Fractionated Ionizing Radiation Exposure Induces Apoptosis through ...

ANTICANCER RESEARCH 26: 4549-4558 (2006)

Fractionated Ionizing Radiation Exposure Induces Apoptosis through Caspase-3 Activation and Reactive Oxygen Species Generation BARBARA BUCCI1, SILVIA MISITI2, ANNAPAOLA CANNIZZARO1, RODOLFO MARCHESE1, GIORGIO H. RAZA3 ROBERTO MICELI3, ANTONIO STIGLIANO2, DONATELLA AMENDOLA1, OLIMPIA MONTI1, MICHELA BIANCOLELLA4, FRANCESCA AMATI4, GIUSEPPE NOVELLI1,4, ALDO VECCHIONE5, ERCOLE BRUNETTI1 and UGO DE PAULA3 1AFAR-Centro

Ricerca S. Pietro and 3Unità di Radioterapia Oncologica S. Pietro, Fatebenefratelli Hospital, 00189 Roma; di Endocrinologia, II Facoltà di Medicina, Università "La Sapienza", 00189 Roma; 4Department of Biopathology and Diagnostic Imaging, Tor Vergata University, 00133 Roma; 5I Istituto Nazionale Tumori, Fondazione G. Pascale, 80131 Naples, Italy

2Cattedra

Abstract. Background: Radiation therapy (RT) is a well established therapeutic modality for the treatment of solid tumors. In particular, post-operative RT is considered the standard treatment adjuvant to surgery since its ability to prolong median survival of patients with malignant astrocytoma has been shown; nevertheless the ionizing radiation (IR) treatment fails in a considerable number of astrocytoma patients. Materials and Methods: Using an ADF human astrocytoma cell line the molecular mechanisms involved in the DNA damage induced by fractionated irradiation (FIR) and single IR treatment have been investigated. Results: FIR and single IR treatment inhibited the growth of the ADF human astrocytoma cell line. FACS analysis revealed that FIR treatment, but not single IR treatment, induced growth inhibition associated with the induction of apoptosis. Apoptosis was related to caspase-3 activation and reactive oxygen species (ROS) generation. ROS formation depends on the up-regulation of the cytochrome P450 enzyme gene. On the contrary, 12.5 Gy induced necrotic cell death up-regulating the HSPD1, HSPCB, HSPCA and HSPB1 genes. Conclusion: FIR treatment induced cell death through caspase-3 and ROS-mediated apoptosis. Post-operative fractionated irradiation (FIR), is actually considered the standard treatment adjuvant to surgery since

Correspondence to: Barbara Bucci, AFAR-Centro Ricerca S. Pietro, Fatebenefratelli Hospital, Via Cassia 600, 00189 Rome, Italy. Tel: +39633582873, Fax: +39633251278, e-mail: [email protected] Key Words: Astrocytoma, radiotherapy, apoptosis, ROS content, microarray.

0250-7005/2006 $2.00+.40

it has been shown to be able to prolong median survival of patients with malignant astrocytoma (1-3), nevertheless the irradiation treatment fails in a considerable number of astrocytoma patients (2, 4). Recent studies suggest that the induction of apoptosis in tumor cells plays an important role in the efficacy of radiation therapy (RT) (5, 6) even though the role of apoptotic cell death in solid tumors is still unclear. The cells respond to ionizing radiation (IR) in a variety of ways, depending on cell type. The tumor suppressor protein p53 activates two opposing cellular pathways, in response to DNA damage, one resulting in cellcycle arrest and one triggering apoptosis. Gene expression studies have revealed the existence of more than one pathway regulating growth inhibition and apoptotic processes (7). This antineoplastic effect can be mediated by activation of different target genes such as p21, caspases, BAX and BCL-2 which act as cross-point regulators able to induce or inhibit apoptosis. Activation of human caspase-1 to -10 have been described to be involved in chemical and physical agent-induced apoptosis (8, 9). In particular, caspase-3 exists as an inactive pro-caspase-3 in the cytoplasm and is proteolytically converted into the caspase-3 active form, by a single cleavage event in cells undergoing apoptosis. After caspase-3 activation a specific substrate for caspase-3, PARP (poly ADP-ribose polymerase-1) protein is degraded by proteolytic cleavage and this is important for the occurrence of apoptosis (10, 11). Moreover, Kroemer et al. (12) have shown that biochemical modifications could represent an alternative apoptotic pathway. Reactive oxygen species (ROS) are per se inducers of apoptosis (13), and RT seems to be able to induce an apoptotic program through ROS generation (14). Our aim was to investigate the molecular mechanisms underlying the DNA damage induced

4549

ANTICANCER RESEARCH 26: 4549-4558 (2006) by single and FIR treatment which seems to activate either genetic repair, irreversible growth arrest or cell death. The role of oxidative and heat shock genes, implicated in the modulation of ionizing radiation induced apoptosis (15, 16) has also been investigated. Microarray gene expression technology (17) permitted the simultaneous analysis of the expression levels of these genes and the identification differentially expressed genes after the two regimes of treatment on an ADF human astrocytoma cell line.

