Molecular Cancer Therapeutics CTRC-AACR San Antonio Breast Cancer Symposium Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Karlsson, J.
Right arrow Articles by Pettersson, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Karlsson, J.
Right arrow Articles by Pettersson, H. M.
Mol Cancer Ther. 2005;4:1128-1135
© 2005 American Association for Cancer Research

Multidrug-resistant neuroblastoma cells are responsive to arsenic trioxide at both normoxia and hypoxia

Jenny Karlsson, Anders Edsjö, Sven Påhlman and Helen M. Pettersson

Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, Malmö, Sweden

Requests for reprints: Helen Pettersson, Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, Entrance 78, 3rd floor, S-205 02 Malmö, Sweden. Phone: 46-40-332285; Fax: 46-40-337322. E-mail: helen.pettersson{at}med.lu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite intensive treatment, the outcome of high-risk neuroblastoma patients is poor with acquired multidrug resistance as an important cause. Previously, our group has shown that arsenic trioxide (As2O3) kills multidrug-resistant neuroblastoma cells in vitro and in vivo at clinically tolerable doses. Regions of tissue hypoxia often arise in aggressive solid tumors, and hypoxic tumors exhibit augmented invasiveness and metastatic ability in several malignancies. Furthermore, hypoxia may impair the treatment efficiency; therefore, we have studied the cytotoxic effect of As2O3 on neuroblastoma cells grown under normoxic as well as hypoxic (1% oxygen) conditions. At both normoxia and hypoxia, 2 and 4 µmol/L As2O3 induced evident cell death in the drug-sensitive SH-SY5Y and IMR-32 cells as well as in the multidrug-resistant SK-N-BE(2)c (with a mutated p53) and SK-N-FI cells after 72 hours of exposure. In contrast, the conventional chemotherapeutic drug etoposide showed lowered efficiency in hypoxic IMR-32 cells. In accordance with our previously published results, although not to the same extent as in their normoxic counterparts, Bax is proteolytically cleaved also in neuroblastoma cells exposed to As2O3 at hypoxia. This suggests that similar molecular mechanisms are involved in As2O3-induced neuroblastoma cell death during hypoxia compared with normoxia. Together, our results support As2O3 as a potential candidate drug as a complement to conventional treatments for high-risk neuroblastoma patients and perhaps also for patients with other multidrug-resistant solid tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neuroblastoma is a childhood malignancy derived from the sympathetic nervous system and accounts for 8% to 10% of all cancers in children. Intensive chemoradiotherapy supported with bone marrow transplantation and followed by 13-cis-retinoic acid treatment has improved the survival for high-risk neuroblastoma patients (1). Still, the majority of neuroblastoma patients eventually develop progressive disease, which is refractory to further therapy. A major contributing factor to chemotherapeutic treatment failure of neuroblastoma is the development of resistance to multiple and functionally unrelated cytotoxic drugs, and as a consequence, the long-term survival rate of this group of neuroblastoma patients is only about 30% (1–3).

As2O3 has been used for therapeutic purposes for centuries, and recently, it has been successfully employed in the treatment of acute promyelocytic leukemia (APL) where several reports have shown that low doses of As2O3 can with minimal toxicity induce complete remission in patients with relapsed APL (4–6). Moreover, the potency of As2O3 to induce cell death in vitro has been shown in a variety of cancer cells including non-APL leukemia and lymphoma cells (7, 8) as well as solid tumors like neuroblastoma (9–11) and tumors originating from prostate, kidney, cervix, and bladder (12). The underlying mechanisms of As2O3-induced cytotoxicity are complex and include induction of apoptosis, cellular differentiation, degradation of the t(15;17)-specific fusion protein PML/retinoic acid receptor-{alpha} seen in APL, growth inhibition, induction of autophagy, and inhibition of angiogenesis (13–16). In different cell lines, As2O3 has been shown to induce apoptosis by down-regulation of Bcl-2 (13), activation of caspases (17), modulation of p53 (17), generation of reactive oxygen species (ROS; ref. 18), and uncoupling of the mitochondrial potential (19).

We and others have shown that clinically relevant concentrations of As2O3 promote cell death in vitro and in vivo not only in drug-sensitive neuroblastoma cell lines but also in the multidrug-resistant p53-mutated/deleted neuroblastoma cell lines, SK-N-BE(2) and LA-N-1 (9–11). These promising results support the possible use of As2O3 in combination therapy in high-risk neuroblastoma patients, and a phase II clinical trial with As2O3 is presently done at the Memorial Sloan-Kettering Cancer Center. We have shown that the As2O3-induced neuroblastoma cell death seems not to involve induced neuronal differentiation but includes increased expression and proteolytic activation of Bax, down-regulation of Bcl-2, and to a slight extent, activation of caspase-3 (9, 10). The activation of Bax seems to be a key event because inhibition of the Bax cleavage prevented As2O3-induced cell death (10). The activation of Bax was also shown to be independent of functional p53 (10), a feature of great interest because deletions or mutations in p53 often are seen in high-risk neuroblastoma patients with relapsed, refractory disease (20, 21). In contrast, drugs commonly used in induction treatment of children with advanced neuroblastoma were only able to induce cell death and hence cleavage of Bax in drug-sensitive neuroblastoma cells (10), indicating that a different cytotoxic pathway is induced upon As2O3 treatment.

