- Email: [email protected]
Increased antitumor efficacy by the combined administration of swainsonine and cisplatin in vivo
Phytomedicine 18 (2011) 1096–1101
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Increased antitumor efficacy by the combined administration of swainsonine and cisplatin in vivo Felipe M. Santos a , Andreia O. Latorre a , Isis M. Hueza b , Daniel S. Sanches a , Luciana L. Lippi a , Dale R. Gardner c , Helenice S. Spinosa a,∗ a
Department of Pathology, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, SP 05508-270, Brazil Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo (UNIFESP), Diadema, SP, Brazil c Poisonous Plant Research Lab, N. Logan, UT 84341, USA b
a r t i c l e
i n f o
Keywords: Cancer chemotherapy Cis-diamminedichloroplatinum (II) Golgi ␣-mannosidase II ␣-Mannosidase inhibitor Astragalus lentiginosus Cell cycle Swainsonine
a b s t r a c t Swainsonine is a natural ␣-mannosidase inhibitor found in numerous poisonous plants, such as Astragalus lentiginosus. Its mechanism of action is through the inhibition of Golgi ␣-mannosidase II activity in the N-glycan biosynthesis pathway. As a result, swainsonine inhibits the production of complex ˇ1,6branched N-linked glycans, which are related to the malignant phenotype of tumor cells. In this study, we investigated whether treatment with swainsonine affects the sensitivity of Ehrlich ascites carcinoma (EAC) cells to cisplatin. To this end, male C57BL/6 mice were treated with swainsonine (SW – 0.5 mg/kg, i.p., twice-daily for ten days) and/or cisplatin (Cis – 0.25 mg/kg, i.p., every other day for a total of five applications) two days after transplantation with EAC cells. The results showed a greater reduction in the ascites volume in mice from the CisSW group (63.5%) than in mice from the Cis group (45.7%), an elevated induction of apoptosis by CisSW treatment when compared to Cis alone, as demonstrated by higher percentage of cells in the subG1 phase in that group (p < 0.0001 Kruskal–Wallis, p < 0.0001 control vs. CisSW, p < 0.001 Co vs. Cis post-test Dunn), and an increase in the median survival from 12.5 days observed in the control group to 27 days in the CisSW group, which corresponds to a 116% survival increase (p = 0.0022 Co vs. CisSW Log-rank test). In addition, the mice from the Cis group had a median survival of only 15 days, an increase of just 20% compared to controls. Our results indicate that swainsonine increases the sensitivity of EAC cells to cisplatin. © 2011 Elsevier GmbH. All rights reserved.
Introduction Swainsonine is a natural ␣-mannosidase inhibitor that is found in numerous poisonous plants, such as Astragalus lentiginosus (Molyneux and James 1982). Swainsonine has been reported to be a promising antitumor agent and its molecular formula is C8 H15 NO3 (Fig. 1). It is an effective inhibitor of both lysosomal ␣-mannosidase and Golgi ␣-mannosidase II (Gerber-Lemaire and Juillerat-Jeanneret 2010). Swainsonine in cancer therapies has been linked to the inhibition of Golgi ␣-mannosidase II by blocking the production of the complex ˇ1,6-branched N-linked glycans (Wrodnigg et al. 2008). In fact, the expression of complex ˇ1,6branched N-linked glycans on the cell surface has been associated with the malignant phenotype (Fuster and Esko 2005). Therefore,
∗ Corresponding author at: Department of Pathology, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Orlando Marques de Paiva 87, São Paulo 05508-270, Brazil. Tel.: +55 11 3091 7656; fax: +55 11 3091 7829. E-mail address: [email protected] (H.S. Spinosa). 0944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2011.06.005
glycosylation pathways are a promising target for novel cancer therapies. Glycoproteins are proteins with one or more covalently bound glycans. These proteins can regulate important cellular functions, such as cell–cell communication, cell growth, migration and signal transduction. Most of the glycoproteins in the cell are Asn-linked (N-linked) oligosaccharides. Integrins, receptors and extracellular matrix (ECM) ligands display altered pattern of N-linked glycosylation in cells undergoing a malignant transformation (Janik et al. 2010). In addition, there is an increasing body of evidence that suggests that tumor cells, which overexpress some glycosyltranferases, synthesize glycans that facilitate processes such as invasion and metastasis (Fuster and Esko 2005; Zhao et al. 2008). Swainsonine has been shown to reduce tumor cell invasion in Phase I human cancer trials and to enhance the natural antitumor defenses of the body, such as lymphokine activated killer cell (LAK) and natural killer cell (NK) cytotoxicity (Galustian et al. 1994; Goss et al. 1994). However, in the Phase II trials, no antitumor activity was observed in patients with locally advanced or metastatic renal cell carcinomas, and the treatment was discontinued due to disease progression and the toxicity of the compound (Shaheen
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
Treatment protocol
N
OH
H
1097
OH
OH
Fig. 1. Chemical structure of swainsonine.
et al. 2005). Analogues of swainsonine have been shown to have a reduced toxicity, but they also have a diminished antitumor effect (Gerber-Lemaire and Juillerat-Jeanneret 2010). However, swainsonine has been shown to increase tumor cell sensitivity to cytotoxic drugs and to reduce resistance to anticancer drugs in multistage colorectal cancer cells in vitro (Hamaguchi et al. 2007). Therefore, the aim of the current study was to determine if swainsonine could increase the sensitivity of Ehrlich carcinoma cells to cisplatin (cis-diamminedichloroplatinum (II)), a platinum-based chemotherapeutic, in vivo. Materials and methods Mice Eighty-seven sixty-day-old male C57BL/6 mice, bred in the Department of Pathology at the School of Veterinary Medicine and Animal Sciences, University of São Paulo, were used. The mice were maintained under controlled temperature conditions (22–25 ◦ C), relative humidity (50–65%) and lighting (12-h/12-h light/dark cycle). Drinking water and standard diet (NuvilabCR1® , Nuvital Nutrientes LTDA) were provided ad libitum. All procedures were performed following the Guide for the Care and Use of Laboratory Animals, NIH publication no. 8523 (http://www.nap.edu/readingroom/books/labrats/), and were reviewed and approved by the Bioethics Committee of the FMVZUSP (process #1161/2007). Chemicals Propidium iodide (PI) was obtained from Sigma Chemical Co. (St. Louis, MO). RNAse A was obtained from Invitrogen (Carlsbad, CA). Dopalen (ketamine) and Anasedan (xylazine) were obtained from VetbrandsTM (São Paulo, Brazil). Platistine® CS (cisplatin) was obtained from Pfizer (São Paulo, Brazil), and EDTA was obtained from Merck (Darmstadt, Germany). Swainsonine Swainsonine (Fig. 1) was purified from dry A. lentiginosus aerial parts using a previously described method presented by Gardner et al. (2003). Tumor model Intraperitoneal Ehrlich ascites carcinoma (EAC) cells syngeneic to BALB/c mice were used in the study. Therefore, EAC cells were transplanted intraperitoneally (i.p.) into the C57BL/6 mice, and just the fluid transplant generations that were passed over 12 times, provoking the formation of regular exudates, as described previously by Klein (1951), were used in all subsequent experiments. In all experiments, tumor transplantation was performed by i.p. injection of 2 × 107 cells suspended in 200 l of sterile PBS.