Materials and Methods Cell culture. The ADF human astrocytoma cell line was maintained as monolayer cultures in DMEM medium supplemented with FCS 10% (Life Technologies, Scotland, United Kingdom), penicillin (100 Ìg/ml), streptomycin (100 Ìg/ml) and L-glutamine (2 mM) at 37ÆC in a 5% CO2 /95% air atmosphere. Radiation exposure. Cells were irradiated by a Varian Clinac 600c/d 6MV photon beam. A scanditronix FC65G farmer ionization chamber was used to evaluate the beam properties in water and in PMMA (polymethylmethacrylate). Homogeneity was about 2% and symmetry was about 0.5%. In all cases homogeneity and symmetry were less than 3%, as prescribed by our quality assurance program. The irradiation of the cells was based on five single irradiation doses (5, 10, 12.5, 15 and 20 Gy), while for FIR the cells were exposed daily to 5 Gy for four consecutive days (4x5 Gy). The fractionated condition was then determined by the RBE relationship using appropriate astrocytoma ·/‚ of about 10 Gy, as reported by Williams et al. (18). All the experiments were repeated three times and each experimental sample was seeded in triplicate. Cell growth and cell cycle analysis. The ADF human astrocytoma cancer cells were seeded in 100-mm Petri dishes at a density of 1x105 cells per dish. Cell viability was determined daily from day 2 (24 h after treatment) to day 8 (168 h after treatment) of culture. For FIR exposure, samples were analyzed at 24, 48, 72 and 96 h after the last dose of 5 Gy. Data were evaluated as percentage of control (i.e., absolute treated cell number/absolute control sample). Trypan blue exclusion test assay was used to evaluate cell growth inhibition. The analysis of cell cycle was performed by flow cytometry (FCM) after propidium iodide (PI) staining, as previously described (19). Samples were then measured by using a FACScan cytofluorimeter (Becton Dickinson, Sunnyvale, CA, USA) at the indicated times for each treatment. PI cytotoxicity assay. The capacity of IR treatments to produce cell death was determined by PI staining exclusion test and FCM, as previously described (19). Apoptotis detection. The induction of apoptosis was studied by using FITC-Annexin-V (Bender Med Systems, Vienna, Austria), TUNEL (Tdt-Utp Nick End Labeling) assays (Roche Diagnostics, Mannheim, Germany) and FCM analysis. Annexin-V assay was used to discriminate between necrotic and apoptotic cell death. The simultaneous staining of cells with FITC-Annexin-V and PI allowed the resolution of viable cells (double negative), apoptotic

4550

cells (Annexin-V positive and PI negative), and necrotic cells (PI positive). Cells were treated, as previously described (19). As to TUNEL assay treated and untreated cells were handled as previously described (20). Western blot. Western blotting was performed as reported previously (19). Forty mg aliquots of protein were subjected to 12% SDS-PAGE. The resolved proteins were blotted to a nitrocellulose membrane and then the blots were incubated with primary antibodies such as: anti-caspase-3 (clone N-19, Santa Cruz, CA, USA), anti-PARP (clone F2, Santa Cruz), anti-Bcl-2 (clone Bcl2/100, Pharmingen, San Diego, CA, USA), anti-Bax (Pharmingen) and anti ‚-actin (clone C-11, Santa Cruz). Peroxidase-labeled antigoat, anti-mouse and anti-rabbit IgG (Amersham Life Science, Arlington Heights, IL, USA) were used as secondary antibodies. The immunoblots were processed for enhanced chemiluminescence detection (Amersham Life Science). Western blotting experiments for Bcl-2 and Bax protein levels were quantified by densitometry analysis (total Lab image analysis solution version-2003-non linear dynamics), normalized for ‚-actin level. Measurement of ROS content. For the ROS content analysis, untreated and IR-treated (12.5 Gy and 4x5 Gy) adherent cells were first assayed for viability by trypan blue dye exclusion and then incubated with 4 ÌM dihydroethidium (Molecular Probes, Eugene, OR, USA) for 45 min at 37ÆC. In the experiments with N-acetyl-Lcysteine (NAC) antioxidant (Sigma, Milan, Italy), cells were preincubated with 5 mM NAC for 6 h. Then cells were washed twice and treated with the different ionizing radiation regimes (12.5 Gy and 4x5 Gy, respectively). NAC was re-added to the cells at the end of each FIR exposure and left in the culture medium until the FCM analysis. Microarray analysis. For RNA extraction and labeling, total RNA was isolated by the TRIZOL standard protocol (Invitrogen Corporation, Carlsbad, CA, USA). Four Ìg of total RNA were retrotranscribed and labeled with 32-dCTP (NEN) using a GEArrayTM Probe Synthesis Kit (SuperArray Bioscience Corporation, USA). Labeled cDNAs were then hybridized on GEArray S Series Human Apoptosis and Cell Cycle Gene Array (HS-603 SuperArray Bioscience Corporation, USA). The GEArray S Series Human Apoptosis and Cell Cycle Gene Array contains 96 key apoptosis genes, 96 key cell cycle regulator genes and 75 stress and toxicity genes. It moreover contains a system of controls, such as negative controls (pUC18 DNA and blanks) and putative housekeeping genes (‚-actin, GAPDH). Labeled cDNAs were denatured at 95ÆC for 5 min and applied directly to the hybridization solution. Microarray hybridization was performed at 60ÆC overnight. Posthybridization washings were made according to GEArray instructions. For statistical analysis of expression data, the acquisition of filter images was carried out by using a STORM apparatus (Amersham Bioscences) after a 90 min exposure. Filter images were then analyzed with GEArray Analyzer software (www.superarray.com). Statistical analysis. The data are expressed as mean values±SD. Statistical significance of differences between groups was tested by paired Student’s t-test or, if there were more than two groups, by one-way ANOVA. A p-value of less than 0.05 was considered significant.

