Induction of Differentiation in Rat C6 Glioma
Cells with Saikosaponins
Yan-Jyu Tsai,1I-Ling Chen,1Lin-Yea Horng2and Rong-Tsun Wu2*
1
Department of Pharmacology, Institute of Medical Research, Taipei Medical College, Taipei, Taiwan, R.O.C.
2Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan, R.O.C.
The effects of saikosaponins (a, b1, b2, c, d), isolated from Bupleurum Radix, on the induction of
differen-tiation in rat C6 glioma cells were studied. Saikosaponins a and d were shown to inhibit cell proliferation and alter cell morphology. In addition to cytostasis, the enzymatic activities of glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) were also noticeably increased after treatment with saikosaponin a. Nevertheless, saikosaponin d only showed an increase of GS activity, no significant changes in CNP activity were found. These results suggest that saikosaponin a can induce the differentia-tion of C6 glioma cells into astrocytes and/or oligodendrocytes, but saikosaponin d can only induce the differentiation of C6 glioma cells into astrocytes. Copyright# 2002 John Wiley & Sons, Ltd.
Keywords: saikosaponins; differentiation induction; C6 glioma cells.
INTRODUCTION
Despite aggressive treatments that include surgical resection, radiotherapy and cytotoxic chemotherapy, the survival rate of patients with malignant glioma, the most common primary tumour of the brain, is still low (Brandes et al., 1991). Human malignant gliomas remain an important, unresolved oncological challenge. Thus, the novel approach of differentiation induction of tumour cells has become especially highlighted.
C6 glioma cells, derived from rat brain treated with N-nitroso-methylurea (Benda et al., 1968), may differenti-ate into either oligodendrocytes and/or type 2 astrocytes (Parker et al., 1980; Kumar et al., 1986; Mangoura et al., 1989). In the CNS, glutamine synthetase (GS) is localized primarily in astrocytes and has long been considered as a specific enzyme marker for astrocytes (Norenberg, 1979). It synthesizes glutamine from glutamate to protect the central nervous system from injury. On the other hand,
2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) is
considered to be a specific enzyme marker for oligoden-drocytes and myelin membranes (Poduslo and Norton, 1972).
Saikosaponins, as the biologically active components, are obtained by extraction from the roots of Bupleurum falcatum L., which has been widely used in Chinese herbal medicine (Kubota and Hinoh, 1968; Shimaoka et al., 1975). In this present study, the effects of saiko-saponins (a, b1, b2, c, d) on the induction of differentia-tion in cultured rat C6 glioma cells have been investigated.
MATERIALS AND METHODS
Materials. Rat C6 glioma cells were purchased from
American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum were procured from Gibco (Gaithersburg, MD). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) and dimethyl sulphoxide (DMSO) were obtained from Sigma (St Louis, MO). N,N-Dimethylfluoramide (DMF) was purchased from Merck (Germany). Saikosaponins (a, b1, b2, c, d) were a gift from Dr H. Abe (University of Kinki, Japan) and were dissolved completely in 70% ethanol. These agents were then sterilized by millipore filtration, dried and stored at
20°C.
Cell culture. C6 cells were maintained as a monolayer
culture in DMEM medium with 10% FCS in the presence
of penicillin (100 U/mL) and streptomycin (100mg/mL)
at 37°C in a humidified atmosphere of 5% CO2 in air.
Subculture was made by using 0.05% trypsin with 0.02% EDTA every 3 to 4 days.
Cell growth and morphology. 135mL of cell suspension
(2.5 104cell/mL) was inoculated into 96-well
micro-plates. Four hours later, 15mL of saikosaponins in a
concentration ranging from 0.1 to 100mg/mL was added.
After 3 days incubation, cultures were fixed for 10 min by 10% formaldehyde and stained with Mayer’s haematoxy-lin for 5–10 min, washed and dyed again with 1% eosin B for 3–5 min. Cultures were observed with a phase contrast microscope.
MTT colorimetric assay. MTT was dissolved at a
concentration of 5 mg/mL in phosphate-buffered saline
(PBSA), sterilized by filtration and stored at 4°C
(Mosmann, 1983; Hansen et al., 1989). After treatment
Phytother. Res. 16, 117–121 (2002)
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.752
* Correspondence to: Dr R.-T. Wu, Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan, R.O.C.
E-mail: [email protected]
Contract/grant sponsor: National Science Council of the Republic of China; Contract/grant number: NSC-85-2331-B-038-020; Contract/grant number: NSC-84-2331-B-010-070.
with saikosaponins for 4 days, the medium was discarded, and the cultures were rinsed with PBS
solution, and replaced with 100mL of fresh medium.
