Carbohydrate Polymers 129 (2015) 50–54
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Anti-tumor activity and the mechanism of SIP-S: A sulfated polysaccharide with anti-metastatic effect Aizhen Zong a,e , Yuhong Liu b , Yan Zhang a , Xinlei Song a , Yikang Shi c , Hongzhi Cao c , Chunhui Liu a,c , Yanna Cheng d , Wenjie Jiang a , Fangling Du e , Fengshan Wang a,c,∗ a Key Laboratory of Chemical Biology (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, PR China b School of Pharmaceutical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong, PR China c National Glycoengineering Research Center, Shandong University, Jinan 250012, Shandong, PR China d Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, PR China e Institute of Agro-Food Science Technology, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 31 January 2015 Received in revised form 1 April 2015 Accepted 12 April 2015 Available online 21 April 2015 Keywords: SIP-S Cyclophosphamide Anti-tumor activity Immunoenhancing activity Apoptosis
a b s t r a c t Our previous studies demonstrated that SIP-S had anti-metastatic activity and inhibited the growth of metastatic foci. Here we report the anti-tumor and immunoregulatory potential of SIP-S. SIP-S could significantly inhibit tumor growth in S180-bearing mice, and the inhibition rates was 43.7% at 30 mg/kg d. Besides, SIP-S could improve the thymus and spleen indices of S180-bearing mice and the mice treated with CTX. The combination of SIP-S (15 mg/kg d) with CTX (12.5 mg/kg d) showed higher anti-tumor potency than CTX (25 mg/kg d) alone. These results indicated that SIP-S had immunoenhancing and anticancer activity, and the immunoenhancing activity might be one mechanism for its anti-tumor activity. Flow cytometry results showed that SIP-S could induce tumor cells apoptosis. Western blot analysis indicated that SIP-S could upregulate the expression of pro-apoptotic proteins, caspase-3, -8, -9 and Bax, and downregulate the expression of anti-apoptotic protein PARP-1 in tumor cells in a dose-dependent manner. In summary, SIP-S has anti-tumor activity, which may be associated with its immunostimulating and pro-apoptotic activity. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Cancer is a leading cause of death worldwide. Chemotherapy is one of the standard therapeutic protocols for cancer. However, most of the chemotherapeutic agents produce non-selective cytotoxicity and cause severe side effects including hemopoetic and immune suppression (Ehrke, 2003). The low immune function of an organism may not only result in the generation and development of a tumor, but also be one of the most important factors that prevent the tumor patients’ recovery (Mitchell, 2003). The enhancement of host immune response has been recognized as a possible means
∗ Corresponding author at: Shandong University, Key Laboratory of Chemical Biology (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, No. 44 Wenhuaxi Road, Jinan 250012, Shandong, PR China. Tel.: +86 531 88382658; fax: +8653188382548. E-mail addresses: [email protected], [email protected] (F. Wang). http://dx.doi.org/10.1016/j.carbpol.2015.04.017 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
of inhibiting tumor growth without harming the host (Yuan, Song, Li, Li, & Dai, 2006). Therefore, searching for new anti-tumor drugs with potential immunoenhancing effect is essential for cancer therapy. Most polysaccharides from natural sources are found to be nontoxic substances with wide variety of biological activities, and have attracted lots of attention in the biochemical and medical areas (Zong, Cao, & Wang, 2012). Thus, natural polysaccharides are ideal candidates for therapeutics with immunomodulatory and antitumor effects and low toxicity (Schepetkin & Quinn, 2006). SIP was a new heteropolysaccharide isolated from the ink of cuttlefish Sepiella maindroni de Rochebruns (Liu et al., 2008). We then prepared the sulfated SIP (SIP-S) by using chlorosulfonic acid (Geresh, Manontov, & Weinstein, 2002). Studies have shown that SIP-S has significant anti-metastatic and anti-angiogenic activities, and can also obviously inhibit metastatic tumor growth, which suggests that SIP-S may be an ideal candidate for cancer therapy (Zong et al., 2013).
A. Zong et al. / Carbohydrate Polymers 129 (2015) 50–54
In the present study, we carried out experiments to investigate the anti-tumor effects of SIP-S alone and its combination with cyclophosphamide(CTX) on Sarcoma 180 (S180)-tumor bearing mice. Meanwhile the immunomodulatory activity of SIP-S was evaluated. The results will be helpful for developing novel antitumor drugs.
51
2. Materials and methods
last administration, mice were weighed and sacrificed by cervical dislocation. Tumors, spleens and thymus were excised and weighted, respectively. The tumor inhibitory rate was calculated as ((A − B)/A) × 100%, where A and B were the average tumor weights of the control group and the treated group, respectively. Thymus index was expressed as the thymus weight relative to body weight. Spleen index was expressed as the spleen weight relative to body weight.
2.1. Reagents and antibodies
2.4. Synergetic anti-tumor activity of SIP-S and CTX
SIP-S was prepared as described previously (Wang et al., 2008), and dissolved in serum-free medium for the in vitro assay and in normal saline (N.S.) for the in vivo assay. Mouse anti-caspase-3 monoclonal antibody (against a full length caspase-3 of human origin, SC-65497), rabbit anti-Bcl-2 polyclonal antibody (against amino acids 1-205 of Bcl-2 of human origin, SC-783), rabbit anti-Bax polyclonal antibody (against the N-terminus of Bax of human origin, SC-493), and mouse anti-PARP1 monoclonal antibody (against amino acids 1–300 mapping at the N-terminus of PARP-1 of human origin, SC-74470) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-caspase-9 polyclonal antibody (9502) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-caspase-8 polyclonal antibody (AP0358) was purchased from Bioworld Technology, Inc. (Louis Park, MN, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit rabbit IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). FITC Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Bestbio. Co. Ltd., (Bestbio. Co. Ltd., Shanghai, China).
