Human recombinant epidermal growth factor (EGF) promotes cell proliferation as well as skin remodeling and healing. However, it’s unclear if EGF is stable in in vitro cell culture medium treatment conditions. Since EGF’s instability in aqueous solutions has resulted in different kinds of challenges, novel treatment approaches that preserve EGF’s stability and usefulness have been developed. The current study examined the effects of the antioxidants sodium selenite (Se), disodium ethylenediaminetetraacetic acid dihydrate (EDTA), zinc chloride (Zn), and ascorbic acid (AA) on the stability of EGF in cell culture using Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI), as well as their ability to scavenge free radicals. In vitro, DMEM and RPMI serum free medium with 2.5 μM of EDTA, Se, AA, and Zn antioxidants were used separately at 37？C for three days to examine the stability of EGF. The stability of EGF was evaluated using reversed phase high-performance liquid chromatography (RP-HPLC). Cells were cultivated for 3 days to further estimate the impact of antioxidants on cell cytotoxicity and proliferation using the MTT assay with NIH-3T3, L929, TF-1, and Sp2/0-Ag14 cell types. The antioxidants EDTA, Se, AA, and Zn at an extracellular concentration of 2.5 μM improved EGF stability in both DMEM and RPMI medium while having no effect on the proliferation of NIH-3T3, L929, TF-1, and SP2/0-Ag14 cells.
References
[1]
Esquirol Caussa, J. and Herrero Vila, E. (2015) Factor de crecimiento epidérmico, innovación y seguridad. Epidermal Growth Factor, Innovation and Safety. Medicina Clínica, 145, 305-312. https://doi.org/10.1016/j.medcli.2014.09.012
[2]
Berlanga, J., Fernández, J.I., López, E., López, P.A., del Río, A., Valenzuela, C., Baldomero, J., Muzio, V., Raíces, M., Silva, R., Acevedo, B.E. and Herrera, L. (2013) Heberprot-P: A Novel Product for Treating Advanced Diabetic Foot Ulcer. MEDICC Review, 15, 11-15. https://doi.org/10.37757/MR2013V15.N1.4
[3]
Bennett, N.T. and Schultz, G.S. (1993) Growth Factors and Wound Healing: Biochemical Properties of Growth Factors and Their Receptors. The American Journal of Surgery, 165, 728-737. https://doi.org/10.1016/S0002-9610(05)80797-4
[4]
Carpenter, G. and Cohen, S. (1979) Epidermal Growth Factor. Annual Review of Biochemistry, 48, 193-216. https://doi.org/10.1146/annurev.bi.48.070179.001205
[5]
Wu, Z., Tang, Y., Fang, H., Su, Z., Xu, B., Lin, Y., Zhang, P. and Wei, X. (2015) Decellularized Scaffolds Containing Hyaluronic Acid and EGF for Promoting the Recovery of Skin Wounds. Journal of Materials Science: Materials in Medicine, 26, Article No. 59. https://doi.org/10.1007/s10856-014-5322-1
[6]
Senderoff, R.I., Wootton, S.C., Boctor, A.M., Chen, T.M., Giordani, A.B., Julian, T.N. and Radebaugh, G.W. (1994) Aqueous Stability of Human Epidermal Growth Factor 1-48. Pharmaceutical Research, 11, 1712-1720. https://doi.org/10.1023/A:1018903014204
[7]
Manning, M.C., Chou, D.K., Murphy, B.M., Payne, R.W. and Katayama, D.S. (2010) Stability of Protein Pharmaceuticals: An Update. Pharmaceutical Research, 27, 544-575. https://doi.org/10.1007/s11095-009-0045-6
[8]
Kamerzell, T.J., Esfandiary, R., Joshi, S.B., Middaugh, C.R. and Volkin, D.B. (2011) Protein-Excipient Interactions: Mechanisms and Biophysical Characterization Applied to Protein Formulation Development. Advanced Drug Delivery Reviews, 63, 1118-1159. https://doi.org/10.1016/j.addr.2011.07.006
[9]
Rayaprolu, B.M., Strawser, J.J. and Anyarambhatla, G. (2018) Excipients in Parenteral Formulations: Selection Considerations and Effective Utilization with Small Molecules and Biologics. Drug Development and Industrial Pharmacy, 44, 1565-1571. https://doi.org/10.1080/03639045.2018.1483392
[10]
Araki, F., Nakamura, H., Nojima, N., Tsukumo, K. and Sakamoto, S. (1989) Stability of Recombinant Human Epidermal Growth Factor in Various Solutions. Chemical and Pharmaceutical Bulletin, 37, 404-406. https://doi.org/10.1248/cpb.37.404
