Toxicology in Vitro 20 (2006) 217–226 www.elsevier.com/locate/toxinvit
Diazene JK-279 induces apoptosis-like cell death in human cervical carcinoma cells S. Jakopec a, K. Dubravcic b, S. Polanc c, J. Kosmrlj c, M. Osmak a
a,*
Department of Molecular Biology, Rudjer Boskovic Institute, Bijenicka cesta 54, HR-10000 Zagreb, Croatia b Zagreb Clinical Hospital Center, Kispaticeva 12, HR-10000 Zagreb, Croatia c Faculty of Chemistry and Chemical Technology, Askerceva 5, SI-1000 Ljubljana, Slovenia Received 26 October 2004; accepted 15 June 2005 Available online 2 August 2005
Abstract Diazene N-phenyl-2-(2-pyridinyl)diazenecarboxamide (JK-279) is a newly synthesized compound, cytotoxic for several tumor cell lines and their drug-resistant sublines. In human cervical carcinoma cells (HeLa), this compound reduced intracellular glutathione content and increased sensitivity to cisplatin. The aim of the present study was to elucidate the molecular mechanisms involved in the cytotoxic effect of diazene JK-279 on HeLa cells. Cytotoxicity was determined by the MTT method. Flow cytometry analysis showed that diazene JK-279 induces G2/M phase arrest, mediated by the increase in p21 expression, and accompanied by an alteration in the expression of survivin. The highest concentration of JK-279 altered nuclear morphology in intact cells, showing ‘‘apoptosis-like’’ features. No cleavage of procaspase-3, procaspase-9 and PARP, or altered expression of apoptotic proteins Bcl-2 and Bax were detected. At the same time, PS externalization and internucleosomal DNA cleavage were observed. Partial necrosis was detected as well. Our results demonstrate that cytotoxicity of diazene JK-279 is mostly the consequence of caspase-independent cell death, which is in some aspects ‘‘apoptosis-like’’. Taking into account the multiplicity of mechanisms used by cancer cells to prevent apoptosis, the drugs (like diazene JK-279) that would activate alternative cell death pathways could provide a useful tool for new types of cancer therapy. 2005 Elsevier Ltd. All rights reserved. Keywords: Diazenes; Tumor cells; Anti-cancer drugs; Apoptosis-like cell death; Caspase-independent cell death
1. Introduction The success of chemotherapy of cancer patients is hampered by the problem of drug-resistance, and a call for discovery of more effective agents to treat cancer is becoming increasingly urgent. For this purpose, new drugs have been synthesized and tested. Abbreviations: JK-279, N-phenyl-2-(2-pyridinyl)diazenecarboxamide; GSH, glutathione; AO, acridine orange; EtBr, ethidium bromide; PBS, phosphate-buffered saline; PS, phosphatidylserine; PI, propidium iodide; PARP, poly(ADP-ribose)polymerase; ERK2, extracellular signal-regulated kinase 2; CDDP, cisplatin; DOX, doxorubicine; NaAC, sodium acetate. * Corresponding author. Tel.: +385 14561145; fax: +385 14561177. E-mail address:
[email protected] (M. Osmak). 0887-2333/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2005.06.008
Insight into tumor cell biology has led to identification of a number of molecules which are important for vital cell function. Increasing evidence has accumulated over the past five decades supporting the central role of glutathione (GSH) in cellular homeostasis. GSH serves diverse physiological functions. Among them, protection of cells from oxidative stress and detoxification of xenobiotics are the most important (Locigno and Castronovo, 2001; Dickinson and Forman, 2002). Protective role of GSH was confirmed by the addition of glutathionedepleting agent buthionine sulfoximine, that increased cell-sensitivity to xenobiotics (such as anti-cancer drugs) (Arrick and Nathan, 1984; Hamilton et al., 1985). There is, however, compelling evidence suggesting that GSH homeostasis has a more fundamental role in
