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Purple Sweet Potato Anthocyanin Inhibits the Proliferation of Human Retinal Pigment Epithelial Cell by Blocking Cell Cycle and Inducing Apoptosis

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Purple Sweet Potato Anthocyanin (PSPA), a class of naturally occurring anthocyanins derived from purple sweet potato storage roots, possesses unique color and multiple bioactivities. This study investigated the anti-proliferative effect of PSPA in human Retinal Pigment Epithelial (RPE) cells, the proliferation of which accounts for Proliferative Vitreoretinopathy (PVR). Blueberry Anthocyanin (BBA) was used as a contrast. PSPA and BBA inhibited RPE proliferation time-and dose-dependently through blocking the cell cycle in G0/G1 phase and inducing apoptosis via ROS accumulation, DNA damage and caspase 3/7 activation. Meanwhile, PSPA showed stronger potential than BBA in inhibiting RPE growth. Hence, we highlighted the importance of dietary supplementation of anthocyanins in PVR prevention and the application of PSPA in health industry.
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Advance Journal of Food Science and Technology 11(8): 561-569, 2016
ISSN: 2042-4868; e-ISSN: 2042-4876
© 2016 Maxwell Scientific Publication Corp.
Submitted: August 5, 2015 Accepted: September 3, 2015 Published: July 15, 2016
Corresponding Author:
Xiaoling Lu, Key Laboratory of Food Nutrition and Safety of the Ministry of Education, College of
Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th
Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, P.R.
China, Tel.: 86-22-60912343; Fax: 86-22-60912343
This work is licensed under a Creative Commons Attribution 4.0 International License (URL:
Research Article
Purple Sweet Potato Anthocyanin Inhibits the Proliferation of Human Retinal Pigment
Epithelial Cell by Blocking Cell Cycle and Inducing Apoptosis
Xiaoling Lu, MinSun, Lei Hao, Tao Wu, Huanjiao Zhao and Chao Wang
Key Laboratory of Food Nutrition and Safety of the Ministry of Education, College of Food Engineering
and Biotechnology, Tianjin University of Science and Technology, Tianjin, China
Abstract: Purple Sweet Potato Anthocyanin (PSPA), a class of naturally occurring anthocyanins derived from
purple sweet potato storage roots, possesses unique color and multiple bioactivities. This study investigated the anti-
proliferative effect of PSPA in human Retinal Pigment Epithelial (RPE) cells, the proliferation of which accounts for
Proliferative Vitreoretinopathy (PVR). Blueberry Anthocyanin (BBA) was used as a contrast. PSPA and BBA
inhibited RPE proliferation time-and dose-dependently through blocking the cell cycle in G0/G1 phase and inducing
apoptosis via ROS accumulation, DNA damage and caspase 3/7 activation. Meanwhile, PSPA showed stronger
potential than BBA in inhibiting RPE growth. Hence, we highlighted the importance of dietary supplementation of
anthocyanins in PVR prevention and the application of PSPA in health industry.
Keywords: Apoptosis, cell cycle, proliferation, purple sweet potato anthocyanin, retinal pigment epithelial
The Retinal Pigment Epithelium (RPE), interposed
between the neural retina and the choroidal blood, is a
monolayer responsible for maintaining the health of the
retina by providing structural and nutritional support
(Adijanto and Philp, 2014; Sparrrow et al., 2010;
Strauss, 2009). In vivo, highly differentiated RPE cells
have a limited proliferation capacity (Hecquet et al.,
2002). However, several pathological insults may
induce RPE cells to dedifferentiate and proliferate,
which accounts for Proliferative Vitreoretinopathy
(PVR), a major cause of failure in retinal detachment
surgery (Garweg et al., 2013). The inhibition of the
proliferation of RPE cells may be helpful in preventing
the recurrence of retinal detachment (Hou et al., 2013).
In vitro, cultured RPE cells can be used to investigate
PVR related pathogenesis and the inhibition of RPE cell
proliferation has been achieved by using statins
(Wu et al., 2011), daunorubicin (Wang et al., 2002) and
hypericin (Zhou et al., 2007), indicating their potential
to prevent PVR. However, the most economic and
convenient way to gain the potential to prevent PVR is
from food rather than medicine. Therefore, it’s more
practical and meaningful to identify protective
components with no side effects in daily diet.
Polyphenolic compounds are the most abundant
antioxidants people can get from dietary and they are
known to be protective to retina (Hanneken et al.,
2006). Meanwhile, anthocyanins are the most available
flavonoid constituents of fruits and vegetables. The
daily intake of anthocyanins is estimated to be 9-fold
higher than that of other dietary flavonoids (Wang and
Stoner, 2008). Anthocyanins have been reported to
inhibit the proliferation of human umbilical vein
endothelial cells and human retinal microvascular
endothelial cells (Matsunaga et al., 2010a, 2010b;
Tanaka et al., 2012), but their effect on RPE cells
hasn’t been proven. Besides, the anthocyanins in these
researches are limited to nonacylated anthocyanins.
Although nonacylated anthocyanins contributed more
than acylated anthocyanins in food intake (Wu et al.,
2006), acylated anthocyanins are more promising in
food processing industry for their better stability
(Netzel et al., 2007). Purple sweet potato is a good
source of acylated anthocyanins. Mainly constituted by
cyanidinacylglucosides and peonidinacylglucosides,
Purple Sweet Potato anthocyanin (PSPA) possesses
multiple bioactivities including attenuating the
proliferation of hepatic stellate cells (Choi et al., 2011)
and inhibiting Sarcoma S180 cell growth in ICR mice
(Zhao et al., 2013). Furthermore, PSPA can be
absorbed directly into rat and human in intact acylated
forms (Harada et al., 2004; Oki et al., 2006; Suda et al.,
2002) and anthocyanins can pass the blood-brain barrier
and blood-retinal barrier and accumulate in eyes, as
observed in vivo (Kalt et al., 2008; Matsumoto et al.,
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
In view of all these considerations, the purpose of
the present study was to evaluate the anti-proliferative
effects of PSPA in RPE cells. Blueberry Anthocyanin
(BBA), well-known to promote vision health (Wang
et al., 2015) and mainly constituted by nonacylated
anthocyanins, was used as a contrast in this study. This
investigation was aimed at providing a new source for
preventing PVR from dietary and increasing the
application of acylated anthocyanins from purple sweet
potato in health industry.
