Polymethoxyflavones as agents that prevent formation of cataract: Nobiletin congeners show potent growth inhibitory effects in human lens epithelial cells

Article abstract of DOI:10.1016/j.bmcl.2012.10.133

Posterior capsular opacification (PCO) is the most frequent complication and the primary reason for visual decrease after extracapsular cataract surgery. The proliferation and migration of leftover lens epithelial cells (LECs) after surgery may contribute to the development of PCO. To prevent PCO, a rational approach would be to inhibit both the proliferation and the migration of LECs using nontoxic xenobiotics. Nobiletin, one of the most abundant polymethoxyflavones (PMFs) in citrus peel, and its synthetic congeners displayed a potent inhibition of LEC proliferation. Structural features which enhance anti-proliferative activity have also been discussed.

Full text of DOI:10.1016/j.bmcl.2012.10.133

Bioorganic & Medicinal Chemistry Letters 23 (2013) 183–187
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Polymethoxyflavones as agents that prevent formation of cataract: Nobiletin
congeners show potent growth inhibitory effects in human lens epithelial cells
Yoshiki Miyata, Tetsuta Oshitari, Yuji Okuyama, Arata Shimada, Hideyo Takahashi, Hideaki Natsugari,
⇑
Hiroshi Kosano
Faculty of Pharma-Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Posterior capsular opacification (PCO) is the most frequent complication and the primary reason for
visual decrease after extracapsular cataract surgery. The proliferation and migration of leftover lens epi-
thelial cells (LECs) after surgery may contribute to the development of PCO. To prevent PCO, a rational
approach would be to inhibit both the proliferation and the migration of LECs using nontoxic xenobiotics.
Nobiletin, one of the most abundant polymethoxyflavones (PMFs) in citrus peel, and its synthetic cong-
eners displayed a potent inhibition of LEC proliferation. Structural features which enhance anti-prolifer-
ative activity have also been discussed.
Received 5 September 2012
Revised 16 October 2012
Accepted 29 October 2012
Available online 9 November 2012
Keywords:
Nobiletin
Polymethoxyflavone
Cataract
Ó 2012 Elsevier Ltd. All rights reserved.
LEC proliferation
Matrix metalloproteinase-9
Cataract is the most common cause of vision impairment in the
world today. It is curable with highly effective surgery, which in-
volves the extracapsular extraction of the natural opaque lens fi-
bers and the implantation of an intraocular lens (IOL).1 However,
many patients gradually develop posterior capsular opacification
(PCO), also known as after-cataract (Fig. 1).2–7 PCO progresses
through three stages, which can be roughly described as: (1) the
improper proliferation of lens epithelial cells (LECs) left behind
after-cataract surgery; (2) the migration of LECs onto the posterior
capsule underlying the intraocular lens and into the light path; (3)
an epithelial–mesenchymal transition (EMT) resulting in the for-
mation of fibroblasts and spindle-like myofibroblasts.8–13 The inci-
dence of PCO within 2 months to 5 years after initial surgery is as
high as 50% in adults and 100% in children.14
Flavonoids are a group of polyphenolic compounds ubiquitously
distributed throughout the plant kingdom. Several studies report
that some flavonoids have protective effects against lens opacifica-
tion in both in vivo and in vitro models of cataract.15–18 However,
the detailed mechanism of action has not been addressed so far.
Recently, we reported that nobiletin (1) (Fig. 2), one of the most
abundant polymethoxyflavones (PMFs) in Citrus species, and its
three metabolites inhibit the production of the pro-matrix metallo-
proteinase (proMMP)-9.19 This enzyme plays an important role in
the migration of LECs, which occurs during the second stage of
PCO development. Therefore, these flavones could be potent lead
compounds for developing chemotherapeutic agents against PCO.
Moreover, the study of divergent nobiletin congeners has unveiled
a
relationship between their structures and their biological
activities.20
On the other hand, the effects of flavones on LEC proliferation,
occurring at the first stage of PCO development, have thus far been
left almost unexplored. With the exception of studies on baica-
lein21 and quercetin,22 no systematic study of the growth inhibi-
tory effects of flavones has been reported. Herein, we examined
the effects of nobiletin (1) and 16 synthetic congeners on the pro-
liferation of LECs, one of the major processes leading to PCO.
We first examined the effects of nobiletin (1) on the prolifera-
tion of the human lens epithelial cell line SRA01/04.23,24 Cell
growth curve assays were performed using Alamar Blue stain25
to monitor the changes in growth over 4 days. As demonstrated
in Figure 3A, the proliferation of SRA01/04 cells in the nobiletin-
treated group was significantly suppressed in a dose-dependent
manner when compared to the control group. This tendency be-
came more apparent over time. On day 4, the inhibitory rates of
Abbreviations: IOL, intraocular lens; PCO, posterior capsule opacification; LEC,
lens epithelial cell; EMT, epithelial–mesenchymal transition; PMF, polymethoxyf-
lavone; proMMP, pro-matrix metalloproteinase; PCNA, proliferating cell nuclear
antigen; MAPK, mitogen-activated protein kinase.
1 at doses of 32 and 64 lM were 47% and 80%, respectively. These
findings strongly suggesting that nobiletin (1) inhibits the prolifer-
ation of LECs were substantiated by western blot analysis of
expression of proliferating cell nuclear antigen (PCNA), a marker
⇑
Corresponding author. Tel.: +81 3 3964 8191; fax: +81 3 3964 8195.
E-mail address: h-kosano@pharm.teikyo-u.ac.jp (H. Kosano).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.133