Bucci et al: FIR Induced ROS-mediated Apoptosis

Figure 1. A) Cells were exposed to increasing doses of ionizing radiation (5 ■, 10 ▲, 12.5 x, 15 * and 20 ● Gy) and the analysis was performed 24, 48, 72, 96, 120, 144 and 168 h from treatment. The arrow indicates the start of gamma radiation treatment. B) Cell growth inhibition of 4x5 Gy and 12.5 Gy. FIR treatment, grey and single treatment, black square. Error bars represents standard deviation of three separate experiments. (C) Cytotoxicity after each treatment was determined by PI staining exclusion test and FCM. The experiments were repeated three times showing similar results. Values are means (bars, SE, Standard Error) of triplicate samples.

Results The total treatment dose to divide in smaller fractions for the FIR treatment was outlined. ADF cells were exposed to 5, 10, 12.5, 15 and 20 Gy doses and examined the effects produced on cell growth and cell cycle at different times after IR exposure (24, 48, 72, 96, 120, 144 and 168 h). At each time cells were harvested and counted by using the trypan blue dye exclusion test. The IR exposure caused inhibition of cell proliferation of the ADF cell line (Figure 1A): a significant cell growth inhibition of about 53%, 59% and 65%, respectively, was already observed 24 h after exposure to 12.5, 15 and 20 Gy. The inhibitory effect increased to 94% for 12.5 and 96% for both 15 and 20 Gy after 168 h. In contrast, when cells were exposed to the lowest IR radiation doses (5 and 10 Gy), the inhibition of the cell proliferation at 24 h was about 20% and 40%, respectively. These effects were partially lost during the following days decreasing to 13% and 25%, respectively, after

168 h. These data indicate a significant and persistent inhibition of the cell growth only with the highest (12.5, 15 and 20 Gy) radiation doses. Conversely, at the lowest doses (5 and 10 Gy) a fraction of the cell population seems to be able to recover from the radiation-induced sub-lethal DNA damage. To evaluate whether the IR-induced cell growth inhibition could be related to the cell cycle perturbation, PI-staining and FACS analysis were performed on the ADF cells exposed to IR doses of 5, 10, 12.5, 15 and 20 Gy. The relative number of cells in each phase of the cell cycle was estimated from DNA content by Cell Quest software analysis. As shown in Table I, the IR exposure induced a dose-dependent accumulation of cells in G2-phase of the cell cycle compared to untreated cells (0 Gy) 24 h after treatment. Forty-eight hours after 20 Gy treatment, the G2 accumulation was still markedly evident (70%), suggesting that the ionizing radiation exposure leaded to irreversible DNA damage promoting cell killing. The marked presence of cell death within the following hours

4551

ANTICANCER RESEARCH 26: 4549-4558 (2006) Table I. Cell cycle phases percentages after ionizing radiation. 24 h IR doses

48 h

cell cycle phases (%)

Gy

G1

S

0 5 10 12.5 15 20

49±2.1 45±3.0 40±2.3 22±0.9 15±2.2 5±2.1

38±2.2 35±1.7 15±0.9 18±1.6 5±1.2 6±1.9

(%)


G2 Toxicity G1 13±0.8 20±0.5 45±1.2 60±2.0 80±2.1 89±1.8

5 5 8 8 9 15

72 h

50±3.0 46±2.7 48±0.7 39±1.5 30±1.1 12±1.3

S

G2

37±1.5 43±0.8 24±1.6 30±2.0 36±0.8 18±1.7

13±0.3 11±1.1 28±2.0 31±3.1 34±2.1 70±0.4

(%)


Toxicity G1 4 4 10 22 18 30

96 h

58±3.1 51±2.9 47±2.2 47±0.6 39±1.1 nv

S 32±0.4 33±0.04 29±1.1 29±1.3 33±0.9 nv

(%)