Hypoxic microenvironments are frequently found in solid tumors as a result of inefficient vascular supply and high oxygen consumption of rapidly proliferating malignant cells. Tumor hypoxia is associated with malignant progression, lower sensitivity to chemotherapy and radiotherapy, increased metastatic potential, and poor prognosis (22, 23). Many of the features in hypoxic tumors are mediated by the hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and HIF-2{alpha}, transcription factors which modulate the expression of several genes involved in both short-term adaptive mechanisms (e.g., glucose transport and switch to anaerobic metabolism) and long-term adaptive mechanisms (e.g., angiogenesis; ref. 24). In addition to these features, we recently showed that hypoxia alters the gene expression in neuroblastoma cells toward an immature phenotype (25, 26). Because there is a correlation in neuroblastoma between low stage of differentiation and poor clinical outcome, hypoxic adaptation could contribute to increased malignancy of the tumor. Hypoxia-induced dedifferentiation is not restricted to neuroblastoma cells but is also seen in other types of tumors like breast cancer (27) and prostate cancer (28). It has been shown that an As2O3-resistant APL cell line became sensitive and underwent apoptosis when the treatment included both As2O3 and all-trans retinoic acid, a combination which greatly accelerated differentiation in these cells (29). It is therefore of great interest to evaluate if hypoxia-induced adaptive mechanisms like dedifferentiation could impair the efficiency of As2O3 treatment of neuroblastoma cells.

In the present study, we investigated the cytotoxic effect of As2O3 on drug-sensitive and multidrug-resistant neuroblastoma cells, respectively. We observed an evident cytotoxic effect of As2O3 in all tested cell lines also when the cells were cultured under hypoxic growth conditions. Although As2O3 to some extent induced growth inhibition, the main effect of As2O3 was induction of cell death. These results support the idea of using As2O3 as an efficient treatment strategy also in solid tumors with hypoxic areas like neuroblastoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Drugs
We used four neuroblastoma cell lines in this study; the SH-SY5Y and SK-N-FI cell lines are gifts from Dr. June Biedler (Memorial Sloan-Kettering Cancer Center); SK-N-BE(2)c and IMR-32 were obtained from American Type Culture Collection (Manassas, VA). SK-N-BE(2)c and IMR-32 cells have a MYCN amplification. The multidrug-resistant SK-N-BE(2)c cell line has a p53 mutation (20, 21). SK-N-BE(2)c and SH-SY5Y were grown in MEM and IMR-32 and SK-N-FI were grown in RPMI 1640. The media were supplemented with 10% FCS, penicillin (100 units/mL), and streptomycin (100 µg/mL; Invitrogen Life Technologies, Paisley, United Kingdom). The cells were cultured at 37°C in a 95% air/5% CO2 humidified incubator. Hypoxic conditions, 1% O2, were created in a Hypoxia workstation 400 (Ruskinn Technology, Leeds, United Kingdom) connected to a Ruskinn gas mixer module.

As2O3 was dissolved in 1.0 mol/L NaOH; vincristine, carboplatin, and doxorubicin were dissolved in distilled H2O and etoposide in DMSO. All cytotoxic drugs were purchased from Sigma (St. Louis, MO).

Cell Viability and Proliferation Assays
The cells were seeded at day 0 under normoxic conditions and the cytotoxic drugs were added at day 1. Thereafter, the cells were grown under normoxia or hypoxia for 72 hours. When indicated, the cells were grown under normoxia or hypoxia for 72 hours before the cytotoxic drugs were added. In these experiments, the cells were reseeded under hypoxic conditions 1 day before initiation of the drug treatments. To assess cell viability, the cells were collected and stained with trypan blue (Sigma). Approximately 200 cells per culture were counted using a light microscope. All treatments were done in triplicates and the experiments were repeated three or four times. The amount of viable cells was compared with untreated normoxic or hypoxic controls, respectively, which were both set to 100%.

The effect of cytotoxic drugs under normoxic conditions was determined in 96-well plates by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (CellTiter 96, Promega, Madison, WI), according to the manufacturer's recommendation. All experiments were done in triplicates and repeated thrice.

To evaluate the proliferative capacity of neuroblastoma cells after As2O3 treatment, SK-N-BE(2)c cells were seeded at day 0 and varying concentrations of As2O3 were added at day 1. After 72 hours of treatment, the cells were replated in 96-well plates and grown for 24 to 72 hours where 0.5 µCi [3H]thymidine (Amersham Bioscience, Bucks, United Kingdom) was added at the last 24 hours of culture. The cells were harvested and proliferation was measured by incorporation of [3H]thymidine in a beta scintillation counter.

