Scabraside D Derived from Sea Cucumber Induces Apoptosis and Inhibits Metastasis via iNOS and STAT-3 Expression in Human Cholangiocarcinoma Xenografts

  • Assawasuparerk, Kanjana (Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University) ;
  • Rawangchue, Thanakorn (Center for Veterinary Diagnosis, Faculty of Veterinary Science, Mahidol University) ;
  • Phonarknguen, Rassameepen (The Monitoring and Surveillance Center for Zoonotic Disease in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University)
  • Published : 2016.06.01


Scabraside D, a sulfated triterpene glycoside, was extracted from the sea cucumber Holothuria scabra. It shows anti-proliferation in many of cancer cell lines, but the function and mechanisms of action of scabraside D in human cholangiocarcinoma (HuCCA) have not previously determined. In this study, we investigated the activity of scabraside D on HuCCA cell apoptosis, lymphangiogenesis and metastasis in a nude mouse model. Scabraside D induced signs of apoptosis, such as cell shrinkage, nuclear condensation, nuclear fragmentation and DNA fragmentation on TUNEL assays, while effectively decreasing expression of BCl-2 but increasing caspase-3 gene level expression. Immunohistochemistry revealed that scabraside D significantly reduced lymphatic vessel density (LVD). Moreover, scabraside D treatment significantly decreased VEGF-C, MMP-9 and uPA gene expression, which play important roles in the lymphangiogenesis and invasion of cancer cells in metastasis processes. Quantitative real-time PCR showed that scabraside D significantly decreased iNOS and STAT-3 gene expression. This study demonstrated that scabraside D plays a role in activation of HuCCA tumor apoptosis and inhibition of lymphangiogenesis, invasion and metastasis through decreasing BCl-2, MMP-9, uPA and VEGF-C and increasing caspase-3 expression by suppression of iNOS and STAT-3 expression. Therefore, scabraside D could be a promising candidate for cholangiocarcinoma treatment.


