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Retinal Cell Biology  |   November 2012
Lectin from Agaricus bisporus Inhibited S Phase Cell Population and Akt Phosphorylation in Human RPE Cells
Author Affiliations & Notes
  • Yiu-Him Cheung
    From the Eye Institute, The University of Hong Kong, Pokfulam, Hong Kong; the
  • Carl M. Sheridan
    Research Centre of Heart, Brain, Hormone and Healthy Aging, The University of Hong Kong, Pokfulam, Hong Kong.
  • Amy C. Y. Lo
    From the Eye Institute, The University of Hong Kong, Pokfulam, Hong Kong; the
    Eye and Vision Science Institute of Aging and Chronic Disease, University of Liverpool, Liverpool, United Kingdom; and the
  • Wico W. Lai
    From the Eye Institute, The University of Hong Kong, Pokfulam, Hong Kong; the
    Eye and Vision Science Institute of Aging and Chronic Disease, University of Liverpool, Liverpool, United Kingdom; and the
  • *Each of the following is a corresponding author: Wico W. Lai, Clinical Associate Professor, Eye Institute, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong; wicolai@yahoo.com
  • Amy C. Y. Lo, Assistant Professor, Eye Institute, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong; amylo@hkucc.hku.hk
Investigative Ophthalmology & Visual Science November 2012, Vol.53, 7469-7475. doi:10.1167/iovs.12-10589
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      Yiu-Him Cheung, Carl M. Sheridan, Amy C. Y. Lo, Wico W. Lai; Lectin from Agaricus bisporus Inhibited S Phase Cell Population and Akt Phosphorylation in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(12):7469-7475. doi: 10.1167/iovs.12-10589.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit cell proliferation of some ocular and cancer cell lines. To elucidate how ABL inhibited RPE cell proliferation, we investigated the changes in cell cycle distribution and cell proliferation-related signaling pathways after ABL treatment.

Methods.: Primary human RPE cells were isolated and grown in DMEM/F12 with or without the ABL (20 or 90 μg/mL) for 3 days. Analysis of cell cycle was performed by flow cytometry. Phosphorylation status of Erk, Jnk, p38, and Akt as well as p53 expression levels were investigated by Western blotting. The role of phosphorylated-Akt in RPE cell proliferation was further evaluated using LY294002.

Results.: After ABL treatment (90 μg/mL), the amount of cells present in the S phase was found to be reduced. These changes were not apparent in cells treated with 20 μg/mL ABL. In addition, Erk and Akt were found to be hyperphosphorylated and hypophosphorylated, respectively. The expression levels of phosphorylated-Jnk, phosphorylated-p38, and p53 were not altered when compared with those of the control cells. When RPE cells were treated with LY294002 and deprived from phosphorylated-Akt expression, cell proliferation rate was reduced. Reduction in the amount of cells present in S phase was also observed.

Conclusions.: Our results showed that ABL hypophosphorylated Akt and this observation is in line with the fact that ABL attenuates cell proliferation. As the level of p53 was not significantly altered by ABL, this indicated that ABL-arrested cell cycle progression was independent of p53 activation.

