- Email: [email protected]
Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii
ARTICLE IN PRESS
JID: PHYMED
[m5G;August 28, 2015;11:15]
Phytomedicine xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii Siran Yu a, Xuewei Ye a, Lu Chen a, Xin Xie b, Qian Zhou b, Xiao-Yuan Lian b, Zhizhen Zhang a,∗
Q1
a b
Ocean College, Zhejiang University, Hangzhou 310058, China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e
i n f o
Article history: Received 6 March 2015 Revised 4 August 2015 Accepted 14 August 2015 Available online xxx Keywords: Holothuria moebii Sulfated saponin Desulfated saponin Inhibition of the proliferation of colorectal cancer cells Anti-colorectal tumor effects
1 2 3 4 5 6 7 8 9 10 11 12 13
a b s t r a c t Background: Whether sulfated saponins from Holothuria moebii inhibit the proliferation of colorectal cancer cells and have anti-colorectal tumor effects in animal model has not been investigated. Purpose: To evaluate the cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii. Method: (1) Column chromatography was used to prepare the total and individual saponins and HPLC was applied to define the components of the total saponins; (2) the activity of the total and individual saponins inhibiting the proliferation of human colorectal cancer cells was determined by SRB assay and the apoptosis induced by the saponins was qualified using cytometric analysis with Annexin V-FITC/PI double staining; and (3) the antitumor effects of the sulfated saponins on colorectal CT-26 tumor-bearing Balb/c mice were tested. Results: The total and individual sulfated saponins significantly inhibited the proliferation of four different human colorectal cancer cells with IC50 values ranging from 1.04 to 4.08 μM (or 1.46 to 3.24 μg/ml for total saponins) and induced late apoptosis at an early treatment time in cancer cells. The total saponins (120 mg/kg) had antitumor activity in colorectal CT-26 tumor-bearing Balb/c mice. Conclusion: The sulfated saponins from H. moebii remarkably inhibited the proliferation of different human colorectal cancer cells and had significant anti-colorectal tumor activity in animal model. © 2015 Elsevier GmbH. All rights reserved.
Introduction Sea cucumbers (class Holothuroidea) are echinoderms, which have 25 families with more than 1400 species (Kim and Himaya 2012). Because of the nutritive values, potential health benefits, and therapeutic uses, the Sea cucumbers have economic importance (Bordbar et al. 2011). Dehydrated sea cucumbers are commercially sold as a human food source in Asian countries, especially China, Korea, Indonesia and Japan. It was estimated that the total production of sea cucumbers was over 12,000 metric tons (dry weight) during the period of 1992−2001 (Lovatelli et al. 2004). Sea cucumbers have been well recognized as a tonic and traditional remedy in China and Malaysia for their activities against hypertension, arteriosclerosis, tumor, rheumatism, diabetes, asthma, cuts and burns, impotence
Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DOX, doxorubicin; FU, fluorouracil; HPLC, high performance liquid chromatography; HRESIMS, high resolution electrospray ionization mass spectroscopy; NMR, nuclear magnetic resonance; ODS, octadecyl-functionalized silica gel; PI, propidium iodide; SRB, sulforhodamine B; TGI, tumor growth inhibition; T. Sap., total saponins. ∗ Corresponding author. Tel./fax: +86 57188208432. E-mail address: [email protected] (Z. Zhang).
and constipation. In addition, a number of biological activities including anticancer, antiangiogenic, anticoagulant, antihypertension and anti-inflammatory are ascribed to various species of sea cucumbers (Bordbar et al. 2011; Guan and Wang 1999; Kim and Himaya 2012). Triterpene glycosides (sea cucumber saponins) are the major and most interesting bioactive metabolites in sea cucumbers. To date, more than 100 saponins have been isolated and identified from sea cucumbers (Kim and Himaya 2012). Accumulated studies demonstrated that most of these sea cucumber saponins had significant cytotoxicity toward cancer cells and multiple mechanisms of action, such as interfering with cell cycle progression, inducing apoptosis, promoting stabilization of microtubule and enhancing the generation of ceramide, were involved in their antitumor effects (Kim and Himaya 2012; Tian et al. 2013; Yun et al. 2012). However, these saponins were difficult to develop as new anticancer drugs because of their haemolytic properties. Fortunately, mature technique of interstitial chemotherapy of glioblastoma in situ used in clinic (Boiardi et al. 1999; Tian et al. 2013; Tomita et al. 1991) has provided us with a successful approach to administrate saponins in situ, which avoids the side effects of hemolytic activity and might overcome the bottleneck of the development of saponins as new drugs (Tian et al. 2013). Several saponins exhibited significant anti-glioblastoma effects in vivo
http://dx.doi.org/10.1016/j.phymed.2015.08.007 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
JID: PHYMED 2
ARTICLE IN PRESS
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
Fig. 1. Structures of compounds 1−4, 3A, and 3B.
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
by in situ administration (interstitial injection or catheter insertion) (Lin et al. 2008; Tian et al. 2013). Although the in vitro activity of sea cucumber saponins against different types of cancer cells has been well documented, the in vivo antitumor efficacy and the molecular antitumor mechanisms of sea cucumber saponins have not been fully characterized (Kim and Himaya 2012; Menchinskaya et al. 2013). Holothuria moebii Ludwig is a species of sea cucumbers in the Holothuriidae family. The chemical constituents and medicinal uses of this species had not been previously described until we recently investigated the anti-glioma constituents of this sea cucumber and discovered a novel saponin moebioside A (Sap. 2, Fig. 1) (Yu et al. 2015; Zhang et al. 2014). Moebioside A significantly inhibited the proliferation of different glioma cells and induced apoptosis in human glioblastoma U87-MG cells. Moebioside A also selectively reduced the expression levels of several glioma metabolic enzymes of glycolysis and glutaminolysis, suggesting that targeting multiple glioma metabolic regulators might be one of the anti-glioma mechanisms of moebioside A. However, whether the saponins from H. moebii have activity inhibiting the proliferation of colorectal cancer cells or antitumor effects on colorectal tumor-bearing animals has not been investigated. The aims of this study were (1) to characterize the chemical profiles of total saponins purified from the sea cucumber H. moebii by identifying the ingredients of the total saponins and determining the concentration of the total saponins and each ingredient and (2) to evaluate the cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber H. moebii.
Materials and methods
62
General experimental procedures
63
Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18-Prep) and Diaion HP-20 (Mitsubishi Chemical) were utilized for column chromatography. TLC analysis was conducted on silica gel 60 RP-18 F254S aluminum TLC plates (Merck). HPLC analysis was performed on an Agilent 1260 HPLC system with a DAD detector using an ODS-2 Hypersil column (150 × 4.6 mm2 , 5 μm, Thermo Scientific). HPLC and analytic grade solvents used for this study were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Standard compounds of holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3), 24-dehydroechinoside B (Sap. 4), 22,25-oxidoholothurinogenin (3A), and desulfated saponin (3B) were prepared by our group (Yu et al. 2015). Doxorubicin (DOX, >98.0%) was obtained from Sigma-Aldrich and 5-fluorouracil (5-FU, >98.0%) from Shanghai Xudong Haipu Pharmaceutical Co. Ltd. A fluorescence microscope (Nikon SMZ1000) was used to detect apoptosis and necrosis in cancer cells doubly stained by DAPI (4,6-diamidino-2phenylindole) and PI (propidium iodide). Flow cytometry (Beckman Coulter, FC500MCL) was applied for quantitation of apoptotic and necrotic cells. Annexin V apoptosis detection kits were obtained from Invitrogen. Human colorectal cancer HCT-8, HCT-15, HCT-116 and SW620 cells and mouse colorectal tumor CT-26 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Human
64
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
JID: PHYMED
ARTICLE IN PRESS
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
87
normal colorectal CCD-18Co cells were obtained from Shanghai Bogoo Biotechnology Co. Ltd.
88
Sea cucumber material
89
Fresh sea cucumbers of H. moebii were collected from the Turtle Islet in the South China Sea close to Shanwei City, Guangdong Province, China in April 2014. A voucher sample (SW-A042014) was authenticated by one of the authors (Z. Zhang) and deposited in the Laboratory at the Institute of Marine Biology, Ocean College, Zhejiang University, China.
86
90 91 92 93 94
95
Preparation of total saponins and individual saponins
96
107
The total saponins (T. Sap., 5.6 g) were prepared from the fresh sea cucumbers of H. moebii (15 kg) using the procedure as described in the previous study (Yu et al. 2015). The individual saponins of Sap. 1 (51.7 mg), Sap. 2 (52.3 mg), Sap. 3 (650.8 mg) and Sap. 4 (60.6 mg) were purified from the total saponins using the same method as reported in the previous study (Yu et al. 2015). The structures of saponins 1−4 were confirmed by HPLC analysis with standard compounds (Yu et al. 2015) and their nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectroscopy (HRESIMS) spectral analyses . Saponins 1−4 had a purity of over 96.0% as determined by high performance liquid chromatography (HPLC).
