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Free Radical Biology & Medicine
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Original Contribution
Promising effects of the 4HPR–BSO combination in neuroblastoma monolayers and spheroids
Roos Cuperus a, André B.P. van Kuilenburg a,⁎, René Leen a, Johannes Bras b, Huib N. Caron a, Godelieve A.M. Tytgat a
aLaboratory of Genetic Metabolic Diseases and Department of Pediatrics/Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands
bDepartment of Pathology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands
a r t i c l e i n f o a b s t r a c t
Article history:
Received 3 February 2011 Revised 25 May 2011 Accepted 9 June 2011 Available online 23 June 2011
Keywords: Neuroblastoma Fenretinide
Buthionine sulfoximine Spheroids
Glutathione
Reactive oxygen species Free radicals
To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5–10 μM, 24 h) resulted in ROS induction (114–633%) and increased GSH levels (68–120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25–100 μM, 24 h). The 4HPR–BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110–150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR–BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR–BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR–BSO combination might be a promising new strategy in the treatment of neuroblastoma.
© 2011 Elsevier Inc. All rights reserved.
Patients suffering from stage 4 neuroblastomahavea poorprognosis, and therefore, new therapeutic options are needed. Fenretinide (N-(4- hydroxyphenyl) retinamide; 4HPR) induces apoptosis in various cancers, including neuroblastoma [1,2]. The precise mechanism under- lying the apoptosis-inducing properties of 4HPR is not yet fully understood. It has been suggested that 4HPR induces apoptosis by both retinoic acid receptor-dependent and reactive oxygen species (ROS)-dependent pathways [3–8]. We have previously shown that Trolox, a radical scavenger, protected 4HPR-treated neuroblastoma cells from ROS and from loss of viability. This indicated that ROS production, induced by low doses of 4HPR, might be involved in the occurrence of apoptosis in neuroblastoma [9].
Glutathione (GSH) was first described by Hopkins as an important cellular reducing agent [10]. In addition to protecting the cell against free radicals, GSH is also involved in detoxification of alkylating agents,
storage and transport of cysteine, cell proliferation and regulation of apoptosis, signal transduction, gene expression, and interaction with nitric oxide [11–16].
The rate-limiting step in the de novo synthesis of GSH is the formation of γ-glutamylcysteine from glutamate and cysteine per- formed by γ-glutamylcysteine synthetase (γ-GCS). Buthionine sulfox- imine (BSO) is a strong γ-GCS inhibitor both in vivo and in vitro [10,17,18]. Conflicting data exist astowhether BSO-inducedcytotoxicity in neuroblastoma cells is associated with ROS production. In some studies the cytotoxic effects of BSO were attenuated by antioxidants, whereas in other studies, the cytotoxicity of BSO was concentration and cell-line dependent [17–19]. A phase I trial with BSO, performed in patients with various tumors, showed minimal toxicity. A significant decrease in GSH, measured in peripheral mononuclear cells, to approximately 30 to 40% of control was achieved in patients receiving BSO. Clinical achievable plasma levels of BSO up to 465 μM (±189) have been reported [20,21].
Abbreviations: 4HPR, N-(4-hydroxyphenyl)retinamide; ROS, reactive oxygen species; BSO, buthionine sulfoximine; GSH, glutathione; DTNB, 5,5′-dithiobis(2-nitrobenzoate); TNB, 5-thio(2-nitrobenzoic) acid; EDTA, ethylenediaminetetraacetic acid; MTS, 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; γ-GCS, γ-glutamylcysteine synthetase.
⁎ Corresponding author. Fax: +31 206962596.
E-mail address: [email protected] (A.B.P. van Kuilenburg). 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2011.06.019
Becauseboth4HPRandBSOareagents withminimalsystemictoxicity, the combination of 4HPR and BSO might be beneficial in neuroblastoma [21,22]. To increase the efficacy of 4HPR we investigated the effect of the combination of 4HPR and BSO on GSH levels, antioxidant enzyme levels, ROS production, and viability in neuroblastoma monolayers and multi- cellular tumor spheroids.