The models of S180 tumor-bearing mice were established and then randomly divided into four groups, each group consisting of 8 animals. The groups were as follows: control group (N.S.); CTX group (25 mg/kg body weight); SIP-S group (15 mg/kg body weight); CTX + SIP-S group (CTX 12.5 mg/kg body weight + SIPS 15 mg/kg body weight). All the groups were administered by intraperitoneal injection of 0.2 mL every day for 10 d, starting from the fifth day after tumor implantation. After the last administration, the mice were sacrificed and treated as mentioned above, and the tumor inhibitory rate, thymus index and spleen index were calculated, respectively.
2.2. Animals and cell lines The use of animals was approved by the Institutional Animal Care Committee of Shandong University, with firm adherence to the Ethical Guidelines for the Care and Use of Animals. Kunming mice (male, 18–22 g) were purchased from the Experimental Animal Center of Shandong University (Jinan, China). Mouse sarcoma cell line S180, human ovarian carcinoma cell line SKOV3 and human umbilical vein cell fusion cell EA.hy926 were obtained from Shanghai Cell Bank, the Institute of Cell Biology, China Academy of Sciences (Shanghai, China). S180 and SKOV3 cells were maintained in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillinstreptomycin (100 IU/mL–100 g/mL), 2 mM glutamine and 10 mM HEPES. EA.hy926 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillinstreptomycin (100 IU/mL–100 g/mL), 2 mM glutamine and 10 mM HEPES. All cells were cultured in a humid atmosphere (5% CO2 , 95% air) at 37 ◦ C, fed every 2 to 3 d and harvested by brief incubation in 0.02% EDTA–0.25% trypsin. 2.3. Anti-tumor activity of SIP-S in vivo S180 cells were maintained as ascites in Kunming mice (6–8 weeks old) for 7 d, and then drawn out from the tumor bearing mice. After being washed and diluted with phosphate-buffered saline (PBS), the S180 cells (0.2 mL, 2 × 106 cells) were injected subcutaneously into the right axilla of Kunming mice. After 24 h, the mice were weighed and randomly divided into five groups, each group consisting of 8 animals. The groups were as follows: control group (normal saline, N.S.); cyclophosphamide (CTX) group (30 mg/kg body weight); three SIP-S groups (30, 20 and 10 mg/kg body weight). All the groups were administered by intraperitoneal injection of 0.2 mL every day for 10 days. 24 h after the
2.5. Detection of apoptosis by flow cytometry Apoptosis analysis was performed by using an Annexin VFITC/PI Apoptosis Detection Kit (Bestbio. Co. Ltd., Shanghai, China) according to the manufacturer’s instructions (Shi, Zhao, Jiao, Shi, & Yang, 2013). Briefly, SKOV3 cells (2 × 105 per well) seeded in 6-well plates were incubated with different doses of SIP-S (500, 100, 20 g/mL) for 36 h. The cells were harvested and washed twice with cold PBS. Then the cells were resuspended in Binding Buffer at a concentration of 1 × 106 cells/mL and stained with 5 L of FITC Annexin V and 10 L PI at 25 ◦ C for 15 min in the dark. The stained cells were analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA) within 1 h. The experiments were repeated three times. 2.6. Western blot analysis Skov3 cells (1 × 105 per well) seeded in 6-well plates were treated with different doses of SIP-S (500, 100, 20 g/mL) for 48 h. The medium was removed and the cells were washed with PBS. The cells were then lysed in 50 L of lysis buffer at 0 ◦ C (icebath) for 30 min with intense shock for 30 s every 10 min. The concentration of total protein was determined using the BCA protein assay method (Pierce, Rockford, IL). Equal amounts of protein (30 g/lane) in the cell extracts were fractionated by SDS-PAGE and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST buffer (20 mM Tris-buffered saline and 0.1% Tween) containing 5% non-fat dry milk for 2 h at room temperature, the membranes were incubated with mouse anti-caspase-3 monoclonal antibody, rabbit anti-Bcl-2 polyclonal antibody, rabbit anti-Bax polyclonal antibody, mouse anti-PARP-1 monoclonal antibody, rabbit anti-caspase-9 polyclonal antibody, and rabbit anti-caspase-8 polyclonal antibody overnight at 4 ◦ C, followed by washing 3 times and reacting with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). The proteins were then detected using chemiluminescence agents (ECL, Amersham). The density of the immunoreactive bands was analyzed using Image-Pro Plus Version 6.0. 2.7. Statistical analysis Data was described as mean ± S.D. and analyzed by Student’s two-tailed t-test. The limit of statistical significance was P < 0.05.
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A. Zong et al. / Carbohydrate Polymers 129 (2015) 50–54
Table 1 Anti-tumor effect of SIP-S in S180-bearing mice (−¯x ± s, n = 8). Group
Body weight (g)
CTX (30 mg/kg d) SIP-S (30 mg/kg d) SIP-S (20 mg/kg d) SIP-S (10 mg/kg d) N.S.
20.33 26.84 23.65 22.83 24.87
± ± ± ± ±
3.93* 2.55# 2.18 3.88 3.29
Tumor weight (g) 0.63 0.89 0.95 1.06 1.58
± ± ± ± ±
0.17*** 0.32** 0.21* 0.56 0.24
Table 2 Anti-tumor effect of SIP-S combined with CTX in S180-bearing mice (¯x ± s, n = 8). Inhibitory rate (%) 60.13 43.67 39.87 32.91 –
CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. *** P < 0.001, ** P < 0.01, and * P < 0.05, compared with control. # P < 0.05, compared with CTX.
