High levels of Mn-superoxide dismutase in serum of patients with neuroblastoma and in human neuroblastoma cell lines

Free Radical Biology & Medicine, Vol. 12, pp. 281-286, 1992 Printed in the USA. All fights reserved.

0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.

Original Contribution HIGH

LEVELS OF MN-SUPEROXIDE DISMUTASE IN SERUM PATIENTS WITH NEUROBLASTOMA AND IN HUMAN NEUROBLASTOMA CELL LINES

NAOHISA KAWAMURA,*~ KEIICHIRO SUZUKI,* M U T S U O ISHIKAWA,~ SUSUMU MUNENORI MIYAKE,'~ MAKOTO MINO,'~ and NAOYUKI TANIGUCHI*

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hZUKA,§

*Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka Suita, Osaka 565, Japan; "tDepartment of Pediatrics, Osaka Medical College, 4-7 Daigakucho, Takatsuki, Japan; :~Department of Obstetrics and Gynecology, Asahikawa Medical College, Kaguraoka, Asahikawa, 078, Japan; §Sapporo National Hospital, Kikusui, Sapporo 030, Japan (Received 4 June 1991; Revised 5 November 1991; Accepted 6 November 1991 ) Abstract--Levels of serum manganese superoxide dismutase (Mn-SOD) in normal children aged from 1 to 14 years and children with various hematological and malignant diseases were determined by enzyme-linked immunosorbent assay (ELISA). In the normal children, the serum Mn-SOD levels gradually increased in proportion to age. By 8 years of age, the Mn-SOD level was nearly at the adult level. The normal values of serum Mn-SOD (mean _+SD) of children below 4 and above 8 years old were 48 _+ 10.2 ng/ml and 84 + 22.5 ng/ml, respectively. Assuming the upper limit of normal Mn-SOD level in serum to be the mean value _+2 SD of children at each age, high serum levels of Mn-SOD were found for 8 of 12 patients with neuroblastoma, three of four patients with Wilms tumor, and four of five patients with acute myeloid leukemia. The patients with neuroblastoma exhibited a transient increase in Mn-SOD following chemotherapy, but after 1 week the levels decreased markedly to the control levels. The changes in serum Mn-SOD levels in the patients with neuroblastoma correlated well with the levels of neuron-specific enolase. Mn-SOD was intensely stained in bone marrow cells of patients whose cancer cells had moved into the bone marrow. High levels of Mn-SOD were also found in cultured human neuroblastoma cells. These data indicate that Mn-SOD is expressed in neuroblastoma cells, may serve as one of the diagnostic and prognostic markers for the neuroblastoma, and may be useful to predict the effectiveness of chemotherapy for neuroblastoma and the recurrence of this disease. Keywords--Neuroblastoma, Mn-SOD, Serum, Cell lines, Neuron-specific enolase, Free radicals

were increased in patients with neuroblastoma as compared to patients with other pediatric tumors. In human neuroblastoma cell lines, the Mn-SOD level was also high. These findings suggest that Mn-SOD can be a helpful tool in discriminating neuroblastomas in this diagnostically difficult group of tumors. We examined the expression of serum Mn-SOD in terms of age at diagnosis, clinical stage, and prognosis to evaluate whether Mn-SOD could be used as a prognostic and diagnostic marker for this disease.

INTRODUCTION

Neuroblastoma is the most frequent type of pediatric tumor arising from embryonal neural crest cells and bears morphological and biochemical similarities to these cells. The embryonal neural crest cells form the adrenal medulla and sympathetic ganglia.l Manganese superoxide dismutase (Mn-SOD) is localized in the mitochondria and catalyzes the dismutation of superoxide anion. The tissues that have large numbers ofmitochondria are rich in the enzyme. The adrenal gland is one such tissue, and indeed its MnSOD level is high. 2 Our previous studies have shown that Mn-SOD is one of the tumor markers for ovarian cancer) In the present study, we found that serum Mn-SOD levels

MATERIALS AND METHODS

Serum samples Serum samples were obtained from healthy child volunteers and from hospitalized patients. Sera were stored at -80°C until used. The sera were obtained from patients prior to any treatment or operation.