Introduction
Mushrooms have long been consumed not only for their dietary value, but also popularly as supplements for their medicinal properties. 1 In recent years, attention has been focused on using mushrooms as antitumor agents and immunoregulators. 13  
Agaricus bisporus, commonly known as button mushroom or table mushroom, is the most widely consumed cultivated mushroom worldwide. 4 Besides containing high amount of vitamins, extracts from the mushroom were found to exhibit chemopreventive, antiproliferative, and anti-inflammatory properties. 58 Specifically, lectin extracted from this edible mushroom, A. bisporus lectin (ABL), was found to inhibit cell proliferation of HT-29 colon cancer cells, primary human subconjunctival fibroblasts, and RPE cells. 911 However, little is known on how ABL suppresses cell proliferation. 
RPE is a single layer of cells that lies between the neuroretina and the choroid. Proliferation of RPE cells is suggested to mediate proliferative vitreoretinopathy (PVR), which is the most common cause of retinal redetachment after successful retinal detachment surgery. Further, RPE cells isolated from patients who had PVR were found to have higher proliferating capacity in vitro compared with that of controls. 12 Therefore, it has been proposed that inhibition of RPE cell proliferation might limit the development of PVR and therefore increase the successful rate of retinal detachment surgery. 
The importance of limiting cell proliferation can be highlighted by the success of using 5-fluorouracil and low molecular weight heparin to reduce the incidence of postoperative PVR. 13 However, concerns have been raised for possible retinal damage as 5-fluorouracil is cytotoxic. 14 In previous studies, lectin extracted from the edible mushroom A. bisporus was shown to suppress the growth of primary RPE cells and HT-29 colon cancer cell line in a dose-dependent manner without apparent cytotoxicity. 10,11,15 However, the mechanism by which ABL retarded cell proliferation was unclear. 
In this study, we performed flow cytometry to investigate how ABL arrested cell cycle progression. We also conducted experiments to identify the possible signaling pathways through which ABL altered cell cycle progression. The antiproliferative effect of ABL on RPE cells was contrasted with that of a phosphorylated-Akt inhibitor LY294002. 
Materials and Methods
Reagents
Pure ABL was obtained from EY Laboratories (San Mateo, CA; Lot number: 190725-2). All chemicals were of analytical grades and obtained from Sigma (St. Louis, MO) unless otherwise indicated. 
Cell Culture
Human postmortem eyes were obtained from Manchester Royal Hospital Eye Bank. Experiments involving the use of postmortem human tissues were approved by the Ethics Committee, National Research Ethics Service, UK. Eyes were collected within 48 hours postmortem and were stored at 4°C before dissection. RPE cell isolation from donor eyes were performed according to previously published procedures. 16,17 Two different RPE cells were obtained and were designated as hRPE1 and hRPE3 cells, respectively. The epithelial nature of the cells was confirmed by positive staining of pan-cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) on all RPE cells. RPE cells were maintained with DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) during routine passages. Human RPE cells at passages 6 to 10 were used in all experiments. 
ABL Treatments on RPE Cells
hRPE1 and hRPE3 cells were seeded into 6-well plates, 60-mm dishes or 96-well plates and grown in DMEM/F12 at reduced serum level (1%) for 24 hours. Cells were cultured with low levels of serum as many serum components like feutin and immunoglobulin were found to possess potential binding sites for ABL. This potentially decreased the availability of free ABL for the RPE cells. 18,19 Except for the cell proliferation assay, seeding densities were adjusted so that the RPE cells reached approximately 80% confluence when they were harvested at the end of the experiments. One day after the initial seeding, cells were maintained with culture medium at a reduced serum level (1%) with or without ABL supplement (20 or 90 μg/mL) thereafter for 3 days. 
Cell Proliferation Assay
Cell proliferation was determined by a tetrazolium salt (MTS) assay (Promega, Madison, WI). It was determined that the optimal seeding densities for hRPE1 and hRPE3 cells were 0.75 × 103 and 0.5 × 103, respectively, at which cell proliferation could be sustained up to 10 days in the 96-well plates (data not shown). Twenty-four hours after the RPE cells were seeded, cells were maintained with culture medium with or without ABL supplement. Proliferation assays were performed 1 and 3 days later. Absorbance at 495 nm was measured by a microplate reader (ELx800; BioTek, Winooski, VT). The MTS assay absorbance value was converted to percentage and absorbance values of the controls at day 1 were denoted as 100% with respect to hRPE1 or hRPE3 cells. 
Cell-Cycle Analysis
RPE cells were harvested and fixed according to an established protocol. 20 Briefly, cells were enzymatically detached from the culture dishes using 0.05% trypsin and then mixed with complete culture medium containing 20% FCS. The cell pellet was collected after centrifugation, resuspended with SFM, and centrifuged for 7 minutes at 200g again. The cell pellet was then dislodged from the centrifuge tube by hitting the tube body gently and cells were resuspended in PBS by repeated up and down pipetting five times. The cells were then fixed with 70% ice-cold ethanol overnight. On the day of analysis, RPE cells were washed and resuspended in PBS. They were then incubated for 30 minutes with propidium iodide solution containing 20 μg/mL propidium iodide, 200 μg/mL DNase-free RNase A, and 0.1% (v/v) Triton X-100 before being analyzed by a flow cytometer (FACSCanto II; BD Biosciences, San Jose, CA). At least 18,000 events were collected for each sample. The obtained raw data were deconvoluted using a software package (FCS Express; De Novo Software, Los Angeles, CA). 
Western Blot Analysis
To obtain cell lysates, cells were washed twice with ice-cold PBS and disrupted in RIPA lysis buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing 2 mM sodium orthovanadate, phosphatase inhibitor cocktail (Merck, Darmstadt, Germany), and protease inhibitor cocktail (Roche, Basel, Switzerland). The cell lysates were centrifuged for 30 minutes at 16,100g at 4°C and the supernatant was collected. Proteins in cell lysates were resolved by SDS-PAGE and then transferred to PVDF membranes. Nonspecific binding sites were blocked according to the manufacturers' instructions or with nonfat-dried milk (Nestle, Switzerland), if not indicated by the manufacturers specifically. The membranes were probed with primary antibodies overnight at 4°C. The following primary antibodies were used to probe the bound proteins: anti-Erk (1:4000); anti-phosphorylated-Erk (1:4000); anti-p38 (1:1000); anti-phosphorylated-p38 (1:1000); anti-JNK (1:1000); anti-phosphorylated-JNK (1:2000); anti-Akt (1:1000); and anti-phosphorylated-Akt (1:1000; Cell Signaling, Danvers, MA) and anti-p53 (1:1000); and anti-GAPDH (1:10,000) antibodies (Santa Cruz, CA). Secondary HRP-conjugated antibodies (1:2000; Vector Laboratories, Burlingame, CA) specific to the primary antibodies were applied followed by an extensive washing procedure again. The targeted proteins were visualized with enhanced chemiluminescence detection kits (Amersham, UK). Semi-quantitative measurements of the Western blotting results were performed using lab analysis software (TotalLab TL120; TotalLab, Tyne and Wear NE1, UK). The expression levels of the proteins were normalized and values of the controls were denoted as 100% with respect to hRPE1 or hRPE3 cells. 
Phosphorylated-Akt Inhibitor Treatment
To deduce the importance of Akt phosphorylation in RPE cell proliferation, primary RPE cells were treated with a PI3 kinase (PI3K) inhibitor (LY294002; Cell Signaling). This PI3K inhibitor was known to suppress Akt from phosphorylation. 21 Cell proliferation rates were reflected by MTS assay. Briefly, RPE cells were seeded into 96-well plates and supplemented with culture medium containing 1% FCS. One day later, the culture medium was replaced with fresh medium containing various concentrations of LY294002 (1, 2, 5, and 10 μM). The control consisted of cells treated with no LY294002 but with vehicle (DMSO). Three days later, the cell proliferation rate was reflected by the MTS assay absorbance. The MTS assay absorbance value was converted to percentage and absorbance values of the controls were denoted as 100% with respect to hRPE1 or hRPE3 cells. 
The effect of LY294002 on cell cycle progression was also determined. RPE cells were treated with various concentrations of LY294002 (1, 2, 5, and 10 μM) for 3 days. The cell cycle distribution was determined using the method as described above. 
Statistical Analysis
Results were expressed as mean ± SEM from at least three independent experiments and were analyzed with ANOVA followed by the Bonferroni test (GraphPad Prism 5.03, San Diego, CA), with statistical significance set at P < 0.05. 
Results
ABL Suppressed RPE Cell Proliferation
When the hRPE1 (Fig. 1A) and the hRPE3 (Fig. 1B) cells were treated with ABL for 3 days, decrease in MTS assay absorbance was observed when the cells were treated with a higher concentration (90 μg/mL) of ABL. A lower ABL concentration (20 μg/mL) had no inhibitory effect (Figs. 1A, 1B). Photomicrographs revealed a decrease in cell count when the RPE cells were treated with ABL at a higher (90 μg/mL; Fig. 1E) but not a lower concentration (20 μg/mL; Fig. 1D) or the control (Fig. 1C). 
Figure 1. 
 
