Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 μM than untreated group, with a significantly lower number of dead cells (15.3±0.4%) were observed compared to the untreated population (20.5±0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 μM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.
References
[1]
Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, et al. (2004) Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells 22: 355–366. doi: 10.1634/stemcells.22-3-355
[2]
Kim HS, Jun Song X, de Paiva CS, Chen Z, Pflugfelder SC, et al. (2004) Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. Exp Eye Res 79: 41–49. doi: 10.1016/j.exer.2004.02.015
[3]
He Y, Pan Z, Luo F (2012) A Novel PAX6 Mutation in Chinese Patients with Severe Congenital Aniridia. Current Eye Research 37: 879–883. doi: 10.3109/02713683.2012.688165
[4]
Smith W, Lange J, Sturm A, Tanner S, Mauger T (2012) Familial peripheral keratopathy without PAX6 mutation. Cornea 31: 130–133. doi: 10.1097/ico.0b013e3182222779
[5]
Le Belle JE, Orozco NM, Paucar AA, Saxe JP, Mottahedeh J, et al. (2011) Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner. Cell Stem Cell 8: 59–71. doi: 10.1016/j.stem.2010.11.028
[6]
Ito K, Hirao A, Arai F, Takubo K, Matsuoka S, et al. (2006) Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med 12: 446–451. doi: 10.1038/nm1388
[7]
Li TS, Marban E (2010) Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells. Stem Cells 28: 1178–1185. doi: 10.1002/stem.438
[8]
Chaudhari P, Ye Z, Jang YY (2012) Roles of Reactive Oxygen Species in the Fate of Stem Cells. Antioxid Redox Signal.
[9]
Pendergrass KD, Boopathy AV, Seshadri G, Maiellaro-Rafferty K, Che PL, et al.. (2013) Acute Preconditioning of Cardiac Progenitor Cells with Hydrogen Peroxide Enhances Angiogenic Pathways Following Ischemia-Reperfusion Injury. Stem Cells Dev.
[10]
Limoli CL, Giedzinski E (2003) Induction of chromosomal instability by chronic oxidative stress. Neoplasia 5: 339–346. doi: 10.1016/s1476-5586(03)80027-1
[11]
Kryston TB, Georgiev AB, Pissis P, Georgakilas AG (2011) Role of oxidative stress and DNA damage in human carcinogenesis. Mutat Res 711: 193–201. doi: 10.1016/j.mrfmmm.2010.12.016
[12]
Song H, Cha MJ, Song BW, Kim IK, Chang W, et al. (2010) Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex. Stem Cells 28: 555–563. doi: 10.1002/stem.302
[13]
Dernbach E, Urbich C, Brandes RP, Hofmann WK, Zeiher AM, et al. (2004) Antioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress. Blood 104: 3591–3597. doi: 10.1182/blood-2003-12-4103
[14]
Urish KL, Vella JB, Okada M, Deasy BM, Tobita K, et al. (2009) Antioxidant levels represent a major determinant in the regenerative capacity of muscle stem cells. Mol Biol Cell 20: 509–520. doi: 10.1091/mbc.e08-03-0274
[15]
Ko E, Lee KY, Hwang DS (2012) Human umbilical cord blood-derived mesenchymal stem cells undergo cellular senescence in response to oxidative stress. Stem Cells Dev 21: 1877–1886. doi: 10.1089/scd.2011.0284
[16]
Gurusamy N, Ray D, Lekli I, Das DK (2010) Red wine antioxidant resveratrol-modified cardiac stem cells regenerate infarcted myocardium. J Cell Mol Med 14: 2235–2239. doi: 10.1111/j.1582-4934.2010.01140.x
[17]
Alves H, Mentink A, Le B, van Blitterswijk CA, de Boer J (2013) Effect of antioxidant supplementation on the total yield, oxidative stress levels, and multipotency of bone marrow-derived human mesenchymal stromal cells. Tissue Eng Part A 19: 928–937. doi: 10.1089/ten.tea.2011.0700
[18]
Mohd Effendy N, Mohamed N, Muhammad N, Mohamad IN, Shuid AN (2012) The effects of tualang honey on bone metabolism of postmenopausal women. Evid Based Complement Alternat Med 2012: 938574. doi: 10.1155/2012/938574
[19]
Khoo YT, Halim AS, Singh KK, Mohamad NA (2010) Wound contraction effects and antibacterial properties of Tualang honey on full-thickness burn wounds in rats in comparison to hydrofibre. BMC Complement Altern Med 10: 48. doi: 10.1186/1472-6882-10-48
[20]
Nasir NA, Halim AS, Singh KK, Dorai AA, Haneef MN (2010) Antibacterial properties of tualang honey and its effect in burn wound management: a comparative study. BMC Complement Altern Med 10: 31. doi: 10.1186/1472-6882-10-31
[21]
Mat Lazim N, Abdullah B, Salim R (2013) The effect of Tualang honey in enhancing post tonsillectomy healing process. An open labelled prospective clinical trial. Int J Pediatr Otorhinolaryngol 77: 457–461. doi: 10.1016/j.ijporl.2012.11.036
[22]
Yaacob NS, Nengsih A, Norazmi MN (2013) Tualang honey promotes apoptotic cell death induced by tamoxifen in breast cancer cell lines. Evid Based Complement Alternat Med 2013: 989841. doi: 10.1155/2013/989841
[23]
Ghashm AA, Othman NH, Khattak MN, Ismail NM, Saini R (2010) Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines. BMC Complement Altern Med 10: 49.