[11]
Yang, C.-H., Huang, Y.-B., Wu, P.-C., and Tsai, Y.-H. (2005) The Evaluation of Stability of Recombinant Human Epidermal Growth Factor in Burn-Injured Pigs. Process Biochemistry, 40, 1661-1665. https://doi.org/10.1016/j.procbio.2004.06.038
[12]
Santana, H., González, Y., Campana, P.T., Noda, J., Amarantes, O., Itri, R., Beldarraín, A. and Páez, R. (2013) Screening for Stability and Compatibility Conditions of Recombinant Human Epidermal Growth Factor for Parenteral Formulation: Effect of pH, Buffers, and Excipients. International Journal of Pharmaceutics, 452, 52-62. https://doi.org/10.1016/j.ijpharm.2013.04.054
[13]
Schreck, R. and Baeuerle, P.A. (1991) A Role for Oxygen Radicals as Second Messengers. Trends in Cell Biology, 1, 39-42. https://doi.org/10.1016/0962-8924(91)90072-H
[14]
Gigli, I., Byrd, D.D. and Fortune, J.E. (2006) Effects of Oxygen Tension and Supplements to the Culture Medium on Activation and Development of Bovine Follicles in Vitro. Theriogenology, 66, 344-353. https://doi.org/10.1016/j.theriogenology.2005.11.021
[15]
Chwa, M., Atilano, S.R., Reddy, V., Jordan, N., Kim, D.W. and Kenney, M.C. (2006) Increased Stress-Induced Generation of Reactive Oxygen Species and Apoptosis in Human Keratoconus Fibroblasts. Investigative Ophthalmology & Visual Science, 47, 1902-1910. https://doi.org/10.1167/iovs.05-0828
[16]
Cassano, E., Tosto, L., Balestrieri, M., Zicarelli, L. and Abrescia, P. (1999) Antioxidant Defense in the Follicular Fluid of Water Buffalo. Cellular Physiology and Biochemistry, 9, 106-116. https://doi.org/10.1159/000016307
[17]
Jr. Feril, L.B., Ogawa, K., Watanabe, A., Ogawa, R., et al. (2017) Anticancer Potential of EDTA: A Preliminary in Vitro Study. Mathews Journal of Cancer Science, 2, Article 009.
[18]
Ebert, R., Ulmer, M., Zeck, S., Meissner-Weigl, J., Schneider, D., Stopper, H., Schupp, N., Kassem, M. and Jakob, F. (2006) Selenium Supplementation Restores the Antioxidative Capacity and Prevents Cell Damage in Bone Marrow Stromal Cells in Vitro. Stem Cells, 24, 1226-1235. https://doi.org/10.1634/stemcells.2005-0117
[19]
Clément, M.V., Ramalingam, J., Long, L.H. and Halliwell, B. (2001) The in Vitro Cytotoxicity of Ascorbate Depends on the Culture Medium Used to Perform the Assay and Involves Hydrogen Peroxide. Antioxidants & Redox Signaling, 3, 157-163. https://doi.org/10.1089/152308601750100687
[20]
Prasad, A.S. (2014) Zinc Is an Antioxidant and Anti-Inflammatory Agent: Its Role in Human Health. Frontiers in Nutrition, 1, Article 14. https://doi.org/10.3389/fnut.2014.00014
[21]
Kim, H., Seo, C., Ji, M., Kim, Y., Kim, J.D., Jeong, D.H., et al. (2018) Monitoring of Epidermal Growth Factor Degradation Products by High‐Performance Liquid Chromatography. Bulletin of the Korean Chemical Society, 39, 864-867. https://doi.org/10.1002/bkcs.11488
[22]
Kim, H., Seo, C., Ji, M., Kim, Y., Kim, J.D., Jeong, D.H., Lee, W. and Paik, M.-J. (2018) Monitoring of Epidermal Growth Factor Degradation Products by High-Performance Liquid Chromatography. Bulletin of the Korean Chemical Society, 39, 864-867. https://doi.org/10.1002/bkcs.11488
[23]
Hayashi, T., Sakamoto, S., Fuwa, T., et al. (1987) Determination of Human Epidermal Growth Factors in Cultured Media of Escherichia coli by High Performance Liquid Chromatography. Analytical Sciences, 3, 445-448. https://doi.org/10.2116/analsci.3.445
[24]
Lee, Y.S., Suh, C.W., Park, S.K. and Lee, E.K. (2003) Purification of Soluble Human Epidermal Growth Factor (hEGF) from Recombinant Escherichia coli Culture Broth by Using Expanded-Bed Adsorption Chromatography. Biotechnology and Applied Biochemistry, 38, 9-13. https://doi.org/10.1042/BA20020113
[25]
Santana, H., García, G., Vega, M., Beldarraín, A. and Páez, R. (2015) Stability Studies of a Freeze-Dried Recombinant Human Epidermal Growth Factor Formulation for Wound Healing. PDA Journal of Pharmaceutical Science and Technology, 69, 399-416.