184
Y. Miyata et al. / Bioorg. Med. Chem. Lett. 23 (2013) 183–187
Figure 1. Pathogenesis of posterior capsule opacification (PCO) and proposed therapeutic roles of polymethoxyflavones (PMFs).
ously,19,20 compounds 7a,28 7b,29 and 1130 were synthesized as
per our previously reported protocol19,20 as depicted in Scheme 1.
Compound 7a proved to be identical to the non-natural flavone
synthesized previously.31 Compound 11 was a flavone obtained
from Scutellaria baicalensis.32 The Alamar Blue assay25 was per-
formed using these compounds.33,34 The results are shown as
growth inhibitory rates compared to the control group on day 4.
As demonstrated in Table 1, tangeretin (6a),35 7a, 8a, and 1035
were found to possess significantly stronger anti-proliferative
activities, unlike that of nobiletin (1). Furthermore, in comparing
the functional groups on the B ring, intriguing patterns emerged.
Compounds 6b, 7b, and 8b, which possess hydroxyl group(s) on
the B ring, showed significantly lower inhibitory activities against
LEC proliferation than compounds 6a (p <0.001), 7a (p <0.001), and
8a (p <0.001), respectively. Similar results can be seen in compar-
isons between nobiletin (1) and 3 (p <0.01), between compounds
4a and 4b (p <0.001), and between compounds 5a and 5b (p
<0.01) as well.27 These observations suggest that a demethylation
at the methoxy group(s) on the B-ring is associated with significant
suppressive effects on the inhibition of LEC proliferation.
Figure 2. Structure of nobiletin, baicalein, and quercetin.
for cell proliferation.26 As shown in Figure 3B, nobiletin (1) signif-
icantly decreased PCNA expression (p <0.05),27 without signifi-
cantly reducing b-actin expression.
Taking these observations into account, we next investigated
the effects of other nobiletin congeners (Fig. 4) on the proliferation
of SRA01/04 cells. In addition to the 14 PMFs reported previ-
We also examined the effects of the 5-demethylated congeners
9–12 on the proliferation of SRA01/04 cells. Among these, com-
pound 10 exhibited considerable inhibitory activity on cell growth.
It is worth noting that compound 10 also exerted marked inhibi-
tory effects (IC50: 0.7
lM) on proMMP-9 production in PMA-trea-
ted cells.20 Similarly, the growth inhibitory rates of compounds
Figure 3. The antiproliferative effects of nobiletin on human lens epithelial cell line SRA01/04. (a) [A]: Cell growth curve assay by means of Alamar Blue stain. Data (n = 4) are
shown as means SD. Asterisks indicate results that were significantly different from untreated control cells at each time (⁄⁄: p <0.01; ⁄⁄⁄: p <0.001). [B]: Western blot analysis
of proliferating cell nuclear antigen (PCNA) expression. Data (n = 4) are shown as means SD (⁄: p <0.05, significantly different from untreated group).

