Journal of Natural Products
Article
History Museum in Washington DC under the accession number
OCDN3255-W.
Extraction and Isolation. The specimens (0.9 kg wet wt) were
Nitrite Determination. RAW 264.7 cells were treated with
indicated concentrations of compound 1 for 2 h and incubated with
LPS (1 μg/mL) for a further 24 h. The NO concentration in the
27
extracted twice with MeOH and then with MeOH/CH Cl (1:1). The
culture supernatants was determined as previously described. After
00 μL of supernatant was added to the empty 96-well plate, 100 μL of
2
2
combined extracts were evaporated in vacuo to remove the solvent.
The extract was subjected to solvent partitioning to give hexane (4.01
1
Griess reagent (1 part 1% sulfanilamide in 5% phosphoric acid plus 1
g), CH Cl (1.44 g), and n-BuOH (1.39 g) soluble fractions. The n-
2
2
part 1% α-naphthylamide in H O) was added to each well and
2
BuOH fraction was fractionated over a Sephadex LH-20 column
incubated at room temperature for 15 min. The absorbance at 550 nm
was read by a scanning multiwell plate reader. The levels of NO2− were
used as an indicator of the amount of NO production.
(
MeOH only) to give fractions F008−F015. Fraction F010 was further
purified over RP-18 vacuum flash column chromatography (25−30%
MeOH/H O with 0.1% TFA) and reversed-phase C HPLC using
2
18
Cytokine Determination by ELISA. RAW 264.7 cells were
treated with compound 1 for 2 h followed by treatment of LPS (1 μg/
mL) for 24 h. The supernatant was then collected and used to
determine the levels of TNF-α and IL-6 secretion by DuoSet ELISA
kits obtained from R&D System.
2
3% MeOH/H O with 0.1% TFA as eluent to yield compound 1 (17
2
mg).
Oscarellin (1): colorless, amorphous solid; UV (MeOH) λmax (log
ε) 210 (4.55), 242 sh (3.83), 318 (3.55); IR ν 3500−2500 (br, NH
max
1
and/or COOH), 1696.6 (CO), 1195; H NMR (CD OD, 500
3
5
Transfection and Reporter Assays. Cells (5 × 10 cells/mL)
MHz) δ 2.18 (2H, p, J = 6.5 Hz, H-2′), 3.19 (2H, t, J = 7.5 Hz, H-3′),
H
were plated into each well of a six-well plate. The cells were transiently
cotransfected with pGL3-NF-κB-Luc or pGL2-AP-1-Luc and pCMV-
β-gal using Lipofectamine Plus according to the manufacturer’s
protocol for use. After transfection for 24 h, the cells were pretreated
with compound 1 for 2 h and then stimulated with LPS (1 μg/mL) for
4
.15 (2H, t, J = 6.0 Hz, H-1′), 6.64 (1H, t, J = 8.0 Hz, H-5), 7.01 (1H,
d, J = 8.0 Hz, H-4), 7.49 (1H, d, J = 8.0 Hz, H-6), (DMSO-d , 500
6
MHz) δ 2.05 (2H, p, J = 6.3 Hz, H-2′), 3.02 (2H, t, J = 7.3 Hz, H-3′),
H
4
.05 (2H, t, J = 6.0 Hz, H-1′), 6.47 (1H, t, J = 7.8 Hz, H-5), 6.92 (1H,
d, J = 7.5 Hz, H-4), 7.32 (1H, d, J = 8.0 Hz, H-6), 8.07 (3H, br s,
NH ); C NMR (CD OD, 125 MHz) δ 28.8 (C-2′), 38.9 (C-3′),
6
13
4
h. Each well was washed with cold phosphate-buffered salin (PBS).
3
3
C
7.3 (C-1′), 113.8 (C-1), 116.3 (C-4), 117.7 (C-5), 125.1 (C-6), 141.5
Luciferase activity was measured in the cell lysates using a Luciferase
assay system according to the manufacturer’s instructions (Promega).
Construction of shRNA-Expressing Plasmid. The pSUPER
plasmid was used to synthesize shRNAs. For construction of the ATF3
shRNA-expressing constructs, oligonucleotide shATF3 5′-
GGAGGCGGCGAGAAAGAAA-3′ was selected to target ATF3
mRNA (GenBank accession no. NM007498.3). To evaluate the effect
of the shRNA, the expression plasmid encoding shRNA was
transfected into RAW 264.7 cells. Stably transfected cells expressing
shRNA, which were selected using puromycin, were used for
subsequent experiments.
+
(
C-2), 148.6 (C-3), 171.9 (C-7); ESIMS m/z 211.1 [M + H] , 233.1
+
+
+
[
[
2
M + Na] , 421.2 [2M + H] , 443.2 [2M + Na] ; FABMS m/z 211.1
M + H] ; HREIMS m/z 210.1001 [M] (calcd for C H N O ,
10.1004).
Synthesis of Compound 1. The mixture of 3-hydroxyanthranilic
+
+
10
14
2
3
acid (0.47 g) in anhydrous MeOH (5.1 mL) and SOCl (0.11 mL) was
2
refluxed for 16 h to give a methyl ester of 3-hydroxyanthranilic acid
(
0.52 g), which further reacted with di-tert-butyl dicarbonate (0.89
mL) in 3 N NaOH (15 mL) to produce methyl 2-(tert-
butoxycarbonylamino)-3-hydroxybenzoate (2, 0.58 g; 71% yield) as
a pale yellow solid. For 3, an anhydrous acetone solution (5.4 mL) of 2
Western Blot Analysis. Western blot analysis was performed as
(
0.29 g) was reacted with K CO (0.5 g) and N-(3-bromopropyl)-
28
2
3
described earlier. Briefly, RAW 264.7 cells were preincubated with
phthalimide (0.38 mg). Subsequently, methyl 2-(tert-butoxycarbony-
lamino)-3-[3-(1,3-dioxoisoindolin-2-yl)propoxy]benzoate (3, 0.32 g)
in 1,4-dioxane (3.5 mL) was subjected to a reaction with concentrated
HCl (1.2 mL). The reaction mixture was stirred for 48 h at 100 °C and
then concentrated under reduced pressure. The residue was purified
by column chromatography and recrystallization (acetone) to afford
compound 1 (0.13 g; mp 206.9−207.4; 75% yield) as its hydrochloride
salt (a white solid).
Chemicals and Reagents. Unless stated otherwise, all reagents
and chemicals were procured from Sigma-Aldrich. pGL3-NF-κB and
the luciferase assay system were from Promega. pGL2-AP-1 (PMA)-
TA-luciferase was from Clontech Laboratories, Inc. Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and
Lipofetamine Plus were purchased from Gibco BRL Life Technologies.
Cytokine DuoSet ELISA kits (TNF-α, IL-6) were from R&D System.
Antibodies for anti-mouse IκB-α, anti-mouse p65, anti-mouse Erk1/2,
anti-mouse p-Erk1/2, anti-mouse p38, anti-mouse p-p38, anti-mouse
JNK, anti-mouse p-JNK, anti-mouse c-Fos, anti-mouse c-Jun, anti-
mouse ATF-3, anti-mouse Lamin A, anti-mouse α-tubulin, and anti-
mouse β-actin were from Santa Cruz Biotechnology.
various concentrations (0.1, 1, and 10 μg/mL) of compound 1 for 2 h.
RAW 264.7 cells were then incubated for 15 min or 4 h with 1 μg/mL
of LPS. After treatment, PBS was used to wash the cells. The cells were
scraped using scrapers and suspended in a lysis buffer. To obtain
cytosolic cell extracts, homogenate was centrifuged for 10 min at 4 °C
at 1500g. Nuclear fraction was recovered by centrifugation at 13000g
for 10 min at 4 °C. Proteins in the samples were resolved by
electrophoresis on an 8% sodium dodecyl sulfate-polyacrylamide gel
and transferred to polyvinylidene difluoride membranes, which were
probed with the appropriate antibodies. Enhanced chemiluminescence
solution was used to detect the protein bands. In all immunoblotting
experiments, membranes wre stripped and reprobed with an anti-β-
actin antibody for protein loading correction.
Statistical Analysis. The results were reported as the mean ±
standard error of the mean (SEM). For comparisons between groups,
the GraphPad program (Software for Science) was used for statistical
analyses. Differences between groups were considered significant at p
< 0.05.
Cell Culture. The macrophage cell line RAW 264.7 was procured
from ATCC and grown in DMEM with 100 IU/mL penicillin, 100
μg/mL streptomycin, and 10% heat-inactivated FBS in a 5% CO2
humidified atmosphere at 37 °C.
ASSOCIATED CONTENT
■
* Supporting Information
S
Cell Proliferation Assay. RAW 264.7 cells were seeded in a 96-
4
well tissue culture plate (5 × 10 cells/well) (Nunc) and treated with
compound 1 (0.1, 1, 10, 50, and 100 μg/mL) for 24 h. After treatment,
cell proliferation was determined by incubating the cells with a mixture
of 125 μL of full DMEM and 25 μL of MTT solution for another 4 h.
Then, the MTT-formazan produced by viable cells was dissolved in
1
All synthetic details and experimental data (R , H and
f
13
C NMR) for synthetic intermediates and product;
1
13
NMR ( H NMR, C NMR, COSY, HMQC, and
HMBC) and mass spectra for 1; and effect of compound
1 on the cell proliferation (PDF)
150 μL of DMSO. The amount of the resulting formazan was assessed
by measuring the optical density at 550 nm using a scanning multiwell
plate reader (Molecular Device).
F
J. Nat. Prod. XXXX, XXX, XXX−XXX