Immunoblot Analysis
Cells were lysed in 10 mmol/L Tris-HCl (pH 7.2), 160 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L EGTA, and 1 mmol/L EDTA in the presence of Complete Protease Inhibitor (Roche Molecular Biochemicals, Mannheim, Germany). For the HIF-1{alpha} Western blot, the cells were harvested under hypoxic conditions. Equal amount of protein was separated on an SDS-PAGE and blotted onto a Hybond C membrane (Amersham Bioscience) for the HIF-1{alpha} Western blot or an Immobilon-P membrane (Millipore Corp., Bedford, MA) for the Bax-Western blot. The following primary antibodies and antiserum were used: anti-actin-antibody (ICN Biomedicals, Aurora, OH) diluted 1:2,000, Bax antiserum (BD PharMingen, San Diego, CA) diluted 1:2,000, anti-caspase-3 antibody (Alexis Biochemicals, San Diego, CA) diluted 1:500, anti-glyceraldehyde-3-phosphate dehydrogenase antibody (Chemicon International, Inc., Temecula, CA) diluted 1:2,000, anti-HIF-1{alpha} antibody (Novus Biologicals, Littleton, CO) diluted 1:1,000, and anti-PKC{alpha} (c-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200. The proteins were visualized using horseradish peroxidase–conjugated anti-mouse antibody from Amersham Pharmacia Biotech (Arlington Heights, IL) and Jackson Immunoresearch Laboratories, Inc. (West Grove, PA) and anti-rabbit antibody from DAKO (Glostrup, Sweden) and a Super Signal detection system (Pierce Chemical Co., Rockford, IL).

Immunocytochemistry
Cell pellets were fixed with 4% buffered paraformaldehyde, paraffin embedded, and cut into 4-µm-thin sections. The sections were microwave treated with a target retrieval solution (DAKO) at pH 9.9 and the cells were stained with an anti-Ki-67 antibody (DAKO) diluted 1:500. Cell staining was done in a DAKO ChemMate 500 using the Envision protocol according to the manufacturer's instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Arsenic Trioxide Induces Cell Death in Hypoxic Multidrug-Resistant Neuroblastoma Cells
We examined three neuroblastoma cell lines, grown under normoxic and hypoxic (1% oxygen) conditions, with regard to their responsiveness to As2O3. In normoxic cultures, the results confirmed our recently published data showing that the p53-mutated and multidrug-resistant neuroblastoma cell line SK-N-BE(2)c is as sensitive to As2O3 as the drug-sensitive SH-SY5Y and IMR-32 cell lines; all cell lines displaying IC50 values in the low micromolar range after 72 hours of treatment (10; Fig. 1A). In addition, As2O3 is toxic to the neuroblastoma cells under hypoxic conditions (Fig. 1A). When treated with 4 µmol/L As2O3 at normoxia and hypoxia, the cultures displayed 45% to 65% of dead cells. At both 1% and 21% oxygen, exposure of the cells to As2O3 gave rise to a concentration-dependent increase of cells with rounded morphology as well as detached floating cells (Fig. 1B). To assure that As2O3 treatment did not prevent induction of a hypoxic response, we investigated the accumulation of the hypoxia-induced transcription factor HIF-1{alpha} in normoxic and hypoxic cells treated with 4 µmol/L As2O3 for 4 hours (Fig. 1C). As expected, at normoxia, no HIF-1{alpha} protein could be detected in either untreated or As2O3-treated cells (Fig. 1C, lanes 1 and 2), whereas a marked accumulation was observed in untreated hypoxic cells (Fig. 1C, lane 3). Upon treatment with As2O3 at hypoxia, a robust HIF-1{alpha} protein stabilization was detected suggesting that an adaptation to the hypoxic environment was induced in these cells (Fig. 1C, lane 4).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 1. As2O3 is toxic to neuroblastoma cells grown under hypoxic conditions. A, the neuroblastoma cell lines SK-N-BE(2)c, SH-SY5Y, and IMR-32 were treated with indicated concentrations of As2O3 and grown under normoxic or hypoxic conditions for 72 h. The cells were harvested and stained with trypan blue and the number of living and dead cells were counted. Points, mean percentage of dead cells or percentage of viable cells compared with untreated controls (n = 9); bars, ±SD. B, phase-contrast photographs showing SK-N-BE(2)c and SH-SY5Y cell morphology after As2O3 treatments for 72 h. C, SK-N-BE(2)c cells were treated with 4 µmol/L As2O3 for 4 h under normoxic or hypoxic conditions. The cells were lysed and 100 µg of protein were subjected to SDS-PAGE and Western blotting. The filter was incubated with an anti-HIF-1{alpha} antibody and subsequently with an anti-PKC{alpha} antibody to show that proteins were present in all lanes. In vitro–translated (iv tr) GST-tagged HIF-1{alpha} was used as a positive control.

