Isolation of Sphingoid Bases of Sea Cucumber Cerebrosides and Their Cytotoxicity against Human Colon Cancer Cells

Bioscience, Biotechnology, and Biochemistry Vol. 70 (2006) , No. 12 pp.2906-2912

Tatsuya SUGAWARA, Nobuhiro ZAIMA, Akiyo YAMAMOTO, Shota SAKAI, Ryoko NOGUCHI and Takashi HIRATA

Sea cucumber is a health-beneficial food, and contains a variety of physiologically active substances including glycosphingolipids. We show here the sphingoid base composition of cerebrosides prepared from sea cucumber and the cytotoxicity against human colon cancer cell lines. The composition of sphingoid bases prepared from sea cucumber was different from that of mammals, and the major constituents estimated from mass spectra had a branched C17–19 alkyl chain with 1–3 double bonds. The viability of DLD-1, WiDr and Caco-2 cells treated with sea cucumber sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. The sphingoid bases induced such a morphological change as condensed chromatin fragments and increased the caspase-3 activity, indicating that the sphingoid bases reduced the cell viability by causing apoptosis in these cells. Sphingolipids of sea cucumber might therefore serve as bioactive dietary components to suppress colon cancer.

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PE, a New sulphated Saponin from Sea Cucumber, Exhibits Anti-Angiogenic and Anti-Tumor Activities in vitro and In Vivo

Cancer Biology & Therapy volume 4 | issue 8 august 2005 Pages: 874 - 882

Fang Tian, Xiongwen Zhang, Yunguang Tong, Yanghua Yi, Shilong Zhang, Ling Li, Peng Sun, Liping Lin and Jian Ding

Here, we examined the in vitro and in vivo anti-angiogenesis and anti-tumor activities of PE, a new marine-derived compound. Inhibition of angiogenesis was assessed in vitro using proliferation, migration, adhesion, tube-formation and apoptosis assays in PE-treated HMECs and HUVECs. In vivo, CAM assays were used to assess inhibition effect of PE on physiological angiogenesis, and immunofluorescent microscopy was used to examine tumor microvessel density and apoptosis in PE-treated mouse tumor models. Finally, Western blotting analyses were performed to examine the effect of PE on VEGF signaling in HMECs. The results showed that PE inhibited proliferation of HMECs and HUVECs with IC50 values of 2.22°¿0.31 ?M and 1.98°¿0.32 ?M, induced endothelial cell apoptosis at concentrations < 2 ?M, induced dose-dependent suppression of cell migration, cell adhesion and tube formation in HMECs and HUVECs, and showed anti-proliferative activities against several tumor cell lines (IC50 values of ~ 4 ?M). In vivo, PE (5 nM/egg) suppressed spontaneous angiogenesis in our CAM assay, and induced marked growth inhibition in mouse sarcoma 180 and hepatoma 22 models. Specifically, PE treatment reduced mouse sarcoma 180 tumor volume by triggering apoptosis of both tumor and tumor-associated endothelial cells, preferentially targeting on endothelial cells comparable with tumor cells. Finally, PE treatment suppressed the active (phosphorylated) forms of VEGFR2, Akt, ERK, FAK and paxillin, which are involved in endothelial cell survival, proliferation, adhesion and migration. Our results indicate that PE exerts an anti-angiogenic activity associated with inhibition of VEGFR2 signaling, and an anti-tumor activity associated with decreased proliferation of tumor cells and increased apoptosis of both endothelial cells and tumor cells.

