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Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo.

Abstract

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer -effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and [IC.sub.50] value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca.sup.2+][sub.i] was measured by LSCM. Expressions of pl6 and p53 protein were evaluated by immunocytochcmical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an [IC.sup.50] value within 24 h of 0.05 [micro]g/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and pi 6 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[[Ca.sup.2+][].sub.i]-induced endoplasmic reticulum stress.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Swainsonine; Glioma cell; Apoptosis; Fluorescene immunohistochemistry [[Ca.sup.2+]]

Introduction

Swainsonine, a water-soluble indole alkaloid and alpha-mannosidase inhibitor which blocks Golgi oligosaccharide processing, represents a new class of compounds that could inhibit tumor growth and metastasis. It has attracted interest due to its potentially therapeutic biological activities. Swainsonine is a potent and specific inhibitor of lysosomal acid and cytosolic [alpha]-mannosidases, as well as Golgi [alpha]-mannosidase II. Its inhibition of the latter enzyme leads to the accumulation of hybrid-type oligosaccharides and a decrease in glycoproteins containing complex side-chains. It has been used as an instrument drug to study glucoprotein N-link oligosaccharide since its initial extraction from the fruit of Australian Swainsona canescens and North America locoweed (including Astragalus and Oxytropis spp.) (Dantas et al. 2007; Li et al. 2004; Ferrara et al. 2006). Swainsonine was shown to inhibit tumor growth and metastasis, enhance lethality of NK cell and LAK cells, reduce the growth rate of human melanoma cells and stimulate proliferation and differentiation of bone marrow cells (Sun et al. 2007; Wang et al. 2003). Initial clinical research has confirmed a clear curative effect against pate malignant tumor and chest and abdominal lymphangioma (Goss et al. 1997). Few reports exist, however, of research on antineoplastic activity and mechanisms of swainsonine. We therefore designed this study to detect the apoptosis of glioma cells induced by swainsonine, and apoptotic-related genes, and also to try to explain antineoplastic activity and mechanisms of swainsonine in vivo and in vitro.

Materials and methods

Materials

RPMI 1640, fetal boving serum was purchased from Gibco. MTT was obtained from Sigma Chemical Co. The Annexin-V-FITC kit: Annexin-V-FITC and pyrimidine of iodinate (PI) were sub-packaged by Department of Immunology. Mouse anti-mouse pl6 and p53 monoclonal IgG, mouse anti-rabbit IgG fluorescent antibody labelled FITC kit, were all the products of ZYMED, USA.

Medicine

Swainsonine (Batch No. 050923), prepared by the manufacturing laboratory of the Institute of Materia Medica in the Fourth Military Medical University (FMMU) with purity over 99%. The molecular formula of the compound is [C.sub.8H.sub.15NO.sub.3]. Cisplatin (CDDP) came from Jiangxi Province East Asia Co. Ltd, Batch No. 050509.

Cell and animals

C6 glioma cells were provided by the Institute of Neurobiology of PLA. Healthy, male SD rats (certificate No. 04-018) and weighting 260g[+ or -]30g each were purchased from the Experimental Animal Center of FMMU at 4-6 weeks of age, and were maintained in the animal facility of the Institute of Materia Medica according to the animal Experimental Ethics Committee guidelines.

Growth inhibition in vitro

C6 glioma cells were seeded in a 96-well plate in logarithmic growth phase; the medium was discarded and replaced with fresh medium containing swainsonine at different concentrations, as follows: 5.12, 2.56, 1.28, 0.64, 0.32, 0.16, 0.08, 0.04, 0.02 and 0.01 [micro]g/ml. The blank group was constituted simultaneously as zero (adding culture medium only), along with a negative control group (inoculating C6 glioma cell and adding culture medium only, no medication), with 8 parallel wells in each group. Cells were incubated at 37[degrees], 5% [CO.sub.2] for 24 h, 48 h and 72 h, respectively, as described previously (Sun et al. 2007).

Cell cycle and apoptosis by flow cytometry

C6 glioma cells in logarithmic growth phase were treated with swainsonine at doses of 1.6, 0.4 and 0.1 [micro]g/ml and CDDP of 3 [micro]g/ml as the positive group. The relative change in the mean fluorescence intensity was detected by FCM (Becton Dickinson) as described previously (Sun et al. 2007).