ABL suppressed cell proliferation for the two human RPE cell-lines. (A, B) Cell proliferation rate was reflected by a tetrazolium salts (MTS) assay. Treating hRPE1 (A) and hRPE3 (B) cells with ABL (90 μg/mL) for 3 days led to a reduction in assay absorbance. (CE) Representative photomicrographs indicated a reduction of hRPE3 cells when the cells were treated with a higher concentration of ABL (90 μg/mL) for 3 days. Mean ± SEM. n = 3–5; *P < 0.05, ***P < 0.001 versus control group; Scale bar, 200 μm.
Figure 1. 
 
ABL suppressed cell proliferation for the two human RPE cell-lines. (A, B) Cell proliferation rate was reflected by a tetrazolium salts (MTS) assay. Treating hRPE1 (A) and hRPE3 (B) cells with ABL (90 μg/mL) for 3 days led to a reduction in assay absorbance. (CE) Representative photomicrographs indicated a reduction of hRPE3 cells when the cells were treated with a higher concentration of ABL (90 μg/mL) for 3 days. Mean ± SEM. n = 3–5; *P < 0.05, ***P < 0.001 versus control group; Scale bar, 200 μm.
ABL Reduced S Phase Cell Population
The distribution of cell populations in various cell cycle phases were altered by ABL (Fig. 2). Decrease in S phase populations was detected in both primary RPE cell lines after treatment with ABL (90 μg/mL) for 3 days (Fig. 2B). ABL at a lower concentration (20 μg/mL) had no effect on S phase population. ABL also reduced the G0/G1 population (Fig. 2A) and increased the G2M population (Fig. 2C) of the hRPE3 cells. Such alterations in G0/G1 and G2M phases were not found in hRPE1 cells. Figures 2D to 2F showed the PI-Area plots with representative data from hRPE1 cells. No prominent sub-G1 population was observed. 
Figure 2. 
 