108
HPLC analysis
97 98 99 100 101 102 103 104 105 106
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133
(1) Standard compounds: Four saponins (Saps.1−4, purity: over 96.0%) isolated and purified from the sea cucumber H. moebii (Yu et al. 2015) were used as standard compounds. (2) Sample of standard compound for HPLC analysis: Each standard compound (5.0 mg) was accurately weighed into a 5.0 ml volumetric flask and dissolved in a mixture of acetonitrile/water (70:30) to make a stock mixture solution (1.0 mg/ml) of the four saponins. The final solution for HPLC analysis was prepared from the stock solution. (3) Calibration equation and calibration curve: The calibration curve and calibration equation were investigated between the peak area (y) and the quantity (x, μg) of each component. Five injections (each 10 μl) with different concentrations (0.05, 0.1, 0.15, 0.25, and 0.5 mg/ml) were performed to obtain the absorption plots. (4) Apparatus: HPLC analysis was performed on the Agilent 1260 HPLC system with the DAD detector using the ODS-2 Hypersil column (150 × 4.6 mm2 , 5 μm, detection: 203 nm; flow rate: 1.0 ml/min; and temperature: 26 °C). Water was employed as mobile phase A, and acetonitrile as mobile phase B. The gradient procedure lasted 0−20 min with 30–36% B, 20.1−28 min with 90% B and 28.1−35 min with 30% B. (5) Sample of total saponin for HPLC analysis: A final solution of 4.8 mg/ml for the total saponins was made and then filtered using a 0.45 μm Econofilter prior to HPLC analysis.
3
Sulforhodamine B (SRB) assay
143
SRB assay was used to evaluate the activity of total saponins (T. Sap.), individual saponins (Saps. 1−4) and hydrolytic products (3A and 3B) suppressing the proliferation of human colorectal cancer HCT-8, HCT-15, HCT-116 and SW620 cells. Doxorubicin (DOX) was used as the positive control. Briefly, cancer cells were plated in a 96-well plate and then treated with tested compound in different concentrations for 72 h. The compound-treated cells were fixed with 50 μl of 10% cold trichloroacetic acid solution for 1 h at 4 °C, washed with distilled water five times, and then dried at room temperature. The dried cells were stained with 50 μl of 0.4% SRB for 10 min and rinsed with 1% acetic acid solution five times. After being dried, dye was dissolved in 10 mM Tris buffer and measured at 515 nm on a microplate reader (Bio-Tech).
144
Morphological analysis of apoptosis
157
DAPI (4,6-diamidino-2-phenylindole) and PI (propidium iodide) double staining were used to detect apoptosis and necrosis in colorectal cancer cells. Cells were treated with tested compounds and then incubated at 37 °C with 5% CO2 or without 5% CO2 (for SW620) for 6 h. The incubated cancer cells were stained by DAPI (10 μg/ml) and PI (5 μg/ml) for 20 min at room temperature. The stained cancer cells were washed with PBS twice and observed under a fluorescence microscope (40× magnification). Apoptotic cells showed bright blue nuclear condensation as stained by DAPI, and necrotic cells stained by PI displayed red fluorescence.
158
145 146 147 148 149 150 151 152 153 154 155 156
159 160 161 162 163 164 165 166 167
Annexin V-FITC/PI double staining assay
168
Cytometric analysis with Annexin V-FITC/PI double staining was applied to qualify the apoptosis and necrosis induced by the tested compounds. Briefly, cells were treated with tested compounds for different times and then 1 × 106 cells were harvested. After being washed with cold PBS buffer, the cells were resuspended in 100 μl 1× binding buffer mixed with 5 μl Annexin V-FITC and 1 μl 100 μg/ml PI working solution. Cells were incubated at room temperature for 15 min and then 400 μl 1× binding buffer was added. The fluorescence, using emission wavelength at 530 nm and 575 nm and excitation wavelength at 488 nm, was determined by flow cytometry.
169
Animals
179
170 171 172 173 174 175 176 177 178
Male Balb/c mice (20 ± 2 g) were purchased from the Shanghai Slac Laboratory Animal Center (Shanghai, China). All animals were housed in a standard environment under controlled conditions (24– 26 °C, a 12 h light/dark cycle) with free access to food and water. The animals were allowed to acclimate for at least one week before their use. All procedures involving animals and their care were approved by the Zhejiang University Animal Experimentation Committee and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All surgeries were performed under urethane anesthesia, and all efforts were made to minimize suffering.
190
180 181 182 183 184 185 186 187 188 189
134
Cell culture
Animal model and drug treatment
191
135
Human colorectal cancer HCT-8 and HCT-15 cells were cultured in RPMI-1640 medium, HCT-116 cells in McCOY’s-5A medium, SW620 cells in Leibovitz’s L-15 medium, CT-26 cells in HDMEM medium with 10% fetal bovine serum and CCD-18Co cells in HDMEM medium with 12.5% fetal bovine serum. SW620 cells were incubated at 37 °C in a humidified incubator without CO2 , while other cells were incubated at 37 °C in a humidified incubator with 5% CO2 . Cells after the third generation were used for the experiment.
One week after the mice acclimated to the environment, 2 × 106 /100 μl CT-26 cells were injected subcutaneously (s.c.) into the armpit of each mouse. All of the procedures were carried out under a pathogen-free environment. After three days of transplantation, all the tumor-bearing mice were divided into vehicle control group (CON, 0.5% tween 80), three total saponins-treated groups (T. Sap.30, T.Sap.60, T.Sap.120, dissolved in 0.5% tween 80), and positive control group (5-FU, dissolved in 0.5%
192
136 137 138 139 140 141 142
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
193 194 195 196 197 198 199
JID: PHYMED 4
ARTICLE IN PRESS
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
Fig. 2. Sulfated saponins inhibited the proliferation of four different colorectal cancer cells. Cancer cells were treated with different concentrations of tested saponins for 72 h and then assayed by SRB.
proliferation of human colorectal cancer cells was determined by SRB assay. Doxorubicin (DOX, one of the chemotherapeutic drugs) (Tacara et al. 2013) was used as the positive control. The results (Table 1 and Fig. 2) showed that total saponins (T. Sap.) and the four individual components (Saps. 1−4) significantly suppressed the proliferation of four different human colorectal cancer cells (HCT-8, HCT-15, HCT-116, and SW620) with IC50 values ranging from 1.04 to 4.08 μM (or 1.46– 3.24 μg/ml for T. Sap.). The activity of these saponins from the sea cucumber H. moebii against colorectal cancer cells was almost equivalent to that of the positive control drug DOX. Compared to the activity of total saponins and saponins 1−4, the activity of desulfated saponin (3B) were significantly decreased (Table 1) with IC50 values in the range of 23.44−76.32 μM. This result implied that the sulfate group at C-4 of xylose might be important for the activity of this type of triterpenoid saponins. The different activities between sulfated saponin and desulfated saponin against four different glioma cell lines were also obtained in the previous report (Yu et al. 2015).
217
tween 80). In order to find an effective dose, animals received total saponins intraperitoneally at dose of 30 mg/kg, 60 mg/kg and 120 mg/kg with interval of 24 h for 12 days. Vehicle controls (CON) received the same volume of 0.5% tween 80 at a 24 h-interval. Positive control groups were treated with 5-FU at a dose of 20 mg/kg (ip) every two days. All the animals were weighed each day during the 13 days of treatment period and sacrificed at the 16th day after transplantation, and then tumors were harvested and weighed. Livers and spleens were also harvested in order to test the hepatotoxicity and the toxicity of the spleen. The tumor growth inhibition (TGI) was calculated by the formula: TGI (%) = [(M − N)/M] × 100%, where M is the tumor weight of the control group, and N is that of the total saponins or 5-FU treated group. The liver/spleen index was calculated by the formula: Liver/Spleen Index = W/B, where W is the liver/spleen weight of every mouse, and B is the whole weight of every mouse. Data are presented as mean ± SD. Differences between groups were analyzed using one way ANOVA, p < 0.05 were considered statistically significant.
218
Results and discussion
Saponins induced apoptosis and necrosis in colorectal cancer cells
259
219
Chemical ingredients of the total saponins
260
220
238
A highly purified sulfated saponin fraction (total saponins, T. Sap.) was prepared from the whole body of sea cucumber H. moebii for bioactive evaluation. Four compounds were isolated from the total saponins. They were identified as holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3) and 24-dehydroechinoside B (Sap. 4) (Fig. 1) based on their NMR and HRESIMS data and Co-TLC (Fig. S1) and Co-HPLC (Fig. S2) analyses with standard compounds. HPLC method was used to determine the contents of total saponins and individual saponins. Linear regression equation and linearity of the standard curves were investigated for holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3) and 24-dehydroechinoside B (Sap. 4). The linearity is expressed in terms of the correlation coefficient (R2 ), which was found to be 0.99926–0.99978 for each saponin in the range of 0.5–0 μg (Table S1). Quantitative analysis of the total saponins indicated that the content of total saponins was 91.22% (Table S2), including 7.48% holothurin A (Sap. 1), 7.53% moebioside A (Sap. 2), 69.29% holothurin B (Sap. 3), 6.92% 24-dehydroechinoside B (Sap. 4), and holothurin B was the major component in the total saponins.