Fifty-five mice were randomly allocated into the following groups: negative control (NC), control (Co), cisplatin alone (Cis), cisplatin and swainsonine (CisSW) and swainsonine alone (SW). Mice from the NC group were neither inoculated with EAC cells nor received any treatment, whereas the mice from all other groups were inoculated with EAC cells, as mentioned above, two days before the start of the different treatments. The mice in the Cis and CisSW groups were i.p. injected with 0.25 mg/kg of cisplatin every other day for a total of five applications. The mice in the SW and CisSW groups were i.p. injected twice a day with 1.0 mg/kg of swainsonine, as proposed by Oredipe et al. (2003), for 10 consecutive days. The mice in the Co group were i.p. injected twice a day with PBS for 10 consecutive days. Throughout the various applications every day, the body weight of each mouse was measured each day, and the dose was adjusted accordingly. Tumor cell evaluation On day 13 of the experiment, control and experimental mice were subjected to intramuscular anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg). Ascites fluid was collected, and the volume was quantified. The EAC cells were counted using the Trypan Blue assay to calculate the total viable and inviable cells. One million EAC cells from each mouse were fixed and preserved in 70% ethanol for up to one week at −20 ◦ C for cell cycle analysis. Cell cycle analysis Cell cycle analysis was performed using one million EAC cells from each mouse that were fixed and preserved in 70% ethanol for up to one week at −20 ◦ C. The cells were washed twice in PBS (5 min, 1200 rpm) and incubated with 200 l of PI solution (20 g/ml propidium iodide, 200 g/ml RNAse A and 0.1% Triton (v/v) in PBS) for 15 min at 37 ◦ C. Then, 10,000 events were acquired from each sample in a FACS Calibur Flow Cytometer equipped with Cell Quest Pro® software (Becton Dickinson [BD] Immunocytometry System). Data analyses were performed using FlowJo 7.2.2® software (Tree Star Inc., Ashland, KY) as shown in Fig. 2A and B. Blood samples, biochemical parameters and histological evaluation On day 13 of the experiment, each mouse was anesthetized (ketamine (100 mg/kg) + xylazine (10 mg/kg)), and ascites fluid was collected prior to blood collection by cardiac puncture. Blood was collected in a 1.0 ml syringe and was separated into two vials, one containing 10 l of EDTA to perform a complete blood count and another to separate the serum for posterior biochemical analyses. The complete blood count was determined using a Vet ABC Animal Blood counter (Horiba ABX Diagnostics). Forty-five minutes after the blood samples were collected, serum was separated by centrifugation at 4000 rpm for 10 min and placed at −20 ◦ C until it was needed. Biochemical analyses (creatinine, urea and alanine transaminase (ALT)) were performed using a CELM SBA-200 (CELM). Liver and kidney were collected for histopathological evaluation. Histopathological analysis was performed on hematoxylin and eosin (HE) stained sections that were five microns thick. Survival analysis For the survival analysis, 32 mice were randomly allocated into the following groups: Co, Cis, CisSW and SW. The tumor inoculation and treatment were performed as mentioned above and the mice
1098
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
Fig. 2. Cell cycle analysis. (A) FL2-Width vs. FL2-Area dot plot showing a single gate, which excludes aggregates. (B) FL2-A DNA histogram of EAC cells, which has been gated to exclude aggregates.
were observed daily until death or for 60 days after the treatments were completed. Statistical analysis The data were analyzed using GraphPad Prism 5.00® software (GraphPad Software, Inc., San Diego, CA) using one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Percentage data from three or more groups and data presenting significant differences among the standard deviations (SDs) were compared using the Kruskal–Wallis test followed by Dunn’s test. Data with two variables were analyzed by two-way ANOVA followed by the Bonferroni test. Survival curves were compared by Log-rank test. Data are expressed as the mean ± SD, and differences were considered statistically significant at p < 0.05.
Effects of swainsonine and/or cisplatin on complete blood count, biochemical parameters and organ histology evaluation of the EAC-bearing mice
Results Effects of swainsonine and/or cisplatin on body weight gain, ascites volume and total EAC cell count of EAC-bearing mice Some mice that were inoculated with EAC cells for this experiment did not develop tumor ascites, and those mice were eliminated from all subsequent analyses. All groups of mice that were inoculated with EAC cells had an increase in body weight when compared to the negative control (NC) group. However, the increase in body weight was smaller in the mice from the CisSW group than in the Cis and SW treatment groups (Fig. 3).
Body weight gain 45
NC Co Cis CisSW SW
g
40 35 30 25
2
3
4
5
6
7
8
9
10
In line with this finding, a significant reduction in ascites volume of 63.5% was observed in mice that were treated with swainsonine plus cisplatin (CisSW group), whereas cisplatin alone (Cis group) reduced the ascites volume by 45.7%, compared to the Co group (p < 0.0001 Kruskal–Wallis test, p < 0.001 Co vs. CisSW, p < 0.01 Co vs. Cis, p < 0.05 CisSW vs. SW post-test Dunn) (Fig. 4A). We observed a significant reduction in total Ehrlich ascitic cell count in mice from the CisSW and Cis groups (p < 0.0001 Kruskal–Wallis test, p < 0.001 Co vs. Cis and Co vs. CisSW, p < 0.01 CisSW vs. SW, p < 0.05 Cis vs. SW post-test Dunn) (Fig. 4B). This reduction in the total count was due to a significant reduction in viable and inviable EAC cells observed in both the CisSW and Cis groups (p < 0.0001 Two-way ANOVA, viable cells p < 0.001 Co vs. Cis and Co vs. CisSW, and inviable cells p < 0.05 Co vs. Cis and Co vs. CisSW post-test Bonferroni) (Fig. 4C).
11
Days Fig. 3. Body weight gain of EAC-bearing mice treated with swainsonine (SW), cisplatin (Cis) and combined therapy (CisSW) for 10 days. Mice treated with the combined therapy (CisSW) showed less body weight gain throughout the treatment period compared with mice from the negative control (NC) group (NC = 11, Co = 10, Cis = 11, CisSW = 8, SW = 10).
Complete blood count was unaffected by treatment with swainsonine and/or cisplatin (Table 1). Furthermore, histological evaluation of the livers and kidneys recovered from each swainsonine and/or cisplatin-treated mouse revealed no differences when compared to the samples obtained from the control mice (data not shown). For the biochemical parameters, a reduction in ALT concentrations in the serum from the mice treated with the combined therapy (CisSW) compared with the mice treated with either swainsonine (SW) or no treatment (NC) (p = 0.0048 Kruskal–Wallis test, p < 0.05 NC vs. CisSW and CisSW vs. SW post-test Dunn) was observed. Moreover, we observed a higher urea level in the mice from the Co group compared with the mice in the NC group (p = 0.002 one-way ANOVA, p < 0.01 NC vs. Co post-test Dunnett) (Table 2). Effects of swainsonine and/or cisplatin on the EAC cell cycle An elevated induction of apoptosis was observed in mice treated with swainsonine and cisplatin compared to mice that received cisplatin alone. This result was based on data showing that the CisSW group had a higher percentage of cells in the subG1 phase (p < 0.0001 Kruskal–Wallis test, p < 0.001 Co vs. Cis, p < 0.0001 Co vs. CisSW post-test Dunn). Consequently, we observed a significant reduction in the percentage of cells in the G0/G1, S and G2 phases in both the CisSW and Cis groups (Fig. 5).