ABL led to reduction in S-phase populations for all RPE cell lines. (A, C) The effects of ABL on G0/G1 and G2M were mixed. No significant effect was detected for the G0/G1 (A) and G2M (C) phases on the hRPE1 cells (open bars). Application of ABL in hRPE3 cells (shaded bars) reduced the G0/G1 population (A) and increased the G2M population (C). (B) S-phase population decreased after the two primary RPE cells were treated with ABL (90 μg/mL) for 3 days. (DF) Representative flow cytometry plots for the cell cycle distribution of hRPE1 cell were shown. The amount of cells in S phase decreased with increasing ABL concentrations (arrows). Mean ± SEM; n = 3–5; *P < 0.05, **P < 0.01 versus control group.
Figure 2. 
 
ABL led to reduction in S-phase populations for all RPE cell lines. (A, C) The effects of ABL on G0/G1 and G2M were mixed. No significant effect was detected for the G0/G1 (A) and G2M (C) phases on the hRPE1 cells (open bars). Application of ABL in hRPE3 cells (shaded bars) reduced the G0/G1 population (A) and increased the G2M population (C). (B) S-phase population decreased after the two primary RPE cells were treated with ABL (90 μg/mL) for 3 days. (DF) Representative flow cytometry plots for the cell cycle distribution of hRPE1 cell were shown. The amount of cells in S phase decreased with increasing ABL concentrations (arrows). Mean ± SEM; n = 3–5; *P < 0.05, **P < 0.01 versus control group.
ABL-Induced Phosphorylated-Erk Expression and Reduced Phosphorylated-Akt Expression in Primary RPE Cells
To deduce the possible signaling pathways in which ABL prevented cells from entering the S phase, three classic mitogen-activated protein kinases (MAPKs), namely Erk, Jnk and p38, along with Akt, were investigated for their possible activation through phosphorylation. 
Phosphorylated-Erk (pErk) expressions were dramatically increased when the cells were treated with ABL (Fig. 3A). The amount of pErk was approximately 100% higher for the primary RPE cells than their corresponding control groups when ABL (90 μg/mL) was introduced. The lower ABL concentration (20 μg/mL) was found to be nonstimulatory. On the other hand, ABL did not alter the phosphorylation levels of Jnk (Fig. 3B) and p38 (Fig. 3C). 
Figure 3. 
 
ABL promoted phosphorylation of Erk but not for the Jnk and p38. (A) ABL at the higher concentration was found to promote phosphorylated-Erk (pErk) expression. (B, C) No significant effect was detected for total phosphorylated-Jnk (pJnk54 and pJnk46; [B]) and phosphorylated-p38 (pp38; [C]). Mean ± SEM; n = 3–5; *P < 0.05, ***P < 0.001 versus control group.
Figure 3. 
 
ABL promoted phosphorylation of Erk but not for the Jnk and p38. (A) ABL at the higher concentration was found to promote phosphorylated-Erk (pErk) expression. (B, C) No significant effect was detected for total phosphorylated-Jnk (pJnk54 and pJnk46; [B]) and phosphorylated-p38 (pp38; [C]). Mean ± SEM; n = 3–5; *P < 0.05, ***P < 0.001 versus control group.
When ABL (90 μg/mL) was applied, an approximately 50% reduction of phosphorylated-Akt (pAkt) was observed in hRPE1 and hRPE3 cells (Fig. 4A). A dose-dependent suppression in pAkt expression was suggested from the graph; however, no significant changes were observed when ABL was applied at the lower concentration (20 μg/mL). We also examined the expression level of p53 (Fig. 4B) which is one of the downstream regulators of Akt. Our results demonstrated that there was no statistically significant alteration in p53 expression after application of ABL to the two primary RPE cell-lines. 
Figure 4. 
 
Phosphorylation level of Akt (pAkt) was found to be reduced after primary RPE cells were treated with ABL. (A) Treating hRPE1 (open bars) and hRPE3 (shaded bars) cells with ABL (90 μg/mL) for 3 days led to reduction in pAkt expression in a significant manner. A dose-dependent inhibition was suggested. (B) On the other hand, ABL at all concentrations did not alter the p53 expression. Mean ± SEM; n = 5–7; ***P < 0.001 versus control group.
Figure 4. 
 