239
Saponins inhibited the proliferation of colorectal cancer cells
240
The activity of total saponins, four individual saponins, the aglycone of saponin (3A), and the desulfated saponin (3B) inhibiting the
The apoptosis and necrosis induced by total saponins, individual saponins, saponin aglycone and desulfated saponin were detected by DAPI and PI double staining. As shown in Fig. 3, T. Sap. (3 μg/ml), Sap. 1 (3 μM), Sap. 2 (3 μM), Sap. 3 (3 μM) and Sap. 4 (3 μM) induced apoptosis and necrosis in HCT-15 cells after early treatment (6 h) and the changes in the morphology of HCT-15 cells with characteristic apoptotic and necrotic appearance were observed. Under fluorescence microscope, the control HCT-15 cells displayed blue fluorescence with consistent nucleus intensity, while apoptotic cells showed bright blue nuclear pyknosis stained by DAPI and necrotic cells stained by PI displayed red fluorescence. However, the apoptosis induced by saponin aglycone (3A, 25 μM) and desulfated saponin (3B, 50 μM) was observed after later treatment (48 h) (Fig. S3). The apoptosis induced by tested compounds was further quantified by flow cytometry using Annexin V-FITC/PI double staining. HCT-15 cells were treated with T. Sap. (3 μg/ml) for 2 h, 4 h, and 6 h, and the total apoptotic cells (early and late apoptotic cells) were increased by 8.17%, 19.82%, and 48.84%, respectively, when compared to the control (CON, Table 2 and Fig. 4). The four individual saponins also significantly induced apoptosis in HCT-15 cells with a 72.23% (Sap. 1, 3 μM), 78.18% (Sap. 2, 3 μM), 28.37% (Sap. 3, 3 μM) and 28.75% (Sap. 4, 3 μM) increase in total apoptotic cells after the early 6 h treatment (Table 2, Figs. 5, S4, and S5). However, the individual Sap. 2 (3 μM) did not induce apoptosis in the normal colorectal CCD-18Co cells (Fig. S6) after 36 h treatment. Treatment with desulfated saponin (3B, 50 μM) also caused a 50.01% increase in total apoptotic cells after
200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237
241
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
ARTICLE IN PRESS
JID: PHYMED
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
5
Table 1 Compounds inhibited the proliferation of colorectal cancer cells (IC50 : μM or μg/ml for T. sap). Cells
T. Sap.
HCT-8 HCT-15 HCT-116 SW620
2.90 3.24 1.46 2.24
± ± ± ±
Sap.1 0.17 0.09 0.19 0.09
4.08 2.84 1.46 2.63
Sap. 2
± ± ± ±
0.15 0.17 0.13 0.07
2.88 2.41 1.66 1.55
± ± ± ±
Sap. 3 0.36 0.04 0.31 0.36
2.92 2.37 2.26 2.14
Sap. 4
± ± ± ±
0.09 0.28 0.41 0.11
4.06 1.04 2.14 2.26
± ± ± ±
3A 0.04 0.01 0.05 0.07
21.63 23.42 37.38 38.19
3B ± ± ± ±
0.66 0.62 0.25 0.62
76.32 56.62 36.34 23.44
DOX ± ± ± ±
6.23 3.55 0.82 0.56
1.50 1.13 0.80 1.33
± ± ± ±
0.12 0.08 0.01 0.06
T. Sap.: total saponins; DOX: doxorubicin, positive control.
Fig. 3. Sulfated saponins induced apoptosis and necrosis in colorectal cancer HCT-15 cells. HCT-15 cells were treated with tested compounds for 6 h and then double stained with DAPI and PI. Apoptotic cells stained by DAPI showed bright blue nuclear condensation and necrotic cells displayed red fluorescence as stained by PI. Table 2 Compounds induced apoptosis in colorectal cancer HCT-15 cells (%). Time
2 h (TAC) 4 h (TAC) 6 h (TAC)
Sap. 1 (3 μM)
Sap. 2 (3 μM)
Sap. 3 (3 μM)
Sap. 4 (3 μM)
CON
1
1−CON
CON
2
2−CON
CON
3
3−CON
CON
4
4−CON
6.52 7.24 8.73
19.59 40.99 80.96
13.07 33.75 72.23
11.10 10.25 13.51
18.45 46.19 91.69
7.35 35.94 78.18
6.52 7.24 8.73
16.23 29.62 37.10
9.71 22.38 28.37
4.05 3.74 4.50
14.94 20.62 33.25
10.89 16.88 28.75
Time
2 or 24 h (TAC) 4 or 48 h (TAC) 6 or 72 h (TAC)
T. Sap. (3 μg/ml)
3A (25 μM)
CON
TS
TS−CON
CON
3A
3A−CON
CON
3B (50 μM) 3B
3B−CON
CON
DOX (1 μM) DOX
4.05 3.74 4.50
12.22 23.56 53.34
8.17 19.82 48.84
8.33 9.63
12.12 15.11
3.79 5.48
12.11 12.23
31.59 62.24
19.48 50.01
8.33 9.63
13.91 5.58 16.40 6.77
DOX−CON
TAC: total apoptotic cells (early apoptotic cells + late apoptotic cells).
295
72 h treatment (Table 2 and Fig. S7). But the apoptosis and necrosis induced by saponin aglycone (3A, 25 μM) and DOX (1 μM) had no significant changes after 72 h treatment when compared to the control (CON) (Table 2 and Fig. S7). It was noted that sulfated saponins (T. Sap. and Saps. 1−4) mainly induced late apoptosis at the early treatment time (2−4 h) in HCT-15 cells (Figs. 4, 5, S4 and S5), while desulfated saponin (3B) mainly induced early apoptosis at the later treatment time (48−72 h) (Fig. S7). The ability to induce apoptosis by total saponins and individual saponins was time-dependent (Table 2, Figs. 4, 5, S4 and S5).
296
Total saponins inhibited the growth of tumor in animal model
286 287 288 289 290 291 292 293 294
297 298 299
The antitumor activity of total saponins was determined in colorectal CT-26 tumor-bearing Balb/c mice. To determine the effective dose, the antitumor activity of the three doses of total saponins (30,
60 and 120 mg/kg/day, n = 5 for each dose) for 12 days was tested in CT-26 tumor-bearing Balb/c mice. At the 13th day after the treatment, all the animals were sacrificed and then the parameters were measured as per the designed protocol. As shown in Table 3, Fig. 6B and C, total saponins at a dose of 120 mg/kg/day (ig) significantly inhibited CT-26 tumor growth with an inhibition rate of 55.08% (p < 0.01) and the positive group 5-FU (20 mg/kg, ip) with an inhibition rate of 69.70% (p < 0.001), when compared to the control group. The total saponins at dose of 30 mg/kg/day (ig) and 60 mg/kg/day (ig) also produced some antitumor efficacy with an inhibition rate of 36.88% and 31.95%, respectively, but there is no statistical significant. All the data suggested that the total saponins from the sea cucumber H. moebii had antitumor effect in colorectal CT-26 tumor-bearing Balb/c mice. The significant changes of body weights of the animals treated with T. Sap. 60 from the 9th to 13th day, and T. Sap. 120 from the 8th to 13th day, and 5-FU from the 5th to 7th day and 9th and 10th
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315
ARTICLE IN PRESS
JID: PHYMED 6
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
Fig. 4. Total saponins induced apoptosis in colorectal cancer HCT-15 cells quantified by cytometric analysis with annexin V-FITC/PI double staining. HCT-15 cells were treated with total saponins (T. Sap., 3.0 μg/ml) for 2 h, 4 h, and 6 h, respectively, stained with annexin-V FITC and PI, and then analyzed by flow cytometry. Fractions B1, B2, B3 and B4 represent necrotic cells, late apoptotic cells, glioma cells, and early apoptotic cells, respectively.
Fig. 5. Moebioside A (Sap. 2) induced apoptosis in colorectal cancer HCT-15 cells quantified by cytometric analysis with annexin V-FITC/PI double staining. HCT-15 cells were treated with Sap. 2 (3.0 μM) for 2 h, 4 h, 6 h, respectively, stained with annexin-V FITC and PI, and then analyzed by flow cytometry. Fractions B1, B2, B3 and B4 represent necrotic cells, late apoptotic cells, glioma cells, and early apoptotic cells, respectively.
