Inhibition of the growth of human gastric carcinoma in vivo and in vitro by swainsonine.
In Europe, swainsonine has been studied widely for prevention of metastasis and cancer therapy. In order to investigate the effects and mechanisms of swainsonine on the human gastric carcinoma SGC-7901 cell, we carried out in vivo and in vitro experiments.
After treatment with swainsonine, an effective dose and [IC.sub.50] value of swainsonine for SGC-7901 cells were examined by MTT assay. Cell-cycle distribution and apoptotic rates were analyzed using FCM, and [[Ca.sup.2+]][.sub.i] was measured using LSCM. The expression of p53, c-myc and Bcl-2 were determined using an immunocytochemical method. Simultaneously, 50 mice were divided randomly into five groups. Three groups were administrated swainsonine at dose of 3, 6 and 12 mg/kg body wt., two control groups were administrated N.S. 20 ml/kg body wt. and 5-Fu 20 mg/kg body wt., respectively, by intraperitoneal injection. The inhibition rate was calculated and pathological sections were observed.
The growth of SGC-7901 cell is inhibited by swainsonine in vitro, with an [IC.sub.50] value at 24 h of 0.84[micro]g/ml, and complete inhibition concentration is 6.2 [micro]g/ml. After treatment with swainsonine at the concentrations of 0.5, 1.5 and 4.5 [micro]g/ml for 24 h, the expression of apoptosis inhibiting gene p53 and bcl-2 decreases, and the apoptotic trigger gene c-myc increases markedly (p<0.05), as well as [[Ca.sup.2+]][.sub.i] overloading, SGC-7901 cell is induced to apoptosis in the end. It is also found that the percentages of S phase are 38.8%, 39.7% and 29.6%, respectively (20.0% in control group and 23.2% in 5-Fu group). The rates of inhibition were 13.2%, 28.9%, 27.3%, respectively, when the nude mice were administered swainsonine (p<0.05 or 0.01). The structure of the tumor showed hemorrhage, necrosis and inflammatory cell infiltration. We therefore conclude that swainsonine could inhibit cell proliferation in vitro and the growth of human gastric carcinoma in vivo. The mechanisms of swainsonine-induced apoptosis may relate to [[Ca.sup.2+]][.sub.i] overloading and the expression of apoptosis-related genes.
[c] 2006 Published by Elsevier GmbH.
Keywords: Swainsonine; Gastric carcinoma; SGC-7901; Apoptosis; [[Ca.sup.2+]][.sub.i]
Swainsonine, a kind of water-soluble indole alkaloid and an [alpha]-mannosidase inhibitor which blocks Golgi oligosaccharide processing, represents a new class of compounds that could inhibit the growth and metastasis of tumors. It has received 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 was used to study glucoprotein N-link oligosaccharide as an instrument drug, since it was separated initially from the fruit of Australian Swainsona canescens and North America locoweed (including Astragalus and Oxytropis spp.) (Li et al., 2004; Douglas et al., 2005; Guetens et al., 2002; William et al., 2001). Swainsonine was verified to inhibit tumor growth and metastasis, enhance lethality of NK and LAK cells, and reduce the growth rate of human melanoma cells (Wang et al., 2003; Dennis, 1986; Dennis et al., 1990; Galustian et al., 1994). Initial clinical research confirmed that in pate malignant tumor and lymphangioma of the chest and abdomen, swainsonine has obviously curative effects (Goss et al., 1994). Nevertheless, research on antineoplastic activity and mechanisms of swainsonine remains scarce. In order to observe the functions mentioned above, we have detected that apoptosis of SGC-7901 cells induced by swainsonine, the apoptotic-related gene and concentration of [[Ca.sup.2+]][.sup.i], and have tried to explain the antineoplastic activity and mechanisms of swainsonine in vivo and in vitro.
Materials and methods
Materials: RPMI1640, fetal bovine serum was purchased from Gibco. MTT was obtained from Sigma. Annexin-V-FITC kit: Annexin-V-FITC and pyrimidine of iodinate (PI), subpackaged by the Department of Immunology. Mouse anti-human p53 monoclonal IgG, mouse anti-human c-myc monoclonal IgG and mouse anti-human bcl-2 monoclonal IgG, peroxidase labeling strepto-antibiotin (streptavidin/peroxidase) dyeing kit, are all the products of ZYMED, USA.
Medicine: Swainsonine (Batch No. 040508; Fig. 1), prepared by the manufacturing laboratory of the Institute of Materia Medica of the Fourth Military Medical University, with purity over 99%. Fluorouracil (5-Fu) was from Shanghai Xudong Haipu Pharmaceutical Co. Ltd., Batch No. 040209.
