Cytotoxicity and modulation of cancer-related signaling by (Z)- and (E)-3,4,3',5'-tetramethoxystilbene isolated from Eugenia rigida

Article abstract of DOI:10.1021/np300893n

Bioassay-guided fractionation of the leaves of Eugenia rigida yielded three stilbenes, (Z)-3,4,3′,5′-tetramethoxystilbene (1), (E)-3,4,3′,5′-tetramethoxystilbene (2), and (E)-3,5,4′- trimethoxystilbene (3). Their structures were determined using 1D- and 2D-NMR spectroscopy and HRESIMS. The sterically hindered Z-stereoisomer 1, a new natural product, was prepared by time-dependent photoisomerization of the E-isomer (2) under UV irradiation at λ₂₅₄ nm, while 2,3,5,7-tetramethoxyphenanthrene (5) was identified at λ₃₆₅ nm by UHPLC/APCI-MS and NMR spectroscopy. Compounds 1-3 were tested against a panel of luciferase reporter gene assays that assess the activity of many cancer-related signaling pathways, and the Z-isomer (1) was found to be more potent than the E-isomer (2) in inhibiting the activation of Stat3, Smad3/4, myc, Ets, Notch, and Wnt signaling, with IC₅₀ values between 40 and 80 μM. However, both compounds showed similar inhibition against Ap-1 and NF-κB signaling. In addition, 1 demonstrated cytotoxic activity toward human leukemia cells, solid tumor cells of epidermal, breast, and cervical carcinomas, and skin melanoma, with IC₅₀ values between 3.6 and 4.3 μM, while 2 was weakly active against leukemia, cervical carcinoma, and skin melanoma cells. Interestingly, 2 showed antioxidant activity by inhibition of ROS generation to 50% at 33.3 μM in PMA-induced HL-60 cells, while 1 was inactive at 100 μM (vs Trolox 1.4 μM).

Full text of DOI:10.1021/np300893n

Article
pubs.acs.org/jnp
Cytotoxicity and Modulation of Cancer-Related Signaling by (Z)- and
(E)‑3,4,3′,5′-Tetramethoxystilbene Isolated from Eugenia rigida
Mohamed A. Zaki,†,‡ Premalatha Balachandran,† Shabana Khan,†,§ Mei Wang,† Rabab Mohammed,‡
Mona H. Hetta,‡ David S. Pasco,*,†,§ and Ilias Muhammad*,†
†National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University
of Mississippi, University, Mississippi 38677, United States
‡Department of Pharmacognosy, School of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
§Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi,
University, Mississippi 38677, United States
S
* Supporting Information
ABSTRACT: Bioassay-guided fractionation of the leaves of Eugenia rigida yielded three stilbenes, (Z)-3,4,3′,5′-
tetramethoxystilbene (1), (E)-3,4,3′,5′-tetramethoxystilbene (2), and (E)-3,5,4′-trimethoxystilbene (3). Their structures were
determined using 1D- and 2D-NMR spectroscopy and HRESIMS. The sterically hindered Z-stereoisomer 1, a new natural
product, was prepared by time-dependent photoisomerization of the E-isomer (2) under UV irradiation at λ254 nm, while 2,3,5,7-
tetramethoxyphenanthrene (5) was identified at λ365 nm by UHPLC/APCI-MS and NMR spectroscopy. Compounds 1−3 were
tested against a panel of luciferase reporter gene assays that assess the activity of many cancer-related signaling pathways, and the
Z-isomer (1) was found to be more potent than the E-isomer (2) in inhibiting the activation of Stat3, Smad3/4, myc, Ets, Notch,
and Wnt signaling, with IC50 values between 40 and 80 μM. However, both compounds showed similar inhibition against Ap-1
and NF-κB signaling. In addition, 1 demonstrated cytotoxic activity toward human leukemia cells, solid tumor cells of epidermal,
breast, and cervical carcinomas, and skin melanoma, with IC50 values between 3.6 and 4.3 μM, while 2 was weakly active against
leukemia, cervical carcinoma, and skin melanoma cells. Interestingly, 2 showed antioxidant activity by inhibition of ROS
generation to 50% at 33.3 μM in PMA-induced HL-60 cells, while 1 was inactive at 100 μM (vs Trolox 1.4 μM).