[26]
Playford, R.J., Marchbank, T., Calnan, D.P., Calam, J., Royston, P., Batten, J.J. and Hansen, H.F. (1995) Epidermal Growth Factor Is Digested to Smaller, Less Active Forms in Acidic Gastric Juice. Gastroenterology, 108, 92-101. https://doi.org/10.1016/0016-5085(95)90012-8
[27]
DiBiase, M.D. and Rhodes, C.T. (1991) The Design of Analytical Methods for Use in Topical Epidermal Growth Factor Product Development. Journal of Pharmacy and Pharmacology, 43, 553-558. https://doi.org/10.1111/j.2042-7158.1991.tb03535.x
[28]
Burgess, A.W., Knesel, J., Sparrow, L.G., Nicola, N.A. and Nice, E.C. (1982) Two Forms of Murine Epidermal Growth Factor: Rapid Separation by Using Reverse-Phase HPLC. Proceedings of the National Academy of Sciences of the United States of America, 79, 5753-5757. https://doi.org/10.1073/pnas.79.19.5753
[29]
Lim, J.Y., Kim, N.A., Lim, D.G., Kim, K.H. and Jeong, S.H. (2015) Effects of Thermal and Mechanical Stress on the Physical Stability of Human Growth Hormone and Epidermal Growth Factor. Archives of Pharmacal Research, 38, 1488-1498. https://doi.org/10.1007/s12272-014-0521-3
[30]
Strober, W. (2015) Trypan Blue Exclusion Test of Cell Viability. Current Protocols in Immunology, 111, A3.B.1-A3.B.3. https://doi.org/10.1002/0471142735.ima03bs111
[31]
Shantha, N.C. and Decker, E.A. (1994) Rapid, Sensitive, Iron-Based Spectrophotometric Methods for Determination of Peroxide Values of Food Lipids. Journal of AOAC International, 77, 421-424. https://doi.org/10.1093/jaoac/77.2.421
[32]
Stine, C.M., Harland, H.A., Coulter, S.T. and Jenness, R. (1954) A Modified Peroxide Test for Detection of Lipid Oxidation in Dairy Products. Journal of Dairy Science, 37, 202-208. https://doi.org/10.3168/jds.S0022-0302(54)70245-X
[33]
Marins, J.S., Sassone, L.M., Fidel, S.R. and Ribeiro, D.A. (2012) In Vitro Genotoxicity and Cytotoxicity in Murine Fibroblasts Exposed to EDTA, NaOCl, MTAD and Citric Acid. Brazilian Dental Journal, 23, 527-533. https://doi.org/10.1590/S0103-64402012000500010
[34]
Abedelahi, A., Salehnia, M. and Allameh, A.A. (2008) The Effects of Different Concentrations of Sodium Selenite on the in Vitro Maturation of Preantral Follicles in Serum-Free and Serum Supplemented Media. Journal of Assisted Reproduction and Genetics, 25, 483-488. https://doi.org/10.1007/s10815-008-9252-z
[35]
Selenius, M., Rundlöf, A.K., Olm, E., Fernandes, A.P. and Björnstedt, M. (2010) Selenium and the Selenoprotein Thioredoxin Reductase in the Prevention, Treatment and Diagnostics of Cancer. Antioxidants & Redox Signaling, 12, 867-880. https://doi.org/10.1089/ars.2009.2884
[36]
Schmidt, R.J., Chung, L.Y., Andrews, A.M. and Turner, T.D. (1993) Toxicity of L-ascorbic Acid to L929 Fibroblast Cultures: Relevance to Biocompatibility Testing of Materials for Use in Wound Management. Journal of Biomedical Materials Research, 27, 521-530. https://doi.org/10.1002/jbm.820270413
[37]