[FIGURE 1 OMITTED]
Cells and animals: SGC-7901 came from the Institute of Digestive Disease of PLA. BALB/c nu/nu mice (SPF grade, Certificate No. 08-014) of either sex weighing 18[+ or -]2 g each were purchased from the Experimental Animal Center.
Growth inhibition in vitro: To culture SGC-7901 cells in logarithmic growth phase, we added swainsonine at different concentrations, as follows: 20, 10, 5, 2.5, 1.25, 0.625, 0.30, 0.15, 0.08 and 0.04 [micro]g/ml; we simultaneously established the blank group as zero (adding culture medium only), negative control group (inoculating SGC-7901 cell and adding culture medium only, no medication) and 5-Fu of 10 [micro]g/ml as the positive group, with eight parallel wells in each group. Cells were incubated at 37 [degrees]C, 5% C[O.sub.2] for 24, 48 and 72 h, respectively. We added MTT (20[micro]l) to each well, and kept them culturing for 4h, after which we removed the culture medium carefully and added 150[micro]l DMSO to each well. The absorbances were measured after 10 min at 570 nm. We calculated the cell survival rate according to following formula: cell survival rate (%) = absorbance of treatment group/absorbance of control group x 100%. We then drew the growth curve and calculated the [IC.sub.50] value.
Cell cycle: We inoculated and cultured the SGC-7901 cell to logarithmic growth phase, treated with swainsonine at the doses of 4.5, 1.5 and 0.5[micro]g/ml respectively, 5-Fu 10[micro]g/ml as the positive group, and a negative control group (inoculated SGC-7901 cells only). After incubating at 37 [degrees]C, 5% C[O.sub.2] for 24 h, the cells were collected, rinsed with PBS twice, suspended again with 75% ethanol and left overnight at 4 [degrees]C. Before examination, cells were centrifuged, the alcohol was removed, and they were rinsed and modulated to 1 x [10.sup.6]/ml We added staining solution containing RNase and PI and left them in a dark place for 30min, then detected by FCM at the wavelength of 488 nm.
Apoptosis: We collected SGC-7901 cells of different groups, modulated the concentration of cells to 1 x [10.sup.6]/ml, washed them with PBS twice and then suspended the cells in 200 [micro]l balanced solution. We added 10[micro]l Annexin-V-FITC and 5[micro]l PI away from light at 4 [degrees]C for 30min, added 300[micro]l balanced solution, then detected by FCM immediately. From every group, 6000 cells were measured.
Immunocytochemistry: We selected SGC-7901 cells in logarithmic growth phase, and using inoculated culture dishes placed with 6 x 6 mm coverslips, treated the cells with swainsonine according to the doses mentioned above for 24 h. These were treated according to the directions of the dyeing kit. Taking brown particles intra-cellular as positive, we counted the rate of positive cells in five fields randomly and compared them with the percentage of positive cells of every group.
[[Ca.sup.2+]][.sub.i]: We cultured the SGC-7901 cells to logarithmic growth phase, and treated the cells as described mentioned for 24 h. We added, drop-wise, 2 ml Fluo-3/AM into 1000 ml DMSO, then poured it to D-Hanks, diluted to a final concentration of 5 [micro]mol/L Fluo-3/AM, incubated for 45 min at 37 [degrees]C away from light, removed the epipolic staining solution before determination, washed twice and added 2 ml D-Hanks again. We chose eight cells with good shape and firm adherence from every group. The cells also had to maintain fluorescence value stably for 5 min, observed at the concentration of [[Ca.sup.2+]][.sub.i] of SGC-7901 cells (the wavelength of excitation wave is 488-514nm).
Tumor implanted into nude mice: Transplanted tumor models were established by injecting 1 x [10.sup.9]/L human gastric cancer cells into subcutaneous tissues of the forelimbs of nude mice. Fifty tumor-bearing mice were divided into five groups randomly, 10 days later. The model group was administrated normal saline at the dose of 0.4ml/20 g body wt. and the positive control group received an intraperitoneal injection of 5-Fu at 15mg/kg body wt. Treatment groups were administrated swainsonine at different concentrations at the doses of 12, 6 and 3mg/kg body wt., respectively. The animals were put to death after being treated for 21 days and pathological changes were observed. We measured the tumor weight [tumor inhibition rate (IR) was calculated according to the following formula: IR = (tumor weight of the control group/tumor weight of treated group-1) x 100%], making serial sections and observing the changes.