Table 3 Total saponins inhibited tumor growth in colorectal CT-26 tumor-bearing mice. Treatment Tumor weight (g) Inhibition (%)
Control 2.64 ± 0.65 0
T. Sap. 30 1.68 ± 0.35 36.88 ± 13.39
T. Sap. 60 1.80 ± 0.68 31.95 ± 25.58
T. Sap. 120
5-FU ∗∗
1.19 ± 0.72 55.08 ± 27.37∗∗
0.80 ± 0.26∗∗∗ 69.70 ± 9.76∗∗∗
The values are presented as mean ± SD (n = 5). ∗∗ p < 0.01. ∗∗∗ p < 0.001 vs control.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
ARTICLE IN PRESS
JID: PHYMED
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
7
Fig. 6. Total saponins inhibited the growth of tumors in colorectal CT-26 tumor-bearing Balb/c mice. (A) Mice body weights of CON, 5-FU, T. Sap. 30, T. Sap. 60, and T. Sap. 120 groups, 1–13 days after administration; (B) mice tumor weights of different groups 13 days after administration; (C) mice tumors harvested from different groups 13 days after administration. The mice body and tumor weights of 5-FU and saponins-treated groups were compared with those of the control groups, ∗∗∗p < 0.001, ∗∗p < 0.01. All data are shown as mean ± SD (n = 5). Table 4 Body weights of colorectal CT-26 tumor-bearing mice. Day
Control
1 2 3 4 5 6 7 8 9 10 11 12 13
21.38 22.36 21.98 21.88 22.76 22.04 21.96 22.10 22.64 22.88 22.68 22.90 22.92
± ± ± ± ± ± ± ± ± ± ± ± ±
T. Sap. 30 0.86 0.92 1.19 1.18 1.28 1.29 1.16 1.45 1.59 1.52 1.71 1.87 1.63
21.14 21.00 20.86 20.86 20.42 20.20 20.22 20.44 20.96 21.14 20.90 20.92 21.06
± ± ± ± ± ± ± ± ± ± ± ± ±
0.93 0.65 0.78 0.69 0.59 0.75∗ 0.57 0.46 0.56 0.73 0.44 0.33 0.36
T. Sap. 60 21.06 21.00 20.46 20.58 20.84 20.40 20.18 19.52 19.86 20.02 19.82 19.92 20.08
± ± ± ± ± ± ± ± ± ± ± ± ±
T. Sap. 120
0.86 1.77 1.90 1.67 1.50 1.61 1.54 1.57 1.54∗∗ 1.66∗∗ 2.23∗∗ 2.72∗ 2.92∗
21.34 21.48 21.28 21.14 21.26 20.06 19.56 18.94 18.92 19.34 19.32 20.06 20.06
± ± ± ± ± ± ± ± ± ± ± ± ±
1.68 1.70 1.52 1.48 1.54 1.69 2.22 1.59∗∗ 1.25∗∗ 1.18∗∗∗ 1.38∗∗ 1.30∗ 1.27∗
5-FU 21.28 21.42 21.14 20.16 19.80 19.44 19.80 20.52 20.68 21.04 20.84 20.94 20.90
± ± ± ± ± ± ± ± ± ± ± ± ±
1.54 1.73 1.68 1.46 1.38∗∗ 1.36∗∗ 1.47∗ 1.53 1.45∗ 1.35∗ 1.27 1.37 1.41
The values are presented as mean ± SD (n = 5). ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001 vs control.
316 317 318 319 320 321 322 323 324 325
day were observed, when compared to the control group (Table 4 and Fig. 6A). The liver index and spleen index of the animals treated with total saponins and control 5-FU were calculated. The results showed that both liver index and spleen index of the animals treated with T. Sap. 120 were significantly lower (∗p < 0.05 or ∗∗p < 0.01, Fig. S8) when compared to the control. The spleen index of the animals treated with 5-FU was also significantly reduced (Fig. S8). Taken together, it seemed that both total saponins and control drug 5-FU might have some side effects on the colorectal CT-26 tumor-bearing Balb/c mice.
326
Conclusion
327
A highly purified sulfated saponin fraction was prepared from the sea cucumber H. moebii. This fraction is enriched with four sulfated saponins of holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3) and 24-dehydroechinoside B (Sap. 4) with total saponins (T. Sap.) over 91%. The activities of total saponins (T. Sap.) and individual saponins against colorectal cancer cells were evaluated. It
328 329 330 331 332
was found that the sulfated saponins and desulfated saponin (3B) displayed differences in their abilities to inhibit the proliferation of cancer cells and to induce apoptosis in cancer cells. The sulfated saponins (total saponins and individual saponins) significantly suppressed the proliferation of four different human colorectal cancer cell lines (HCT8, HCT-15, HCT-116 and SW620) with IC50 values ranging from 1.04 to 4.08 μM (or 1.46– 3.24 μg/ml for T. sap.), while the inhibitory activity of the desulfated saponin was significantly lowered with IC50 values of 23.44–76.32 μM. The sulfated saponins mainly induced late apoptosis at the early treatment time (2−6 h) in HCT-15 cells, while desulfated saponin (3B) mainly induced early apoptosis at the later treatment time (48−72 h). Further investigation demonstrated that the total saponins had antitumor effects in colorectal CT-26 tumorbearing Balb/c mice.
Conflict of interest The authors declare no conflict of interest.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
333 334 335 336 337 338 339 340 341 342 343 344 345 346
347 348
JID: PHYMED 8
ARTICLE IN PRESS S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
349
Acknowledgments
350
355
This work was supported by grants from the National Natural Science Foundation of China (No. 81273428), the Zhejiang Provincial Natural Science Foundation (LY15H300002) and the CrossDisciplinary Research for Ocean Science of Zhejiang University (No. 2012HY018B). The authors thank Mr. Zhifeng Zhang and Mrs. Fangxia Du at the Shanwei High School for their help with sample collection.
356
Supplementary Materials
357 358
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.08.007.
359
References
360 361 362 363 364 365 366 367 368
Boiardi, A., Silvani, A., Pozzi, A., Fariselli, L., Broggi, G., Salmaggi, A., 1999. Interstitial chemotherapy plus systemic chemotherapy for glioblastoma patients: improved survival in sequential studies. J. Neuro-Oncol 41, 151–157. Bordbar, S., Anwar, F., Saari, N., 2011. High-value components and bioactives from sea cucumbers for functional foods – a review. Mar. Drugs 9, 1761–1805. Guan, H.S., Wang, S.G., 1999. Chinese Marine Materia Medica, vol. III. Shanghai Scientific and Technical Publishers, Shanghai, China, pp. 561–598. Kim, S.K., Himaya, S.W., 2012. Triterpene glycosides from sea cucumbers and their biological activities. Adv. Food Nutr. Res. 65, 297–319.
351 352 353 354
[m5G;August 28, 2015;11:15]
Lin, H., Zhang, X., Cheng, G., Tang, H.F., Zhang, W., Zhen, H.N., Chen, J.X., Liu, B.L., Cao, W.D., Dong, W.P., Wang, P., 2008. Apoptosis induced by ardipusilloside III through BAD dephosphorylation and cleavage in human glioblastoma U251MG cells. Apoptosis 13, 247–257. Lovatelli, A., Conand, C., Purcell, S., Uthicke, S., Hamel, J.-F., Mercier, A., 2004. Advances in Sea Cucumber Aquaculture and Management, FAO Fisheries Technical Paper No. 463. Food and Agriculture Organization of the United Nations, Rome, Italy, p. 425. Menchinskaya, E.S., Pislyagin, E.A., Kovalchyk, S.N., Davydova, V.N., Silchenko, A.S., Avilov, S.A., Kalinin, V.I, Aminin, D.L., 2013. Antitumor activity of cucumarioside A2-2. Chemotherapy 59, 181–191. Tacara, O., Sriamornsak, P., Dass, C.R., 2013. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J. Pharm. Pharmacol. 65, 157–170. Tian, X., Tang, H., Lin, H., Cheng, G., Wang, S., Zhang, X., 2013. Saponins: the potential chemotherapeutic agents in pursuing new anti-glioblastoma drugs. Mini-Rev. Med. Chem. 13, 1709–1724. Tomita, T., 1991. Interstitial chemotherapy for brain tumors: review. J. Neuro-Oncol. 10, 57–74. Yu, S.R., Ye, X.W., Huang, H.C., Peng, R., Su, Z.H., Lian, X.Y., Zhang, Z.Z., 2015. Bioactive sulfated saponins from sea cucumber Holothuria moebii. Planta Med. 81, 152–159. Yun, S.H., Park, E.S., Shin, S.W., Na, Y.W., Han, J.Y., Jeong, J.S., Shastina, V.V., Stonik, V.A., Park, J.I., Kwak, J.Y., 2012. Stichoposide C induces apoptosis through the generation of ceramide in leukemia and colorectal cancer cells and shows in vivo antitumor activity. Clin. Cancer Res. 18, 5934–5948. Zhang, Z.Z., Yu, S.R., Chen, L., Lian, X.Y., 2014. Method for extracting moebioside A from Holothuria moebii and its application for treating brain glioblastoma. Patent CN 104045682 A. Faming Zhuanli Shenqing, 2014/09/17.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395
JID: PHYMED
[m5G;August 28, 2015;11:15]
Phytomedicine xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii Siran Yu a, Xuewei Ye a, Lu Chen a, Xin Xie b, Qian Zhou b, Xiao-Yuan Lian b, Zhizhen Zhang a,∗
Q1
a b
Ocean College, Zhejiang University, Hangzhou 310058, China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e
i n f o
Article history: Received 6 March 2015 Revised 4 August 2015 Accepted 14 August 2015 Available online xxx Keywords: Holothuria moebii Sulfated saponin Desulfated saponin Inhibition of the proliferation of colorectal cancer cells Anti-colorectal tumor effects
1 2 3 4 5 6 7 8 9 10 11 12 13
a b s t r a c t Background: Whether sulfated saponins from Holothuria moebii inhibit the proliferation of colorectal cancer cells and have anti-colorectal tumor effects in animal model has not been investigated. Purpose: To evaluate the cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii. Method: (1) Column chromatography was used to prepare the total and individual saponins and HPLC was applied to define the components of the total saponins; (2) the activity of the total and individual saponins inhibiting the proliferation of human colorectal cancer cells was determined by SRB assay and the apoptosis induced by the saponins was qualified using cytometric analysis with Annexin V-FITC/PI double staining; and (3) the antitumor effects of the sulfated saponins on colorectal CT-26 tumor-bearing Balb/c mice were tested. Results: The total and individual sulfated saponins significantly inhibited the proliferation of four different human colorectal cancer cells with IC50 values ranging from 1.04 to 4.08 μM (or 1.46 to 3.24 μg/ml for total saponins) and induced late apoptosis at an early treatment time in cancer cells. The total saponins (120 mg/kg) had antitumor activity in colorectal CT-26 tumor-bearing Balb/c mice. Conclusion: The sulfated saponins from H. moebii remarkably inhibited the proliferation of different human colorectal cancer cells and had significant anti-colorectal tumor activity in animal model. © 2015 Elsevier GmbH. All rights reserved.