Phosphorylation level of Akt (pAkt) was found to be reduced after primary RPE cells were treated with ABL. (A) Treating hRPE1 (open bars) and hRPE3 (shaded bars) cells with ABL (90 μg/mL) for 3 days led to reduction in pAkt expression in a significant manner. A dose-dependent inhibition was suggested. (B) On the other hand, ABL at all concentrations did not alter the p53 expression. Mean ± SEM; n = 5–7; ***P < 0.001 versus control group.
Inhibiting Akt from Phosphorylation Suppressed RPE Cell Proliferation
After the primary RPE cells were treated with LY294002 for 3 days, a dose-dependent inhibition on MTS assay absorbance was observed (Fig. 5). This indicated a reduction in cell proliferation rate. LY294002 at concentrations of 5 and 10 μM was found to suppress hRPE1 (Fig. 5A) and hRPE3 (Fig. 5B) cell proliferation. In addition, the pAkt inhibitor was found to suppress S phase population of the RPE cells (Fig. 6). A dose-dependent inhibition on S phase populations were observed while the required concentrations to give a statistically significant inhibition were 2 μm and 1 μm for the hRPE1 (Fig. 6B) and hRPE3 cells (Fig. 6E), respectively. The G2M phase was found to be unaffected by the inhibitor (Figs. 6C, 6F) and changes were observed in the G0/G1 phase for the hRPE3 cells only (Fig. 6D). 
Figure 5. 
 
Phosphorylated-Akt inhibitor LY294002 suppressed RPE cell proliferation in a dose-dependent manner. (A, B) Primary RPE cell hRPE1 (open bars) and hRPE3 (shaded bars) were treated with LY294002 for 3 days and the degree of cell proliferation were reflected by a tetrazolium salts (MTS) assay. The amount of cells was found suppressed by LY294002 in a dose-dependent manner. Mean ± SEM. n = 3; **P < 0.01, ***P < 0.001 versus control group.
Figure 5. 
 
Phosphorylated-Akt inhibitor LY294002 suppressed RPE cell proliferation in a dose-dependent manner. (A, B) Primary RPE cell hRPE1 (open bars) and hRPE3 (shaded bars) were treated with LY294002 for 3 days and the degree of cell proliferation were reflected by a tetrazolium salts (MTS) assay. The amount of cells was found suppressed by LY294002 in a dose-dependent manner. Mean ± SEM. n = 3; **P < 0.01, ***P < 0.001 versus control group.
Figure 6. 
 
PI3 kinase inhibitor LY294002 suppressed S-phase population of the RPE cells. RPE cells were treated with LY294002 for 3 days and the cellular distributions in various cell cycle phases were investigated. (A, D) The G0/G1 phase was found unaffected for hRPE1 cells (A), while LY294002 promoted cell population in G0/G1 phase for hRPE3 cells (D). (B, E) Decreases in S-phase populations were observed for hRPE1 (B) and hRPE3 (E) cells in a dose-dependent manner. (C, F) The G2M phase was found unaffected by LY294002 for hRPE1 (C) and hRPE3 (F) cells. Mean ± SEM; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus control group.
Figure 6. 
 
PI3 kinase inhibitor LY294002 suppressed S-phase population of the RPE cells. RPE cells were treated with LY294002 for 3 days and the cellular distributions in various cell cycle phases were investigated. (A, D) The G0/G1 phase was found unaffected for hRPE1 cells (A), while LY294002 promoted cell population in G0/G1 phase for hRPE3 cells (D). (B, E) Decreases in S-phase populations were observed for hRPE1 (B) and hRPE3 (E) cells in a dose-dependent manner. (C, F) The G2M phase was found unaffected by LY294002 for hRPE1 (C) and hRPE3 (F) cells. Mean ± SEM; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus control group.
Discussion
In line with previous studies, we found that ABL suppressed RPE cell proliferation in a dose-dependent manner. 15,22 Previous studies indicated that such suppression did not result in cell death. 9,15,23 This finding was further confirmed by the absence of sub-G1 (apoptotic) population in our flow cytometry studies. Through analyzing the cell cycle patterns, we demonstrated that ABL specifically suppressed the amount of cells present in S phase. This suggested that cells were less able to transit into S phase from G1 phase when ABL was present. Alternatively, it could be argued that such a reduction in the S phase population was due to a change in the cell cycle dynamics whereby cells were facilitated to leave the S phase and enter the G2M phase. This is very unlikely, however, as such event would promote cell proliferation and ABL was shown to be antiproliferative. 
The MAPKs control different kinds of cellular events like cell proliferation, migration, adhesion, and cell death. 24 Currently, five distinct families of MAPKs have been identified, and Erk, Jnk, and p38 are the most studied MAPKs to date. 25 Erk is well-known to mediate cell proliferation and persistent activation of Erk 1/2 is required for the cells to complete G1 phase and enter into S phase. 26 While activation of p38 is largely linked to inflammatory response, p38 is also known to suppress cyclin D1 expression and therefore prevents cells from entering into S phase. 27 Similar to p38, Jnk responses mainly to cellular stress. In addition to the classic knowledge that Jnk can trigger apoptosis, Jnk has also been implicated in the regulation of G1/S phase transition. 28  
Akt, also known as protein kinase B, regulates diverse cellular events. These include cell growth, survival, and cell cycle progression. 29 Akt is known to positively regulate G1/S phase transition by downregulating glycogen synthase kinase (GSK) 3α and β expressions. Upregulation of Akt also activates MDM2, which subsequently suppresses the action of p53. 30 In contrast, downregulation of Akt can promote the action of forkhead box protein O1 (Foxo1) and therefore leads to cell cycle arrest or apoptosis. 31  
As both MAPKs and Akt are critically involved in cell proliferation, we examined these kinases for their activation status (phosphorylation). We found that ABL activated Erk. While ABL suppressed Akt from activation, the expression of its downstream effector, p53, remain unchanged. 
Classically, activation of the Erk signaling pathway is considered to be mitogenic with pErk promoting G1/S transition and regulating G2M progression. 26,32 Surprisingly, an increase in Erk 1/2 phosphorylation was detected after ABL was applied to the RPE cells. Another Thomsen-Friedenreich (TF) antigen binding lectin, Arachis hypogaea , was known to promote Erk phosphorylation through activation of c-met. 33 At this moment, we are not sure whether such an increase has any contribution in the antiproliferative effect of ABL. Cell death was observed when we treated our RPE cells with ABL together with a phosphorylated-Erk inhibitor (result not shown). Thus, the increase in pErk could be a feedback signal to maintain RPE cell survival and not related to cell proliferation. 34 It could also be possible that the suppression in Akt phosphorylation simply overrides the mitogenic effect of the Erk pathways. 
Since the level of pAkt was found to be decreased for the primary hRPE cells after ABL treatment, we suggested that the potency of ABL on inhibiting cell proliferation was in part determined by the level in which pAkt could be suppressed. The evidence that supported such idea was the finding that pAkt expression could regulate RPE cell proliferation. Through using a pAkt inhibitor LY294002, primary RPE cell proliferation was found to be suppressed in a dose-dependent manner. We also compared the potency of ABL and LY294002 in terms of their inhibitory effects on pAkt expression. We found that ABL at 90 μg/mL gave equivalent pAkt suppression when the primary RPE cells were treated with 2 to 5 μM of LY294002 (result not shown). Furthermore, through flow cytometry analysis, we found that S phase populations of the RPE cells were suppressed by the LY294002 inhibitor. Therefore, the antiproliferative effect of ABL could be at least partially attributed to the suppression of pAkt expression. It is known that inhibiting Akt from activation could lead to the loss of inhibitory effect on GSK3α/β and ultimately causes a decrease in cyclin D production. The action of cyclin-dependent kinase inhibitor (p27Kip1) and mammalian target of rapamycin complex 1 (mTORC1) are also found to be affected by the phosphorylation status of Akt. 29 As Cyclin D, p27Kip1 and mTOR complex 1 are known to regulate cell proliferation, further research could be done to investigate how ABL affect the actions of these molecules. 
On the other hand, we cannot exclude the possibility that an increase in pErk expression may have a role in limiting cell proliferation. Such hypothesis is not unprecedented as it is documented that elevated pErk level can lead to cell cycle arrest through induction of p21Waf1/cip1, which is a potent cyclin-dependent kinase inhibitor. 35 We are now investigating the roles of pErk in the ABL treated RPE cells. 
In summary, our results suggest that RPE cell proliferation is suppressed by the reduction of pAkt. Further, we hypothesize that the lack of cytotoxicity of ABL could be a result of a balance between pErk and pAkt expressions (Fig. 7). 
Figure 7. 
 
Proposed signaling pathways through which ABL affects cell proliferation and viability. The arrest of G1/S phase transition could be mediated by an increase of GSKα/β when the pAkt expression was suppressed. The noncytotoxic nature of ABL could be a result of a balance between survival signals mediated by pErk and death signals from Akt. Arrow: stimulatory event. Line with blunt end: inhibitory event. Solid line: our experimental finding. Dashed line: proposed signaling event.
Figure 7. 
 
