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. 2008 Jan 17;134(7):761–768. doi: 10.1007/s00432-007-0338-2

Increased chemosensitivity and elevated reactive oxygen species are mediated by glutathione reduction in glutamine deprived neuroblastoma cells

Sakurako Izaki 1,✉, Hiroaki Goto 1, Shumpei Yokota 1
PMCID: PMC12160717  PMID: 18202857

Abstract

Purpose

Glutamine is an essential amino acid for the synthesis of glutathione (GSH), the major endogenous antioxidant which protects cells from oxidative injury. To evaluate the effects of glutamine concentrations, cell growth, GSH levels, oxidative stress, and chemosensitivity were evaluated in neuroblastoma cell lines.

Methods

Three human neuroblastoma cell lines (SMS-KCNR, SMS-KANR, SMS-LHN) were cultured with different concentrations of glutamine (2, 0.2 and 0 mM) under hypoxic (5% O2) or normoxic (20% O2) condition. Cell proliferation and chemosensitivity were determined by MTT assay, and the levels of intracellular GSH were measured by DTNB-GSSG reductase method. Cellular reactive oxidative species (ROS) were quantified by flow cytometry.

Results

There was a significant decrease of cell growth in low glutamine (0.2 and 0 mM) compared with control (2 mM) in all three cell lines (P < 0.01), while adding GSH partially restored the reduced cell proliferation by low glutamine. The levels of GSH in neuroblastoma cells decreased significantly in low glutamine compared with the levels of control cells cultured in 2 mM glutamine (P < 0.05), and the accumulation of cellular ROS was significantly higher in 0 mM glutamine compared to the control. Moreover, glutamine deprivation significantly enhanced cytotoxicity of L-PAM in all three cell lines, which was abolished after addition of GSH.

Conclusion

Glutamine deprivation decreased cell proliferation and enhances cell chemosensitivity in neuroblastoma, which is presumably associated with GSH depletion.

Keywords: Neuroblastoma, Glutamine, Glutathione (GSH), Chemosensitivity, L-PAM, Hypoxia, Oxidative stress

Introduction

Glutamine is the most abundant free amino acid in the bloodstream, accounting for 30–50% of the amino acid nitrogen in the plasma. It serves as a metabolic intermediate by contributing carbon and nitrogen to the synthesis of other amino acids, nucleic acids, fatty acids and proteins. Glutamine also regulates cellular proliferation and apoptosis, and promotes the synthesis of specific proteins (Neu et al. 1996; Wasa et al. 1996; Chang et al. 1999; Curi et al. 2005). Moreover, glutamine, via glutamate, is an essential amino acid for the synthesis of glutathione (GSH), the major endogenous antioxidant that protects cells from oxidative injury and detoxifies alkylating agents (Meister 1991; Roth et al. 2002). Previous studies showed that starving cells of glutamine results in energy depletion, decreased immune defense, increased apoptosis and a reduced level of GSH (Oehler and Roth 2003). It is also known that high intracellular GSH levels in cancer cells are associated with high cellular proliferation rates and drug resistance (Green et al. 1984; Fernandes and Cotter 1994, Anderson et al. 1997).

In contrast, some in vivo studies demonstrated that glutamine-enriched diets enhance tumor death and diminish GSH levels. It is not clear whether glutamine improves or impairs the efficacy of chemotherapeutic treatments, nor whether particular types of cancer patient might benefit from glutamine administration.

Neuroblastoma is the most frequent solid extracranial neoplasia in children, and is responsible for about 15% of all pediatric cancer deaths (Gurney et al. 1997). It is a tumor of the sympathetic nervous system and has been reported to produce increased amounts of oxygen intermediates during synthesis of catecholamines and to store high amounts of reduced iron that can interact with hydrogen peroxide to produce hydroxyl radical via the Fenton reaction (Anderson et al. 1999). In order to survive this oxidative stress, neuroblastomas depend on the presence of antioxidants, particularly GSH. Furthermore, GSH conjugates with alkylating agents, such as L-PAM, and detoxifies by transporting these alkylating agents out of cells (Tew et al. 1993; Schroder et al. 1996). Buthionine sulfoximine (BSO), which inhibits the synthesis of GSH, is known to be beneficial in patients with neuroblastoma and has been used in a clinical trial in combination with L-PAM (Anderson et al. 1998).