G2 Toxicity G1 10±1.1 16±1.9 24±1.3 24±1.8 28±2.2 nv

6 7 11 30 35 60

63±3.2 59±2.2 50±2.4 nv nv nv

S 25±0.2 26±1.7 30±1.1 nv nv nv

(%)

G2 Toxicity 12±0.1 5 15±0.04 7 20±1.3 13 nv 55 nv 64 nv 96

Abbreviations: nv = not valuable. Cells were treated with 5, 10, 12.5, 15 and 20 Gy ionizing radiation. At the indicated times they were harvested, fixed in ethanol 70% and stained with PI. The percentages in the cell cycle compartment were estimated by applying the MODFIT software to each DNA histogram. Data are means±SE of three separate experiments. Toxicity represents the percentage of PI-positive cells evaluated by cytotoxicity assay.

supported this hypothesis. The cell death effect was principally due to toxicity. Indeed, already at 48 h after treatment the toxicity percentage evaluated as % of PI stained cells was 30% and 60% at 72 h and reached values of 96% at 96 h. The PI exclusion test indicated the loss of structural integrity of the cell plasma membrane which represents one of the key markers distinguishing necrotic cell killing from living cells. Using 12.5 and 15 Gy doses, a partial G2 recovery was observed during the following 48 and 72 h from treatment, the G2-phase % were: 31% and 34%, 24% and 28%, respectively. The G2-induced ionizing radiation arrest was apparently recovered, but within the following hours the cells died suggesting that the G2 reversible arrest becomes irreversible with a delay of 48 h. This anti-proliferative effect was also associated with a high toxicity percentage, 55% and 64%, respectively, for the 12.5 Gy and 15 Gy at 96 h, reaching values of 85% at 168 h (data not shown). Conversely, with the treatment of 5 and 10 Gy, the ability of cells to survive to the IR DNA damage was more evident, the IR-induced G2 accumulation, indeed, was completely recovered within the following 96 h (15% and 20%, respectively) concomitantly at the increased G1 (59% and 50%, respectively). Moreover, the exposure to 5 and 10 Gy produced only a moderate cytotoxic effect within 96 h from treatment, with the percentages of PI positive cells less than 10% and 15%, respectively. These percentages of toxicity persisted even at 168 h after treatment (data not shown). These data indicated that the 12.5, 15 and 20 Gy doses produced an equal persistent inhibitory effect on ADF cell growth associated with the same cytostatic effect during the 168 h after IR exposure, consequently the 12.5 Gy dose was chosen as the total dose for the unique treatment. The effect of IR, using a single dose of 12.5 Gy radiation (unique administration) was compared with that of fractionated 4x5 Gy (exposure daily to 5 Gy for 4 consecutive

4552

days) and the mechanism of action underlying the antitumor activity was investigated. As shown in Figure 1B, the FIR exposure (4x5 Gy) already drastically inhibited cell proliferation to about 70% compared to unique treatment (about 50%), at 24 h after the last dose of 5 Gy. This antineoplastic effect increased at 96 h after treatment reaching about 89%. The antiproliferative effect was associated with a moderate cytotoxicity percentage, being less than 25% at 96 h and suggesting that the ADF cells exposed to FIR showed an increased tolerance to IR induced cytotoxicity (Figure 1C). The cells irradiated with 12.5 Gy showed a similar cell inhibitory effect to the FIR exposure (82%) at 96 h, but this antineoplastic effect was associated with a marked cytotoxicity percentage. Indeed, it was more than 50%, compared to FIR after 96 h, making this IR treatment unsuitable even thought there was a significant antineoplastic effect. In order to determine the role of apoptosis in the decrease of cell growth after the two different treatments (12.5 and 4x5 Gy), Annexin-V assay and FACS analysis were used. The analysis was performed 24, 48 and 72 h after each treatment. As shown in Figure 2A, the dose of 4x5 Gy produced the highest amount of apoptosis associated with a lower percentage of necrosis than that induced by the 12.5 Gy dose. After 24 h from 4x5 Gy treatment the percentage of apoptosis was 11% and the number of necrotic cells was still negligible (5%), compared to 3% apoptosis and 8% of necrosis produced by the 12.5 Gy dose. At 72 h apoptotic cell percentage increased up to 30% with a still low percentage of necrosis, indicating that the FIR treatment provoked cell killing activating apoptotic cell death. On the contrary, after single radiation treatment the number of necrotic cells increased to 35%, with a low amount of apoptosis confirming that the antiproliferative effect was likely due to necrosis. To evaluate whether the different types of cell death observed


Figure 2. A) Cytofluorimetric analysis of the Annexin-V versus PI staining assay. Apoptotic cells, grey and necrotic cells, black square. B, C and D) Western blot analysis of caspase-3, PARP, Bcl-2 and Bax proteins in the cell lysates of untreated (0 Gy) and treated cells performed at 48 h. All the experiments were repeated three times showing similar results.