Introduction Sea cucumbers (class Holothuroidea) are echinoderms, which have 25 families with more than 1400 species (Kim and Himaya 2012). Because of the nutritive values, potential health benefits, and therapeutic uses, the Sea cucumbers have economic importance (Bordbar et al. 2011). Dehydrated sea cucumbers are commercially sold as a human food source in Asian countries, especially China, Korea, Indonesia and Japan. It was estimated that the total production of sea cucumbers was over 12,000 metric tons (dry weight) during the period of 1992−2001 (Lovatelli et al. 2004). Sea cucumbers have been well recognized as a tonic and traditional remedy in China and Malaysia for their activities against hypertension, arteriosclerosis, tumor, rheumatism, diabetes, asthma, cuts and burns, impotence
Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DOX, doxorubicin; FU, fluorouracil; HPLC, high performance liquid chromatography; HRESIMS, high resolution electrospray ionization mass spectroscopy; NMR, nuclear magnetic resonance; ODS, octadecyl-functionalized silica gel; PI, propidium iodide; SRB, sulforhodamine B; TGI, tumor growth inhibition; T. Sap., total saponins. ∗ Corresponding author. Tel./fax: +86 57188208432. E-mail address: [email protected] (Z. Zhang).
and constipation. In addition, a number of biological activities including anticancer, antiangiogenic, anticoagulant, antihypertension and anti-inflammatory are ascribed to various species of sea cucumbers (Bordbar et al. 2011; Guan and Wang 1999; Kim and Himaya 2012). Triterpene glycosides (sea cucumber saponins) are the major and most interesting bioactive metabolites in sea cucumbers. To date, more than 100 saponins have been isolated and identified from sea cucumbers (Kim and Himaya 2012). Accumulated studies demonstrated that most of these sea cucumber saponins had significant cytotoxicity toward cancer cells and multiple mechanisms of action, such as interfering with cell cycle progression, inducing apoptosis, promoting stabilization of microtubule and enhancing the generation of ceramide, were involved in their antitumor effects (Kim and Himaya 2012; Tian et al. 2013; Yun et al. 2012). However, these saponins were difficult to develop as new anticancer drugs because of their haemolytic properties. Fortunately, mature technique of interstitial chemotherapy of glioblastoma in situ used in clinic (Boiardi et al. 1999; Tian et al. 2013; Tomita et al. 1991) has provided us with a successful approach to administrate saponins in situ, which avoids the side effects of hemolytic activity and might overcome the bottleneck of the development of saponins as new drugs (Tian et al. 2013). Several saponins exhibited significant anti-glioblastoma effects in vivo
http://dx.doi.org/10.1016/j.phymed.2015.08.007 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
JID: PHYMED 2
ARTICLE IN PRESS
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
Fig. 1. Structures of compounds 1−4, 3A, and 3B.
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
by in situ administration (interstitial injection or catheter insertion) (Lin et al. 2008; Tian et al. 2013). Although the in vitro activity of sea cucumber saponins against different types of cancer cells has been well documented, the in vivo antitumor efficacy and the molecular antitumor mechanisms of sea cucumber saponins have not been fully characterized (Kim and Himaya 2012; Menchinskaya et al. 2013). Holothuria moebii Ludwig is a species of sea cucumbers in the Holothuriidae family. The chemical constituents and medicinal uses of this species had not been previously described until we recently investigated the anti-glioma constituents of this sea cucumber and discovered a novel saponin moebioside A (Sap. 2, Fig. 1) (Yu et al. 2015; Zhang et al. 2014). Moebioside A significantly inhibited the proliferation of different glioma cells and induced apoptosis in human glioblastoma U87-MG cells. Moebioside A also selectively reduced the expression levels of several glioma metabolic enzymes of glycolysis and glutaminolysis, suggesting that targeting multiple glioma metabolic regulators might be one of the anti-glioma mechanisms of moebioside A. However, whether the saponins from H. moebii have activity inhibiting the proliferation of colorectal cancer cells or antitumor effects on colorectal tumor-bearing animals has not been investigated. The aims of this study were (1) to characterize the chemical profiles of total saponins purified from the sea cucumber H. moebii by identifying the ingredients of the total saponins and determining the concentration of the total saponins and each ingredient and (2) to evaluate the cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber H. moebii.
Materials and methods
62
General experimental procedures
63
Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18-Prep) and Diaion HP-20 (Mitsubishi Chemical) were utilized for column chromatography. TLC analysis was conducted on silica gel 60 RP-18 F254S aluminum TLC plates (Merck). HPLC analysis was performed on an Agilent 1260 HPLC system with a DAD detector using an ODS-2 Hypersil column (150 × 4.6 mm2 , 5 μm, Thermo Scientific). HPLC and analytic grade solvents used for this study were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Standard compounds of holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3), 24-dehydroechinoside B (Sap. 4), 22,25-oxidoholothurinogenin (3A), and desulfated saponin (3B) were prepared by our group (Yu et al. 2015). Doxorubicin (DOX, >98.0%) was obtained from Sigma-Aldrich and 5-fluorouracil (5-FU, >98.0%) from Shanghai Xudong Haipu Pharmaceutical Co. Ltd. A fluorescence microscope (Nikon SMZ1000) was used to detect apoptosis and necrosis in cancer cells doubly stained by DAPI (4,6-diamidino-2phenylindole) and PI (propidium iodide). Flow cytometry (Beckman Coulter, FC500MCL) was applied for quantitation of apoptotic and necrotic cells. Annexin V apoptosis detection kits were obtained from Invitrogen. Human colorectal cancer HCT-8, HCT-15, HCT-116 and SW620 cells and mouse colorectal tumor CT-26 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Human
64
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
JID: PHYMED
ARTICLE IN PRESS
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
87
normal colorectal CCD-18Co cells were obtained from Shanghai Bogoo Biotechnology Co. Ltd.
88
Sea cucumber material
89
Fresh sea cucumbers of H. moebii were collected from the Turtle Islet in the South China Sea close to Shanwei City, Guangdong Province, China in April 2014. A voucher sample (SW-A042014) was authenticated by one of the authors (Z. Zhang) and deposited in the Laboratory at the Institute of Marine Biology, Ocean College, Zhejiang University, China.
86
90 91 92 93 94
95
Preparation of total saponins and individual saponins
96
107
The total saponins (T. Sap., 5.6 g) were prepared from the fresh sea cucumbers of H. moebii (15 kg) using the procedure as described in the previous study (Yu et al. 2015). The individual saponins of Sap. 1 (51.7 mg), Sap. 2 (52.3 mg), Sap. 3 (650.8 mg) and Sap. 4 (60.6 mg) were purified from the total saponins using the same method as reported in the previous study (Yu et al. 2015). The structures of saponins 1−4 were confirmed by HPLC analysis with standard compounds (Yu et al. 2015) and their nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectroscopy (HRESIMS) spectral analyses . Saponins 1−4 had a purity of over 96.0% as determined by high performance liquid chromatography (HPLC).