Methods Chemicals
4HPR (Sigma, St. Louis, MO, USA) was dissolved in 100% ethanol, stored at 4 °C, and protected from light. L-BSO (Sigma) was dissolved in H2O at a 0.1 M stock concentration and stored at 4 °C. Serial dilutions were prepared from the stock solutions with growth medium just before use. Glutathione reductase and NADPH were obtained from Roche (Mannheim, Germany). All other chemicals were purchased from Sigma.
Cell culture
Three MYCN-single-copy neuroblastoma cell lines (FISK, NASS, SY5Y) and three MYCN-amplified neuroblastoma cell lines (IMR32, SJ8, SJNB10) were cultured in RPMI 1640 culture medium supple- mented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin/streptomycin, 4 mM glutamine (Gibco, Invitrogen, Carls- bad, CA, USA), and 5 mg/L plasmocin (Invivogen, San Diego, CA, USA). Cells were grown at 37 °C, 5% CO2 in 95% humidified air; culture fl asks and plates were obtained from Corning (Corning, NY, USA). All cell lines were a generous gift from Professor R. Versteeg (Department of Human Genetics, Academic Medical Center, Amsterdam, The Nether- lands). Spheroids were prepared from IMR32 cells and spheroid growth was analyzed as described before [9]. The spheroids were treated with 1 μM 4HPR and/or 12.5 μM BSO for 2 weeks, without changing the culture medium. Spheroids for immunohistochemical analysis were cultured without drugs for 1 week, after which incubation with 10 μM 4HPR and/or 200 μM BSO was started. All experiments were performed in quadruplicate unless stated otherwise.
Measurement of cell viability and apoptosis
Cells were plated (30×103/ml) in 100 μl in 96-well plates and allowed to adhere overnight, after which the medium was replaced by medium containing various concentrations of 4HPR (0–50 μM) or BSO (0–1000 μM). After 24, 48, and 72 h the viability of the cells was measured using an MTS assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. MTS incubation lasted for 4 h at 37 °C. The dose–absorption curves were used to calculate the IC50 values.
Cells (125×103/ml) were plated in 4 ml in 25-cm2 flasks and allowed to adhere overnight. Subsequently, the cells were preincubated with BSO (12.5 or 25 μM) for 24 h, followed by co-incubation with 4HPR (0–10 μM) and BSO. After 48 h 4HPR–BSO co-incubation, the cells were harvested and the total amount of cells was counted using a cell counter (Coulter Counter; Beckman Coulter, Fullerton, CA, USA). The percentage of viable cells was determined using the trypan blue assay. A cell suspension was resuspended 1:1 with 0.4% trypan blue and incubated for 1 min. The viable cells were counted with the microscope using a Bürker–Türk counting chamber. The amount of viable cells was calculated from the total amount of cells and the percentage of trypan blue-negative cells. Incubation times and concentrations for the combination experiments were chosen according to the knowledge that BSO and 4HPR induced time- and dose-dependent cytotoxicity in our cell lines (Table 1).
Apoptosis was measured using the Caspase Glo assay (Promega). Cells were plated in 96-well plates as described above. After incubation for 24 h with BSO (0–50 μM) and/or 4HPR (0–20 μM), the cells were incubated for 30 min with 100 μl Caspase Glo reagent per well, prepared according to the manufacturer’s protocol. Luminescence was measured using a plate reader (Fluostar Optima; BMG).