Table II. Single and FIR effects on the cell cycle in the ADF cell line. Time after treatment (h)

Cell cycle phases (%) G1

S

G2

24 0 Gy 12.5 Gy 0 Gy 4x5 Gy

49±0.3 22±1.2 41±2.5 41±0.9

38±1.1 18±2.2 45±1.4 23±1.7

13±2.0 60±2.0 14±3.1 36±1.4

0 Gy 12.5 Gy 0 Gy 4x5 Gy

50±0.2 39±0.9 63±0.8 40±1.2

37±1.2 30±1.1 28±1.1 27±1.4

13±2.1 31±2.2 9±2.3 33±2.0

0 Gy 12.5 Gy 0 Gy 4x5 Gy

58±1.2 47±1.1 77±1.2 47±3.1

32±1.3 29±1.1 11±2.3 27±1.1

10±2.2 24±2.0 12±2.0 26±2.1

48

could be related to cell cycle perturbations induced by the treatments, and to identify the cell cycle compartment from which the apoptotic cells derive, cell cycle distribution of untreated (0 Gy) and treated ADF cells was analyzed by PIstaining and FCM analysis. The relative number of cells in each phase of the cell cycle was estimated from DNA content by CellQuest software analysis. The results, presented in Table II, showed that, the percentages of cells in the G2-phase increased markedly to about 60% after 24 h from treatment with 12.5 Gy and to about 40% with 4x5 Gy (24 h after the last dose of 5 Gy). However during this time the different treatments produced a very similar cell cycle distribution,

72

Cells were exposed to single (12.5 Gy) and FIR (4x5 Gy; 5 Gy for 4 consecutive days) treatment. The cell cycle analysis was performed at 24, 48 and 72 h from the end of treatment for 12.5 Gy and 24, 48 and 72 h after the last dose of 5 Gy. The percentages in the cell cycle compartment were estimated by applying the MODFIT software to each DNA histogram. Data are means ±SE of three separate experiments.

4553

ANTICANCER RESEARCH 26: 4549-4558 (2006)

Figure 3. A and B) Flow cytometric analysis of ROS content. C) Effect of the NAC antioxidant on ROS generation after 4x5 Gy exposure evaluated at 72 h. D). Apoptosis as evaluated by TUNEL assay at 72 h in NAC+ and NAC– 4x5 Gy exposure. Data are representative of three separate experiments.

suggesting that the different forms of cell death occurring after single and FIR exposure, cannot be explained by the different cell cycle profiles. To elucidate the mechanisms by which FIR treatment induces apoptosis, the caspase activation was investigated, testing the caspase-3 (CPP32/Yama) and PARP proteins. Figure 2 (B and C) shows the protein expression levels of caspase-3 protease and the processing of PARP in the treated and untreated cells evaluated at 48 h for both FIR and 12.5 Gy treatments. The results demonstrated that the caspase-3 protein procession is involved in FIR induced apoptosis treatment. The production of the active cleaved fragment, caspase-3-active, after FIR treatment supported the involvement of this protease in apoptotic cell death. On the contrary, no caspase-3 processing was observed after 12.5 Gy exposure. Considering that after caspase-3 activation, the specific substrate PARP protein is cleaved and this proteolytic cleavage is an important event for the occurrence of apoptosis, the behavior of PARP in ADF cells exposed to FIR treatment was evaluated. With exposure to FIR, degradation of 116 kDa PARP into an 85 kDa fragmentation protein was observed suggesting that FIR induces the activation of caspase-3 and proteolytic PARP cleavage. No processing of the PARP substrate was observed when the

4554

cells were treated with 12.5 Gy irradiation, indeed, only the 116 kDa form was evident like the control cells. Moreover, Bcl-2 family proteins, important apoptotic regulators, located upstream of caspase activation, were analyzed and a significant decrease in Bcl-2 expression of about 85% was found after FIR exposure but not after 12.5 Gy (Figure 2D). No appreciable changes were observed in the expression of Bax protein both in the FIR and the 12.5 Gy treatments. Several studies have demonstrated that biochemical modifications could represent an alternative apoptotic pathway including ROS production. To test whether the FIR treatment was also able to activate the apoptotic program by generating ROS, these were measured by FCM analysis. No changes in the ROS content were evident in the relative fluorescence shift in the untreated (0 Gy) or the 12.5 Gy treated ADF cells (Figure 3A). On the contrary, ROS content increased markedly in cells receiving FIR. Indeed, the FIR treatment induced ROS production at 48 h and the percentages of ROS progressively increased from 15% (48 h) to 45% (72 h) (Figure 3B). These data suggest that FIR treatment induced ROS generation stimulating the apoptotic process. To confirm the evidence of ROS production in the FIR induced apoptosis, the effect of NAC antioxidant inhibitor on ROS production and apoptosis was evaluated by