 
To further show the efficiency of As2O3 on hypoxic neuroblastoma cells, we wanted to include an additional multidrug-resistant neuroblastoma cell line. We have previously compared several neuroblastoma cell lines with regard to their sensitivity to vincristine, etoposide, doxorubicin, and carboplatin (9, 10), all of which are chemotherapeutic drugs commonly used in the induction treatment of high-risk neuroblastoma. In comparison with all previously tested cells, we have now identified the MYCN-amplified SK-N-FI cells to be one of the most drug-insensitive cell line (Fig. 2A), displaying resistance to all of the abovementioned drugs at clinically relevant concentrations (30–33). However, despite their multidrug-resistant phenotype and in accordance with the results obtained with the SK-N-BE(2)c cells, the amount of viable SK-N-FI cells was markedly reduced after treatment with As2O3 for 72 hours, displaying an IC50 value in the low micromolar range (Fig. 2A). As2O3 induced a pronounced cell death in SK-N-FI cells also under hypoxic growth conditions (Fig. 2B). At hypoxia after 72 hours of treatment with 4 µmol/L As2O3, the proportion of dead SK-N-FI cells was about 55% (Fig. 2B), a level comparable with those seen in As2O3-treated SH-SY5Y, IMR-32, and SK-N-BE(2)c cells (Fig. 1A).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. The drug-resistant SK-N-FI neuroblastoma cells are sensitive to As2O3 under normoxic and hypoxic conditions. A, SK-N-FI cells were treated with different cytotoxic drugs for 72 h at normoxia. The amount of viable cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Points, mean percentage of viable cells compared with untreated controls (n = 9); bars, ±SD. B, SK-N-FI cells were grown in the presence of indicated concentrations of As2O3 under normoxic or hypoxic conditions. The cells were collected after 72 h and stained with trypan blue. Columns, mean percentage of dead cells or percentage of viable cells compared with untreated controls (n = 9); bars, ±SD.

 
Arsenic Trioxide Responsiveness in Hypoxic Neuroblastoma Cells Is Due to Induction of Cell Death and to Some Extent Inhibition of Proliferation
It has been shown in different types of cancer cell lines that treatment with As2O3 induces cell death as well as growth inhibition (34). To investigate the possible involvement of growth inhibition upon As2O3 treatment in neuroblastoma cells, normoxic as well as hypoxic paraffin-embedded SK-N-BE(2)c, SH-SY5Y, and SK-N-FI cells were immunostained for the proliferation marker Ki-67. All three untreated cell lines grown under normoxic conditions showed about ≥50% Ki-67-positive cells (Fig. 3A). At normoxia after 72 hours of treatment with 2 µmol/L As2O3, the proportions of proliferating cells were decreased in all cell lines, indicating that the reduction of viable neuroblastoma cells to some extent is due to decreased proliferation. In the SK-N-BE(2)c cells, showing the highest percentage of Ki-67-positive cells in the normoxic untreated control, the reduction of Ki-67 positivity was much more marked upon As2O3 treatment at normoxia (Fig. 3A). However, in all cell lines, the combination of As2O3 treatment and hypoxic growth conditions did not increase the growth inhibition seen with either of the treatments alone (Fig. 3A). To address the possibility that As2O3 induces a selection of cells with low proliferative capacity, cells treated with As2O3 for 72 hours were replated and grown in the absence of As2O3. As shown in Fig. 3B, there was a reduction in number of surviving replated SK-N-BE(2)c cells from cultures previously treated with As2O3; however, the proliferation rates of As2O3-treated cells were comparable with that of untreated cells. These results together with the results presented in Figs. 1 and 2 suggest that the As2O3 responsiveness in normoxic and hypoxic neuroblastoma cells is due to induction of cell death and only to some extent inhibition of proliferation and in addition, no selection of slow proliferating cells seems to occur during treatment.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 3. The proliferating capacity of neuroblastoma cells is to some extent influenced by As2O3 and/or hypoxia. A, the cells were harvested after 72 h of As2O3 treatment at normoxia or hypoxia, fixed in paraformaldehyde, and stained immunocytochemically for the proliferation marker Ki-67. In each section, 200 cells were counted and the average percentage of Ki-67-positive cells from two separate experiments is shown. Bars, the two different measure points. B, SK-N-BE(2)c cells were treated with indicated concentrations of As2O3 for 72 h under normoxic conditions. Thereafter, the cells were replated and grown in absence of As2O3 for 24, 48, or 72 h at normoxia. [3H]thymidine was added to the growth medium 24 h before the cells were harvested, the incorporation was measured in a beta scintillation counter. The experiment was repeated three times in triplicates. Columns, means (n = 9); bars, ±SD.