108
HPLC analysis
97 98 99 100 101 102 103 104 105 106
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133
(1) Standard compounds: Four saponins (Saps.1−4, purity: over 96.0%) isolated and purified from the sea cucumber H. moebii (Yu et al. 2015) were used as standard compounds. (2) Sample of standard compound for HPLC analysis: Each standard compound (5.0 mg) was accurately weighed into a 5.0 ml volumetric flask and dissolved in a mixture of acetonitrile/water (70:30) to make a stock mixture solution (1.0 mg/ml) of the four saponins. The final solution for HPLC analysis was prepared from the stock solution. (3) Calibration equation and calibration curve: The calibration curve and calibration equation were investigated between the peak area (y) and the quantity (x, μg) of each component. Five injections (each 10 μl) with different concentrations (0.05, 0.1, 0.15, 0.25, and 0.5 mg/ml) were performed to obtain the absorption plots. (4) Apparatus: HPLC analysis was performed on the Agilent 1260 HPLC system with the DAD detector using the ODS-2 Hypersil column (150 × 4.6 mm2 , 5 μm, detection: 203 nm; flow rate: 1.0 ml/min; and temperature: 26 °C). Water was employed as mobile phase A, and acetonitrile as mobile phase B. The gradient procedure lasted 0−20 min with 30–36% B, 20.1−28 min with 90% B and 28.1−35 min with 30% B. (5) Sample of total saponin for HPLC analysis: A final solution of 4.8 mg/ml for the total saponins was made and then filtered using a 0.45 μm Econofilter prior to HPLC analysis.
3
Sulforhodamine B (SRB) assay
143
SRB assay was used to evaluate the activity of total saponins (T. Sap.), individual saponins (Saps. 1−4) and hydrolytic products (3A and 3B) suppressing the proliferation of human colorectal cancer HCT-8, HCT-15, HCT-116 and SW620 cells. Doxorubicin (DOX) was used as the positive control. Briefly, cancer cells were plated in a 96-well plate and then treated with tested compound in different concentrations for 72 h. The compound-treated cells were fixed with 50 μl of 10% cold trichloroacetic acid solution for 1 h at 4 °C, washed with distilled water five times, and then dried at room temperature. The dried cells were stained with 50 μl of 0.4% SRB for 10 min and rinsed with 1% acetic acid solution five times. After being dried, dye was dissolved in 10 mM Tris buffer and measured at 515 nm on a microplate reader (Bio-Tech).
144
Morphological analysis of apoptosis
157
DAPI (4,6-diamidino-2-phenylindole) and PI (propidium iodide) double staining were used to detect apoptosis and necrosis in colorectal cancer cells. Cells were treated with tested compounds and then incubated at 37 °C with 5% CO2 or without 5% CO2 (for SW620) for 6 h. The incubated cancer cells were stained by DAPI (10 μg/ml) and PI (5 μg/ml) for 20 min at room temperature. The stained cancer cells were washed with PBS twice and observed under a fluorescence microscope (40× magnification). Apoptotic cells showed bright blue nuclear condensation as stained by DAPI, and necrotic cells stained by PI displayed red fluorescence.
158
145 146 147 148 149 150 151 152 153 154 155 156
159 160 161 162 163 164 165 166 167
Annexin V-FITC/PI double staining assay
168
Cytometric analysis with Annexin V-FITC/PI double staining was applied to qualify the apoptosis and necrosis induced by the tested compounds. Briefly, cells were treated with tested compounds for different times and then 1 × 106 cells were harvested. After being washed with cold PBS buffer, the cells were resuspended in 100 μl 1× binding buffer mixed with 5 μl Annexin V-FITC and 1 μl 100 μg/ml PI working solution. Cells were incubated at room temperature for 15 min and then 400 μl 1× binding buffer was added. The fluorescence, using emission wavelength at 530 nm and 575 nm and excitation wavelength at 488 nm, was determined by flow cytometry.
169
Animals
179
170 171 172 173 174 175 176 177 178
Male Balb/c mice (20 ± 2 g) were purchased from the Shanghai Slac Laboratory Animal Center (Shanghai, China). All animals were housed in a standard environment under controlled conditions (24– 26 °C, a 12 h light/dark cycle) with free access to food and water. The animals were allowed to acclimate for at least one week before their use. All procedures involving animals and their care were approved by the Zhejiang University Animal Experimentation Committee and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All surgeries were performed under urethane anesthesia, and all efforts were made to minimize suffering.
190
180 181 182 183 184 185 186 187 188 189
134
Cell culture
Animal model and drug treatment
191
135
Human colorectal cancer HCT-8 and HCT-15 cells were cultured in RPMI-1640 medium, HCT-116 cells in McCOY’s-5A medium, SW620 cells in Leibovitz’s L-15 medium, CT-26 cells in HDMEM medium with 10% fetal bovine serum and CCD-18Co cells in HDMEM medium with 12.5% fetal bovine serum. SW620 cells were incubated at 37 °C in a humidified incubator without CO2 , while other cells were incubated at 37 °C in a humidified incubator with 5% CO2 . Cells after the third generation were used for the experiment.
One week after the mice acclimated to the environment, 2 × 106 /100 μl CT-26 cells were injected subcutaneously (s.c.) into the armpit of each mouse. All of the procedures were carried out under a pathogen-free environment. After three days of transplantation, all the tumor-bearing mice were divided into vehicle control group (CON, 0.5% tween 80), three total saponins-treated groups (T. Sap.30, T.Sap.60, T.Sap.120, dissolved in 0.5% tween 80), and positive control group (5-FU, dissolved in 0.5%
192
136 137 138 139 140 141 142
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
193 194 195 196 197 198 199
JID: PHYMED 4
ARTICLE IN PRESS
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
Fig. 2. Sulfated saponins inhibited the proliferation of four different colorectal cancer cells. Cancer cells were treated with different concentrations of tested saponins for 72 h and then assayed by SRB.
proliferation of human colorectal cancer cells was determined by SRB assay. Doxorubicin (DOX, one of the chemotherapeutic drugs) (Tacara et al. 2013) was used as the positive control. The results (Table 1 and Fig. 2) showed that total saponins (T. Sap.) and the four individual components (Saps. 1−4) significantly suppressed the proliferation of four different human colorectal cancer cells (HCT-8, HCT-15, HCT-116, and SW620) with IC50 values ranging from 1.04 to 4.08 μM (or 1.46– 3.24 μg/ml for T. Sap.). The activity of these saponins from the sea cucumber H. moebii against colorectal cancer cells was almost equivalent to that of the positive control drug DOX. Compared to the activity of total saponins and saponins 1−4, the activity of desulfated saponin (3B) were significantly decreased (Table 1) with IC50 values in the range of 23.44−76.32 μM. This result implied that the sulfate group at C-4 of xylose might be important for the activity of this type of triterpenoid saponins. The different activities between sulfated saponin and desulfated saponin against four different glioma cell lines were also obtained in the previous report (Yu et al. 2015).
217
tween 80). In order to find an effective dose, animals received total saponins intraperitoneally at dose of 30 mg/kg, 60 mg/kg and 120 mg/kg with interval of 24 h for 12 days. Vehicle controls (CON) received the same volume of 0.5% tween 80 at a 24 h-interval. Positive control groups were treated with 5-FU at a dose of 20 mg/kg (ip) every two days. All the animals were weighed each day during the 13 days of treatment period and sacrificed at the 16th day after transplantation, and then tumors were harvested and weighed. Livers and spleens were also harvested in order to test the hepatotoxicity and the toxicity of the spleen. The tumor growth inhibition (TGI) was calculated by the formula: TGI (%) = [(M − N)/M] × 100%, where M is the tumor weight of the control group, and N is that of the total saponins or 5-FU treated group. The liver/spleen index was calculated by the formula: Liver/Spleen Index = W/B, where W is the liver/spleen weight of every mouse, and B is the whole weight of every mouse. Data are presented as mean ± SD. Differences between groups were analyzed using one way ANOVA, p < 0.05 were considered statistically significant.
218
Results and discussion
Saponins induced apoptosis and necrosis in colorectal cancer cells
259
219
Chemical ingredients of the total saponins
260
220
238
A highly purified sulfated saponin fraction (total saponins, T. Sap.) was prepared from the whole body of sea cucumber H. moebii for bioactive evaluation. Four compounds were isolated from the total saponins. They were identified as holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3) and 24-dehydroechinoside B (Sap. 4) (Fig. 1) based on their NMR and HRESIMS data and Co-TLC (Fig. S1) and Co-HPLC (Fig. S2) analyses with standard compounds. HPLC method was used to determine the contents of total saponins and individual saponins. Linear regression equation and linearity of the standard curves were investigated for holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3) and 24-dehydroechinoside B (Sap. 4). The linearity is expressed in terms of the correlation coefficient (R2 ), which was found to be 0.99926–0.99978 for each saponin in the range of 0.5–0 μg (Table S1). Quantitative analysis of the total saponins indicated that the content of total saponins was 91.22% (Table S2), including 7.48% holothurin A (Sap. 1), 7.53% moebioside A (Sap. 2), 69.29% holothurin B (Sap. 3), 6.92% 24-dehydroechinoside B (Sap. 4), and holothurin B was the major component in the total saponins.