Address correspondence to Dr. Naoyuki Taniguchi, Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. 281

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The sera of several patients with neuroblastoma were collected both before and after the operation and at regression. The neuroblastomas were divided into clinical stages I-IV according to the International Neuroblastoma Staging System.4

Cell lines The following cell lines were used in this study: three human neuroblastoma cell lines (NB- 1, GOTO, and IMR-32); Wilms tumor cell line (G-401); cervical cancer cell line (Hela cell); ovarian epithelial cancer cell line (Kuramochi); and fatal lung fibroblast cell line (WI-38). All cell lines were obtained from the Japanese Cancer Research Resources Bank. Human epithelial cells were separated from normal umbilical cord vein. Cells except G-401 and epithelial cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 in 45% Roswell Park Memorial Institute (RPMI) 1640, 45% minimum Eagle's essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum containing penicillin and streptomycin. G-401 cells were maintained in McCoy's 5A supplemented with 10% fetal calf serum and plated at 1 × 10 6 cells per dish (Falcon), and cell lines were grown in monolayer. Epithelial cells were maintained in E-GM UV (Clonetics Corp., USA). The viable cells were counted by the trypan blue dyeexclusion method. New cultures were tested for Mycoplasma-free frozen stocks every 3 months, and cultures were tested for Mycoplasma prior to being replaced by newly thawed stocks.

Enzyme-linked immunosorbent assay (ELISA) for Mn-SOD and assay of neuron-specific enolase ELISA for serum Mn-SOD was carried out using a sandwich Mn-SOD ELISA kit (Ube Industries) that had been developed in our group as described. 3'5 In brief, each sample was thawed at room temperature and then sonicated. After centrifugation at 3000 revolutions per minute (rpm) for 15 min, the supernatant was diluted 50- to 100-fold with 10 mM phosphate buffer, pH 7.4, containing 0.5 M NaC1 and 0.1% bovine serum albumin. Neuron-specific enolase was determined by the method described6 using a radioimmunoassay kit (SRL Co. Ltd., Japan) according to the supplier's instruction manual. These studies were performed after informed consent had been obtained from the parents.

Immunocytochemistry of Mn-SOD in bone marrow cells Bone marrow cells were fixed with ice-cold acetone/methanol (1/1, vol/vol) for 5 min at -15 °C and then incubated with the anti-Mn-SOD polyclonal IgG antibody that had been prepared by injecting the purified human Mn-SOD 7 in rabbits in our laboratory. The preparation was washed and then stained with the LSAB method (DAKO, Japan) using a technique of strepto-avidin-biotin according to the manufacturer's instructions.

Statistical analysis Statistical analysis was carried out using the Student t test and a one-way variance test. RESULTS

Developmental changes in serum Mn-SOD levels in children Serum Mn-SOD levels of children under 4 years old were 47.6 + 10.2 ng/ml (mean + SD) and 84.0 _+ 22.5 ng/ml above 8 years old. By 8 years of age, the value was nearly at the adult level (Fig. 1).

Serum Mn-SOD levels in various pediatric tumors As shown in Figure 2, serum Mn-SOD levels of patients with various pediatric tumors were examined. High Mn-SOD levels were found in patients with neuroblastoma. Sera that gave values above the mean _+ 2 SD of normal children at each age in the ELISA were scored as positive, and the incidence of positive cases (positive/number of cases) was calculated. Eight of twelve patients with neuroblastoma were found to have high Mn-SOD levels. Slightly high Mn-SOD levels and a high incidence of positive cases were also found in patients with Wilms tumor and acute myeloid leukemia.