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
1099
Table 1 Complete blood count of the EAC-bearing mice treated with swainsonine (SW) and/or cisplatin (Cis) for 10 days. NC Hematocrit (%) Hemoglobin (g/dl) RBC (106 /mm3 ) WBC (103 /mm3 ) Platelet (103 /mm3 ) MCV (fm3 ) MCH (pg) MCHC (g/dl)
30.2 12.8 8.3 12.4 364.5 36.4 15.4 42.7
Co ± ± ± ± ± ± ± ±
3.8 1.0 0.9 5.2 174.9 1.4 0.7 2.6
27.8 12.3 7.7 10.7 438.2 36.1 15.9 44.4
Cis ± ± ± ± ± ± ± ±
2.7 0.9 0.7 5.1 259.6 1.0 0.7 1.8
27.3 12.1 7.6 10.5 391.5 36.0 16.1 44.8
CisSW ± ± ± ± ± ± ± ±
4.4 1.1 1.2 3.1 193.7 1.2 1.1 3.8
27.5 11.9 7.6 8.9 334.0 36.1 15.7 43.7
SW ± ± ± ± ± ± ± ±
5.5 1.8 1.4 3.2 192.2 1.3 1.0 3.8
28.3 12.4 7.9 8.8 467.2 35.8 15.8 44.2
± ± ± ± ± ± ± ±
4.9 1.7 1.3 4.4 157.2 1.3 0.5 1.8
Data are presented as mean ± SD (NC = 11, Co = 10, Cis = 11, CisSW = 8, SW = 10); NC = negative control; Co = control; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration.
Table 2 Biochemical parameters of the EAC-bearing mice treated with swainsonine (SW) and/or cisplatin (Cis) for 10 days.
Creatinine (mg/dl) Urea (mg/dl) ALT (U/l)
NC
Co
Cis
CisSW
SW
0.55 ± 0.21 71.2 ± 10.3 15.3 ± 4.7
0.51 ± 0.24 92.2 ± 16.1* 13.5 ± 5.5
0.49 ± 0.17 71.2 ± 13.5 9.5 ± 4.5
0.36 ± 0.23 64.9 ± 7.8 8.3 ± 3.4#
0.40 ± 0.24 78.0 ± 11.2 17.5 ± 9.1
Data are presented as mean ± SD (NC = 11, Co = 10, Cis = 11, CisSW = 8, SW = 10); NC = negative control; Co = control; ALT = alanine transaminase. * p < 0.01 NC vs. Co, post-test Dunnett. # p < 0.05 NC vs. CisSW and CisSW vs. SW, post-test Dunn.
Effects of swainsonine and/or cisplatin on the survival of the EAC-bearing mice The median survival in mice in the CisSW group was 27 days, which corresponds to an increase of 116% compared to 12.5-day survival observed in mice in the Co group (p = 0.0022 Co vs. CisSW Log-rank test) (Fig. 6). In mice in the Cis group, the median survival was only 15 days, which correspond to an increase of just 20% compared to 12.5-day survival observed in the Co group. Discussion Cisplatin is considered to be one of the most potent chemotherapeutic agents discovered to date; however, severe side effects in normal tissues and acquired resistance to cisplatin in tumor cells has limited its use (Pabla and Dong 2008). Therefore, combination therapies that enhance the antitumor effectiveness of cisplatin without increasing the toxicity are highly desirable. Our study demonstrates, for the first time, that swainsonine enhances the sensitivity of Ehrlich carcinoma cells to cisplatin and suggests that swainsonine could be useful in therapies that utilize cisplatin. The antitumor effects of swainsonine are linked to the inhibition of ␣-mannosidase II activity in the Golgi and the blockade of ˇ1,6-branched N-linked glycan complex production in tumor cells. Swainsonine was also verified to decrease expression of apoptosisinhibiting genes in different tumor cells, which culminated with the induction of apoptosis in vivo and in vitro (Sun et al. 2007, 2009). In the present study, it was observed that the application of swainsonine twice-a-day did not show any antitumor effect when administered alone. However, two injections of swainsonine along with cisplatin every other day resulted in an antitumor effectiveness that was higher than that observed with cisplatin alone. The response to irreparable DNA damage in mammalian cells often results in permanent cell cycle arrest or, in many cases, cell death by apoptosis (Morgan 2007). Cell cycle analysis revealed that a higher percentage of cells were in the subG1 phase in tumor samples from mice treated with the combined therapy (CisSW) than with cisplatin alone. Taking into account that the cytotoxic mechanism of cisplatin is related to the induction of intrastrand DNA adducts that ultimately trigger apoptosis (Siddik 2003), it is likely that the reduction in expression of apoptosis inhibiting genes by
swainsonine may facilitate the induction of apoptosis provoked by cisplatin. Consequently, we observed a smaller percentage of cells in the other cell cycle phases (G0/G1, S and G2/M) in tumor samples from mice treated with swainsonine and cisplatin than those from mice treated with cisplatin alone. ATP binding cassette (ABC)-transport proteins such as Pglycoproteins (P-gp) play a key role in the resistance to anticancer drugs (Gottesman et al. 2002). The P-gp is a broad-spectrum multidrug efflux pump and its functional activity depends on Nglycosylation (Draheim et al. 2010). Considering swainsonine, an inhibitor of N-glycan biosynthesis, it is likely that swainsonine may interfere with P-gp function reducing drug efflux. However, until now the resistance to cisplatin was not correlated with this mechanism of drug resistance (Gottesman et al. 2002; Veneroni et al. 1994). Mice treated with the combined therapy (CisSW) showed less body weight gain throughout the treatment period compared with mice from the negative control (NC) group due to reduced tumor progression in that group. In addition, a smaller ascites volume was observed, and the total EAC cell count and total viable EAC cell count were less in the EAC-bearing mice treated with swainsonine and cisplatin than in the EAC-bearing mice from other groups. One of the adverse effects of swainsonine observed during the human cancer trials was an elevated level of serum aspartate aminotransferase (AST) (Goss et al. 1994, 1997; Shaheen et al. 2005), and one of the most common side effects of cisplatin use is nefrotoxicity, which is manifested by high serum creatinine levels (Pabla and Dong 2008). In the present study, creatinine levels were unaffected by the combined therapy (CisSW), and the other biochemical parameters analyzed, complete blood count and histopathological evaluation of the liver and kidney, were also unaffected. It was important to determine if swainsonine and cisplatin could enhance the median survival of EAC-bearing mice. Therefore, we repeated the schedule of treatments and observed the mice daily until death or for up to 60 days after the treatments were completed. We observed a significant increase in median survival (116%) of mice treated with the combined therapy (CisSW), whereas the mice treated with cisplatin alone showed only a slight increase (20%) when compared to the median survival from the control group.
1100
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
Fig. 6. Effects of swainsonine and/or cisplatin on the survival of the EAC-bearing mice. We observed a significant increase in median survival of mice treated with the combined therapy (CisSW) when compared to the median survival from the control group (p = 0.0022 Co vs. CisSW Log-rank test). N = eight mice per group.