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Basic and clinical study on the antithrombotic mechanism of glycosaminoglycan extracted from sea cucumber

Chinese Medical Journal 2000; Volume 113 Number 8: pages 706-711

LI Zhiguang 李志广, WANG Hongli 王鸿利, LI Jiazeng 李家增, ZHANG Guangshen 张广森, GAO Cunji 高存记

Objective To investigate the antithrombotic mechanism of glycosaminoglycan (GAG) extracted from sea cucumber.
Methods We studied the effects of GAG on the coagulant pathway by measuring clotting time. The antithrombin mechanism of GAG was checked by assaying its effects on the thrombin activity in normal human pooled plasma, purified human heparin cofactor Ⅱ system and antithrombin Ⅲ system. The effects of GAG on the assembly, dispersion, and structure of fibrin gels as well as on the activity of plasmin were studied by means of turbidimetry, electron microscopy, and chromogenic substrate assay. We studied the effect of GAG on the expression and transcription of tissue factor (TF) and thrombomodulin (TM) in LPS (lipopolysaccharide)-stimulated human umbilical vein endothelial cells (HUVECs), and used heparin as a control. HUVECs were treated with different concentrations of GAG (1 μg/ml, 5 μg/ml, and 10 μg/ml respectively) and 5 μg/ml heparin as a control together with LPS (1 μg/ml). After incubation for 6 hours, TF and TM were investigated by ELISA and the mRNA study was carried out by RT-PCR. In a clinical trail, a series of variables were observed before and after treatment with GAG in patients recovering from cerebral ischemic stroke or suffering from ischemic heart disease.
Results The TT and APTT were significantly prolonged by GAG (0.1 μg/ml). GAG inhibited thrombin activity in the presence of HCII with a second order rate constant of 1.14×107 m-1 ·min-1 , which was 4.6 times higher than that of ATIII. GAG significantly inhibited the polymerisation of fibrin monomer and enhanced the activity of plasmin in a concentration dependent manner. GAG could impair TF mRNA expression and up-regulate TM mRNA expression. The result of clinical trail showed that the fat metabolism was enhanced in addition to the anticoagulant and the blood viscosity reducing effects. No side-effect was found.
Conclusions GAG mainly affected on the intrinsic pathway of blood coagulation. GAG was similar to dermatan sulphate both in the efficiency and in the mechanism of antithrombin. The acceleration of colt lysis by GAG depended on its ability to increase the activity of plasmin, to inhibit the polymerising of fibrin monomer, and consequently, to alter the architecture of the fibrin net work. This effect on HUVECs appears to be at a transcriptional level and might be relevant for the antithrombotic action of GAG. GAG possess anticoagulant activity in vivo and it is a promising drug for antithrombotic therapy.

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Philinopside A, a novel marine-derived compound possessing dual anti-angiogenic and anti-tumor effects

Yunguang Tong 1 2, Xiongwen Zhang 1, Fang Tian 1 2, Yanghua Yi 3, Qiangzhi Xu 3, Ling Li 3, Linjiang Tong 1, Liping Lin 1, Jian Ding 1 *

1 Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science, Chinese Academy of Sciences; Shanghai, People's Republic of China 2 Graduate School of Chinese Academy of Sciences, Beijing, People's Republic of China 3 Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China

email: Jian Ding (jding@mail.shcnc.ac.cn)

* Correspondence to Jian Ding, Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science, Chinese Academy of Sciences; Shanghai 201203, People's Republic of China

Funded by:

• High Tech Research and Development Program; Grant Number: 2002AA2Z346A, 2001AA624100
• Knowledge Innovation Program of Chinese Academy of Sciences; Grant Number: KSCX2-SW-202, No. KSCX2-3-07-8
• National Natural Science Foundation; Grant Number: 30228032

Philinopside A is a novel sulfated saponin isolated from the sea cucumber, Pentacta quadrangulari. The effects of philinopside A on angiogenesis and tumor growth were assessed in a series of models in vitro and in vivo. Our results demonstrated that philinopside A significantly inhibited the proliferation, migration and tube formation of human microvascular endothelial cells (HMECs) in a dose-dependent manner, with average IC50 values of 1.4 ± 0.17, 0.89 ± 0.23 and 0.98 ± 0.19 M, respectively. Rat aortas culture assay provides a close imitation of in vivo angiogenesis process and 2-10 M philinopside A suppressed the formation of new microvessels in cultured rat aortas. Philinopside A 2-10 nmol/egg obviously inhibited angiogenesis in chick embryo chorioallantoic membrane assay. In addition, philinopside A manifested strong anti-tumor activities both in vitro and in vivo. Through immunofluorescent analysis, we found the compound reduced mouse sarcoma 180 tumor volume by inducing apoptosis of tumor and tumor-associated endothelial cells. An examination of the effects of philinopside A on the angiogenesis-related receptor tyrosine kinases (RTKs) showed that philinopside A broadly inhibited all tested RTKs, including vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor-1, platelet-derived growth factor (PDGF) receptor- and epithelial growth factor (EGF) receptor, with IC50 values ranging from 2.6-4.9 M. These results suggest that philinopside A is a promising anti-cancer agent that possesses dual cytotoxic and anti-angiogenic effects that were at least partly due to its inhibitory effects on RTKs. © 2004 Wiley-Liss, Inc.