Resveratrol is postulated as a potential modulator of signal
transduction pathways for cancer and the carcinogen response;
therefore, this collective activity may contribute an important
role in the anticancer properties of resveratrol2b and its
analogues.
Piceatannol [(E)-3,4,3′,5′-tetrahydroxystilbene], a natural
resveratrol analogue with antileukemic activity,4 was isolated
from Euphorbia lagascae Spreng. (Euphorbiaceae)1 and, like
resveratrol, is also present in grapes and red wine.1 Piceatannol
was reported as a metabolite of resveratrol, transformed with
the hydroxylation by the CYP1B1 enzyme, which is overex-
pressed in a wide range of human tumors.5 Piceatannol and
tilbenes are naturally occurring phytoalexins that generally
Sexist as their more stable E-isomers. The most well-known
stilbene is resveratrol [(E)-3,5,4′-trihydroxystilbene], which is
present in a wide range of plants, notably Vitis vinifera L.
(Vitaceae) and Arachis hypogaea L. (Fabaceae).1 During the
past decade, an array of important biological activities, including
the potential cancer chemopreventive effect of resveratrol, has
prompted investigations on the modulation of cancer-related
signaling pathways2 as well as the development of potent
natural and synthetic anticancer analogues.3 Since transcription
factors and the signaling pathways that control their activity are
involved in modulating the expression of many cancer-related
genes, the identification of molecular targets within these
pathways for such compounds or analogues is required to
understand how they affect the chemoprevention process.
Received: December 21, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
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(E)-3,4,3′,5′-tetramethoxystilbene were also found to be
equally effective as etoposide against the gastric carcinoma
cell line with the classical MDR phenotype EPG85-257RDB,
while piceatannol was more potent than (E)-3,4,3′,5′-
tetramethoxystilbene against the pancreatic carcinoma cell
line with the classical EPP85-181RDB6MDR phenotype.6 On
the other hand, the Z-form of 3,4,3′,5′-tetramethoxystilbene
showed >10-fold more activity than the E-form against the
SW480 human colorectal tumor cell line.7
The genus Eugenia is the largest in the family Myrtaceae, with
up to 2000 species distributed from the south of Mexico, Cuba,
and the Antilles, to Uruguay and Argentina, with a small
number of species in Africa.8 Some species, with edible fruits,
have been cultivated in tropical and subtropical regions.9
Eugenia rigida DC. is a shrub or small tree producing
characteristic brown and green fruits, which turn black when
mature. This plant, used folklorically for leukemia in Argentina,
has not been subjected previously to either chemical or
biological investigations, but the genus has exhibited a wide
array of secondary metabolites on phytochemical analysis.
However, the only stilbene reported from the family Myrtaceae
is piceatannol, isolated from Callistemon rigidus R. Br.9 In this
study, we report the isolation of (Z)- (1) and (E)-3,4,3′,5′-
tetramethoxystilbene (2) and (E)-3,5,4′-trimethoxystilbene (3)
from E. rigida and the photoisomerization of 2 to 1. Their
cytotoxicity is described against selected human tumor cells, as
well as their effects on modulation of cancer-related signaling
pathways and inhibition of reactive oxygen species (ROS) and
nitrite generation.
RESULTS AND DISCUSSION
■
Bioassay-guided fractionation, based on luciferase reporter
assays, of the active n-hexane extract of E. rigida, using
centrifugal preparative thin-layer chromatography with custom-
made C18 and normal-phase silica gel rotors (see Experimental
Section), resulted in the isolation of three stilbenes (1−3), of
which 110 was found to be more cytotoxic than 2 and 3.