Statistical analysis was done using SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA). 3. Results and discussion 3.1. SIP-S inhibits tumor growth and enhances immune function in tumor-bearing mice The anti-tumor and immunoregulatory activity of SIP-S was evaluated in the S180-bearing mice model. As shown in Table 1, after administration for 10 d, the body weights of CTX-treated mice were significantly decreased compared with those treated with N.S. and SIP-S (30 mg/kg d) (P < 0.05), while SIP-S groups showed no significant difference compared with control group. The growth of S180 tumors in SIP-S-treated mice was significantly suppressed compared with those treated with normal saline (P < 0.01 or P < 0.05). The inhibition rates of 10, 20 and 30 mg/kg d of SIP-S were 32.9%, 39.9% and 43.7%, respectively. There was no significant difference between the tumor weights of the mice treated with high-dose SIP-S (30 mg/kg d) and those treated with CTX. Moreover, CTX considerably decreased the spleen and thymus indices in S180bearing mice compared with control group and SIP-S (30 mg/kg d) group (P < 0.05 or P < 0.01) (Fig. 1). In contrast, SIP-S significantly improved the thymus and spleen indices in tumor bearing mice compared with N.S. and CTX (Fig. 1). The immune system is fundamental in suppressing both the initiation of carcinogenesis and the progression of established tumors (Beavis, Stagg, Darcy, & Smyth, 2012). The great majorities of chemotherapy drugs including CTX caused non-selective cytotoxicity and immunosuppression. Thus discovery of new
Group
Body weight (g)
N.S. CTX (25 mg/kg d) SIP-S (15 mg/kg d) CTX (12.5 mg/kg d) + SIP-S (15 mg/kg d)
25.27 22.23 24.51 23.28
± ± ± ±
3.13 2.60 2.88 2.17
Tumor weight (g) 2.37 1.39 1.59 1.32
± ± ± ±
0.52 0.67* 0.62* 0.45*
Inhibitory rate (%) – 41.35 32.91 44.30
CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. * P < 0.05, compared with control.
safe compounds, capable of potentiating immune function, has become an important goal of research in the biomedical sciences (Sun et al., 2013). The result of this experiment showed that SIPS had significant antitumor and immunomodulatory activity, and increased body weight of the tumor-bearing mice at a high dose, which indicated that SIP-S had no obvious toxicity in vivo. 3.2. The combination treatment of SIP-S and CTX shows higher anticancer activity As shown in Table 2, after administration for 10 d, the body weights of mice in drug groups showed no obvious difference compared with those in control group. SIP-S and CTX significantly inhibited tumor growth in S180-bearing mice (P < 0.05). Moveover, the combination treatment of SIP-S (15 mg/kg d) and CTX (12.5 mg/kg d) showed higher anti-tumor activity than both SIP-S (15 mg/kg d) and CTX (25 mg/kg d) alone. Compared with the control, the inhibition rates of SIP-S (15 mg/kg d), CTX (25 mg/kg d) and SIP-S (15 mg/kg d) + CTX (12.5 mg/kg d) were 32.9%, 41.4% and 44.3%, respectively. In addition, the combination treatment of SIPS and CTX cause no significant damage to the spleen or thymus in mice (Fig. 2). Combination therapy is frequently used in cancer treatment to reduce drug resistance, alleviate adverse effects and enhance anticancer efficacy. In recent years, accumulated evidence has demonstrated that some biological macromolecules, including polysaccharides, can increase the efficacy of conventional chemotherapy drugs (Zhang et al., 2015). Here we found that the combination treatment of SIP-S and CTX showed higher antitumor activity and lower toxicity in vivo. The result suggested that
Fig. 1. Effect of SIP-S on thymus and spleen indices of S180-bearing mice(−¯x ± s, n = 8). (A) The spleen index of tumor-bearing mice. (B) The thymus index of tumor-bearing mice. CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. *** P < 0.001, ** P < 0.01, and * P < 0.05, compared with control; ## P < 0.01 and # P < 0.05, compared with CTX.
A. Zong et al. / Carbohydrate Polymers 129 (2015) 50–54
53
Fig. 2. Effects of SIP-S combined with CTX on thymus and spleen indices of S180-bearing mice (−¯x ± s, n = 8). (A) The spleen index of tumor-bearing mice. (B) The thymus index of tumor-bearing mice. CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. * P < 0.05, compared with control.
combination of chemotherapy drugs with SIP-S was a potential treatment for cancer. 3.3. SIP-S induces tumor cells apoptosis in vitro The effect of SIP-S on tumor cells apoptosis was assessed by flow cytometry after staining the cells with annexin V-FITC and PI. As shown in Fig. 3, SIP-S significantly induced SKOV3 cells apoptosis in vitro, and mainly increased the late apoptosis rates. The apoptosis rates of SKOV3 cells under the concentrations of 500, 100, 20 and 0 g/mL of SIP-S for 36 h were 25.95%, 10.10%, 7.55% and 7.08%, respectively. Compared with vehicle treated group, the apoptotic
rate of SKOV3 cells was significantly increased by 36 h pretreatment with 500 g/mL of SIP-S (P < 0.05). Carcinogenesis can be viewed as a process that involves accelerated, and abnormal, cellular changes in which the genes controlling proliferation, differentiation, and apoptosis are transformed under selective environmental pressures (Bertram, 2000). Apoptosis is the mechanism used by metazoans to regulate tissue homeostasis through the elimination of redundant or potentially deleterious cells (Sun, Hail, & Lotan, 2004). Malignant cells have the capacity to evade apoptosis to form a tumor (de Bruin & Medema, 2008). Apoptosis induction is one of the most potent therapeutic strategies against cancer (Hassan, Watari, AbuAlmaaty, Ohba, & Sakuragi,
Fig. 3. Effect of SIP-S treatment on apoptosis of SKOV3 cells. Apoptosis of SKOV3 cells treated with SIP-S for 36 h was detected by annexin V and PI staining using flow cytometry analysis. Results visualized as a representative experiment (A) or mean values ± SEM of three experiments (B), each performed in triplicate. * P < 0.05 versus control (untreated cells).