Measurement of GSH
Total GSH levels (reduced and oxidized) were measured according to the enzymatic method described by Neumann et al. and Tietze [23,24], which relies on the fact that GSH can cleave DTNB to the yellow-colored TNB (412 nm). By the addition of glutathione reductase to the reaction mix the oxidized GSSG is reduced to GSH and the total amount of GSH can be measured. Cells were cultured and allowed to adhere and grow for 48 h before harvest or incubation. To measure the effect of BSO on the GSH levels, cells were incubated with BSO (0–200 μM) for 24 h. To study the effect of the 4HPR–BSO combination on GSH levels, IMR32, NASS, and SJNB10 cells were incubated for 3, 6, or 24 h with 4HPR (0–20 μM) and/or BSO (0– 100 μM), after which the cells were harvested with trypsin and lysed in 0.1% Triton X-100 for 10 min at room temperature. The homoge- nates were centrifuged at 10,000g, 2 min. at 4 °C, and the supernatant was frozen in liquid nitrogen and stored at – 80 °C until use. The reaction mix contained 0.06 mM DTNB, 0.2 mM NADPH, and 5 mM EDTA in a 100 mM sodium phosphate buffer, pH 7.4, and an aliquot of supernatant. The reaction was started by the addition of a 0.1 mg/ml glutathione reductase solution in 100 mM sodium phosphate buffer, pH 7.4. TNB formation was measured spectrophotometrically at 412 nm using a COBAS FARA centrifugal analyzer (Roche, Mannheim, Germany). All experiments were performed in triplicate.
Measurement of antioxidants: catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD)
For measurement of baseline antioxidant levels, cells of each cell line were cultured in triplicate in 75-cm2 flasks and harvested after 48 h, and the cell pellets were frozen in liquid nitrogen and stored at
- 80 °C. For measurement of the effects of BSO and 4HPR on antioxidant levels, SJNB10 cells were cultured in 162-cm2 flasks and allowed to adhere overnight after which they were preincubated with BSO (25 μM) for 24 h followed by co-incubation with 4HPR (0–5 μM) and BSO for 48 h. Cells were harvested and the cell pellets were frozen in liquid nitrogen and stored at – 80 °C. Catalase activity was measured spectrophotometrically, following the decomposition of H2O2 at 240 nm in a 0.1 M potassium phosphate buffer, pH 7.0, at 37 °C [25]. GPx activity and SOD activity were measured using commercial assay kits (Calbiochem, San Diego, CA, USA, and Fluka, Buchs, Switzerland, respectively) according to the manufacturer’s protocol.
Measurement of ROS
Cells were cultured and allowed to adhere overnight. Subsequently, cells were preincubated with BSO (50 μM) for 24 h followed by co- incubation with 4HPR (0–10 μM) for 4 h. Previously, we showed that ROS induction is an early phenomenon after 4HPR administration, occurring 1–6 h after incubation [9]. ROS were detected using the ROS-responsive dye CM-H2DCFDA (Invitrogen, Molecular Probes) as described before [9]. Protein determination was performed using bicinchoninic acid reagents according to the manufacturer’s protocol, using bovine serum albumin as a standard (Thermo Scientific, Rockford, IL, USA).
Immunohistochemistry
Spheroids were grown, and incubation with 4HPR (10 μM) and/or BSO (200 μM) for 2 weeks was started. Immunohistochemistry was performed as described before [9]. Apoptosis was detected using anti- cleaved caspase-3 1:100 (Bioke, Leiden, The Netherlands), and proliferation was detected using anti-Ki-67 1:200 (Dako, Carpinteria, CA, USA), both incubated at room temperature for 1 h. Sections were examined using a Zeiss microscope and photographed using a Leica camera. The assessment of the sections was performed qualitatively.
Table 1
(A)IC50 values for 4HPR and BSO and baseline ROS production in six neuroblastoma cell lines.
Cell line IC50 4HPR (μM)a IC50 BSO (μM) ROSb
24 h 48 h 72 h 24 h 48 h 72 h
(DCFDA/μg protein)
FISK 10.0 5.0 4.3 N 1000 N 1000 N 1000 4.0±0.2
NASS 34.0 11.5 8.3 N 1000 N 1000 978 3.4±0.2
SY5Y 10.2 4.7 4.1 N 1000 8 8 1.4±0.1
IMR32c 3.7 1.0 1.0 N 1000 80 26 5.5±0.4
SJ8c 7.7 5.9 5.3 N 1000 556 94 3.2±0.1
SJNB10c 8.6 4.8 4.6 N 1000 N 1000 37 2.4±0.1
(B)Baseline antioxidant levels in six neuroblastoma cell lines.