Intercedensides A-C, Three New Cytotoxic Triterpene Glycosides from the Sea Cucumber Mensamaria intercedens Lampert

J. Nat. Prod., 66 (8), 1055 -1060, 2003. 10.1021/np030064y S0163-3864(03)00064-8
Web Release Date: July 25, 2003

Zheng-Rong Zou, Yang-Hua Yi,* Hou-Ming Wu, Jiu-Hong Wu, Chih-Chaung Liaw, and Kuo-Hsiung Lee

Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People's Republic of China, State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Science, 354 Feng-Lin Road, Shanghai 200032, People's Republic of China, Department of Pharmacy, 306 Hospital, Beijing, 100101, People's Republic of China, and Natural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360

Received February 14, 2003

Abstract:

Three new triterpene glycosides, intercedensides A (1), B (2), and C (3), were isolated from the sea cucumber Mensamria intercedens Lampert, which is found in the South China Sea, and their structures have been elucidated by spectroscopic analysis (NMR and ESIMS) and chemical transformations. Intercedensides A (1) and C (3) have a conjugated double bond (22E,24-diene) in the side chain of the aglycon. Intercedenside B (2) has two -D-xylose and two sulfate groups in the carbohydrate chain. All three glycosides showed significant cytotoxicity against 10 human tumor cell lines with ED50 in the range 0.6-4.0 g/mL. Intercedenside A (1) exhibited significant in vivo antineoplastic activity against mouse Lewis lung cancer and mouse S180 sarcoma. On the basis of these initially promising results, intercedensides A-C merit further study as potential anticancer agents.

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* Correspondence to Robert A. Newman, Pharmaceutical Development Center, BOX 601, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030-4009.

Funded by:

Background

Branched-chain fatty acids or fatty alcohols have been reported to possess anti-tumor activity in various tumor models. Here we study 12-methyltetradecanoic acid (12-MTA), a branched-chain fatty acid, isolated from a sea cucumber extract, on the growth of prostate cancer cells and investigate the underlying mechanisms of its effect.

Methods

12-MTA was evaluated by MTT assay for its ability to inhibit cell proliferation in various cancer types. The ability of 12-MTA to induce apoptosis of PC3 cells was examined by morphologic changes, propidium iodide (PI) staining, and caspase-3 activation. Furthermore, alteration of eicosanoid metabolism by 12-MTA was examined in PC3 and RBL-1 cells and in purified lipoxygenase (LOX) and cyclooxygenase (COX) enzymes.

Results

12-MTA inhibited proliferation of various cell lines, with IC50s ranging from 17.99 to 35.44 g/ml. PI staining clearly showed that 12-MTA caused PC3 cell death through induction of apoptosis. At 50 g/ml, 12-MTA increased caspase-3 activity four to seven-fold compared with that in control cells. Examination of cellular arachidonate metabolism showed that at 25 g/ml, 12-MTA reduced the level of 5-hydroxyeicosatetraenoic acid (5-HETE) by 45%. Furthermore, exogenous 5-HETE protects PC3 cells from 12-MTA induced cell death.

Conclusions

12-MTA inhibited proliferation of cancer cells via apoptosis, in which caspase-3 may play a role. At relevant concentrations, 12-MTA can selectively inhibit the formation of 5-HETE, a metabolite of 5-lipoxygenase. This agent may be a novel adjunctive therapy for selected malignancies including prostate cancer.

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