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the activation or inhibition of proteins crucial for cell survival and cell death (Slater et al., 1996; Hampton et al., 1998; Filomeni et al., 2002). Many proteins have thiol moieties whose redox status is essential for their function (Lin et al., 1995), and glutathione may modify their redox state (Pognonec et al., 1992; Filomeni et al., 2002). A strong relationship between intracellular glutathione levels and mitochondria-dependent apoptotic pathway has been observed, showing that reduction in GSH level may initiate apoptosis (Sandstrom et al., 1994; Filomeni et al., 2002). The essential role of glutathione in the control of processes pivotal for cell survival has focused the anti-cancer drug research to compounds that may be targeted to GSH. Recently we have synthesized new compounds, diazenecarboxamides (short name—diazenes). They are selective oxidants for a mild transformation of various thiols to the corresponding disulfides (Kosmrlj et al., 1998). Diazenes easily oxidize cysteamine, thiosalicylic acid derivatives, dithiothreitol and other thiols in methanol at room temperature. Under quasi-physiological conditions, they interact also with GSH (Kosmrlj et al., 1998). If diazenes could reduce the intracellular level of GSH, this might cause the inhibition of tumor cellÕs growth. Therefore we determined the cytotoxic activity of diazenes against various tumor cell lines (Osmak et al., 1999a). We previously showed that N-phenyl-2-(2-pyridinyl)diazenecarboxamide (JK-279) was cytotoxic to several tumor cell lines of different origin, including drug-resistant sublines (Osmak et al., 1999a). The aim of the present study was to elucidate the molecular mechanisms involved in this process. As a model system we chose human cervical carcinoma (HeLa) cells because we found that: (a) diazene JK-279 was very cytotoxic to HeLa cells (Osmak et al., 1999a), (b) it reduced intracellular accumulation of GSH in these cells (Osmak et al., 1999b), (c) it partially reversed the resistance of HeLa cells to cisplatin (Osmak et al., 1999b).
2. Materials and methods 2.1. Cell culture Human cervical carcinoma (HeLa) cells were used in this study. They were grown as a monolayer culture in DulbeccoÕs medium (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL) and antibiotics in a humid atmosphere containing 5% CO2.
procedure described earlier for the synthesis of diazenes (Kosmrlj et al., 1996, 1998). Structure of this compound is given in Fig. 1. Diazene JK-279 was dissolved in water:ethanol (1:1), sterilized by filtration and stored at 20 C. Stock solution was diluted with the growth medium immediately before use. 2.3. Cytotoxicity assay The MTT assay was performed in order to examine the cytotoxic effect of diazene JK-279. 4.5 · 103 cells were seeded per well in 96 well-plates. Different concentrations of this compound were added to the cells 24 h after seeding. The cells were continuously treated for 72 h and then the MTT assay was done as described by Mickisch et al. (1990). 2.4. Cellular and nuclear morphology and membrane permeability Morphology and membrane permeability of treated cells were determined by epifluorescence microscopy (Axiovert 35, Opton). For this purpose cells were seeded in 96 well-plates. At certain time points after treatment with JK-279, 4 ll of acridine orange (AO, 15 lg/ml in PBS, Serva, Germany) and 4 ll of ethidium bromide (EtBr, 50 lg/ml in PBS, Serva, Germany) were added in 50 ll of medium per well. For nuclear morphology experiments, cells were seeded in 10 cm petri dish and after JK-279 treatment they were trypsinized, centrifuged and washed. Thereafter 10 ll of cell suspension was labeled with 4 ll AO and 4 ll of EtBr. Living cells were green due to the DNA staining with AO, while dead cells were red due to the DNA staining with EtBr (this dye enters only in death cells with damaged plasma cell membrane). Fluorescence was detected through the BP 450-490, FT 510, LP 520 filter, and images were taken with camera Pixera Pro150ES. 2.5. Flow cytometry Flow cytometry was used for the cell cycle analysis. Cells were trypsinized, counted, centrifuged, and fixed in ethanol at certain time points after the treatment. Thereafter cells were washed twice in PBS and centrifuged. Pellet was resuspended in a solution of RNAse (0.02 mg/ml, Sigma, USA) and propidium iodide (PI, 0.02 mg/ml, Sigma, USA), and incubated at 4 C for 30 min. Fluorescence of stained cells was measured for approximately 10,000–20,000 cells. Data were collected O
2.2. Diazene JK-279 Diazene JK-279 was prepared by oxidation of the corresponding 1,4-disubstituted semicarbazide (N-phenyl-2-(2-pyridinyl)hydrazinecarboxamide), following the
N NH
C
N
N
Fig. 1. Structure of diazene JK-279.