Compounds 2 and 3 have been isolated previously,11,12 and
their structures were suggested by comparison of spectroscopic
data with those published.11−13 The HRESIMS of compound 1
showed a molecular ion at m/z 301.1357 [M + H]+ (calcd for
C18H21O4, 301.1362), attributed to the molecular formula
C18H20O4. The 1H NMR data of 1 were reported previously as
a synthetic compound,10 but only at low field, and the 13C
NMR data were not published. In order to assign the 13C NMR
data unambiguously to substantiate the structure of 1, a
complete set of 2D-NMR experiments was carried out, with the
data compared with those assigned for 2 (see Table S1,
Supporting Information). Thus, the HMBC spectrum of 1
confirmed the unambiguous assignments of carbon signals by
showing correlations between H-α (δ 6.52) and C-1 (δ 129.7),
C-2 (δ 111.8), C-6 (δ 122.0), C-β (δ 128.8), and C-1′ (δ
139.5) and between H-β (δ 6.47) and C-1′, C-2′/6′ (δ 106.6),
C-α (δ 130.3), and C-1. In addition, the signal at δ 6.46 (H-2′/
6′) showed correlations with resonances at δ 99.6 (C-4′) and
128.8 (C-β), and that at δ 6.32 (H-4′) with the C-2′/6′ signal
(δ 106.6), indicating the presence of methoxy groups at the C-
1
3′ and C-5′ positions. Finally, the H−1H NOESY spectrum
revealed cross-peaks between δ 6.75 (H-5) and 3.86 (OMe-4)
and between δ 6.83 (H-2) and 3.64 (OMe-3), confirming the
position of the methoxy groups at C-3 and C-4.
In order to supplement the low yield of the sterically
hindered Z-isomer (1), an additional quantity of this
compound was prepared from the more stable 2 by
photoisomerization at λ254 nm.14 A time-dependent UV
irradiation was carried out to study the conversion of (E)-2
to the (Z)-1 isomer over a time range of 0−1000 min, where
the yield of product 1 was quantified by UHPLC/APCI-MS. A
linear increase in the Z-isomer, with 80% conversion, was noted
at λ254 nm during the first 250 min of irradiation (Figure 2A).
Figure 2D depicts a first-order rate plot of (E)-2 photo-
isomerization within a 250 min time interval, using data from
Figure 2A, which showed clearly good linearity, as reflected by
an R2 of 0.9937. In contrast, a sharp increase of 1 was noted in
the first 30 min of irradiation at λ365 nm, followed by a steady
decrease of 1 (Figure 2B) with the formation of 5, which was
Figure 1. Photoisomerization of (E)-2 into (Z)-1 and compound 5 by UV irradiation at λ254 nm and λ365 nm, respectively, in tetrahydrofuran.
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Figure 2. Photoisomerization of (E)-2 into (Z)-1 by UV irradiation in tetrahydrofuran (A) at λ254 nm and (B) at λ365 nm. (C) Assessment of the
ratio of (E)-2 into (Z)-1 inversion by UHPLC at λ254 nm or λ365 nm. (D) Determination of apparent rate constant (kz) and half-life (t1/2) of (E)-2
photoisomerization based on pseudo-first-order kinetics by UHPLC.
Table 1. Cytotoxic Activities of Compounds 1−4
a
cytotoxic activity (IC50 in μM)
b
c
d
e
f
g
h
i
compound
HL-60
KB
4.3
NA
17.7
NA
3.8
BT-549
SK-OV-3
SK-MEL
HeLa
LLC-PK-1
Vero
j
1
4.3
4.0
NA
NA
4.3
60.0
12.2
47.3
1.5
3.6
8.0
6.0
NA
20
>33.3
NA
2
33.3
NA
55.5
113.0
k
3
NT
15.6
NA
13.3
26.7
73.6
>9.2
4
31.8
0.2
NA
3.9
52.5
1.6
doxorubicin
3.7
2.6
a
b−g
IC50 is the concentration that affords 50% inhibition of cell growth.
Human cell lines of leukemia, epidermal carcinoma, breast carcinoma,
i j
h
ovarian carcinoma, skin melanoma, cervical carcinoma. Pig kidney epithelial cells. African green monkey kidney cell line. No activity at 100 μM.
k
Not tested.
identified from the mixture as 2,3,5,7-tetramethoxyphenan-
determine their isomer composition and/or byproduct (5, 6)
formation is relevant to their therapeutic promise.
1
threne by UHPLC/MS (M+ 298; C18H19O4), UV, and H
NMR spectra15 (Figures S22−S27, Supporting Information).