Then 25mL of MTT stock solution was added, and the
cultures were incubated for an additional 2 h. Then
100mL of lysis buffer (20% SDS–50% DMF) was added
and mixed thoroughly for overnight incubation at 37°C.
The absorbance of each well at 570 nm was measured with an ELISA multiscanner (Bio-Rad model 450).
Preparation of GS and CNP extracts. 2 105 cells/ well were inoculated to 6-well plates. 4 h later, the cultures were treated with a different concentration of
saikosaponin a or d for 3 days at 37°C, in a 5% CO2
incubator. Then the cells were harvested and lysed by a
repeated freezing and thawing technique in 100mL of
deionized water. Cell pellets were centrifuged at 40 000 g for 1 h at 4°C. The supernatants were prepared for
GS assay. The precipitates were solubilized with 100mL
of extraction solution (0.15MNaCl, 0.5% Triton X-100
and 10 mMTris-HCl, pH.7.5) and sonicated twice for 15 s
at 0°C and centrifuged. The supernatants were collected
for CNP assay.
Measurement of GS activity. GS activity was
deter-mined according to the method described by Iqbal and
Ottaway (1970). 40mL of the GS extracts prepared as
above were assayed with 60mL of reaction mixture
containing 0.2MMgCl2, 0.5Mglutamate, 1M
hydroxyl-amine, 0.25M 2-mercaptoethanol, 19 mM ATP, 0.1M
2-phosphoenol pyruvate and 1 mg/mL pyruvate kinase
(pH 7.2). The reaction mixture was incubated at 37°C for
15 min, then 150mL of 0.37M ferric chloride (0.67M
HCl-5% TCA) was added to terminate the reaction. After centrifugation at 3000 rpm for 5 min, the absorbance of the supernatant at 535 nm was detected using synthetic r-glutamylhydroxamate as a standard.
Measurement of CNP activity. The assay for CNP
activity was carried out essentially as described by
Prohaska et al. (1973). 5 to 25mg of protein of CNP
extracts was added to 7.5 mM2',3'-c-AMP, 50 mM
Tris-maleate buffer (pH 6.2) to make a final volume of
200mL. The initial reaction was carried out at 30°C for
10 min, and terminated by 90°C for 1 min. 100 mL of
21 mM MgCl2 in 0.3M Tris-HCl buffer (pH 9.0) was
added with 60mg of 0.72 U alkaline phosphatase (pH 8.5)
at 30°C for 20 min. Then 0.7 mL of 10% ascorbic acid:
0.42% ammonium molybdate in 1NH2SO4(1:6) mixture
was added at 45°C for 20 min. After centrifugation at
3000 rpm for 5 min, the absorbance of supernatants at
820 nm was detected using 2'-AMP as a standard (Ames,
1966).
RESULTS
Effect of saikosaponins on cell growth
After treatment with saikosaponins in concentrations of
0.1–100mg/mL for 4 days, the proliferation of C6 glioma
cells was studied by the MTT colorimetric method (Fig.
1). At lower concentrations of 0.1 or 1.0mg/mL, none of
the saikosaponins showed an effect on the growth of C6
glioma cells. But at a concentration of 10mg/mL,
saiko-saponins a and d caused not only a cytostatic effect, but also an alteration in cell morphology (Fig. 2). At a higher
concentration of 100mg/mL, saikosaponins a, b1 and d
showed unexpected cytotoxic effects and caused cell death.
Effect of saikosaponins on cell morphology
After saikosaponin treatment for 3 days, an alteration of cell morphology was observed. The results showed that no significant differences were observed between the
control and 0.1 or 1.0mg/mL treatment (data not shown).
Saikosaponins a and d at 10mg/mL (Fig. 2B, F), showed
not only growth inhibition but also alteration in cell morphology. Figure 2B, F shows a decrease of cell density and an extension of long cytoplasmic processes. These results indicate that saikosaponins (especially a, d) may have the ability to induce cultured C6 glioma cells to differentiate into phenotypes.
Changes in the enzymatic activity of GS and CNP
After treatment of C6 cells with 0, 2.5, 5.0, 10mg/mL of
saikosaponins a or d for 3 days, the activities of GS and CNP were measured. As seen in Fig. 3, a significant increase in the specific GS activity was observed at
10mg/mL of saikosaponin a. In addition, saikosaponin a
can induce an increase of CNP enzyme activity (Fig. 4).
A maximal effect was found at 5mg/mL concentration.