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2.6. Western blot analysis Protein extracts of cells treated with diazene JK-279 were prepared by lysing cells in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris HCl, pH 8), 10 mM EDTA (Kemika, Croatia) and 1 mM PMSF (Sigma, USA) for 30 min at 4 C. Samples were then centrifuged 15 min at 15,000g. Protein concentration in supernatant was determined according to the Lowry method (Lowry et al., 1951). For each sample, 60 lg of protein were loaded on 12.5% SDS-polyacrylamide gel, electrophoresed, and transferred to nitrocellulose membrane (0.22 lm, Protran, Schleicher and Schuell). Membrane was blocked for 1 h at the room temperature with blocking buffer (TBS containing 0.1% Tween 20 (w/v) (Sigma, USA) and 5% milk (w/v) (Sirela, Croatia)). Primary antibodies (applied for 1 h at room temperature, or overnight at 4 C) were: anti-PARP (mouse monoclonal C2-10, Pharmingen, USA), anti-caspase-9 p10 (rabbit polyclonal H-83, Santa Cruz Biotechnology, Germany), anti-caspase-3 (mouse monoclonal E-8, Santa Cruz), anti-bcl-2 (mouse monoclonal Ab-1, Oncogene Research Products, UK), anti-p21 (WAF1) (mouse monoclonal Ab-1, Oncogene), anti-Bax (rabbit polyclonal N-20, Santa Cruz), anti-survivin (rabbit polyclonal FL-142, Santa Cruz), anti-bcl-2 (mouse monoclonal Ab-1, Oncogene), anti-actin (mouse monoclonal C-2, Santa Cruz), anti-ERK2 (rabbit polyclonal C-14, Santa Cruz). They were diluted 1:1000, except: anti-PARP (1:4000), antiERK2 (1:3000), anti-actin (1:500), and anti-p21 (1:300). Thereafter, membranes were incubated for 1 h with HRP-labeled secondary antibodies (Amersham Pharmacia Biotech, Sweden): sheep anti-mouse NA 931 (diluted 1:2500), and donkey anti-rabbit NA 934 (diluted 1:5000), and then developed by an ECL system according to the manufacturerÕs instructions (Amersham).
2.7. Analysis of DNA fragmentation Cells were collected at certain time points after the treatment. DNA fragments were isolated according to the standard protocol for DNA isolation (Ausubel et al., 1992). Briefly, cells were harvested, washed and pelleted by centrifugation. Thereafter they were lysed (0.1 M NaCl, 10 mM Tris HCl, 25 mM EDTA, pH 8.0; 500 ll per 5 · 106 cells). Lysates were incubated with SDS (25 ll, 10%) and proteinase K (5 ll, 20 mg/ml in dH20, Sigma, USA) for 3 h at 56 C, and then left at 37 C overnight. RNase was then added (5 ll, 10 mg/ml) and incubated for 1.5 h at 37 C. DNA was isolated by the standard method using phenol and chloroform isoamylalcohol solution. DNA was precipitated after addition of NaAC (sodium acetate, 3 M, Merck, Germany), pH 5.2 and 96% ethanol ( 20 C precold), left overnight, and centrifuged for 45 min at 15,000g, washed again with 70% ethanol ( 20 C precold), centrifuged and dried. DNA pellet was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and RNase (100 lg/ml final concentration) and left for 1 h at 37 C, or additionally overnight at 4 C. Thereafter, samples were loaded on 1.5% agarose gel, separated by electrophoresis (50 V, 1.5 h) and visualized by staining with ethidium bromide. 3. Results 3.1. Cytotoxicity Diazene JK-279 inhibited the growth of cervical carcinoma (HeLa) cells in dose-dependent manner, as shown in Fig. 2. For further experiments three doses 100 90 80 70
% control
and analysed with ModFitLTTM program (Verity Software House Inc., Topsham, Maine, USA). Results were expressed as a plot of fluorescence intensity vs cell number. The appearance of phosphatidylserine (PS) on the extracellular side of membrane was evaluated with annexinV/PI method. At certain time points after the treatment, cells were trypsinized, counted, washed twice in ice-cold PBS and resuspended in 1· binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Thereafter, 5 ll of annexin V-FITC (BD Pharmingen) and 10 ll of propidium iodide (50lg/ml) were added to 100 ll of cell suspension and incubated for 15 min at room temperature protected from light. Thereafter 400 ll of binding buffer were added to the samples and handled ice-cold until they were analysed on FACSCalibur (Becton Dickinson) flow cytometer. Ten thousand cells were analysed per sample.