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after administration for 10 d at the doses of 10, 20 and 30 mg/kg d. Besides, the combination treatment of SIP-S (15 mg/kg d) and CTX (12.5 mg/kg d) showed higher anti-tumor activity than both SIP-S (15 mg/kg d) and CTX (25 mg/kg d). CTX is an immune suppressive agent and considerably decreased the spleen and thymus indices in mice. In contrast, SIP-S showed significant immunoenhancing activity, which might be one of the mechanisms of its anti-tumor activity and enhanced effect in combination with CTX. Results of in vitro experiments showed that SIP-S induce SKOV3 cells apoptosis through suppressing the expression of PARP-1 and increasing the expression of caspase-3, -8, -9 and Bax in tumor cells in a dosedependent manner. The apoptosis inducing activity can be another mechanism of action of SIP-S to inhibit tumor growth. Since SIP-S has been demonstrated to have significant anti-metastatic activity in vivo (Zong et al., 2013), it is believed that SIP-S can be a candidate of anti-tumor drugs that affect tumor growth and metastasis, and deserves further research. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 30973678), the Natural Science Foundation of Shandong Province (ZR2009CZ010) and the Jinan Science and Technology Development Funds For Youths (No. 20080212). Fig. 4. Effects of SIP-S treatment on expression of apoptosis associated proteins in SKOV3 cells. The protein expression were detected by Western blot analysis using lysates from SKOV3 cells treated with varying doses of SIP-S for 48 h. Each of the blots shown was demonstrated to have equal protein loading by re-probing with the monoclonal antibody for -actin.
2014). Therefore, the apoptosis-inducting activity of SIP-S could be one important mechanism of its anti-tumor effect. 3.4. SIP-S regulates apoptosis-related proteins expression in tumor cells In order to understand the mechanism of SIP-S-induced apoptosis, the expression of apoptosis proteins in SKOV3 cells was evaluated using Western blot analysis. As shown in Fig. 4, SIP-S could suppress the expression of PARP-1 and increase the expression of caspase-3, -8, -9 and Bax in tumor cells and the effect was associated with concentration, while it had no significant influence on the expression of Bcl-2 and caspase-10. PARP-1 is the most abundant member in PARP family, and plays a role in the repair of DNA breaks. Through binding on DNA damaged structures, PARP-1 recruits repair enzymes and serves as a survival factor (Diamantopoulos et al., 2014). The caspase family and Bcl-2 family play important roles in the regulation of apoptosis (Wong, 2011). Caspases play the central role in the transduction of death receptor apoptotic signals. Bcl-2 members are localized in the mitochondria and have either proapoptotic (Bax, Bak, Bid, and Bim) or antiapoptotic (Bcl-2, Bcl-xL, and Bcl-W) functions (Hassan et al., 2014). SIP-S induced apoptosis of ovarian cancer cells through down-regulating the expression of PARP-1 and up-regulating the expression of caspase-3, -8, -9 and Bax in tumor cells. 4. Conclusion In the present study, we report, for the first time, the anticancer and immunomodulating activity of SIP-S, and the anticancer effect of combination treatment of SIP-S and CTX on mice transplanted with S180. SIP-S significantly inhibited the tumor growth and increased the spleen and thymus indices in S180-bearing mice
References Beavis, P. A., Stagg, J., Darcy, P. K., & Smyth, M. J. (2012). CD73: A potent suppressor of antitumor immune responses. Trends in Immunology, 33, 231–237. Bertram, J. S. (2000). The molecular biology of cancer. Molecular Aspects of Medicine, 21, 167–223. de Bruin, E. C., & Medema, J. P. (2008). Apoptosis and non-apoptotic deaths in cancer development and treatment response. Cancer Treatment Reviews, 34, 737–749. Diamantopoulos, P. T., Sofotasiou, M., Papadopoulou, V., Polonyfi, K., Iliakis, T., & Viniou, N. A. (2014). PARP1-driven apoptosis in chronic lymphocytic leukemia. BioMed Research International, 2014, 106713. Ehrke, M. J. (2003). Immunomodulation in cancer therapeutics. International Immunopharmacology, 3, 1105–1119. Geresh, S., Manontov, A., & Weinstein, J. (2002). Sulfation of extracellular polysaccharides of red microalgae: Preparation, characterization and properties. The Journal of Biochemical and Biophysical Methods, 50, 179–187. Hassan, M., Watari, H., AbuAlmaaty, A., Ohba, Y., & Sakuragi, N. (2014). Apoptosis and molecular targeting therapy in cancer. BioMed Research International, 2014, 150845. Liu, C. H., Li, X. D., Li, Y. H., Feng, Y., Zhou, S., & Wang, F. S. (2008). Structural characterisation and antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink. Food Chemistry, 110, 807–813. Mitchell, M. S. (2003). Combinations of anticancer drugs and immunotherapy. Cancer Immunology Immunotherapy, 52, 686–692. Schepetkin, I. A., & Quinn, M. T. (2006). Botanical polysaccharides: Macrophage immunomodulation and therapeutic potential. International Immunopharmacology, 6, 317–333. Shi, X. L., Zhao, Y., Jiao, Y. D., Shi, T. R., & Yang, X. B. (2013). ROS-dependent mitochondria molecular mechanisms underlying antitumor activity of Pleurotus abalonus acidicpolysaccharides in human breast cancer MCF-7 cells. PLoS ONE, 8, e64266. Sun, S. Y., Hail, N., Jr., & Lotan, R. (2004). Apoptosis as a novel target for cancer chemoprevention. Journal of the National Cancer Institute, 96, 662–672. Sun, Y., Sun, T. W., Wang, F., Zhang, J., Li, C., Chen, X. N., et