Assessment of the Inhibitory Effects of Ficin-hydrolyzed Gelatin Derived from Squid (Uroteuthis duvauceli) on Breast Cancer Cell Lines and Animal Model
AbstractMarine novel natural products have been applied for cancer therapy. Enzyme-digested gelatin hydrolysates have proven to serve as promising sources of potent biologically active peptides. Potential anti-breast cancer properties of the extracted Ficin-digesterd gelatin hydrolysate from Indian squid (Uroteuthis duvauceli) was extensively characterized by cellular and animal models. Gelatin was extracted from squid skin, hydrolyzed by Ficin, and characterized by standard physico-chemical methods. Ficin-digested gelatin hydrolysate was used at various doses of 0-0.1 mg/mL for assessment of MCF-7 and MDA-MB-231 breast cancer cells versus HUVEC normal cells. Cytotoxicity, phase-contrast morphological examination, apoptosis/necrosis, clonal-growth, cell-migration, Matrix-metalloproteinases (MMPs) zymography, and Western blotting were used for cellular assessments. For animal studies, breast tumor-induced BALB/c mice received hydrolyzed gelatin regimen, followed by tumor size/growth and immune-histochemical analyses. Significant inhibition of MCF-7 and MDA-MB-231 with no cytotoxicity on HUVEC cells were detected. Apoptosis was increased in cancer cells, as revealed by elevated ratio of cleaved caspase-3 and PARP. MMP-2 and MMP-9 activities in both cancer cells were diminished. In mice, gelatin hydrolysate prevented weight loss, decreased tumor size, induced p53, and down-regulated Ki67 levels. These findings suggest that Ficin-digested gelatin hydrolysate could be a beneficial candidate for novel breast cancer therapies.
1. Rao, G.S., Current status and prospects of fishery resources of the Indian continental shelf. 2010.2. Ahmad, M. and S. Benjakul, Characteristics of gelatin from the skin of unicorn leatherjacket (Aluterus monoceros) as influenced by acid pretreatment and extraction time. Food Hydrocolloids, 2011. 25(3): p. 381-388.3. Karim, A.A. and R. Bhat, Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food hydrocolloids, 2009. 23(3): p. 563-576.4. Wilesmith, J., J. Ryan, and M. Atkinson, Bovine spongiform encephalopathy: epidemiological studies on the origin. The veterinary record, 1991. 128(9): p. 199-203.5. Gómez-Guillén, M., et al., Structural and physical properties of gelatin extracted from different marine species: a comparative study. Food Hydrocolloids, 2002. 16(1): p. 25-34.6. Muyonga, J., C. Cole, and K. Duodu, Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin. Food hydrocolloids, 2004. 18(4): p. 581-592.7. Balti, R., et al., Extraction and functional properties of gelatin from the skin of cuttlefish (Sepia officinalis) using smooth hound crude acid protease-aided process. Food Hydrocolloids, 2011. 25(5): p. 943-950.8. Cho, S., et al., Processing optimization and functional properties of gelatin from shark (Isurus oxyrinchus) cartilage. Food Hydrocolloids, 2004. 18(4): p. 573-579.9. Aewsiri, T., S. Benjakul, and W. Visessanguan, Functional properties of gelatin from cuttlefish (Sepia pharaonis) skin as affected by bleaching using hydrogen peroxide. Food Chemistry, 2009. 115(1): p. 243-249.10. Nagarajan, M., et al., Characteristics and functional properties of gelatin from splendid squid (Loligo formosana) skin as affected by extraction temperatures. Food Hydrocolloids, 2012. 29(2): p. 389-397.11. Gómez-Guillén, M., et al., Antioxidant and antimicrobial peptide fractions from squid and tuna skin gelatin. Sea by-products as a real material: New ways of application, 2010: p. 89-115.12. Giménez, B., et al., Antioxidant and functional properties of gelatin hydrolysates obtained from skin of sole and squid. Food Chemistry, 2009. 114(3): p. 976-983.13. Lin, L. and B.f. Li, Radical scavenging properties of protein hydrolysates from Jumbo flying squid (Dosidicus eschrichitii Steenstrup) skin gelatin. Journal of the Science of Food and Agriculture, 2006. 