[24]
Fauzi AN, Norazmi MN, Yaacob NS (2011) Tualang honey induces apoptosis and disrupts the mitochondrial membrane potential of human breast and cervical cancer cell lines. Food Chem Toxicol 49: 871–878. doi: 10.1016/j.fct.2010.12.010
[25]
Albietz JM, Lenton LM (2006) Effect of antibacterial honey on the ocular flora in tear deficiency and meibomian gland disease. Cornea 25: 1012–1019. doi: 10.1097/01.ico.0000225716.85382.7b
[26]
Mansour AM (2002) Epithelial corneal oedema treated with honey. Clinical & experimental ophthalmology 30: 149–150. doi: 10.1046/j.1442-6404.2002.00482.x
[27]
Uwaydat S, Jha P, Tytarenko R, Brown H, Wiggins M, et al. (2011) The use of topical honey in the treatment of corneal abrasions and endotoxin-induced keratitis in an animal model. Current eye research 36: 787–796. doi: 10.3109/02713683.2010.544441
[28]
Bashkaran K, Zunaina E, Bakiah S, Sulaiman SA, Sirajudeen K, et al. (2011) Anti-inflammatory and antioxidant effects of Tualang honey in alkali injury on the eyes of rabbits: experimental animal study. BMC Complement Altern Med 11: 90. doi: 10.1186/1472-6882-11-90
[29]
Ahmad I, Jimenez H, Yaacob NS, Yusuf N (2012) Tualang honey protects keratinocytes from ultraviolet radiation-induced inflammation and DNA damage. Photochem Photobiol 88: 1198–1204. doi: 10.1111/j.1751-1097.2012.01100.x
[30]
Kannan TP, Ali AQ, Abdullah SF, Ahmad A (2009) Evaluation of Tualang honey as a supplement to fetal bovine serum in cell culture. Food Chem Toxicol 47: 1696–1702. doi: 10.1016/j.fct.2009.04.020
[31]
Mohamed SA, Khan JA (2013) Antioxidant capacity of chewing stick miswak Salvadora persica. BMC Complementary and Alternative Medicine 13: 40. doi: 10.1186/1472-6882-13-40
[32]
Frankel EN (1985) Chemistry of autoxidation: mechanism, products and flavor significance. American Oil Chemists' Society, Champaign.
[33]
Tan HT, Rahman RA, Gan SH, Halim AS, Hassan SA, et al. (2009) The antibacterial properties of Malaysian tualang honey against wound and enteric microorganisms in comparison to manuka honey. BMC Complement Altern Med 9: 34. doi: 10.1186/1472-6882-9-34
[34]
Sukur SM, Halim AS, Singh KK (2011) Evaluations of bacterial contaminated full thickness burn wound healing in Sprague Dawley rats Treated with Tualang honey. Indian J Plast Surg 44: 112–117. doi: 10.4103/0970-0358.81459
[35]
Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, et al. (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7: 1028–1034.