al. (2013). A polysaccharide from the fungi of Huaier exhibits anti-tumor potential and immunomodulatory effects. Carbohydrate Polymers, 92, 577–582. Wang, S. B., Cheng, Y. N., Wang, F. S., Sun, L. R., Liu, C. H., Chen, G. J., et al. (2008). Inhibition activity of sulfated polysaccharide of Sepiella maindroni ink on matrix metalloproteinase (MMP)-2. Biomedicine & Pharmacotherapy, 62, 297–302. Wong, R. S. (2011). Apoptosis in cancer: From pathogenesis to treatment. Journal of Experimental & Clinical Cancer Research, 30, 87. Yuan, H., Song, J., Li, X., Li, N., & Dai, J. (2006). Immunomodulation and antitumor activity of kappa-carrageenan oligosaccharides. Cancer Letters, 243, 228–234. Zhang, G. W., Qin, G. F., Han, B., Li, C. X., Yang, H. G., Nie, P. H., et al. (2015). Efficacy of Zhuling polyporus polysaccharide with BCG to inhibit bladder carcinoma. Carbohydrate Polymers, 118, 30–35. Zong, A. Z., Cao, H. Z., & Wang, F. S. (2012). Anticancer polysaccharides from natural resources: A review of recent research. Carbohydrate Polymers, 90, 1395–1410. Zong, A. Z., Zhao, T., Zhang, Y., Song, X. L., Shi, Y. K., Cao, H. Z., et al. (2013). Antimetastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni ink. Carbohydrate Polymers, 91, 403–409.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Anti-tumor activity and the mechanism of SIP-S: A sulfated polysaccharide with anti-metastatic effect Aizhen Zong a,e , Yuhong Liu b , Yan Zhang a , Xinlei Song a , Yikang Shi c , Hongzhi Cao c , Chunhui Liu a,c , Yanna Cheng d , Wenjie Jiang a , Fangling Du e , Fengshan Wang a,c,∗ a Key Laboratory of Chemical Biology (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, PR China b School of Pharmaceutical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong, PR China c National Glycoengineering Research Center, Shandong University, Jinan 250012, Shandong, PR China d Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, PR China e Institute of Agro-Food Science Technology, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 31 January 2015 Received in revised form 1 April 2015 Accepted 12 April 2015 Available online 21 April 2015 Keywords: SIP-S Cyclophosphamide Anti-tumor activity Immunoenhancing activity Apoptosis
a b s t r a c t Our previous studies demonstrated that SIP-S had anti-metastatic activity and inhibited the growth of metastatic foci. Here we report the anti-tumor and immunoregulatory potential of SIP-S. SIP-S could significantly inhibit tumor growth in S180-bearing mice, and the inhibition rates was 43.7% at 30 mg/kg d. Besides, SIP-S could improve the thymus and spleen indices of S180-bearing mice and the mice treated with CTX. The combination of SIP-S (15 mg/kg d) with CTX (12.5 mg/kg d) showed higher anti-tumor potency than CTX (25 mg/kg d) alone. These results indicated that SIP-S had immunoenhancing and anticancer activity, and the immunoenhancing activity might be one mechanism for its anti-tumor activity. Flow cytometry results showed that SIP-S could induce tumor cells apoptosis. Western blot analysis indicated that SIP-S could upregulate the expression of pro-apoptotic proteins, caspase-3, -8, -9 and Bax, and downregulate the expression of anti-apoptotic protein PARP-1 in tumor cells in a dose-dependent manner. In summary, SIP-S has anti-tumor activity, which may be associated with its immunostimulating and pro-apoptotic activity. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Cancer is a leading cause of death worldwide. Chemotherapy is one of the standard therapeutic protocols for cancer. However, most of the chemotherapeutic agents produce non-selective cytotoxicity and cause severe side effects including hemopoetic and immune suppression (Ehrke, 2003). The low immune function of an organism may not only result in the generation and development of a tumor, but also be one of the most important factors that prevent the tumor patients’ recovery (Mitchell, 2003). The enhancement of host immune response has been recognized as a possible means
∗ Corresponding author at: Shandong University, Key Laboratory of Chemical Biology (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, No. 44 Wenhuaxi Road, Jinan 250012, Shandong, PR China. Tel.: +86 531 88382658; fax: +8653188382548. E-mail addresses: [email protected], [email protected] (F. Wang). http://dx.doi.org/10.1016/j.carbpol.2015.04.017 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
of inhibiting tumor growth without harming the host (Yuan, Song, Li, Li, & Dai, 2006). Therefore, searching for new anti-tumor drugs with potential immunoenhancing effect is essential for cancer therapy. Most polysaccharides from natural sources are found to be nontoxic substances with wide variety of biological activities, and have attracted lots of attention in the biochemical and medical areas (Zong, Cao, & Wang, 2012). Thus, natural polysaccharides are ideal candidates for therapeutics with immunomodulatory and antitumor effects and low toxicity (Schepetkin & Quinn, 2006). SIP was a new heteropolysaccharide isolated from the ink of cuttlefish Sepiella maindroni de Rochebruns (Liu et al., 2008). We then prepared the sulfated SIP (SIP-S) by using chlorosulfonic acid (Geresh, Manontov, & Weinstein, 2002). Studies have shown that SIP-S has significant anti-metastatic and anti-angiogenic activities, and can also obviously inhibit metastatic tumor growth, which suggests that SIP-S may be an ideal candidate for cancer therapy (Zong et al., 2013).