 
Proteolytic Cleavage of Bax and a Slight Activation of Caspase-3 in Hypoxic Neuroblastoma Cells upon Treatment with As2O3
Previously, we have shown that at normoxia As2O3 provoked Bax expression in neuroblastoma cells and that p21 Bax was proteolytically cleaved into a p18 Bax form (10), which is more proapoptotic (35, 36). In addition, As2O3 treatment only induced a slight activation of caspase-3 and inhibition of caspase-3 did not prevent cell death, whereas inhibition of Bax cleavage was associated with a decreased As2O3-induced cytotoxicity (10). This suggests that activation of Bax is a key event in As2O3-induced cell death in normoxic neuroblastoma cells and that As2O3 activates a cytotoxic pathway different from that induced by the conventionally used drugs. To clarify whether As2O3 induces a similar cell death pathway in hypoxic neuroblastoma cells, we did Western blot analyses on cell lysates from normoxic and hypoxic cells treated with 2 or 4 µmol/L As2O3 for 72 hours. In accordance with the normoxic samples, the proteolytic activation of Bax was induced in hypoxic SK-N-BE(2)c cells with increasing concentrations of As2O3 (Fig. 4). However, the proportion of cleaved Bax was not as large in the As2O3-treated hypoxic cells as in the normoxic cells (Fig. 4). Caspase-3 was activated to a slight extent in a dose-dependent manner upon As2O3 treatment and the levels of activation were comparable in the normoxic and hypoxic SK-N-BE(2)c cells (Fig. 4).



View larger version (61K):
[in this window]
[in a new window]
  		Figure 4. Bax and caspase-3 is proteolytically activated during As2O3 treatment at normoxia and hypoxia. SK-N-BE(2)c cells were treated with 2 and 4 µmol/L As2O3 for 72 h under normoxic or hypoxic conditions. The cells were lysed and 35 µg protein were subjected to SDS-PAGE and Western blotting. The filter was incubated with an antiserum recognizing the p18 and p21 Bax forms and an anti-caspase-3 antibody recognizing full-length caspase-3 (data not shown) and the active 17- and 13-kDa cleavage products. An anti–glyceraldehyde-3-phosphate dehydrogenase antibody and an anti-actin antibody were used as loading controls. Representative blots of two experiments.

 
Neuroblastoma Cells Preexposed to Hypoxia Are Sensitive to As2O3
In the experiments described in Figs. 1 and 2, As2O3 treatment was initiated at the same time as the hypoxic growth condition was induced. However, in tumor environments, there are most likely areas exposed to hypoxia for varying periods of time. Experimental systems where the cells already have been adapted to a hypoxic environment mimics tumor areas exposed to hypoxia for longer periods and allow hypoxia-mediated dedifferentiation (25, 26) and stabilization of proapoptotic as well as antiapoptotic proteins (37) to occur before drug exposure. To further evaluate the potency of As2O3 in hypoxic cells, the multidrug resistant SK-N-BE(2)c cells and the drug-sensitive IMR-32 cells were thus preexposed to normoxic or hypoxic conditions for 72 hours before induction of As2O3 treatment under normoxia or hypoxia for 72 hours. In accordance with the results presented in Fig. 1, both the SK-N-BE(2)c cells and the IMR-32 cells showed a sustained sensitivity to As2O3 under hypoxic growth conditions, although the cells already had adapted to hypoxia when the As2O3 treatment was initiated (Fig. 5). In both cell lines, the percentage of dead cells reached about 45% after treatment with 4 µmol/L As2O3 at normoxia as well as hypoxia and the cell death was consistent with the cell viability data.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. As2O3 is toxic to cells that have been adapted to hypoxic conditions before drug treatment. SK-N-BE(2)c and IMR-32 cells were grown under normoxia or hypoxia for 72 h before addition of indicated concentrations of As2O3. After another 72 h of incubation under normoxic or hypoxic conditions in the presence of As2O3, the cells were collected and stained with trypan blue and the amount of cell death was evaluated. Points, mean percentage of dead cells or percentage of viable cells compared with untreated controls, for SK-N-BE(2)c cells (n = 9) and for IMR-32 cells (n = 12); bars, ±SD.