[36]
de Paiva CS, Chen Z, Corrales RM, Pflugfelder SC, Li DQ (2005) ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells 23: 63–73. doi: 10.1634/stemcells.2004-0093
[37]
Kubota M, Shimmura S, Miyashita H, Kawashima M, Kawakita T, et al. (2010) The anti-oxidative role of ABCG2 in corneal epithelial cells. Invest Ophthalmol Vis Sci 51: 5617–5622. doi: 10.1167/iovs.10-5463
[38]
Priya CG, Prasad T, Prajna NV, Muthukkaruppan V (2013) Identification of human corneal epithelial stem cells on the basis of high ABCG2 expression combined with a large N/C ratio. Microscopy research and technique 76: 242–248. doi: 10.1002/jemt.22159
[39]
Arpitha P, Prajna NV, Srinivasan M, Muthukkaruppan V (2005) High expression of p63 combined with a large N/C ratio defines a subset of human limbal epithelial cells: implications on epithelial stem cells. Investigative ophthalmology & visual science 46: 3631–3636. doi: 10.1167/iovs.05-0343
[40]
Chen Z, Evans WH, Pflugfelder SC, Li DQ (2006) Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells. Stem Cells 24: 1265–1273. doi: 10.1634/stemcells.2005-0363
[41]
Mavric E, Wittmann S, Barth G, Henle T (2008) Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Mol Nutr Food Res 52: 483–489. doi: 10.1002/mnfr.200700282
[42]
Cheng G, He Y, Xie L, Nie Y, He B, et al. (2012) Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier. Int J Nanomedicine 7: 3991–4006. doi: 10.2147/ijn.s32961
[43]
Chen Y, Li M, Li B, Wang W, Lin A, et al. (2013) Effect of reactive oxygen species generation in rabbit corneal epithelial cells on inflammatory and apoptotic signaling pathways in the presence of high osmotic pressure. PloS one 8: e72900. doi: 10.1371/journal.pone.0072900
[44]
Brudzynski K, Kim L (2011) Storage-induced chemical changes in active components of honey de-regulate its antibacterial activity. Food Chem 126: 1155–1163. doi: 10.1016/j.foodchem.2010.11.151
[45]
Daniels-Wells TR, Helguera G, Rodriguez JA, Leoh LS, Erb MA, et al. (2013) Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1. Toxicol In Vitro 27: 220–231. doi: 10.1016/j.tiv.2012.10.006
[46]
Cho KA, Woo SY, Seoh JY, Han HS, Ryu KH (2012) Mesenchymal stem cells restore CCl4-induced liver injury by an antioxidative process. Cell Biol Int 36: 1267–1274. doi: 10.1042/cbi20110634
Pan Q, Qiu WY, Huo YN, Yao YF, Lou MF (2011) Low levels of hydrogen peroxide stimulate corneal epithelial cell adhesion, migration, and wound healing. Investigative ophthalmology & visual science 52: 1723–1734. doi: 10.1167/iovs.10-5866
[49]
Palma M, Taylor LT (2001) Supercritical fluid extraction of 5-hydroxymethyl-2-furaldehyde from raisins. J Agric Food Chem 49: 628–632. doi: 10.1021/jf001070s
[50]
Kulkarni A, Suzuki S, Etoh H (2008) Antioxidant compounds from Eucalyptus grandis biomass by subcritical liquid water extraction. J Wood Sci 54: 153–157. doi: 10.1007/s10086-007-0916-6
[51]
Kishore RK, Halim AS, Syazana MS, Sirajudeen KN (2011) Tualang honey has higher phenolic content and greater radical scavenging activity compared with other honey sources. Nutr Res 31: 322–325. doi: 10.1016/j.nutres.2011.03.001
[52]
Liu AM, Qu WW, Liu X, Qu CK (2012) Chromosomal instability in in vitro cultured mouse hematopoietic cells associated with oxidative stress. Am J Blood Res 2: 71–76.