A. Zong et al. / Carbohydrate Polymers 129 (2015) 50–54
In the present study, we carried out experiments to investigate the anti-tumor effects of SIP-S alone and its combination with cyclophosphamide(CTX) on Sarcoma 180 (S180)-tumor bearing mice. Meanwhile the immunomodulatory activity of SIP-S was evaluated. The results will be helpful for developing novel antitumor drugs.
51
2. Materials and methods
last administration, mice were weighed and sacrificed by cervical dislocation. Tumors, spleens and thymus were excised and weighted, respectively. The tumor inhibitory rate was calculated as ((A − B)/A) × 100%, where A and B were the average tumor weights of the control group and the treated group, respectively. Thymus index was expressed as the thymus weight relative to body weight. Spleen index was expressed as the spleen weight relative to body weight.
2.1. Reagents and antibodies
2.4. Synergetic anti-tumor activity of SIP-S and CTX
SIP-S was prepared as described previously (Wang et al., 2008), and dissolved in serum-free medium for the in vitro assay and in normal saline (N.S.) for the in vivo assay. Mouse anti-caspase-3 monoclonal antibody (against a full length caspase-3 of human origin, SC-65497), rabbit anti-Bcl-2 polyclonal antibody (against amino acids 1-205 of Bcl-2 of human origin, SC-783), rabbit anti-Bax polyclonal antibody (against the N-terminus of Bax of human origin, SC-493), and mouse anti-PARP1 monoclonal antibody (against amino acids 1–300 mapping at the N-terminus of PARP-1 of human origin, SC-74470) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-caspase-9 polyclonal antibody (9502) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-caspase-8 polyclonal antibody (AP0358) was purchased from Bioworld Technology, Inc. (Louis Park, MN, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit rabbit IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). FITC Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Bestbio. Co. Ltd., (Bestbio. Co. Ltd., Shanghai, China).
The models of S180 tumor-bearing mice were established and then randomly divided into four groups, each group consisting of 8 animals. The groups were as follows: control group (N.S.); CTX group (25 mg/kg body weight); SIP-S group (15 mg/kg body weight); CTX + SIP-S group (CTX 12.5 mg/kg body weight + SIPS 15 mg/kg body weight). All the groups were administered by intraperitoneal injection of 0.2 mL every day for 10 d, starting from the fifth day after tumor implantation. After the last administration, the mice were sacrificed and treated as mentioned above, and the tumor inhibitory rate, thymus index and spleen index were calculated, respectively.
2.2. Animals and cell lines The use of animals was approved by the Institutional Animal Care Committee of Shandong University, with firm adherence to the Ethical Guidelines for the Care and Use of Animals. Kunming mice (male, 18–22 g) were purchased from the Experimental Animal Center of Shandong University (Jinan, China). Mouse sarcoma cell line S180, human ovarian carcinoma cell line SKOV3 and human umbilical vein cell fusion cell EA.hy926 were obtained from Shanghai Cell Bank, the Institute of Cell Biology, China Academy of Sciences (Shanghai, China). S180 and SKOV3 cells were maintained in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillinstreptomycin (100 IU/mL–100 g/mL), 2 mM glutamine and 10 mM HEPES. EA.hy926 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillinstreptomycin (100 IU/mL–100 g/mL), 2 mM glutamine and 10 mM HEPES. All cells were cultured in a humid atmosphere (5% CO2 , 95% air) at 37 ◦ C, fed every 2 to 3 d and harvested by brief incubation in 0.02% EDTA–0.25% trypsin. 2.3. Anti-tumor activity of SIP-S in vivo S180 cells were maintained as ascites in Kunming mice (6–8 weeks old) for 7 d, and then drawn out from the tumor bearing mice. After being washed and diluted with phosphate-buffered saline (PBS), the S180 cells (0.2 mL, 2 × 106 cells) were injected subcutaneously into the right axilla of Kunming mice. After 24 h, the mice were weighed and randomly divided into five groups, each group consisting of 8 animals. The groups were as follows: control group (normal saline, N.S.); cyclophosphamide (CTX) group (30 mg/kg body weight); three SIP-S groups (30, 20 and 10 mg/kg body weight). All the groups were administered by intraperitoneal injection of 0.2 mL every day for 10 days. 24 h after the
2.5. Detection of apoptosis by flow cytometry Apoptosis analysis was performed by using an Annexin VFITC/PI Apoptosis Detection Kit (Bestbio. Co. Ltd., Shanghai, China) according to the manufacturer’s instructions (Shi, Zhao, Jiao, Shi, & Yang, 2013). Briefly, SKOV3 cells (2 × 105 per well) seeded in 6-well plates were incubated with different doses of SIP-S (500, 100, 20 g/mL) for 36 h. The cells were harvested and washed twice with cold PBS. Then the cells were resuspended in Binding Buffer at a concentration of 1 × 106 cells/mL and stained with 5 L of FITC Annexin V and 10 L PI at 25 ◦ C for 15 min in the dark. The stained cells were analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA) within 1 h. The experiments were repeated three times. 2.6. Western blot analysis Skov3 cells (1 × 105 per well) seeded in 6-well plates were treated with different doses of SIP-S (500, 100, 20 g/mL) for 48 h. The medium was removed and the cells were washed with PBS. The cells were then lysed in 50 L of lysis buffer at 0 ◦ C (icebath) for 30 min with intense shock for 30 s every 10 min. The concentration of total protein was determined using the BCA protein assay method (Pierce, Rockford, IL). Equal amounts of protein (30 g/lane) in the cell extracts were fractionated by SDS-PAGE and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST buffer (20 mM Tris-buffered saline and 0.1% Tween) containing 5% non-fat dry milk for 2 h at room temperature, the membranes were incubated with mouse anti-caspase-3 monoclonal antibody, rabbit anti-Bcl-2 polyclonal antibody, rabbit anti-Bax polyclonal antibody, mouse anti-PARP-1 monoclonal antibody, rabbit anti-caspase-9 polyclonal antibody, and rabbit anti-caspase-8 polyclonal antibody overnight at 4 ◦ C, followed by washing 3 times and reacting with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). The proteins were then detected using chemiluminescence agents (ECL, Amersham). The density of the immunoreactive bands was analyzed using Image-Pro Plus Version 6.0. 2.7. Statistical analysis Data was described as mean ± S.D. and analyzed by Student’s two-tailed t-test. The limit of statistical significance was P < 0.05.