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60 50 40 30 20 10 0 0
0.108
0.13
0.18
0.216
0.324
JK-279 concentration (mM) Fig. 2. Dose–response curve of human cervical carcinoma (HeLa) cells following 72 h treatment with diazene JK-279, measured by the MTT assay. Results are shown as mean values of three experiments (±SD).
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of JK-279 were selected that reduced cell survival to 80% (IC80 = 0.13 mM), 40% (IC40 = 0.18 mM) and 20% (IC20 = 0.216 mM). 3.2. Flow cytometry analysis Flow cytometry analysis of DNA content was performed to investigate if diazene JK-279 induces changes
in the cell cycle distribution. Results showed that treatment with 0.13, 0.18 and 0.216 mM of JK-279 induced G2/M arrest (Fig. 3). The percentages of cells in G1/ G0 phase decreased, while the number of cells in G2/ M phase of cell cycle increased after 24 h treatment. It is important to note that the highest concentration of JK-279 (0.216 mM) induced a significant increase in ‘‘sub-G1 peak’’ cell-fraction (Fig. 3) that represented
Fig. 3. Cell cycle changes after JK-279 treatment. Effects of 6 and 24 h treatment with 0.13, 0.18 and 0.216 mM of JK-279 on cell cycle distribution of HeLa cells. Cell cycle distribution was analysed by flow cytometry and the data are presented as histograms in which the cell number (y-axis) is plotted against DNA content (x-axis). Percentages of cells distributed in G1/G0, S, G2/M and sub-G1 region of histograms are indicated at each histogram.
Fig. 4. Cell cycle changes and the expression of survivin and p21. (A) Time-dependent alterations in cell cycle phases during treatment of HeLa cells with 0.18 mM of JK-279 analysed by flow cytometry. Untreated cells collected after 6, 24, 48 and 72 h were used as corresponding controls. (B) Western blot analysis of survivin and p21 protein expression in HeLa cells at different time points following treatment with 0.18 and 0.216 mM JK279. Untreated cells analysed after 6 h (C), 48 h (C48) and 72 h (C72) of growth in culture were used as corresponding controls (C was used as control for 3, 6, 12 and 24 h treated cells). Human HEK-293 cell line (‘‘293’’) was used as positive control for p21 expression.
