http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2015; 38(1): 16–21 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.900067
RESEARCH ARTICLE
Dose dependent cytotoxicity of pranoprofen in cultured human corneal endothelial cells by inducing apoptosis Yi-Han Li1,2*, Qian Wen1*, Ting-Jun Fan1, Yuan Ge1, Miao-Miao Yu1, Ling-Xiao Sun1, and Yu Zhao1 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 06/16/15 For personal use only.
1
Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao, China and 2School of Medicine, Shanghai Jiaotong University, Shanghai, China Abstract
Keywords
Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.
Apoptosis, cytotoxicity, human corneal endothelial cell, pranoprofen
Introduction Keratitis, one of the most common corneal diseases, is the predominant factor leading to impaired vision or blindness if not treated timely (Agrawal et al., 1994; Jones, 1981). Non-steroidal anti-inflammatory drugs (NSAIDs) which can eliminate inflammation in cornea by controlling the secretion of prostaglandin are often used in keratitis treatment in eye clinic. Amongst NSAIDs, pranoprofen (PPF), as a kind of propanoic acid with both hydrophilic and hydrophobic properties, is often used clinically in therapy of inflammation in the exterior and anterior segments of the eye (AkyolSalman et al., 2007; Qu et al., 2011; Zhao et al., 2000). At the same time, there have also been reports on side effects of PPF, including irritation, conjunctival congestion, itching, and palpebral edema (Zhu et al., 2009). And it has been found that eyedrops containing NSAIDs exhibited greater cytotoxicity to rabbit and bovine corneal epithelial cells even than
*Both the first two authors contributed equally to this paper. Address for correspondence: Ting-Jun Fan, Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China. Tel: +86 532 82031637. Fax: +86 532 82031793. E-mail: [email protected]
History Received 16 March 2013 Revised 10 February 2014 Accepted 18 February 2014 Published online 18 March 2014
those containing steroids with benzalkonium at comparable concentrations (Ayaki et al., 2012). Monolayer human corneal endothelium (HCE), as the barrier between cornea and aqueous humor, plays an indispensible role in maintaining cornea transparency, thickness and physiological functions (Hassell & Birk, 2010). HCE cells lose their ability of proliferation in adult, therefore local damage to these cells can be repaired solely by enlargement of cells immediately adjacent to the wound to manage HCE monolayer integrality, resulting in age-related decrease of HCE cell density and corneal transparency (Joyce, 2003). Damage by surgical wounds, infection, long-term high eye pressure and drugs can result in excessive loss of HCE cells and lead to decompensation of the endothelium and eventual loss of visual acuity (Joyce, 2003; Schierho¨lter & Honegger, 1975). Since NSAIDs are considered to be a major causative factor of ocular surface disorders during treatment of inflamed eyes, such as postoperatively or for inflammatory eye diseases (Ayaki et al., 2012), it becomes more and more important to access their endothelial cytotoxicity for us to be aware of their toxicity as well as efficacy. Unfortunately, little is known about the potential endothelial toxicity of PPF till now (Ayaki et al., 2010). With an established nontransfected HCE cell line (Fan et al., 2011), this study was intended to assess the cytotoxicity of PPF and its underlying
DOI: 10.3109/01480545.2014.900067
mechanism to HCE cells in vitro, for the purpose of providing theoretical safeguards for clinical prescription of PPF and protecting us from damage of HCE cells and visual acuity as well.
Cytotoxicity of pranoprofen to human corneal endothelial cells
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in total. Apoptotic ratio was calculated using the following quotation: Apoptotic ratio (%) ¼ apoptotic cell numbers/ (normal cell numbers + apoptotic cell numbers) 100. DNA agarose gel electrophoresis
Materials and methods
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 06/16/15 For personal use only.