Materials: The human retinal pigment epithelial (RPE)
cell line (no. D407) was purchased from the Animal
Experiment Center of Sun Yat-sen University
(Guangzhou, China). D407 is a spontaneous
immortalized cell line retaining many of the metabolic
and morphologic characteristics of RPE cells in vivo
(Davis et al., 1995) and the same cell line was also used
in other research conducted by Xu et al. (2010). PSPA
used in the study were supplied by Huludao Maohua
Biology Co., Ltd. (Liaoning, China), the major
components of PSPA by HPLC-MS analysis are
cyanidinacylglucosides and peonidinacylglucosides
(Sun et al., 2015). BBA were supplied by Tianjin
Jianfeng Natural Product R and D Co., Ltd (Tianjin,
China), the major components of BBA are cyanidin and
petunidin glucosides (Peng et al., 2012). Dulbecco’s
modified Eagle’s Medium (DMEM), penicillin,
streptomycin, 0.5 % (vol/vol) trypsin/EDTA and Fetal
Bovine Serum (FBS) were purchased from Gibco Life
Technologies (Grand Island, NY, USA).3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide (MTT), 2’,7’-dichlorofluorescin diacetate
(DCFDA), Hoechst 33342 (HOE) and Propidiumiodide
(PI) were bought from Sigma-Aldrich, Inc. (St. Louis,
MO, USA). Muse
count and viability assay kit, cell
cycle kit, Ki67 proliferation kit, Annexin and dead
cell kit, oxidative stress kit, multicolor DNA damage kit
and Caspase-3/7 kit were purchased from Merck
Millipore (Billerica, MA, USA).
Cell culture and treatment: The RPE cells were
grown in whole culture medium, namely, DMEM with
10 FBS and a 1% antibiotic mixture of penicillin (100
U/mL) and streptomycin (100 mg/mL). Cells were
incubated at 37°C under a humidified 5% CO
atmosphere. Before treating with anthocyanins, the cells
were incubated in FBS-free DMEM medium for 1 day.
PSPA and BBA were dissolved in DMEM at a
concentration of 125 µg/mL
(Cyanidin 3-O-glucoside
equivalent) as a stock solution and stored at -20°C.
Before all experiments, the stock solution was sterilized
by processing through a 0.1 µm filter and then it was
diluted with DMEM to certain concentrations.
Evaluation of proliferation: RPE cells were seeded in
96-well plates at a concentration of 2×10
allowed to attach for 1 day. The cells were then
incubated in FBS-free DMEM medium for another day
followed by anthocyanins treatments for 1, 2 and 3 days
at concentrations ranging from 6.25 to 18.75 µg/mL.
The control groups were processed exactly the same,
just with 0 µg/mL anthocyanins. Then MTT assay (Wu
et al., 2009) was used to detect the Optical Density
(OD) values of each group and the proliferation was
calculated as % of the control group in each day.
Assay of cell viability: RPE cells were seeded in 6-
well plates at a concentration of 5×10
allowed to attach for 1 day. The cells were then
incubated in FBS-free DMEM medium for another day
followed by 12.5 µg/mL anthocyanins treatments for 1,
2 and 3 days. (The same process was used in the
following measurements, except where noted.) Then the
cells were digested and collected. The viabilities were
performed according to the count and viability assay kit
user’s manual, using Muse
Cell Analyser (Merck-
Millipore, Germany), 2, 000 cells were acquired for
each sample. The viable cells and total cells were each
counted and viability was expressed as a percentage of
the viable cells.
Assessment of apoptosis and necrosis: Morphological
changes in cells treated with PSPA and BBA were
assessed by double staining with HOE and PI. Having
been incubated on cover slips with anthocyanins for 2
d, the cells were washed twice with PBS and then 0.5
mL HOE (10 µg/mL) was added to each slip and
incubated for 10 min at 37°C, followed by another 10-
min incubation with PI (10 µg/mL) in total darkness.
Then the cells were washed twice with PBS and
observed by fluorescence microscopy (Olympus
CKX41, Japan).
Annexin and dead cell kit was also used
to measure the percentages of apoptosis and necrosis
after 12.5 µg/mL
anthocyanins treatment for 1, 2 and 3
days according to the user’s manual.
Analysis of cell cycle: After being treated with 12.5
µg/mL anthocyaninsfor 1 and 2 days, RPE cells were
collected to analyze cell cycle distribution using
Muse™ cell cycle kit. 20, 000 cells were recorded for
each sample. The results were analyzed using Modfit
3.2 (Verity Software House, USA).
Detection of Ki67 expression: RPE cells treated with
12.5 µg/mL anthocyaninsfor 1, 2 and 3 days were
conducted the assay of Ki67 according to the
Ki67 proliferation kit user’s guide with the
Cell Analyser.
Observation of ROS generation: The generation of
Reactive Oxygen Species (ROS) in the cells with
anthocyanins treatment for 2 days was observed by
fluorescence microscopy using DCFDA assay (Lu
et al., 2011). The production of ROS was also evaluated
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
by Muse
oxidative stress kit after incubating with
anthocyanins for 1, 2 and 3 days as described in user’s
Determination of DNA damage: Treated with 12.5
µg/mL PSPA and BBA for 1, 2 and 3 days, DNA
damage of RPE cells in each group was determined by
multicolor DNA damage kit at the end of each day.
Examination of caspase-3/7 expression: The
activation of Caspase-3/7 in RPE cells treated with
anthocyanins was examined by Muse™ Caspase-3/7
kit, utilizing a novel Muse™ Caspase-3/7 reagent
NucView™ for the detection.
Statistical analysis: All experiments were performed in
triplicate; results were given in means ± standard
deviation. One-way ANOVA with Duncan’s multiple
comparison tests were performed with SPSS software,
version 18.0 (SPSS Inc., Chicago, IL, USA). Results
were considered statistically significant at p<0.05.