[53]
Richter T, von Zglinicki T (2007) A continuous correlation between oxidative stress and telomere shortening in fibroblasts. Exp Gerontol 42: 1039–1042. doi: 10.1016/j.exger.2007.08.005
[54]
Abasi M, Massumi M, Riazi G, Amini H (2012) The synergistic effect of beta-boswellic acid and Nurr1 overexpression on dopaminergic programming of antioxidant glutathione peroxidase-1-expressing murine embryonic stem cells. Neuroscience 222: 404–416. doi: 10.1016/j.neuroscience.2012.07.009
[55]
Sakata H, Niizuma K, Wakai T, Narasimhan P, Maier CM, et al. (2012) Neural stem cells genetically modified to overexpress cu/zn-superoxide dismutase enhance amelioration of ischemic stroke in mice. Stroke 43: 2423–2429. doi: 10.1161/strokeaha.112.656900
[56]
Chatterjee S, Browning EA, Hong N, DeBolt K, Sorokina EM, et al. (2012) Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS. Am J Physiol Heart Circ Physiol 302: H105–114. doi: 10.1152/ajpheart.00298.2011
[57]
Urtasun R, Lopategi A, George J, Leung TM, Lu Y, et al. (2012) Osteopontin, an oxidant stress sensitive cytokine, up-regulates collagen-I via integrin alpha(V)beta(3) engagement and PI3K/pAkt/NFkappaB signaling. Hepatology 55: 594–608. doi: 10.1002/hep.24701
[58]
Schauen M, Spitkovsky D, Schubert J, Fischer JH, Hayashi J, et al. (2006) Respiratory chain deficiency slows down cell-cycle progression via reduced ROS generation and is associated with a reduction of p21CIP1/WAF1. J Cell Physiol 209: 103–112. doi: 10.1002/jcp.20711
[59]
Esteban MA, Wang T, Qin B, Yang J, Qin D, et al. (2010) Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6: 71–79. doi: 10.1016/j.stem.2009.12.001
[60]
Ragupathi Raja Kannan R, Arumugam R, Anantharaman P (2012) Chemical composition and antibacterial activity of Indian seagrasses against urinary tract pathogens. Food chemistry 135: 2470–2473. doi: 10.1016/j.foodchem.2012.07.070
[61]
Kumar PP, Kumaravel S, Lalitha C (2010) Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. African Journal of Biochemistry Research 4: 191–195.
[62]
Xiangying Y, Zhao M, Fei L, Shitong Z, Jun H (2013) Identification of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-py？ran-4-oneas a strong antioxidant in glucose–histidine Maillard reaction products. Food Research International 51: 397–403. doi: 10.1016/j.foodres.2012.12.044
[63]
？echovská L, Cejpek K, Kone？ny M, Velí？ek J (2011) On the role of 2,3-dihydro-3,5-dihydroxy-6-methyl-(4H)-？pyran-4-onein antioxidant capacity of prunes. European Food Research and Technology 233: 367–376. doi: 10.1007/s00217-011-1527-4
[64]
Osada Y, Shibamoto T (2006) Antioxidative activity of volatile extracts from Maillard model systems. Food chemistry 98: 522–528. doi: 10.1016/j.foodchem.2005.05.084
[65]
Nam S, Jang HW, Shibamoto T (2012) Antioxidant Activities of Extracts from Teas Prepared from Medicinal Plants, Morus alba L., Camellia sinensis L. and Cudrania tricuspidata and Their Volatile Components. Journal of Agricultural and Food Chemistry 60: 9097–9105. doi: 10.1021/jf301800x
[66]
Westman J, Wiman K, Mohell N (2007) Levoglucosenone derivatives for the treatment of disorders such as cancer, autoimmune diseases and heart diseases. In: W. I. P. Organization, editor editors. Geneva Switzerland.
[67]
Ruiz CM, Gomes JC (2000) Effects of ethanol, acetaldehyde, and acetic acid on histamine secretion in guinea pig lung mast cells. Alcohol 20: 133–138. doi: 10.1016/s0741-8329(99)00065-8
[68]
Wei A, Mura K, Shibamoto T (2001) Antioxidative activity of volatile chemicals extracted from beer. Journal of Agricultural and Food Chemistry 49: 4097–4101. doi: 10.1021/jf010325e
[69]
Fuster MD, Mitchell AE, Ochi H, Shibamoto T (2000) Antioxidative activities of heterocyclic compounds formed in brewed coffee. Journal of Agricultural and Food Chemistry 48: 5600–5603. doi: 10.1021/jf000605e
[70]
Yanagimoto K, Lee KG, Ochi H, Shibamoto T (2002) Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by Maillard reaction. Journal of Agricultural and Food Chemistry 50: 5480–5484. doi: 10.1021/jf025616h
[71]
Suh DY, Han YN, Han BH (1996) Maltol, an antioxidant component of Korean red ginseng, shows little prooxidant activity. Archives of Pharmacal Research 19: 112–115. doi: 10.1007/bf02976844
[72]
Lee KG, Shibamoto T (2000) Antioxidant properties of aroma compounds isolated from soybeans and mung beans. Journal of Agricultural and Food Chemistry 48: 4290–4293. doi: 10.1021/jf000442u
[73]
Aoyagi N, Kimura R, Murata T (1974) Studies on Passiflora incarnata dry extract. I. Isolation of maltol and pharmacological action of maltol and ethyl maltol. Chemical & pharmaceutical bulletin 22: 1008–1013. doi: 10.1248/cpb.22.1008
[74]
Choi KT (2008) Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng CA Meyer. Acta Pharmacologica Sinica 29: 1109–1118. doi: 10.1111/j.1745-7254.2008.00869.x