We evaluated the effects of glutamine deprivation in three neuroblastoma cell lines cultured under both normoxic and hypoxic conditions. We found that cellular proliferation rates, intracellular GSH levels and chemosensitivity of neuroblastoma cell lines varied depending on the concentration of extracellular glutamine. Additionally, we suggest that the decreased rates of cell proliferation and reduction in L-PAM resistance seen under conditions of low glutamine are mediated by GSH. Our data indicate that lowering systemic glutamine levels may be of therapeutic value to patients with neuroblastoma.

Methods

Cell culture

Human neuroblastoma cell lines (SMS-KCNR, SMS-KANR, SMS-LHN) were continuously cultured in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) with 10% fetal bovine serum (FBS) and supplemented with 2 mM l-glutamine (GIBCO, Invitrogen Co., Carsbad, CA, USA) at 37°C in 5% CO2. Cells were grown without antibiotics. Puck’s Saline A plus EDTA was used to detach cells from culture plates or flasks. The culture medium was changed every 3 days until cells were confluent. Experiments were carried out at passages 15–30.

Cell growth

The cells (7,000 cells/well) were cultured in 96-well microtiter plates with various concentrations of glutamine (2 mM as control, 0.2 and 0 mM) under hypoxic (5% O2) or normoxic (20% O2) conditions in replicates of 8–16 wells per treatment group. Additionally, to examine if the effects of low glutamine could be restored by adding GSH, 1 mM GSH was added to the culture media. After 6 days of culture, cell growth rate was measured by the 3-(4-5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT; Sigma) assay. The percent survival was calculated by dividing the OD reading of cells of a given treatment group by the OD reading for the same cells cultured in 2 mM glutamine (control), and multiplying by 100.

Cell viability was assessed using the trypan blue exclusion technique. The cells (1 × 105 cells/well) were cultured in 12-well plates with different concentrations of glutamine. After 6 days of culture, trypan blue isotonic solution was added and the total numbers of cells were counted in four replicates. Deeply stained cells were counted as dead cells.

Microphotographs of SMS-KCNR cells cultured in the absence or presence (2 mM) of glutamine for 6 days were visualized and photographed using an Olympus phase contrast microscope.

Drug sensitivity assay

Prior to the assay, the cells were preincubated in culture media with 2, 0.2 or 0 mM glutamine for 7 days. The cells were seeded at a density of 7,000 cells/well in 96-well microtiter plates, with replicates of 16 wells per treatment group. At the end of the preincubation period, L-PAM (Sigma), at a concentration equivalent to the clinically achievable plasma level (0.0192–12 μg/ml), was added on day 0 and the cells were incubated for 6 days. Cell viability was determined by the MTT assay and calculated by dividing the OD reading of cells treated with a given concentration of L-PAM by the OD reading for the same cells cultured without L-PAM at each glutamine concentration (2, 0.2 and 0 mM), and multiplying by 100.

MTT assay

Cell proliferation rate and drug sensitivity were determined by means of the MTT assay. The cell culture medium was removed and replaced by serum-free-medium containing 50 μg/ml of MTT. The cells were then incubated for 2 h. This medium was removed and, after solubilization of the formazan crystals with dimethyl sulfoxide (Wako, Osaka, Japan), absorption at 570 nm was measured in a spectrophotometer. Cell viability was considered to be linearly proportional to the color intensity measurements. Each treatment group was tested in replicates of eight or more microwells.

Measurement of glutathione

Cells were seeded into 3.5 cm2 tissue culture flasks at 1 × 105 cells/flask. The cells were cultured in various concentration of glutamine (2, 0.2 and 0 mM) for 6 days in 5% O2 (hypoxia) or 20% O2 (normoxia). For the SMS-KCNR cell line, GSH was added to the culture media to a final concentration of 1 mM. Cells were harvested using Puck’s EDTA, washed in phosphate-buffered saline (PBS), and lysed in 1% Triton-X in 10 mM HCl. Total GSH content was measured by the DTNB 5,5′-dithiobis-(2-nitrobenzoic acid)-GSSG reductase method (Vandeputte et al. 1994) using 96-well plates and a microplate reader (Bio-Rad, Hercules, CA, USA) for 2 min at 405 nm. OD values were converted to nanomoles of total GSH by the use of a standard curve generated from OD measurements of samples containing known concentrations of GSH (Sigma). The total amount of protein in the sample was determined and the level of GSH was expressed as the value per mg protein. Each determination was performed in triplicate.

