined by a standard MTT method23 with some modifications. Logarithmically growing cells (5 × 104 cells/well) were seeded into a 96-well plate and 24 h later incubated with a series of drug concentrations for 2 h. After two washings of cell monolayers with PBS, 200 µl of fresh medium were added to each well and cells were allowed to grow for additional 72 h. Then the medium in each well was replaced with 50 µl of MTT (3-[4,5-dimethylthiazol2-yl]-2,3-diphenyltetrazolium bromide), 5 mg/ml final concentration, and four hours later 100 µl DMSO/well were added in order to dissolve the formed violet formazan crystals within metabolically viable cells. The plates were gently shaken for 5 min at room temperature to allow complete dissolving of the formazan and read at 545 nm with a microplate reader. Cytotoxicity of the drugs was compared on the basis of their IC50 values, the inhibitory drug concentration resulting in a 50% reduction of cell survival relative to the survival of untreated (control) cells, taken as 100%. Identification of DNA fragmentation 5–6 × 106 cells were lysed at 4◦ C for 10 min with TET buffer (5 mM Tris pH 8.0, 20 mM EDTA and 0.5% Triton X-100). The supernatant fractions were collected by centrifugation at 12 000 × g for 30 min and the DNA was extracted with phenol-chlorophorm-isoamyl alcohol (24:24:1, v/v) and then precipitated with 3 M sodium acetate and 100% ethanol at –20◦ C overnight. DNA pellets were resuspended in 20 µl TE buffer (10 mM Tris,

Mechanisms of induction of apoptosis

1 mM EDTA pH 8.0), RNAse A was added to the final concentration of 10 µg/ml and samples were incubated at 37◦ C for 2 h. DNA were analyzed by electrophoresis in a 1.8% agarose gel stained with 0.5 µg/ml ethidium bromide. PCR marker (Sigma) was used as a reference for DNA size. DNA electrophoresis was performed in TBE buffer (0.1 M Tris-HCl, 0.1 M boric acid and 1.5 mM EDTA), pH 8.0, at 38 V for approximately 16 h. The resulting DNA fragmentation pattern was visualized and photographed under UV illumination.

Flow cytometric analysis of DNA Percentage of apoptotic cells was estimated by a flow cytometry method on the basis of cell fraction in the subG1 peak on DNA histograms, considered as an indicator of the onset of apoptosis.24–26 Exponentially growing cells were incubated for 2 h in culture conditions with 0.5, 1 and 2 µM drug concentrations chosen on the basis of a drug cytotoxicity assay in order to include both lower and higher values than IC50 . Then drug-containing medium was removed, cell monolayers were washed twice with pre-warmed (37◦ C) PBS, fresh medium was added and cells were further cultured for up to 72 h. Cells were harvested at defined time points for flow cytometric analysis: directly after the treatment (0 h time point) and after 24, 48 and 72 h. Cells were pelleted by centrifugation at 200 × g and after two washings with cold (4◦ C) PBS, resuspended in PBS at the final concentration of 106 cells/ml/sample, fixed in cold 70% ethanol and stored at −20◦ C until analysis. Before the measurement of their DNA content, cells were washed twice with PBS, resuspended in 0.5 ml PBS and stained with 1 µg/ml DAPI (4 –6 diamidino-2-phenylindole), which dyes DNA in blue, and 20 µg/ml of sulforhodamine, which dyes protein in red. Dyes were dissolved in 10 mM piperazine-N, N-bis-2-ethanesulfonic acid buffer, containing 100 mM NaCl, 2 mM MgCl2 and 1% Triton X-100 (pH 6.8) at 0–4◦ C. The multicycle program (Becton Dickinson) was used for the analysis of DNA and protein content on a FACS Vantage laser flow cytometer (Beckton Dickinson) using Cell-Quest software. For each sample at least 1 × 104 cells were analyzed.

Cell morphology assessment Cells designated for the evaluation of the morphological changes, associated with apoptosis, were prepared in the same way as for the flow cytometry analysis. Cell morphology was evaluated using an Olympus BX light 60 fluorescence microscope under 400 × magnification. Photographs were taken under UV light on a Fuji 200 slide film.

Morphological assessment of apoptosis and necrosis: Double staining with Hoechst 33258-Propidium iodide (PI) For the demonstration the ratio between live, apoptotic and necrotic cell fractions, we applied simultaneous cell staining with Hoechst 33258 and propidium iodide (PI). Both fluorescent dyes vary in their spectral characteristics and ability to penetrate cells. The blue-fluorescent Hoechst 33258 stains the condensed chromatin of apoptotic cells more brightly than the looser chromatin of normal cells. The red-fluorescent propidium iodide is permeant only to necrotic cells. Thus, the double staining method allows one to estimate quantitatively the percentage of apoptotic and necrotic cells in the same cell population. After drug-treatment, cells were cultured in fresh medium for a further 12–72 h. At defined time points the cells were removed from the culture dishes by trypsynization, centrifuged and suspended in PBS at the concentration 106 cells/ml. To 100 µl of cell suspension 1 µl Hoechst 33258 (0,13 mM) and 1 µl PI (0,23 mM) were added and the cells were incubated at room temperature, for 5 min, in the dark. The analysis was done on a fluorescence microscope (Olympus IX70, Japan) under 400 × magnification. At least 300 cells were counted on each slide and each experiment was done in triplicate. Cells were classified as live, apoptotic or necrotic on the basis of their morphological and staining characteristics27,28 and the percentages of particular cell types were determined from the total number of cells.

Estimation of caspase-3 activity A caspase-3 activity assay was performed under the same conditions as the apoptosis assessment. Control and drug treated cells were harvested and collected in phosphate buffered saline (PBS) (2 × 106 cells/sample), and cell pellets after short centrifugation at 200 × g were kept frozen at −80◦ C until analysis. Caspase 3 activity was estimated by means of a fluorimetric method with a Caspase ZDEVD-AMC Assay Kit #1 (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s protocol. Microtiter wells were set in 4–8 repeats for controls, blanks and drug-treated cells. The measurement was done on a Fluoroskan Ascent FL microplate reader (Labsystems, Sweden), using 355 nm excitation and 450 nm emission wavelengths. Caspase-3 activity was expressed as a ratio of the fluorescence of the drug-treated samples relative to the corresponding untreated controls taken as 100%. The Ac-DEVD-CHO inhibitor was used in the control experiments to confirm that the observed fluorescence in both the control and the drug-treated cells is due to caspase-3 presence in the samples. Apoptosis · Vol 10 · No 6 · 2005

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Evaluation of the effect of the caspase-3 inhibitor Ac-DEVD-CHO on drug cytotoxicity and the induction of apoptosis

Figure 1. Cytotoxicity of aclarubicin (ACL), doxorubicin (DOX) and mitoxantrone (MTX) in B14 and NIH 3T3 cells.

In our experiments involving the influence of the reversible aldehyde caspase-3 Ac-DEVD-CHO inhibitor29 on drug cytotoxicity and the induction of apoptosis, prior to drug-treatment, cells were pretreated for 1 h with the inhibitor (40 µM/100 µM) and then incubated with a drug for an additional 2 h in the presence of the inhibitor.