Materials Non-transfected human corneal endothelial cell line (utHCEC01), established previously in our laboratory (Fan et al., 2011), was cultured in Dulbecco’s modified Eagle medium: Ham’s nutrient mixture F-12 (1:1) medium (DMEM/F12, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT). Pranoprofen (PPF) powder (Purity: 99%; CAS# 52549-17-4) was purchased from Jinan Boss Chemical Ltd. (Jinan, China). Before usage, 20 mg PPF powder was dissolved into 10 ml serumfree DMEM/F12 culture medium to prepare a 2.0 g/l PPF solution and used as a 2-fold stock solution. And serial dilutions from the stock solution were prepared also in the serum-free DMEM/F12 medium (Invitrogen). Light microscopy HCE cells, cultured in 25 cm2 culture flask using 10% FBSDMEM/F12 culture medium, were harvested using 2.5 g/l trypsin digestion, pipetting, and centrifugation methods as described previously once confluent monolayer established (Fan et al., 2012). Harvested cells were resuspended in 24 ml 10% FBS-DMEM/F12 culture medium, seeded into a 24 wells plate for 1 ml/well, and cultured at 37 C in a 5% CO2 incubator till cells cover 80% area of the well bottom. After the old medium in each well was discarded, 2-fold PPF stock solution diluted in 10% FBS-DMEM/F12 culture medium was added into wells to final PPF concentrations varying from 0.015625 to 1.0 g/l and then cultured under the same condition. The growth status and cell morphology were monitored with an Eclipse TS100 inverted microscope (Nikon, Tokyo, Japan). Acridine orange/ethidium bromide double fluorescence staining HCE cells under the same culture and treatment condition were collected as described above at 1–3 h intervals. HCE cells from 3 wells were collected from each time point and centrifuged at 1500 r/min for 10 min. Cell pellets were fully resuspended in 0.1 ml serum-free DMEM/F12 medium and 4 ml acridine orange/ethidium bromide (AO/EB, 100 mg/l AO solution: 100 mg/l EB solution ¼ 1:1, v/v) staining solution was added and mixed thoroughly for 1 min at room temperature. Labelled cell suspension was dropped on glass slide, covered with a coverslip and then observed under a Ti-S fluorescent microscope (Nikon, Tokyo, Japan). According to the accepted criterion, cells with their plasma membrane permeability elevated and their nuclei exhibiting red or orange fluorescence were counted as apoptotic cells while cells without plasma membrane permeability elevation and with green fluorescent nuclei were counted as non-apoptotic cells (Leite et al., 1999). At least 5 fields were selected randomly and counted from each group, no less than 400 cells
For DNA isolation and electrophoresis, HCE cells were seeded, cultured, PPF treated for 2–24 h, and harvested as described above. Genomic DNA isolation was carried out with a cell/tissue DNA rapid isolation kit (Dongsheng Biotech Ltd., Guangzhou, China) and manipulated according to its user manual. Each of genomic DNA preparation was loaded onto a 1% agarose gel and electrophoresed (200 mA, 255 min). The gel was then stained with 0.5 mg/l EB solution for 10 min and observed with an EC3 Imaging System (UVP, LLC, Upland, CA). Transmission electron microscopy For transmission electron microscope (TEM) analysis, HCE cells were seeded into 25 cm2 culture flask and cultured in a 37 C incubator. Till cells proliferate to cover 80% area of flask bottom, the culture medium was replaced with same culture medium containing 0.125 g/l PPF solution and cultured in a 37 C incubator for 24 h. The cells harvested by using the same method as stated above were fixed using 40 g/l glutaraldehyde-sucrose-dimethylarsine buffer and then refixed, embedding, ultrathin sectioning and dying. Samples were observed under H-700 TEM (Hitachi, Tokyo, Japan). Statistical analysis HCE cells cultured in plates or flasks without PPF were set as negative controls. Each experiment was repeated 3 times independently. Data were presented as mean ± SE in triplicates and analyzed for statistical significance with ANOVA single factor. Changes were considered statistically significant if p50.05.
Results Morphology of HCE cells Inversed light microscopic observations showed that HCE cells treated by 0.0625–1.0 g/l PPF exhibited retarded growth and proliferation, shrinkage, loss of attachment, and eventual death, which developed in strong relationship to the concentration and time of treatment (Figure 1). As expected, 0.25– 1.0 g/l PPF demonstrated the severest toxicity to the cells, few being alive 2–4 h after treatment, which was significantly different from those of negative controls. PPF at a concentration of 0.125 g/l killed lots of cells after 24 h, and had relatively low toxicity at a concentration of 0.0625 g/l, while no significant toxicity was found at concentrations of 0.03125 g/l when compared with those of negative controls. From these results, we could conclude that PPF at concentrations of 0.0625–1.0 g/l has cytotoxicity to HCE cells in a dose- and time-dependent manner. Plasma membrane permeability of HCE cells Results of AO/EB double-fluorescent staining showed that PPF at concentrations of 0.0625–1.0 g/l could significantly
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Drug Chem Toxicol, 2015; 38(1): 16–21
Figure 1. Pranoprofen exhibits cytotoxicity on HCE cells in vitro. Cultured HCE cells were treated with or without pranoprofen at doses and times indicated. One representative photograph from three independent experiments is shown. Bars: 50 mm.