Measurement of reactive oxygen species production

Intracellular redox levels were measured using the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Frenkel and Gleichauf 1991). DCFDA becomes oxidized to the fluorescent compound DCF in the presence of H2O2. SMS-KCNR cells (1 × 106 cells in 25 cm2 flask) were incubated with either 2, 0.2 or 0 mM glutamine for 6 days in 20% O2. Cells were then washed with PBS, and resuspended in 500 μl PBS containing 5 μM H2DCFDA. Cellular reactive oxygen species (ROS) was analyzed using a FACS Caliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA): 1 × 104 cells were analyzed in list mode and dead cells excluded by light scatter. The mean fluorescence intensity of control (2 mM) cells was set to 1, and the level of intracellular ROS was expressed relative to the mean fluorescence intensity of cells cultured with reduced (0.2 and 0 mM) glutamine. ROS measurements were performed in five replicates.

Statistical analysis

All data are expressed as mean ± standard errors. Two-sided Student’s t tests (Microsoft Excel 2000 software) were used to identify statistically significant differences. P < 0.05 was taken as statistically significant.

Results

Cell growth

A higher rate of proliferation was found under hypoxia compared to normoxia in all three cell lines. At glutamine concentrations of 0.2 and 0 mM, there was a statistically significant decrease in the rate of cellular proliferation compared with the control in all three cell lines.

After 6 days culture with 0.2 or 0 mM glutamine under normoxia (20% O2), the rate of cellular proliferation was reduced to 57.4 ± 6.3 or 47.7 ± 5.5%, respectively, of the control (2 mM glutamine) in SMS-KCNR, 84.3 ± 10.1 or 80.5 ± 8.5%, respectively, of control in SMS-KANR, and 80.1 ± 9.6 and 70.6 ± 7.3%, respectively, of control in SMS-LHN. In all cell lines, the reductions at 0.2 and 0 mM glutamine were highly significant (P < 0.01). Under hypoxia (5% O2), proliferation of SMS-KCNR cells cultured with 0.2 or 0 mM glutamine was reduced to 77.3 ± 5.9 or 51.6 ± 3.1%, respectively, of control (2 mM), 95.0 ± 5.2 or 76.2 ± 8.9%, respectively, in SMS-KANR, and 82.3 ± 4.6 or 64.8 ± 8.0%, respectively, of control in SMS-LHN. Statistical analyses showed these reductions to be significant (P < 0.01, except P < 0.05 for 0.2 mM in SMS-KANR). Thus, the effect of reducing glutamine concentration on cellular proliferation was greater under normoxia than hypoxia. Adding GSH to culture media in cells cultured with reduced glutamine (0.2 or 0 mM) partially restored the rate of cellular proliferation. Except 0 mM glutamine cultured at hypoxia in SMS-KCNR (P = 0.3), additional GSH improved the proliferation rate significantly (P < 0.05, 0.2 mM SMS-KCNR at hypoxia; P < 0.01, SMS-KCNR at normoxia; P < 0.01, SMS-KANR; P < 0.01, SMS-LHN) at the range between 4 and 27% (Fig. 1).

Fig. 1.

Fig. 1

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Effect of glutamine concentration on cell growth in SMS-KCNR, SMS-KANR and SMS-LHN. Cells were cultured in 2, 0.2 or 0 mM glutamine, with or without GSH under hypoxic (5% O2) or normoxic (20% O2) conditions. Bars show the mean OD, and error bars indicate SD. * P < 0.05, ** P < 0.01 compared to the control (2 mM glutamine)

The numbers of live cells were significantly lower in cells cultured with reduced glutamine (0.2 and 0 mM) compared to the control (P < 0.05, control vs. 0.2 mM; P < 0.01, control vs. 0 mM). In contrast, the numbers of dead cells were significantly higher in cells cultured with 0.2 and 0 mM glutamine compared to the control (P < 0.05, control vs. 0.2 mM; P < 0.01, control vs. 0 mM) (Fig. 2). Microphotographs of SMS-KCNR cells revealed that cells cultured in 2 mM glutamine appeared well spread whereas those cultured without glutamine appeared spherical or partially spread (Fig. 3). Thus, compared to controls, glutamine deprivation resulted in a decrease in cell numbers and a loss of typical cell shape.

Fig. 2.

Fig. 2

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Effect of glutamine concentration on cell viability in SMS-KCNR. The cells (1 × 105 cells/well) were cultured in 12-well plates with 2, 0.2 or 0 mM glutamine for 6 days. The total numbers of cells of live and dead were assessed by using the trypan blue exclusion technique and counted in triplicates. Bars show the mean cell count, and error bars indicate SD. * P < 0.05, ** P < 0.01 compared to the control (2 mM glutamine)

Fig. 3.