Statistical analysis The data are expressed as a mean ± S.D. An analysis of variance with a Tuckey post hoc test was used for multiple comparisons. All statistics were calculated using a statistical program STATISTICA (StatSoft, Tulsa, OK, USA). A P value of DOX > ACL in the B14 cells and MTX > ACL > DOX in the NIH 3T3 cells. No significant differences in the cell survival curves and the IC50 drug concentrations were found between cells preincubated with the caspase-3 Ac-DEVD-CHO inhibitor and cells treated with a drug alone (Figure 2).

Induction of apoptosis To evaluate the possible role of apoptosis in drug cytotoxicity as well as the mode of cell death triggered by individual drugs, we investigated the time course of apop1500 Apoptosis · Vol 10 · No 6 · 2005

tosis and its dependence on drug concentration. Using different methods, enabling qualitative and quantitative assessments of molecular events connected with apoptosis, we investigated the average effects of aclarubicin, doxorubicin and mitoxantrone on cell population, as well as morphological changes at the single cell level. Additionally, we employed the caspase-3 Ac-DEVD-CHO inhibitor to evaluate the involvement of caspase-3 protease in the pathway of apoptosis and the role of this enzyme in the mode of cell death triggered by the investigated drugs.

Time-course of apoptosis: Dependence of DNA fragmentation on the post-treatment time duration The time-course of apoptosis was estimated after incubation of cells for 2 h with an equal concentration of each

Mechanisms of induction of apoptosis Figure 3. Drug-induced DNA fragmentation detected by agarose gel electrophoresis of DNA isolated from B14 cells. Fibroblasts were treated with 1 µM of doxorubicin (A), aclarubicin (B) or mitoxantrone (C) for 2 h and then cultured in drug-free medium for 6, 12, 24, 36 and 48 h. DNA isolated from 6 × 106 cells were loaded in each line.

drug (1 µM) followed by a further cell culture in drug-free medium for up to 48 h. The appearance of the oligonucleosomal fragmentation, typical of apoptotic cells, was observed in B14 cells. Internucleosomal DNA cleavage was detected at the earliest (after 12 h) in cells treated with aclarubicin. 12 h later a DNA ladder appeared in cells exposed to doxorubicin and after another 12 h in cells incubated with mitoxantrone. Whenever apoptosis was triggered the extent of DNA damage was rather independent of the type of the drug and the duration of post-treatment time. Cells contained predominantly oligonucleosomal sized DNA, in the midsize range of 3–6 nucleosome multiples (600–1200 bp) (Figure 3) Under the same conditions, no apoptosis-like DNA ladder pattern was found in NIH 3T3 cells.

Dependence of DNA fragmentation on drug concentration With the purpose of establishing the lowest drug concentration sufficient to induce detectable DNA cleavage, we exposed B14 cells to a wide range of drug concentrations in the subtoxic and toxic ranges (0.01–1 µM) for 2 h and then cultured the cells in drug-free medium for an additional 48 h. This period of time was chosen on the basis of experiments on the time-course of apoptosis, described above, which showed that the slowest acting mitoxantrone, at the concentration of 1 µM, did not induce DNA fragmentation earlier than 36 h after the treatment (Figure 4). Therefore, we assumed that probably the same, or even longer, amount of time would be necessary for this drug to cause DNA fragmentation at lower concentration. As shown in Figure 4, subtoxic con-

centrations of the investigated compounds were sufficient to induce pronounced DNA cleavage at this time point. The appearance of the DNA ladder was dependent on both the drug type and its concentration. Mitoxantrone induced DNA fragmentation at the concentration lower by an order of magnitude (0.01 µM) than aclarubicin (0.2 µM) and doxorubicin (0.3 µM). Since a similar relation was found between the cytotoxicity (IC50 ) of mitoxantrone and anthracyclines, these data suggest that DNA fragmentation might be an important factor for the cytotoxicity of mitoxantrone in B14 cells. The effect of the caspase-3 Ac-DEVD-CHO inhibitor on DNA fragmentation In order to investigate the importance of caspase-3 activation for the apoptotic changes induced by the investigated drugs on DNA level, we pretreated B14 cells for 1 h with the inhibitor and then incubated the cells with an equal concentration (1 µM) of each drug for an additional 2 h in the presence of the inhibitor. DNA fragmentation was estimated 24 and 48 h following the treatment. Ac-DEVDCHO could not prevent the apoptotic DNA degradation at both 40 and 100 µM concentrations, independently on the length of the post-treatment period. What was observed was oligonucleosomal DNA fragmentation without noticeable differences in the extent of DNA cleavage between cells exposed to the investigated drugs in the absence or in the presence of Ac-DEVD-CHO (Figure 5).

Flow cytometric analysis Flow cytometry analysis confirmed the results obtained from the DNA fragmentation study. A small number (less Apoptosis · Vol 10 · No 6 · 2005

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A. Koceva-Chyła et al. Figure 4. Evidence of apoptosis appearing as a DNA ladder in agarose gel electrophoresis. B14 cells were treated with different concentrations of DOX (A), ACL (B) or mitoxantrone (C, D) for 2 h and cultured in drug-free medium for the additional 48 h. M—PCR marker; 1—control (untreated cells); 2—0.1 µM; 3—0.2 µM; 4—0.3 µM; 5—0.4 µM; 6—0.5 µM; 7—1.0 µM; 8—0.001 µM; 9—0. 002 µM; 10—0.003 µM; 11—0.005 µM; 12—0.01 µM; 13—0.05 µM. DNA isolated from 6 × 106 cells were loaded in each line.

Figure 5. The effect of Ac-DEVD-CHO caspase-3 inhibitor on fragmentation of DNA isolated from control and drug-treated B14 cells: M—marker C—control cells; 1—ACL (1 µM), 48 h; 2—ACL (1 µM + 100 µM Ac-DEVD-CHO), 48 h; 3—DOX (1 µM), 48 h; 4—DOX (1 µM + 100 µM Ac-DEVD-CHO), 48 h; 5—MTX (1 µM), 36 h; 6—MTX (1 µM + 100 µM Ac-DEVD-CHO), 36 h; 7—MTX (1 µM), 48 h; 8—MTX (1 µM + 100 µM Ac-DEVD-CHO), 48 h; 9—DOX (1 µM), 24 h; 10—DOX (1 µM + 100 µM Ac-DEVDCHO), 24 h; 11—ACL (1 µM), 24 h; 12—ACL (1 µM + 100 µM Ac-DEVD-CHO), 24 h.