Inhibitory effects of anthocyanins on RPE cell
proliferation and viability: The inhibition in RPE cell
proliferation induced by anthocyanins was assayed
following treatment with different doses of PSPA and
BBA for 1~3 days (Fig. 1A). Both anthocyanins
exhibited inhibitory effects in the dose-and time-
dependent manner, as observed at each time point
analyzed. PSPA showed an obviously stronger activity
than BBA at the same concentrations (cyanidin 3-O-
glucosideequivalent). Anthocyanins at the concentration
of 12.5 µg mL had significantly better inhibitory effect
than that of 6.25 µg/mL, but there were no very
dramatic differences between the groups of 12.5 and
18.25 µg/mL. Thus, 12.5 µg/mL anthocyanins were
selected for the following study.
When given the individual cell rather than cell
population, viabilities of RPE cells treated with 12.5
µg/mL PSPA and BBA were detected by counting the
numbers of living and dead cells (Fig. 1B). The control
group maintained 84.73% living cells after 3-day
incubation, while the viabilities of PSPA and BBA
groups decreased marvelously during the treatment.
Notably, the preserved percentages of viability (Fig.
1B) were higher than that of proliferation (Fig. 1A) at
the end of each day’s treatment with anthocyanins,
which suggested that the loss of cell viability partially
accounted for the decrease in proliferation treated by
Effects of anthocyanins on RPE cell apoptosis and
necrosis: Double staining with Hoechst 33342 and PI
was used to observe the morphological changes in cells
treated with anthocyanins for 2-day (Fig. 2A).
Anthocyanins treated cells showed more early apoptotic
cells (intensive bright-blue fluorescence) and late
apoptotic cells (blue-violet fluorescence). BBA
treatment resulted in more apoptotic cells than PSPA
treatment, showing the typical apoptosis-like
morphological changes like chromatin condensation
and fragmentation (Fig. 2A). The percentages of
apoptosis and necrosis after 12.5 µg/mL
treatment for 1, 2 and 3 days were also measured by
Annexinand dead cell kit (Fig. 2B). Numbers of late
apoptotic and dead cells increased sharply after PSPA
and BBA treatment for 2 days. BBA had better
apoptosis-inducing effect than PSPA, while PSPA led
to more cell death in all at the end of third day (47.76%
vs 38.98%).
(a) (b)
Fig. 1: PSPA and BBA inhibited RPE cell proliferation and viability; (a): Time-dependent and dose-dependent effect of PSPA
and BBA on cell proliferation assayed by MTT; (b): time course of 12.5 µg/mL PSPA and BBA inhibition in cell viability
determined by count and viability assay kit. The means of the values marked with different lowercase letters are
significantly different (p<0.05) relative to others in each day of incubation
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
Fig. 2: PSPA and BBA induced apoptosis and necrosis in RPE cells; (A): Fluorescence micrographs stained with Hoechst 33342
and PI of RPE cells treated with 12.5 µg/mL PSPA and BBA for 2-day. Scale bar: 25 µm. * intensive bright-blue
fluorescence, early apoptotic cells;blue-violet fluorescence, late apoptotic cells;→chromatin condensation; and ▲
fragmentation; (B): apoptosis of RPE cell assessed by Annex in and dead cell kit. The means of the values marked
with different lowercase letters are significantly different (p<0.05) relative to others in each day of incubation
Fig. 3: PSPA and BBA induced cell cycle arrest in RPE cells. (A, B) Cell cycle distribution of RPE cells treated with PSPA and
BBA analyzed by cell cycle kit; (C): percentages of Ki67-positive cells treated with PSPA and BBA for 1~3 days, The
means of the values marked with different lowercase letters are significantly different (p<0.05) relative to others in each
day of incubation
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
Effects of anthocyanins on RPE cell cycle and Ki67
expression: Cell cycle analysis revealed the RPE cell
cycle distribution after anthocyanins treatment (Fig. 3A
and B). PSPA and BBA incubation both resulted in
striking increases of G1/G0 stage cells whereas the
percentages of G2/M phase decreased significantly at
the end of the first day. Similar changes accompanied
by declines in the relative amount of S-phase cells were
recognized on the second day. Altogether, anthocyains
blocked PRE cells in G0/G1 phase and led to the cell
cycle arrest. And it was remarkable that an obvious sub-
G1 phase was detected after 2-day treatment by BBA,
which indicating the noteworthy apoptosis in BBA
group. This finding was in line with the results in HOE-
PI staining (Fig. 2A) and Annexin and dead analysis
(Fig. 2B).
Ki67 is a prototypic cell cycle-related nuclear
protein, expressed by proliferating cells in all phases of
the active cell cycle (G1, S, G2, M phases), but it is
absent in the resting G0 phase (Scholzen and Gerdes,
2000). In Fig. 3C, anthocyanins treatment diminished
the percentages of Ki67+ cells observably in each day,
which meant fewer cells were undergoing mitosis after
incubating with anthocyanins. And PSPA exhibited a
more intense effect than BBA in giving rise to G0 phase
cells. With the extension of incubation time, an evident
decline of Ki67+ percentages in the control group was
also found. Ki67 assay together with cell cycle analysis
revealed that anthocyanins induced a decrease of
continuously cycling cells and a blockage in G0/G1
phase, contributing to cell proliferation inhibition.
Effects of anthocyanins on ROS generation in RPE
cells: Anthocyanins induced ROS generation in RPE
cells could be seen in fluorescence microscopical
photos (Fig. 4A). More green fluorescence stained cells
were observed in anthocyanins treated groups. The data
measured by Muse oxidative stress kit (Fig. 4B) also
proved that the production of ROS was significantly
(p<0.05) increased by anthocyanins treatment,
especially from the second day of incubation. In
addition, PSPA led to a larger augmentation of the ROS
generation than BBA did on the third day (44.31% vs
34.38%, p<0.05). Notably, the control group also
showed a sharp rise in ROS generation on the third day.
Effects of anthocyanins on DNA damage in RPE
cells: Ataxia-Telangiectasia Mutated kinase (ATM) is a
DNA damage-inducible protein kinase (Tichy et al.,
2010) and once activated, ATM phosphorylates a
number of downstream factors, including histone
H2A.X (Burma et al., 2001). The activation of ATM
was easily detected in anthocyanins treatment in time
course of incubation, while phospho-H2A.X didn’t
change a lot (Fig. 5). And there were significant
differences between PSPA and BBA treatment,
resulting in 17.6% p-ATM in PSPA group and 9.57% in
BBA group on the third day.