 
Influence of Hypoxia on Cell Death Induced by Etoposide in Drug-Sensitive Neuroblastoma Cells
Several reports indicate that some cytotoxic drugs are less efficient in a hypoxic environment (38, 39), but the results described above suggest that As2O3 does not belong to that group of substances, at least not for treatment of neuroblastoma cells. To compare the efficiency of As2O3 with other drugs, we examined IMR-32 cells, grown under both normoxic and hypoxic (1% O2) conditions, with regard to its responsiveness to the conventional chemotherapeutic drug etoposide, a drug previously shown to have decreased efficiency under hypoxic conditions (22, 39). The effects of etoposide were tested both without (Fig. 6A) and with (Fig. 6B) preexposure to hypoxia. In agreement with our previous results (10), 72 hours of treatment with 0.5 µmol/L etoposide induced an evident cell death in IMR-32 cells grown under normoxia (Fig. 6). However, a significant difference in cell death as well as amount of viable cells was shown in the hypoxic IMR-32 cells compared with their normoxic counterparts upon etoposide treatment (Fig. 6). At 0.2 µmol/L etoposide, the cytotoxic effect was markedly reduced at hypoxia, which was seen both when the etoposide treatment was initiated at the same time as the hypoxic growth condition was induced (Fig. 6A) and even more pronounced when the cells were preexposed to hypoxia 3 days before the addition of etoposide (Fig. 6B). These results show that in contrast to As2O3, etoposide exhibits a clearly reduced efficacy under hypoxic conditions in IMR-32 cells.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 6. The drug-sensitive IMR-32 cells are less sensitive to etoposide under hypoxic conditions, especially in cells adapted to hypoxia before treatment initiation. A, IMR-32 cells were treated with indicated concentrations of etoposide and grown under normoxia or hypoxia for 72 h. B, IMR-32 cells were first preincubated at normoxia or hypoxia for 72 h before initiation of etoposide treatment for another 72 h at normoxic or hypoxic growth conditions. The cells were collected and stained with trypan blue, and the amount of cell death was evaluated. Points, mean percentage of dead cells or percentage of viable cells compared with untreated controls, n = 9 in (A) and n = 12 in (B); bars, ±SD. *, P < 0.05, statistically significant differences between the normoxic and hypoxic counterparts according to ANOVA followed by Duncan's multiple range test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show a potent cytotoxicity of As2O3 in two drug-sensitive as well as two multidrug-resistant neuroblastoma cell lines both under normoxic and hypoxic growth conditions. We also show that the As2O3 responsiveness seen in hypoxic neuroblastoma cells is only to some extent due to hypoxia- or As2O3-induced growth inhibition but mainly depends on induction of cell death. These results together show that the cytotoxic efficiency of As2O3 is sustained under low oxygen pressures, which is important from a clinical point of view; that is, As2O3 is a potential candidate drug in treatment of solid tumors with hypoxic areas like neuroblastoma. The fact that As2O3 seems to activate a cytotoxic pathway different from that induced by the conventionally used drugs during induction treatment of neuroblastoma and as a result is able to kill multidrug-resistant neuroblastoma cells (10) lends support to the ongoing efforts to introduce the use of As2O3 in combination with existing treatment protocols for patients with multidrug-resistant neuroblastomas.

The precise molecular mechanisms by which As2O3 induces neuroblastoma cell death are yet to be determined. Our previously published results suggest that proteolytically activation of the proapoptotic protein Bax is an important mechanism in normoxic neuroblastoma cells (10). Here we show that cleavage of Bax also occurred in As2O3-treated hypoxic cells but was not as pronounced as in the normoxic cells. This may indicate that induction of p18 Bax is less important for As2O3-induced cell death under hypoxic conditions and that complementary cell death mechanisms may be activated. Further analyses are required to be able to establish to what extent the mechanisms involved in As2O3-induced cell death differ in neuroblastoma cells grown under normoxic or hypoxic conditions. In addition to oxygen pressure, other differences like genotypically changes may influence the mechanisms of As2O3-induced cytotoxicity. For instance, published data indicate that As2O3 induces cell death of myeloma cells by two different mechanisms depending on the p53 status of the cell (40, 41). It is not known whether this is true also for neuroblastoma cells. However, we do show here and in our previous study (10) that As2O3 is a potent cytotoxic drug in wild-type p53 expressing as well as in p53-mutated/deleted multidrug-resistant neuroblastoma cells and that Bax is proteolytically activated by As2O3 treatment in all these different cells.

Pronounced As2O3-induced cell death is still seen when the neuroblastoma cells were preexposed to hypoxia for 3 days before induction of treatment, which shows that the potency of As2O3 is still intact in cells with a well-developed hypoxic phenotype (25, 26). It is possible that the hypoxic environment contributes to cellular conditions supporting the cytotoxic effect of As2O3. For instance, oxidative stress with generation of ROS is believed to be one of the major pathways for arsenic-mediated cytotoxicity (18, 42, 43), although the mechanism(s) and pathway(s) whereby these ROS are generated remain largely unknown. There is also growing evidence that mitochondrial ROS are produced during hypoxia (44). Thus, the combination of hypoxia and As2O3-derived ROS may contribute to the induced cell death seen in hypoxic neuroblastoma cells. Based on the literature, there is a formal possibility that hypoxic growth conditions may lead to cellular adaptations, which impair the efficiency of cytotoxic drugs (22, 38, 39). For instance, growth at hypoxia dedifferentiates neuroblastoma cells (25, 26) and a low differentiation stage is correlated to more aggressive neuroblastoma tumors and a poor response to treatment. In contrast, it has been shown that a resistant APL cell line underwent apoptosis upon combined treatment of As2O3 and all-trans retinoic acid, which greatly accelerated differentiation in the cells (29). However, our group has previously shown that As2O3, alone or in combination with retinoic acid, does not induce neuronal differentiation in neuroblastoma cells (9), and although we have not investigated the differentiation stage of the hypoxic As2O3-treated cells, we show that the cytotoxic potency of As2O3 is sustained at hypoxia, despite that hypoxia promotes a stem cell–like phenotype in neuroblastoma cells (25, 26).