than 4%) of cells with spontaneous DNA degradation was identified in the sub-G1 DNA peak of the untreated (control) cells (Table 1). 2 h following the incubation of the cells with drugs (time point 0), the sub-G1 fraction 1502 Apoptosis · Vol 10 · No 6 · 2005

did not exceed that of the control cells. An increase in the percentage of hypodiploid cells, dependent on the cell line and the drug type, was detected 24–72 h following the treatment. In B14 cells, aclarubicin triggered apoptosis at the earliest and caused the most pronounced changes. After 24 h, a subtoxic concentration of this drug (0.5 µM) induced a 17.7% increase in the sub-G1 cell population, whereas at this time point no perceptible DNA degradation occurred in cells exposed to the same concentration of doxorubicin or mitoxantrone (Table 1). While only toxic concentrations (1–2 µM) of doxorubicin were sufficient to induce a visible increase in the number of hypodiploid cells 24 h after the treatment, mitoxantrone, irrespective of its concentration, was unable to trigger any apoptotic changes until 48 h. After a prolonged time (48 and 72 h), aclarubicin, at the concentrations of 1–2 µM, caused the most excessive augmentation of the apoptotic cells (about 80% on average). At the same time points, a considerably lower amount of apoptotic cells were found in cells treated with doxorubicin or mitoxantrone. An increase in the fraction of hypodiploid cells in the B14 cell line followed the same kinetics as DNA fragmentation: ACL > DOX > MTX. The NIH 3T3 cell line, in contrast to B14 cells, was rather resistant to the induction of apoptosis by the investigated anthraquinone drugs. The percentage of hypodiploid cells after the drug treatment did not differ much from that found in untreated cells and was dependent to a small degree on drug concentration and the duration of the post-treatment period (Table 1). Doxorubicin and mitoxantrone, even at a toxic concentration (2 µM), did not induce substantial rise in the apoptotic cell fraction. Aclarubicin was the only drug, which caused

Mechanisms of induction of apoptosis Table 1. Time- and concentration-dependent apoptosis in B14 and NIH 3T3 fibroblasts after DOX, ACL or MTX treatment Percentage of apoptotic cells has been calculated as sub G1 peak in flow cytometric cell cycle analysis. The values are the mean ± SD of three independent experiments in 3–5 repeats each ACL

c [µM]

Posttreatment time [h]

1.0

2.0

MTX

B14 2.3 ± 0.65

Control 0.5

DOX

0

3.2 ± 0.57

2.0 ± 0.55

4.5 ± 0.68

24

17.7 ± 2.21

4.5 ± 0.49

6.4 ± 0.72

48

30.2 ± 3.40

19.0 ± 3.06

35.9 ± 3.78

72

47.1 ± 5.22

25.7 ± 6.77

39.0 ± 4.60

0

4.8 ± 0.44

4.1 ± 0.55

2.5 ± 0.33

24

52.2 ± 6.23

15.8 ± 1.96

4.5 ± 0.59

48

69.8 ± 7.25

36.7 ± 4.03

41.7 ± 4.56

72

83.7 ± 7.99

47.9 ± 5.01

53.5 ± 5.82

0

6.1 ± 0.68

6.5 ± 0.49

3.7 ± 0.32

24

67.8 ± 7.26

24.1 ± 3.35

3.7 ± 0.45

48

79.4 ± 6.15

49.2 ± 6.20

39.5 ± 2.78

72

84.2 ± 9.02

69.2 ± 5.55

62.3 ± 5.05

NIH 3T3 3.9 ± 0.42 0.5

1.0

2.0

0

4.2 ± 0.26

4.0 ± 0.32

2.7 ± 0.18

24

5.1 ± 0.66

4.3 ± 0.47

7.2 ± 0.63

48

17.6 ± 1.89

12.3 ± 0.99

9.8 ± 1.35

72

17.5 ± 1.55

14.3 ± 1.26

5.7 ± 0.68

0

4.0 ± 0.42

2.6 ± 0.33

8.8 ± 0.79

24

8.8 ± 0.68

8.3 ± 0.75

15.2 ± 1.46

48

19.5 ± 2.08

9.2 ± 1.57

21.0 ± 2.45

72

21.7 ± 2.12

14.2 ± 1.89

24.0 ± 2.65

0

4.3 ± 0.67

4.2 ± 0.55

7.1 ± 0.73

24

38.8 ± 4.45

6.6 ± 0.88

17.9 ± 2.54

48

70.3 ± 6.66

11.1 ± 2.63

15.3 ± 1.15

72

85.0 ± 7.79

14.2 ± 1.12

16.5 ± 1.44

noteworthy cleavage of DNA, but no more than at the toxic concentration and after a prolonged post-treatment time. While about an 80% increase in the sub-G1 cells on average, comparable to that found in B14 cells, was present 48 and 72 h following the cell treatment with 2 µM aclarubicin, the apoptotic fraction in the cells incubated with lower concentrations of this drug (0.5 and 1 µM) was rather small and did not exceed 22%.

Morphological changes in drug treated cells In view of the different patterns of time- and concentration-dependent DNA fragmentation triggered

by the individual drugs in B14 cells, lack of DNA fragmentation in NIH 3T3 fibroblasts, and substantial differences in the number of hypodiploid cells between these cell lines, we further aimed at estimating the apoptotic events induced by the investigated anthraquinone drugs at the single cell level. Numerous changes in cell morphology, typical either for apoptosis or necrosis were detected (Figures 6 and 7). Alterations in the structure, size and shape of the cell nucleus, and a significant number of polyploid cells containing two nuclei was observed 24 h following the treatment of B14 cells with doxorubicin. After a prolonged post-incubation time (48–72 h), we observed chromatin condensation, cell shrinkage and nuclear fragmentation Apoptosis · Vol 10 · No 6 · 2005

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A. Koceva-Chyła et al. Figure 6. Fluorescent microscopy images of B14 fibroblasts treated with 1 µM doxorubicin (DOX), aclarubicin (ACL) or mitoxantrone (MTX) for 2 h and then cultured in drug-free medium for 24, 48 and 72 h (C—control cells). Note the typical morphological features of apoptosis: loss of the structural framework of the nuclei, condensation of chromatin, cell shrinkage, nuclear fragmentation and detachment of apoptotic bodies (white arrows). Numerous cells with two nuclei (blue arrow) were observed as early as 24 h after cell treatment with DOX or ACL. Swollen enlarged cells typical for necrosis (light grey arrow) were present 24 h after cell treatment with MTX; cell disintegration (dark grey arrow).

as well as a reduction in cell volume, formation and detachment of apoptotic bodies and cell disintegration. Although some of the nuclear changes in doxorubicintreated cells appeared relatively early, cell proliferation was not considerably inhibited and most cells still possessed ability to proliferate. Some cells were polyploid and contained two nuclei 24 h after their exposure to aclarubicin, while others were shrunken with highly condensed chromatin. After a prolonged post-incubation time, nuclear fragmentation, apoptotic bodies and cell disintegration was evident, too. Compared to anthracyclines, mitoxantrone induced apoptotic changes and the disintegration of apoptotic cells considerably later (after 48 h), but enlarged necrotic cells were observed as early as 24 h after treatment with this drug (Figure 6). In the case of NIH 3T3 cell line polyploidy (two nuclei) was found only in cells treated with aclarubicin. 1504 Apoptosis · Vol 10 · No 6 · 2005

After a prolonged post-incubation time (72 h), formation of apoptotic bodies was also observed. Swollen enlarged necrotic cells were present 48 h after exposure to doxorubicin or mitoxantrone (Figure 7). Sequence in the appearance of apoptotic changes in cell morphology as well as their intensity showed similarity with the sequence in the appearance of DNA fragmentation and with the increase in the number of the hypodiploid cell fraction as well. Mitoxantrone was the slowest acting drug, inducing biochemical and morphological apoptotic symptoms considerably later than aclarubicin and doxorubicin. Aclarubicin, compared to doxorubicin and mitoxantrone, generated the most intense apoptotic changes and within the shortest time. In cells treated with doxorubicin or mitoxantrone evidence for the appearance of necrotic cell death was also present as early as 24 after the treatment (MTX in B14 cells).