We collected glioma cells of different groups, and modulated the concentration of cells to 1 x [10.sup.9] cells [l-.sub.1], washing with PBS twice and then suspended the cells in 200 [micro]l balanced solution, added 10 [micro]l Annexin-V-FITC and 5[micro]l PI away from light at 4[degrees]C for 30 min, together with 300 [micro]l balanced solution. In each measurement, a minimum of 6000 cells was analyzed by FCM.

Fluorescene immunohistochemistry

Selected C6 glioma cells in logarithmic growth phase and inoculated culture dishes, fitted with 6 mm x 6 mm coverslips, were treated with swainsonine according to the doses above for 24 h. We prepared the dyeing kit according to manufacturer's instructions. Taking green fluorescene intra-cellular particles as positive, we counted the rate of positive cells in 5 fields randomly and compared the percentage of positive cells of every group.

Measurement of the concentration of [Ca.sup.2+].sub.i]] of C6 glioma cells by LSCM

We cultured the C6 glioma cells to logarithmic growth phase, and treated the cells as described above for 24 h. Dropwise were added 2 ml Fluo-3/AM into 1000 ml DMSO, after which the cells were poured into D-Hanks, diluted to final concentrations of 5 [micro]mol/1 Fluo-3/AM. Culture medium was replaced, followed by incubation at 37 [degrees]C away from light for 45 min. We then removed the epipolic staining solution before determination, washing with 2 ml D-Hanks twice, and adding 2 ml D-Hanks again. After reaction, we chose 8, C6 glioma cells with good shape and adherence from each group, as the cells must maintain fluorescence stably for 5 min in order to observe the concentration of [Ca.sup.2+].sub.i]] of C6 glioma cells by LSCM (the wavelength of excitation wave is 488 514 nm).

C6 glioma model

A healthy, male SD rat model was established by injecting 2 x [10.sup.6] /[mu]l C6 glioma cells into rats' right caudate nuclei, using a stereotaxis instrument according to Barker's method. 50 tumor-bearing rats were randomly divided into five groups of 10 rats each, l0 d later. Rats of the model group were administered normal saline and the positive control group received CDDP (2 mg/kg body wt., i.p., twice weekly). Treatment groups were administered swainsonine at doses of 8, 4 and 2 mg/kg body wt. All animals were put to death after being treated for 21 days. After measuring tumor weight, the tumor inhibition rate was calculated according to the following formula: IRTW = (tumor weight of control group/tumor weight of treated group-1)x 100%, along with observing the pathological changes of the tumor tissue using serial sections.

Statistic analysis

Data were expressed as mean [+ or -] S.D. Statistical analysis was performed using statistical software SPSS 10.0. ANOVA was used for analyzing statistical differences between groups under different conditions. p < 0.05 was considered statistically significant.

Results

Suppressive effect of swainsonine on glioma cells in vitro

An MTT experiment confirmed that C6 glioma cells were sensitive to swainsonine, and swainsonine inhibited the growth and proliferation of C6 glioma cells significantly. The suppressive effect of swainsonine was dose-dependent. The dose of complete suppression at 24 h was 1.28[micro]g/ml, with an [IC.sub.50] value after 24 h of 0.05 [micro]g/ml.

Inhibition of swainsonine on glioma cell cycle and apoptosis

The proportion of S phase in the normal growth of glioma cells was low. Treated with swainsonine for 24 h, the proportion of S phase increased substantially over that of the control group. With the increase in swainsonine's concentration, it was found that the percentages of S phase were 11.3%, 11.6% and 12.4%, respectively (7.7% in control group and 8.5% in CDDP group), and also that the expression of apoptosis inhibiting [p.sup 53] and [p.sup.l6] protein decreased gradually (p < 0.05 orp < 0.01).

A cytogram made up of four quadrants was obtained using FCM. The results suggested living cells and a few apoptotic and damaged necrotic cells in the control group. Treated with swainsonine at different dosages, the normal cells decreased remarkably, while the proportion of apoptotic cells and necrotic cells increased significantly. Along with the increased dosage, viable apoptotic cells decreased, but the proportion of nonviable apoptotic and necrotic cells increased obviously. The group treated with CDDP at doses of 0.4 and 1.6 [micro]g/ml showed activity similar to the group treated with swainsonine.