FCM. As shown in Figure 3C, the percentage of ROS evaluated at 72 h after FIR treatment reduced from 40% to about 6% indicating that NAC prevents ROS generation. Moreover, pre-treatment of the cells with NAC overcame FIR apoptosis. Figure 3D shows no evidence of apoptosis analyzed by TUNEL assay, after NAC pretreatment while the percentage of apoptosis is 40% in NAC pretreated cells, suggesting that the inhibition of apoptosis by the NAC antioxidant is principally caused by the effect of antioxidant on ROS generation. Since it has recently been reported that oxidative and heat shock genes and their products might be involved both in the apoptotic pathway after IR exposure and in the intracellular redox state (10), the analysis of 96 apoptotic, 96 cell cycle regulator and 75 stress and toxicity related genes was performed at 24 h after each treatment using a GEArray S Series Human Apoptosis and Cell Cycle gene Array. One hundred and sixty out of 267 (60%) genes of the 12.5 Gy treated cells and 188 (70%) genes of the 4x5 Gy treated cells were modulated. Twenty-three genes were downregulated and 42 up-regulated in the 12.5 Gy treated cells, 36 genes were down-regulated and 44 up-regulated after 4x5 Gy exposure. The data analysis considered significant only those genes whose differential expression had a threshold >±2.0. Focusing on genes involved in heat shock and related stress: HSPB1 (Heat shock 27 kDa protein 1), HSPCA (Heat shock 90 kDa protein 1, alpha), HSPCB (Heat shock 90 kDa protein 1, beta) and HSPD1 (Heat shock 60 kDa protein 1, chaperon in) were up-regulated after a single 12.5 Gy treatment. Moreover, regarding the genes involved in the oxidative and metabolic stress a gene, CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) was found to be significantly upregulated in FIR treated cells, suggesting that the modulation of these genes could play a role in the formation of ROS generation (Figure 4).

Discussion In the present study the involvement of apoptosis in the FIR treatment sensitivity of human astrocytoma cell line was demonstrated. It was also shown that FIR induces apoptosis by caspase-3 activation and ROS generation. The radiation doses of 5 and 10 Gy were not toxic, and were able to inhibit cell growth (about 13 and 25% growth inhibition respectively). On the other hand, the doses of 12.5, 15 and 20 Gy, which caused the highest and persistent inhibition of cell growth (more than 90%) appeared to be highly toxic as evidenced by PI-staining (about 95% 168 h after treatment). Our data are in agreement with Louagie et al. (21) who have evidenced that 20 Gy IR mainly induces necrosis on lymphocytes. Although some investigators have indicated apoptosis as an important mechanism, by which radiotherapy kills cells (5, 6) others have argued that apoptosis is not the predominant form of cell death caused by exposure to IR (22).

Figure 4. Histogram representing the expression levels of five differentially expressed genes at 24 h after 4x5 Gy (5 Gy for 4 consecutive days) and 12.5 Gy, respectively. 12.5 Gy treatment (black bars) and 4x5 Gy (grey bars).

On the other hand, IR might delay the progression rate of cells through the different phases of the cell cycle, (23) causing G2-phase accumulation (24), and keeping cells from undergoing mitotic division (25). In agreement with the literature, our data showed a dose-dependent G2 accumulation after ionizing radiation treatment (23, 24, 26). Using fractionated doses (4x5 Gy), we have observed an antiproliferative effect and a decreased toxicity with respect to a single dose suggesting that at 12.5 Gy the process of apoptotic cell death can be preferentially inactivated. This hypothesis is also supported by Tsoncheva et al. (27) who have observed that at high doses (50 and 100 Gy) the cells showed less apoptosis than the cells irradiated with a lower IR dose (8 Gy). Our data clearly showed that the highest radiation dose promoted cell killing with a high percentage of necrosis, while using the lowest dose of 5 Gy given daily for four days consecutive, tumor cells responded to IR by activating the apoptotic pathway. Although Liqun et al. (28) reported a decrease of apoptosis after fractionated treatment in a MCF7 cell line via a p53-dependent repair system, however in our cells, p53 is mutant confirming the role of p53 in DNA repair process. In order to identify the cell cycle compartment from which the apoptotic cells derive, cell cycle profile in the two treatments has been studied. 4x5 Gy treatment determined a transient G2 arrest, but the protective response process, which is activated at the G2 checkpoint after the IR DNA damage, was not induced. On the other hand, it is generally reported that fractionated treatment is more effective if it is administered at the G2 cell cycle phase (26). Although it would be likely that these cells progressively escaped from the G2 block to undergo apoptosis thus eliminating the damaged cells, in our data

