[Frontiers in Bioscience 17, 1461-1468, January 1, 2012]

Anti-apoptotic effects of melatonin in retinal pigment epithelial cells

Yang Fu1, Min Tang1, Ying Fan1, Haidong Zou1, Xiaodong Sun1, Xun Xu1

1Department of Ophthalmology, Shanghai Jiaotong University Affiliated First People's Hospital, Shanghai, China, 200080

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture
3.2. TUNEL apoptosis assay
3.3. Annexin V/PI/Flow cytometry apoptosis assay
3.4. Transmission electron microscopy (TEM) observation
3.5. Intracellular ROS measurements
3.6. Mitochondrial DNA damage measurement
3.6.1. Melatonin treatment groups
3.6.2. Melatonin pretreatment groups
3.7. Measurement of cytochrome C release
3.8. Measurement of apoptosis inducing factor release
3.9. Statistical analysis
4. Results
4.1. Melatonin reduced apoptotic cells in models by TUNEL apoptosis assay
4.2. Melatonin decreased the percentage of apoptotic cells in models by annexin V/PI/Flow cytometry apoptosis assay
4.3. Melatonin alleviated the structural damage of RPE cells
4.4. Melatonin reduced intracellular ROS
4.5. Melatonin reduced mtDNA damage
4.6. Melatonin inhibited cytochrome C release
4.7. Melatonin did not inhibit AIF release
5. Discussion
6. Conclusion
7. Acknowledgements
8. References

1. ABSTRACT

Recent studies have revealed that melatonin exerts strong anti-apoptotic effects. Retina secretes melatonin, and melatonin receptors are distributed in almost all the layers of retina, including the layer of retinal pigment epithelial (RPE) cells. However, it is not known whether melatonin inhibits apoptosis through its anti-oxidant effects and how it works in RPE cells. Here, we show that melatonin decreases H2O2-induced apoptosis in RPE cells partially through protection of mitochondria. Melatonin decreased reactive oxygen species in mitochondria and mitochondrial DNA damage, alleviated structural damage and inhibited cytochrome C release.

2. INTRODUCTION

Apoptosis can cause impaired visual function, which is a pathological change that is commonly seen in clinical practice. Because of the specific anatomic and physiological characteristics of retinal pigment epithelial (RPE) cells, apoptosis can be induced in RPE cells by various factors (1-3). Apoptosis of RPE cells occurs in eye diseases including light damage (4) and age-related macular degeneration (5-7). Although the underlying apoptosis mechanism is not yet clear, change in mitochondrial function might play a key role in this process. For instance, various apoptosis signals can be transduced to mitochondria, and subsequently promote or inhibit apoptosis (8); this phenomenon appears more obvious since mitochondria are abundant in the RPE cytoplasm.

Recent studies have revealed that melatonin, an anti-oxidant, can protect mitochondria and exert strong anti-apoptotic effect (9-13). Retinas can secrete melatonin, and melatonin receptors are distributed in almost all the layers of retina, including the layer of RPE cells (14-16). But it remains unknown whether it inhibits apoptosis via its anti-oxidant effects in mitochondria. The purpose of this study was to investigate the anti-apoptotic effects of melatonin in RPE cells in vitro, by focusing on the processes occurring in the mitochondria.

Evidence from our present study demonstrated that melatonin decreased H2O2-induced apoptosis in RPE cells, partially by protecting mitochondria. In fact, melatonin decreased reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) damage, and alleviated the structural damage and inhibited cytochrome C release though it could not inhibit the release of apoptosis inducing factor (AIF) in the mitochondria. Thus, the process of melatonin-protected mitochondria is incomplete and might be selective.

3. MATERIALS AND METHODS

3.1. Cell culture

RPE cells were purchased from ATCC (American Type Cell Culture, Manassas, VA, USA), thawed as a suspension, and intracellular keratin identification was performed. RPE cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (1:1) containing 10% fetal bovine serum and were incubated at 37° C with 5% CO2 until the cells reached 80-85% confluence. Melatonin (M5250, Sigma, St. Louis, MO, USA) was prepared separately in 0.5% ethanol in normal saline buffer.

3.2. TUNEL apoptosis assay

Culture medium was removed, RPE cells were washed twice with PBS, and returned to serum-free DMEM/F12 medium (1:1), containing various concentrations of H2O2 (0 or 500�M solutions were prepared from 30% H2O2 stock; Sigma, St. Louis, MO, USA) and melatonin (0 or 100�M). After incubation of the cells for 24 or 48 hours, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL Apoptosis Detection Kit; Promega, Madison, WI, USA) assay was performed and cells analyzed by fluorescence microscope (AxioVert S100, Zeiss, exposure time: 1 200ms). Six microscopic visual fields in each sample were examined for quantitative analysis of apoptotic cells.