increase the plasma membrane permeability of HCE cells, and nuclei of these cells condensed into round shape and were stained into even red or orange fluorescence. Contrarily, PPF at the concentration of 0.03125 g/l had no obvious effects on the plasma membrane permeability of HCE cells, and nuclei of these cells did not condensed and were stained into uneven green fluorescence, similar to those of control cells (Figure 2). And these imply that PPF at concentrations of 0.0625–1.0 g/l might be able to induce apoptosis in HCE cells in vitro. Quantification of the effects of PPF on plasma membrane permeability of HCE cells was shown in Figure 3. It was found that 0.0625–1.0 g/l PPF had statistically significant effects on inducing HCE cell apoptosis (p50.01), and the apoptotic ratio of the cells treated 1–2 h with 0.5–1.0 g/l PPF was as high as 100%. However, PPF at 0.03125 g/l
had no statistically significant effect on inducing HCE cell apoptosis (Figure 3). Conclusion could be made from above results that PPF from 0.0625 g/l to 1.0 g/l might have an apoptosis inducing effect on HCE cells which is dose and time dependent. DNA fragmentation of HCE cells Electrophoresis in 1% agarose gel showed that DNA from HCE cells treated 1 h by 0.5 g/l PPF was decomposed into a highly dispersed state, and typical DNA ladders appeared for cells treated by 0.25 g/l PPF for 4 h and by 0.125 g/l PPF for 24 h, while no DNA ladder was found in those of the negative control (Figure 4). It indicates that PPF can induce DNA fragmentation in HCE cells, which proving its apoptosis inducing capability on HCE cells.
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DOI: 10.3109/01480545.2014.900067
Cytotoxicity of pranoprofen to human corneal endothelial cells
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Figure 2. Pranoprofen induces elevation of plasma membrane permeability in HCE cells. Cultured HCE cells were treated with or without pranoprofen at doses and times indicated. Plasma membrane permeability was analyzed by AO/EB double staining. Even and red or orange fluorescence represents apoptotic cell, and uneven and green fluorescence indicates non-apoptotic cell. Bars: 10 mm.
while some demonstrated characteristics associated with late stage, e.g. nuclear envelope breakdown and apoptotic body formation. These results demonstrate that PPF can elicit typical apoptotic morphological and structural features in HCE cells, which proving the apoptosis inducing ability of PPF on HCE cells.
Discussion
Figure 3. Apoptotic ratios of HCE cells treated with pranoprofen at different doses for different times. Data are mean ± SEM from three independent experiments. **p50.01, *p50.05 versus negative control.
Ultrastructure of HCE cells Results of TEM observation on HCE cells at different stage of apoptosis after treatment by 0.125 g/l PPF for 24 h were illustrated in Figure 5. Compared with the negative control, some of HCE cells demonstrated morphological and structural characteristics associated with early stage of apoptosis such as chromatin condensation in nuclei, some demonstrated characteristics associated with intermediate stage, including cytoplasm shrinkage and vacuolisation, ultrastructure disorder, advanced chromatin condensation and marginalization,
Damage by cytotoxic drugs can result in excessive loss of HCE cells which may lead to decompensation of the endothelium and loss of visual acuity (Schierho¨lter & Honegger, 1975). PPF, one of NSAIDs, is considered to be a major causative factor of ocular surface disorders during keratitis treatment (Ayaki et al., 2012). Therefore, it will be of great importance to access its endothelial cytotoxicity. The present study was intended to access the corneal endothelial cytotoxicity of PPF by using the non-transfected HCE cells as an in vitro experiment model (Fan et al., 2011). Our light microscopic observation indicates that PPF at 0.625–1.0 g/l can result in retarded cell growth, shrinkage, and even death in a dose- and time-dependent manner. The morphological changes of HCE cells resulted from PPF treatment resembles those of apoptotic cells (Vaux, 2002), suggesting the possible apoptosis inducing role of PPF towards HCE cells. To indentify the underlying cytotoxic mechanism of PPF on HCE cells, we utilized AO/EB double fluorescence staining to detect the membrane permeability of HCE cells after various PPF treatment since the elevation in membrane permeability is one of the typical hallmarks
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Figure 4. Pranoprofen induces DNA fragmentation in HCE cells. Cultured HCE cells were treated with or without pranoprofen at doses and times indicated, and their DNA preparations were analyzed by agarose gel electrophoresis. Marker, D2000 DNA marker.
Drug Chem Toxicol, 2015; 38(1): 16–21
in apoptotic cells (Leite et al., 1999). We discover from the AO/EB staining results that PPF from 0.0625 to 1.0 g/l can elevate the membrane permeability of HCE cells, in a concentration and time dependent manner, indicating PPF is very likely to induce HCE cells apoptosis. This conclusion is consistent to light microscopic observation and previously published results (Fan et al., 2012). Since DNA fragmentation is another signature of apoptotic cells (Oberhammer et al., 1993), agarose gel electrophoresis of DNA from PPF treated HCE cells was carried out to confirm the apoptosis inducing effect of PPF. Our results confirmed that 0.125–0.5 g/l PPF can induce DNA fragmentation, and possibly apoptosis in HCE cells. The formation of apoptotic bodies is the most representative hallmark in structural alteration in apoptotic cells, which is the most significant feature to distinguish apoptosis from necrosis (Ziegler & Groscurth, 2004). In order to verify the apoptosis inducing capability of PPF on HCE cells, we also carried out ultrastructural characterization of PPF treated HCE cells, which showing apoptotic morphological features including cytoplasm shrinkage and vacuolisation, ultrastructural disorder, chromatin condensation and inner-nuclear marginalization, nuclear membrane breakdown, and apoptotic body formation. These results are consistent to reported features of apoptotic cells (Fan et al., 2012; Takemura et al., 2001). All the results verify sufficiently that PPF can induce apoptosis in HCE cells.
Figure 5. Pranoprofen induces apoptosis in HCE cells. Cultured HCE cells were treated with or without pranoprofen at the dose of 0.125 g/l for times indicated, and their ultrastructures were analyzed by TEM. Note the extended vacuolisation of the cytoplasm (C), alterations of mitochondria (C), the enlargement of the space between the inner and outer nuclear membranes (C), and the appearance of apoptotic bodies (D) in pranoprofen-treated HCE cells. N, cell nucleus. V, vacuole. H, mitochondrion. *, apoptotic body. Bars: 1 mm.
DOI: 10.3109/01480545.2014.900067
PPF at the concentration of 1.0 g/l, used in topical application, is very deleterious for HCE cells. Even the final exposure concentration of PPF after dilution in the corneal endothelium is lower, potential cytotoxicity of PPF is most probably valid because PPF at a concentration of 0.0625 g/l, 1/16 of the topical concentration, also has obvious cytotoxicity to HCE cells, i.e. inducing apoptosis in HCE cells. Development of a new-style and non-cytotoxic PPF substitute is now undertaken in our laboratory.
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Conclusions PPF at concentrations ranging from 0.0625 to 1.0 g/l has strong cytotoxicity to HCE cells in vitro in a dose- and timedependent manner, and its cytotoxicity is realized by inducing apoptosis in HCE cells. As a consequence, prolonged and repeated exposure to PPF can result in excessive loss of HCE cells and lead to decompensation of corneal endothelium which will reduce our visual acuity. This study provides a guideline for prescribing PPF in eye clinic, and suggests the necessity to develop new and nontoxic substitutes for PPF.
Declaration of interest The authors declare that there is no conflict of interest. This work was supported by National High Technology Research and Development Program (‘‘863’’ Program) of China (No. 2006AA02A132).
References Agrawal V, Biswas J, Madhavan HN, et al. (1994). Current perspectives in infectious keratitis. Indian J Ophthalmol 42:171–191. Akyol-Salman I, Lec¸e-Serto¨z D, Baykal O. (2007). Topical pranoprofen 0.1% is as effective anti-inflammatory and analgesic agent as diclofenac sodium 0.1% after strabismus surgery. J Ocul Pharmacol Ther 3:280–283. Ayaki M, Iwasawa A, Niwano, Y. (2012). Comparative assessment of the cytotoxicity of six anti-inflammatory eyedrops in four cultured
Cytotoxicity of pranoprofen to human corneal endothelial cells
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ocular surface cell lines, as determined by cell viability scores. Clin Ophthalmol 6:1879–1884. Ayaki M, Taguchi Y, Soda M, et al. (2010). Cytotoxicity of topical medications used for infection and inflammation control after cataract surgery in cultured corneal endothelial cells. Biocontrol Sci 15: 97–102. Fan T, Zhao J, Ma X, et al. (2011). Establishment of a continuous untransfected human corneal endothelial cell line and its biocompatibility to denuded amniotic membrane. Mol Vis 17:469–480. Fan TJ, Wen Q, Yu MM, et al. (2012). Experimental studies on the effect of oxybuprocaine hydrochloride on human corneal endothelial cells. Int Eye Sci 8:1442–1446. Hassell JR, Birk DE. (2010). The molecular basis of corneal transparency. Exp Eye Res 3:326–335. Jones DB. (1981). Decision-making in the management of microbial keratitis. Ophthalmology 8:814–820. Joyce NC. (2003). Proliferative capacity of the corneal endothelium. Prog Retin Eye Res 22:359–389. Leite M, Quinta-Costa M, Leite PS, Guimara˜es JE. (1999). Critical evaluation of techniques to detect and measure cell death – study in a model of UV radiation of the leukaemic cell line HL60. Anal Cell Pathol 3–4:139–151. Oberhammer F, Wilson JW, Dive C, et al. (1993). Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 9: 3679–3684. Qu M, Wang Y, Yang L, Zhou Q. (2011). Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components. Mol Vis 17:3147–3155. Schierho¨lter R, Honegger H. (1975). Morphology of the corneal endothelium under normal conditions and during regeneration after mechanical injury. Adv Ophthalmol 31:34–99. Takemura G, Kato S, Aoyama T, et al. (2001). Characterization of ultrastructure and its relation with DNA fragmentation in Fas-induced apoptosis of cultured cardiac myocytes. J Pathol 4:546–556. Vaux DL. (2002). Apoptosis timeline. Cell Death Differ 4:349–354. Zhao J, Li X, Dong D, et al. (2000). Clinical assessment of Pranoprofen eyedrops on postoperative inf lammation of cataract extraction and non-infection inf lammation of the anterior segments of the eye. Chin Ophthal Res 6:560–564. Zhu G, Wang Q, Cai C, Zhang S. (2009). Research progress in the use of NSAIDS in ophthalmology. Chin J Optom Ophthalmol 2:157–160. Ziegler U, Groscurth P. (2004). Morphological features of cell death. News Physiol Sci 19:124–128.
RESEARCH ARTICLE
Dose dependent cytotoxicity of pranoprofen in cultured human corneal endothelial cells by inducing apoptosis Yi-Han Li1,2*, Qian Wen1*, Ting-Jun Fan1, Yuan Ge1, Miao-Miao Yu1, Ling-Xiao Sun1, and Yu Zhao1 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 06/16/15 For personal use only.
1
Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao, China and 2School of Medicine, Shanghai Jiaotong University, Shanghai, China Abstract
Keywords
Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.
Apoptosis, cytotoxicity, human corneal endothelial cell, pranoprofen
Introduction Keratitis, one of the most common corneal diseases, is the predominant factor leading to impaired vision or blindness if not treated timely (Agrawal et al., 1994; Jones, 1981). Non-steroidal anti-inflammatory drugs (NSAIDs) which can eliminate inflammation in cornea by controlling the secretion of prostaglandin are often used in keratitis treatment in eye clinic. Amongst NSAIDs, pranoprofen (PPF), as a kind of propanoic acid with both hydrophilic and hydrophobic properties, is often used clinically in therapy of inflammation in the exterior and anterior segments of the eye (AkyolSalman et al., 2007; Qu et al., 2011; Zhao et al., 2000). At the same time, there have also been reports on side effects of PPF, including irritation, conjunctival congestion, itching, and palpebral edema (Zhu et al., 2009). And it has been found that eyedrops containing NSAIDs exhibited greater cytotoxicity to rabbit and bovine corneal epithelial cells even than
*Both the first two authors contributed equally to this paper. Address for correspondence: Ting-Jun Fan, Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China. Tel: +86 532 82031637. Fax: +86 532 82031793. E-mail: [email protected]
History Received 16 March 2013 Revised 10 February 2014 Accepted 18 February 2014 Published online 18 March 2014
those containing steroids with benzalkonium at comparable concentrations (Ayaki et al., 2012). Monolayer human corneal endothelium (HCE), as the barrier between cornea and aqueous humor, plays an indispensible role in maintaining cornea transparency, thickness and physiological functions (Hassell & Birk, 2010). HCE cells lose their ability of proliferation in adult, therefore local damage to these cells can be repaired solely by enlargement of cells immediately adjacent to the wound to manage HCE monolayer integrality, resulting in age-related decrease of HCE cell density and corneal transparency (Joyce, 2003). Damage by surgical wounds, infection, long-term high eye pressure and drugs can result in excessive loss of HCE cells and lead to decompensation of the endothelium and eventual loss of visual acuity (Joyce, 2003; Schierho¨lter & Honegger, 1975). Since NSAIDs are considered to be a major causative factor of ocular surface disorders during treatment of inflamed eyes, such as postoperatively or for inflammatory eye diseases (Ayaki et al., 2012), it becomes more and more important to access their endothelial cytotoxicity for us to be aware of their toxicity as well as efficacy. Unfortunately, little is known about the potential endothelial toxicity of PPF till now (Ayaki et al., 2010). With an established nontransfected HCE cell line (Fan et al., 2011), this study was intended to assess the cytotoxicity of PPF and its underlying
DOI: 10.3109/01480545.2014.900067
mechanism to HCE cells in vitro, for the purpose of providing theoretical safeguards for clinical prescription of PPF and protecting us from damage of HCE cells and visual acuity as well.
Cytotoxicity of pranoprofen to human corneal endothelial cells
17
in total. Apoptotic ratio was calculated using the following quotation: Apoptotic ratio (%) ¼ apoptotic cell numbers/ (normal cell numbers + apoptotic cell numbers) 100. DNA agarose gel electrophoresis
Materials and methods
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 06/16/15 For personal use only.
Materials Non-transfected human corneal endothelial cell line (utHCEC01), established previously in our laboratory (Fan et al., 2011), was cultured in Dulbecco’s modified Eagle medium: Ham’s nutrient mixture F-12 (1:1) medium (DMEM/F12, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT). Pranoprofen (PPF) powder (Purity: 99%; CAS# 52549-17-4) was purchased from Jinan Boss Chemical Ltd. (Jinan, China). Before usage, 20 mg PPF powder was dissolved into 10 ml serumfree DMEM/F12 culture medium to prepare a 2.0 g/l PPF solution and used as a 2-fold stock solution. And serial dilutions from the stock solution were prepared also in the serum-free DMEM/F12 medium (Invitrogen). Light microscopy HCE cells, cultured in 25 cm2 culture flask using 10% FBSDMEM/F12 culture medium, were harvested using 2.5 g/l trypsin digestion, pipetting, and centrifugation methods as described previously once confluent monolayer established (Fan et al., 2012). Harvested cells were resuspended in 24 ml 10% FBS-DMEM/F12 culture medium, seeded into a 24 wells plate for 1 ml/well, and cultured at 37 C in a 5% CO2 incubator till cells cover 80% area of the well bottom. After the old medium in each well was discarded, 2-fold PPF stock solution diluted in 10% FBS-DMEM/F12 culture medium was added into wells to final PPF concentrations varying from 0.015625 to 1.0 g/l and then cultured under the same condition. The growth status and cell morphology were monitored with an Eclipse TS100 inverted microscope (Nikon, Tokyo, Japan). Acridine orange/ethidium bromide double fluorescence staining HCE cells under the same culture and treatment condition were collected as described above at 1–3 h intervals. HCE cells from 3 wells were collected from each time point and centrifuged at 1500 r/min for 10 min. Cell pellets were fully resuspended in 0.1 ml serum-free DMEM/F12 medium and 4 ml acridine orange/ethidium bromide (AO/EB, 100 mg/l AO solution: 100 mg/l EB solution ¼ 1:1, v/v) staining solution was added and mixed thoroughly for 1 min at room temperature. Labelled cell suspension was dropped on glass slide, covered with a coverslip and then observed under a Ti-S fluorescent microscope (Nikon, Tokyo, Japan). According to the accepted criterion, cells with their plasma membrane permeability elevated and their nuclei exhibiting red or orange fluorescence were counted as apoptotic cells while cells without plasma membrane permeability elevation and with green fluorescent nuclei were counted as non-apoptotic cells (Leite et al., 1999). At least 5 fields were selected randomly and counted from each group, no less than 400 cells
For DNA isolation and electrophoresis, HCE cells were seeded, cultured, PPF treated for 2–24 h, and harvested as described above. Genomic DNA isolation was carried out with a cell/tissue DNA rapid isolation kit (Dongsheng Biotech Ltd., Guangzhou, China) and manipulated according to its user manual. Each of genomic DNA preparation was loaded onto a 1% agarose gel and electrophoresed (200 mA, 255 min). The gel was then stained with 0.5 mg/l EB solution for 10 min and observed with an EC3 Imaging System (UVP, LLC, Upland, CA). Transmission electron microscopy For transmission electron microscope (TEM) analysis, HCE cells were seeded into 25 cm2 culture flask and cultured in a 37 C incubator. Till cells proliferate to cover 80% area of flask bottom, the culture medium was replaced with same culture medium containing 0.125 g/l PPF solution and cultured in a 37 C incubator for 24 h. The cells harvested by using the same method as stated above were fixed using 40 g/l glutaraldehyde-sucrose-dimethylarsine buffer and then refixed, embedding, ultrathin sectioning and dying. Samples were observed under H-700 TEM (Hitachi, Tokyo, Japan). Statistical analysis HCE cells cultured in plates or flasks without PPF were set as negative controls. Each experiment was repeated 3 times independently. Data were presented as mean ± SE in triplicates and analyzed for statistical significance with ANOVA single factor. Changes were considered statistically significant if p50.05.
Results Morphology of HCE cells Inversed light microscopic observations showed that HCE cells treated by 0.0625–1.0 g/l PPF exhibited retarded growth and proliferation, shrinkage, loss of attachment, and eventual death, which developed in strong relationship to the concentration and time of treatment (Figure 1). As expected, 0.25– 1.0 g/l PPF demonstrated the severest toxicity to the cells, few being alive 2–4 h after treatment, which was significantly different from those of negative controls. PPF at a concentration of 0.125 g/l killed lots of cells after 24 h, and had relatively low toxicity at a concentration of 0.0625 g/l, while no significant toxicity was found at concentrations of 0.03125 g/l when compared with those of negative controls. From these results, we could conclude that PPF at concentrations of 0.0625–1.0 g/l has cytotoxicity to HCE cells in a dose- and time-dependent manner. Plasma membrane permeability of HCE cells Results of AO/EB double-fluorescent staining showed that PPF at concentrations of 0.0625–1.0 g/l could significantly
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Drug Chem Toxicol, 2015; 38(1): 16–21
Figure 1. Pranoprofen exhibits cytotoxicity on HCE cells in vitro. Cultured HCE cells were treated with or without pranoprofen at doses and times indicated. One representative photograph from three independent experiments is shown. Bars: 50 mm.
increase the plasma membrane permeability of HCE cells, and nuclei of these cells condensed into round shape and were stained into even red or orange fluorescence. Contrarily, PPF at the concentration of 0.03125 g/l had no obvious effects on the plasma membrane permeability of HCE cells, and nuclei of these cells did not condensed and were stained into uneven green fluorescence, similar to those of control cells (Figure 2). And these imply that PPF at concentrations of 0.0625–1.0 g/l might be able to induce apoptosis in HCE cells in vitro. Quantification of the effects of PPF on plasma membrane permeability of HCE cells was shown in Figure 3. It was found that 0.0625–1.0 g/l PPF had statistically significant effects on inducing HCE cell apoptosis (p50.01), and the apoptotic ratio of the cells treated 1–2 h with 0.5–1.0 g/l PPF was as high as 100%. However, PPF at 0.03125 g/l
had no statistically significant effect on inducing HCE cell apoptosis (Figure 3). Conclusion could be made from above results that PPF from 0.0625 g/l to 1.0 g/l might have an apoptosis inducing effect on HCE cells which is dose and time dependent. DNA fragmentation of HCE cells Electrophoresis in 1% agarose gel showed that DNA from HCE cells treated 1 h by 0.5 g/l PPF was decomposed into a highly dispersed state, and typical DNA ladders appeared for cells treated by 0.25 g/l PPF for 4 h and by 0.125 g/l PPF for 24 h, while no DNA ladder was found in those of the negative control (Figure 4). It indicates that PPF can induce DNA fragmentation in HCE cells, which proving its apoptosis inducing capability on HCE cells.
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DOI: 10.3109/01480545.2014.900067
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Figure 2. Pranoprofen induces elevation of plasma membrane permeability in HCE cells. Cultured HCE cells were treated with or without pranoprofen at doses and times indicated. Plasma membrane permeability was analyzed by AO/EB double staining. Even and red or orange fluorescence represents apoptotic cell, and uneven and green fluorescence indicates non-apoptotic cell. Bars: 10 mm.
while some demonstrated characteristics associated with late stage, e.g. nuclear envelope breakdown and apoptotic body formation. These results demonstrate that PPF can elicit typical apoptotic morphological and structural features in HCE cells, which proving the apoptosis inducing ability of PPF on HCE cells.
Discussion
Figure 3. Apoptotic ratios of HCE cells treated with pranoprofen at different doses for different times. Data are mean ± SEM from three independent experiments. **p50.01, *p50.05 versus negative control.
