Nicholas J. Macedo, Catherine C. Neto, Anne M. Liberty, Tracie L. Ferreira^*

Show more
Department of Bioengineering, The University of Massachusetts Dartmouth, North Dartmouth, USA.
Department of Chemistry/Biochemistry, The University of Massachusetts Dartmouth, North Dartmouth, USA.
Show less

Abstract: Antioxidants have been widely studied in various naturally occurring substances as a bioavailable cancer prevention treatment. Proanthocyanidins (PACs), which are abundant polyphenols in Early Black (EB) Cranberry (Vaccinium macrocarpon), are readily available and we have shown their anticancer activity in several cancer cell lines. This work focused on the activity of these compounds when incorporated into the zebrafish (Danio rerio) system. We began investigating the in vivo effect of these phytochemicals, the protective role of several other cranberry compounds, and the metabolic activity of the vertebrate model organism. Proanthocyanidin fractions were separated from fresh EB Cranberry fruit by chromatography on Sephadex LH-20 in order to acquire a workable stock solution in DMSO. Various concentrations of proanthocyanidins in solution were tested against fish ranging in age from 1-cell stage to adult level of growth. Acridine orange apoptosis indicator dye was incorporated into the treatment protocol, and it was observed that irregular epithelial cell death was occurring in treated embryos but not in the control group. Further apoptosis assays were carried out utilizing Dihydroethidium (DHE) superoxide sensitive dye in the treatment protocol. Fluorescing red nuclei were visible along the outer surface of the epithelium cell layer; an indication of superoxide release within cells leading to the nicking of DNA within the nucleus. It was also possible to screen for superoxide release in PACs treated CCD-CO18 and HT-29 cells using confocal microscopy and cell apoptosis was investigated by trypan blue cytotoxicity assay; cell apoptosis results were statistically significant as confirmed by ANOVA analysis. Results indicate that the phytochemicals may induce apoptosis in rapidly dividing cells.

Keywords: Zebrafish; In Vivo; Proanthocyanidins

Cite this paper: J. Macedo, N. , C. Neto, C. , M. Liberty, A. and L. Ferreira, T. (2014) Zebrafish as an in Vivo Screen for Early Black Cranberry Proanthocyanidin Biomolecular Activity. American Journal of Molecular Biology, 4, 37-48. doi: 10.4236/ajmb.2014.42006.

References

[1]   Dohadwala, M.M., Holbrook, M., Hamburg, N.M., Shenouda, S.M., Chung, W.B., Titas, M., Kluge, M.A., Wang, N., Palmisano, J., Milbury, P.E., Blumberg, J.B. and Vita, J.A. (2011) Effects of Cranberry Juice Consumption on Vascular Function in Patients with Coronary Artery Disease. The American Journal of Clinical Nutrition, 93, 934-940.
http://dx.doi.org/10.3945/ajcn.110.004242

[2]   Cesoniene, L., Jasutiene, I. and Sarkinas, A. (2009) Phenolics and Anthocyanins in Berries of European Cranberry and Their Antimicrobial Activity. Medicina (Kaunas), 45, 992-999.

[3]   Caillet, S., Côté, J., Sylvain, J.F. and Lacroix, M. (2012) Antimicrobial Effects of Fractions from Cranberry Products on the Growth of Seven Pathogenic Bacteria. Food Control, 23, 419-428.
http://dx.doi.org/10.1016/j.foodcont.2011.08.010

[4]   Neto, C.C. (2007) Cranberry and Its Phytochemicals: A Review of in Vitro Anticancer Studies. The Journal of Nutrition, 137, 186S-193S.

[5]   Kim, K.K., Singh, A.P., Singh, R.K., DeMartino, A., Brard, L., Vorsa, N., Lange, T.S. and Moore, R.G. (2012) AntiAngiogenic Activity of Cranberry Proanthocyanidins and Cytotoxic Properties in Ovarian Cancer Cells. International Journal of Oncology, 40, 227-235. http://dx.doi.org/10.3892/ijo.2011.1198

[6]   Nandakumar, V., Singh, T. and Katiyar, S.K. (2008) Multi-Targeted Prevention and Therapy of Cancer by Proanthocyanidins. Cancer Letters, 269, 378-387. http://dx.doi.org/10.1016/j.canlet.2008.03.049

[7]   Neto, C.C., Krueger, C.G., Lamoureaux, T.L., Kondo, M., Vaisberg, A.J., Hurta, R.A.R., Curtis, S., Matchett, M.D., Yeung, H., et al. (2006) MALDI-TOF MS Characterization of Proanthocyanidins from Cranberry Fruit (Vaccinium macrocarpon) That Inhibit Tumor Cell Growth and Matrix Metalloproteinase Expression in Vitro. Journal of the Science of Food and Agriculture, 86, 18-25. http://dx.doi.org/10.1002/jsfa.2347

[8]   Yokota, K., Kimura, H., Ogawa, S. and Akihiro, T. (2013) Analysis of A-Type and B-Type Highly Polymeric Proanthocyanidins and Their Biological Activities as Nutraceuticals. Journal of Chemistry, 2013, Article ID: 352042.
http://dx.doi.org/10.1155/2013/352042