In summary, these results showed that swainsonine enhances Ehrlich carcinoma cell sensitivity to cisplatin, as demonstrated by the improvement in the antitumor effectiveness. Contributors FMS participated in the concept and design of the experiments, cared for the experimental animals and interpreted the results; AOL conceived and performed the cell cycle and statistical analyses and drafted the manuscript; IMH participated in the concept and design of the experiments; DSS performed the histopathological analyses; LLL participated in the experiments; DRG purified swainsonine from A. lentiginosus; HSS participated in the concept and design of the experiments, supervised the research and edited the final version of the manuscript. Acknowledgments
Fig. 4. Effects of swainsonine and/or cisplatin on ascites volume and total EAC cell count of EAC-bearing mice. (A) Ascites volume at day 13. The combined therapy (CisSW) reduced ascites volume more than cisplatin alone (***p < 0.001 Co vs. CisSW, **p < 0.01 Co vs. Cis, # p < 0.05 CisSW vs. SW, post-test Dunn). (B) Total Ehrlich ascitic cell number. It is observed that both treatments the CisSW and the Cis reduced the total number of EAC cells (***p < 0.001 Co vs. Cis and Co vs. CisSW, ## p < 0.01 CisSW vs. SW, # p < 0.05 Cis vs. SW, post-test Dunn). (C) Total Ehrlich ascitic cell viability. We observed a significant reduction in viable and consequently in inviable EAC cells with both treatments the CisSW and the Cis (***p < 0.001, # p < 0.05, Co vs. Cis and Co vs. CisSW, post-test Bonferroni). Data are expressed as the mean ± SD (Co = 10, Cis = 11, CisSW = 8, SW = 10).
Cell Cycle a
100
Co Cis CisSW SW
b
80
%
60 40 c
20
b
a a
c b
0 sub G1
G0/G1
S
G2/M
Fig. 5. Effects of swainsonine and/or cisplatin on the EAC cell cycle. It is observed a higher percentage of cells were in the subG1 phase in tumor samples from mice treated with the combined therapy (CisSW) than with cisplatin alone. It is represented only the differences from experimental groups compared to control group (a p < 0.001, b p < 0.001, c p < 0.05, post-test Dunn). Data are expressed as the mean ± SD (Co = 10, Cis = 11, CisSW = 8, SW = 10).
This research was supported by grants from FAPESP (Fundac¸ão de Amparo a Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and is part of the Ms. dissertation of F.M.S., presented to the Programa de Pós-Graduac¸ão em Patologia Experimental e Comparada da Universidade de São Paulo. References Draheim, V., Reichel, A., Weitschies, W., Moenning, U., 2010. N-glycosylation of ABC transporters is associated with functional activity in sandwich-cultured rat hepatocytes. Eur. J. Pharm. Sci. 41, 201–209. Fuster, M.M., Esko, J.D., 2005. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat. Rev. Cancer 5, 526–542. Galustian, C., Foulds, S., Dye, J.F., Guillou, P.J., 1994. Swainsonine, a glycosylation inhibitor, enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity. Immunopharmacology 27, 165–172. Gardner, D.R., Lee, S.T., Molyneux, R.J., Edgar, J.A., 2003. Preparative isolation of swainsonine from locoweed: extraction and purification procedures. Phytochem. Anal. 14, 259–266. Gerber-Lemaire, S., Juillerat-Jeanneret, L., 2010. Studies toward new anti-cancer strategies based on ␣-mannosidase inhibition. Chimia 64, 634–639. Goss, P.E., Baptiste, J., Fernandes, B., Baker, M., Dennis, J.W., 1994. A phase I study of swainsonine in patients with advanced malignancies. Cancer Res. 54, 1450–1457. Goss, P.E., Reid, C.L., Bailey, D., Dennis, J.W., 1997. Phase IB clinical trial of the oligosaccharide processing inhibitor swainsonine in patients with advanced malignancies. Clin. Cancer Res. 3, 1077–1086. Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2, 48–58. Hamaguchi, J., Nakagawa, H., Takahashi, M., Kudo, T., Kamiyama, N., Sun, B., Oshima, T., Sato, Y., Deguchi, K., Todo, S., Nishimura, S.I., 2007. Swainsonine reduces 5fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines. Mol. Cancer 6, 1–9. ´ Janik, M.E., Litynska, A., Vereecken, P., 2010. Cell migration – the role of integrin glycosylation. Biochim. Biophys. Acta: Gen. Subjects 1800, 545–555.
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101 Klein, G., 1951. Comparative studies of mouse tumors with respect to their capacity for growth as “ascites tumors” and their average nucleic acid content per cell. Exp. Cell Res. 2, 518–573. Molyneux, R.J., James, L.F., 1982. Loco intoxication: indolizidine alkaloids of spotted locoweed (Astragalus lentiginosus). Science 216, 190–191. Morgan, D.O., 2007. The DNA damage response. In: Lawrence, E. (Ed.), The Cell Cycle. Principles of Control. New Science Press Ltd., London, pp. 228–244. Oredipe, O., Furbert-Harris, P., Green, W., White, S., Olden, K., Laniyan, I., ParishGause, D., Vaughn, T., Griffin, W., Sridhar, R., 2003. Swainsonine stimulates bone marrow cell proliferation and differentiation in different strains of inbred mice. Pharmacol. Res. 47, 69–74. Pabla, N., Dong, Z., 2008. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int. 73, 994–1007. Shaheen, P.E., Stadler, W., Elson, P., Knox, J., Winquist, E., Bukowski, R.M., 2005. Phase II study of the efficacy and safety of oral GD0039 in patients with locally advanced or metastatic renal cell carcinoma. Invest. New Drugs 23, 577–581. Siddik, Z.H., 2003. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22, 7265–7279.
1101
Sun, J.Y., Yang, H., Miao, S., Li, J.P., Wang, S.W., Zhu, M.Z., Xie, Y.H., Wang, J.B., Liu, Z., Yang, Q., 2009. Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo. Phytomedicine 16, 1070–1074. Sun, J.Y., Zhu, M.Z., Wang, S.W., Miao, S., Xie, Y.H., Wang, J.B., 2007. Inhibition of the growth of human gastric carcinoma in vivo and in vitro by swainsonine. Phytomedicine 14, 353–359. Veneroni, S., Zaffaroni, N., Daidone, M.G., Benini, E., Villa, R., Silvestrini, R., 1994. Expression of P-glycoprotein and in vitro or in vivo resistance to doxorubicin and cisplatin in breast and ovarian cancers. Eur. J. Cancer Part A: Gen. Top. 30, 1002–1007. Wrodnigg, T.M., Steiner, A.J., Ueberbacher, B.J., 2008. Natural and synthetic iminosugars as carbohydrate processing enzyme inhibitors for cancer therapy. Anti-Cancer Agents Med. Chem. 8, 77–85. Zhao, Y.Y., Takahashi, M., Gu, J.G., Miyoshi, E., Matsumoto, A., Kitazume, S., Taniguchi, N., 2008. Functional roles of N-glycans in cell signaling and cell adhesion in cancer. Cancer Sci. 99, 1304–1310.