4555

ANTICANCER RESEARCH 26: 4549-4558 (2006) there is no evident association with a loss of cells from the G2 phase. Using 12.5 Gy a partial G2 recovery was observed, and, within the following hours the cells died suggesting that the G2 reversible arrest becomes irreversible inducing necrotic cell death. Thus, the apoptotic effect elicited by the FIR treatment should be ascribed to a different mechanism of action from that induced by a single treatment. In our data, apoptosis induced by the FIR treatment proceeded through a process involving caspase-3 activation and ROS generation. In fact, the PARP substrate is cleaved exclusively in the FIR treatment suggesting a different regulation of this protease in the two schedules of treatments. This is in agreement with other findings that have reported a specific cleavage of the 116 kDa PARP to a 85 kDa proteolytic fragment occurring in apoptotic processes in several systems (29). Moreover, it is well known that ROS generation is common and an important mediator of apoptosis (12, 13). In our study, the induction of apoptosis was exclusively observed in the FIR treated cells in which caspase-3 activation was associated with ROS generation. In fact, a significant percentage of ROS formation was exclusively observed in the FIR treated cells indicating that ROS induced apoptosis occured when the amount of ROS generated could not be handled by radical scavenging cellular antioxidant (12, 30). This was confirmed in our experiments, by the use of NAC antioxidant which clearly protected the ADF cells from the FIR induced apoptosis by inhibiting ROS generation. Bcl-2 protein, which represents one of the most common proteins with antioxidant function was analyzed, and it has been found to increase cell resistance to ROS or inhibit ROS generation by regulating the opening of permeability transition pore (31). In our data, ROS generation induced by the FIR treatment was associated with a decrease in the Bcl2 expression levels suggesting that the massive apoptotic cell death could be due to a synergism of the two different mechanisms of action. IR works via DNA damage and ROS generation could be induced by different genes expression (32), although the genes involved in the apoptosis triggered by ROS formation have to be identified. Recently, it has been reported that the mitochondrial cytochrome P450 enzyme is able to induce apoptosis by ROS-generation (15). In our data it was observed that ROS induced apoptosis after the FIR treatment occured via an up-regulation of the CYP1A1 enzyme gene. It has been suggested recently, that the heat shock genes and their products are able to protect cells from apoptotic cell death, triggered by a variety of stimuli including IR (16). A significant up-regulation of the heat shock genes HSPD1, HSPCB, HSPCA and HSPB1 was observed in the single treated cells in which apoptosis did not occur. These data support the hypothesis that specific gene modulation induced by FIR or single IR treatment, could explain the differences observed in cell death types

4556

(apoptosis or necrosis). In particular, the FIR treatment induced ROS mediated-apoptosis through the up regulation of the CYP1A1 gene.

Acknowledgements We thank Nicoletta Giuliani for her technical assistance. This work was supported by AFaR (Associazione Fatebenefratelli per la Ricerca).

References 1 Kristiansen K, Hagen S, Kollevold T, Torvik A, Holme I, Nesbakken R, Hatlevoll R, Lindgren M and Elgen K: Combined modality therapy of operated astrocytomas grade III and IV. Confirmation of the value of postoperative irradiation and lack of potentiation of bleomycin on survival time: a prospective multi center trial of the Scandinavian Glioblastoma Study Group. Cancer 47: 649-652, 1981. 2 Nelson DF, Diener-West M, Weinstein AS, Schoenfeld D, Nelson JS, Sause WT, Chang CH and Carabell S: A randomized comparison of misonidazole sensitized radiotherapy plus BCNU and radiotherapy plus BCNU for treatment of malignant glioma after surgery: final report of an RTOG study. Int J Radiat Oncol Biol Phys 12: 1793-1800, 1986. 3 Deutch M, Green SB, Strike TA, Burger PC, Robertson JT, Selker RG, Shapiro WR, Mealey J Jr, Ransohoff J II and Paoletti P: Results of randomized trial comparing BCNU plus radiotherapy, streptozocin plus radiotherapy, BCNU plus hyper fractionated radiotherapy, and BCNU following misonidasole plus radiotherapy in the post-operative treatment of malignant glioma. Int J Radiat Oncol Biol Phys 16: 1389-1396, 1989. 4 Withers HR: Biologic basis for altered fractionated schemes. Cancer 55: 2086-2095, 1985. 5 Reed JC: Regulation of apoptosis by BCL-2 family proteins and its role in cancer and chemo resistance. Curr Opin Oncol 7: 541-546, 1995. 6 Meyn RE, Stephens LC and Milas L: Programmed cell death and radio resistance. Cancer Metastasis Rev 15: 119-131, 1996. 7 Chendil D, Oakesm R, Alcock RA, Patel N, Mayhew C, Mohiuddin M, Gallicchio VS and Ahmed MM: Low dose fractionated radiation enhances the radio sensitization effect of paclitaxel in colorectal tumor cells with mutant p53. Cancer 89: 1893-1900, 2000. 8 Whyte M and Evan G: Apoptosis. The last cut is the deepest. Nature 376: 17-18, 1995. 9 Marsden VS, O’Connor L, O’Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K and Strasser A: Apoptosis initiated by Bcl-2-regulated caspase activation independently of cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 419: 634-637, 2002. 10 Essmann F, Wieder T, Otto A, Muller EC, Dorken B and Daniel PT: GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis. Biochem J 346: 777-783, 2000. 11 Soldani C and Scovassi AI: Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. 2002. Apoptosis 7: 321328, 2002. 12 Kroemer G: The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 3: 614-620, 1997.