Cell line GSH
(nmol/mg protein)
Catalase
(μmol/min/mg protein)
GPx
(nmol/min/mg protein)
SOD
(U/mg protein)
FISK 57.1±2.4 1.0±0.01 9.1±0.2 10.3
NASS 54.0±3.0 5.0±0.30 7.1±0.6 7.3
SY5Y 25.1±0.2 1.0±0.02 9.6±1.0 11.2
IMR32c 26.4±2.3 5.2±0.20 7.6±1.1 11.4
SJ8c 40.2±0.4 1.6±0.07 9.9±1.0 6.2
SJNB10c 74.0±2.9
aIC50 values of 4HPR were obtained from Cuperus et al. [9].
bArbitrary units.
cMYCN-amplifi ed cell lines.
1.1±0.03 1.8±1.8 11.8
Statistics
Differences in cytotoxicity between two groups were analyzed with the two-sample t test (SPSS 16.0). Correlation between ROS production and sensitivity for the different drugs was studied by linear regression and determination of Pearson correlation coefficient.
Results
Evaluation of antioxidant baseline levels and sensitivity to 4HPR and BSO
In six cell lines the IC50 values for 4HPR and BSO were determined in addition to baseline ROS production, baseline levels of GSH, and the activity of the antioxidant enzymes catalase, GPx, and SOD (Table 1). No clear correlation was observed between sensitivity to 4HPR or BSO and the baseline levels of ROS production. Furthermore, the endogenous levels of GSH and the baseline levels of the various antioxidant enzymes did not correlate with the sensitivity to 4HPR or BSO (Table 1). Nevertheless, the levels of GSH, catalase, and GPX were high in NASS cells, which are relatively insensitive to 4HPR, compared to other cell lines.
significant increase in ROS production in NASS, SJ8, SJNB10, and SY5Y (17–160%) but not in FISK or IMR32. Incubation with the 4HPR–BSO combination increased the ROS production significantly in SJ8 and SJNB10 (31–62%) compared to 4HPR incubation alone, depending on the concentration of 4HPR. In NASS a marginally increased ROS production (30%, p =0.03) was observed, and in FISK, IMR32, and SY5Y no significant increase in ROS was observed after incubation with the 4HPR–BSO combination compared to incubation with 4HPR alone (Fig. 3). In cell lines with a high production of ROS, as a result of 4HPR incubation, GSH was strongly upregulated (IMR32, SJNB10). When 4HPR-induced ROS production was low, no increase in GSH was observed (NASS; Fig. 2).
Viability and apoptosis after incubation with 4HPR and BSO
To reduce GSH levels, all cell lines were preincubated with BSO for 24 h, followed by co-incubation for 48 h with 4HPR and BSO. 4HPR reduced the number of viable cells in all cell lines. BSO as a single drug significantly reduced the number of viable cells only in IMR32 (22% reduction, p =0.015). However, in four of the six cell lines a significant
Effects of BSO and 4HPR on cellular GSH levels
After 24 h incubation with 12.5 μM BSO a decrease of at least 75% in GSH levels was observed in all six cell lines. Increasing BSO concentration up to 100 μM resulted in a reduction of at least 82% of GSH levels in all cell lines tested (Fig. 1). In addition, a time- dependent decrease in GSH levels was observed in three cell lines tested when incubated with BSO only. Treatment with 4HPR for 24 h resulted in an increase of 120 and 68% in GSH compared to untreated cells in IMR32 and SJNB10, respectively. In contrast, GSH levels dropped to 86% in NASS after incubation with 4HPR for 24 h. In IMR32 and SJNB10, the addition of BSO to 4HPR annulled the effect of 4HPR, and the GSH levels observed after 4HPR–BSO incubation were comparable to the GSH levels of the cells treated with BSO alone
120
100
80
60
40
20
0
0 25 50 75 100
(Fig. 2).