Compound 1 showed cytotoxic activity against human
leukemia (HL-60) cells, human solid tumor cells of epidermal
(KB), breast (BT-459), and cervical (HeLa) carcinomas, and
skin melanoma (SK-MEL), with IC50 values of 4.3, 4.3, 4.0, 3.6,
and 4.3 μM, respectively, while 2 was weakly active against HL-
60, HeLa, and SK-MEL cells (IC50 values of 33.3, 8.0, and 60.0
μM) (Table 1). In addition, 3 was also found to be weakly
cytotoxic toward KB, BT-459, HeLa, and SK-MEL cells (IC50
17.7, 15.6, 13.3, and 12.2 μM, respectively). Compound 1 was
inactive against a noncancerous cell line (monkey kidney
fibroblast; Vero) up to 33.3 μM, thus exhibiting selectivity
toward the tumor cells. Since compound 1 was more potent
than 2 with respect to human cancer cell line cytotoxicity, it was
1
The H NMR spectrum showed doublets at δ 7.53 and 7.62
(each J = 8.5 Hz; H-9, H-10) and δ 6.88 and 6.74 (each J = 2.0
Hz; H-6, H-8) and singlets at δ 7.21 and 9.11 (H-1, H-4),
thereby suggesting the position of cyclization at C-4a and C-4b.
Four methoxy groups at δ 3.95, 4.03, 4.08, and 4.10 were
assigned at the C-2, -3, -5, and -7 positions, respectively, of 5.
The formation of 5 suggested that 1 was likely cyclized to an
unstable intermediate, 2,3,5,7-tetramethoxy-4a,4b-dihydrophe-
nanthrene (6), which rapidly undergoes oxidative trans-
formation to yield 5 (Figure 1).16 Finally, since the geometrical
isomerism of (E)-1 or (Z)-2 can influence bioactivity (vide
infra), knowledge of photostability together with methods to
C
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a
Table 2. Activity of Compounds 1, 2, and 4 (IC50 values in μM) against Cancer-Related Signaling Pathways in HeLa Cells
Stat3
IL-6
Smad3/4
TGF-β
Ap-1 PMA NF-κB PMA E2F PMA Myc PMA Ets PMA Notch PMA FoxO Wnt wnt3a Hedgehog PMA
pTK
1
2
4
50/77
−/−
70/53
40/55
100/77
50/33
80/80
80/100
−/−
80/80
90/-
−/−
−/−
−/−
60/65
−/−
60/55
40/65
−/−
50/55
40/40
−/−
40/40
−/−
−/−
−/−
40/50
80/80
60/70
−/−
−/−
40/40
−/−
−/−
−/−
50/60
a
Values (from two independent experiments) are the IC50 or lowest concentration (both in μmol/L) that maximally inhibited luciferase induction by
50−60%. A dash indicates that luciferase induction was not inhibited more than 40% at 100 μM. Compounds (final concentrations of 40, 60, 80, or
100 μM) were added to cells 30 min before the addition of the indicated inducer and were harvested for the luciferase assay 4 or 6 h (Notch, FoxO,
Wnt, and Hedgehog) later. No inducer was added to cells transfected with FoxO vector or pTK control vector.
injection using a Bruker Bioapex-FTMS with electrospray ionization
(ESI). UHPLC/APCI-MS was performed using an Agilent 1290
UHPLC system coupled with an Agilent 6120 single quadrupole mass
spectrometer. UV irradiation was carried out with a Spectroline UV
lamp ENF 240C (Spectromics Corporation, NY, USA) in a sealed
chamber. TLC was conducted on precoated silica gel 60 F254 (EMD
Chemicals Inc., Darmstadt, Germany) using toluene−EtOAc (9:1) as
solvent. Centrifugal preparative TLC (CPTLC, using a Chromatotron,
Harrison Research Inc., Palo Alto, CA, USA; model 8924, tagged with
a fraction collector) was carried out on 6 mm reversed-phase C18 silica
gel (Chromatorotor)18 or a 2 mm silica gel P254 (Analtech) disk, using
H2O in MeOH, and Me2CO in n-hexane as eluents, respectively.
Samples were dried using a Savant Speed Vac Plus SC210A
concentrator. The compounds were visualized by spraying the TLC
plates with 1% vanillin−H2SO4 as spray reagent. The reference
compounds (E)-resveratrol and (E)-3,5,4′-trimethoxystilbene were
purchased from TCI America (Waltham, MA, USA; purity >98.0%,
GC), and reference standard doxorubicin (purity ≥98.0%) was
procured from Sigma-Aldrich. Details of vectors for transfection and
luciferase assays are listed in Table S2, Supporting Information.
