162
G.-Y. Liu et al. / European Journal of Pharmacology 786 (2016) 161–168
with fluorine-substituted groups attaching to 2-, 3- or 4-position
on the aromatic ring (Fig. 1). Thus we focus on the SAR (structure-
acitivity relationships) underlying cytotoxicity and explore the
apoptotic mechanism associated with curcumin analogs.
2.3. Cell culture
Human lung cancer cells (NCI-H460), Human lung carcinoma
cells (A549), Human liver hepatocellular carcinoma (HepG2) and
normal liver cells (Chang'sliver) were purchased from the Shang-
hai Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences and cultivated in RPMI 1640 at 37 °C in a humidified
atmosphere with 5% CO2.
2. Materials and methods
2.1. Materials
2.4. MTT assay
Roswell Park Memorial Institute (RPMI)-1640 was from GIBCO.
2′,7′-dichlorofluorescein diacetate, rhodamine 123, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Glu-
tathione reductase (GR), the reduced (GSH) and oxidized (GSSG)
glutathione, N-acetylcysteine (NAC), 2-vinylpyridine (97%) and
thiobarbituric acid were obtained from Sigma. All other chemicals
were of the highest quality available.
Cell viability was determined by a colorimetric assay using
MTT. NCI-H460, HepG2, A549 and Chang'sliver cells were seeded
at a density of 3 ꢀ 103/well in a complete growth medium in
96-well plates and incubated for 24 h. Then cells were treated for
another 24 or 48 h with compounds at the selected concentration
before the MTT assay.
2.5. Stability assay
2.2. Synthesis of the curcumin analogs
Stability of curcumin and 1a (25 M) in RPMI 1640 supple-
μ
The mono-carbonyl curcumin analogs were synthesized ac-
cording to the published procedure (Weber et al., 2005). Briefly,
Aqueous NaOH (20 wt%, 5 ml) was added dropwise to a vigorously
stirred solution of modified benzaldehyde (51 mmol) and ketone
(25 mmol) in ethanol (8 ml). After 48 h stirring at room tem-
perature, distilled water (40 ml) was added to the reaction mixture
followed by the neutralization with HCl. The precipitating yellow
solid was filtered off, washed with distilled water and dried under
vacuum. The crude products were directly charged onto a silica gel
column and eluted with a mixture of ethyl acetate/petroleum to
afford the pure product. Their structures were confirmed by 1H
and 13C NMR spectroscopy.
mented with 10% (v/v) heat-inactivated fetal calf serum at 25 °C
(The common international guideline for long-term stability stu-
dies specifies 2572 °C (ICH, Q1A(R2), 2003)) was monitored at
their band maximum for 150 min (at 10-min intervals) by using
UV/Vis spectroscopy.
2.6. Cell apoptosis analysis
NCI-H460 cells (5 ꢀ 105/well) were plated in six-well plates and
incubated for 24 h to allow exponential growth, then treated with
the test compounds for 24 h at the indicated concentration. When
necessary, the cells were pretreated with GSH or NAC for 1 h
before adding test compounds. The treated cells were harvested
and labeled with annexin V-FITC/PI. A total of 10,000 cells per
sample were collected and analyzed by FACSDiva software.
2.2.1. 1,5-Bis(2-fluorophenyl)-penta-1,4-dien-3-one (1a)
Yellow solid; m.p.: 65–69 °C; Yield: 71.4%; 1H NMR (400 MHz,
CD3Cl),
7.42 (m, 2 H), 7.12–7.23 (m, 6 H); 13C NMR (100 MHz, CD3Cl),
189.0, 162.9, 160.4, 136.1, 131.9, 129.4, 127.6, 124.5, 122.9, 116.4.
δ 7.85 (d, J¼16.4 Hz, 2 H), 7.62 (dt, J¼7.6, 1.6 Hz, 2 H), 7.37–
2.7. Intracellular reactive oxygen species measurement
δ
Generation of reactive oxygen species was measured by the
oxidative-sensitive fluorescent probe DCFH-DA. After 6 of treat-
ment with the test compounds in the absence or presence of GSH
2.2.2. 1,5-Bis(3-fluorophenyl)-penta-1,4-dien-3-one (1b)
Yellow solid; m.p.: 93–95 °C; Yield: 62%; 1H NMR 400 MHz
(CD3Cl),
δ 7.68 (d, J¼16.0 Hz, 2 H), 7.60–7.63 (m, 4 H), 7.10
or NAC, NCI-H460 cells were incubated with 3 M DCFH-DA for
μ
(t, J¼7.6 Hz, 4 H), 6.98 (d, J¼16.0 Hz, 2 H); 13H NMR 100 MHz
30 min at 37 °C in the dark. Then cells were washed with PBS and
analyzed immediately for 2′,7′-dichlorofluorescein fluorescence
intensity by flow cytometry.
(CD3Cl),
δ
188.4, 165.3, 162.8, 142.1, 131.0, 130.3, 125.1, 116.3, 116.0.
2.2.3. 1,5-Bis(4-fluorophenyl)-penta-1,4-dien-3-one (1c)
Yellow solid; m.p.: 147–149 °C; Yield: 71%; 1H NMR 400 MHz
2.8. Uptake and metabolic stability assay
(CD3Cl),
δ 7.69 (d, J¼16.0 Hz, 2 H ), 7.55 (d, J¼8.7 Hz, 4 H), 6.89
(d, J¼8.7 Hz, 4 H), 6.64 (d, J¼15.9 Hz, 2 H), 5.98 (s, 1 H); 13H NMR
NCI-H460 cells were seeded in six-well plates at a density of
5ꢀ 105 cells/well and incubated for 24 h. After 0.5, 1, 2, 4 or 6 h of
treatment with the test compounds, the cells were extracted with
ice-cold methanol (1 ml/well) for 10 h at 4 °C. Then the suspension
was centrifuged for 5 min (9400 g, at 4 °C), and the supernatant was
then scanned using a UV/Visible spectrophotometer (Dai et al., 2015).
100 MHz (CD3Cl),
δ
184.6, 160.6, 141.1, 131.0, 127.7, 122.1, 116.9, 101.8.
2.9. Measurement of GSH and GSSG levels
NCI-H460 cells at a density of 5 ꢀ 105 per well were grown in
six-well plates for 24 h, after 6 h of treatment with 1a at the
indicated concentrations, the cells were collected, resuspended
in 500
freezing and thawing. The remaining protein was subsequently
precipitated by adding 120 of ice-cold 5-sulfosalicylic
μ
l of ice-cold HCl (10 mM) and lysed by three cycles of
μ
l
acid (SSA, 6.5%, w/v) for 10 min and removed by centrifugation
Fig. 1. Molecular structures of curcumin and its analogs.
for 15 min (8000 g, at 4 °C). The resulting supernatant was