239
Saponins inhibited the proliferation of colorectal cancer cells
240
The activity of total saponins, four individual saponins, the aglycone of saponin (3A), and the desulfated saponin (3B) inhibiting the
The apoptosis and necrosis induced by total saponins, individual saponins, saponin aglycone and desulfated saponin were detected by DAPI and PI double staining. As shown in Fig. 3, T. Sap. (3 μg/ml), Sap. 1 (3 μM), Sap. 2 (3 μM), Sap. 3 (3 μM) and Sap. 4 (3 μM) induced apoptosis and necrosis in HCT-15 cells after early treatment (6 h) and the changes in the morphology of HCT-15 cells with characteristic apoptotic and necrotic appearance were observed. Under fluorescence microscope, the control HCT-15 cells displayed blue fluorescence with consistent nucleus intensity, while apoptotic cells showed bright blue nuclear pyknosis stained by DAPI and necrotic cells stained by PI displayed red fluorescence. However, the apoptosis induced by saponin aglycone (3A, 25 μM) and desulfated saponin (3B, 50 μM) was observed after later treatment (48 h) (Fig. S3). The apoptosis induced by tested compounds was further quantified by flow cytometry using Annexin V-FITC/PI double staining. HCT-15 cells were treated with T. Sap. (3 μg/ml) for 2 h, 4 h, and 6 h, and the total apoptotic cells (early and late apoptotic cells) were increased by 8.17%, 19.82%, and 48.84%, respectively, when compared to the control (CON, Table 2 and Fig. 4). The four individual saponins also significantly induced apoptosis in HCT-15 cells with a 72.23% (Sap. 1, 3 μM), 78.18% (Sap. 2, 3 μM), 28.37% (Sap. 3, 3 μM) and 28.75% (Sap. 4, 3 μM) increase in total apoptotic cells after the early 6 h treatment (Table 2, Figs. 5, S4, and S5). However, the individual Sap. 2 (3 μM) did not induce apoptosis in the normal colorectal CCD-18Co cells (Fig. S6) after 36 h treatment. Treatment with desulfated saponin (3B, 50 μM) also caused a 50.01% increase in total apoptotic cells after
200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237
241
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
ARTICLE IN PRESS
JID: PHYMED
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
5
Table 1 Compounds inhibited the proliferation of colorectal cancer cells (IC50 : μM or μg/ml for T. sap). Cells
T. Sap.
HCT-8 HCT-15 HCT-116 SW620
2.90 3.24 1.46 2.24
± ± ± ±
Sap.1 0.17 0.09 0.19 0.09
4.08 2.84 1.46 2.63
Sap. 2
± ± ± ±
0.15 0.17 0.13 0.07
2.88 2.41 1.66 1.55
± ± ± ±
Sap. 3 0.36 0.04 0.31 0.36
2.92 2.37 2.26 2.14
Sap. 4
± ± ± ±
0.09 0.28 0.41 0.11
4.06 1.04 2.14 2.26
± ± ± ±
3A 0.04 0.01 0.05 0.07
21.63 23.42 37.38 38.19
3B ± ± ± ±
0.66 0.62 0.25 0.62
76.32 56.62 36.34 23.44
DOX ± ± ± ±
6.23 3.55 0.82 0.56
1.50 1.13 0.80 1.33
± ± ± ±
0.12 0.08 0.01 0.06
T. Sap.: total saponins; DOX: doxorubicin, positive control.
Fig. 3. Sulfated saponins induced apoptosis and necrosis in colorectal cancer HCT-15 cells. HCT-15 cells were treated with tested compounds for 6 h and then double stained with DAPI and PI. Apoptotic cells stained by DAPI showed bright blue nuclear condensation and necrotic cells displayed red fluorescence as stained by PI. Table 2 Compounds induced apoptosis in colorectal cancer HCT-15 cells (%). Time
2 h (TAC) 4 h (TAC) 6 h (TAC)
Sap. 1 (3 μM)
Sap. 2 (3 μM)
Sap. 3 (3 μM)
Sap. 4 (3 μM)
CON
1
1−CON
CON
2
2−CON
CON
3
3−CON
CON
4
4−CON
6.52 7.24 8.73
19.59 40.99 80.96
13.07 33.75 72.23
11.10 10.25 13.51
18.45 46.19 91.69
7.35 35.94 78.18
6.52 7.24 8.73
16.23 29.62 37.10
9.71 22.38 28.37
4.05 3.74 4.50
14.94 20.62 33.25
10.89 16.88 28.75
Time
2 or 24 h (TAC) 4 or 48 h (TAC) 6 or 72 h (TAC)
T. Sap. (3 μg/ml)
3A (25 μM)
CON
TS
TS−CON
CON
3A
3A−CON
CON
3B (50 μM) 3B
3B−CON
CON
DOX (1 μM) DOX
4.05 3.74 4.50
12.22 23.56 53.34
8.17 19.82 48.84
8.33 9.63
12.12 15.11
3.79 5.48
12.11 12.23
31.59 62.24
19.48 50.01
8.33 9.63
13.91 5.58 16.40 6.77
DOX−CON
TAC: total apoptotic cells (early apoptotic cells + late apoptotic cells).
295
72 h treatment (Table 2 and Fig. S7). But the apoptosis and necrosis induced by saponin aglycone (3A, 25 μM) and DOX (1 μM) had no significant changes after 72 h treatment when compared to the control (CON) (Table 2 and Fig. S7). It was noted that sulfated saponins (T. Sap. and Saps. 1−4) mainly induced late apoptosis at the early treatment time (2−4 h) in HCT-15 cells (Figs. 4, 5, S4 and S5), while desulfated saponin (3B) mainly induced early apoptosis at the later treatment time (48−72 h) (Fig. S7). The ability to induce apoptosis by total saponins and individual saponins was time-dependent (Table 2, Figs. 4, 5, S4 and S5).
296
Total saponins inhibited the growth of tumor in animal model
286 287 288 289 290 291 292 293 294
297 298 299
The antitumor activity of total saponins was determined in colorectal CT-26 tumor-bearing Balb/c mice. To determine the effective dose, the antitumor activity of the three doses of total saponins (30,
60 and 120 mg/kg/day, n = 5 for each dose) for 12 days was tested in CT-26 tumor-bearing Balb/c mice. At the 13th day after the treatment, all the animals were sacrificed and then the parameters were measured as per the designed protocol. As shown in Table 3, Fig. 6B and C, total saponins at a dose of 120 mg/kg/day (ig) significantly inhibited CT-26 tumor growth with an inhibition rate of 55.08% (p < 0.01) and the positive group 5-FU (20 mg/kg, ip) with an inhibition rate of 69.70% (p < 0.001), when compared to the control group. The total saponins at dose of 30 mg/kg/day (ig) and 60 mg/kg/day (ig) also produced some antitumor efficacy with an inhibition rate of 36.88% and 31.95%, respectively, but there is no statistical significant. All the data suggested that the total saponins from the sea cucumber H. moebii had antitumor effect in colorectal CT-26 tumor-bearing Balb/c mice. The significant changes of body weights of the animals treated with T. Sap. 60 from the 9th to 13th day, and T. Sap. 120 from the 8th to 13th day, and 5-FU from the 5th to 7th day and 9th and 10th
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315
ARTICLE IN PRESS
JID: PHYMED 6
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
Fig. 4. Total saponins induced apoptosis in colorectal cancer HCT-15 cells quantified by cytometric analysis with annexin V-FITC/PI double staining. HCT-15 cells were treated with total saponins (T. Sap., 3.0 μg/ml) for 2 h, 4 h, and 6 h, respectively, stained with annexin-V FITC and PI, and then analyzed by flow cytometry. Fractions B1, B2, B3 and B4 represent necrotic cells, late apoptotic cells, glioma cells, and early apoptotic cells, respectively.
Fig. 5. Moebioside A (Sap. 2) induced apoptosis in colorectal cancer HCT-15 cells quantified by cytometric analysis with annexin V-FITC/PI double staining. HCT-15 cells were treated with Sap. 2 (3.0 μM) for 2 h, 4 h, 6 h, respectively, stained with annexin-V FITC and PI, and then analyzed by flow cytometry. Fractions B1, B2, B3 and B4 represent necrotic cells, late apoptotic cells, glioma cells, and early apoptotic cells, respectively.
