Apoptosis inducing capacity of Holothuria arenicola in CT26 colon carcinoma cells in vitro and in vivo

Document Type: Original Article


1 Department of Biology, Research Center for Applied Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

2 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

3 MSc Student of Developmental Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

4 Medical Genetics Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

5 Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

6 Cancer Genetics Research Unit, Reza Radiation Oncology Center, Mashhad, Iran


Objective(s):Sea cucumber is one of the classes of echinoderms, which is considered as a health marine product and possess various biological characteristics with therapeutic application. The present investigation attempted to evaluate the potential of anti-cancer Persian Gulf sea cucumber species Holothuria arenicola (H. arenicola) aqueous extract on mice colon carcinoma cells in vitro and in vivo.
Materials and Methods: The CT26 carcinoma cells were treated with various concentrations of extract in 24 and 48 hr, and then its anti-proliferative effect was measured by MTT assay and morphological observations. The apoptotic effect was examined by fluorescence microscopy (DNA fragmentation assay), Flow cytometry, caspase-3 and -9 colorimetric assays. The in vivo anti-tumor efficacy of sea cucumber extract on CT26 tumor cells transplanted in BALB/c mice was also investigated.
Results: The results showed that the water extract of sea cucumber revealed remarkable anti-proliferative effect on CT26 tumor cells with IC50= 31 µg/ml with recruitment of intrinsic apoptotic pathway in vitro. In addition, the colon tumor volume in treated groups remarkably reduced in homozygous mice. Histopathological examination elucidated that sea cucumber extract attenuated tumor size and volume along with apoptosis characteristics. Moreover, RT-PCR analysis revealed that sea cucumber extract induced intrinsic apoptosis in vivo through suppression of Bcl-2 expression.
Conclusion: Our data confirmed this notion that sea cucumber administrates anti-cancer effect that can be used as complementary in preclinical experiments, so further characterization are recommended for detection sea cucumber metabolites and clinical application.


1.Aliabadi  A, Eghbalian E, Kiani A. Synthesis and evaluation of the cytotoxicity of a series of 1,3,4-Thiadiazole based compounds as anticancer agents. Iran J Basic Med Sci 2013; 16:1133-1138.                          

2.Murthy KNC, Jayaprakasha GK, Patil BS. The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells. Eur J Pharmacol 2012; 688:14–21.

3.Lai LR, Hsieh SCh, Huang HY, Chou Ch. Effect of lactic fermentation on the total phenolic, saponin and phytic acid contents as well as anti-colon cancer cell proliferation activity of soymilk. J Biosci Bioeng 2013; 115:552–566.

4.Varshosaz J, Hassanzadeh F, Sadeghi-aliabadi H, Firozian F. Uptake of Etoposide in CT-26 cells of colorectal cancer using folate targeted dextran stearate polymeric micelles. Biomed Res Int 2014; 2014:1–11.

5. Zhou R, Huang WJ, Ma C, Zhou Y, Yao YQ, Wang YX, et al. HOCl oxidation-modified CT26 cell vaccine inhibits colon tumor growth in a mouse model. Asian Pacific J Cancer Prev 2012; 13:4037–4043.

6. Deng Sh, Hu B, An HM, Du Q, Xu L, Shen K, et al. Teng-Long-Bu-Zhong-Tang, a Chinese herbal formula, enhances anticancer effects of 5 - Fluorouracil in CT26 colon carcinoma. BMC Complement Altern Med 2013; 13:1-11.

7.Momtazi-borojeni  A, Behbahani M, Sadeghi Aliabadi H. Antiproliferative activity and apoptosis induction of crude extract and fractions of Avicennia marina. Iran J Basic Med Sci 2013; 16:1203-1208.

8. Siu D. Natural products and their role in cancer therapy Vasopressin derivative. Med Oncol 2011; 28:888–900.

9. Anter J, Fernández-bedmar Z, Villatoro-pulido M, Demyda-peyras S, Moreno-millán M, Alonso-moraga Á, et al. A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts. Mutat Res 2011; 723:165-170.

10. Portt L, Norman G, Clapp C, Greenwood M, Greenwood MT. Anti-apoptosis and cell survival: a review. Biochim Biophys Acta 2011;  1813:238-259.

11. Thangam R, Sathuvan M, Poongodi A, Suresh V, Pazhanichamy K, Sivasubramanian S, Kanipandian N, et al. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions. Carbohydr Polym 2014; 107:138-150.

12. Xia Y, Liu Z, Li Z. Effects of high hydrostatic pressure treatment on physicochemical characteristics of Sea cucumber. J Food Sci Eng 2012; 2:227–238.

13. Soltani M, Parivar K, Baharara J, Kerachian MA, Asili j. Putative mechanism for apoptosis-inducing properties of crude saponin isolated from sea cucumber (Holothurialeucospilota) as an antioxidant compound. Iran J Basic Med Sci 2015; 18:180-187.

14. Popov A, Artyukov A, Krivoshapko O, Kozlovskaya E. Biological activities of collagen peptides obtained by enzymic hydrolysis from Far-Eastern holothurians. Am J Biomed Life Sci 2013; 1:17–26.