Plant Material. The leaves of Eugenia rigida were collected from
Guanica, Puerto Rico, in March 2006. The sample was identified by
Mr. F. Axelrod, and a voucher specimen (no. 3008783; collection no.
Gust 1116) was deposited at the Herbarium of the Missouri Botanical
Garden, St. Louis, MO, USA.
determined if the Z-isomer (1) was also a more effective
modulator of cancer-related signaling pathways than the E-
isomer (2). Modulation of the activity of these pathways was
assessed using a battery of luciferase reporter gene vectors, in
which luciferase expression is driven by the binding of
transcription factors to multiple copies of synthetic enhancers
within each vector. Compound 1 was more potent than 2 at
inhibiting the activation of Stat3, Smad3/4, myc, Ets, Notch,
and Wnt signaling. Ap-1 and NF-κB signaling were inhibited by
both compounds similarly, and neither compound inhibited
E2F or Hedgehog pathway activation at the tested concen-
trations. Similarly, the activation of the apoptotic mediator
FoxO was not observed with either compound. Compound 1
was similar in potency to (E)-resveratrol (4) for inhibiting
signaling mediated by Stat3, Smad3/4, myc, Ets, and Notch,
while resveratrol was more potent for NF-κB and Hedgehog.
Compound 3 was also tested and found inactive up to 100 μM.
None of the compounds at the concentrations tested inhibited
luciferase expression driven by the minimal thymidine kinase
promoter (pTK), indicating the lack of general cytotoxicity or
luciferase enzyme inhibition (Table 2). Finally, the inhibition of
intracellular generation of reactive oxygen and nitrite species
was measured to determine the potentials of the test
compounds against oxidative and inflammatory stress in the
cellular environment. The results obtained showed that the E-
isomer (2) exhibited an inhibition of 50% in ROS generation at
33.3 μM in phorbol-12-myristate-13-acetate (PMA)-induced
HL-60 cells, while the Z-isomer (1) did not exhibit any effect at
the doses tested (100 μM), which might be due to the
enhanced conjugation of the olefinic bond with the aromatic
ring in the E-isomer (1), compared to the Z-isomer. However,
compounds 1 and 2 did not show any effect on inducible nitric
oxide synthase (iNOS) activity in LPS-induced macrophages
(RAW264.7) up to 33.3 μM (vs parthenolide at 8 μM).
Extraction and Isolation. Air-dried, powdered leaves (107 g)
were soaked in n-hexane and sonicated (600 mL × 3 × 2 h each). The
combined extract was filtered and dried (2.5 g), and the residue was
extracted with CH2Cl2, followed by EtOAc and MeOH, yielding
extracts of 3.8 g (CH2Cl2), 1.1 g (EtOAc), and 14.0 g (MeOH). The
n-hexane extract (2 g) was subjected to centrifugal preparative TLC
(CPTLC, Chromatotron), using a 6 mm custom-made reversed- phase
Chromatorotor packed with binder-free C18 silica gel.18 The rotor was
mounted on a Chromatotron and packed under slow rotation (100
rpm) by applying a slurry of C18 silica gel (100 g) impregnated with
UV 254 and 365 nm fluorescent indicators (0.5% each) in H2O−
MeOH (1:9; 300 mL). The sample, dissolved in Me2CO, was applied
to the rotor under a rotation of 700 rpm, and then the rotor was
removed from the instrument and left to dry in a desiccator. The dried
rotor was mounted on a Chromatotron and eluted with H2O−MeOH
(3:7), which afforded fr. 12−21 (174 mg) containing a mixture of
compounds 1−3. This fraction was subjected to CPTLC, using a 2
mm silica gel rotor, and eluted with 0.5−20% Me2CO in n-hexane,
which furnished compound 1 (3 mg), followed by 2 (30 mg) and 3 (1
mg), respectively, as monitored and pooled by TLC analysis (silica gel;
solvent: toluene−EtOAc, 9:1).
This is the first report of (Z)-3,4,3′,5′-tetramethoxystilbene
(1) from a natural source, although it has been synthesized
previously together with its E-isomer,7,10 and also the first
report of stilbenoids from the genus Eugenia. Interestingly, the
potent inhibitory activity of 1 against HL-60 cells supports the
reported use of E. rigida in Argentina for leukemia,17 although
this needs to be confirmed by additional experimental work.