Effects of anthocyanins on the expression of caspase
3/7: The effect of anthocyanins on the expression of
caspase 3/7 was demonstrated in Fig. 6. Increases of
caspase 3/7 activation in early apoptotic RPE cells were
only observed when cells were treated with
anthocyanins for 3 days, with percentages less than 3%
in each group. While the expression of caspase 3/7 in
late apoptotic or dead cell elevated prominently by
Fig. 4: PSPA and BBA induced ROS generation; (A): Fluorescence micrographs stained with DCFDA of RPE cells treated with
12.5 µg/mL PSPA and BBA for 1 and 2-day. Scale bar: 25 µm; (B): ROS generation after 12.5 µg/mL PSPA and BBA
treatment detected by oxidative stress kit. The means of the values marked with different lowercase letters are
significantly different (p<0.05) relative to others in each day of incubation
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
Fig. 5: PSPA and BBA induced DNA damage. The distribution of phosphor-ATM, H2A.X and double-strand breaks measured
by multicolor DNA damage kit after 12.5 µg/mL PSPA and BBA treatment. The means of the values marked with
different lower case letters for each indicator are significantly different (p<0.05) relative to others in each day of
Fig. 6: PSPA and BBA induced caspase 3/7 activation. The caspase 3/7 activation in RPE cells treated by 12.5 µg/mL PSPA and
BBA was examined by Caspase-3/7 kit. The means of the values marked with different lowercase letters for each
indicator are significantly different (p<0.05) relative to others in each day of incubation
anthocyanins treatment, reaching the percentage of 30%
at the end of the second day. PSPA showed a distinct
stronger enhancement in caspase 3/7 activation than
Pure anthocyanins and anthocyanin-rich extracts
from fruits and vegetables have exhibited anti-
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
proliferative activity towards multiple cell types in vitro
(Choi et al., 2011; Matsunaga et al., 2010a; Shih et al.,
2005; Tanaka et al., 2012; Wang and Stoner, 2008;
Zhang et al., 2005). The non-toxic anthocyanins from
purple sweet potato and blueberry used in this study
demonstrated potent growth inhibitory properties in
RPE cells, the proliferation of which might cause PVR.
Both PSPA and BBA showed the time- and dose-
dependently anti-proliferation effect through cell cycle
arrest and induced cell apoptosis.
Notably, enhanced reductions in proliferation rate
(Fig. 1A) and Ki67 positive percentage (Fig. 3C), more
pronounced cell cycle arrest (Fig. 3A and B); ROS
generation (Fig. 4); DNA damage (Fig. 5) and Caspase
3/7 activation (Fig. 6) were found in PSPA group when
compared to BBA group at the same concentration.
And BBA treatment resulted in more remarkable early
apoptosis than PSPA (Fig. 2 and 3A). Since the
anthocyanins used in this study are compounds, it’s
difficult to put the discrepant effect between PSPA and
BBA treatment down to the structure differences of
their aglycone anthocyanidins. But the acylation of
aglycon in PSPA might responsible for the discrepancy
partly. Compared to BBA, cyanidindiacyl glucosides
and peonidindiacyl glucosides in PSPA have higher
molecular weights, which makes the PSPA group
received more anthocyanins than BBA group did when
the concentration was calculated as cyanid in 3-O-
glucoside equivalent. Furthermore, coffeoyl and
feruloyl in PSPA molecules might contribute to the
anti-proliferative effect as well, which could be
supported by the inhibitory effect of caffeic and ferulic
acid in cancer cells (Eitsuka et al., 2014; Rajendra
Prasad et al., 2011; Serafim et al., 2011).
As reported, anti-proliferation activity in RPE cells
is mainly regulated by inhibition in growth and
induction of apoptosis and many signaling pathways are
involved (Sawada et al., 2014; Sun and You, 2014; Wu
et al., 2011). Our present study tried to shed light on the
role of ROS mediated pathway in PSPA induced
growth inhibition and apoptosis. ROS have crucial roles
in normal physiological processes; either too low or too
high level of ROS could cause health problems (Brieger
et al., 2012). Anthocyanins are known to act as ROS
scavengers in many bioactivities, whereas they also
could lead to accumulations of ROS and subsequent
apoptosis in certain cells (Cvorovic et al., 2010; Feng
et al., 2007). The paradoxical effect of anthocyanins
might due to different cell redox state and the double-
edged sword property of ROS in determining cell fate
(Wang and Yi, 2008). Increased ROS generations were
observed in anthocyanins treated RPE cells, especially
from the second day of incubation (Fig. 4). Many
studies have demonstrated that ROS is responsible for
DNA damage (Dhillon et al., 2014; Lv et al., 2013),
while the data in our present work seemed that DNA
damage was partially independent on ROS production,
since more aggravated increases in DNA damage than
ROS generation were observed on the first day of
treatment (Fig. 5). Cleavage of the DNA backbone
during DNA base excision repair might account for
DNA strand break in this stage (Watt et al., 2007). And
anthocyanin might induce oxidative DNA cleavage, as
reported by Webb et al. (2008). Within DNA damage,
the activation of ATM was easily detected in
anthocyanins treated RPE cells while histone H2AX
phosphorylation and DNA double-strand breaks were
limited (Fig. 5). Besides, PSPA treatment led to
distinguished phospho-ATM than BBA, accompanied
by the severer cell cycle arrest in G0/G1 phase and
decrease in Ki67 positive cells. DNA damage and
repairmen in addition to ROS generation might
responsible for the cell cycle arrest and inhibition of
quiescent RPE cells transiting into cell cycle (Surova
and Zhivotovsky, 2013).
Although it’s been reported that necrosis, but not
apoptosis, is a major type of cell death in RPE cells in
response to oxidative stress (Hanus et al., 2013), we
drew a conclusion here that anthocyanins induced RPE
cell death mainly through caspase 3/7 mediated
apoptosis, when taken the results from cell
apoptosis/necrosis analysis (Fig. 2) and the outcome of
caspase 3/7 detection (Fig. 6) together. Caspase 3
activation has been demonstrated to be involved in RPE
cell apoptosis (Yang et al., 2007) and anthocyanins
induced cell death (Forbes-Hernandez et al., 2014),
which aid the reliability of our conclusion. Both ROS
and DNA damage could act as intracellular death
signals to trigger the intrinsic cell death pathway,and
then activate executioner caspases (caspase 3, 6, 7),
which finally induce cell apoptosis (Radogna et al.,
To conclude, anthocyanins from purple sweet
potato and blueberry inhibited RPE cell proliferation
through cell cycle arrest and the induction of apoptosis,
which involved in ROS generation, DNA damage and
caspase 3/7 activation. The findings from our study
have highlighted the important role PSPA can play in
inhibiting RPE cell proliferation, which is responsible
for the progression of PVR. Dietary supplementation
with fruits and vegetables rich in anthocyanins can be
an effective strategy for prevention of eye diseases like
PVR. And we believe that acylated anthocyanins from
purple sweet potato are worth to be popularized and
applied in food and health industry.