When the efficiency of As2O3 and the conventionally used drug etoposide was compared at hypoxia, it became clear that their different mechanisms of action influenced their ability to kill hypoxic neuroblastoma cells. In contrast to As2O3, etoposide showed a reduced cytotoxicity in hypoxic neuroblastoma cells, results that are in keeping with those reported by Unruh et al., which show that cells with no or low levels of HIF-1{alpha} are more sensitive to etoposide treatment than cells with higher HIF-1{alpha} proteins levels (22). These observations and the successful use of As2O3 in treating refractory APL with minimal toxicity encourage attempts to broaden the application of As2O3 to treatment of other cancer forms as well. The results presented in this study indicate that As2O3 also could be a drug with high potential for efficient treatment of neuroblastoma patients with aggressive tumors, which invariably display areas of severe hypoxia.


   Acknowledgments
 
We thank Siv Beckman and Elise Nilsson for skillful technical assistance.


   Footnotes
 
Grant support: Children Cancer Foundation of Sweden, the Swedish Cancer Society, HKH Kronprinsessan Lovisas Förening för Barnasjukvård, Hans von Kantzows Stiftelse, the Crafoord Foundation, and the Malmö University Hospital Research Funds.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/14/05; revised 4/ 5/05; accepted 4/29/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Matthay KK, Villablanca JG, Seeger RC, et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group. N Engl J Med 1999;341:1165–73.[Abstract/Free Full Text]
  2. Philip T, Ladenstein R, Lasset C, et al. 1070 myeloablative megatherapy procedures followed by stem cell rescue for neuroblastoma: 17 years of European experience and conclusions. European Group for Blood and Marrow Transplant Registry Solid Tumour Working Party. Eur J Cancer 1997;33:2130–5.
  3. Keshelava N, Seeger RC, Groshen S, Reynolds CP. Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy. Cancer Res 1998;58:5396–405.[Abstract/Free Full Text]
  4. Shen ZX, Chen GQ, Ni JH, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997;89:3354–60.[Abstract/Free Full Text]
  5. Niu C, Yan H, Yu T, et al. Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood 1999;94:3315–24.[Abstract/Free Full Text]
  6. Soignet SL, Frankel SR, Douer D, et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol 2001;19:3852–60.[Abstract/Free Full Text]
  7. Puccetti E, Guller S, Orleth A, et al. BCR-ABL mediates arsenic trioxide-induced apoptosis independently of its aberrant kinase activity. Cancer Res 2000;60:3409–13.[Abstract/Free Full Text]
  8. Zhu XH, Shen YL, Jing YK, et al. Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations. J Natl Cancer Inst 1999;91:772–8.[Abstract/Free Full Text]
  9. Øra I, Bondesson L, Jönsson C, et al. Arsenic trioxide inhibits neuroblastoma growth in vivo and promotes apoptotic cell death in vitro. Biochem Biophys Res Commun 2000;277:179–85.[CrossRef][Medline]
  10. Karlsson J, Øra I, Pörn-Ares I, Påhlman S. Arsenic trioxide-induced death of neuroblastoma cells involves activation of Bax and does not require p53. Clin Cancer Res 2004;10:3179–88.[Abstract/Free Full Text]
  11. Akao Y, Nakagawa Y, Akiyama K. Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro. FEBS Lett 1999;455:59–62.[CrossRef][Medline]
  12. Murgo AJ. Clinical trials of arsenic trioxide in hematologic and solid tumors: overview of the National Cancer Institute Cooperative Research and Development Studies. Oncologist 2001;6 Suppl 2:22–8.[Abstract/Free Full Text]
  13. Chen GQ, Zhu J, Shi XG, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR {alpha}/PML proteins. Blood 1996;88:1052–61.[Abstract/Free Full Text]
  14. Shao W, Fanelli M, Ferrara FF, et al. Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR {alpha} protein in acute promyelocytic leukemia cells. J Natl Cancer Inst 1998;90:124–33.[Abstract/Free Full Text]
  15. Roboz GJ, Dias S, Lam G, et al. Arsenic trioxide induces dose- and time-dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis. Blood 2000;96:1525–30.[Abstract/Free Full Text]
  16. Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 2003;63:2103–8.[Abstract/Free Full Text]
  17. Jiang XH, Wong BC, Yuen ST, et al. Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3. Int J Cancer 2001;91:173–9.[CrossRef][Medline]
  18. Jing Y, Dai J, Chalmers-Redman RM, Tatton WG, Waxman S. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 1999;94:2102–11.[Abstract/Free Full Text]