Bozym, R.A., Chimienti, F., Giblin, L.J., Gross, G.W., Korichneva, I., Li, Y., Libert, S., Maret, W., Parviz, M., Frederickson, C.J. and Thompson, R.B. (2010) Free Zinc Ions Outside a Narrow Concentration Range Are Toxic to a Variety of Cells in Vitro. Experimental Biology and Medicine, 235, 741-750. https://doi.org/10.1258/ebm.2010.009258
[38]
Du, Y., Guo, D., Wu, Q., Liu, D. and Bi, H. (2014) Zinc Chloride Inhibits Human Lens Epithelial Cell Migration and Proliferation Involved in TGF-β1 and TNF-α Signaling Pathways in HLE B-3 Cells. Biological Trace Element Research, 159, 425-433. https://doi.org/10.1007/s12011-014-9979-6
[39]
Di Fiore, P.P., Pierce, J.H., Fleming, T.P., Hazan, R., Ullrich, A., King, C.R., Schlessinger, J. and Aaronson, S.A. (1987) Overexpression of the Human EGF Receptor Confers an EGF-Dependent Transformed Phenotype to NIH 3T3 Cells. Cell, 51, 1063-1070. https://doi.org/10.1016/0092-8674(87)90592-7
[40]
Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y.F., Miyazono, K., Urabe, A. and Takaku, F. (1989) Establishment and Characterization of a Unique Human Cell Line that Proliferates Dependently on GM-CSF, IL-3, or Erythropoietin. Journal of Cellular Physiology, 140, 323-334. https://doi.org/10.1002/jcp.1041400219
[41]
Ali, M., Kwak, S.H., Byeon, J.Y. and Choi, H.J. (2023) In Vitro and in Vivo Evaluation of Epidermal Growth Factor (EGF) Loaded Alginate-Hyaluronic Acid (AlgHA) Microbeads System for Wound Healing. Journal of Functional Biomaterials, 14, Article 403. https://doi.org/10.3390/jfb14080403
[42]
Ray, P.D., Huang, B.W. and Tsuji, Y. (2012) Reactive Oxygen Species (ROS) Homeostasis and Redox Regulation in Cellular Signaling. Cellular Signalling, 24, 981-990. https://doi.org/10.1016/j.cellsig.2012.01.008
[43]
Rock, C.L., Jacob, R.A. and Bowen, P.E. (1996) Update on the Biological Characteristics of the Antioxidant Micronutrients: Vitamin C, Vitamin E, and the Carotenoids. Journal of the Academy of Nutrition and Dietetics, 96, 693-702. https://doi.org/10.1016/S0002-8223(96)00190-3
[44]
Prakasha Gowda, A.S., Schaefer, A.D. and Schuck, T.K. (2021) Effect of Excipients on Recombinant Interleukin-2 Stability in Aqueous Buffers. American Journal of Analytical Chemistry, 12, 347-372. https://doi.org/10.4236/ajac.2021.1210022
[45]
Son, D.H., Yang, D.J., Sun, J.S., Kim, S.K., Kang, N., Kang, J.Y., Choi, Y.H., Lee, J.H., Moh, S.H., Shin, D.M. and Kim, K.W. (2018) A Novel Peptide, Nicotinyl-Isoleucine-Valine-Histidine (NA-IVH), Promotes Antioxidant Gene Expression and Wound Healing in HaCaT Cells. Marine Drugs, 16, Article 262. https://doi.org/10.3390/md16080262
[46]
Wang, W. (1999) Instability, Stabilization, and Formulation of Liquid Protein Pharmaceuticals. International Journal of Pharmaceutics, 185, 129-188. https://doi.org/10.1016/S0378-5173(99)00152-0
[47]
Halliwell, B. and Gutteridge, J.M.C. (1999) Free Radicals in Biology and Medicine. 3rd Edition, Oxford University Press.