Adijanto, J. and N.J. Philp, 2014. Cultured primary
human fetal retinal pigment epithelium (hfRPE) as
a model for evaluating RPE metabolism. Exp. Eye
Res., 126: 77-84.
Brieger, K., S. Schiavone, F.J. Miller Jr. and K.H.
Krause, 2012. Reactive oxygen species: From
health to disease. Swiss Med. Wkly., 142: w13659.
Burma, S., B.P. Chen, M. Murphy, A. Kurimasa and
D.J. Chen, 2001. ATM phosphorylates histone
H2AX in response to DNA double-strand breaks. J.
Biol. Chem., 276(45): 42462-42467.
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
Choi, J.H., Y.P. Hwang, B.H. Park, C.Y. Choi, Y.C.
Chung and H.G. Jeong, 2011. Anthocyanins
isolated from the purple-fleshed sweet potato
attenuate the proliferation of hepatic stellate cells
by blocking the PDGF receptor. Environ. Toxicol.
Phar., 31(1): 212-219.
Cvorovic, J., F. Tramer, M. Granzotto, L. Candussio, G.
Decorti and S. Passamonti, 2010. Oxidative stress-
based cytotoxicity of delphinidin and cyanidin in
colon cancer cells. Arch. Biochem. Biophys.,
501(1): 151-157.
Davis, A.A., P.S. Bernstein, D. Bok, J. Turner, M.
Nachtigal and R.C. Hunt, 1995. A human retinal
pigment epithelial cell line that retains epithelial
characteristics after prolonged culture. Invest.
Ophth. Vis. Sci., 36(5): 955-964.
Dhillon, H., S. Chikara and K.M. Reindl, 2014.
Piperlongumine induces pancreatic cancer cell
death by enhancing reactive oxygen species and
DNA damage. Toxicol. Rep., 1: 309-318.
Eitsuka, T., N. Tatewaki, H. Nishida, T. Kurata, K.
Nakagawa and T. Miyazawa, 2014. Synergistic
inhibition of cancer cell proliferation with a
combination of delta-tocotrienol and ferulic acid.
Biochem. Bioph. Res. Co., 453(3): 606-611.
Feng, R., H.M. Ni, S.Y. Wang, I.L. Tourkova, M.R.
Shurin, H. Harada et al., 2007. Cyanidin-3-
rutinoside: A natural polyphenol antioxidant,
selectively kills leukemic cells by induction of
oxidative stress. J. Biol. Chem., 282(18):
Forbes-Hernandez, T.Y., F. Giampieri, M. Gasparrini,
L. Mazzoni, J.L. Quiles, J.M. Alvarez-Suarez
et al., 2014. The effects of bioactive compounds
from plant foods on mitochondrial function: A
focus on apoptotic mechanisms. Food Chem.
Toxicol., 68: 154-182
Garweg, J.G., C. Tappeiner and M. Halberstadt, 2013.
Pathophysiology of proliferative vitreoretinopathy
in retinal detachment. Surv. Ophthalmol., 58(4):
Hanneken, A., F.F. Lin, J. Johnson and P. Maher, 2006.
Flavonoids protect human retinal pigment
epithelial cells from oxidative-stress-induced death.
Invest. Ophth. Vis. Sci., 47(7): 3164-3177.
Hanus, J., H. Zhang, Z. Wang, Q. Liu, Q. Zhou and S.
Wang, 2013. Induction of necrotic cell death by
oxidative stress in retinal pigment epithelial cells.
Cell Death Dis., 4: e965.
Harada, K., M. Kano, T. Takayanagi, O. Yamakawa
and F. Ishikawa, 2004. Absorption of acylated
anthocyanins in rats and humans after ingesting an
extract of Ipomoea batatas purple sweet potato
tuber. Biosci. Biotech. Bioch., 68(7): 1500-1507.
Hecquet, C., G. Lefevre, M. Valtink, K. Engelmann and
F. Mascarelli, 2002. Activation and role of MAP
kinase-dependent pathways in retinal pigment
epithelial cells: ERK and RPE cell proliferation.
Invest. Ophth. Vis. Sci., 43(9): 3091-3098.
Hou, Q., J. Tang, Z. Wang, C. Wang, X. Chen, L. Hou
et al., 2013. Inhibitory effect of microRNA-34a on
retinal pigment epithelial cell proliferation and
migration. Invest. Ophth. Vis. Sci., 54(10):
Kalt, W., J.B. Blumberg, J.E. McDonald, M.R.
Vinqvist-Tymchuk, S.A.E. Fillmore, B.A. Graf
et al., 2008. Identification of anthocyanins in the
liver, eye and brain of blueberry-fed pigs. J. Agr.
Food Chem., 56(3): 705-712
Lu, X., J. Liu, X. Cao, X. Hou, X. Wang, C. Zhao et al.,
2011. Native low density lipoprotein induces
pancreatic beta cell apoptosis through generating
excess reactive oxygen species. Lipids Health Dis.,
10: 123.
Lv, L., L. Zheng, D. Dong, L. Xu, L. Yin, Y. Xu et al.,
2013. Dioscin, a natural steroid saponin, induces
apoptosis and DNA damage through reactive
oxygen species: A potential new drug for treatment
of glioblastoma multiforme. Food Chem. Toxicol.,
59: 657-669.
Matsumoto, H., Y. Nakamura, H. Iida, K. Ito and H.
Ohguro, 2006. Comparative assessment of
distribution of blackcurrant anthocyanins in rabbit
and rat ocular tissues. Exp. Eye Res., 83(2):
Matsunaga, N., Y. Chikaraishi, M. Shimazawa, S.