[9]   Kresty, L.A., Howell, A.B. and Baird, M. (2011) Cranberry Proanthocyanidins Mediate Growth Arrest of Lung Cancer Cells through Modulation of Gene Expression and Rapid Induction of Apoptosis. Molecules, 16, 2375-2390.
http://dx.doi.org/10.3390/molecules16032375

[10]   Howe, K., Clark, M.D., Torroja, C.F., Torrance, J., Berthelot, C., Muffato, M., Collins, J.E., et al. (2013) The Zebrafish Reference Genome Sequence and Its Relationship to the Human Genome. Nature, 496, 498-503
http://dx.doi.org/10.1038/nature12111

[11]   Kim, D.-S., Huh, J.-W., Kim, Y.-H., Park, S.-J., Kim, H.-S. and Chang, K.-T. (2010) Bioinformatic Analysis of TESpliced New Exons within Human, Mouse and Zebrafish Genomes. Genomics, 96, 266-271.
http://dx.doi.org/10.1016/j.ygeno.2010.08.004

[12]   Santoriello, C. and Zon, L.I. (2012) Hooked! Modeling Human Disease in Zebrafish. JCI: The Journal of Clinical Investigation, 122, 2337-2343. http://dx.doi.org/10.1172/JCI60434

[13]   Steffen, L.S., Guyon, J.R., Vogel, E.D., Beltre, R., Pusack, T.J., Zhou, Y., Zon, L.I. and Kunkel, L.M. (2007) Zebrafish Orthologs of Human Muscular Dystrophy Genes. BMC Genomics, 8, 79. http://dx.doi.org/10.1186/1471-2164-8-79

[14]   Drummond, I.A. (2005) Kidney Development and Disease in the Zebrafish. JASN Journal of the American Society of Nephrology, 16, 299-304. http://dx.doi.org/10.1681/ASN.2004090754

[15]   Field, H.A., Ober, E.A., Roeser, T. and Stainier, D.-Y.R. (2003) Formation of the Digestive System in Zebrafish. I. Liver Morphogenesis. Developmental Biology, 253, 279-290. http://dx.doi.org/10.1016/S0012-1606(02)00017-9

[16]   Feitsma, H. and Cuppen, E. (2008) Zebrafish as a Cancer Model. The American Association for Cancer Research. Molecular Cancer Research, 6, 685-694. http://dx.doi.org/10.1158/1541-7786.MCR-07-2167

[17]   Morash, M.G., Douglas, S.E., Robotham, A., Ridley, C.M., Gallant, J.W. and Soanes, K.H. (2011) The Zebra Fish Embryo as a Tool for Screening and Characterizing Pleurocidin Host-Defense Peptides as Anti-Cancer Agents. Disease Models & Mechanics, 4, 622-633. http://dx.doi.org/10.1242/dmm.007310

[18]   Patel, K.D., Scarano, F.J., Kondo, M., Hurta, R.A.R. and Neto, C.C. (2011) Proanthocyanidin-Rich Extracts from Cranberry Fruit (Vaccinium macrocarpon, Ait.) Selectively Inhibit the Growth of Human Pathogenic Fungi Candida spp. and Cryptococcus neoformans. Journal of Agricultural and Food Chemistry, 59, 12864-12873.
http://dx.doi.org/10.1021/jf2035466

[19]   Kent, M.L., Bishop-Stewart, J.K., Matthews, J.L. and Spitsbergen, J.M. (2002) Pseudocapillariatomentosa, a Nematode Pathogen, and Associated Neoplasms of Zebrafish (Daniorerio) Kept in Research Colonies. Comparative Medicine, 52, 354-358.

[20]   Gu, L., Kelm, M.A., Hammerstone, J.F., Beecher, G., Holden, J., Haytowitz, D., Gebhardt, S. and Prior, R.L. (2004) Concentrations of Proanthocyanidins in Common Foods and Estimations of Normal Consumption. The Journal of Nutrition, 134, 613-617.

[21]   Chang, C.T., Chung, H.Y., Su, H.T., Tseng, H.P., Tzou, W.S. and Hu, C.H. (2013) Regulation of Zebrafish CYP3A65 Transcription by AHR2. Toxicology and Applied Pharmacology, 270, 174-184.
http://dx.doi.org/10.1016/j.taap.2013.04.010

[22]   Silva, T.L., Cox, A.A., Boominathan, V.P., Jezewski, P.A. and Ferreira, T.L. (2012) Early Expression of the tbx22 Gene in Zebrafish Influences Positioning of Pharyngeal Arch Cartilages. American Journal of Molecular Biology, 2, 318-331. http://dx.doi.org/10.4236/ajmb.2012.24033

[23]   Frisch, S.M., Schaller, M. and Cieply, B. (2013) Mechanisms That Link the Oncogenic Epithelial-Mesenchymal Transition to Suppression of Anoikis. Journal of Cell Science, 126, 21-29. http://dx.doi.org/10.1242/jcs.120907

[24]   Grossmann, J. (2002) Molecular Mechanisms of “Detachment-Induced Apoptosis—Anoikis”. Apoptosis, 7, 247-260.
http://dx.doi.org/10.1023/A:1015312119693

[25]   Choi, E.M., Kwak, S.J., Kim, Y.M., et al. (2005) COX-2 Inhibits Anoikis by Activation of the PI-3K/Akt Pathway in Human Bladder Cancer Cells. Experimental and Molecular Medicine, 37, 199-203.
http://dx.doi.org/10.1038/emm.2005.27