Table 3 Total saponins inhibited tumor growth in colorectal CT-26 tumor-bearing mice. Treatment Tumor weight (g) Inhibition (%)
Control 2.64 ± 0.65 0
T. Sap. 30 1.68 ± 0.35 36.88 ± 13.39
T. Sap. 60 1.80 ± 0.68 31.95 ± 25.58
T. Sap. 120
5-FU ∗∗
1.19 ± 0.72 55.08 ± 27.37∗∗
0.80 ± 0.26∗∗∗ 69.70 ± 9.76∗∗∗
The values are presented as mean ± SD (n = 5). ∗∗ p < 0.01. ∗∗∗ p < 0.001 vs control.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
ARTICLE IN PRESS
JID: PHYMED
[m5G;August 28, 2015;11:15]
S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
7
Fig. 6. Total saponins inhibited the growth of tumors in colorectal CT-26 tumor-bearing Balb/c mice. (A) Mice body weights of CON, 5-FU, T. Sap. 30, T. Sap. 60, and T. Sap. 120 groups, 1–13 days after administration; (B) mice tumor weights of different groups 13 days after administration; (C) mice tumors harvested from different groups 13 days after administration. The mice body and tumor weights of 5-FU and saponins-treated groups were compared with those of the control groups, ∗∗∗p < 0.001, ∗∗p < 0.01. All data are shown as mean ± SD (n = 5). Table 4 Body weights of colorectal CT-26 tumor-bearing mice. Day
Control
1 2 3 4 5 6 7 8 9 10 11 12 13
21.38 22.36 21.98 21.88 22.76 22.04 21.96 22.10 22.64 22.88 22.68 22.90 22.92
± ± ± ± ± ± ± ± ± ± ± ± ±
T. Sap. 30 0.86 0.92 1.19 1.18 1.28 1.29 1.16 1.45 1.59 1.52 1.71 1.87 1.63
21.14 21.00 20.86 20.86 20.42 20.20 20.22 20.44 20.96 21.14 20.90 20.92 21.06
± ± ± ± ± ± ± ± ± ± ± ± ±
0.93 0.65 0.78 0.69 0.59 0.75∗ 0.57 0.46 0.56 0.73 0.44 0.33 0.36
T. Sap. 60 21.06 21.00 20.46 20.58 20.84 20.40 20.18 19.52 19.86 20.02 19.82 19.92 20.08
± ± ± ± ± ± ± ± ± ± ± ± ±
T. Sap. 120
0.86 1.77 1.90 1.67 1.50 1.61 1.54 1.57 1.54∗∗ 1.66∗∗ 2.23∗∗ 2.72∗ 2.92∗
21.34 21.48 21.28 21.14 21.26 20.06 19.56 18.94 18.92 19.34 19.32 20.06 20.06
± ± ± ± ± ± ± ± ± ± ± ± ±
1.68 1.70 1.52 1.48 1.54 1.69 2.22 1.59∗∗ 1.25∗∗ 1.18∗∗∗ 1.38∗∗ 1.30∗ 1.27∗
5-FU 21.28 21.42 21.14 20.16 19.80 19.44 19.80 20.52 20.68 21.04 20.84 20.94 20.90
± ± ± ± ± ± ± ± ± ± ± ± ±
1.54 1.73 1.68 1.46 1.38∗∗ 1.36∗∗ 1.47∗ 1.53 1.45∗ 1.35∗ 1.27 1.37 1.41
The values are presented as mean ± SD (n = 5). ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001 vs control.
316 317 318 319 320 321 322 323 324 325
day were observed, when compared to the control group (Table 4 and Fig. 6A). The liver index and spleen index of the animals treated with total saponins and control 5-FU were calculated. The results showed that both liver index and spleen index of the animals treated with T. Sap. 120 were significantly lower (∗p < 0.05 or ∗∗p < 0.01, Fig. S8) when compared to the control. The spleen index of the animals treated with 5-FU was also significantly reduced (Fig. S8). Taken together, it seemed that both total saponins and control drug 5-FU might have some side effects on the colorectal CT-26 tumor-bearing Balb/c mice.
326
Conclusion
327
A highly purified sulfated saponin fraction was prepared from the sea cucumber H. moebii. This fraction is enriched with four sulfated saponins of holothurin A (Sap. 1), moebioside A (Sap. 2), holothurin B (Sap. 3) and 24-dehydroechinoside B (Sap. 4) with total saponins (T. Sap.) over 91%. The activities of total saponins (T. Sap.) and individual saponins against colorectal cancer cells were evaluated. It
328 329 330 331 332
was found that the sulfated saponins and desulfated saponin (3B) displayed differences in their abilities to inhibit the proliferation of cancer cells and to induce apoptosis in cancer cells. The sulfated saponins (total saponins and individual saponins) significantly suppressed the proliferation of four different human colorectal cancer cell lines (HCT8, HCT-15, HCT-116 and SW620) with IC50 values ranging from 1.04 to 4.08 μM (or 1.46– 3.24 μg/ml for T. sap.), while the inhibitory activity of the desulfated saponin was significantly lowered with IC50 values of 23.44–76.32 μM. The sulfated saponins mainly induced late apoptosis at the early treatment time (2−6 h) in HCT-15 cells, while desulfated saponin (3B) mainly induced early apoptosis at the later treatment time (48−72 h). Further investigation demonstrated that the total saponins had antitumor effects in colorectal CT-26 tumorbearing Balb/c mice.
Conflict of interest The authors declare no conflict of interest.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
333 334 335 336 337 338 339 340 341 342 343 344 345 346
347 348
JID: PHYMED 8
ARTICLE IN PRESS S. Yu et al. / Phytomedicine xxx (2015) xxx–xxx
349
Acknowledgments
350
355
This work was supported by grants from the National Natural Science Foundation of China (No. 81273428), the Zhejiang Provincial Natural Science Foundation (LY15H300002) and the CrossDisciplinary Research for Ocean Science of Zhejiang University (No. 2012HY018B). The authors thank Mr. Zhifeng Zhang and Mrs. Fangxia Du at the Shanwei High School for their help with sample collection.
356
Supplementary Materials
357 358
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.08.007.
359
References
360 361 362 363 364 365 366 367 368
Boiardi, A., Silvani, A., Pozzi, A., Fariselli, L., Broggi, G., Salmaggi, A., 1999. Interstitial chemotherapy plus systemic chemotherapy for glioblastoma patients: improved survival in sequential studies. J. Neuro-Oncol 41, 151–157. Bordbar, S., Anwar, F., Saari, N., 2011. High-value components and bioactives from sea cucumbers for functional foods – a review. Mar. Drugs 9, 1761–1805. Guan, H.S., Wang, S.G., 1999. Chinese Marine Materia Medica, vol. III. Shanghai Scientific and Technical Publishers, Shanghai, China, pp. 561–598. Kim, S.K., Himaya, S.W., 2012. Triterpene glycosides from sea cucumbers and their biological activities. Adv. Food Nutr. Res. 65, 297–319.
351 352 353 354
[m5G;August 28, 2015;11:15]
Lin, H., Zhang, X., Cheng, G., Tang, H.F., Zhang, W., Zhen, H.N., Chen, J.X., Liu, B.L., Cao, W.D., Dong, W.P., Wang, P., 2008. Apoptosis induced by ardipusilloside III through BAD dephosphorylation and cleavage in human glioblastoma U251MG cells. Apoptosis 13, 247–257. Lovatelli, A., Conand, C., Purcell, S., Uthicke, S., Hamel, J.-F., Mercier, A., 2004. Advances in Sea Cucumber Aquaculture and Management, FAO Fisheries Technical Paper No. 463. Food and Agriculture Organization of the United Nations, Rome, Italy, p. 425. Menchinskaya, E.S., Pislyagin, E.A., Kovalchyk, S.N., Davydova, V.N., Silchenko, A.S., Avilov, S.A., Kalinin, V.I, Aminin, D.L., 2013. Antitumor activity of cucumarioside A2-2. Chemotherapy 59, 181–191. Tacara, O., Sriamornsak, P., Dass, C.R., 2013. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J. Pharm. Pharmacol. 65, 157–170. Tian, X., Tang, H., Lin, H., Cheng, G., Wang, S., Zhang, X., 2013. Saponins: the potential chemotherapeutic agents in pursuing new anti-glioblastoma drugs. Mini-Rev. Med. Chem. 13, 1709–1724. Tomita, T., 1991. Interstitial chemotherapy for brain tumors: review. J. Neuro-Oncol. 10, 57–74. Yu, S.R., Ye, X.W., Huang, H.C., Peng, R., Su, Z.H., Lian, X.Y., Zhang, Z.Z., 2015. Bioactive sulfated saponins from sea cucumber Holothuria moebii. Planta Med. 81, 152–159. Yun, S.H., Park, E.S., Shin, S.W., Na, Y.W., Han, J.Y., Jeong, J.S., Shastina, V.V., Stonik, V.A., Park, J.I., Kwak, J.Y., 2012. Stichoposide C induces apoptosis through the generation of ceramide in leukemia and colorectal cancer cells and shows in vivo antitumor activity. Clin. Cancer Res. 18, 5934–5948. Zhang, Z.Z., Yu, S.R., Chen, L., Lian, X.Y., 2014. Method for extracting moebioside A from Holothuria moebii and its application for treating brain glioblastoma. Patent CN 104045682 A. Faming Zhuanli Shenqing, 2014/09/17.
Please cite this article as: S. Yu et al., Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber holothuria moebii, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.08.007
369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395