(Z)-3,4,3′,5′-Tetramethoxystilbene (1): yellow oil; UV (MeOH)
λmax (log ε) 286 (0.80) nm; IR (KBr) νmax 2999, 1588, 1456, 1203
EXPERIMENTAL SECTION
■
1
cm−1; H and 13C NMR data, see Table S1, Supporting Information;
General Experimental Procedures. Melting points were
determined on a Mettler FP 51 apparatus. UV spectra were obtained
in MeOH using a Hewlett-Packard 8453 UV/vis spectrometer. IR
spectra were obtained using a Bruker Tensor 27 instrument. NMR
spectra were acquired on a Varian Mercury 400 MHz spectrometer at
400 (1H) and 100 (13C) MHz in CDCl3, using the residual solvent as
an internal standard. Multiplicity determinations (DEPT) and 2D-
NMR spectra (HMQC, HMBC, NOESY) were obtained using
standard Bruker pulse programs. HRESIMS were obtained by direct
HRESIMS m/z 301.1357 [M + H]+ (calcd for C18H21O4, 301.1362).
(E)-3,4,3′,5′-Tetramethoxystilbene (2): colorless solid (CHCl3);
mp 67−71 °C (lit.13 mp 67−68 °C); UV (MeOH) λmax (log ε) 323
1
(1.49) nm; IR (KBr) νmax 2932, 1591, 1458, 1203 cm−1; H and 13C
NMR data, see Table S1, Supporting Information; HRESIMS m/z
301.1358 [M + H]+ [calcd for C18H21O4, 301.1362]).
(E)-3,5,4′-Trimethoxystilbene (3): colorless solid; 1H and 13C
NMR spectroscopic data were in agreement with those reported for
D
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(E)-3,5,4′-trimethoxystilbene.12 The identity of compound 3 was
confirmed by direct comparison with an authentic sample of (E)-
3,5,4′-trimethoxystilbene.
GA, USA) and 1% penicillin/streptomycin in a humidified atmosphere
of 5% CO2 at 37 °C. Luciferase vectors are summarized in Table S2,
Supporting Information. Cells were grown to 60−80% confluence,
trypsinized with 0.05% trypsin-EDTA (Gibco Life Technologies,
Grand Island, NY, USA), and plated in 96-well plates at a density of
0.015 × 106 cells/well in 100 μL of growth medium with 10% fetal
bovine serum (FBS). After 24 h, the growth medium was replaced with
DMEM containing 1% FBS. The cells were transfected with the
appropriate plasmid DNA(s) using X-tremeGENE HP transfection
reagent (Roche Applied Science, Indianapolis, IN, USA). After 24 h of
transfection, the agents to be tested were added to the transfected
cells, followed 30 min later by an inducing agent (IL-6 and TGF-beta
were from R&D Systems, Inc., Minneapolis, MN, USA; m-wnt3a was
from Peprotech Corporation, Rocky Hill, NJ, USA; and phorbol 12-
myristate 13-acetate was from Sigma Chemical Company, St. Louis,
MO, USA). After 4 or 6 h of induction, the medium was aspirated and
the cells were lysed by the addition of a 1:1 mixture of One-Glo
luciferease assay system (Promega Corporation, Madison, WI, USA)
and phosphate-buffered saline (PBS). The light output was detected in
a Glomax Multi+ detection system with Instinct Software (Promega
Corporation, Madison, WI, USA).