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Increased antitumor efficacy by the combined administration of swainsonine and cisplatin in vivo Felipe M. Santos a , Andreia O. Latorre a , Isis M. Hueza b , Daniel S. Sanches a , Luciana L. Lippi a , Dale R. Gardner c , Helenice S. Spinosa a,∗ a
Department of Pathology, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, SP 05508-270, Brazil Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo (UNIFESP), Diadema, SP, Brazil c Poisonous Plant Research Lab, N. Logan, UT 84341, USA b
a r t i c l e
i n f o
Keywords: Cancer chemotherapy Cis-diamminedichloroplatinum (II) Golgi ␣-mannosidase II ␣-Mannosidase inhibitor Astragalus lentiginosus Cell cycle Swainsonine
a b s t r a c t Swainsonine is a natural ␣-mannosidase inhibitor found in numerous poisonous plants, such as Astragalus lentiginosus. Its mechanism of action is through the inhibition of Golgi ␣-mannosidase II activity in the N-glycan biosynthesis pathway. As a result, swainsonine inhibits the production of complex ˇ1,6branched N-linked glycans, which are related to the malignant phenotype of tumor cells. In this study, we investigated whether treatment with swainsonine affects the sensitivity of Ehrlich ascites carcinoma (EAC) cells to cisplatin. To this end, male C57BL/6 mice were treated with swainsonine (SW – 0.5 mg/kg, i.p., twice-daily for ten days) and/or cisplatin (Cis – 0.25 mg/kg, i.p., every other day for a total of five applications) two days after transplantation with EAC cells. The results showed a greater reduction in the ascites volume in mice from the CisSW group (63.5%) than in mice from the Cis group (45.7%), an elevated induction of apoptosis by CisSW treatment when compared to Cis alone, as demonstrated by higher percentage of cells in the subG1 phase in that group (p < 0.0001 Kruskal–Wallis, p < 0.0001 control vs. CisSW, p < 0.001 Co vs. Cis post-test Dunn), and an increase in the median survival from 12.5 days observed in the control group to 27 days in the CisSW group, which corresponds to a 116% survival increase (p = 0.0022 Co vs. CisSW Log-rank test). In addition, the mice from the Cis group had a median survival of only 15 days, an increase of just 20% compared to controls. Our results indicate that swainsonine increases the sensitivity of EAC cells to cisplatin. © 2011 Elsevier GmbH. All rights reserved.
Introduction Swainsonine is a natural ␣-mannosidase inhibitor that is found in numerous poisonous plants, such as Astragalus lentiginosus (Molyneux and James 1982). Swainsonine has been reported to be a promising antitumor agent and its molecular formula is C8 H15 NO3 (Fig. 1). It is an effective inhibitor of both lysosomal ␣-mannosidase and Golgi ␣-mannosidase II (Gerber-Lemaire and Juillerat-Jeanneret 2010). Swainsonine in cancer therapies has been linked to the inhibition of Golgi ␣-mannosidase II by blocking the production of the complex ˇ1,6-branched N-linked glycans (Wrodnigg et al. 2008). In fact, the expression of complex ˇ1,6branched N-linked glycans on the cell surface has been associated with the malignant phenotype (Fuster and Esko 2005). Therefore,
∗ Corresponding author at: Department of Pathology, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Orlando Marques de Paiva 87, São Paulo 05508-270, Brazil. Tel.: +55 11 3091 7656; fax: +55 11 3091 7829. E-mail address: [email protected] (H.S. Spinosa). 0944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2011.06.005
glycosylation pathways are a promising target for novel cancer therapies. Glycoproteins are proteins with one or more covalently bound glycans. These proteins can regulate important cellular functions, such as cell–cell communication, cell growth, migration and signal transduction. Most of the glycoproteins in the cell are Asn-linked (N-linked) oligosaccharides. Integrins, receptors and extracellular matrix (ECM) ligands display altered pattern of N-linked glycosylation in cells undergoing a malignant transformation (Janik et al. 2010). In addition, there is an increasing body of evidence that suggests that tumor cells, which overexpress some glycosyltranferases, synthesize glycans that facilitate processes such as invasion and metastasis (Fuster and Esko 2005; Zhao et al. 2008). Swainsonine has been shown to reduce tumor cell invasion in Phase I human cancer trials and to enhance the natural antitumor defenses of the body, such as lymphokine activated killer cell (LAK) and natural killer cell (NK) cytotoxicity (Galustian et al. 1994; Goss et al. 1994). However, in the Phase II trials, no antitumor activity was observed in patients with locally advanced or metastatic renal cell carcinomas, and the treatment was discontinued due to disease progression and the toxicity of the compound (Shaheen
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
Treatment protocol
N
OH
H
1097
OH
OH
Fig. 1. Chemical structure of swainsonine.
et al. 2005). Analogues of swainsonine have been shown to have a reduced toxicity, but they also have a diminished antitumor effect (Gerber-Lemaire and Juillerat-Jeanneret 2010). However, swainsonine has been shown to increase tumor cell sensitivity to cytotoxic drugs and to reduce resistance to anticancer drugs in multistage colorectal cancer cells in vitro (Hamaguchi et al. 2007). Therefore, the aim of the current study was to determine if swainsonine could increase the sensitivity of Ehrlich carcinoma cells to cisplatin (cis-diamminedichloroplatinum (II)), a platinum-based chemotherapeutic, in vivo. Materials and methods Mice Eighty-seven sixty-day-old male C57BL/6 mice, bred in the Department of Pathology at the School of Veterinary Medicine and Animal Sciences, University of São Paulo, were used. The mice were maintained under controlled temperature conditions (22–25 ◦ C), relative humidity (50–65%) and lighting (12-h/12-h light/dark cycle). Drinking water and standard diet (NuvilabCR1® , Nuvital Nutrientes LTDA) were provided ad libitum. All procedures were performed following the Guide for the Care and Use of Laboratory Animals, NIH publication no. 8523 (http://www.nap.edu/readingroom/books/labrats/), and were reviewed and approved by the Bioethics Committee of the FMVZUSP (process #1161/2007). Chemicals Propidium iodide (PI) was obtained from Sigma Chemical Co. (St. Louis, MO). RNAse A was obtained from Invitrogen (Carlsbad, CA). Dopalen (ketamine) and Anasedan (xylazine) were obtained from VetbrandsTM (São Paulo, Brazil). Platistine® CS (cisplatin) was obtained from Pfizer (São Paulo, Brazil), and EDTA was obtained from Merck (Darmstadt, Germany). Swainsonine Swainsonine (Fig. 1) was purified from dry A. lentiginosus aerial parts using a previously described method presented by Gardner et al. (2003). Tumor model Intraperitoneal Ehrlich ascites carcinoma (EAC) cells syngeneic to BALB/c mice were used in the study. Therefore, EAC cells were transplanted intraperitoneally (i.p.) into the C57BL/6 mice, and just the fluid transplant generations that were passed over 12 times, provoking the formation of regular exudates, as described previously by Klein (1951), were used in all subsequent experiments. In all experiments, tumor transplantation was performed by i.p. injection of 2 × 107 cells suspended in 200 l of sterile PBS.