13 Biroccio A , Benassi B, Amodei S, Gabellini C, Del Bufalo D and Zupi G: c-Myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in M14 human melanoma cells. Mol Pharmacol 60: 174-182, 2001. 14 Guo G, Yan-Sanders Y, Lyn-Cook BD, Wang T, Tamae D, Ogi J, Khaletskiy A, Li Z, Weydert C and Li JJ: Manganese superoxide dismutase-mediated gene expression in radiationinduced adaptive responses. Mol Cell Biol 23: 2362-2378, 2003. 15 Derouet-Humbert E, Roemer K and Bureik M: Adrenoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by generation of reactive oxygen species in mitochondria. Biol Chem 386: 453461, 2005. 16 Lee YJ, Lee DH, Cho CK, Chung HY, Bae S, Jhon GJ, Soh JW, Jeoung DI, Lee SJ and Lee YS: HSP25 inhibits radiationinduced apoptosis through reduction of PKC delta-mediated ROS production. Oncogene 24: 3715-3725, 2005. 17 De Risi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA and Trent JM: Use of a cDNA micro array to analyze gene expression patterns in human cancer. Nat Genet 14: 457-460, 1996. 18 Williams MV, Denekamp J and Fowler JF: A review of ·/‚ ratios for experimental tumors. Int J Radiat Oncol Biol Phys 11: 87-96, 1985. 19 Bucci B, D'Agnano I, Amendola D, Citti A, Raza GH, Miceli R, De Paula U, Marchese R, Albini S and Vecchione A: Myc downregulation sensitizes melanoma cells to radiotherapy by inhibiting MLH1 and MSH2 mismatch repair proteins. Clin Cancer Res 11: 2756-2767, 2005. 20 D'Agnano I, Antonelli A, Bucci B, Marcucci L, Petrinelli P, Ambra R, Zupi G and Elli R: Effects of poly (ADP-ribose) polymerase inhibition on cell death and chromosome damage induced by VP16 and bleomycin. Environ Mol Mutagen 32: 5663, 1998. 21 Louagie H, Cornelissen M, Philippe J, Vral A, Thierens H and De Ridder L: Flow cytometric scoring of apoptosis compared to electron microscopy in Á irradiated lymphocytes. Cell Biol Int 22: 277-283, 1998. 22 Steel GG: The case against apoptosis. Acta Oncol 40: 968-975, 2001.

23 Ngo FQ, Blakely EA, Tobias CA, Chang PY and Lommel L: Sequential exposures of mammalian cells to low- and high-LET radiations. II. As a function of cell cycle stages. Radiat Res 115: 54-69, 1998. 24 Kal HB, Hatfield M and Hahn GM: Cell cycle progression of murine sarcoma cells after X irradiation or heat shock. Radiology 117: 215-217, 1975. 25 Lucke-Huhle C, Blakely EA, Chang PY and Tobias CA: Drastic G2 arrest in mammalian cells after irradiation with heavy-ion beams. Radiat Res 79: 97-112, 1979. 26 Geldof AA, Plaizier MA, Duivenvoorden I, Ringelberg M, Versteegh RT, Newling DW and Teule GJ: Cell cycle perturbations and radio sensitization effects in a human prostate cancer cell line. J Cancer Res Clin Oncol 129: 175-182, 2003. 27 Tsoncheva VL, Kirov KS, Valkova CA and Milchev GI: Evaluation of delayed apoptotic response in lethally irradiated human melanoma cell lines. Z Naturforsch [C] 56: 660-665, 2001. 28 Liqun X, Aimee P and Li JJ: p53 Activation in chronic radiationtreated breast cancer cells: regulation of MDM2/p14ARF. Cancer Research 64: 221-228, 2004. 29 Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE and Poirier GG: Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 53: 3976-3985, 1993. 30 Lennon SV, Martin SJ and Cotter TG: Dose-dependent induction of apoptosis in human tumor cell lines by widely diverging stimuli. Cell Prolif 24: 203-214, 1991. 31 Hockenbery H, Oltvai ZN, Yin XM, Milliman CL and Korsmeyer SJ: Bcl-2 functions as an anti-oxidant pathway to prevent apoptisis. Cell 75: 241-251, 1993. 32 Park WY, Hwang CI, Im CN, Kang MJ, Woo JH, Kim JH, Kim YS, Kim JH, Kim H, Kim KA, Yu HJ, Lee SJ, Lee YS and Seo JS: Identification of radiation-specific responses from gene expression profile. Oncogene 21: 8521-8528, 2002.

Received July 21, 2006 Accepted October 2, 2006

4557