Antioxidant Assays. Antioxidant activity was assessed by the
DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) method, as
described earlier.20 Myelomonocytic HL-60 cells (0.8 × 106 cells/mL;
ATTC, Manassas, VA, USA) were suspended in RPMI-1640 medium
containing 10% fetal bovine serum, penicillin (50 units/mL), and
streptomycin (50 μg/mL). The cell suspension (150 μL) was added to
the wells of a 96-well plate. After treatment with different
concentrations (0.4−100 μM) of the test compounds for 30 min,
cells were treated with phorbol-12-myristate-13-acetate (PMA, 100
ng/mL) for 30 min. DCFH-DA (5 μg/mL) was added, and cells were
further incubated for 15 min. Levels of fluorescent DCF [produced by
ROS-catalyzed oxidation of DCFH] were measured on a SpectraMax
plate reader with an excitation wavelength of 485 nm and an emission
wavelength of 530 nm. DCFH-DA is a nonfluorescent probe that
diffuses into the cells, where cytoplasmic esterases hydrolyze it to the
nonfluorescent 2′,7′-dichlorodihydrofluorescein (DCFH). ROS gen-
erated within HL-60 cells oxidize DCFH to the fluorescent dye 2′,7′-
dichlorofluorescein (DCF). The ability of the test compounds to
inhibit ROS-mediated oxidation of DCFH in PMA-treated HL-60 cells
was measured in comparison to the vehicle control. Trolox (IC50 1.4
μM) was used as a positive control. The cytotoxicity to HL-60 cells
was also determined after incubation for 48 h of cells (2 × 104 cells/
well in 225 μL) with test samples by the XTT method, as described
earlier.20
Photoisomerization of 2. Compound 2 (10 mg) was dissolved in
tertrahydrofuran (THF; 5 mL) and subjected to UV irradiation at λ254
nm at room temperature. The reaction mixture was monitored by
running TLC of aliquots at 30 min intervals. The irradiation was
stopped after 250 min (i.e., initially determined by a time-dependent
study, see below), and the solution was dried and then dissolved in
CH2Cl2 (5 mL). The sample was loaded on a Chromatotron, using a 1
mm silica gel rotor to separate the Z-isomer (1) from the residual E-
isomer (2), following a similar procedure to that described above to
afford 8 mg of 1, showing an 80% conversion.
UHPLC/APCI-MS Analysis. Analysis was performed on an Agilent
1290 Infinity liquid chromatograph coupled with an Agilent 6120
single quadrupole mass spectrometer. The LC column was a Waters
Acquity UPLC BEH RP-C18 column (1.7 μm, 2.1 × 150 mm). The
mobile phase consisted of A (acetonitrile with 0.05% formic acid) and
B (water with 0.05% formic acid) at a flow rate of 0.2 mL/min. The
gradient elution started with 55% A for 8 min, and then it was
increased linearly to 100% A in 9 min and held for 3 min. The column
temperature was maintained at 30 °C. The compounds of interest
were analyzed by ESI and APCI in both the positive and negative
modes. The APCI positive mode was selected for analysis because it
produced a better ion signal for the test compounds than the other
ionization mode. The drying gas flow was 10 L/min, and the nebulizer
pressure was 30 psi. The drying gas temperature and vaporizer
temperature were set to 250 and 200 °C, respectively. The capillary
voltage was 3000 V, and the corona current was 4.0 μA. The MS was
operated in a selected ion monitoring (SIM) mode; m/z 301 [M +
H]+ was selected to monitor compounds 1 and 2, and m/z 299 [M
+H]+ was selected for compound 5.
Stock solutions of compounds 1 and 2 were prepared separately at a
concentration of ∼1.0 mg/mL in THF. A series of calibration standard
solutions within the concentration range of 10−550 μg/mL was
prepared. The calibration curves were linear over the full concentration
range. Compound 2 (2.0 mg) was dissolved in 4 mL of THF, and the
solution was divided into two vials. One was exposed to short- (λ254
nm) and the other to long-wavelength (λ365 nm) UV light. An aliquot
(100 μL) from each reaction mixture was taken at 30 min intervals and
analyzed by UHPLC/MS as discussed above. The last aliquot was
taken from λ254 nm UV irradiation reaction after 1000 min, which
showed compounds 1 (tR 6.929 min; [M + H]+ m/z 301) and 2 (tR
6.397 min; [M + H]+ m/z 301). Compound 5 (tR 6.593 min; [M +
H]+ m/z 299; C18H19O4) was identified as the major product from the
reaction mixture, irradiated at λ365 nm, in addition to residual amounts
of compounds 1 and 2. The 1H NMR spectroscopic data of compound
5 were in agreement with those reported for 2,3,5,7-tetramethox-
yphenanthrene.15
Cytotoxicity Assays. Cytotoxic activity was determined against six
human cancer cell lines (HL-60, SK-MEL, KB, BT-549, SKOV-3, and
HeLa) and two noncancerous kidney cell lines (LLC-PK1 and Vero).