Yokota and H. Hara, 2010a. Vaccinium myrtillus
(Bilberry) extracts reduce angiogenesis In vitro and
In vivo. Evid-Based Compl. Alt., 7(1): 47-56.
Matsunaga, N., K. Tsuruma, M. Shimazawa, S. Yokota
and H. Hara, 2010b. Inhibitory actions of bilberry
anthocyanidins on angiogenesis. Phytother. Res.,
24(Suppl. 1): S42-S47.
Netzel, M., G. Netzel, D.R. Kammerer, A. Schieber, R.
Carle, L. Simons et al., 2007. Cancer cell
antiproliferation activity and metabolism of black
carrot anthocyanins. Innov. Food Sci. Emerg., 8(3):
Oki, T., I. Suda, N. Terahara, M. Sato and M.
Hatakeyama, 2006. Determination of acylated
anthocyanin in human urine after ingesting a
purple-fleshed sweet potato beverage with various
contents of anthocyanin by LC-ESI-MS/MS.
Biosci. Biotech. Bioch., 70(10): 2540-2543.
Peng, C., Y. Zuo, K.M. Kwan, Y. Liang, K.Y. Ma,
H.Y. Chan et al., 2012. Blueberry extract prolongs
lifespan of Drosophila melanogaster. Exp.
Gerontol., 47(2): 170-178.
Radogna, F., M. Dicato and M. Diederich, 2015.
Cancer-type-specific crosstalk between autophagy,
necroptosis and apoptosis as a pharmacological
target. Biochem. Pharmacol., 94(1): 1-11.
Rajendra Prasad, N., A. Karthikeyan, S. Karthikeyan
and B.V. Reddy, 2011. Inhibitory effect of caffeic
acid on cancer cell proliferation by oxidative
mechanism in human HT-1080 fibrosarcoma cell
line. Mol. Cell. Biochem., 349(1-2): 11-19.
Adv. J. Food Sci. Technol., 11(8): 561-569, 2016
Sawada, O., L. Perusek, H. Kohno, S.J. Howell, A.
Maeda, S. Matsuyama et al., 2014. All-trans-retinal
induces Bax activation via DNA damage to
mediate retinal cell apoptosis. Exp. Eye Res., 123:
Scholzen, T. and J. Gerdes, 2000. The Ki-67 protein:
From the known and the unknown. J. Cell.
Physiol., 182(3): 311-322.
Serafim, T.L., F.S. Carvalho, M.P.M. Marques, R.
Calheiros, T. Silva, J. Garrido et al., 2011.
Lipophilic caffeic and ferulic acid derivatives
presenting cytotoxicity against human breast
cancer cells. Chem. Res. Toxicol., 24(5): 763-774.
Shih, P.H., C.T. Yeh and G.C. Yen, 2005. Effects of
anthocyanidin on the inhibition of proliferation and
induction of apoptosis in human gastric
adenocarcinoma cells. Food Chem. Toxicol.,
43(10): 1557-1566.
Sparrrow, J.R., D. Hicks and C.P. Hamel, 2010. The
retinal pigment epithelium in health and disease.
Curr. Mol. Med., 11(1): 802-823.
Strauss, O., 2009. The role of retinal pigment
epithelium in visual functions. Ophthalmologe,
106(4): 299-304.
Suda, I., T. Oki, M. Masuda, Y. Nishiba, S. Furuta, K.
Matsugano et al., 2002. Direct absorption of
acylated anthocyanin in purple-fleshed sweet
potato into rats. J. Agr. Food Chem., 50(6):
Sun, M., X. Lu, L. Hao, T. Wu, H. Zhao and C. Wang,
2015. The influences of purple sweet potato
anthocyanin on the growth characteristics of
human retinal pigment epithelial cells. Food Nutr.
Res., 59: 27830-3402.
Sun, Y. and Z.P. You, 2014. Curcumin inhibits human
retinal pigment epithelial cell proliferation. Int. J.
Mol. Med., 34(4): 1013-1019.
Surova, O. and B. Zhivotovsky, 2013. Various modes
of cell death induced by DNA damage [Review].
Oncogene, 32(33): 3789-3797.
Tanaka, J., S. Nakamura, K. Tsuruma, M. Shimazawa,
H. Shimoda and H. Hara, 2012. Purple rice (Oryza
sativa L.) extract and its constituents inhibit
VEGF-induced angiogenesis. Phytother. Res.,
26(2): 214-222.
Tichy, A., J. Vavrova, J. Pejchal and M. Rezacova,
2010. Ataxia-telangiectasia mutated kinase (ATM)
as a central regulator of radiation-induced DNA
damage response. Acta Med., 53(1): 13-17.
Wang, J. and J. Yi, 2008. Cancer cell killing via ROS
To increase or decrease, that is the question.
Cancer Biol. Ther., 7(12): 1875-1884.
Wang, L.S. and G.D. Stoner, 2008. Anthocyanins and
their role in cancer prevention. Cancer Lett.,
269(2): 281-290.
Wang, Y.S., Y.N. Hui and P. Wiedemann, 2002. Role
of apoptosis in the cytotoxic effect mediated by
daunorubicin in cultured human retinal pigment
epithelial cells. J. Ocul. Pharmacol. Th., 18(4):
Wang, Y., D. Zhang, Y. Liu, D. Wang, J. Liu and B. Ji,
2015. The protective effects of berry-derived
anthocyanins against visible light-induced damage
in human retinal pigment epithelial cells. J. Sci.
Food Agr., 95(5): 936-944.
Watt, N.T., M.N. Routledge, C.P. Wild and N.M.
Hooper, 2007. Cellular prion protein protects
against reactive-oxygen-species-induced DNA
damage. Free Radical. Bio. Med., 43(6): 959-967.
Webb, M.R., K.M. Min and S.E. Ebeler, 2008.
Anthocyanin interactions with DNA: Intercalation,
topoisomerase I inhibition and oxidative reactions.
J. Food Biochem., 32(5): 576-596.
Wu, L.Y., X.Q. Zheng, J.L. Lu and Y.R. Liang, 2009.
Protective effect of green tea polyphenols against
ultraviolet B-induced damage to HaCaT cells.
Hum. Cell, 22(1): 18-24.