Ultrastructure of HCE cells Results of TEM observation on HCE cells at different stage of apoptosis after treatment by 0.125 g/l PPF for 24 h were illustrated in Figure 5. Compared with the negative control, some of HCE cells demonstrated morphological and structural characteristics associated with early stage of apoptosis such as chromatin condensation in nuclei, some demonstrated characteristics associated with intermediate stage, including cytoplasm shrinkage and vacuolisation, ultrastructure disorder, advanced chromatin condensation and marginalization,
Damage by cytotoxic drugs can result in excessive loss of HCE cells which may lead to decompensation of the endothelium and loss of visual acuity (Schierho¨lter & Honegger, 1975). PPF, one of NSAIDs, is considered to be a major causative factor of ocular surface disorders during keratitis treatment (Ayaki et al., 2012). Therefore, it will be of great importance to access its endothelial cytotoxicity. The present study was intended to access the corneal endothelial cytotoxicity of PPF by using the non-transfected HCE cells as an in vitro experiment model (Fan et al., 2011). Our light microscopic observation indicates that PPF at 0.625–1.0 g/l can result in retarded cell growth, shrinkage, and even death in a dose- and time-dependent manner. The morphological changes of HCE cells resulted from PPF treatment resembles those of apoptotic cells (Vaux, 2002), suggesting the possible apoptosis inducing role of PPF towards HCE cells. To indentify the underlying cytotoxic mechanism of PPF on HCE cells, we utilized AO/EB double fluorescence staining to detect the membrane permeability of HCE cells after various PPF treatment since the elevation in membrane permeability is one of the typical hallmarks
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Y.-H. Li et al.
Figure 4. Pranoprofen induces DNA fragmentation in HCE cells. Cultured HCE cells were treated with or without pranoprofen at doses and times indicated, and their DNA preparations were analyzed by agarose gel electrophoresis. Marker, D2000 DNA marker.
Drug Chem Toxicol, 2015; 38(1): 16–21
in apoptotic cells (Leite et al., 1999). We discover from the AO/EB staining results that PPF from 0.0625 to 1.0 g/l can elevate the membrane permeability of HCE cells, in a concentration and time dependent manner, indicating PPF is very likely to induce HCE cells apoptosis. This conclusion is consistent to light microscopic observation and previously published results (Fan et al., 2012). Since DNA fragmentation is another signature of apoptotic cells (Oberhammer et al., 1993), agarose gel electrophoresis of DNA from PPF treated HCE cells was carried out to confirm the apoptosis inducing effect of PPF. Our results confirmed that 0.125–0.5 g/l PPF can induce DNA fragmentation, and possibly apoptosis in HCE cells. The formation of apoptotic bodies is the most representative hallmark in structural alteration in apoptotic cells, which is the most significant feature to distinguish apoptosis from necrosis (Ziegler & Groscurth, 2004). In order to verify the apoptosis inducing capability of PPF on HCE cells, we also carried out ultrastructural characterization of PPF treated HCE cells, which showing apoptotic morphological features including cytoplasm shrinkage and vacuolisation, ultrastructural disorder, chromatin condensation and inner-nuclear marginalization, nuclear membrane breakdown, and apoptotic body formation. These results are consistent to reported features of apoptotic cells (Fan et al., 2012; Takemura et al., 2001). All the results verify sufficiently that PPF can induce apoptosis in HCE cells.
Figure 5. Pranoprofen induces apoptosis in HCE cells. Cultured HCE cells were treated with or without pranoprofen at the dose of 0.125 g/l for times indicated, and their ultrastructures were analyzed by TEM. Note the extended vacuolisation of the cytoplasm (C), alterations of mitochondria (C), the enlargement of the space between the inner and outer nuclear membranes (C), and the appearance of apoptotic bodies (D) in pranoprofen-treated HCE cells. N, cell nucleus. V, vacuole. H, mitochondrion. *, apoptotic body. Bars: 1 mm.
DOI: 10.3109/01480545.2014.900067
PPF at the concentration of 1.0 g/l, used in topical application, is very deleterious for HCE cells. Even the final exposure concentration of PPF after dilution in the corneal endothelium is lower, potential cytotoxicity of PPF is most probably valid because PPF at a concentration of 0.0625 g/l, 1/16 of the topical concentration, also has obvious cytotoxicity to HCE cells, i.e. inducing apoptosis in HCE cells. Development of a new-style and non-cytotoxic PPF substitute is now undertaken in our laboratory.
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Conclusions PPF at concentrations ranging from 0.0625 to 1.0 g/l has strong cytotoxicity to HCE cells in vitro in a dose- and timedependent manner, and its cytotoxicity is realized by inducing apoptosis in HCE cells. As a consequence, prolonged and repeated exposure to PPF can result in excessive loss of HCE cells and lead to decompensation of corneal endothelium which will reduce our visual acuity. This study provides a guideline for prescribing PPF in eye clinic, and suggests the necessity to develop new and nontoxic substitutes for PPF.
Declaration of interest The authors declare that there is no conflict of interest. This work was supported by National High Technology Research and Development Program (‘‘863’’ Program) of China (No. 2006AA02A132).
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