  19. Larochette N, Decaudin D, Jacotot E, et al. Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp Cell Res 1999;249:413–21.[CrossRef][Medline]
  20. Keshelava N, Zuo JJ, Chen P, et al. Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines. Cancer Res 2001;61:6185–93.[Abstract/Free Full Text]
  21. Tweddle DA, Malcolm AJ, Bown N, Pearson AD, Lunec J. Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line. Cancer Res 2001;61:8–13.[Abstract/Free Full Text]
  22. Unruh A, Ressel A, Mohamed HG, et al. The hypoxia-inducible factor-1 {alpha} is a negative factor for tumor therapy. Oncogene 2003;22:3213–20.[CrossRef][Medline]
  23. Le QT, Denko NC, Giaccia AJ. Hypoxic gene expression and metastasis. Cancer Metastasis Rev 2004;23:293–310.[CrossRef][Medline]
  24. Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol 2002;64:993–8.[CrossRef][Medline]
  25. Jögi A, Øra I, Nilsson H, et al. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci U S A 2002;99:7021–6.[Abstract/Free Full Text]
  26. Jögi A, Vallon-Christersson J, Holmquist L, et al. Human neuroblastoma cells exposed to hypoxia: induction of genes associated with growth, survival, and aggressive behavior. Exp Cell Res 2004;295:469–87.[CrossRef][Medline]
  27. Helczynska K, Kronblad A, Jögi A, et al. Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ. Cancer Res 2003;63:1441–4.[Abstract/Free Full Text]
  28. Ghafar MA, Anastasiadis AG, Chen MW, et al. Acute hypoxia increases the aggressive characteristics and survival properties of prostate cancer cells. Prostate 2003;54:58–67.[CrossRef][Medline]
  29. Gianni M, Koken MH, Chelbi-Alix MK, et al. Combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells. Blood 1998;91:4300–10.[Abstract/Free Full Text]
  30. de Graaf SS, Bloemhof H, Vendrig DE, Uges DR. Vincristine disposition in children with acute lymphoblastic leukemia. Med Pediatr Oncol 1995;24:235–40.[Medline]
  31. Relling MV, Mahmoud HH, Pui CH, et al. Etoposide achieves potentially cytotoxic concentrations in CSF of children with acute lymphoblastic leukemia. J Clin Oncol 1996;14:399–404.[Abstract/Free Full Text]
  32. Frost BM, Eksborg S, Bjork O, et al. Pharmacokinetics of doxorubicin in children with acute lymphoblastic leukemia: multi-institutional collaborative study. Med Pediatr Oncol 2002;38:329–37.[CrossRef][Medline]
  33. Riccardi R, Riccardi A, Lasorella A, et al. Clinical pharmacokinetics of carboplatin in children. Cancer Chemother Pharmacol 1994;33:477–83.[Medline]
  34. Rojewski MT, Baldus C, Knauf W, Thiel E, Schrezenmeier H. Dual effects of arsenic trioxide (As2O3) on non-acute promyelocytic leukaemia myeloid cell lines: induction of apoptosis and inhibition of proliferation. Br J Haematol 2002;116:555–63.[CrossRef][Medline]
  35. Wood DE, Newcomb EW. Cleavage of Bax enhances its cell death function. Exp Cell Res 2000;256:375–82.[CrossRef][Medline]
  36. Cao X, Deng X, May WS. Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax. Blood 2003;102:2605–14.[Abstract/Free Full Text]
  37. Erler JT, Cawthorne CJ, Williams KJ, et al. Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. Mol Cell Biol 2004;24:2875–89.[Abstract/Free Full Text]
  38. Teicher BA. Hypoxia and drug resistance. Cancer Metastasis Rev 1994;13:139–68.[CrossRef][Medline]
  39. Shannon AM, Bouchier-Hayes DJ, Condron CM, Toomey D. Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies. Cancer Treat Rev 2003;29:297–307.[CrossRef][Medline]
  40. Akay C, Gazitt Y. Arsenic trioxide selectively induces early and extensive apoptosis via the APO2/caspase-8 pathway engaging the mitochondrial pathway in myeloma cells with mutant p53. Cell Cycle 2003;2:358–68.[Medline]
  41. Liu Q, Hilsenbeck S, Gazitt Y. Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL. Blood 2003;101:4078–87.[Abstract/Free Full Text]
  42. Chen YC, Lin-Shiau SY, Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 1998;177:324–33.[CrossRef][Medline]
  43. Gao F, Yi J, Yuan JQ, Shi GY, Tang XM. The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species. Cell Res 2004;14:81–5.[CrossRef][Medline]
  44. Chandel NS, McClintock DS, Feliciano CE, et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1{alpha} during hypoxia: a mechanism of O2 sensing. J Biol Chem 2000;275:25130–8.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Karlsson, J.
Right arrow Articles by Pettersson, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Karlsson, J.
Right arrow Articles by Pettersson, H. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation