Abstract
Background
Photodynamic inactivation (PDI) may be a potential alternative in cases of therapy-resistant infectious keratitis. The purpose of our study was to determine the impact of PDI using the photosensitizer chlorin e6 (Ce6) on viability, apoptosis, and proliferation of human keratocytes, in vitro.
Methods
Primary human keratocytes were isolated by digestion in collagenase (1 mg/ml) from human corneal buttons, and cultured in DMEM/Ham’s F12 medium supplemented with 10 % FCS. Keratocyte cell cultures underwent illumination using red (670 nm) light for 13 min following exposure to 50 nM to 64 μM concentrations of Ce6 in the culture medium. Twenty-four hours after PDI, cell viability was evaluated by the Alamar blue assay, total DNA content of the cells and apoptosis using the APO-DIRECT™ Kit, and cell proliferation by the BrdU Cell Proliferation Assay Kit.
Results
Using Ce6 or illumination only, we did not detect significant changes of cell viability, apoptosis, and proliferation. Using illumination, viability of keratocytes decreased significantly above 100 nM (P < 0.01), and proliferation at 250 nM Ce6 concentration (P = 0.01) and the percentage of apoptotic keratocytes increased significantly at 500 nM (P < 0.01) concentration.
Conclusions
In the short term, photodynamic inactivation using Ce6 decreases viability and proliferation, and also triggers apoptosis of human keratocytes, in vitro.
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References
Jones DB (1981) Decision-making in the management of microbial keratitis. Ophthalmology 88:814–820
Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q (1998) Photodynamic therapy. J Natl Cancer Inst 90:889–905
Moore C, Wallis C, Melnick JL, Kuns MD (1972) Photodynamic treatment of herpes keratitis. Infect Immun 5:169–171
Ito T (1981) Photodynamic action of hematoporphyrin on yeast cells - a kinetic approach. Photochem Photobiol 34:521–524
Li Z, Jhanji V, Tao X, Yu H, Chen W, Mu G (2013) Riboflavin/ultraviolet light-mediated crosslinking for fungal keratitis. Br J Ophthalmol 97:669–671
Fimple JL, Fontana CR, Foschi F, Ruggiero K, Song X, Pagonis TC, Tanner AC, Kent R, Doukas AG, Stashenko PP, Soukos NS (2008) Photodynamic treatment of endodontic polymicrobial infection in vitro. J Endod 34:728–734
Martins SA, Combs JC, Noguera G, Camacho W, Wittmann P, Walther R, Cano M, Dick J, Behrens A (2008) Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest Ophthalmol Vis Sci 49:3402–3408
Khan YA, Kashiwabuchi RT, Martins SA, Castro-Combs JM, Kalyani S, Stanley P, Flikier D, Behrens A (2011) Riboflavin and ultraviolet light a therapy as an adjuvant treatment for medically refractive Acanthamoeba keratitis: report of 3 cases. Ophthalmology 118:324–331
Goto B, Iriuchishima T, Horaguchi T, Tokuhashi Y, Nagai Y, Harada T, Saito A, Aizawa S (2011) Therapeutic effect of photodynamic therapy using Na-pheophorbide a on osteomyelitis models in rats. Photomed Laser Surg 29:183–189
Roberts WG, Shiau FY, Nelson JS, Smith KM, Berns MW (1988) In vitro characterization of monoaspartyl chlorin e6 and diaspartyl chlorin e6 for photodynamic therapy. J Natl Cancer Inst 80:330–336
Kostenich GA, Zhuravkin IN, Furmanchuk AV, Zhavrid EA (1993) Sensitivity of different rat tumour strains to photodynamic treatment with chlorin e6. J Photochem Photobiol B 17:187–194
Kostenich GA, Zhuravkin IN, Zhavrid EA (1994) Experimental grounds for using chlorin e6 in the photodynamic therapy of malignant tumors. J Photochem Photobiol B 22:211–217
Moon YH, Kwon SM, Kim HJ, Jung KY, Park JH, Kim SA, Kim YC, Ahn SG, Yoon JH (2009) Efficient preparation of highly pure chlorin e6 and its photodynamic anti-cancer activity in a rat tumor model. Oncol Rep 22:1085–1091
Wang J, Stachon T, Eppig T, Langenbucher A, Seitz B, Szentmary N (2013) Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human corneal endothelial cells. Graefes Arch Clin Exp Ophthalmol 251:1199–1204
Seitz B, Hayashi S, Wee WR, LaBree L, McDonnell PJ (1996) In vitro effects of aminoglycosides and fluoroquinolones on keratocytes. Invest Ophthalmol Vis Sci 37:656–665
Cubitt CL, Tang Q, Monteiro CA, Lausch RN, Oakes JE (1993) IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes. Invest Ophthalmol Vis Sci 34:3199–3206
Finke M, Bleses K, Winkler K, Wang J, Szentmáry N, Eppig T, Foth H-J, Hüttenberger D, Langenbucher A, Seitz B, Bischoff M (2012) Die photodynamische Therapie: eine neue Perspektive für die Behandlung der bakteriell induzierten infektiösen Keratitis. Ophthalmologe 109(suppl 1):59, abstract PDo06-046
Wong TW, Tracy E, Oseroff AR, Baumann H (2003) Photodynamic therapy mediates immediate loss of cellular responsiveness to cytokines and growth factors. Cancer Res 63:3812–3818
Oleinick NL, Morris RL, Belichenko I (2002) The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 1:1–21
Lavie G, Kaplinsky C, Toren A, Aizman I, Meruelo D, Mazur Y, Mandel M (1999) A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: possible relevance to photodynamic therapy. Br J Cancer 79:423–432
Kessel D, Poretz RD (2000) Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME). Photochem Photobiol 71:94–96
Wollensak G, Sporl E, Seiler T (2003) Treatment of keratoconus by collagen cross linking. Ophthalmologe 100:44–49
Makdoumi K, Mortensen J, Sorkhabi O, Malmvall BE, Crafoord S (2012) UVA-riboflavin photochemical therapy of bacterial keratitis: a pilot study. Graefes Arch Clin Exp Ophthalmol 250:95–102
Price MO, Tenkman LR, Schrier A, Fairchild KM, Trokel SL, Price FW Jr (2012) Photoactivated riboflavin treatment of infectious keratitis using collagen cross-linking technology. J Refract Surg 28:706–713
Wollensak G, Spoerl E, Reber F, Seiler T (2004) Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro. Eye (Lond) 18:718–722
Mencucci R, Marini M, Paladini I, Sarchielli E, Sgambati E, Menchini U, Vannelli GB (2010) Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea. Clin Exp Ophthalmol 38:49–56
Acknowledgments
We thank the supports of the China Scholarship Council (CSC) for the author’s study (J Wang), and the Alexander von Humboldt Foundation for supporting the work of Dr. N. Szentmáry at the Department of Ophthalmology of Saarland University, Homburg/Saar, Germany. This project was also supported by “Zentrales Innovationsprogramm Mittelstand (ZIM)” of the German Federal Ministry of Economics and Technology (grant number: KF2152004MD0).
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Wang, J., Stachon, T., Eppig, T. et al. Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human keratocytes in vitro. Graefes Arch Clin Exp Ophthalmol 251, 2725–2731 (2013). https://doi.org/10.1007/s00417-013-2470-9
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DOI: https://doi.org/10.1007/s00417-013-2470-9