86(14): p. 2290-2295.14. Mendis, E., N. Rajapakse, and S.-K. Kim, Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. Journal of agricultural and food chemistry, 2005. 53(3): p. 581-587.15. Giménez, B., et al., Improvement of the antioxidant properties of squid skin gelatin films by the addition of hydrolysates from squid gelatin. Food hydrocolloids, 2009. 23(5): p. 1322-1327.16. Kaufmann, S.H. and W.C. Earnshaw, Induction of apoptosis by cancer chemotherapy. Experimental cell research, 2000. 256(1): p. 42-49.17. DeSantis, C., et al., Breast cancer statistics, 2013. CA: a cancer journal for clinicians, 2014. 64(1): p. 52-62.18. Recht, A. and L.J. Solin. Breast-conserving surgery and radiotherapy in early-stage breast cancer: the importance of local control. in Seminars in radiation oncology. 2011. Elsevier.19. Hochdörffer, K., et al., Development of novel bisphosphonate prodrugs of doxorubicin for targeting bone metastases that are cleaved pH dependently or by cathepsin B: synthesis, cleavage properties, and binding properties to hydroxyapatite as well as bone matrix. Journal of medicinal chemistry, 2012. 55(17): p. 7502-7515.20. Lu, B., et al., Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases: I: Formulation and in vitro characterization. International journal of pharmaceutics, 2006. 307(2): p. 168-174.21. Chaffer, C.L. and R.A. Weinberg, A perspective on cancer cell metastasis. Science, 2011. 331(6024): p. 1559-1564.22. Bedi, A., et al., Inhibition of apoptosis during development of colorectal cancer. Cancer research, 1995. 55(9): p. 1811-1816.23. Aewsiri, T., et al., Surface activity and molecular characteristics of cuttlefish skin gelatin modified by oxidized linoleic acid. International journal of biological macromolecules, 2011. 48(4): p. 650-660.24. Uriarte-Montoya, M.H., et al., Giant squid skin gelatin: chemical composition and biophysical characterization. Food research international, 2011. 44(10): p. 3243-3249.25. Gómez-Guillén, M., et al., Fish gelatin: a renewable material for developing active biodegradable films. Trends in Food Science & Technology, 2009. 20(1): p. 3-16.26. Haard, N.F., Enzymic modification of proteins in food systems. Chemical and functional properties of food proteins, 2001: p. 155-190.27. Kim, S.-K., et al., Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin. Journal of Agricultural and Food Chemistry, 2001. 49(4): p. 1984-1989.28. Alemán, A., et al., Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. Food Research International, 2011. 44(4): p. 1044-1051.29. Kunnumakkara, A.B., P. Anand, and B.B. Aggarwal, Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer letters, 2008. 269(2): p. 199-225.30. Grossman, S. and M. Bergman, Process for the production of gelatin from fish skins. 1992, Google Patents.31. Horwitz, W., Official methods of analysis of the AOAC International. Vol. 18. 2000: The Association.32. Barrett, A.J., et al., L-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochemical Journal, 1982. 201(1): p. 189-198.33. Adler-Nissen, J., Enzymic hydrolysis of food proteins. 1986: Elsevier Applied Science Publishers.34. Bollag, D.M., M.D. Rozycki, and S.J. Edelstein, Protein methods. 1996: Wiley-Liss New York.35. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature, 1970. 227(5259): p. 680-685.36. Liu, L., et al., Curcumin loaded polymeric micelles inhibit breast tumor growth and spontaneous pulmonary metastasis. International journal of pharmaceutics, 2013. 443(1): p. 175-182.37. Kumar, D., et al., Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells. Biomedicine & Pharmacotherapy, 2016. 81: p. 31-37.38. Ogle, A.E., ETHIDIUM BROMIDE/ACRIDINE ORANGE VIABILITY STAINING METHOD. 2010.39. Franken, N., et al., Clonogenic assay of cells in vitro. Nature Protoc 2006; 1: 2315-9; PMID: 17406473. 2006.40. Bimonte, S., et al., Curcumin inhibits tumor growth and angiogenesis in an orthotopic mouse model of human pancreatic cancer. BioMed research international, 2013. 