15. Wang Y, Su W, Zhang C, Xue C, Chang Y, Wu X, et al. Protective effect of sea cucumber (Acaudina molpadioides) fucoidan against ethanol-induced gastric damage. Food Chem 2012; 133:1414–1419.

16. Baharara J, Amini E, Kerachian MA, Soltani M. The osteogenic differentiation stimulating activity of sea cucumber methanolic crude extraction on rat bone marrow mesenchymal stem cells. Iran J Basic Med Sci 2014; 17:626-631.

17. Keshavarz M, Mohammadkia D, Dabbagh AR. The echinoderms fauna in intertidal zone of southern oli village coast (Boushehr, Persian Gulf). J Anim Sci Adv 2012; 2:495–498.

18. Althunibat OY, Ridzwan BH, Taher M, Daud JM, Qaralleh H. Antioxidant and cytotoxic properties of two sea cucumber Holothuria edulis Leson and Stichopus horrens Selenka. Acta Biologica Hungarica 2013; 64:10–20.

19. Mahendran G, Bai VN. Antioxidant and antiproliferative activity of Sewertia corymbosa (GRISEB) wight EX C.B. CLARKE.  Int J Pharm Pharmaceutical Sci 2013; 5:551-558.

20. Namvar F, Sulaiman Rahman H, Mohamad R, Baharara J, Mahdavi M, Amini E, et al. Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract. Int J Nanomedicine 2014; 9:2479–2488.

21. Thangam R, Sathuvan M, Poongodi A, Suresh V, Pazhanichamy K, Sivasubramanian S, et al. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions. Carbohydr Polym 2014; 107:138–150.

22. Tayarani-najaran Z, Mousavi SH, Tajfard F, Asili J, Soltani S. Cytotoxic and apoptogenic properties of three isolated diterpenoids from Salvia chorassanica through bioassay-guided fractionation. Food Chem Toxicol 2013; 57:346-351.

23. Roy S, Besra SE, De T, Banerjee B, Mukherjee J, Vedasiromoni JR. Induction of apoptosis in human leukemic cell lines U937 , K562 and HL-60 by Litchi chinensis leaf extract via activation of mitochondria mediated caspase cascades. Open Leukemia J 2008; 1:1–14.

24. Tong Q, Qing Y, Shu D, He Y, Zhao Y, Li Y, et al. Deltonin , a steroidal saponin , inhibits colon cancer cell growth in vitro and tumor growth in vivo via induction of apoptosis and antiangiogenesis. Cell Physiol Biochem Biochem 2011; 27:233–242.

25. Kuppusamy P, Yusoff MM, Maniam GP, Ichwan SJ. Nutraceuticals as potential therapeutic agents for colon cancer : a review. Acta Pharm Sin B 2014; 4:173–181.

26. Serra AT, Poejo J, Matias AA, Bronze MR, Duarte CMM. Evaluation of Opuntia spp . derived products as antiproliferative agents in human colon cancer cell line ( HT29 ). Food Res Int 2013; 54:892–901.

27. Ogushi M, yoshie-stark YY, Suzuki T. Cytostatic activity of hot water extracts from the sea cucumber in Caco- 2. Food Sci Technol Res 2005; 11:202–206.

28. Wijesinghe WAJP, Jeon YJ, Ramasamy P, Wahid
MF, Vairappan CS. Anticancer activity and mediation of apoptosis in human HL-60 leukaemia cells by edible sea cucumber (Holothuria edulis) extract. Food Chem 2013; 139:326–331.

29. Althunibat OY, Hashim R Bin, Taher M, Mohddaud J, Ikeda MA. In vitro antioxidant and antiproliferative activities of three Malaysian sea cucumber species. Eur J Sci Res 2009; 37:376–387.

30. Imai M, Kikuchi H, Denda T, Ohyama K, Hirobe C, Toyoda H. Cytotoxic effects of flavonoids against a human colon cancer derived cell line , COLO 201 : A potential natural anti-cancer substance. Cancer Lett 2009; 276:74–80.

31. Athukorala Y, Kim KN JY. Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava. Food ChemToxicol 2006; 44:1065–1074.

32. Hajiaghaalipour F, Kanthimathi MS, Sanusi J, Rajarajeswaran J. White tea (Camellia sinensis) inhibits proliferation of the colon cancer cell line , HT-29, activates caspases and protects DNA of normal cells against oxidative damage. Food Chem 2015; 169:401–410.

33. Kim EJ, Park SY, Lee JY, Park JHY. Fucoidan present in brown algae induces apoptosis of human colon cancer cells. BMC Gastroenterol 2010; 10:1–11.

34. Silva-Stenico ME, Kaneno R, Zambuzi FA, Vaz MG, Alvarenga DO FM. Natural products from cyanobacteria with antimicrobial and antitumor activity. Curr Pharm Biotechnol 2013; 14:820–828.

35. Arghiani N, Matin MM, Reza A, Iranshahi M, Sazgarnia A, Rassouli FB. Investigating anticancer properties of the sesquiterpene ferutinin on colon carcinoma cells, in vitro and in vivo. Life Sci 2014; 109:87–94.