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Table 1 Anti-tumor effect of SIP-S in S180-bearing mice (−¯x ± s, n = 8). Group
Body weight (g)
CTX (30 mg/kg d) SIP-S (30 mg/kg d) SIP-S (20 mg/kg d) SIP-S (10 mg/kg d) N.S.
20.33 26.84 23.65 22.83 24.87
± ± ± ± ±
3.93* 2.55# 2.18 3.88 3.29
Tumor weight (g) 0.63 0.89 0.95 1.06 1.58
± ± ± ± ±
0.17*** 0.32** 0.21* 0.56 0.24
Table 2 Anti-tumor effect of SIP-S combined with CTX in S180-bearing mice (¯x ± s, n = 8). Inhibitory rate (%) 60.13 43.67 39.87 32.91 –
CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. *** P < 0.001, ** P < 0.01, and * P < 0.05, compared with control. # P < 0.05, compared with CTX.
Statistical analysis was done using SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA). 3. Results and discussion 3.1. SIP-S inhibits tumor growth and enhances immune function in tumor-bearing mice The anti-tumor and immunoregulatory activity of SIP-S was evaluated in the S180-bearing mice model. As shown in Table 1, after administration for 10 d, the body weights of CTX-treated mice were significantly decreased compared with those treated with N.S. and SIP-S (30 mg/kg d) (P < 0.05), while SIP-S groups showed no significant difference compared with control group. The growth of S180 tumors in SIP-S-treated mice was significantly suppressed compared with those treated with normal saline (P < 0.01 or P < 0.05). The inhibition rates of 10, 20 and 30 mg/kg d of SIP-S were 32.9%, 39.9% and 43.7%, respectively. There was no significant difference between the tumor weights of the mice treated with high-dose SIP-S (30 mg/kg d) and those treated with CTX. Moreover, CTX considerably decreased the spleen and thymus indices in S180bearing mice compared with control group and SIP-S (30 mg/kg d) group (P < 0.05 or P < 0.01) (Fig. 1). In contrast, SIP-S significantly improved the thymus and spleen indices in tumor bearing mice compared with N.S. and CTX (Fig. 1). The immune system is fundamental in suppressing both the initiation of carcinogenesis and the progression of established tumors (Beavis, Stagg, Darcy, & Smyth, 2012). The great majorities of chemotherapy drugs including CTX caused non-selective cytotoxicity and immunosuppression. Thus discovery of new
Group
Body weight (g)
N.S. CTX (25 mg/kg d) SIP-S (15 mg/kg d) CTX (12.5 mg/kg d) + SIP-S (15 mg/kg d)
25.27 22.23 24.51 23.28
± ± ± ±
3.13 2.60 2.88 2.17
Tumor weight (g) 2.37 1.39 1.59 1.32
± ± ± ±
0.52 0.67* 0.62* 0.45*
Inhibitory rate (%) – 41.35 32.91 44.30
CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. * P < 0.05, compared with control.
safe compounds, capable of potentiating immune function, has become an important goal of research in the biomedical sciences (Sun et al., 2013). The result of this experiment showed that SIPS had significant antitumor and immunomodulatory activity, and increased body weight of the tumor-bearing mice at a high dose, which indicated that SIP-S had no obvious toxicity in vivo. 3.2. The combination treatment of SIP-S and CTX shows higher anticancer activity As shown in Table 2, after administration for 10 d, the body weights of mice in drug groups showed no obvious difference compared with those in control group. SIP-S and CTX significantly inhibited tumor growth in S180-bearing mice (P < 0.05). Moveover, the combination treatment of SIP-S (15 mg/kg d) and CTX (12.5 mg/kg d) showed higher anti-tumor activity than both SIP-S (15 mg/kg d) and CTX (25 mg/kg d) alone. Compared with the control, the inhibition rates of SIP-S (15 mg/kg d), CTX (25 mg/kg d) and SIP-S (15 mg/kg d) + CTX (12.5 mg/kg d) were 32.9%, 41.4% and 44.3%, respectively. In addition, the combination treatment of SIPS and CTX cause no significant damage to the spleen or thymus in mice (Fig. 2). Combination therapy is frequently used in cancer treatment to reduce drug resistance, alleviate adverse effects and enhance anticancer efficacy. In recent years, accumulated evidence has demonstrated that some biological macromolecules, including polysaccharides, can increase the efficacy of conventional chemotherapy drugs (Zhang et al., 2015). Here we found that the combination treatment of SIP-S and CTX showed higher antitumor activity and lower toxicity in vivo. The result suggested that
Fig. 1. Effect of SIP-S on thymus and spleen indices of S180-bearing mice(−¯x ± s, n = 8). (A) The spleen index of tumor-bearing mice. (B) The thymus index of tumor-bearing mice. CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. *** P < 0.001, ** P < 0.01, and * P < 0.05, compared with control; ## P < 0.01 and # P < 0.05, compared with CTX.