[FIGURE 2 OMITTED]
Statistic analysis: Data were expressed as mean [+ or -] [??] s.e.m. Statistical analysis was performed using the statistical software SPSS10.0. ANOVA was used to analyze statistical differences between groups under different conditions. p< 0.05 was considered statistically significant.
Suppressing effect of swainsonine on SGC-7901 cells in vitro
The MTT experiment confirmed that SGC-7901 cells were sensitive to swainsonine, and swainsonine inhibited the growth and proliferation of SGC-7901 cells significantly. The suppressing effect of swainsonine showed to be dose dependent (Fig. 2). The dose of complete suppression at 24 h was 6.2 [micro]g/ml; the [IC.sub.50] value at 24 h was 0.84 [micro]g/ml.
Inhibition of swainsonine on SGC-7901 cell cycle and apoptosis: The proportion of S phase in the normal growth of SGC-7901 was low. Treated with swainsonine for 24 h, the proportion of S phase increased substantially more than that of the control group (p<0.05). Especially at the dosage of 1.5 [micro]g/ml, the percent of S phase increased to 39.7%. The cell population in the [G.sub.0]/[G.sub.1] phase reduced to some extent. Compared to the control group, the cell population of treated groups in the M/[G.sub.2] phase was also clearly reduced. The proportion of the M/G2 phase reduced to 1.7% (Fig. 3).
The cytogram in Fig. 4, 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 doses, the normal cells decreased clearly, while the proportion of apoptotic and necrotic cells increased significantly. Along with the dosage increased, viable apoptotic cells decreased, and the proportion of nonviable apoptotic and necrotic cells increased obviously. The group treated with 5-Fu showed similar activity on SGC-7901 cells to the group treated with swainsonine at the doses of 1.5 and 4.5 [micro]g/ml.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Effect on p53, c-myc and Bcl-2 gene of SGC-7901 induced by swainsonine: It was clear that the nucleolus or cytoplasm of the positive cell showed yellowish-brown, and the non-staining cells only showed coeruleus nucleolus by microscopic examination. Treated with swainsonine for 24 h, the expression of p53 decreased significantly, and took on a dose-effect relationship. Compared with the control group, the expression of p53 in the 5-Fu group was evidently different (p<0.05). The loci appearing yellowish-brown induced by c-myc gene were situated mainly in the nucleolus and slightly in the cytoplasm. Treated with swainsonine and 5-Fu, the proportion of positive cells increased greatly, their color darkened, and the standard of positive expression for Bcl-2 was a yellowish-brown particle diameter on cytoplasm, karyolemma and a few cell membranes. It was thus evident that a proportion of positive expression for Bcl-2 decreased obviously after medication, and the proportion between swainsonine groups showed no statistical difference (Fig. 5).
[FIGURE 5 OMITTED]
Effect of [[Ca.sup.2+][.sub.i] of SGC-7901 induced by swainsonine: Experimental results indicated that [[Ca.sup.2+]][.sub.i] raised significantly (p<0.01), and a dose-effect relationship was clear. As compared to the control group, 10min after addition of swainsonine at dosage of 1.5[micro]g/ml [[Ca.sup.2+]][.sub.i]was significantly decreased (p<0.01).
Suppression of SGC-7901 by swainsonine in vivo: The general state of the mice inoculated with SGC-7901 cells at the hypodermic was fine. The accrescence of tumor volume in treatment groups was reduced compared to that of the control group. Dissection showed that the peplos of tumor tissue was integrated and showed no evidence of infestation or metastasis. Compared with the control group, the tumor weight decreased clearly (p<0.05 or 0.01). Simultaneously, a peripheral hemogram in 5-Fu group decreased significantly. Dissection of the tumor showed that its model group color was ruddy and the texture was stiff, with no obvious bleeding or necrosis. HE dyeing suggested a small quantity of phlegmonosis cell infiltration and no obviously bleeding band. Tumor tissue of all treated groups showed gray, the texture was soft, necrosis and hemorrhagic were internally focused. HE dyeing showed a great quantity of phlegmonosis cell infiltration and clear bleeding band.
Swainsonine has been confirmed to have inhibitory and antimetastatic effects in human tumors including hepatoma, spongioblastoma cell, breast carcinoma, melanoma cell and pate malignant tumor (Rooprai et al., 2001; Mohla et al., 1990; Humphries et al., 1986; Newton et al., 1989; Van den Elsen et al., 2001) and [S.sub.180] ascites tumor (Kino et al., 1985). OA Oredipe (Oredipe et al., 2003) verified that prophylactic treatment with swainsonine by continuous administration to rats for 10 days could decrease the death rate significantly when administered with chemotherapeutics (adriamycin at the dose of the [LD.sub.50] value) and 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, and suggesting that swainsonine is also a commendable immunomodulator.