The effect of 4HPR and BSO on ROS production
4HPR induced the production of ROS (14–533%) in a dose- dependent manner in all cell lines. However, the 4HPR-induced ROS production was minimal in FISK and NASS cells (Fig. 3). BSO induced a
[BSO] ( M)
Fig. 1. The effect of BSO on GSH levels. IMR32 cells were incubated for 24 h with a BSO concentration range of 0–100 μM. After incubation, GSH concentration was measured (control: 36.0 nmol/mg protein). Error bars represent standard deviation. The inset shows the decrease in GSH at the lower BSO concentration range. The decline in GSH in the IMR32 cell line is representative of the other cell lines of our panel. The GSH levels are depicted as percentage of the control.
250
200
150
100
50
0
decreasein number of viablecells wasobserved aftertreatmentwith the 4HPR–BSO combination, compared to that observed for 4HPR alone (Fig. 4). In the two cell lines (FISK and NASS) that show minimal 4HPR- induced ROS production, no additional effect of BSO was observed on the viability of the cells.
Previously, we showed that 4HPR induced concentration- and time- dependent apoptosis in neuroblastoma cell lines [9]. BSO, as a single drug, induced apoptosis in IMR32 and SJNB10 cells (55%, p =0.002, and 85%, p =0.012, respectively) and no significant apoptosis in NASS cells. In contrast, an increase in apoptosis of 90, 46, and 24% (p =0.007, 0.002, and 0.032, respectively) was observed when BSO was combined with 4HPR in IMR32, SJNB10, and NASS cells, respectively, compared with
IMR32 NASS SJNB10 incubation with 4HPR alone.
Fig. 2. Time-dependent effects of 4HPR and BSO on GSH levels. GSH levels were measured in three cell lines treated for 24 h with control medium (black), BSO (dark gray), 4HPR (light gray), 4HPR–BSO combination (white). Concentrations: IMR32, 2.5 μM 4HPR, 25 μM BSO; NASS, 20 μM 4HPR, 100 μM BSO; SJNB10, 5 μM 4HPR, 50 μM BSO. The GSH levels are depicted as percentage of the control. *p b 0.05; **p ≤ 0.001.
Activity of catalase, GPx, and SOD after 4HPR–BSO combination
The activity of various antioxidant enzymes was measured in SJNB10 after incubation with the 4HPR–BSO combination to investigate whether their activity was increased because of the ROS induction.
800
600
400
200
0
FISK
0 M BSO 50 M BSO
800
600
400
200
0
SJ8
0 2.5 5 10 0 2.5 5 10
800
600
400
IMR32
800
600
400
SJNB10
*
*
200
0
200
0
0 2.5 5 10 0 2.5 5 10
800
600
400
NASS
800
600
400
SY5Y
*
200
0
*
0
2.5
5
*
10
200
0
0
2.5
5
10
Fig. 3. The effects of 4HPR and BSO on ROS production. ROS production was measured using the ROS-responsive dye CM-H2DCFDA. Cells were preincubated with BSO (50 μM) for 24 h followed by a 4-h co-incubation with a range of concentrations of 4HPR (0–10 μM). Black bars represent ROS production with 4HPR without BSO, gray bars represent ROS production with 4HPR and BSO. The ROS production is depicted as percentage of the control. *p b 0.05.
8
6
FISK
control + BSO
4
3
SJ8
4
2
0
2
1
0
*
0 5 10 0 1 5
6
4
2
0
IMR32
16
12
8
4
0
SJNB10
0 1.25 2.5 0 2.5 5
4
3
2
1
0
NASS
4
3
2
1
0
SY5Y
0
5
conc 4HPR (µM)
10
0
2.5
conc 4HPR (µM)
5
Fig. 4. The effects of 4HPR and BSO on viability. Cells were counted and the percentage of viable cells was measured using the trypan blue exclusion assay. Cells were preincubated 24 h with BSO (FISK, NASS, SJ8, and SJNB10, 25 μM; IMR32 and SY5Y, 12.5 μM) followed by a 48-h co-incubation with a range of concentrations of 4HPR (0–10 μM, depending on the sensitivity to 4HPR of each cell line). Black bars represent the amount of viable cells with 4HPR without BSO, gray bars represent the amount of viable cells with 4HPR and BSO. *p b 0.05.