Wu, X., G.R. Beecher, J.M. Holden, D.B. Haytowitz,
S.E. Gebhardt and R.L. Prior, 2006. Concentrations
of anthocyanins in common foods in the United
States and estimation of normal consumption. J.
Agr. Food Chem., 54(11): 4069-4075.
Wu, W.C., Y.H. Lai, M.C. Hsieh, Y.C. Chang, M.H.
Wu, H.J. Wu et al., 2011. Pleiotropic role of
atorvastatin in regulation of human retinal pigment
epithelial cell behaviors in vitro. Exp. Eye Res.,
93(6): 842-851.
Xu, J.Y., L.Y. Wu, X.Q. Zheng, J.L. Lu, M.Y. Wu and
Y.R. Liang, 2010. Green tea polyphenols
attenuating ultraviolet B-induced damage to human
retinal pigment epithelial cells in vitro. Invest.
Ophth. Vis. Sci., 51(12): 6665-6670.
Yang, P., J.J. Peairs, R. Tano, N. Zhang, J. Tyrell and
G.J. Jaffe, 2007. Caspase-8-mediated apoptosis in
human RPE cells. Invest. Ophth. Vis. Sci., 48(7):
Zhang, Y., S.K. Vareed and M.G. Nair, 2005. Human
tumor cell growth inhibition by nontoxic
anthocyanidins, the pigments in fruits and
vegetables. Life Sci., 76(13): 1465-1472.
Zhao, J.G., Q.Q. Yan, L.Z. Lu and Y.Q. Zhang, 2013.
In vivo antioxidant, hypoglycemic and anti-tumor
activities of anthocyanin extracts from purple
sweet potato. Nutr. Res. Pract., 7(5): 359-365.
Zhou, G., J. Wu and Q. Gao, 2007. Study of the effects
of hypericin on proliferation of cultured human
retinal pigment epithelial cells. Chinese J. Optom.
Ophthalmol., 9(6): 387-390.
... Interestingly, purple sweet potato showed an inhibitory effect on breast cancer, gastric cancer, bladder cancer cell, and colon adenocarcinoma proliferation1. [17][18][19] This suggests that purple sweet potato might both stimulate or inhibit cell proliferation depending on the type of cells. ...
... We observed that the PSPA inhibited the BC cell viability in a dose-dependent manner. This effect was largely attributed to the acylated anthocyanins in PSPA, which was supported by a previous study (27). Caffeic and ferulic acid also demonstrated anti-proliferative abilities in cancer cells (28,29), therefore, coffeoyl and feruloyl in PSPA provided the beneficial antitumor effect. ...
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Bladder cancer (BC) is the most common malignant disease. The developing of economically sustainable and available agents for the treatment of BC is required. Purple sweet potato anthocyanin (PSPA) has been shown to have antitumor abilities. The present study aimed to evaluate the potential role of PSPA in BC treatment. CCK-8 assay was used to assess the viability of BC cells. Flow cytometry assays were performed to evaluate the mitochondrial membrane potential (MMP), cell apoptosis and cell-cycle distribution. Real-time PCR (RT-PCR) and western blot analysis were performed to determine the expression of the target genes. The results of this study revealed that PSPA reduced the viability of BC in a dose-dependent manner. The MMP collapse was aggravated by the PSPA treatment. The apoptosis rate was higher in the PSPA groups than that in the control group. The expression of the pro-apoptosis genes, including cleaved caspase-3, Fas, Fasl, Bcl-2-associated X proteins (Bax) and anti-apoptotic gene (Bcl-2) was induced and decreased by PSPA, respectively. The cell-cycle progression was suppressed by the presence of PSPA. The activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt (PI3K/Akt) signaling pathway was suppressed by PSPA treatment during BC treatment. The PI3K/Akt signaling was closely related to the antitumor effect of PSPA in BC. The present study provided evidence regarding the treatment of BC and enhanced the understanding of the potential role that PSPA plays in cancer prevention.
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Anthocyanins have been proven to be beneficial to the eyes. However, information is scarce about the effects of purple sweet potato (Ipomoea batatas, L.) anthocyanin (PSPA), a class of anthocyanins derived from purple sweet potato roots, on visual health. The aim of this study was to investigate whether PSPA could have influences on the growth characteristics (cellular morphology, survival, and proliferation) of human retinal pigment epithelial (RPE) cells, which perform essential functions for the visual process. The RPE cell line D407 was used in the present study. The cytotoxicity of PSPA was assessed by MTT assay. Then, cellular morphology, viability, cell cycle, Ki67expression, and PI3K/MAPK activation of RPE cells treated with PSPA were determined. PSPA exhibited dose-dependent promotion of RPE cell proliferation at concentrations ranging from 10 to 1,000 µg/ml. RPE cells treated with PSPA demonstrated a predominantly polygonal morphology in a mosaic arrangement, and colony-like cells displayed numerous short apical microvilli and typical ultrastructure. PSPA treatment also resulted in a better platform growing status, statistically higher viability, an increase in the S-phase, and more Ki67+ cells. However, neither pAkt nor pERK were detected in either group. We found that PSPA maintained high cell viability, boosted DNA synthesis, and preserved a high percentage of continuously cycling cells to promote cell survival and division without changing cell morphology. This paper lays the foundation for further research about the damage-protective activities of PSPA on RPE cells or human vision.
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Proliferative vitreoretinopathy (PVR) is a common cause of intraoperative failure following retinal reattachment surgery and is mediated in part through the migration, de-differentiation and proliferation of retinal pigment epithelial (RPE) cells. Given the cytotoxic effects of curcumin on epithelial and endothelial cells, in this study, we assessed the effects of curcumin on human RPE (hRPE) cell proliferation. WST-1 analysis revealed that curcumin significantly inhibited primary hRPE cell proliferation in a dose- and time-dependent manner (P<0.001) with the greatest inhibition observed at the dose of 15 µg/ml curcumin. Flow cytometric analysis indicated that the cytotoxic effects of curcumin on hRPE cell proliferation were mediated by cell cycle arrest at the G0/G1 phase and the induction of apoptosis (both P<0.001), which was confirmed by ultrastructural analysis using transmission electron microscopy. Furthermore, western blot analysis revealed that curcumin induced p53 and p21WAF1/CIP1 expression with a concomitant decrease in proliferating cell nuclear antigen protein levels (P<0.05). Curcumin effectively inhibited primary hRPE cell proliferation, which may be mediated by the p53 pathway. Further in vivo studies are required in order to fully explore the therapeutic potential of curcumin for PVR.