Since swainsonine was isolated, study has concentrated mostly on its toxicity and seldom on its antineoplastic activity, mechanisms or clinical application. The mechanisms of swainsonine may inhibit Golgi [alpha]-mannosidase II, change the structure of oligosaccharide of the cell surface, and then change 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; Yagita et al., 1992; Dimitroff et al., 2003). This article and our prior research indicate that the antitumor action of swainsonine may result from co-operation of many pathways.
Using Annexin-V and PI together, we 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.
Apoptosis is gene-regulated. We observed mainly the expression of p53, c-myc and bcl-2 in this study. p53 could make the impaired deoxyribonucleic acid blockage at [G.sub.1] phase and reentrance cell cycle recover. If the damage is not recovered, the expression level of p53 increased, switching on the point of apoptosis. The function of c-myc gene is much more complicated. It can stimulate cell growth and induce cell apoptosis; its function is decided by obtaining pivotal growth factor. Bcl-2 is believed to inhibit or delay cell apoptosis caused by many factors, and the function may be to clean up active oxygen and free radicals and alleviate damage by oxygen and free-radical-induced apoptosis through changing the function of chondriosome (Heiser et al., 2004; Piro, 2004).
[[Ca.sup.2+]][.sub.i], as the second intra-cellular messenger of signal transduction, participates in many kinds of physiological activity, such as muscle contraction, nerve conduction, cell proliferation and differentiation. High level [[Ca.sup.2+]][.sub.i] interferes severely with the mitochondrial oxidative phosphorylation, down-regulates production of adenosine triphosphate, and even leads to mitochondrium disorganization, activating phospholipase and encouraging membrane phospholipid hydrolization. This in turn causes cause cellular membrane and cellular organ damage, activating proteinase free radical multiplication, or expression of gene, induce to apoptosis or death. Therefore, [[Ca.sup.2+]][.sub.i] overloading is the final passageway in cell death. In addition, the level of [[Ca.sup.2+]][.sub.i] regulates the intercellular communication induced by promoter and growth factor. The relation of intercellular communication to tumor development is intimate. Some oncogenes and chemical materials switch on the target of development of tumors. In combination with LSCM, this experiment selected Fluo-3 as the fluorescent indicator. The results show that swainsonine makes [[Ca.sup.2+]][.sub.i] overload in the SGC-7901 cells, and the overloading of [[Ca.sup.2+]][.sub.i] is probably one of the important apoptotic mechanisms induced by swainsonine.
Most medicines, especially cycle-non-specific medicines such as alkylating agents, antitumor antibiotics, DDP, etc., chiefly influence [G.sup.1] phase. The S phase, as a limited period of DNA synthesis and replication, is of extremely active metabolism, in which many enzymatic systems participate. These characteristics provide good targets for medication, but this kind of drug is still deficient due to short duration of positive effects and severe chemical side effects. Swainsonine applied in our test proved that the SGC-7901 cell regulated mainly at the S-G2 transition. This makes the tumor cell accumulate largely during the S phase. Our results agree with reports by Myc et al. (1989). The mechanisms of swainsonine are possibly the results of [[Ca.sup.2+]][.sub.i] overloading and the expression of the apoptotic trigger gene and inhibiting gene induced to apoptosis. The study of cell-cycle inhibition for SGC-7901 cells provides a few ideas for others in researching related antitumor medicines.
We are grateful to Yun-xin CAO for her technical assistance and extend special thanks to Yong-quan SHI for the donation of SGC-7901.
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J.-Y. Sun (a), M.-Z. Zhu (b), S.-W. Wang (a,*), S. Miao (a), Y.-H. Xie (a), J.-B. Wang (a)
(a) Institute of Materia Medica, The Fourth Military Medical University, Xi'an, 710032 Shaanxi, China
(b) Department of Physiology, The Fourth Military Medical University, Xi'an, Shaanxi, China
Received 2 May 2006; accepted 21 June 2006
*Corresponding author. Tel.: +8629 8477 4748; fax: +8629 8322 4779.
E-mail address: firstname.lastname@example.org (S.-W. Wang).
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|Author:||Sun, J.-Y.; Zhu, M.-Z.; Wang, S.-W.; Miao, S.; Xie, Y.-H.; Wang, J.-B.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Article Type:||Clinical report|
|Date:||May 1, 2007|
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