2013.41. Khorramizadeh, M.R., et al., Interaction of CpG-oligodeoxynucleotides with Toll like receptor 9 induces apoptosis and modulates metaloproteinase-2 activity in human intestinal epithelium. Iranian Journal of Allergy, Asthma and Immunology, 2007. 6(3): p. 107-114.42. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 1976. 72(1-2): p. 248-254.43. Mokhtarian, K., et al., Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens. Parasitology research, 2016. 115(8): p. 2957-2965.44. Liu, Y.X., et al., Attempt towards a novel classification of triple-negative breast cancer using immunohistochemical markers. Oncology letters, 2016. 12(2): p. 1240-1256.45. Mariant, M., et al., A new enzymatic method to obtain high-yield F (ab) 2 suitable for clinical use from mouse IgGl. Molecular Immunology, 1991. 28(1-2): p. 69-77.46. Milenic, D.E., J.M. Esteban, and D. Colcher, Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72. 3) reactive with human carcinomas. Journal of immunological methods, 1989. 120(1): p. 71-83.47. Creighton, T., Disulphide bonds between cysteine residues. Protein Structure—a Practical Approach, 1989: p. 155-167.48. Guerard, F., L. Guimas, and A. Binet, Production of tuna waste hydrolysates by a commercial neutral protease preparation. Journal of Molecular Catalysis B: Enzymatic, 2002. 19: p. 489-498.49. Jang, A. and M. Lee, Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Science, 2005. 69(4): p. 653-661.50. Moreno, M.M. and V.F. Cuadrado, Enzymic hydrolysis of vegetable proteins: mechanism and kinetics. Process biochemistry, 1993. 28(7): p. 481-490.51. Diniz, F.M. and A. Martin, Influence of process variables on the hydrolysis of shark muscle protein/Influencia de las variables de proceso en la hidrólisis de proteína del músculo de tiburón. Food Science and Technology International, 1998. 4(2): p. 91-98.52. Giménez, B., et al., Physico-chemical and film forming properties of giant squid (Dosidicus gigas) gelatin. Food Hydrocolloids, 2009. 23(3): p. 585-592.53. Guerard, F., et al., Enzymatic hydrolysis of proteins from yellowfin tuna (Thunnus albacares) wastes using Alcalase. Journal of Molecular Catalysis B: Enzymatic, 2001. 11(4): p. 1051-1059.54. Gómez-Estaca, J., et al., Physico-chemical and film-forming properties of bovine-hide and tuna-skin gelatin: a comparative study. Journal of Food Engineering, 2009. 90(4): p. 480-486.55. Arnesen, J.A. and A. Gildberg, Preparation and characterisation of gelatine from the skin of harp seal (Phoca groendlandica). Bioresource Technology, 2002. 82(2): p. 191-194.56. Voigt, M.N. and J.R. Botta. Advances in fisheries technology and biotechnology for increased profitability. in Atlantic Fisheries Technology Conference 1989: St. John's, Nfld.); Seafood Biotechnology Workshop (1989: St. John's, Nfld.). 1990. Technomic Pub. Co.57. Giménez, B., et al., Use of lactic acid for extraction of fish skin gelatin. Food hydrocolloids, 2005. 19(6): p. 941-950.58. Morales, J., P. Montero, and A. Moral, Isolation and partial characterization of two types of muscle collagen in some cephalopods. Journal of agricultural and food chemistry, 2000. 48(6): p. 2142-2148.59. Kim, J.S. and J.W. Park, Partially purified collagen from refiner discharge of Pacific whiting surimi processing. Journal of food science, 2005. 70(8).60. Lazebnik, Y.A., et al., Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE. Nature, 1994. 371(6495): p. 346-347.61. Mondal, S., et al., Natural products: promising resources for cancer drug discovery. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 2012. 12(1): p. 49-75.62. Benjakul, S. and M.T. Morrissey, Protein hydrolysates from Pacific whiting solid wastes. Journal of Agricultural and Food Chemistry, 1997. 45(9): p. 3423-3430.63. Siefert, S.A. and R. Sarkar, Matrix metalloproteinases in vascular physiology and disease. Vascular, 2012. 20(4): p. 210-216.64. Rodríguez, D., C.J. Morrison, and C.M. Overall, Matrix metalloproteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2010. 1803(1): p. 39-54.