Fifty-five mice were randomly allocated into the following groups: negative control (NC), control (Co), cisplatin alone (Cis), cisplatin and swainsonine (CisSW) and swainsonine alone (SW). Mice from the NC group were neither inoculated with EAC cells nor received any treatment, whereas the mice from all other groups were inoculated with EAC cells, as mentioned above, two days before the start of the different treatments. The mice in the Cis and CisSW groups were i.p. injected with 0.25 mg/kg of cisplatin every other day for a total of five applications. The mice in the SW and CisSW groups were i.p. injected twice a day with 1.0 mg/kg of swainsonine, as proposed by Oredipe et al. (2003), for 10 consecutive days. The mice in the Co group were i.p. injected twice a day with PBS for 10 consecutive days. Throughout the various applications every day, the body weight of each mouse was measured each day, and the dose was adjusted accordingly. Tumor cell evaluation On day 13 of the experiment, control and experimental mice were subjected to intramuscular anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg). Ascites fluid was collected, and the volume was quantified. The EAC cells were counted using the Trypan Blue assay to calculate the total viable and inviable cells. One million EAC cells from each mouse were fixed and preserved in 70% ethanol for up to one week at −20 ◦ C for cell cycle analysis. Cell cycle analysis Cell cycle analysis was performed using one million EAC cells from each mouse that were fixed and preserved in 70% ethanol for up to one week at −20 ◦ C. The cells were washed twice in PBS (5 min, 1200 rpm) and incubated with 200 l of PI solution (20 g/ml propidium iodide, 200 g/ml RNAse A and 0.1% Triton (v/v) in PBS) for 15 min at 37 ◦ C. Then, 10,000 events were acquired from each sample in a FACS Calibur Flow Cytometer equipped with Cell Quest Pro® software (Becton Dickinson [BD] Immunocytometry System). Data analyses were performed using FlowJo 7.2.2® software (Tree Star Inc., Ashland, KY) as shown in Fig. 2A and B. Blood samples, biochemical parameters and histological evaluation On day 13 of the experiment, each mouse was anesthetized (ketamine (100 mg/kg) + xylazine (10 mg/kg)), and ascites fluid was collected prior to blood collection by cardiac puncture. Blood was collected in a 1.0 ml syringe and was separated into two vials, one containing 10 l of EDTA to perform a complete blood count and another to separate the serum for posterior biochemical analyses. The complete blood count was determined using a Vet ABC Animal Blood counter (Horiba ABX Diagnostics). Forty-five minutes after the blood samples were collected, serum was separated by centrifugation at 4000 rpm for 10 min and placed at −20 ◦ C until it was needed. Biochemical analyses (creatinine, urea and alanine transaminase (ALT)) were performed using a CELM SBA-200 (CELM). Liver and kidney were collected for histopathological evaluation. Histopathological analysis was performed on hematoxylin and eosin (HE) stained sections that were five microns thick. Survival analysis For the survival analysis, 32 mice were randomly allocated into the following groups: Co, Cis, CisSW and SW. The tumor inoculation and treatment were performed as mentioned above and the mice
1098
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
Fig. 2. Cell cycle analysis. (A) FL2-Width vs. FL2-Area dot plot showing a single gate, which excludes aggregates. (B) FL2-A DNA histogram of EAC cells, which has been gated to exclude aggregates.
were observed daily until death or for 60 days after the treatments were completed. Statistical analysis The data were analyzed using GraphPad Prism 5.00® software (GraphPad Software, Inc., San Diego, CA) using one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Percentage data from three or more groups and data presenting significant differences among the standard deviations (SDs) were compared using the Kruskal–Wallis test followed by Dunn’s test. Data with two variables were analyzed by two-way ANOVA followed by the Bonferroni test. Survival curves were compared by Log-rank test. Data are expressed as the mean ± SD, and differences were considered statistically significant at p < 0.05.
Effects of swainsonine and/or cisplatin on complete blood count, biochemical parameters and organ histology evaluation of the EAC-bearing mice
Results Effects of swainsonine and/or cisplatin on body weight gain, ascites volume and total EAC cell count of EAC-bearing mice Some mice that were inoculated with EAC cells for this experiment did not develop tumor ascites, and those mice were eliminated from all subsequent analyses. All groups of mice that were inoculated with EAC cells had an increase in body weight when compared to the negative control (NC) group. However, the increase in body weight was smaller in the mice from the CisSW group than in the Cis and SW treatment groups (Fig. 3).
Body weight gain 45
NC Co Cis CisSW SW
g
40 35 30 25
2
3
4
5
6
7
8
9
10
In line with this finding, a significant reduction in ascites volume of 63.5% was observed in mice that were treated with swainsonine plus cisplatin (CisSW group), whereas cisplatin alone (Cis group) reduced the ascites volume by 45.7%, compared to the Co group (p < 0.0001 Kruskal–Wallis test, p < 0.001 Co vs. CisSW, p < 0.01 Co vs. Cis, p < 0.05 CisSW vs. SW post-test Dunn) (Fig. 4A). We observed a significant reduction in total Ehrlich ascitic cell count in mice from the CisSW and Cis groups (p < 0.0001 Kruskal–Wallis test, p < 0.001 Co vs. Cis and Co vs. CisSW, p < 0.01 CisSW vs. SW, p < 0.05 Cis vs. SW post-test Dunn) (Fig. 4B). This reduction in the total count was due to a significant reduction in viable and inviable EAC cells observed in both the CisSW and Cis groups (p < 0.0001 Two-way ANOVA, viable cells p < 0.001 Co vs. Cis and Co vs. CisSW, and inviable cells p < 0.05 Co vs. Cis and Co vs. CisSW post-test Bonferroni) (Fig. 4C).
11
Days Fig. 3. Body weight gain of EAC-bearing mice treated with swainsonine (SW), cisplatin (Cis) and combined therapy (CisSW) for 10 days. Mice treated with the combined therapy (CisSW) showed less body weight gain throughout the treatment period compared with mice from the negative control (NC) group (NC = 11, Co = 10, Cis = 11, CisSW = 8, SW = 10).
Complete blood count was unaffected by treatment with swainsonine and/or cisplatin (Table 1). Furthermore, histological evaluation of the livers and kidneys recovered from each swainsonine and/or cisplatin-treated mouse revealed no differences when compared to the samples obtained from the control mice (data not shown). For the biochemical parameters, a reduction in ALT concentrations in the serum from the mice treated with the combined therapy (CisSW) compared with the mice treated with either swainsonine (SW) or no treatment (NC) (p = 0.0048 Kruskal–Wallis test, p < 0.05 NC vs. CisSW and CisSW vs. SW post-test Dunn) was observed. Moreover, we observed a higher urea level in the mice from the Co group compared with the mice in the NC group (p = 0.002 one-way ANOVA, p < 0.01 NC vs. Co post-test Dunnett) (Table 2). Effects of swainsonine and/or cisplatin on the EAC cell cycle An elevated induction of apoptosis was observed in mice treated with swainsonine and cisplatin compared to mice that received cisplatin alone. This result was based on data showing that the CisSW group had a higher percentage of cells in the subG1 phase (p < 0.0001 Kruskal–Wallis test, p < 0.001 Co vs. Cis, p < 0.0001 Co vs. CisSW post-test Dunn). Consequently, we observed a significant reduction in the percentage of cells in the G0/G1, S and G2 phases in both the CisSW and Cis groups (Fig. 5).