Saikosaponin d at 10mg/mL can also significantly
increase the activity of GS enzyme (Fig. 3). Nevertheless, by comparison with the control group, there was no obviously change in CNP activity in saikosaponin d treated cells (Fig. 4). Therefore, it seems that
saikosapo-nin a at 5–10mg/mL may induce C6 glioma cells to
differentiate into astrocytes and/or oligodendrocytes, but saikosaponin d only can induce C6 glioma cells to differentiate into astrocytes.
Figure 1. Effects of saikosaponins on the proliferation of C6 glioma cells. C6 glioma cells were cultured with each extract for 4 days and detected by MTT assay (*** p < 0.001, ** p < 0.02, * p < 0.01).
DISCUSSION
Because the number of glial cells is much greater than neural cells in the central nervous system, glioma is the most common malignant tumour in the human brain. Its
prognosis is very poor because of the blood–brain barrier to drugs, the low sensitivity of the immune response, and the heterogeneity of glioma. Most clinically used cytotoxic antitumour drugs are unsatisfactory for glioma treatment since they would cause unnecessary harm to healthy brain cells.
Figure 2. Effects of saikosaponins on the morphologic changes of C6 glioma cells. C6 glioma cells (2 104cells/mL) were cultured with (A) control, (B) saikosaponin a (10 mg/mL), (C) saikosaponin b1 (10 mg/mL), (D) saikosaponin b2 (10 mg/mL), (E) saikosaponin c (10 mg/mL), (F) saikosaponin d (10 mg/mL). After 3 days, the morphology was observed by phase-contrast microscopy.
Recent studies have suggested that cancer, including brain tumours, can be considered a disorder of cell differentiation. Some chemical agents, such as retinoic acid (Sidell, 1982; Thiele et al., 1985), sodium butyrate (Hargreaves et al., 1989), dibutyryl cyclic AMP (Pishak and Phillips, 1980; Jackson et al., 1995), cytosine arabinoside (Ponzoni et al., 1989), hexamethylene bisacetamide (Li et al., 1996) and phenylacetate (Samid et al., 1992;1994), have been found to induce certain kinds of tumour cells to differentiate with a loss of proliferative capacity and the expression of certain characteristics of mature cells.
Phenylacetate (PA), a naturally occurring plasma component, induces cytostasis and the reversal of malignant phenotypes in a variety of cultured human cancer cells, including malignant glioma (Stockhammer et al., 1995). The in vitro and in vivo antitumour activity was noted at millimolar concentration levels. Since the effective doses of saikosaponins in our experiments are
micromolar level (12.8mM), saikosaponins may be
valuable in the brain in vivo.
Many reports have indicated that saikosaponins have multiple biological activities including antihepatotoxicity (Abe et al., 1980;1985), antiinflammatory action (Yama-moto et al., 1975), immunomodulatory activity (Kato et al., 1995) and antihepatoma activity (Qian et al., 1995). It is interestering to note that saikosaponins a, b1, b2, c, and d have similar chemical structures in the aglycone
framework (oleanene skeleton). The difference of chemical structure between saikosaponins a, d group and b group is the ether linkage between C13 and C28, and this linkage might cause the difference in biological activity between saikosaponins a, d, and saikosaponin b. Kohno (1995) showed a melanogenesis effect of saikosaponin b1 and b2 on cultured melanoma cells. It is likely to be due to the cell type specificity of these saikosaponins.
Parker et al. (1980) reported that C6 glioma cells have both astrocytic and oligodendrocytic glial properties with passage of the cells as shown by the presence of specific enzyme activities, GS and CNP, respectively. From our results, saikosaponin a can induce the differentiation of C6 glioma cells into astrocytes and/or oligodendrocytes, but saikosaponin d can only induce the differentiation of C6 glioma cells into astrocytes. Based on these results, we propose that saikosaponins may be applied in cancer therapy as a noncytotoxic differentiation inducer.
Acknowledgements
The authors are grateful to Dr H. Abe for the supply of saikosaponins and to the National Science Council of the Republic of China for the financial support (NSC-85-2331-B-038-020) and NSC-84-2331-B-010-070).
Figure 3. Effect of saikosaponin a and saikosaponin d on the GS activity. C6 glioma cells were cultured with saikosaponin a and saikosaponin d for 3 days (* p < 0.01). Values are the mean SEM (n = 3±6).
Figure 4. Effect of saikosaponin a and saikosaponin d on CNP activity. C6 glioma cells were treated with saikosaponin a and saikosaponin d for 3 days. Values are the mean SEM (n = 3, * p < 0.01).
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