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cells with the DNA content lower than the basic one, seen in G1 phase. This decrease in the DNA amount was caused by the cleavage of DNA and subsequent loss of small DNA fragments during the preparation of cells for flow cytometry analysis. Treatment with 0.18 mM of JK-279 induced the most obvious changes in the cell cycle. Therefore, time-dependence of this process was investigated on a wider time scale (Fig. 4A). After 12 h treatment, cells started to arrest in G2/M phase, and reached the highest value in the next 12 h (totally 24 h treatment). At this time, a fraction of cells that accumulated in G2/M phase (as compared to control cells) was almost doubled. In the following 24 h (48 h treatment) G2/M arrest began to disappear, with a simultaneous increase in ‘‘sub-G1
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peak’’. After 72 h treatment the cell cycle perturbation disappeared, but the ‘‘sub-G1 peak’’ was still present and was the highest. This ‘‘sub-G1 peak’’ had a different pattern than the one seen after treatment with 0.216 mM JK-279 (Fig. 3). 3.3. Expression of proteins involved in cell cycle regulation Expression of proteins involved in cell cycle regulation, p21 and survivin, was analysed by Western blot. Treatment with 0.18 mM concentration of JK-279 for 6, 12 and 24 h increased p21 expression (Fig. 4B). After 48 and 72 h treatment with 0.18 mM JK-279 (when G2/ M arrest disappeared, and ‘‘sub-G1 peak’’ appeared),
Fig. 5. Membrane permeability, morphological changes and nuclear alterations in HeLa cells following treatment with JK-279. Images were taken under the epifluorescence microscope. (A) Cells were treated with 0.13, 0.18 and 0.216 mM of JK-279 for 6 and 24 h, and thereafter stained with acridine orange (AO) and ethidium bromide (EtBr) (100· magnification). Images of cells stained with fluorescent dyes are presented in the upper row for each concentration/time point. Phase-contrast images of the same cells are presented below. (B) Cells were treated with 0.216 mM of JK-279 for 6 and 24 h and then stained with AO and EtBr (1000· magnification). Four different types of nuclear alterations were observed: (a, c) apoptosis-like; (b) highly condensed, on one side inverted nucleus; (d) necrosis. As positive control for characteristic apoptotic nuclear morphology HeLa cells were collected 24 h after 1 h treatment with 80 lM of cisplatin.
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p21 expression returned to the control value (C48, C72) (Fig. 4B). The highest concentration of JK-279 (0.216 mM) briefly increased the expression of p21 (after 12 h treatment) (Fig. 4B). Survivin expression also changed during treatment with JK-279. After 12 h treatment with 0.18 mM concentration of JK-279 minimal expression of survivin was detected (Fig. 4B), followed by an increase after 24 h treatment. Survivin levels remained increased even after 72 h (Fig. 4B). The highest concentration of JK279 (0.216 mM) progressively downregulated the expression of survivin during 24 h treatment (Fig. 4B). 3.4. Morphological alterations Fig. 6. Western blot analysis of HeLa cells treated with 0.216, 0.18 and 0.13 mM of JK-279 for 6 and 24 h (C = control). Cleavage of PARP, procaspase-3 and -9 and expression of Bcl-2 and Bax protein were assayed. 60 lg of proteins were loaded per sample, and actin was used as equal loading control. HeLa cells treated with cisplatin (collected 24 h after 1 h treatment with 80 lM cisplatin, CDDP) were used as positive control for PARP. HeLa cells treated with doxorubicin (collected following 24 h treatment with 2.6 lM of doxorubicin, DOX) were used as positive control for cleavage of caspases.
Morphological analysis of cell characteristics observed under epifluorescence microscope showed that cells started to change their shape (they shrunk and started to round up) 3 h after the continuous treatment with JK-279 (data not shown). These alterations were even more expressed following 6 and 24 h treatment (Fig. 5A). At the same time (6 and 24 h treatment) we also noticed changes in nuclear morphology (Fig. 5B). Four different types of alterations were detected: (a)
Fig. 7. PS externalization and DNA cleavage. (A) HeLa cells were treated for 3, 6, 12 and 24 h with 0.216 mM of JK-279. PS externalization was determined by combined annexin V/PI assay. Cells stained positive for annexin and negative for PI represent those with intact membrane and externalised PS (percentages are indicated in the lower right panel). Cells stained positive with annexin and PI are those that lost membrane integrity (percentages are indicated in the upper right panel). (B) HeLa cells were treated for 3, 6, 12 and 24 h with 0.216 mM of JK-279. Isolated DNA was analysed in agarose gel electrophoresis as described under Section 2. Extracts from 2 · 106 cells were loaded per well. 1 Kb DNA ladder marker (Gibco, SAD) was used as marker (M) of DNA fragment size. C denotes untreated cells.