[48]
Halliwell, B. (2003) Oxidative Stress in Cell Culture: An Under-Appreciated Problem? FEBS Letters, 540, 3-6. https://doi.org/10.1016/S0014-5793(03)00235-7
[49]
Stadtman, E.R. and Oliver, C.N. (1991) Metal-Catalyzed Oxidation of Proteins. Physiological Consequences. The Journal of Biological Chemistry, 266, 2005-2008. https://doi.org/10.1016/S0021-9258(18)52199-2
[50]
Ji, J.A., Zhang, B., Cheng, W. and Wang, Y.J. (2009) Methionine, Tryptophan, and Histidine Oxidation in a Model Protein, PTH: Mechanisms and Stabilization. Journal of Pharmaceutical Sciences, 98, 4485-4500. https://doi.org/10.1002/jps.21746
[51]
Halliwell, B. (1995) How to Characterize an Antioxidant: An Update. Biochemical Society Symposia, 61, 73-101. https://doi.org/10.1042/bss0610073
[52]
Ribeiro, D.A., Matsumoto, M.A., Duarte, M.A., Marques, M.E. and Salvadori, D.M. (2005) In Vitro Biocompatibility Tests of Two Commercial Types of Mineral Trioxide Aggregate. Brazilian Oral Research, 19, 183-187. https://doi.org/10.1590/S1806-83242005000300005
[53]
Kasugai, S., Hasegawa, N. and Ogura, H. (1990) A Simple in Vito Cytotoxicity Test Using the MTT (3-(4,5)-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide) Colorimetric Assay: Analysis of Eugenol Toxicity on Dental Pulp Cells (RPC-C2A). Japanese Journal of Pharmacology, 52, 95-100. https://doi.org/10.1016/S0021-5198(19)37772-8
[54]
Wang, H.P., Qian, S.Y., Schafer, F.Q., Domann, F.E., Oberley, L.W. and Buettner, G.R. (2001) Phospholipid Hydroperoxide Glutathione Peroxidase Protects against Singlet Oxygen-Induced Cell Damage of Photodynamic Therapy. Free Radical Biology and Medicine, 30, 825-835. https://doi.org/10.1016/S0891-5849(01)00469-5
[55]
Hartikainen, Xue, T.L. and Piironen, V. (2000) Selenium as an Anti-Oxidant and Pro-Oxidant in Ryegrass. Plant and Soil, 225, 193-200. http://www.jstor.org/stable/42950858 https://doi.org/10.1023/A:1026512921026
[56]
De Laurenzi, V., Melino, G., Savini, I., Annicchiarico-Petruzzelli, M., Finazzi-Agrò, A. and Avigliano, L. (1995) Cell Death by Oxidative Stress and Ascorbic Acid Regeneration in Human Neuroectodermal Cell Lines. European Journal of Cancer, 31, 463-466. https://doi.org/10.1016/0959-8049(95)00059-R
[57]
Park, C.H., Bergsagel, D.E. and McCulloch, E.A. (1971) Ascorbic Acid: A Culture Requirement for Colony Formation by Mouse Plasmacytoma Cells. Science, 174, 720-722. https://doi.org/10.1126/science.174.4010.720
[58]
Holmes, A.M., Mackenzie, L. and Roberts, M.S. (2020) Disposition and Measured Toxicity of Zinc Oxide Nanoparticles and Zinc Ions against Keratinocytes in Cell Culture and Viable Human Epidermis. Nanotoxicology, 14, 263-274. https://doi.org/10.1080/17435390.2019.1692382
[59]
Lobo, V., Patil, A., Phatak, A. and Chandra, N. (2010) Free Radicals, Antioxidants and Functional Foods: Impact on Human Health. Pharmacognosy Reviews, 4, 118-126. https://doi.org/10.4103/0973-7847.70902
[60]
Garg, S., Rose, A.T. and Waite, T.D. (2011) David Waite, Photochemical Production of Superoxide and Hydrogen Peroxide from Natural Organic Matter. Geochimica et Cosmochimica Acta, 75, 4310-4320. https://doi.org/10.1016/j.gca.2011.05.014
[61]
Stohs, S.J. and Bachi, D. (1995) Oxidative Mechanisms in the Toxicity of Metal Ions. Free Radical Biology and Medicine, 18, 321-336. https://doi.org/10.1016/0891-5849(94)00159-H
[62]
Dean, R.T., Fu, S., Stocker, R. and Davies, M.J. (1997) Biochemistry and Pathology of Radical-Mediated Protein Oxidation. Biochemical Journal, 324, 1-18. https://doi.org/10.1042/bj3240001