Mechanisms of induction of apoptosis Figure 7. Photographs of NIH 3T3 cells exposed to 1.0 µM DOX, ACL and MTX for 2 h and incubeated in drug-free medium for 24, 48 and 72 h. Apoptotic bodies were observed only in ACL-treated and after the longest of the postincubation times (white arrow). In the case of DOX- and MTX-treated cells only cell shrinkage and condensation of chromatin were observed (white arrow). Swollen enlarged cells typical for necrosis (light grey arrow) were found 48 h after cell treatment with MTX.

Assessment of apoptosis and necrosis: Double staining with Hoechst 33258-Propidium iodide (PI) Given the presence of morphological changes typical for either apoptosis or necrosis, we subsequently evaluated the involvement of each of these processes in cell death induced by the investigated drugs. For this purpose, we applied simultaneous staining of cells with Hoechst 33258 and propidium iodide (PI). These fluorescent dyes, when exited, emit different fluorescence and possess a different ability to penetrate cells. The blue-fluorescent Hoechst 33258 can cross the intact membrane of live cells, stains the condensed chromatin of apoptotic cells more brightly than the looser chromatin of normal cells and enables monitoring nuclear changes associated with apoptosis such as chromatin condensation and nuclear fragmentation. Propidium iodide is excluded from viable and early apoptotic cells. Thus, its uptake indicates loss of membrane integrity, characteristic of late apoptotic and

necrotic cells (membrane altered cells). In combination with a fluorescence microscopy technique, the selective uptake of the two dyes allows one to monitor the induction of apoptosis in intact cell cultures and to distinguish it from nonapoptotic cell death (necrosis). Necrotic cells were characterized by nuclear PI uptake without chromatin condensation or nuclear fragmentation. Thus, on the basis of the applied staining procedure we were able to identify four types of cells: (i) viable cells (bright blue fluorescence); (ii) early apoptotic cells (cells showing intensive blue fluorescence and the presence of apoptotic features); (iii) late apoptotic cells (blue-violet stained cells with concomitant apoptotic morphology) and (iv) dead (necrotic) cells (prevalence of red fluorescence and also very intensive blue fluorescence). All counted cells were taken as 100% and the cell number in each group was calculated as a percentage of the whole. We found B14 cells more susceptible to apoptosis and necrosis induced by the investigated drugs than NIH 3T3 cells (Figure 8). Although overall percentage of the Apoptosis · Vol 10 · No 6 · 2005

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A. Koceva-Chyła et al. Figure 8. Fractions of the early apoptotic, late apoptotic and necrotic B14 (A) and NIH 3T3 (B) cells at different time following the treatment with 1 µM ACL, DOX or MTX; C–F—cells pretreated for 1 h with 100 µM of Ac-DEVD-CHO caspase-3 inhibitor and then incubated for 2 h with 1 µM ACL, DOX or MTX in the presence of the inhibitor. After the treatment the cells were cultured in drug-free medium for 24 h (C, D) and 48 h (E, F).

apoptotic cells in this cell line remained at a low level during the first 24 h following the treatment, even then aclarubicin, as compared to doxorubicin and mitoxantrone, induced a higher level of apoptosis. A pronounced increase (by about 30%) in the number of apoptotic cells (early and late) was found 48 h following ex1506 Apoptosis · Vol 10 · No 6 · 2005

posure to aclarubicin, and a respectively smaller increase (by about 20% and 15%) was observed in cells treated with doxorubicin or mitoxantrone. During the following 48–72 h post-treatment period, the percentage of the apoptotic fraction after aclarubicin or doxorubicin treatment strikingly decreased, whereas the apoptotic fraction

Mechanisms of induction of apoptosis

in cells treated with mitoxantrone continued to increase. The maximal increase in the percentage of apoptotic cells was noted 48 h after treatment with aclarubicin and 72 h after treatment with MTX. Dead or membrane-altered cells, emitting an intensive red fluorescence signal, were detected in all cases and were the consequence of cellular damage, induced by the technique, combined with the effect of the drugs on the cell membrane. At 37◦ C, dead or membrane-altered cells in controls represented on average 4–8% of the total cell population. After doxorubicin or mitoxantrone treatment the fraction of the necrotic B14 cells outnumbered the fraction of the apoptotic cells at any of the post-treatment time points investigated (Figure 8A). These results may imply that immortalized B14 cells treated with a 1 µM concentration of doxorubicin or mitoxantrone died prevalently by necrosis, although in other publication it has been suggested that in tumor cells doxorubicin causes necrosis at doses higher than 1 µM.31 A decrease in the apoptotic cell fraction at the expense of an increase in the necrotic cell fraction after a prolonged post-treatment period (72 h) pointed to the fact that a part of the apoptotic B14 cells after a prolonged post-incubation time (48–72 h) might have switched to the necrotic mode of death. Under the same conditions a steady state-increase in the amount of apoptotic and necrotic cells was observed in NIH 3T3 cells. Similarly to B14 cells, in this cell line aclarubicin induced a slightly higher level of apoptosis than doxorubicin and mitoxantrone (Figure 8B), but the differences were considerably lesser than those observed in the B14 cell line. Generally, the percentage of apoptotic and necrotic cells in NIH 3T3 cells was lower than that in B14 cells. To investigate whether caspase-3 activation is essential for the mode of cell death induced by the investigated anticancer drugs, we examined the effect of the cell permeable caspase-3 Ac-DEVD-CHO inhibitor on the size of the apoptotic and necrotic cell fractions at 24 and 48 h time points following drug exposure. Preincubation of B14 cells with the caspase-3 AcDEVD-CHO inhibitor (40/100 µM) led to a decrease in the percentage of apoptotic cells, mostly after treatment with aclarubicin. At the 24 h time point, a lower concentration of the inhibitor (40 µM) was sufficient to partly reduce the percentage of the apoptotic fraction in cells treated with aclarubicin, while pre-incubation with a higher concentration (100 µM) of the inhibitor was adequate to diminish the number of the early apoptotic fraction also in cells incubated with doxorubicin or mitoxantrone and additionally the number of necrotic cells after ACL treatment (Figure 8C and E, data shown only for 100 µM concentration). At the 48 h time point the Ac-DEVD-CHO inhibitor caused a significant decrease in the amount of apoptotic and necrotic cells following treat-

ment with aclarubicin or doxorubicin and in the number of apoptotic cells after exposure to mitoxantrone. The inhibitor, however, did not influence the level of necrosis in cells incubated with mitoxantrone. In NIH 3T3 cells, pretreatment with Ac-DEVDCHO, irrespective of the length of the post-incubation period, resulted in a decrease in the level of apoptotic and necrotic cells following treatment with aclarubicin, but did not show any influence on necrosis induced by doxorubicin or mitoxantrone (Figure 8D and F). In cells treated with DOX or MTX the caspase-3 inhibitor caused a decrease only in the fraction of the early apoptotic cells and after a prolonged postincubation time (48 h).