3.3. Annexin V/PI/Flow cytometry apoptosis assay

Culture medium was removed, RPE cells were washed twice with PBS, and serum-free DMEM/F12 medium (1:1), containing H2O2 (0 or 500�M) and melatonin (0 or 100�M) was added. The number of apoptotic cells was determined 24 or 48 hours post-treatment, using Annexin V/Propidium iodide (Molecular Probes, Eugene, OR, USA) flow cytometry (FACScan Becton Dickinson, USA) assay.

3.4. Transmission electron microscopy (TEM) observation

Culture medium was removed, RPE cells were washed twice with PBS, and then cultured in serum-free DMEM/F12 medium (1:1) containing various concentrations of H2O2 (0 or 500�M) and melatonin (0 or 100�M). RPE cell ultrastructure was examined 24 or 48 hours post-treatment, using transmission electron microscopy.

3.5. Intracellular ROS measurement

When RPE cells, initially seeded onto slides within culture dishes, reached 80% confluence, culture medium was removed, cells were washed twice with PBS and then cultured in serum-free DMEM/F12 medium (1:1) containing various concentrations of H2O2 (0 or 500�M) and melatonin (0 or 100 �M). After incubation for 1 or 2 hours, cells were washed twice with PBS, treated for 1 hour with 2nM 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA, Fluka, St. Paul, MN, USA) and 500nM MitoTracker Red CM-H2XRos (MTR; Molecular Probes, Eugene, OR, USA), then washed twice with PBS, and then examined by fluorescent microscope (AxioVert S100, Zeiss, Exposure time: 500ms).

3.6. Mitochondrial DNA damage measurement

3.6.1. Melatonin treatment groups

Culture medium was removed. RPE cells were washed twice with PBS, and then cultured in DMEM/F12 medium, containing 200 �M H2O2 and various concentrations of melatonin (0.1, 1, 10, or 100�M). Control cells were treated with DMEM/F12 medium containing 200�M H2O2 Only. Another control solvent cell group was treated with DMEM/F12 medium, 200 �M H2O2 and 0.5% ethanol in normal saline buffer. After incubation for 1 hour, culture medium was removed and RPE cells were washed twice with PBS.

3.6.2. Melatonin pretreatment groups

RPE cells of different densities were seeded onto 6-well plates. DMEM/F12 medium with 10% fetal bovine serum (2.5 ml), containing various concentrations of melatonin (0.1, 1, 10, or 100�M) was added to each well. Two cell control groups were also prepared in the same manner. The medium was changed every 24 hours with addition of melatonin; this melatonin pretreatment was stopped after 1, 3, 5, 7 or 14 days (all the cells reached about 80% confluence). After the culture medium was discarded, RPE cells were washed twice with PBS and cultured with serum-free DMEM/F12 medium, containing 200μM H2O2 for 1 hour. The culture medium was discarded and RPE cells were washed twice with PBS.

The cell mtDNA was extracted from melatonin treatment and pretreatment cell groups according to the manufacturer's instructions (Mitochondrial DNA Isolation Kit, K280-50, Biovision, USA). PCR was performed aiming at a reference gene, a deleted fragment gene, and a reserved fragment gene using the primers shown in Table 1. For deleted fragments, mtDNA sequence between the pair of primers, mtDNA locations 8,150~8,169bp and 13,650~13,631bp, was evaluated. Residual fragment of 524 bp could be PCR-extended if a 4,977-bp deletion existed. For reserved fragments, a sequence of 4,262 bp between mtDNA locations 12,279~12,98bp and 16,540~16,521bp was the target sequence. A 111-bp reference gene was used as a control.

3.7. Detection of cytochrome C release

RPE cells were cultured in serum-free DMEM/F12 medium (1:1) with various concentrations of H2O2 (0 or 500μM) and melatonin (0or 100 μM) for 1 or 2 hours. Mitochondrial and cytosolic protein lysates were extracted according to the manufacturer's instructions (Mitochondria/Cytosol Fractionation Kit, Biovision, USA). Western blot analysis was performed to evaluate cytochrome C levels using mouse monoclonal anti-cytochrome C antibody (ab13575, Abcam, Cambridge, MA, USA).