Serum Mn-SOD levels during the clinical courses of patients with neuroblastoma At stage I, serum levels of Mn-SOD were within normal range (Table 1). At stage IV and in recurrence, a definite increase in Mn-SOD levels in serum was observed. Changes in serum Mn-SOD were followed during the clinical courses of two patients with neuroblastoma. The level of serum Mn-SOD increased right after treatment with chemotherapy (Figs. 3A and 3B). Figure 4 indicates the changes in serum Mn-SOD

Mn-SOD in neuroblastoma

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Fig. l. Developmental changes in serum Mn-SOD levels in normal children. The bar indicates mean _+ SD. *Statistically significant (p < .01 ) values as compared to those of ages 0 to 4 years.

of immunoreactive Mn-SOD, as shown in Figure 6. This supports the evidence that bone marrow tissue is a likely candidate for tumor cell invasion in neuroblastoma patients.

levels before and after treatments in nine patients with neuroblastoma. All of the patients were found to have decreased Mn-SOD levels after the treatments. In addition, the patients with recurrence had high serum Mn-SOD levels, which suggests that serum Mn-SOD is useful in monitoring the disappearance or recurrence of these types of tumors. Levels of serum Mn-SOD and neuron-specific enolase were determined for the two patients with neuroblastoma during their clinical courses. Statistically significant correlation was found between the changes in neuron-specific enolase and Mn-SOD, as shown in Figure 5 (n = 16, r = .70, p < .01).

Mn-SOD levels in human neuroblastoma and Wilms tumor cell lines Three human neuroblastoma cell lines and various normal and cancer cell lines, including Wilms tumor cell line, were examined for expression of Mn-SOD. As shown in Table 2, all of the h u m a n neuroblastoma cell lines were found to have relatively high Mn-SOD levels as compared to the other cancer cell lines as well as normal cell lines.

Immunochemical staining of Mn-SOD in bone marrow tissues

DISCUSSION

Bone marrow cells of one patient whose tumor cells had metastasized were found to have a high level

In the present study, we found that the serum MnSOD levels of normal children were slightly lower

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Fig. 2. Serum Mn-SOD levels of patients with various pediatric tumors. AML, acute myeloid leukemia. ALL, acute lymphocytic leukemia. An upper limit of Mn-SOD levels in serum of normal children at each age was defined as mean _+2SD. Sera that gave values above the upper limit in the ELISA were scored as positive. The incidence of positive cases was expressed as positive/ number of cases and indicated in the right column. Numbers in the upper column indicate the stage of the patients as described in Materials and Methods.

284

N. KAWAMURAet al. Table 1. Serum Mn-SOD Level and Clinical Stage of Neuroblastoma

Stage

Total No. Tested

Mn-SOD level* (ng/ml)

2 1 4 10 3

44 _+ 8 94 115 + 37 197 + 151 140 + 11

I II III IV Recurrence

study, we found that patients with neuroblastoma have high serum Mn-SOD levels. The high expression of Mn-SOD in neuroblastoma cells seems to result from a similar mechanism. Our preliminary experiments have indicated that addition of TNF-a slightly induces Mn-SOD mRNA and protein levels in the neuroblastoma cell lines (data not shown). Another possible explanation for the increased expression of Mn-SOD in neuroblastoma is that the neuroblastoma is rich in mitochondria, as was observed in adrenal glands whose origin is also neural crest.l A third possible explanation is suggested by the recent observation that a large amount of hydrogen peroxide is present in the neuroblastoma cell lines) 2 The neuroblastoma cells may produce superoxide anion, which is then converted to hydrogen peroxide in the presence of metal ions by the Haber-Weiss reaction. ]3 To scavenge the superoxide anion generated by the tumor cells, an adaptive increase in Mn-SOD expression may occur. In the present study, we found that serum Mn-SOD is temporarily increased right after chemotherapy, as shown in Figure 3. This is likely due to the release of Mn-SOD from tumor cell mitochondria damaged by the treatment. Recently, o u r g r o u p 14 also found that phorbol ester induced Mn-SOD in TNF-resistant cell lines, suggesting that protein kinase C is implicated in the induction mechanism. The intracellular signal-transducing mechanism for the induction needs further study.