Fig. 3

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Microphotographs of SMS-KCNR cultured in the absence (0 mM, left) or presence (2 mM, right) of glutamine for 6 days, visualized and photographed using an Olympus phase contrast microscope. Control cells incubated with 2 mM glutamine reveals typical shape with extended neurites (right), whereas cells incubated without glutamine appears partially spread (left)

Glutathione levels

In all three cell lines, cells with reduced glutamine had significantly decreased levels of GSH under normoxia (P < 0.05) compared to controls (2 mM glutamine). In SMS-KCNR, the intracellular GSH level in the control (2 mM) was 125.1 ± 4.5 nmol/mg protein; this was significantly higher than 112.5 ± 6.7 or 94.8 ± 4.5 nmol/mg protein detected, respectively, in 0.2 or 0 mM glutamine (P < 0.05, control vs. 0.2 mM; P < 0.01, control vs. 0 mM). When GSH was added to the culture medium, intracellular GSH levels increased significantly. Similarly, GSH levels were higher in control cultures of SMS-KANR and SMS-LHN cell lines compared to those with reduced glutamine concentrations. In SMS-KANR, an intracellular GSH level in 2 mM of 144.2 ± 8.7 was obtained; this was significantly higher than the levels of 126.2 ± 1.0 or 110.9 ± 5.8 nmol/mg protein found, respectively, in those cultured with 0.2 or 0 mM glutamine. The reductions were statistically significant (P < 0.05, 2 vs. 0.2 mM; P < 0.01, 2 vs. 0 mM). In SMS-LHN, we observed 112.2 ± 1.1, 92.5 ± 0.8, or 85.5 ± 3.5 nmol/mg protein, respectively, in those cultured with 2, 0.2 or 0 mM of glutamine (P < 0.01 for both) (Fig. 4).

Fig. 4.

Fig. 4

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Effects of extracellular glutamine on intracellular GSH levels in SMS-KCNR, SMS-KANR and SMS-LHN cell lines. In all three cell lines, intracellular GSH levels were dependent on glutamine concentrations of the culture media. In SMS-KCNR, 1 mM GSH was added to the culture media and were incubated under hypoxia (5% O2) or normoxia (20% O2). Data are mean ± SD. * P < 0.05, ** P < 0.01 compared to the control (2 mM glutamine)

To examine GSH levels after culture under conditions with lower oxidative stress, SMS-KCNR cells were cultured at hypoxia (5% O2). Intracellular GSH levels were lower at hypoxia compared to normoxia, regardless of glutamine concentration. In these conditions we observed GSH levels of 79.7 ± 2.9, 64.3 ± 2.4 or 60.7 ± 2.8 nmol/mg protein in cultures with 2, 0.2, or 0 mM of glutamine, respectively (P < 0.05, 2 vs. 0.2 mM; P < 0.01, 2 vs. 0 mM) (Fig. 4).

Cytotoxicity assay

We examined the effect of glutamine deprivation on L-PAM cytotoxicity in all three cell lines. When SMS-KCNR cells were cultured with 0.096 μg/ml L-PAM, cell viability in cells cultured with 0 mM glutamine decreased to 51.9 ± 10.2% compared to 81.2 ± 15.9% in 2 mM glutamine (P < 0.01). At 0.48 μg/ml L-PAM, viability rates of 18.7 ± 3.75 or 3.7 ± 1.5%, respectively, were found in 0.2 or 0 mM, respectively, while 48.5 ± 7.8% survived in control (2 mM) (P < 0.01 for both). Similarly, viability of SMS-KANR and SMS-LHN cells was decreased in those cultured with reduced glutamine. At 0.48 μg/ml L-PAM, viability of SMS-KANR cells decreased to 9.4 ± 3.4% in 0 mM glutamine compared to 48.8 ± 5.4% in 2 mM (P < 0.01). For SMS-LHN cells exposed to 0.48 μg/ml L-PAM, reduced viability of 63.7 ± 3.3 or 52.0 ± 6.8% were observed at 0.2 or 0 mM glutamine, respectively, compared to 83.6 ± 4.4% in the control (2 mM) (P < 0.01 for both) (Fig. 5).

Fig. 5.