Effects on [p.sup.l6] and p53 gene of glioma induced by swainsonine

It was clear that the nucleolus or cytoplasm of positive cells appeared yellowish-brown, and the non-stained cells showed only coeruleus nucleolus under microscopic examination. Treated with swainsonine for 24h, the expression of [p.sup.l6] and p53 decreased significantly, especially at the dosage of 0.4([micro]g/ml. Compared with the control group, the expression of [p.sup.16] and p53 in the CDDP group was evidently different, and the proportion of positive cells increased markedly.

[[Ca.sup.2.sup.+]sub.i] in C6 glioma cell by LSCM induced by swainsonine

Experimental results indicated that [[Ca.sup.2.sup.+]sub.i], raised significantly, and a dose-effect relationship was clear. Compared to the control group 10 min after addition of swainsonine at dosage of 1.5mg/ml, [[Ca.sup.2.sup.+].sub.i] decreased significantly (p < 0.01).

In vivo suppression of swainsonine in rats C6 glioma cells

The general state of the rats inoculated with C6 glioma cells was fine. The tumor volume and weight decreased clearly in treatment groups compared to that of the control group. Inhibition rates of tumor weight (IRTW) of every group administrated with swainsonine at the doses of 1.6, 0.4 and 0.1 [micro]g/ml and with CDDP were 50.0%, 54.7%, 48.6% and 49.3%, respectively. Dissection showed the peplos of tumor tissue was integrated and showed no evidence of infestation or metastasis. Simultaneously, its model color was ruddy and the texture was stiff, with no obvious bleeding or necrosis. HE dyeing suggested a small quantity of phlegmonosis cells infiltrating and no obviously bleeding band. Tumor tissue of all treated groups showed gray, the texture was soft, with necrosis and hemorrhagic internally focused. HE dyeing showed a great quantity of phlegmonosis cell infiltration and a clear bleeding band.

Discussion

Swainsonine has been confirmed to have inhibitory and antimetastatic effects in human tumors including hepatoma, spongioblastoma cell, breast carcinoma, melanoma cell and pate malignant tumors (Rooprai et al. 2001; Van den Elscn et al. 2001) as well as [S.sub 180] ascites tumors (Kino et al. 1985). Oredipe et al. (2003) verified that prophylactic treatment of rats with swainsonine by continuous administration for 10 days decreased the death rate significantly when swainsonine was administered along with chemotherapeutics (adria-mycin at the dosage of the [LD.sub.50] value). The same authors confirmed that the effect is owed to accelerated proliferation and differentiation of haemopoietic stem cells, upgrading the level of WBC, maintaining the balance of each strain cell in the blood, which together suggest that swainsonine is also a significant immuno-modulator. Huxtable and Dorling (1985) observed that rats treated with high doses of swainsonine (about 15 mg/day) for 100-200 days developed neuronal mannosidase storage disease only in areas of the brain not protected by the blood/brain barrier.

Since swainsonine was isolated, research has focused mainly on its toxicity and only seldom on its antineoplastic activity, mechanisms or clinical application. The toxic mechanism of the action of swainsonine is related to the biological activity of calystegines. It has only been recognized that calystegines [B.sub.2] and [C.sub.1] are inhibitors of [beta]-glucosidase and a- and [beta]-galactosidases, respectively. In addition, it has been reported that these inhibitory activities of calystegines [B.sub.2] and [C.sub.1] could produce phenocopies of the genetic lysosomal storage (Gaucher's and Fabry's) diseases. Swainsonine may inhibit Golgi [alpha]-mannosidasc II and change the structure of oligosaccharides of the cell surface, together with the expression of special membrane glucoproteins (Suzuki et al. 2004). A few researchers have also suggested that swainsonine may produce a marked effect indirectly through immunological regulation (Yagita and Saksela 1990). This article and our prior research indicate that the antitumor action of swainsonine may result from the co-operation of many pathways.