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weak and irregularly shaped marginal chromatin condensation; (b) highly condensed chromatin of nucleus that was inverted in one side; (c) relatively compact and irregularly shaped marginal chromatin condensation; (d) decondensed chromatin colored red1 because of plasma membrane lysis. None of these changes were typical for apoptosis which is characterized by the condensation of chromatin to compact and simple globular geometric figures. However, some of them (like alterations described under (a) and (c)) were similar to ‘‘apoptosis-like’’ alterations described in the literature (Leist and Ja¨a¨ttela¨, 2001). Cells that lost membrane integrity died via necrosis (the nuclei stained red with EtBr) (Fig. 5A and B). 3.5. Type of cell death To confirm that morphological changes induced by diazene JK-279 are not the consequence of apoptosis, we analysed the expression of proteins involved in the apoptotic process by Western blot. For this purpose, HeLa cells were again treated with the same concentrations of JK-279 (0.13, 0.18 and 0.216 mM) for 6 and 24 h. Procaspase-9, procaspase-3 and PARP cleavage was analysed in these samples, as well as the expression of Bcl-2 and Bax proteins. HeLa cells treated with cisplatin were used as positive control for PARP cleavage, and HeLa cells treated with doxorubicin were used as positive control for the cleavage of procaspases. Results showed that, at the time when ‘‘apoptosis-like’’ morphological changes were observed (6 and 24 h treatment), no cleavage of procaspase-9, procaspase-3 or PARP was detected. Also, the levels of Bcl-2 and Bax proteins did not change (Fig. 6). Thus, our results suggest that JK279 does not induce typical apoptosis (a caspase-dependent process), but some alternative type of programmed cell death that is caspase-independent. Externalization of phosphatidylserine and cleavage of DNA, the hallmarks of apoptosis, were also recently found in alternative types of programmed cell death. To determine whether such processes were induced due to JK-279 treatment, HeLa cells were treated for 3, 6, 12, and 24 h with 0.216 mM of JK-279 (this concentration of JK-279 induced ‘‘apoptosis-like’’ changes). The result of annexin V/PI assay showed that PS externalization began after 12 h treatment, and then increased in the following 12 h (Fig. 7A). Also, ‘‘DNA ladders’’ were detected at these time points (Fig. 7B). Analysis of annexin V/PI assay revealed that some cells lost their membrane integrity at the time when PS externalization occurred. This observation confirmed the data obtained
1 For interpretation of color in Figs. 3–5, the reader is referred to the web version of this article.
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by morphological analysis (the nuclei stained red, Fig. 5A and B).
4. Discussion We previously showed that a newly synthesized compound, diazene JK-279, was cytotoxic for several tumor cell lines, among them human cervical carcinoma (HeLa) cells (Osmak et al., 1999a). In this study, we examined the molecular mechanisms involved in this process. Mammalian cells have evolved a complex defence network to maintain genomic integrity by inhibiting the fixation of permanent damage. Cell-cycle check points prevent cells with damaged genomes from undergoing DNA replication or mitosis (Shackelford et al., 1999). Therefore we analysed the influence of JK-279 treatment on the distribution of cells through the cell cycle. Following treatment with JK-279, cell-arrest in the G2/M phase of the cell cycle was observed (Figs. 3 and 4A), most probably caused by the increased expression of p21. p21 is a member of cyclin kinase inhibitors (CKI), a protein family involved in G1/S and G2/M cell cycle arrest (Niculescu et al., 1998). Lower concentration (0.18 mM) of JK-279 induced the prolonged expression of p21 (Fig. 4B), while the higher one (0.216 mM) increased only briefly the level of p21 protein (Fig. 4B). The upregulation of p21 may protect cells from apoptosis through binding and enabling activation of procaspase-3 (Suzuki et al., 1998, 1999), and may protect cells from some forms of caspase-independent cell death as well (Ussat et al., 2002). However, the detailed mechanism of this process has not yet been resolved. We speculate that the prolonged p21 expression induced G2/M arrest and protected cells from caspaseindependent cell death, while brief induction of p21 also arrested cells in G2/M (to a lesser extent, as we noticed), but did not last long enough to protect cells from caspase-independent cell death. The changes in expression of p21 were accompanied