All cell lines were obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA). Each assay was performed
in 96-well tissue culture-treated microplates. Cells were seeded at a
density of 25 000 cells/well and incubated for 24 h (except HL-60
cells, which were incubated for 3 h). Samples at different
concentrations were added, and cells were again incubated for 48 h.
At the end of incubation, the cell viability was determined using
Neutral Red dye according to a modification of the procedure of
Borenfreund et al.19 In the case of HL-60 cells, viability was
determined by an XTT method, as described earlier.20 IC50 values
were determined from dose−response curves of percent growth
inhibition against test concentrations. Doxorubicin was used as a
positive control, while DMSO was used as the negative (vehicle)
control.
Assay for Inhibition of iNOS. Inhibition of intracellular NO
production as a result of iNOS activity was assayed in mouse
macrophages (RAW 264.7 cells), as described before.21 Cells were
seeded at a density of 50 000 cells/well in 96-well plates and grown for
24 h. Test samples were added to the cells after incubating with
samples for 30 min, LPS (5 μg/mL) was added, and cells were further
incubated for 24 h. The activity of iNOS was determined by measuring
the level of nitrite in the cell culture supernatant with Griess reagent.
The degree of inhibition of nitrite production was calculated in
comparison to the vehicle control. IC50 values were obtained from
dose−response curves. Parthenolide was used as a positive control
(IC50 8 μM). Cytotoxicity of test samples to macrophages was also
determined in parallel to check if the inhibition of iNOS was due to
cytotoxic effects.
ASSOCIATED CONTENT
■
S
* Supporting Information
Transfection and Luciferase Assays. HeLa cells (ATCC,
Bethesda, MD, USA) were maintained in Dulbecco’s modified Eagle’s
medium (Gibco Life Technologies, Grand Island, NY, USA)
containing 10% fetal bovine serum (Atlanta Biologicals Inc., Atlanta,
Spectroscopic data of compounds 1 and 2; H and 13C NMR
1
spectra of 3; and photoisomerization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
E
dx.doi.org/10.1021/np300893n | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(20) (a) Choi, Y. W.; Takamatsu, S.; Khan, S. I.; Srinivas, P. V.;
Ferreira, D.; Zhao, J. J. Nat. Prod. 2006, 69, 356−359. (b) Reddy, M.
K.; Gupta, S. K.; Jackob, M. R.; Khan, S. I.; Ferreira, D. Planta Med.
2007, 73, 461−467.
AUTHOR INFORMATION
■
Corresponding Author
*(I.M.) Tel: (662) 915-1051. Fax: (662) 915-7989. E-mail:
milias@olemiss.edu. (D.S.P.) Tel: (662) 915-7130. Fax: (662)
915-7062. E-mail: dpasco@olemiss.edu.
(21) Quang, D. N.; Harinantenaina, L.; Nishizawa, T.; Hashimoto,
T.; Kohchi, C.; Soma, G.; Asakawa, Y. Biol. Pharm. Bull. 2006, 29, 34−
37.
Notes
(22) Chang, C. C.; Zhang, J.; Lombardi, L.; Neri, A.; Dalla-Favera, R.
The authors declare no competing financial interest.
Oncogene 1994, 9, 923−933.
(23) Subbaramaiah, K.; Bulic, P.; Lin, Y.; Dannenberg, A. J.; Pasco, D.
S. J. Biomol. Screen. 2001, 6, 101−110.
ACKNOWLEDGMENTS
■
(24) Laherty, C. D.; Yang, W. M.; Sun, J. M.; Davie, J. R.; Seto, E.;
Eisenman, R. N. Cell 1997, 89, 349−356.
The authors sincerely thank Dr. V. Raman, Dr. B. Avula, Mr. F.
Wiggers, Ms. K. Martin, and Mr. J. Trott, NCNPR, School of
Pharmacy, for providing information on the plant, recording
mass spectra, running NMR spectra, and performing the
cytotoxicity assays, respectively. This work was supported in
part by a contract from the University of Mississippi Medical
Center Cancer Institute and the USDA Agricultural Research
Service Specific Cooperative Agreement No. 58-6408-2-0009.
M.A. Zaki is thankful to the Egyptian Government for a
scholarship.
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