Neither GPx nor SOD activity was increased after 4HPR and/or BSO incubation. Catalase activity was increased (1.5-fold, p =0.003) after 4HPR–BSO incubation compared to untreated, whereas 4HPR incuba- tion alone was followed by a lower increase in catalase activity (1.3-fold, p =0.000; data not shown). However, this increase was apparently not sufficient to protectthe cells against the cell death induced by the4HPR– BSO combination (Fig. 4).
Synergistic growth retardation in IMR32 spheroids after incubation with 4HPR and BSO
The benefi cial effects of the BSO–4HPR combination in monolayers prompted us to investigate the efficacy of this combination in IMR32 spheroids (Fig. 5). After spheroids were incubated 2 weeks with both 4HPR (1 μM) and BSO (12.5 μM), a decrease in cross-sectional area of about 20% was observed compared to the untreated control spheroids. Surprisingly, in spheroids treated with the combination of the two agents a decrease of 90% of the cross-sectional area was observed compared to untreated spheroids (Fig. 5).
Combined treatment with 4HPR and BSO decreases proliferation and increases apoptosis in IMR32 spheroids
Histological sections of IMR32 spheroids treated with 4HPR, BSO, or the 4HPR–BSO combination were stained with a proliferation marker, anti-Ki67, and an apoptosis marker, anti-cleaved caspase-3 (Fig. 6). In untreated control spheroids a classical organized pattern of an outer proliferative rim and an inner apoptotic and necrotic core was observed; fuzzy cells with disrupted membranes represent either apoptosis or necrosis. Apoptotic cells were represented by both fragmentation of the nuclei and staining with the apoptosis marker. Morphological assess- ment of the spheroids showed little effect of BSO; the cell structure of the rim was looser than that of the untreated control. In contrast, in 4HPR-treated spheroids the organized cell structure disappeared and the cell–cell pattern was looser than the rim of the control spheroids. Spheroids treated with the 4HPR–BSO combination showed a similar structure compared to the 4HPR-treated spheroids. However, at the cellular level an enormous change in morphology was observed; in a substantial amount of cells no nucleus was observed, indicating that these were apoptotic or necrotic cells.
A
*
Apoptosis (Caspase)
Proliferation
(Ki67)
120
100
80
60
40
20
0
control 4HPR BSO combi
control
10 µM 4HPR
10x
20x
B
Day 1
20x
Day 14
Fig. 5. The effects of 4HPR and BSO on spheroid growth. IMR32 spheroids were treated with 4HPR (1 μM) and/or BSO (12.5 μM) and photographed weekly to monitor the
40x
increase in the cross-sectional area. (A) The calculated cross-sectional area of spheroids treated with 4HPR and/or BSO for 2 weeks, depicted as percentage compared to the untreated control. Each bar represents the mean area±SD of four experiments. (B) IMR32 spheroids treated with 4-HPR and/or BSO. *p b 0.05; **p ≤ 0.001.
200µM BSO
Discussion
10x
4HPR is known to induce oxidative stress, which induces apoptosis in neuroblastoma cells [2,9,26–28]. Therefore, the cellular redox state, reflecting the intracellular antioxidant defense against free radicals, might be a key factor in protecting the cells from ROS-induced cytotoxicity. To test this hypothesis, on the one hand, we induced ROS by incubating cells with 4HPR and, on the other hand, we reduced the capacity to scavenge these ROS by reducing the GSH levels with BSO,
20x
in a panel of neuroblastoma cell lines. In line with our hypothesis is the observation that GSH levels increased in cell lines that showed profound 4HPR-induced ROS production, whereas in cell lines that did not produce ROS after 4HPR incubation, no upregulation of GSH was detected. This suggests that GSH is an important component of the
combination
cellular defense mechanism against 4HPR-induced cytotoxicity in neuroblastoma. With respect to other tumors, in Ewing sarcoma a decreased level of GSH as a result of 4HPR incubation was observed depending on the cell line; in cell lines with no ROS induction, no change in GSH levels as a result of 4HPR incubation was observed [29]. In leukemia cells, a decrease in GSH was observed after 4HPR incubation, whereas increased levels were observed after retinoic acid incubation. This indicates that modulatory effects of retinoic acids on intracellular antioxidant levels seem to be cell-type specifi c [30].