Induction of caspase-3 activation Caspase-3 activation was determined on the basis of the proteolytic cleavage of the Z-DEVD-AMC substrate. Although caspase-3 may be the major enzyme to cleave at the DEVD sequences, other caspases can also recognize this sequence (e.g. caspases 6, 7, 8 and 10). Hence, DEVD-ase activity is often referred to as “caspase-3-like.” Caspase-3 activation was considerably greater in B14 cells where a significant increase in the enzyme activity was found during the 12–48 h post-treatment period. The foremost changes were found in cells exposed to mitoxantrone and the least ones in cells incubated with aclarubicin. Caspase-3 activation, independently on the type of the drug studied, reached maximal level 24 h after the treatment, followed by a decrease at 48 h (Figure 9A). Caspase-3 activation was not observed during the first 6 h following drug exposure. In NIH 3T3 cells, caspase-3 activation showed a considerably lesser and slower steady-state increase during the entire post-treatment period. Similarly to B14 cells, mitoxantrone induced the major changes and aclarubicin – the least. Maximal increase in caspase-3 activation, observed 48 h following the treatment with aclarubicin, did not exceed 26%. Doxorubicin and mitoxantrone caused similar increase in the activity of caspase-3—about 1.5fold after 24 h and about 2-fold after 48 h (Figure 9B). Taking into account that investigated drugs showed considerably different cytotoxicity in both cell lines, and thus considerably dissimilar 1 µM/IC50 ratio, varied from 0.89 (B14) to 0.92 (NIH 3T3) for ACL, through 1.92 (B14)–0.37 (NIH 3T3) for DOX to 20 (B14)–11.1 (NIH 3T3) for MTX, we aimed at the comparing the activation of caspase-3 induced by the IC50 and 1 µM drug concentrations to test out for any dose and cytotoxicity dependence. In NIH 3T3 cells, a comparable, steady-state increase in caspase-3 activity was observed irrespective of the drug concentrations (IC50 or 1 µM), while B14 cells demonstrated some variations dependent on the drug Apoptosis · Vol 10 · No 6 · 2005

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A. Koceva-Chyła et al. Figure 9. A–D: Time-dependent changes in caspase-3 activity in B14 and NIH 3T3 cells treated with 1 µM or IC50 doses of ACL, DOX or MTX for 2 h and then cultured in drug-free medium for 3–48 h. E: The effect of the caspase-3 inhibitor Ac-DEVD-CHO on caspase-3 activity in B14 and NIH 3T3 cells treated with 1 µM ACL, DOX or MTX. Cells were pretreated for 1 h with 100 µM of Ac-DEVD-CHO caspase-3 inhibitor and then incubated with 1 µM of ACL, DOX AND MTX for additional 2 h in the presence of the inhibitor. Further the cells were cultured in drug-free medium for 24 h.

type (Figure 9C and D). After the treatment with aclarubicin, which IC50 was approximately equal to 1 µM, similar increase in caspase-3 activity was observed for both concentrations (about 2.5-fold after 24–48 h postincubation time). In cells treated with doxorubicin or mitoxantrone 24 h following the treatment greater increase in caspase-3 activation was observed for 1 µM drug

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concentration—3.8-fold (DOX) and 4.4-fold (MTX) than for IC50 concentrations−3.4-fold and 3.7-fold, respectively. This implies positive correlation between caspase3 activation and drug concentration in this cell line. Mitoxantrone, which was characterized by the highest 1 µM/IC50 ratio caused the highest increase in caspase 3-activation when was applied at 1 µM concentration.

Mechanisms of induction of apoptosis

The effect of the caspase-3 Ac-DEVD-CHO inhibitor on caspase-3 activity in drug-treated cells The preincubation of both cell lines with 100 µM AcDEVD-CHO inhibitor prior their treatment with drugs almost completely suppressed DEVD-specific caspase activity at the 24 h time point following drug exposure. At a lower (40 µM) concentration of the inhibitor only partial inhibition was observed (Figure 9E and F, data shown for the 100 µM concentration only). The preincubation of cell lysates with 40 µM AcDEVD-CHO before the addition of the Z-DEVD-AMC substrate partially inhibited caspase-3 activity, whereas almost complete blockage was observed at 100 µM of Ac-DEVD-CHO (data not shown). These results confirm that caspase-3 activation indeed took place during the course of apoptosis induced by aclarubicin, doxorubicin or mitoxantrone and that an increase in the fluorescence of the Z-DEVD-AMC substrate was directly related to caspase-3 activity.

Discussion Apoptosis has been suggested to play an important role in the therapeutic effects of anthraquinone drugs on tumor cells. Although the molecular mechanisms of cytostatic and cytotoxic action of anthraquinones are intensively investigated, the role of apoptosis in cell kill by these drugs is still under debate. It has been suggested that the cytotoxic effect of anthracyclines is directly related to the extent of apoptosis they induce in tumor cells.16–19 At the same time, in other research no positive correlation between the induction of apoptosis and anthracycline cytotoxicity has been found.20,21 Moreover, the evidence has been provided for the role of necrosis in cytotoxicity of some anticancer drugs and necrotic cell death has been proposed as a more advantageous and favorable mode of cell kill in effective chemotherapy.13,30,31 Therefore, the elucidation of the type of cell death induced by anthraquinone drugs and the role of apoptosis/necrosis in drug cytotoxicity is very important for understanding the pharmacological activity of these compounds and for the development of the most effective therapeutic strategies. With the purpose of investigating the importance of programmed cell death in the cytotoxicity of the anthraquinone drugs aclarubicin and mitoxantrone, we used a range of methods, which enabled us to estimate the mode of cell death and the course of apoptosis induced by these drugs. We compared their mechanisms of action with doxorubicin—the most intensively investigated anthracycline. Cells were examined for biochemical hallmarks of apoptosis such as DNA oligonucleosomal fragmentation, hypodiploid sub-G1 (apoptotic) cell population, and