by an altered expression of survivin (Fig. 4B). In adults, survivin is expressed only in tumor cells. It enables them to grow indefinitely by suppressing apoptosis (Tamm et al., 1998; Verhagen et al., 2001) and promoting cell proliferation as a chromosomal passenger protein (Temme et al., 2003). During treatment with lower concentration (0.18 mM) of JK-279, at the time when cells entered G2/M arrest (12 h treatment), p21 expression was upregulated and survivin was downregulated. These data could be explained by the fact that p21 could, by the inhibition of cdc2 kinase activity (Taylor and Stark, 2001; OÕConnors et al., 2002), downregulate survivin expression by inhibiting its phopshorylation and enhancing its degradation (Wall et al., 2003). The
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increase in survivin expression after 24 h treatment, while p21 was still upregulated and cells were still in G2/M arrest, might suggest that some other kinases (except cdc2) may also be involved in survivin phosphorylation and stabilization in G2/M phase (Wall et al., 2003). The higher concentration of JK-279 (0.216 mM) induced G2/M arrest and caspase-independent cell death (see further in text) and downregulated the expression of survivin (as shown in Fig. 4B). These data are in accordance with those previously published, showing that the downregulation of survivin promotes apoptosis and other types of cell death (Shankar et al., 2001). Recent publications have demonstrated that chemotherapeutic agents exert their cytotoxicity, at least partially, by inducing apoptosis (Hannum, 1997; Kaufmann and Earnshaw, 2000). Further, the reported evidence emphasized the crucial role of GSH in the programmed cell death (Sandstrom et al., 1994; Filomeni et al., 2002). As diazene JK-279 was cytotoxic for HeLa cells and reduced intracellular level of GSH, we investigated the induction of apoptosis following treatment with this compound. Morphological changes in the cell shape and chromatin condensation are basic and the oldest criteria for identification of apoptotic cells (Kerr et al., 1972). These changes are caused by activation of proteolytic enzymes, caspases, the central executioners of apoptosis (Earnshaw et al., 1999). Members of the caspase family cleave specific substrates involved in DNA repair, cytoskeletal organization, nuclear integrity and survival (Ha¨cker, 2000). The specificity of caspases for these targets results in a highly controlled and efficient removal of damaged or unwanted cells. However, inhibition of caspases demonstrated that alternative types of programmed cell death could occur as well (Leist and Ja¨a¨ttela¨, 2001). According to nuclear changes in dying cells, such alternative death programs are divided into ‘‘apoptosis-like’’ and ‘‘necrosis-like’’ (Leist and Ja¨a¨ttela¨, 2001). Treatment of cervical carcinoma cells with diazene JK-279 (0.216 mM) induced four different types of nuclear morphological changes (Fig. 5B). Some of them were ‘‘apoptosis-like’’. Also, we noticed that some cells lost membrane integrity (Fig. 5A and B) and died via necrosis. To prove that morphological alterations induced by diazene JK-279 are indeed not the consequence of apoptosis, we examined the activation of caspase-9 (initiator caspase) and caspase-3 (executioner caspase) following treatment with diazene JK-279 by Western blot analysis. No cleavage of these caspases was found, as shown in Fig. 6. Also, PARP cleavage (PARP is the substrate of caspase-3) was absent. However, after 24 h treatment with 0.216 mM of JK-279, the amount of intact PARP protein (113 kD) was reduced, but the smaller fragment (89 kD), which is the product of caspase cleavage, did
not appear. Nevertheless, this fragment was present in positive control (HeLa cells treated with cisplatin), in which typical apoptosis was detected. Reduced amount of intact PARP protein in JK-279 treated cells could be explained by the data of Duriez and Shah (1997). They showed that PARP can also be cleaved with other proteases, some of which are involved in caspase-independent cell death as well (Mathiasen and Ja¨a¨ttela¨, 2002). It should be mentioned, that we detected PARP cleavage in HeLa cells treated with cisplatin (that were used as positive control for apoptosis induction), but procaspase-3 cleavage was absent. Similar data were published very recently, showing PARP cleavage to 89 and 24 kD subunits even when caspase-3 was inhibited (Del Bello et al., 2004). Therefore as positive control for cleavage of caspases, we used HeLa cells treated with doxorubicin (Fig. 6). Our results indicate that JK-279 does not induce