Previously, a correlation between high GSH levels and low sensitivity to ROS was observed in neuroblastoma [31,32], whereas a relation between high GSH baseline levels and low sensitivity to 4HPR has been observed only in leukemia cells [33,30]. In our study, no correlation between baseline levels of GSH and other antioxidants and sensitivity to 4HPR was observed. Nevertheless, in NASS cells, GSH, catalase, GPx, and SOD levels are high compared to other cell lines. This phenomenon might underlie the lack of ROS induction in NASS after 4HPR incubation. In SJNB10, which show low endogenous levels of ROS, GSH levels are high and other antioxidant levels are
10x
20x
Fig. 6. The effects of 4HPR and BSO on proliferation and apoptosis in spheroids. Immunohistochemical stained sections of spheroids treated with control medium, 4HPR (10 μM), BSO (200 μM), or the 4HPR–BSO combination for 2 weeks are shown. An apoptosis marker, anti-caspase-3 (brown nuclei, left), and a proliferation marker, anti- Ki67 (brown nuclei, right), were used to stain the cells. Assessment of the sections was performed at two magnifications, 10× and 20×.
relatively low. Therefore, GSH might be important in protection against ROS-induced cytotoxicity in neuroblastoma cells.
The degree of cytotoxicity induced by BSO as a single drug was dependent on the cell line. ROS were increased after BSO incubation in
cell lines sensitive to BSO, which is in line with earlier studies and most likely due to inhibition of GSH levels [34–36]. Anderson et al. claimed that formation of ROS is the underlying apoptosis-inducing mechanism of BSO [17,19,20,34]. This ROS induction resulted in reduction of the levels of the antiapoptotic protein Bcl-2 and activation of the levels of protein kinase C, p38, JNK MAPK, and caspase-3 [21,37–39]. No correlation was observed between MYCN amplification and sensitivity to BSO, baseline GSH levels, or antioxidant enzyme levels. This is in contrast with the findings of others, who suggested that MYCN- amplified cell lines might have lower GSH levels and be more sensitive to BSO than MYCN-single-copy cells [18,34,35,40].
A moderate upregulation of catalase was observed after incubation with the 4HPR–BSO combination (in SJNB10 cells). However, this effect did not attenuate the benefi cial effect of the 4HPR–BSO combination. The other antioxidant enzyme activities (SOD and GPx) were not affected by 4HPR–BSO treatment. Cornelissen et al. observed an increase in both catalase and GPx in neuroblastoma cells suffering from ROS as a result of MIBG incubation and Lee suggested an upregulation of catalase and SOD and a downregulation of GPx after induction of ROS in neuroblastoma cells [41,31,42]. This indicates that regulation of antioxidant levels as a result of ROS induction might depend on the cell lines that are involved.
The depletion of GSH by BSO resulted in a profound cytotoxic effect of the 4HPR–BSO combination in ROS-producing cell lines, although the additional ROS induction in these cell lines after incubation with BSO and 4HPR was only moderate. The GSH depletion by BSO also enhanced the cytotoxicity of 4HPR in both leukemia cells and Ewing sarcoma cells [29,30]. This benefi cial effect was confi rmed in spheroids; the 4HPR–BSO combination showed profound growth retardation, as a result of decreased proliferation and increased apoptosis, compared to either BSO-treated spheroids or 4HPR-treated spheroids. As spheroids resemble micrometastases, these results indicate that the combination might be successful in clinical use.
Over all, we conclude that the combination of 4HPR and BSO has a beneficial effect on cytotoxicity in neuroblastoma monolayers as well as in spheroids in cell lines with ROS-producing properties. The sensitivity to BSO in ROS-producing cell lines is within the range of clinically achievable plasma levels for BSO [23]. Because both 4HPR and BSO have been tested in clinical trials and no dose-limiting toxicity has been observed [21,22], the 4HPR–BSO combination could be a potential combination therapy for neuroblastoma.
Acknowledgment
This study was supported by the Stichting Kindergeneeskundig Kankeronderzoek.
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