caspase-3 activation, and for morphological changes connected with both apoptosis/necrosis. We have found that the immortalized cell lines B14 and NIH 3T3 used in the experiments exhibit markedly different sensitivity to the studied drugs. Aclarubicin indicated similar cytotoxicity towards both cell lines. The effect of mitoxantrone, similarly to that of doxorubicin, was dependent on the cell line but in both cell lines this drug was noticeably more toxic than aclarubicin and doxorubicin. These results are consistent with other data obtained in vivo and in vitro with neoplastic cell lines, where mitoxantrone was reported to be considerably more toxic than anthracyclines.32,33 Using agarose gel electrophoresis, flow cytometry and fluorescence microscopy methods we have demonstrated that the investigated drugs can trigger both apoptosis and necrosis. The extent and the timing of apoptotic responses were dependent on the kind of cell line, the type of drug studied, and its dose. Under studied conditions, DNA fragmentation was not detected in NIH 3T3 cells up to 72 h after drug treatment. In B14 fibroblasts, under the same conditions, an apoptosis-like DNA ladder was observed 12 h and 24 h following cell exposure to aclarubicin and doxorubicin, respectively. Mitoxantrone induced oligonucleosomal DNA fragmentation considerably later (after 36 h), regardless of its notably greater cytotoxicity. These results are in agreement with our earlier study showing that mitoxantrone was an effective inducer of apoptosis in vivo, with a relatively long lag phase.34 Different time- and dose-dependent increase in the apoptotic cell population was found in B14 and NIH 3T3 cell lines. The percentage of apoptotic cells in the sub-G1 DNA peak was remarkably higher in ACL-treated cells than in cells incubated with DOX or MTX. These results were confirmed by double staining the drug-treated cells with Hoechst 33258 and PI. The observed increase in the apoptotic cell fraction was mainly due to the direct interaction of the investigated drugs with nuclear DNA. In B14 cells, an increase in the amount of the apoptotic cells was positively correlated with the appearance of DNA fragmentation. Aclarubicin caused the earliest and the greatest increase in the apoptotic cell fraction, and the earliest appearance of DNA ladder. Apoptosis-like morphological events, such as apoptotic body formation, alterations in the structure of the cell nucleus, as well as polyploidal cells with two nuclei were observed 24 h after DOX administration. These morphological changes were correlated with the internucleosomal degradation of DNA induced by this drug by that time point. In NIH 3T3 fibroblasts, apoptotic bodies, similarly to an increase in the apoptotic cell population, appeared considerably later (at 72 h), and after ACL treatment only. The presence of numerous cells with two nuclei evidences a post-mitotic arrest in part of the drug-treated cells and Apoptosis · Vol 10 · No 6 · 2005

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cytokinesis failure, resulting in polyploidy. This observation was confirmed by a flow cytometry analysis, which revealed a progressive G2/M block in both B14 and NIH 3T3 cells after their treatment with any of the investigated drugs (data not shown). A fluorescence microscopy examination of drug-treated cells revealed numerous changes in their morphology, typical also for necrosis. The fraction of necrotic cells increased with the length of post-treatment time. This suggests that part of the apoptotic cells could switch to necrotic mode of death after a prolonged post-treatment time. We have found that in both immortalized cell lines, doxorubicin and mitoxantrone induced prevalently necrotic cell death, while aclarubicin caused mainly apoptosis. In B14 cells, the extent of apoptosis was partly inhibited by the caspase-3 Ac-DEVD-CHO inhibitor irrespectively of the drug type. A decrease in the necrotic cell population was, however, observed only in cells exposed to aclarubicin (after 24 and 48 h) or doxorubicin (after 48 h). Ac-DEVD-CHO showed the greatest inhibitory effect on the apoptotic and necrotic cell fractions after aclarubicin treatment. In NIH 3T3 cells, Ac-DEVD-CHO partially inhibited apoptosis and necrosis only after treatment with aclarubicin. In cells exposed to doxorubicin or mitoxantrone, the inhibitor decreased only the fraction of apoptotic cells 48 h after drug treatment. These results suggest the involvement of caspase-3 protease in the pathway of apoptosis triggered by the investigated anthraquinone drugs in immortalized fibroblasts. We also showed that a considerably lower level of caspase-3 activity in NIH 3T3 cells, as compared to B14 cells, closely corresponds to the noticeably lower percentage of the apoptotic cells in this cell line. The major evidence for the central role of caspases in apoptosis comes from those experiments in which caspase inhibitors block the appearance of apoptotic features. The same caspase inhibitors applied in other studies failed to inhibit some of the apoptotic events, showing that, the inactivation of caspase-3 is not sufficient to suppress entirely all hallmarks of apoptosis. At present it is generally accepted that caspase inhibitors can prevent the appearance of certain, but not all, features of apoptosis in certain model systems.35–37 Moreover, several forms of cell death seem to be caspase-independent, or even to be accelerated by caspase inhibitors.38–40 In numerous studies it has been shown that caspase-3 activation is responsible for determining the mode of cell death—apoptosis or necrosis—but at the same time a caspase-independent pathway has been demonstrated for apoptosis.41–46 It has been found that the activation of caspases can occur without inducing cell death, and other protease families including serine proteases, cathepsin and calpains have been implicated in apoptosis as well.47–49 1510 Apoptosis · Vol 10 · No 6 · 2005

In some experimental models caspase inhibition did not alter the extent of cell death, but rather its mode.41 In vivo, under pathological conditions, apoptosis and necrosis may often coexist. Cells committed to undergo apoptosis can be forced to die by necrosis when intracellular ATP is depleted.50 Recently, evidence has been provided that when intracellular energy levels were compromised early in dying neurons, cell death progresses in a caspase3 independent way.51,52 The apoptosis-inducing factor (AIF), a 57 kDa protein, can directly cause some apoptotic events in these cells.53 Chromatin condensation and DNA fragmentation in high molecular fragments were present in neurons treated with the caspase inhibitor ZVAD-fmk.52 We have found that in immortalized B14 cells DNA fragmentation to the smaller oligonucleosomal fragments occurred in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Caspase-3 activation was sufficient to mediate some of the hallmarks of apoptosis in Jurkat cells but it was not responsible for the entire apoptotic phenotype.42 It has been found that the caspase-3 Ac-DEVD-CHO inhibitor, when added directly to the medium, did not inhibit DNA ladder formation in HL60 cells because of its poor penetration.25,43,44 A complete suppression of DNA fragmentation was achieved with 500 µM concentration of the inhibitor but only when it was loaded into the cells using the osmotic lysis of pinosomes. In our work we used much lesser concentrations of Ac-DEVD-CHO (40 and 100 µM), added directly to the cell medium. Therefore, we cannot rule out the possibility that these concentrations were insufficient to inhibit DNA fragmentation in the investigated immortalized cells under these conditions. Lack of any inhibitory effect of Ac-DEVD-CHO on the survival of drug-treated cells suggests that despite the participation of caspase-3 in the induction of apoptosis by aclarubicin, doxorubicin and mitoxantrone, the activation of this enzyme is not directly involved in the main processes responsible for their cytotoxicity towards the investigated immortalized cells. Another possibility is that the enzyme activation might occur downstream of other key molecular events through which these drugs demonstrate their cytotoxicity. Lack of evidence for the importance of caspase-3 activation for cell survival after treatment with aclarubicin, doxorubicin or mitoxantrone supports the view that the induction of apoptosis does not play a key role in the cytotoxicity of these drugs. The relation between the cytotoxicity of drugs and their pro-apoptotic activity is rather complex. In B14 cells, morphological as well as biochemical changes were dependent on the type of drug, its concentration and the duration of post-treatment time. Mitoxantrone induced DNA fragnentation at the latest, but at a much lower concentration than aclarubicin and doxorubicin. On the other hand, aclarubicin, which showed the lowest cytotoxicity among the investigated drugs, induced