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Pancreatic cancer is one of the most deadly cancers with a nearly 95% mortality rate. The poor response of pancreatic cancer to currently available therapies and the extremely low survival rate of pancreatic cancer patients point to a critical need for alternative therapeutic strategies. The use of reactive oxygen species (ROS)-inducing agents has emerged as an innovative and effective strategy to treat various cancers. In this study, we investigated the potential of a known ROS inducer, piperlongumine (PPLGM), a bioactive agent found in long peppers, to induce pancreatic cancer cell death in cell culture and animal models. We found that PPLGM inhibited the growth of pancreatic cancer cell cultures by elevating ROS levels and causing DNA damage. PPLGM-induced DNA damage and pancreatic cancer cell death was reversed by treating the cells with an exogenous antioxidant. Similar to the in vitro studies, PPLGM caused a reduction in tumor growth in a xenograft mouse model of human pancreatic cancer. Tumors from the PPLGM-treated animals showed decreased Ki-67 and increased 8-OHdG expression, suggesting PPLGM inhibited tumor cell proliferation and enhanced oxidative stress. Taken together, our results show that PPLGM is an effective inhibitor for in vitro and in vivo growth of pancreatic cancer cells, and that it works through a ROS-mediated DNA damage pathway. These findings suggest that PPLGM has the potential to be used for treatment of pancreatic cancer.
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Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.
Cell death plays an essential role in the development of organs, homeostasis, and cancer. Apoptosis and programmed necrosis are two major types of cell death, characterized by different cell morphology and pathways. Accumulating evidence shows autophagy as a new alternative target to treat tumour resistance. Besides its well-known pro-survival role, autophagy can be a physiological cell death process linking apoptosis and programmed necrosis cell death pathways, by various molecular mediators. Here, we summarize the effects of pharmacologically active compounds as modulators of different types of cancer cell death depending on the cellular context. Indeed, current findings show that both natural and synthetic compounds regulate the interplay between apoptosis, autophagy and necroptosis stimulating common molecular mediators and sharing common organelles. In response to specific stimuli, the same death signal can cause cells to switch from one cell death modality to another depending on the cellular setting. The discovery of important interconnections between the different cell death mediators and signalling pathways, regulated by pharmacologically active compounds, presents novel opportunities for the targeted treatment of cancer. The aim of this review is to highlight the potential role of these compounds for context-specific anticancer therapy. Copyright © 2015. Published by Elsevier Inc.
Rice bran consists of many functional compounds and thus much attention has been focused on the health benefits of its components. Here, we investigated the synergistic inhibitory effects of its components, particularly δ-tocotrienol (δ-T3) and ferulic acid (FA), against the proliferation of an array of cancer cells, including DU-145 (prostate cancer), MCF-7 (breast cancer), and PANC-1 (pancreatic cancer) cells. The combination of δ-T3 and FA markedly reduced cell proliferation relative to δ-T3 alone, and FA had no effect when used alone. Although δ-T3 induced G1 arrest by up-regulating p21 in PANC-1 cells, more cells accumulated in G1 phase with the combination of δ-T3 and FA. This synergistic effect was attributed to an increase in the cellular concentration of δ-T3 by FA. Our results suggest that the combination of δ-T3 and FA may present a new strategy for cancer prevention and therapy.
BACKGROUND Studies have shown that anthocyanins (ACNs) in berries contribute to eye health. However, information on the relationship between the chemical structures and visual functions of ACNs is scarce. This study investigated the protection effects of ACNs with different structures against visible light-induced damage in human retinal pigment epithelial (RPE) cells.RESULTSFour ACNs with different aglycones, namely, pelargonidin-3-glucoside (Pg-3-glu), cyanidin-3-glucoside (Cy-3-glu), delphinidin-3-glucoside, and malvidin-3-glucoside (Mv-3-glu), were isolated from three berries (blueberry, blackberry and strawberry). Of these ACNs, Cy-3-glu exhibited the highest reactive oxygen species inhibitory capacity in RPE cells, with 40 µg mL−1 Cy-3-glu showing a ROS clearance of 57.5% ± 4.2%. The expression of vascular endothelial growth factor levels were significantly (P < 0.05) down-regulated by Cy-3-glu and Mv-3-glu in a visible light-induced damage RPE cell model. Cy-3-glu and Pg-3-glu treatments significantly (P < 0.05) inhibited the increase in β-galactosidase during the RPE cell ageing caused by visible light exposure.CONCLUSION Our findings suggest that the biological properties of different ACNs significantly vary. Cy-3-glu, which contains an ortho hydroxyl group in its B ring, possibly exerts multiple protective effects (antioxidant, anti-angiogenic and anti-ageing) in RPE cells. Therefore, Cy-3-glu may prove useful as a prophylactic health food for the prevention of retinal diseases. © 2014 Society of Chemical Industry
The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated X protein (Bax) in retinal cell death induced by all-trans-retinal (atRAL). Cellular events which activate Bax, such as DNA damage by oxidative stress and phosphorylation of p53, were evaluated by immunochemical and biochemical methods using ARPE-19 cells, 661W cells, cultured neural retinas and a retinal degeneration model, Abca4(-/-)Rdh8(-/-) mice. atRAL-induced Bax activation in cultured neural retinas was examined by pharmacological and genetic methods. Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser 46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4(-/-)Rdh8(-/-) mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions. Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53. This cascade is closely associated with an apoptotic cell death mechanism rather than necrosis.
Mitochondrial dysfunction has been shown to contribute to age-related and proliferative retinal diseases. Over the past decade, the primary human fetal RPE (hfRPE) culture model has emerged as an effective tool for studying RPE functionand mechanisms of retinal diseases. This model system has been rigorously characterized and shown to closely resemble native RPE cells at the genomic and protein level, and that they are capable of accomplishing the characteristic functions of a healthy native RPE (e.g., rod phagocytosis, ion and fluid transport, and retinoid cycle). In this review, we demonstrated that the metabolic activity of the RPE is an indicator of its health and state of differentiation, and present the hfRPE culture model as a valuable in vitro system for evaluating RPE metabolism in the context of RPE differentiation and retinal disease.