  1. Aggarwal BB, Sethi G, Ahn KS, et al (2006). Targeting signaltransducer- and-activator-of-transcription-3 for prevention and therapy of cancer: modern target but ancient solution. Ann N Y Acad Sci, 1091, 151-69.
  2. Akira S, Nishio Y, Inoue M, et al (1994). Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp 130-mediated signaling pathway. Cell, 77, 63-71.
  3. Antonina H (2005). Progression molecular mechanisms of tumor metastasis and angiogenesis. Anticancer Res, 25, 3327-34.
  4. Attoub S, Arafat K, G?laude A, et al (2013). Frondoside A suppressive effects on lung cancer suuvival, tumor growth, angiogenesis, invasion and metastasis. Plos One, 8, 1-10.
  5. Azad N, Iyer AK, Wang L, et al (2010). Nitric oxide-mediated bcl-2 stabilization potentiates malignant transformation of human lung epithelial cells. Am J Respir Cell Mol Biol, 42, 578-85.
  6. Bahrami Y, Zhang W, Chataway T, et al (2014). Structural elucidation of novel saponins in the sea cucumber Holothuria lessoni. Mar Drugs, 12, 4439-73.
  7. Carpenter RL, Lo HW (2014). STAT-3 target genes relevant to human cancers. Cancers, 6, 897-925.
  8. Caulier G, Dyck SV, Gerbaux P, et al (2011). Review of saponin diversity in sea cucumbers belonging to the family Holothuriidae. SPC Beche-de-mer Inf Bull, 31, 48-54.
  9. Chludil HD, Muniain CC, Seldes AM, et al (2002). Cytotoxic and antifungal triterpene glycosides from the Patagonian sea cucumber Hemoiedema spectabilis. J Nat Prod, 65, 860-65.
  10. Choi HJ, Han JS (2012). Overexpression of phospholipase D enhances Bcl-2 expression by activating STAT-3 through independent activation of ERK and p38MAPK in HeLA cells. Biochim Biophys Acta, 1823, 1082-91.
  11. Dano k, Behrendt N, Hoyer-Hansen G, et al (2005). Plasminogen activation and cancer. Thromb Haemost, 93, 676-81.
  12. Dass K, Ahmad A, Azmi AS, et al (2007). Evolving role of uPA/uPAR system in human cancers. CancerTreat Rev, 34, 122-36.
  13. Dineen SP, Sullivan LA, Beck Aw, et al (2008). The adnectin CT-322 is novel VEGF receptor 2 inhibitor that decrease tumor burden in an orthotopic mouse model of pancreatic cancer. BMC Cancer, 8, 1-10.
  14. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell. Biol, 119, 493-501.
  15. Gores GJ (2003). Cholangiocarcinoma: current concepts and insights. Hepatol, 37, 961-69.
  16. Gritsko T, Williams A, Turkson J, et al (2006). Persistent activation of STAT-3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells. Clin Cancer Res, 12, 11-19.
  17. Han H, Li L, Yi YH, et al (2012). Triterpene glycosides from sea cucumber Holothuria scabra with cytotoxic activity. Chin Herb Med, 4, 183-88.
  18. Harold FD (2002). Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and potential target for diagnosis and therapy. J Clin Oncol, 20, 4368-80.
  19. Haswell-Elkins MR, Mairiang E, Mairiang P, et al (1994). Cross-sectional study of Opisthorchis viverrini infection and cholangiocarcinoma in communities within a high-risk area in northeast Thailand. Int J Cancer, 59, 505-9.
  20. Hoeben A, Landuyt B, Highley M, et al (2004). Vascular endothelial growth factor and angiogenesis. Pharmacol Rev, 56, 549-80.
  21. Janakiram NB, Rao CV (2012). Inos-selective inhibitors for cancer prevention: promise and progress. Future Med Chem, 4, 2193-204.
  22. Karadayi N, Kandemir NO, Yavuzer D, et al (2013) Inducible nitric oxide synthase expression in gastric adenocarcinoma: impact on lymphangiogenesis and lymphatic metastasis. Diagn Pathol, 8, 1-12.
  23. Kerr RG, Chen Z (1995). In vivo and in vitro biosynthesis of saponins in sea cucumbers. J Nat Prod, 58, 172-76.
  24. Kitagawa I, Kobayashi M, Hori M, et al (1989). Marine natural products. XVIII. Four lanostane-type triterpene oligoglycosides, bivittosides A, B, C, and D, from the okinawan sea cucumber bohadschia bivittata mitsukuri. Chem Pharma Bull, 37, 61-7.
  25. Kloz T, Bloch W, Volberge C, et al (1998). Selective expression of inucible nitric oxide synthase in human prostate carcinoma. Cancer (phila), 82, 1897-903.<1897::AID-CNCR12>3.0.CO;2-O
  26. Kojima M, Morisaki T, Tsukahara Y, et al (1999). Nitric oxide synthase expression and nitric oxide production in human colon carcinoma tissue. J Surg Oncol, 70, 222-29.<222::AID-JSO5>3.0.CO;2-G
  27. Lechner M, Lirk P, Rieder J (2005). Inducible nitric oxide synthase (iNOS) in tumor biology: the two sides of the same coin. Semin Cancer Biol, 15, 227-89.
  28. Li H, Huang D, Gao Z, et al (2013). Scutellarin inhibits the growth and invasion of human tongue squamous carcinoma through the inhibition of matrix metalloproteinase-2 and -9 and ${\alpha}v{\beta}6$ integrin. Int J Oncol, 42, 1674-81.
  29. McCawley L, Matrisian L (2000). Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol Med Today, 6, 149-56.
  30. Nabeshima K, Inoue T, Shimao Y, et al (2002). Matrix metalloproteinases in tumor invasion: role for cell migration. Pathol Int, 52, 255-64.
  31. Niu G, wright KL, Huang M, et al (2002). Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene, 21, 2000-08.
  32. Pasco S, Brassart B, Ramont L, et al (2005). Control of melanoma cell invasion by type IV collagen. Cancer Detect Prev, 29, 260-66.
  33. Plengsuriyakarn T, Viyanant V, Eursitthichai V, et al (2012). Anticancer activities against cholangiocarcinoma, toxicity and pharmacological activities of Thai medicinal plants in animal models. BMC Complement Altern Med, 12, 1-19.
  34. Prakobwong S, Gupta SC, Kim JH, et al (2011). Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways. Carcinogen, 32, 1372-80.
  35. Sanceau J, Truchet S, Bauvois B (2003). Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing’s sarcoma cells. J Biol Chem, 278, 36537-46.
  36. Song Z-J, Gong P, Wu G-Y (2002). Relationship between the expression of iNOS, VEGF, tumor angiogenesis and gastric cancer. World J Gastroenterol, 8, 591-95.
  37. Stonix VA, Kalinin VI, Avilov SA (1999). Toxin from sea cucumbers (Holothuroids): chemical structures, properties, taxonomic distribution, biosynthesis and evolution. J Nat Toxins, 8, 235-48.
  38. Storr SJ, Safuan S, Mitra A, et al (2012). Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma. Mod Pathol, 25, 493-504.
  39. Straume O, Jackson DG, Akslen LA (2003). Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma. Clin Cancer Res, 9, 250-56.
  40. Swana HS, Smith SD, Perrotta PL, et al (1999). Inducible nitric oxide synthase with transitional cell carcinoma of the bladder. J Urol, 161, 630-34.
  41. Thomsen LL, Lawton FG, Knowles RG, et al (1994). Nitric oxide synthase activity in human gynecological cancer. Cancer Res, 54, 1352-54.
  42. Thummarati P, Wijitburaphat S, Prasopthum A, et al (2012). High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis. World J Gastoenterol, 18, 244-50.
  43. Tian F, Zhang X, Tong Y, et al (2005). PE, a new sulfated saponin from sea cucumber, exhibits anti-angiogenic and anti-tumor activities in vitro and in vivo. Cancer Biol Ther, 4, 874-82.
  44. Tong Y, Zhang X, Tian F, et al, (2005). Philinopside A, a novel marine-derived compound possessing dual anti-angiogenenic and anti-tumor effects. Int J Cancer. 114, 843-53.
  45. Vakkala M, Kahlos K, Lakari E, et al (2000). Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas. Clin Cancer Res, 6, 2408-16.
  46. Westermarck J, Kahari V (1999). Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J, 31, 781-92.
  47. Xu W, Liu LZ, Loizdou M, et al (2002). The role of nitric oxide in cancer. Cell Res, 12, 311-20.
  48. Xue Y, Chen F, Zhang D, et al (2009). Tumor-derived VEGF modulates hematopoiesis. J Angiogenes Res, 1, 1-9.
  49. Yan L, Li L, Li Q, et al (2015). Expression of signal transducer and activator of transcription 3 and its phosphorylated form is significantly upregulated in patients with papillary thyroid cancer. Exp Ther Med, 9, 2195-201.
  50. Ye J, Coulouris G, Zaretskaya I, et al (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13, 134.
  51. Yu H, Zhang S, Zhang R, et al (2009). The role of VEGF-C/D and Flt-4 in the lymphatic metastasis of early-stage invasive cervical carcinoma. J Exp Clin Canc Res, 28, 1-6.

Cited by

  1. (Thunb) DC. as a Promising Candidate for Cholangiocarcinoma Chemotherapeutics vol.2017, pp.1741-4288, 2017,