3.8. Detection of apoptosis inducing factor release

RPE cells were cultured in serum-free DMEM/F12 medium (1:1) with various concentrations of H2O2 (0 or 500μM) and melatonin (0or 100μM) for 3 or 6 hours. Mitochondrial and cytosolic protein lysates were isolated as described above. Western blot analysis was performed to evaluate AIF levels using monoclonal anti-AIF antibody (Cell Signaling Technology, USA).

3.9. Statistical analysis

Statistical analysis system 6.12 software (SAS Institute Inc., Cary, NC, USA) was used for data analysis. When P<0.05 diferences were considered significant.

4. RESULTS

4.1. Melatonin inhibited apoptosis

In TUNEL assay, red PI fluorescence identifies dead cells and green fluorescence identifies TUNEL-positive apoptotic cells. Using TUNEL assay, our results showed that most of the dead cells exhibited both red and green fluorescence, meaning that cell death was mainly due to apoptosis. More interestingly, melatonin treatment (100 μM) decreased the percentage of positive staining cells in each group (Figure 1).

Using Annexin V/PI/Flow cytometry apoptosis assay, the percentage of apoptotic cells increased when the duration and the concentration of H2O2 increased. However, the percentage of apoptotic cells decreased when melatonin (100 μM) was added to culture medium (Figure 2).

4.2. Melatonin alleviated the structural damage of RPE cells

n identification showed positive results. Compared to cells treated with H2O2 only, the percentage of dark cells (early apoptosis) and dead cells significantly decreased when RPE cells were cultured with both H2O2 and melatonin (100 μM). TEM analysis showed lower mitochondrial density, few vacuoles, few lipid droplets, and normal mitochondrial structure in melatonin-treated cells (Figure 3).

4.4. Melatonin reduced intracellular ROS

Compared to control cells, melatonin treatment (100μM) significantly decreased intracellular ROS levels induced by H2O2 (50 0μM) in RPE cells (Figure 4).

4.5. Melatonin reduced mtDNA damage

The amount of mtDNA deleted fragments and reserved fragments were not affected by any dose of concurrent melatonin treatment, when melatonin was added within 5days prior to H2O2 treatment. However, when cells were pretreated with high concentrations of melatonin (10μM or 100μM) for 7 days prior to H2O2 treatment, mtDNA deletion decreased while the reserved fragments were increased; the effects became more obvious when the pretreatment duration was extended to 14 days. The incidence of deleted mtDNA fragments decreased and reserved fragments increased, with a dose-dependent relationship to melatonin concentration (Figure 5). Thus, long melatonin pretreatment periods (14 days) can inhibit oxidative damage to mtDNA caused by H2O2 in RPE cells.

4.6. Melatonin inhibited cytochrome C release

In the control group, cytochrome C was seen only in the mitochondrial fraction of RPE cells. However, cytochrome C released from the mitochondria to the cytosol following H2O2 treatment (500 μM, 1 or 2 hours) was inhibited by melatonin treatment (100 μM) (Figure 6).

4.7. Melatonin did not inhibit AIF release

In the control group, AIF was seen in the RPE mitochondrial fraction only. However, melatonin treatment (100 μM) did not inhibit H2O2 (500 μM, 3 or 6 hours)-induced AIF release (Figure 7).

5. DISCUSSION

Melatonin, an indole hormone, was recently shown to exert powerful anti-apoptotic and anti-oxidant effects (17). Melatonin alleviated ischemia-induced apoptosis in RPE cells (17). Our results confirmed the anti-apoptotic effect of melatonin, which inhibited H2O2-induced apoptosis in RPE cells in vitro.

How melatonin inhibits apoptosis was the main question to address in this study. The concentration of melatonin in mitochondria is higher than in cytosol (12), and specific binding sites for melatonin might exist on mitochondria. In brain astrocytes, melatonin decreased mitochondrial production of ROS, alleviated calcium overload, improved mitochondrial permeability transition pore (MPTP) function, reduced cytochrome C release, and inhibited apoptosis (11). Our study confirmed that melatonin has protective effects on mitochondria. Indeed, melatonin decreased mitochondrial ROS and mtDNA damage, alleviated the structural damage, and inhibited cytochrome C release.