Y. Miyata et al. / Bioorg. Med. Chem. Lett. 23 (2013) 183–187
185
11 and 12 tended to augment compared to those of the corre-
sponding 5-methoxy congeners 4b and 6b. On the other hand,
the anti-proliferative activity of compound 9 was significantly low-
er (p <0.01) than that of nobiletin (1). This finding seems to be con-
sistent with the report by Akao et al.36 that the methoxy group at
C5 in nobiletin (1) contributed to its anti-proliferative effect on hu-
man neuroblastoma SH-SY5Y cells.
6a, 7a, 8a, and 10 showed potent growth inhibitory activities.
Compound 10 exhibited both potent anti-proliferative effects
on LEC cells and a strong inhibitory activity against proMMP-9
production. This compound would be a suitable lead for target-
ing multiple stages of PCO development. Our findings indicate
the potential of such PMFs not only for medical treatment but
also for a reasonable preventive measure against PCO. We hope
that their effective use will replace laser capsulotomy in the near
future.
In summary, we analyzed the growth inhibitory activity of
nobiletin (1) and its 16 congeners on SRA01/04 cells. Compounds
Figure 4. Structures of the nobiletin congeners.

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Y. Miyata et al. / Bioorg. Med. Chem. Lett. 23 (2013) 183–187
Scheme 1. Representative synthetic procedure of compounds 7a, 7b and 11: Reagents and conditions: (a) Et3N (1.2 equiv), DMAP (0.1 equiv), CH2Cl2; 46% (82% based on
recovered 13); (b) t-BuOK (1.1 equiv), THF, reflux, 1 h; (c) p-TsOHꢀH2O (0.25 equiv), benzene, reflux (Dean–Stark), 10 h; 81% in two steps; (d) H2 (1 atm), 20% Pd(OH)2/C (cat.),
EtOAc/EtOH (1:1), 1 h; 86%; (e) CH3I (5 equiv), K2CO3 (3 equiv), DMF; 63%; (f) (i) BCl3 (2 equiv.), CH2Cl2, ꢁ78 °C to rt; (ii) H2 (1 atm), 20% Pd(OH)2/C (cat.), EtOAc/EtOH (1:1),
1 h; 82% in two steps.
Table 1
Growth inhibitory ratesa and IC50 values for proMMP-9 production on PMA-treated SRA01/04 cellsb
Compound
Substituent(s) on B ring
Growth inhibitory rate (%) at 32
l
Ma
IC50 (l
M) for proMMP-9 productionb
1: Nobiletin
2
3
4a
4b
5a
5b
30,40-Dimethoxy
30-Methoxy-40-Hydroxy
30,40-Dihydroxy
20-Methoxy
47.4 5.3
30.7 4.1
26.5 5.5
23.3 4.2
4.2 3.8
42.0 5.5
18.3 7.1
75.9 6.5
32.0 8.4
75.9 4.8
5.1 4.3
59.9 7.8
11.5 6.8
17.2 8.0
64.1 9.2
30.7 10.9
42.3 6.8
20.9 6.5
3.7 0.3
12.3 1.2
13.7 5.8
0.4 0.1
5.5 4.0
2.5 3.4
6.8 3.9
2.6 1.4
> 64
55.0 2.0
12.0 6.6
8.1 4.4
3.0 2.7
0.7 0.9
>64
20-hydroxy
30-Methoxy
30-Hydroxy
6a: Tangeretin
40-Methoxy
6b
7a
7b
8a
8b
9
10
11
12
40-Hydroxy
20,60-Dimethoxy
20,60-Dihydroxy
30,50-Dimethoxy
30,50-Dihydroxy
30,40-Dimethoxy
30-Methoxy-40-hydroxy
20-Hydroxy
40-Hydroxy
3.5 3.1
a
Data (n = 4) are shown as means SD.
b
IC50 values (lM) of 7a, 7b, and 11 for pro-MMP-9 production on PMA-treated SRA01/04 cells were newly determined. Other IC50 values were previously reported in Refs.
19 and 20. For experimental details, see also Ref. 20.

Y. Miyata et al. / Bioorg. Med. Chem. Lett. 23 (2013) 183–187
187
confluence, the SRA01/04 cells were subcultured (3 ꢂ 103 cells/well) and then
treated with DMEM in the presence of various concentrations of nobiletin or
the congeners for up to 4 days. The cell proliferation was analyzed with the
Alamar Blue assay. Briefly, SRA01/04 cells were treated with flavonoids for up
to 4 days in 96-well multiplates. For the last 2 h of the treatment, Alamar BlueÒ
(Invitrogen) was added to the cells, after which the fluorescence intensity (FI)
of the incorporated reagent was measured at 530 nm (excitation) and 590 nm
(emission). The results were expressed as the inhibition of cell proliferation
calculated as the ratio [(FI530/590 treated (Day 4) ꢁ FI530/590 treated (Day 0)/
FI530/590 control (Day 4) ꢁ FI530/590 control (Day 0)) ꢂ 100].
Acknowledgments
We are grateful to Dr. Nobuhiro Ibaraki (IBARAKI eye clinic,
Tochigi, Japan) for the generous gift of the human lens epithelial
cell line SRA01/04. We thank Ms. Erina Ikebe, Mr. Syunma Takaza-
wa, and Mr. Masaya Nagao for experimental support.
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151.0, 148.9, 148.3, 143.7, 138.0, 132.0, 129.7, 128.8, 115.6, 115.1, 111.2, 103.9,
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d6) d 9.85 (2H, br s), 7.11 (1H, t, J = 8.0 Hz), 6.43 (2H, d, J = 8.0 Hz), 6.10 (1H, s),
4.00 (3H, s), 3.85 (3H, s), 3.84 (3H, s), 3.78 (3H, s); 13C NMR (DMSO-d6) d 176.3,
159.6, 157.3, 151.3, 148.7, 148.0, 143.9, 138.2, 132.2, 115.0, 114.9, 108.8, 107.1,
62.4, 62.2, 62.1, 61.9; HRMS calcd. for [M+H]+ of C19H18O8: 375.1074; found:
375.1070.
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(1H, s), 10.25 (1H, s), 7.96 (1H, d, J = 8.0 Hz), 7.71 (1H, s), 7.42 (1H, ddd,
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24. Two striking similarities can be found between this cell line and the non-
immortalized lens epithelial cells in artificially induced pathological
conditions, that is, (1) their proliferative ability, and (2) their potential for
matrix metalloproteinase (MMP)-9 production. We found that the inhibitors of
mitogen-activated protein kinase (MAPK), such as SP600125 and SB203580,
inhibit both cell proliferation and MMP-9 production in SRA01/04 cells. Since
MAPKs (e.g., ERK, p38, and JNK) are also known to be involved both in the
migration of other human lens epithelial cell lines37–40 and in the proliferation
of non-immortalized LECs stimulated by cytokines,41,42 these phenomena may
be because of closely related molecular mechanisms.
25. Experimental procedure in Figure 3A: The human lens epithelial cell line
SRA01/04 was a kind gift from Dr. Nobuhiro Ibaraki (IBARAKI eye clinic,
Tochigi, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) (Invitrogen, Carlsbad, CA) supplemented with 20% (v/v) heat-
inactivated (56 °C for 30 min) fetal bovine serum (Biowest, Nuaille, France)
and PSN antibiotic mixture (Invitrogen, penicillin/streptomycin/neomycin:
100 lg/mL each) at 37 °C in a humidified 5% CO2 atmosphere. Upon reaching