typical apoptosis (a caspase-dependent process), but some alternative type of programmed cell death that is caspase-independent. The possibility that some other caspase might be involved in this process is not likely, because it would involve the occurrence of typically apoptotic nuclear morphology which was not observed. An increase in the permeability of the outer mitochondrial membrane is central for programmed cell death (Donovan and Cotter, 2004). It leads to release of different factors into the cytoplasm that activate downstream death programs (both caspase-dependent and -independent). Mitochondrial membrane permeability is directly controlled by Bcl-2 family members. Some of the members increase permeability (such as Bax, Bak), while others prevent it (such as Bcl-2) (Tsujimoto, 2003). Caspase-independent cell death has been discovered recently and the proteins involved in its induction and regulation are rather unknown. It is therefore not surprising that the data about the role of Bcl-2 protein family in this process are rare. During this process, Bax cleavage and activation were detected (Choi et al., 2001), while overexpression of Bcl-2 protein prevented this type of cell death (Amarante-Mendes et al., 1998; Okuno et al., 1998). In our study, we did not find any change either in Bcl-2 or Bax expression following treatment with diazene JK-279. The discrepancy of our results with those mentioned before could be explained by different cell origin. Further, our HeLa cells lacked the functional p53 protein (Vukovic et al., 2004), and it is known that wild type p53 may downregulate Bcl-2 levels and upregulate Bax (Miyashita et al., 1994). Caspase-dependent cell death is a very complex process based on altered activity of many genes and regulated by overlapping signalling pathways (SchulzeBergkamen and Krammer, 2004). It is reasonable to assume that caspase-independent cell death pathway will
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be complex as well, involving the altered expression of several genes. Externalization of phosphatidylserine is one of the major hallmarks of apoptotic process that was also found recently in the alternative types of programmed cell death (Leist and Ja¨a¨ttela¨, 2001). PS externalization is a dominant phagocytosis uptake signal, uncoupled from caspase activation in many model systems (Berndt et al., 1998; Hirt et al., 2000; Maianski et al., 2003). Subsequent event in apoptosis is internucleosomal DNA cleavage. It results from caspase-3 activation of caspase activated DNAse (CAD nuclease). However, there is accumulating evidence of a caspase-independent mechanism of the internucleosomal cleavage of DNA. It involves endonuclease G, a mitochondriaspecific nuclease that translocates to the nucleus during apoptosis and other types of programmed cell death (Lily et al., 2001). In our study, treatment with the high concentration of JK-279 induced PS externalization and internucleosomal DNA cleavage (Fig. 7A and B), although there was no caspase activation (Fig. 6), suggesting a caspase-independent mechanism of these processes. In summary, we showed that JK-279 is cytotoxic for human cervical carcinoma cells. It induced G2/M arrest that was mediated by an increase in p21 expression, and accompanied by the altered expression of survivin. Diazene JK-279 caused caspase-independent type of programmed cell death, which was in some aspects ‘‘apoptosis-like’’. At the same time some of the treated cells lost membrane integrity and died via necrosis. Taking into account the multiplicity of mechanisms used by cancer cells to prevent apoptosis, drugs that would activate alternative cell death pathways could provide a useful tool for new types of cancer therapy. This makes diazene JK-279 interesting for further investigations as a potential anti-cancer drug, especially for treatment of cervical carcinomas. Acknowledgments The authors thank Asst. Prof. B. Uzarevic for helpful suggestion in flow cytometry analysis and Mrs. Lj. Krajcar for her valuable technical assistance. Ministry of Science, Education, and Sport of the Republic of Croatia (project No. 0098076) and the Ministry of Education, Science and Sport of the Republic of Slovenia (Project P1-0230-0103 and Joint Project BI-HR/04-05-3) are gratefully acknowledged for financial support. References Amarante-Mendes, G.P., Finucane, D.M., Martin, S.J., Cotter, T.G., Salvesen, G.S., Green, D.R., 1998. Anti-apoptotic oncogenes prevent caspase-dependent and -independent commitment for cell death. Cell Death and Differentiation 5, 298–306.
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