* Each value is expressed as means +_ SD.

than those of normal adults 5 and gradually increased in proportion to age. By 10 years of age, the Mn-SOD levels were nearly at the adult level, as shown in Figure 1. Hayashibe et al. reported that in fetal rat tissues, antioxidant enzymes (including Mn-SOD) increased in proportion to gestational age. 8 Similar changes may occur in human fetal tissues. In a previous study, we found that Mn-SOD is highly expressed in the epithelial type of ovarian cancer and that assessment of serum levels of MnSOD provides a useful tumor marker for the disease. 3 Several cytokines, such as intedeukin- 1 (IL- 1), tumor necrosis factor (TNF), and lipopolysaccharide, have been reported to induce Mn-SOD. 9'~° The increase in expression of Mn-SOD in ovarian cancer tissues is considered to be due to the induction of Mn-SOD mRNA and protein by TNF or IL-1 .~ In the present

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Fig. 3. Serum Mn-SOD levels during the course of treatment of two patients with Stage II (A) and Stage IV (B) neuroblastomas. Arrow indicates the chemotherapy. VP-16, etoposide; CDDP, cysplatin; VCR, vincristine; CPR, cyclophosphamide; ADR, adriamydcin; NSE, neuron-specific enolase.

Mn-SOD in neuroblastoma

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Fig. 4. Changes in serum Mn-SOD levels before and after treatments in nine patients with neuroblastoma. Each number indicates the stage of each patient. 1, 2, 3, and 4 depict stages I, II, III, and IV, respectively. 0 indicates the day of treatments. Abscissa indicates months after treatments on which the patients' sera were taken for analysis.

Clinically, the term neuroblastoma is used for a group of tumors of neural crest derivation. The tumor cells are arrested at various stages of neural differen-

700 600

Fig. 6. Immunochemical staining of Mn-SOD in the bone marrow of a patient whose tumor has metastasized (magnification 1000×).

tiation, and the grade of differention correlates with the clinical course of the disease.15,'6 Several antigens, such as p p 6 0 c'sRc (Ref. 17), s m g p 2 5 A (Ref. 18),/~2m i c r o g l o b u l i n , 19 S-100 p r o t e i n , 2° a n d n e u r o n - s p e c i f i c enolase, 2] h a v e b e e n i d e n t i f i e d as t u m o r m a r k e r s for n e u r o b l a s t o m a . T h e n e u r o b l a s t o m a s o f t e n consist o f u n d i f f e r e n t i a t e d , small, r o u n d cells a n d c a n s o m e t i m e s be c o n f u s e d w i t h o t h e r p e d i a t r i c solid t u m o r s , such as l y m p h o m a , E w i n g ' s s a r c o m a , n e u r o e p i t h e l i o m a , a n d W i l m s t u m o r . 22,23 I n t h e p r e s e n t study, we f o u n d t h a t s e r u m M n - S O D was high in m o s t o f t h e p a t i e n t s with n e u r o b l a s t o m a a n d t h a t t h e p a t i e n t s with stages III a n d I V h a d c o n s i s t e n t l y high s e r u m M n - S O D levels. T h i s i n d i c a t e s t h a t t h e m e a s u r e m e n t o f s e r u m M n - S O D c o u l d be u s e d as o n e o f t h e prognostic a n d d i a g n o s t i c m a r k e r s for this disease.

Acknowledgements--This work was supported in part by grants-in5OO

aid for cancer research from the Ministry of Education, Science,

400

Table 2. Mn-SOD Levels in Various Normal and Cancer Cell Lines

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100 200 300 400 500 M n - S O D levels ( n g / m l )

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Fig. 5. Relationship between serum Mn-SOD and neuron-specific enolase levels. Samples tested included two patients with neuroblav tomas described in Figure 3. During the treatments, the patients' sera were taken and Mn-SOD and neuron-specific enolase levels were simultaneously measured. NSE, neuron-specific enolase.

n

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6 6

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5 5 5

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Each value is expressed as mean _+SD.

286

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and Culture, Japan, and from the Princess Takamatsu Cancer Fund. We acknowledge Mr. Y. Tani, Dako Japan Co. Ltd., for performing immunochemical staining of Mn-SOD.

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ELISA--enzyme-linked immunosorbent assay Mn-SOD--manganese superoxide dismutase TNF--tumor necrosis factor TPA--ohorbol 12-myristate 13-acetate (phorbol ester) IL- 1--interleukin- 1