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
1099
Table 1 Complete blood count of the EAC-bearing mice treated with swainsonine (SW) and/or cisplatin (Cis) for 10 days. NC Hematocrit (%) Hemoglobin (g/dl) RBC (106 /mm3 ) WBC (103 /mm3 ) Platelet (103 /mm3 ) MCV (fm3 ) MCH (pg) MCHC (g/dl)
30.2 12.8 8.3 12.4 364.5 36.4 15.4 42.7
Co ± ± ± ± ± ± ± ±
3.8 1.0 0.9 5.2 174.9 1.4 0.7 2.6
27.8 12.3 7.7 10.7 438.2 36.1 15.9 44.4
Cis ± ± ± ± ± ± ± ±
2.7 0.9 0.7 5.1 259.6 1.0 0.7 1.8
27.3 12.1 7.6 10.5 391.5 36.0 16.1 44.8
CisSW ± ± ± ± ± ± ± ±
4.4 1.1 1.2 3.1 193.7 1.2 1.1 3.8
27.5 11.9 7.6 8.9 334.0 36.1 15.7 43.7
SW ± ± ± ± ± ± ± ±
5.5 1.8 1.4 3.2 192.2 1.3 1.0 3.8
28.3 12.4 7.9 8.8 467.2 35.8 15.8 44.2
± ± ± ± ± ± ± ±
4.9 1.7 1.3 4.4 157.2 1.3 0.5 1.8
Data are presented as mean ± SD (NC = 11, Co = 10, Cis = 11, CisSW = 8, SW = 10); NC = negative control; Co = control; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration.
Table 2 Biochemical parameters of the EAC-bearing mice treated with swainsonine (SW) and/or cisplatin (Cis) for 10 days.
Creatinine (mg/dl) Urea (mg/dl) ALT (U/l)
NC
Co
Cis
CisSW
SW
0.55 ± 0.21 71.2 ± 10.3 15.3 ± 4.7
0.51 ± 0.24 92.2 ± 16.1* 13.5 ± 5.5
0.49 ± 0.17 71.2 ± 13.5 9.5 ± 4.5
0.36 ± 0.23 64.9 ± 7.8 8.3 ± 3.4#
0.40 ± 0.24 78.0 ± 11.2 17.5 ± 9.1
Data are presented as mean ± SD (NC = 11, Co = 10, Cis = 11, CisSW = 8, SW = 10); NC = negative control; Co = control; ALT = alanine transaminase. * p < 0.01 NC vs. Co, post-test Dunnett. # p < 0.05 NC vs. CisSW and CisSW vs. SW, post-test Dunn.
Effects of swainsonine and/or cisplatin on the survival of the EAC-bearing mice The median survival in mice in the CisSW group was 27 days, which corresponds to an increase of 116% compared to 12.5-day survival observed in mice in the Co group (p = 0.0022 Co vs. CisSW Log-rank test) (Fig. 6). In mice in the Cis group, the median survival was only 15 days, which correspond to an increase of just 20% compared to 12.5-day survival observed in the Co group. Discussion Cisplatin is considered to be one of the most potent chemotherapeutic agents discovered to date; however, severe side effects in normal tissues and acquired resistance to cisplatin in tumor cells has limited its use (Pabla and Dong 2008). Therefore, combination therapies that enhance the antitumor effectiveness of cisplatin without increasing the toxicity are highly desirable. Our study demonstrates, for the first time, that swainsonine enhances the sensitivity of Ehrlich carcinoma cells to cisplatin and suggests that swainsonine could be useful in therapies that utilize cisplatin. The antitumor effects of swainsonine are linked to the inhibition of ␣-mannosidase II activity in the Golgi and the blockade of ˇ1,6-branched N-linked glycan complex production in tumor cells. Swainsonine was also verified to decrease expression of apoptosisinhibiting genes in different tumor cells, which culminated with the induction of apoptosis in vivo and in vitro (Sun et al. 2007, 2009). In the present study, it was observed that the application of swainsonine twice-a-day did not show any antitumor effect when administered alone. However, two injections of swainsonine along with cisplatin every other day resulted in an antitumor effectiveness that was higher than that observed with cisplatin alone. The response to irreparable DNA damage in mammalian cells often results in permanent cell cycle arrest or, in many cases, cell death by apoptosis (Morgan 2007). Cell cycle analysis revealed that a higher percentage of cells were in the subG1 phase in tumor samples from mice treated with the combined therapy (CisSW) than with cisplatin alone. Taking into account that the cytotoxic mechanism of cisplatin is related to the induction of intrastrand DNA adducts that ultimately trigger apoptosis (Siddik 2003), it is likely that the reduction in expression of apoptosis inhibiting genes by
swainsonine may facilitate the induction of apoptosis provoked by cisplatin. Consequently, we observed a smaller percentage of cells in the other cell cycle phases (G0/G1, S and G2/M) in tumor samples from mice treated with swainsonine and cisplatin than those from mice treated with cisplatin alone. ATP binding cassette (ABC)-transport proteins such as Pglycoproteins (P-gp) play a key role in the resistance to anticancer drugs (Gottesman et al. 2002). The P-gp is a broad-spectrum multidrug efflux pump and its functional activity depends on Nglycosylation (Draheim et al. 2010). Considering swainsonine, an inhibitor of N-glycan biosynthesis, it is likely that swainsonine may interfere with P-gp function reducing drug efflux. However, until now the resistance to cisplatin was not correlated with this mechanism of drug resistance (Gottesman et al. 2002; Veneroni et al. 1994). Mice treated with the combined therapy (CisSW) showed less body weight gain throughout the treatment period compared with mice from the negative control (NC) group due to reduced tumor progression in that group. In addition, a smaller ascites volume was observed, and the total EAC cell count and total viable EAC cell count were less in the EAC-bearing mice treated with swainsonine and cisplatin than in the EAC-bearing mice from other groups. One of the adverse effects of swainsonine observed during the human cancer trials was an elevated level of serum aspartate aminotransferase (AST) (Goss et al. 1994, 1997; Shaheen et al. 2005), and one of the most common side effects of cisplatin use is nefrotoxicity, which is manifested by high serum creatinine levels (Pabla and Dong 2008). In the present study, creatinine levels were unaffected by the combined therapy (CisSW), and the other biochemical parameters analyzed, complete blood count and histopathological evaluation of the liver and kidney, were also unaffected. It was important to determine if swainsonine and cisplatin could enhance the median survival of EAC-bearing mice. Therefore, we repeated the schedule of treatments and observed the mice daily until death or for up to 60 days after the treatments were completed. We observed a significant increase in median survival (116%) of mice treated with the combined therapy (CisSW), whereas the mice treated with cisplatin alone showed only a slight increase (20%) when compared to the median survival from the control group.
1100
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101
Fig. 6. Effects of swainsonine and/or cisplatin on the survival of the EAC-bearing mice. We observed a significant increase in median survival of mice treated with the combined therapy (CisSW) when compared to the median survival from the control group (p = 0.0022 Co vs. CisSW Log-rank test). N = eight mice per group.