During cell cycle progression, pRb-assembled complexes are disrupted by CDKs/cychns-mediated phosphorylation, leading to release of E2F. The free transcription factor E2F then activates the genes responsible for cellular proliferation by progression through the Gl. Mitogenic signals start the upward regulation of CDK4(6)/cyclin D complexes that lead to pRb phosphorylation in mid-G sub 1. Later, the action of CDK sub 2/cycIin E results in the complete hyperphosphor-ylation of [p.sup.Rb]. [p.sup.l6],[p.sup.21] and p sup 27 block CDK4/cyclin D and CDK2/cyclin E activities, leading to the accumulation of hypophosphorylated [p.sup.Rb] (Harbour and Dean 2000). They also regulate the activity of CDKs, which in turn regulate the phosphorylation of [p.sup.Rb] and generally inhibit cell cycle progression. P16 gene is a kind of multiple tumor suppressor gene, with a negative regulation effect on cell growth. Its deletion and mutation in relation to the genesis and development of malignant tumor is currently a hot research topic. As a group of anti-oncogenes, [p.sup.l6] is more impressionable than the Rb, [sup. 53] gene in the cell cycle. Many experiments have demonstrated that the depletion rate of [p.sup.16] protein increases along with the extent of brain malignant tumor. More recent studies have also shown that [p sup l6, another key cell cycle regulator and important tumor suppressor, is overexpressed in C sub 6 glioma (Kuo et al. 2006).

We primarily observed the expression of p 16 in this study. It has all too frequent deletion and mutation in many tumor cells and specimens in clinic, especially in glioma. Our experiments showed the important role of glioma in malignant transformation: p sup 53] could make the impaired deoxyribonucleic acid blockage at [G.sub.1] phase and reentrance cell cycle after recovered. If the damage is not recovered, the expression level of p53 increased, switching on the point of apoptosis. By using Annexin-V and PI together, we also distinguished the normal, apoptotic and necrotic cells. Using this method, we demonstrated that swainsonine could induce apoptosis at low doses and kill cells at high doses.

Most current medicines, and particularly non-cycle-specific medicines such as aklylating agents, antitumor antibiotics and DDP, influence the [G.sub.1] phase chiefly. S phase, as a limited period of DNA synthesis and replication, is of extremely active metabolism, in which many enzymatic systems participate. These characteristics make the S phase a good target for medication, but available drugs are deficient because of short-lived effect and/or severe side-effects. Swainsonine applied in this test proved that glioma cells are regulated mainly in the process of [S-G.sub.2] transition. That the tumor cells accumulate largely during the S phase correlates well with the reports of Myc et al. (1989). The mechanisms of swainsonine are possibly the results of [[Ca sup 2+]sub.i] overloading, along with the expression of apoptotic trigger gene and inhibiting gene induced to apoptosis. In addition, our study of the glioma cell cycle may point to further research directions for others interested in related antitumor medicines.

In conclusion, our results suggest that swainsonine is a promising new medicine for treating carcinoma, especially pate malignant tumor. As for the mechanism, we presume that swainsonine could promote the overloading of [[Ca sup 2 + ]sub.i] and the expression of apoptotic trigger gene and inhibiting gene, and then induce ER stress, mediate apoptosis of glioma cell in the final (Biagioli M et al. 2008; Ferreiro et al. 2008). Further studies are needed to better determine the contribution of these factors and the relationship among them.

Acknowledgements

The authors have no conflict of interest of any kind related to the work presented in this publication. The authors are very grateful to Yun-xin CAO (Dept of Immunology, FMMU) for her technical assistance. This study was supported by the Technology Department of Shaanxi Province.

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Ji-Yuan Sun (a), Hao Yang (b), Shan Miao (a), Ji-Peng Li (c), Si-Wang Wang (a), * Miao-Zhang Zhu (d), Yan-Hua Xie (a), Jian-Bo Wang (a), Zhe Liu (a), Qian Yang (a)

(a) Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi'an, PR China

(b) Institute of Neurobiology, Fourth Military Medical University, Xi'an, PR China

(c) Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, PR China

(d) Department of Physiology, Fourth Military Medical University, Xi'an, PR China

* Corresponding author. Tel.: + 86 2984772227; fax: + 186 29 83224790.

E-mail address: siwang_wang@ynhoo.com (S.-W. Wang).

0944-7113/$-see front matter [c] 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phynied.2009.02.012
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Title Annotation:SHORT COMMUNICATION
Author:Sun, Ji-Yuan; Yang, Hao; Miao, Shan; Li, Ji-Peng; Wang, Si-Wang; Zhu, Miao-Zhang; Xie, Yan-Hua; Wang
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Nov 1, 2009
Words:3650
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