Mechanisms of induction of apoptosis

DNA fragmentation several hours earlier than DOX or MTX when the drugs were applied at equal concentration (1 µM). In summary, although mitoxantrone was able to induce apoptotic DNA laddering at much lower concentrations than aclarubicin or doxorubicin, it acted as the slowest inducer of apoptosis, requiring a relatively long lag period to show any pro-apoptotic effect at the DNA level. At 1 µM concentration, this drug caused the same (after 12 h) or even higher (after 24 h) activation of caspase-3 than aclarubicin. The most cytotoxic mitoxantrone induced also a considerably lower level of apoptosis than aclarubicin, as measured by flow cytometric and double staining methods. Therefore we rather suggest that cytotoxicity of the discussed drugs is not directly related to their pro-apoptotic activity. A comparison of drug cytotoxicity with the corresponding m.e.d. (minimal effective dose) sufficient for the induction of apoptosis revealed dose dependence and positive correlation only between the induction of DNA fragmentation and the sensitivity of B14 cells to mitoxantrone. Compared to B14 cells, NIH 3T3 displayed a considerably lower level of apoptosis. An increase in the hypodiploid sub-G1 DNA peak was found only in cells exposed to a high concentration of aclarubicin and after a prolonged post-incubation time (72 h). In drugtreated cells double stained with Hoechst 33258 and PI, an increase in the apoptotic cell fraction with the post-incubation time was considerably lower than in B14 cells. In NIH3T3 cells, morphological changes and an increase in the apoptotic cell population were not accompanied by formation of DNA oligonucleosomal fragments. This suggests that in these cells DNA fragmentation is not required for the triggering of apoptosis, and that aclarubicin, doxorubicin and mitoxantrone might induce apoptotic events through a different pathway or program. Several accounts of apoptosis in the absence of an internucleosomal DNA cleavage pattern have been reported in other cell types.14,25,45,53,54 DNA laddering in normal and transformed NIH3T3 cells has been reported by Kuo et al.,55 but only after prolonged treatment (8– 16 h) with 1 mM methylmethane sulfonate, which concentration is 1000 times higher than the doses of the anthraquinone drugs applied in our work. This suggests that in this cell line, like in other cell types, DNA fragmentation into nucleosomal-sized fragments is not required for the triggering of apoptosis by anthracyclines and mitoxantrone, and that these drugs might induce apoptotic events through a different pathway or program in different cell types. The researched anthraquinone drugs differ in their structure, which may impact their activity as apoptotic inducers. Doxorubicin belongs to the classical group of monosaccaride anthracyclines, whereas aclarubicin is the newer N,N-dimethylated oligosaccharide anthracycline containing an aclavinone aglycone and three

different sugar residues. Moreover, DOX and ACL have different methylation state of their aminosugar, and thus also diverse electrical charges. The drugs display different lipophilicity, too.46 MTX is a synthetic anthracenedione derivative with no sugar residue in its molecules. Although it is structurally related to anthracyclines through its basic quinone structure, MTX, as compared to DOX, displays a significantly reduced potential to undergo oneelectron and enzymatic reduction.56 This drug is capable of inhibiting initiation and propagation reactions in the peroxidative cascade in carcinogenesis being oxidatively metabolized and of inhibiting doxorubicin-induced peroxidation,57,58 which may suggest the occurrence of anti-oxidative properties of MTX molecule/metabolites under certain conditions. ACL and DOX have been shown to induce differentiation in myeloid and elytroid leukemias through different pathways: granulocytic for ACL and monocytic for DOX.59 These drugs have already been shown to affect cell proliferation by different processes. In general, the mechanisms of toxicity of DOX, ACL and MTX are consistent with their direct interaction with DNA.60,61 All three drugs intercalate into DNA and stabilize topoisomerase II–DNA ternary complex.62,63 Among them only trisacharide anthracycline (ACL) is a catalytic inhibitor of both topoisomerase I and II.64 These drugs also produce reactive oxygen species, however, by different types of redox-active groups.65 The quinone moiety of DOX and ACL undergo, above all, one electron reduction to a semiquinone radical, which generates reactive oxygen species (ROS) in the presence of oxygen.60,66 In contrast, the synthetic analogue of the anthracyclines, MTX, produces damaging radicals during the oxidation of the hydroquinone group by peroxidase /H2 O2 system.67 It seems that the toxicity of the drugs used in this study towards immortalized cells depends mainly on the drug structure and on its interaction with DNA and formation of damaging radicals. Our results are in agreement with the data of Dartsch et al.,22 who investigated the mode of cell death induced by DOX, ACL and daunorubicin (DNR) in human leukemia cell lines. They found that the concentrations and times necessary for the inducing of apoptotic events by these drugs were different in HL60 and Jurkat cells. Aclarubicin was the fastest acting drug, as compared to doxorubicin and daunorubicin. Moreover, ACL induced DNA fragmentation in HL60 cells at lower concentration than DOX did. It has been suggested that in human leukemic cells DOX and DNR induced cell death by necrosis, while ACL induced programmed cell death.22 In this work, we have shown that ACL is the most potent apoptotic inducer also in immortalized cells. 0.2 µM of this drug was able to induce DNA fragmentation 12 h earlier than 0.3 µM of DOX. It cannot be excluded that the different effects of these anthracyclines on the cellular processes can influence their ability to induce apoptosis. ACL and DOX Apoptosis · Vol 10 · No 6 · 2005

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have been shown to display different effect on the synthesis of cell macromolecules and expression of some genes (c-myc, c-myb) related to cell differentiation, as well as to possess a different ability to induce differentiation in leukemic cells.59,68 The comparison of our data with results obtained with other cell lines exposed to anthracycline drugs is difficult to interpret. Thus far, research on the induction of apoptosis has been essentially performed on leukemic cell lines employing mainly doxorubicin and daunorubicin (DNR). It has been shown that DOX and DNR induced the apoptosis of hematopoetic cell lines.69 DOX also induced apoptosis in TVM-A12 and ME18 melanoma cells, in K562 erythroleukemia human cell lines.21,70 and in breast cancer cells.71 Despite these extensive studies, the apoptosis-signaling pathway triggered by anthracyclines and MTX is still unknown. Recently, Maestre and co-workers72 have revealed that apoptosis in the leukemia cell line V937 is induced through intracellularmediated signaling pathways and requires DOX and DNR internalization. Moreover, it has been suggested that ROS may serve as an intracellular signal of apoptotic events. The increased production of free radicals induced by DOX and DNR was found in leukemic cell lines,73 cardiomyocytes74 and in activated peripheral lymphocytes.58 By contrast, another group using leukemic cells reported that DNR encapsulated in liposomes diminished ROS generation and increased the apoptosis inducing effect of anthracycyline.75 In summary, we have shown that ACL and MTX induce cell death in immortalized B14 and NIH 3T3 cells by different mechanisms. In NIH 3T3 cells, a slow, steady-state increase in caspase-3 activity, and morphological changes in the cell nucleus without visible DNA oligonucleosomal degradation were observed. In B14 cells, a significant concentration-dependent increase in caspase-3 activity, morphological changes and DNA fragmentation were found. The preincubation of cells with the caspase-3 Ac-DEVD-CHO inhibitor completely inhibited caspase3 activity, decreased the number of apoptotic cells, but did not reduce drug cytotoxicity and did not prevent DNA oligonucleosomal degradation. These results suggest that although caspase-3 activation was necessary for the induction of apoptotic changes in cell morphology, the process of DNA degradation occurred in the presence of other proteolytic enzymes. From our study it can be concluded that ACL, MTX and DOX can induce apoptosis in immortalized fibroblasts and, at higher concentration, also necrotic death.