In summary, these results showed that swainsonine enhances Ehrlich carcinoma cell sensitivity to cisplatin, as demonstrated by the improvement in the antitumor effectiveness. Contributors FMS participated in the concept and design of the experiments, cared for the experimental animals and interpreted the results; AOL conceived and performed the cell cycle and statistical analyses and drafted the manuscript; IMH participated in the concept and design of the experiments; DSS performed the histopathological analyses; LLL participated in the experiments; DRG purified swainsonine from A. lentiginosus; HSS participated in the concept and design of the experiments, supervised the research and edited the final version of the manuscript. Acknowledgments
Fig. 4. Effects of swainsonine and/or cisplatin on ascites volume and total EAC cell count of EAC-bearing mice. (A) Ascites volume at day 13. The combined therapy (CisSW) reduced ascites volume more than cisplatin alone (***p < 0.001 Co vs. CisSW, **p < 0.01 Co vs. Cis, # p < 0.05 CisSW vs. SW, post-test Dunn). (B) Total Ehrlich ascitic cell number. It is observed that both treatments the CisSW and the Cis reduced the total number of EAC cells (***p < 0.001 Co vs. Cis and Co vs. CisSW, ## p < 0.01 CisSW vs. SW, # p < 0.05 Cis vs. SW, post-test Dunn). (C) Total Ehrlich ascitic cell viability. We observed a significant reduction in viable and consequently in inviable EAC cells with both treatments the CisSW and the Cis (***p < 0.001, # p < 0.05, Co vs. Cis and Co vs. CisSW, post-test Bonferroni). Data are expressed as the mean ± SD (Co = 10, Cis = 11, CisSW = 8, SW = 10).
Cell Cycle a
100
Co Cis CisSW SW
b
80
%
60 40 c
20
b
a a
c b
0 sub G1
G0/G1
S
G2/M
Fig. 5. Effects of swainsonine and/or cisplatin on the EAC cell cycle. It is observed a higher percentage of cells were in the subG1 phase in tumor samples from mice treated with the combined therapy (CisSW) than with cisplatin alone. It is represented only the differences from experimental groups compared to control group (a p < 0.001, b p < 0.001, c p < 0.05, post-test Dunn). Data are expressed as the mean ± SD (Co = 10, Cis = 11, CisSW = 8, SW = 10).
This research was supported by grants from FAPESP (Fundac¸ão de Amparo a Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and is part of the Ms. dissertation of F.M.S., presented to the Programa de Pós-Graduac¸ão em Patologia Experimental e Comparada da Universidade de São Paulo. References Draheim, V., Reichel, A., Weitschies, W., Moenning, U., 2010. N-glycosylation of ABC transporters is associated with functional activity in sandwich-cultured rat hepatocytes. Eur. J. Pharm. Sci. 41, 201–209. Fuster, M.M., Esko, J.D., 2005. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat. Rev. Cancer 5, 526–542. Galustian, C., Foulds, S., Dye, J.F., Guillou, P.J., 1994. Swainsonine, a glycosylation inhibitor, enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity. Immunopharmacology 27, 165–172. Gardner, D.R., Lee, S.T., Molyneux, R.J., Edgar, J.A., 2003. Preparative isolation of swainsonine from locoweed: extraction and purification procedures. Phytochem. Anal. 14, 259–266. Gerber-Lemaire, S., Juillerat-Jeanneret, L., 2010. Studies toward new anti-cancer strategies based on ␣-mannosidase inhibition. Chimia 64, 634–639. Goss, P.E., Baptiste, J., Fernandes, B., Baker, M., Dennis, J.W., 1994. A phase I study of swainsonine in patients with advanced malignancies. Cancer Res. 54, 1450–1457. Goss, P.E., Reid, C.L., Bailey, D., Dennis, J.W., 1997. Phase IB clinical trial of the oligosaccharide processing inhibitor swainsonine in patients with advanced malignancies. Clin. Cancer Res. 3, 1077–1086. Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2, 48–58. Hamaguchi, J., Nakagawa, H., Takahashi, M., Kudo, T., Kamiyama, N., Sun, B., Oshima, T., Sato, Y., Deguchi, K., Todo, S., Nishimura, S.I., 2007. Swainsonine reduces 5fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines. Mol. Cancer 6, 1–9. ´ Janik, M.E., Litynska, A., Vereecken, P., 2010. Cell migration – the role of integrin glycosylation. Biochim. Biophys. Acta: Gen. Subjects 1800, 545–555.
F.M. Santos et al. / Phytomedicine 18 (2011) 1096–1101 Klein, G., 1951. Comparative studies of mouse tumors with respect to their capacity for growth as “ascites tumors” and their average nucleic acid content per cell. Exp. Cell Res. 2, 518–573. Molyneux, R.J., James, L.F., 1982. Loco intoxication: indolizidine alkaloids of spotted locoweed (Astragalus lentiginosus). Science 216, 190–191. Morgan, D.O., 2007. The DNA damage response. In: Lawrence, E. (Ed.), The Cell Cycle. Principles of Control. New Science Press Ltd., London, pp. 228–244. Oredipe, O., Furbert-Harris, P., Green, W., White, S., Olden, K., Laniyan, I., ParishGause, D., Vaughn, T., Griffin, W., Sridhar, R., 2003. Swainsonine stimulates bone marrow cell proliferation and differentiation in different strains of inbred mice. Pharmacol. Res. 47, 69–74. Pabla, N., Dong, Z., 2008. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int. 73, 994–1007. Shaheen, P.E., Stadler, W., Elson, P., Knox, J., Winquist, E., Bukowski, R.M., 2005. Phase II study of the efficacy and safety of oral GD0039 in patients with locally advanced or metastatic renal cell carcinoma. Invest. New Drugs 23, 577–581. Siddik, Z.H., 2003. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22, 7265–7279.
1101
Sun, J.Y., Yang, H., Miao, S., Li, J.P., Wang, S.W., Zhu, M.Z., Xie, Y.H., Wang, J.B., Liu, Z., Yang, Q., 2009. Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo. Phytomedicine 16, 1070–1074. Sun, J.Y., Zhu, M.Z., Wang, S.W., Miao, S., Xie, Y.H., Wang, J.B., 2007. Inhibition of the growth of human gastric carcinoma in vivo and in vitro by swainsonine. Phytomedicine 14, 353–359. Veneroni, S., Zaffaroni, N., Daidone, M.G., Benini, E., Villa, R., Silvestrini, R., 1994. Expression of P-glycoprotein and in vitro or in vivo resistance to doxorubicin and cisplatin in breast and ovarian cancers. Eur. J. Cancer Part A: Gen. Top. 30, 1002–1007. Wrodnigg, T.M., Steiner, A.J., Ueberbacher, B.J., 2008. Natural and synthetic iminosugars as carbohydrate processing enzyme inhibitors for cancer therapy. Anti-Cancer Agents Med. Chem. 8, 77–85. Zhao, Y.Y., Takahashi, M., Gu, J.G., Miyoshi, E., Matsumoto, A., Kitazume, S., Taniguchi, N., 2008. Functional roles of N-glycans in cell signaling and cell adhesion in cancer. Cancer Sci. 99, 1304–1310.