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Fig. 2. Effects of SIP-S combined with CTX on thymus and spleen indices of S180-bearing mice (−¯x ± s, n = 8). (A) The spleen index of tumor-bearing mice. (B) The thymus index of tumor-bearing mice. CTX, cyclophosphamide. N.S., normal saline (control). The values are presented as mean ± S.D. * P < 0.05, compared with control.
combination of chemotherapy drugs with SIP-S was a potential treatment for cancer. 3.3. SIP-S induces tumor cells apoptosis in vitro The effect of SIP-S on tumor cells apoptosis was assessed by flow cytometry after staining the cells with annexin V-FITC and PI. As shown in Fig. 3, SIP-S significantly induced SKOV3 cells apoptosis in vitro, and mainly increased the late apoptosis rates. The apoptosis rates of SKOV3 cells under the concentrations of 500, 100, 20 and 0 g/mL of SIP-S for 36 h were 25.95%, 10.10%, 7.55% and 7.08%, respectively. Compared with vehicle treated group, the apoptotic
rate of SKOV3 cells was significantly increased by 36 h pretreatment with 500 g/mL of SIP-S (P < 0.05). Carcinogenesis can be viewed as a process that involves accelerated, and abnormal, cellular changes in which the genes controlling proliferation, differentiation, and apoptosis are transformed under selective environmental pressures (Bertram, 2000). Apoptosis is the mechanism used by metazoans to regulate tissue homeostasis through the elimination of redundant or potentially deleterious cells (Sun, Hail, & Lotan, 2004). Malignant cells have the capacity to evade apoptosis to form a tumor (de Bruin & Medema, 2008). Apoptosis induction is one of the most potent therapeutic strategies against cancer (Hassan, Watari, AbuAlmaaty, Ohba, & Sakuragi,
Fig. 3. Effect of SIP-S treatment on apoptosis of SKOV3 cells. Apoptosis of SKOV3 cells treated with SIP-S for 36 h was detected by annexin V and PI staining using flow cytometry analysis. Results visualized as a representative experiment (A) or mean values ± SEM of three experiments (B), each performed in triplicate. * P < 0.05 versus control (untreated cells).
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after administration for 10 d at the doses of 10, 20 and 30 mg/kg d. Besides, the combination treatment of SIP-S (15 mg/kg d) and CTX (12.5 mg/kg d) showed higher anti-tumor activity than both SIP-S (15 mg/kg d) and CTX (25 mg/kg d). CTX is an immune suppressive agent and considerably decreased the spleen and thymus indices in mice. In contrast, SIP-S showed significant immunoenhancing activity, which might be one of the mechanisms of its anti-tumor activity and enhanced effect in combination with CTX. Results of in vitro experiments showed that SIP-S induce SKOV3 cells apoptosis through suppressing the expression of PARP-1 and increasing the expression of caspase-3, -8, -9 and Bax in tumor cells in a dosedependent manner. The apoptosis inducing activity can be another mechanism of action of SIP-S to inhibit tumor growth. Since SIP-S has been demonstrated to have significant anti-metastatic activity in vivo (Zong et al., 2013), it is believed that SIP-S can be a candidate of anti-tumor drugs that affect tumor growth and metastasis, and deserves further research. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 30973678), the Natural Science Foundation of Shandong Province (ZR2009CZ010) and the Jinan Science and Technology Development Funds For Youths (No. 20080212). Fig. 4. Effects of SIP-S treatment on expression of apoptosis associated proteins in SKOV3 cells. The protein expression were detected by Western blot analysis using lysates from SKOV3 cells treated with varying doses of SIP-S for 48 h. Each of the blots shown was demonstrated to have equal protein loading by re-probing with the monoclonal antibody for -actin.
2014). Therefore, the apoptosis-inducting activity of SIP-S could be one important mechanism of its anti-tumor effect. 3.4. SIP-S regulates apoptosis-related proteins expression in tumor cells In order to understand the mechanism of SIP-S-induced apoptosis, the expression of apoptosis proteins in SKOV3 cells was evaluated using Western blot analysis. As shown in Fig. 4, SIP-S could suppress the expression of PARP-1 and increase the expression of caspase-3, -8, -9 and Bax in tumor cells and the effect was associated with concentration, while it had no significant influence on the expression of Bcl-2 and caspase-10. PARP-1 is the most abundant member in PARP family, and plays a role in the repair of DNA breaks. Through binding on DNA damaged structures, PARP-1 recruits repair enzymes and serves as a survival factor (Diamantopoulos et al., 2014). The caspase family and Bcl-2 family play important roles in the regulation of apoptosis (Wong, 2011). Caspases play the central role in the transduction of death receptor apoptotic signals. Bcl-2 members are localized in the mitochondria and have either proapoptotic (Bax, Bak, Bid, and Bim) or antiapoptotic (Bcl-2, Bcl-xL, and Bcl-W) functions (Hassan et al., 2014). SIP-S induced apoptosis of ovarian cancer cells through down-regulating the expression of PARP-1 and up-regulating the expression of caspase-3, -8, -9 and Bax in tumor cells. 4. Conclusion In the present study, we report, for the first time, the anticancer and immunomodulating activity of SIP-S, and the anticancer effect of combination treatment of SIP-S and CTX on mice transplanted with S180. SIP-S significantly inhibited the tumor growth and increased the spleen and thymus indices in S180-bearing mice
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