Proposed signaling pathways through which ABL affects cell proliferation and viability. The arrest of G1/S phase transition could be mediated by an increase of GSKα/β when the pAkt expression was suppressed. The noncytotoxic nature of ABL could be a result of a balance between survival signals mediated by pErk and death signals from Akt. Arrow: stimulatory event. Line with blunt end: inhibitory event. Solid line: our experimental finding. Dashed line: proposed signaling event.
In the clinical setting, we propose that the ABL may be delivered by intravitreal injection or through the infusion fluid during vitrectomy surgery for retinal detachment. We acknowledge that oral administration would be the most convenient way to deliver the potential therapeutic agent. It has been reported that intact peanut lectin (PNA) could enter the bloodstream through oral intake. 36 Further, intake of some lectins in high quantities is known to have deleterious effects. 3739 To the best of our knowledge, there is very limited knowledge regarding the pharmacological kinetics and dynamics of ABL regardless of the routes of administration. These would be very interesting and important areas for further investigations. 
Conclusion
In conclusion, we showed that ABL may be a potent antiproliferative agent by preventing human RPE cells from entering the S phase of the cell cycle. ABL stimulated expression of pErk. However, ABL also suppressed Akt from phosphorylation. The inhibition in cell proliferation could be explained by the hypophosphorylation of Akt, which prevented RPE cells from staying at the S phase. Further, we found that the level of p53 was not significantly altered by ABL. This suggested that ABL-arrested cell cycle progression was independent of p53 activation. 
Acknowledgments
The authors thank David Wong for his comments on the research design and also the manuscript. The authors thank the Faculty Core Facility of the Li Ka Shing Faculty of Medicine, The University of Hong Kong, for provision of the flow cytometer. 
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Footnotes
 Supported by the University Development Fund and the Seed Funding for Basic Research from The University of Hong Kong.
Footnotes
 Disclosure: Y.-H. Cheung, None; C.M. Sheridan, None; A.C.Y. Lo, None; W.W. Lai, None
Figure 1. 
 
ABL suppressed cell proliferation for the two human RPE cell-lines. (A, B) Cell proliferation rate was reflected by a tetrazolium salts (MTS) assay. Treating hRPE1 (A) and hRPE3 (B) cells with ABL (90 μg/mL) for 3 days led to a reduction in assay absorbance. (CE) Representative photomicrographs indicated a reduction of hRPE3 cells when the cells were treated with a higher concentration of ABL (90 μg/mL) for 3 days. Mean ± SEM. n = 3–5; *P < 0.05, ***P < 0.001 versus control group; Scale bar, 200 μm.
Figure 1. 
 
ABL suppressed cell proliferation for the two human RPE cell-lines. (A, B) Cell proliferation rate was reflected by a tetrazolium salts (MTS) assay. Treating hRPE1 (A) and hRPE3 (B) cells with ABL (90 μg/mL) for 3 days led to a reduction in assay absorbance. (CE) Representative photomicrographs indicated a reduction of hRPE3 cells when the cells were treated with a higher concentration of ABL (90 μg/mL) for 3 days. Mean ± SEM. n = 3–5; *P < 0.05, ***P < 0.001 versus control group; Scale bar, 200 μm.
Figure 2. 
 
ABL led to reduction in S-phase populations for all RPE cell lines. (A, C) The effects of ABL on G0/G1 and G2M were mixed. No significant effect was detected for the G0/G1 (A) and G2M (C) phases on the hRPE1 cells (open bars). Application of ABL in hRPE3 cells (shaded bars) reduced the G0/G1 population (A) and increased the G2M population (C). (B) S-phase population decreased after the two primary RPE cells were treated with ABL (90 μg/mL) for 3 days. (DF) Representative flow cytometry plots for the cell cycle distribution of hRPE1 cell were shown. The amount of cells in S phase decreased with increasing ABL concentrations (arrows). Mean ± SEM; n = 3–5; *P < 0.05, **P < 0.01 versus control group.
Figure 2. 
 
ABL led to reduction in S-phase populations for all RPE cell lines. (A, C) The effects of ABL on G0/G1 and G2M were mixed. No significant effect was detected for the G0/G1 (A) and G2M (C) phases on the hRPE1 cells (open bars). Application of ABL in hRPE3 cells (shaded bars) reduced the G0/G1 population (A) and increased the G2M population (C). (B) S-phase population decreased after the two primary RPE cells were treated with ABL (90 μg/mL) for 3 days. (DF) Representative flow cytometry plots for the cell cycle distribution of hRPE1 cell were shown. The amount of cells in S phase decreased with increasing ABL concentrations (arrows). Mean ± SEM; n = 3–5; *P < 0.05, **P < 0.01 versus control group.
Figure 3. 
 
ABL promoted phosphorylation of Erk but not for the Jnk and p38. (A) ABL at the higher concentration was found to promote phosphorylated-Erk (pErk) expression. (B, C) No significant effect was detected for total phosphorylated-Jnk (pJnk54 and pJnk46; [B]) and phosphorylated-p38 (pp38; [C]). Mean ± SEM; n = 3–5; *P < 0.05, ***P < 0.001 versus control group.
Figure 3. 
 
ABL promoted phosphorylation of Erk but not for the Jnk and p38. (A) ABL at the higher concentration was found to promote phosphorylated-Erk (pErk) expression. (B, C) No significant effect was detected for total phosphorylated-Jnk (pJnk54 and pJnk46; [B]) and phosphorylated-p38 (pp38; [C]). Mean ± SEM; n = 3–5; *P < 0.05, ***P < 0.001 versus control group.
Figure 4. 
 
Phosphorylation level of Akt (pAkt) was found to be reduced after primary RPE cells were treated with ABL. (A) Treating hRPE1 (open bars) and hRPE3 (shaded bars) cells with ABL (90 μg/mL) for 3 days led to reduction in pAkt expression in a significant manner. A dose-dependent inhibition was suggested. (B) On the other hand, ABL at all concentrations did not alter the p53 expression. Mean ± SEM; n = 5–7; ***P < 0.001 versus control group.
Figure 4. 
 
Phosphorylation level of Akt (pAkt) was found to be reduced after primary RPE cells were treated with ABL. (A) Treating hRPE1 (open bars) and hRPE3 (shaded bars) cells with ABL (90 μg/mL) for 3 days led to reduction in pAkt expression in a significant manner. A dose-dependent inhibition was suggested. (B) On the other hand, ABL at all concentrations did not alter the p53 expression. Mean ± SEM; n = 5–7; ***P < 0.001 versus control group.
Figure 5. 
 
Phosphorylated-Akt inhibitor LY294002 suppressed RPE cell proliferation in a dose-dependent manner. (A, B) Primary RPE cell hRPE1 (open bars) and hRPE3 (shaded bars) were treated with LY294002 for 3 days and the degree of cell proliferation were reflected by a tetrazolium salts (MTS) assay. The amount of cells was found suppressed by LY294002 in a dose-dependent manner. Mean ± SEM. n = 3; **P < 0.01, ***P < 0.001 versus control group.
Figure 5. 
 
Phosphorylated-Akt inhibitor LY294002 suppressed RPE cell proliferation in a dose-dependent manner. (A, B) Primary RPE cell hRPE1 (open bars) and hRPE3 (shaded bars) were treated with LY294002 for 3 days and the degree of cell proliferation were reflected by a tetrazolium salts (MTS) assay. The amount of cells was found suppressed by LY294002 in a dose-dependent manner. Mean ± SEM. n = 3; **P < 0.01, ***P < 0.001 versus control group.
Figure 6. 
 
PI3 kinase inhibitor LY294002 suppressed S-phase population of the RPE cells. RPE cells were treated with LY294002 for 3 days and the cellular distributions in various cell cycle phases were investigated. (A, D) The G0/G1 phase was found unaffected for hRPE1 cells (A), while LY294002 promoted cell population in G0/G1 phase for hRPE3 cells (D). (B, E) Decreases in S-phase populations were observed for hRPE1 (B) and hRPE3 (E) cells in a dose-dependent manner. (C, F) The G2M phase was found unaffected by LY294002 for hRPE1 (C) and hRPE3 (F) cells. Mean ± SEM; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus control group.
Figure 6. 
 
PI3 kinase inhibitor LY294002 suppressed S-phase population of the RPE cells. RPE cells were treated with LY294002 for 3 days and the cellular distributions in various cell cycle phases were investigated. (A, D) The G0/G1 phase was found unaffected for hRPE1 cells (A), while LY294002 promoted cell population in G0/G1 phase for hRPE3 cells (D). (B, E) Decreases in S-phase populations were observed for hRPE1 (B) and hRPE3 (E) cells in a dose-dependent manner. (C, F) The G2M phase was found unaffected by LY294002 for hRPE1 (C) and hRPE3 (F) cells. Mean ± SEM; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus control group.
Figure 7. 
 
Proposed signaling pathways through which ABL affects cell proliferation and viability. The arrest of G1/S phase transition could be mediated by an increase of GSKα/β when the pAkt expression was suppressed. The noncytotoxic nature of ABL could be a result of a balance between survival signals mediated by pErk and death signals from Akt. Arrow: stimulatory event. Line with blunt end: inhibitory event. Solid line: our experimental finding. Dashed line: proposed signaling event.
Figure 7. 
 
Proposed signaling pathways through which ABL affects cell proliferation and viability. The arrest of G1/S phase transition could be mediated by an increase of GSKα/β when the pAkt expression was suppressed. The noncytotoxic nature of ABL could be a result of a balance between survival signals mediated by pErk and death signals from Akt. Arrow: stimulatory event. Line with blunt end: inhibitory event. Solid line: our experimental finding. Dashed line: proposed signaling event.
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