Fig. 5

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Dose response of SMS-KCNR, SMS-KANR and SMS-LHN cells to L-PAM cultured with 2, 0.2 or 0 mM glutamine. Cells were cultured with 0 mM (triangle), 0.2 mM (closed circle) or 2 mM glutamine (control, open circle) prior to the assay. L-PAM (0.0192–12 μg/ml) was added to the media at concentrations relevant to clinically achievable levels. After 6-day incubation, chemosensitivity was determined by the MTT assay. Data are mean ± SD.

To determine if enhanced chemosensitivity in cells cultured with reduced glutamine is mediated by GSH, 1 mM of GSH was added to the culture media. The increase in L-PAM sensitivity seen in SMS-KCNR cells cultured with reduced glutamine was abolished by adding GSH. At 0.2 μg/ml L-PAM, addition of GSH increased survival rate from 26.4 ± 14.2 to 62.8 ± 13.9% in 0 mM, and 50.9 ± 11.7 to 91.0 ± 19.2% in 0.2 mM (Fig. 6).

Fig. 6.

Fig. 6

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Effect of GSH supplementation on L-PAM-induced cytotoxicity in SMS-KCNR cells. Cells were preincubated with 2, 0.2 or 0 mM glutamine prior to the assay. L-PAM (0.2–5 μg/ml) was added to the culture media with a 0 mM glutamine (triangle) and b 0.2 mM glutamine (diamond) either with or without GSH. Cytotoxicity was analyzed with an MTT assay after 6-day incubation. Data are mean ± SD

ROS

We measured ROS by flow cytometry using H2DCFDA to determine whether low glutamine increased ROS. SMS-KCNR cells were incubated with 2, 0.2 or 0 mM glutamine and then examined for ROS content by flow cytometry of DCF fluorescence. The increase in DCF relative to that of the control (2 mM glutamine) was 1.3-fold (P = 0.3) in 0.2 mM glutamine and 1.7-fold in 0 mM glutamine (P < 0.5) (Fig. 7).

Fig. 7.

Fig. 7

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Measurement of reactive oxygen species (ROS) in SMS-KCNR. SMS-KCNR was cultured with 2, 0.2 or 0 mM glutamine for 6 days, and the cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA). Dichlorofluorescein (DCF) intensity after H2DCFDA staining, which reflects total level of intracellular ROS, was measured with flow cytometry. ROS measurements were performed in five replicates. Bars show the mean fluorescence intensity, and error bars indicate SD. * P < 0.05

Discussion

This study showed that in neuroblastoma cell lines, the rate of cellular proliferation and intracellular GSH level was proportional to, and intracellular ROS is inversely proportional to the concentration of glutamine in the culture medium. Additionally, we demonstrated that adding GSH could ameliorate the reduced cell growth and chemosensitivity seen under conditions of low glutamine. From these results, we conclude that the decreased cell growth and chemosensitivity caused by glutamine deprivation was mediated by GSH reduction and, thereby, oxidative stress to some degree.

This is well supported by the evidence that cell growth was apparently increased at all conditions under hypoxia, where the effect of ROS is reduced. Conversely, it indicates how oxidative status has direct and great effect on cell growth. Recent studies indicate that the dosage of the redox signal is important in the modulation of cell function (Herrlich and Bohmer 2000). While high concentrations of ROS are pathogenic, moderate levels of oxidative stress act as mediators and signal transduction molecules and arrests cell cycle, whereas very low doses of ROS stimulate cell proliferation. Jogi et al. recently reported that hypoxia alters the expression of differentiation marker genes and affects the differentiation of human neuroblastoma cells, which develop an immature phenotype when grown under hypoxic conditions (Jögi et al. 2002, 2003). They proposed that dedifferentiation of neuroblastoma cells in hypoxic tumor regions contributes to the malignancy of the tumor (Jögi et al. 2003) and the expression of hypoxia-regulated genes such as tyrosine hydroxilase (TH), the rate-limiting enzyme in catecholamine synthesis, may contribute to cellular proliferation.

In this study, we also examined the GSH level under hypoxia in relation to cell proliferation rate. Under hypoxia, intracellular GSH was lower than normoxia at all glutamine concentrations. We suspect that this is because environmental oxidative stress is limited, and necessity and production of GSH may be kept low. On the other hand, intracellular GSH level correlated with environmental glutamine concentration even at hypoxia. Although hypoxia of 5% O2, it still has oxidative stress which is influenced by glutamine concentration dependent GSH production resulting in growth suppression.