Moreover, this study was designed to investigate two more aspects related melatonin protection of mitochondria, by focusing on direct effects of melatonin. Melatonin can bind free radicals (such as the hydroxyl free radical) to form cyclic 3-hydroxymelatonin, then N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N-acetyl-5-methoxykynuramine (AMK), which are also strong anti-oxidants (18). AMK can eliminate not only ROS, but also reactive nitrogen species (19). The direct effects of melatonin were demonstrated in the present study, as it effectively decreased mitochondrial oxidative stress by down-regulating intracellular ROS levels; this phenomenon appeared mainly in mitochondria, the main producers of ROS. Oxidative damage is one of the causing factors of the MPTP dysfunction (8), which can lead to the release of intracellular active components (cytochrome C and AIF) and subsequently apoptosis (20, 21). In the present study, while melatonin effectively decreased cytochrome C release, it had no effect on AIF release. Whether the protective effect of melatonin on MPTP is selective and/or the mechanisms regulating cytochrome C release and AIF are different remain to be studied.

The second interesting aspect was the indirect effects of melatonin, which can bolster mitochondrial protection by affecting intracellular components. In the present study, the indirect effects of melatonin were assessed by measuring the damage of mtDNA, which is more sensitive to oxidative stress than nuclear DNA (nDNA) (22). Detectable fragments are found in the mtDNA sequence, usually between 7,900 to 16,500 bp, with 4,977 bp being the most commonly deleted fragment; this deletion is positively correlated with age in humans (23). In fact, genes that code for ATPase and NADH dehydrogenase are located within the 4,977 bp fragment. These enzymes are necessary for oxidative phosphorylation, and deletion of these genes can impact mitochondrial function directly and lead to enhanced ROS production (24). In addition to deleted fragments, other markers of mtDNA damage include mtDNA breaks as well as nucleotide mutations (deletion, insertion, or substitution) which can disrupt normal reading by DNA polymerase, and subsequently affect gene translation and transcription. Only relatively normal DNA templates can be extended (25). Theoretically, longer DNA templates have less probability of being extended completely, as long sequences are more susceptible to mutations. More deleted fragments or fewer reserved fragments are considered as an indication of serious mtDNA damage, and vice-versa. Thus, our findings indicate that melatonin protects mitochondria by decreasing mtDNA damage.

To investigate the indirect effects of melatonin, RPE cells were pretreated with various concentrations of melatonin at different time points, the cells were washed twice with PBS to remove melatonin from the culture medium, so that the final effect depends on intracellular melatonin only. Our results revealed existence of great amount of deleted fragments, but reserved fragments of mtDNA was not affected by any dose of melatonin treatment or by melatonin pretreatment (5 days prior to H2O2 treatment). However, the effects of melatonin were apparent when pretreatment duration was extended to 7 or 14 days considering that all cell groups should have similar concentrations of intracellular melatonin. These findings indicate that melatonin exerted indirect effects in our experimental model.

Melatonin is known to up-regulate anti-oxidant enzymes (e.g. glutathione peroxidase, glutathione reductase, superoxide dismutases, and glucose-6-phosphate dehydrogenase) and can down-regulate oxidative enzymes (such as 5- or 12-lipoxygenase and nitric oxide synthase) (26-28). Although the resulting balance from the effects of these enzymes plays a key role, the main enzymes that contributed to the effect of melatonin in the present study were not investigated.

In the present study, mitochondrial protection and anti-apoptotic effects of melatonin were demonstrated, although the protection due to melatonin was incomplete and selective. Many issues are yet to be addressed, including mechanism(s) of melatonin-regulated MPTP, the intracellular components affected by melatonin treatment, other melatonin-induced antiapoptotic pathways besides mitochondrial pathway. Further investigation is needed to provide data required to prove the relevance of melatonin to the retina. The clinical application of the anti-apoptotic effect of melatonin in retinal protection deserves further investigation.

6. CONCLUSIONS

The present study demonstrated that melatonin decreased H2O2-induced apoptosis in RPE cells partially through its protection of mitochondria. However, its mitochondrial protection activity is incomplete and selective.

7. ACKNOWLEDGEMENTS

We thank Medica Communicus Asia for translation of the manuscript.

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Abbreviations: RPE: retinal pigment epithelial, ROS: reactive oxygen species, mtDNA: mitochondrial DNA, AIF: apoptosis inducing factor, DMEM/F12: Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, TEM: Transmission electron microscopy, MPTP: mitochondrial permeability transition pore, AFMK: N1-acetyl-N2-formyl-5-methoxykynuramine, AMK: N-acetyl-5-methoxykynuramine, nDNA: nuclear DNA

Key Words: Apoptosis, Melatonin, Retinal Pigment Epithelial Cell

Send correspondence to: Min Tang, Shanghai Jiaotong University Affiliated First People's Hospital, Ophthalmology, Shanghai, 200080, China, Tel: 86-021-33011075, Fax: 86-021-33011075, E-mail:fuyang9377@sina.com.cn