Conclusion This present study provides further information to depict the nuclear events induced by aclarubicin and mi1512 Apoptosis · Vol 10 · No 6 · 2005

toxantrone. We have shown that new derivatives of anthraquinone drugs can trigger both apoptosis and necrosis in immortalized cells but none of these processes was positively correlated with drug cytotoxicity measured using the MTT assay. Apoptosis was the prevalent form of cell kill by aclarubicin, while necrosis was the main mode of cell death induced by doxorubicin and mitoxantrone. The apoptotic process triggered by these drugs was mediated by caspase-3 activation and strongly depended on drug concentration and the type of cell line. References 1. Murphy GP, Lawrence W. Jr, Lenhard RE, eds. American society textbook of clinical oncology, 2nd ed. Atlanta, GA: American Cancer Society, 1995. 2. Doroshow JH. Anthracyclines and anthracenediones. In: Chabner BA, Longo DL, eds. Cancer chemotherapy and biotherapy, 2nd ed., New York: Lippincott-Raven Publishers, Philadelphia, 1996: 409–434. 3. Faulds D, Balfour JA, Chrisp P, Langtry HD. Mitoxantrone, a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in the chemotherapy of cancer. Drugs 1991; 3: 400–449. 4. Nicholson DW, Thornberry NA. Caspases:Killer proteases. Trends Biochem Sci 1997; 22: 299–306. 5. Leist M, J¨aa¨ ttel¨a M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001; 2: 589–598. 6. Woo M, Hakem R, Soengas MS, et al. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev 1998; 12: 806–819. 7. J¨anicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 1998; 273: 9357– 9360. 8. Susin SA, Douglas E, Ravagnan L, et al. Two distinct pathways leading to nuclear apoptosis. J Exp Med 2000; 192: 571–579. 9. Joza N, Susin SA, Douglas E, et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 2001; 410: 549–554. 10. Li LY, Luo X, Wang X. Endonuclease G is an apoptotic Dnase when released from mitochondria. Nature 2001; 412: 95– 99. 11. Nakano H, Shinohara K. Time sequence analysis of caspase3 independent programmed cell death and apoptosis in Xirradiated human leukaemic MOLT-4 cells. Cell Tissue Res 2001; 310: 305–311. 12. Coelho D, Holl V, Weltin D, et al. Caspase-3-like activity determines the type of cell death following ionising radiationin MOLT-4 human leukaemia cells. Br J Cancer 2000; 83: 642– 649. 13. Blagosklonny MV. Cell death beyond apoptosis. Leukemia 2000; 14: 1502–1508. 14. Schulze-Osthoff K, Walczak H, Droge W, Krammer PH. Cell nucleus and DNA fragmentation are not required for apoptosis. J Cell Biol 1994; 127: 15–20. 15. Johnson DE. Programmed cell death regulation: Basic mechanisms and therapeutic opportunities. Leukemia 2000; 14: 1340–1344. 16. Skladanowski A, Konopa J. Adriamycin and daunomycin induce programmed cell death (apoptosis) in tumor cells. Biochem Pharmacol 1993; 46: 375–382.

Mechanisms of induction of apoptosis 17. Ling Y, Priebe W, Perez-Soler R. Apotosis induced by anthracycline antibiotics in P388 parent and multidrug-resistant cells. Cancer Res 1993; 53: 1845–1852. 18. Han J, Dionne CA, Kedersha NL, Goldmacher VS. p53 status affects the rate of the onset but not the overall extent of doxorubicin-induced death in Rat-1 fibroblasts constitutively expressing c-myc. Cancer Res 199757: 176–182. 19. Thakkar NS, Potten CS. Abrogation of adriamycin toxicity in vivo by cycloheximide. Biochem Pharmacol 1992; 43: 1683– 1691. 20. Fornari Jr, FA, Jarvis WD, Grant S, Orr MS, Randolph JK, White FKH, Gewitz DA. Growth arrest and non-apoptotic cell death associated with the suppression of c-myc expression in MCF-7 breast tumor cells following acute exposure to doxorubicin. Biochem Pharmacol 1996; 51: 931–940. 21. Gruber BM, Anuszewska EL, Skierski JS. Activation of programmed cell death (apoptosis) by adriamycin in human neoplastic cells. Mutation Res 2001; 484: 87–93. 22. Dartsch DC, Schaefer A, Boldt S, Kolch W, Marquardt H. Comparison of anthracycline-induced death of human leukaemia cells: Programmed cells death versus necrosis. Apoptosis 2000; 7: 537–548. 23. Mossman T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55–63. 24. Ormerod M, Collins M, Rodriguez-Jarduchy G, Robertson D. Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. J Immunol Methods 1992; 153: 57–65. 25. Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotic cells measured by flow cytometry. Cytometry 1992; 13: 795–808. 26. Hotz MA, Gong J, Traganos F, Darzynkiewicz Z. Flow cytometric detection of apoptosis: Comparison of the assay oof in situ DNA degradation and chromatin changes. Cytometry 1994; 15: 237–244. 27. Elstein KH, Zucker RM. Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations. Exp Cell Res 1994; 211: 322–331. 28. Gasiorowski K, Brokos B, Kulma A, Ogorzalek A, Skorkowska K. A comparison of the methods applied to detect apoptosis in genotoxically-damaged lymphocytes cultured in the presence of four antimutagens. Cell Biol Mol Lett 2001; 6: 141–159. 29. Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376: 37–43. 30. Gonzales VM, Fuertes MA, Alonso C, Perez JM. Is cisplatininduced cell death always produced by apoptosis? Mol Pharmacol 2001; 59: 657–663. 31. Kiaris H, Schally AV. Apoptosis versus necrosis: Which should be the aim of cancer therapy? Proc Soc Exp Biol Med 1999; 221: 87–88. 32. Fukushima T, Kawai Y, Nakayama T, et al. Superior cytotoxic potency of mitoxantrone in interaction with DNA: Comparison with that of daunorubicin. Oncology Res 1996; 8: 95–100. 33. Kaspers GJL, Veerman AJP, Pieters R, et al. In vitro cytotoxicity of mitoxantrone, daunorubicin, and doxorubicin in untreated childhood acute leukaemia. Leukemia 1994; 8: 24– 29. 34. Van der Graaf WTA, de Vries ECE. Mitoxantrone bluebeard for malignancies. Anti-Cancer Drugs 1990; 1: 109–125. 35. Borner C, Money L. Apoptosis without caspases: An inefficient molecular guillotine? Cell Death & Diff 1999; 6: 497–507. 36. McCarthy NJ, Whyte MKB, Gilbert CS, Evan GI. Inhibition of Ced-3/ICE-related proteases does not prevent cell death

37. 38.

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