However, decrease in GSH or increase of ROS may not be the only determinant that mediates glutamine deprivation induced suppression in cellular proliferation. For instance, glutamine may directly affect gene expression. Recent studies suggest that glutamine regulates the expression of several genes and activates intracellular signaling pathways in various cell cultures (Curi et al. 2005). Cell type specific apoptosis signaling mechanisms have been suggested to be an induced response to glutamine deprivation (Fuchs and Bode 2006). To date, however, no studies have yet been carried out on neuroblastomas.

Furthermore, adding GSH in cell culture may affect other mechanisms that might explain the present results. Firstly, it was shown that in A549 cells cultured under glutamine deficient conditions, the effect of extracellular GSH was correlated with a high level of gamma-glutamyltransferase activity. This enzyme converts glutamine to glutamate and thereby increases the intracellular level of glutamate; in turn, this enhances intracellular glutamine synthesis and thereby stimulates cell proliferation (Kang et al. 1994). Secondly, adding GSH might spare constituent amino acids, particularly glutamate, and thereby stimulate cell proliferation. In a previous report on the inhibition of GSH synthesis by BSO, increased cellular glutamate levels were suggested to be responsible for the BSO growth stimulating effect (Kang 1993).

However, previous in vivo studies showed that glutamine supplementation significantly decreased tumor GSH levels. In breast cancers of rats, it was suggested that dietary glutamine supplementation decreased tumor GSH levels and suppressed cells by activation of apoptosis in tumor cells (Todorova et al. 2004). Studies on Ehrlich ascites tumors (Carretero et al. 2000) and melanomas (Benlloch et al. 2006) demonstrated that a glutamine enriched diet increases glutaminase activity, promotes accumulation of cytosolic glutamate and inhibits GSH transport into mitochondria. These changes render tumor cells more susceptible to oxidative stress, and enhance the selectivity of antitumor drugs by protecting normal tissues. Although there may be a difference between cells growing in vivo and in vitro, another possibility is that the type of cell may be of relevance, for it is known that GSH content differs among different types of tumor cell. Because neuroblastomas have been reported to produce increased amounts of oxygen intermediates during synthesis of catecholamines and to store high amounts of reduced iron (Anderson et al. 1997), their GSH levels seem to be higher than those of other cell types reported previously. To date, there have been no in vivo studies of neuroblastoma cell lines, nor has it been shown that dietary glutamine decreases intracellular GSH in neuroblastoma cell lines. At least, with regard to neuroblastomas, another in vitro study of glutamine deprivation also identified decreased intracellular GSH (Soh et al. 2005). Recently, Fuchs et al. reviewed the roles of glutamine and GSH in various cancer cells in detail (Fuchs and Bode 2006), and concluded that an adequate supply of glutamine is required for the maintenance of the GSH economy in several cell types and that glutamine deprivation ultimately elicits apoptosis, depending on cell type.

Numbers of studies have shown that the high level of intracellular GSH is associated with a drug-resistant phenotype, and that the inhibition of GSH synthesis enhances chemosensitivity in neuroblastoma or other cancer cells (Green et al. 1984; Fernandes and Cotter 1994; Anderson et al. 1997). In this study, neuroblastoma cells were more sensitive to L-PAM in the condition of lower glutamine, and such effect of glutamine was abolished by adding GSH, suggesting that the change of biology in neuroblastoma under low environmental glutamine is mediated by GSH suppression.

Here, we studied three different cell lines: the MYCN-amplified cell lines SMS-KCNR and SMS-KANR; and the nonamplified cell line, SMS-LHN. Although a previous report suggested that the MYCN-amplified cell lines were more sensitive to GSH depletion than MYCN-nonamplified neuroblastoma (Anderson et al. 1997), our results showed that all were in fact similar.

Incidentally, we have to note that 10% of FBS used in this study contains a small amount of glutamine. To put it concretely, glutamine, together with glutamic acid, contains about 0.3 mM in the FBS. In brief, culture conditions may contain 0.03 mM glutamine at most even in 0 mM glutamine.

In conclusion, we have demonstrated that glutamine deprivation decreased cell proliferation and intracellular GSH, and enhanced cell chemosensitivity in three neuroblastoma cell lines. Moreover, adding GSH to the culture media partially restored the reduced rate of cell proliferation and chemosensitivity. From these results, we suggest that the decreased cell growth, increased oxidative stress and enhanced chemosensitivity induced by glutamine deprivation are mediated by GSH reduction. Lowering systemic glutamine levels may be of therapeutic value to patients with neuroblastoma.

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