B. Sun et al. / International Immunopharmacology 40 (2016) 400–409
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that the potential anticancer effect and mechanisms of SZC015 on
2.4. Cell culture
human lung cancer cell line H322.
The human non-small-cell lung cancer cell lines H322, H1299, A549
and the human bronchial epithelial cell line HBE were kindly provided
by Dalian Medical University Cancer Center. All cell lines were cultured
in RPMI 1640 medium containing 10% fetal bovine serum, penicillin
(100 units/ml) and streptomycin (100 μg/ml) in 95% humidified air at-
mosphere with 5% CO2 at 37 °C.
2. Materials and methods
2.1. Chemicals
Oleanolic acid (OA), SZC009, SZC014, SZC015 were friendly provided
by Professor Shisheng Wang from Dalian University of Technology.
Roswell Park Memorial Institute 1640 (RPMI1640), fetal bovine
serum, and 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyl-tetrazolium
bromide (MTT) were purchased from Gibco (Gaithersburg, MD, USA).
N-Acetyl-L-Cysteine (NAC) was obtained from Sigma-Aldrich
(St Louis, MO, USA). The 6-carboxy-2′,7′-dichlorodihydrofluorescein
diacetate (DCFH-DA) and 4′,6-diamidino-2-phenylindole (DAPI) were
purchased from the Beyotime Institute of Biotechnology (Haimen,
Jiangsu, China). The Annexin V-FITC Apoptosis Detection Kit and
Propidium Iodide were purchased from the Nanjing Jiancheng
Bioengineering Institute (Haimen, Jiangsu, China). The primary
antibodies against Akt, p-Akt, p65, p-p65, p-IκBα, procaspase-9,
procaspase-3, Bax, Bcl-2, LC3B, β-actin, Histone H3 and the second
antibody goat anti-rabbit IgG-horseradish peroxidase were purchased
from the Proteintech (Chicago, IL, USA).
2.5. MTT assay
Cell viability was estimated by MTT assay as previously
introduced [7]. Briefly, cells were seeded in 96-well plates at a
density of (1 × 105 cells/ml) and then incubated at 37 °C for 24 h.
After treatment with different concentrations of OA, SZC009,
SZC014 and SZC015, 15 μl MTT stock solution (5 mg/ml) was added
to each well. After 4 h of incubation, 100 μl SDS-isobutanol-HCl
solution (10% SDS, 5% isobutanol and 12 mM HCl) was added to dissolve
the formazan crystals and the plates were further incubated at 37 °C for
overnight. The light absorption was recorded at a wavelength of 570 nm
with an Enzyme mark instrument (Thermo-354, USA).
2.6. DAPI staining
To determine the inhibition effect of SZC015, the 4′,6-diamidino-2-
phenylindole (DAPI) assay was performed. Briefly, H322 cells
(2 × 105 cells/ml) were cultured in 6-well plate, incubated at 37 °C
for 24 h. After an exposure to SZC015 for 24 h, cells were washed with
PBS and fixed with 10% formaldehyde for 10 min. Cells were
subsequently stained with DAPI (1 μg/ml) for 8 min in the darkness at
room temperature. Then washed with PBS for three times. At last, the
pictures were taken by using a fluorescence microscope (Nikon, Japan).
2.2. 3-Oxo-olean-12-en-28-oic acid (OA)
OA 2.0 g (4.4 mmol) was dissolved in 100 ml mixed solvent of di-
chloromethane and acetone (volume ratio of 1:1). The solution was
cooled to 0 °C, and 2.0 ml Jones reagent was added dropwise within
30 min. The reaction mixture was stirred for 30 min and 2 ml
isopropanol was added. After 10 min, the mixture was filtered, and
the filtrate was evaporated under reduced pressure to yield a solid res-
idue, which was dissolved in 50 ml ethyl acetate, then washed with sat-
urated brine (50 ml × 3). The organic phase was dried with anhydrous
sodium sulfate and concentrated in vacuo. The crude product was puri-
fied by silica gel column chromatography eluting with petroleum ether/
ethyl acetate (20:1, V: V) to afford 3-oxo-OA as a white solid (1.97 g,
2.7. Transmission electron microscopy
H322 cells (2 × 105 cells/ml) were plated into a 6-well plate and further
incubated at 37 °C for 24 h. After treatment with SZC015 (20 μmol/l) for
24 h, cells were washed, collected, and prefixed in 2.5% glutaraldehyde
overnight at 4 °C. Cells were then post fixed in osmium tetroxide for
30 min, dehydrated in a 10% graded series of 50%–100% ethanol, and em-
bedded in the Epon 812 resin. The ultrathin sections were cut and stained.
Finally, the ultrathin sections were stained and imaged using a transmission
electron microscope (JEM-2000EX; JEOL Co; Japan).
98.2% yield). m p. 181–182 °C. Positive ESI-TOF-MS: m/z 499.2864
+
(M + 2Na-H)
.
1H NMR (CDCl3, 400 MHz): 0.81, 0.91, 0.93, 1.03,
1.05, 1.08, 1.15 (each s, 3H, 7 × CH3), 5.30 (1H, t, J = 3.3 Hz, H-12),
2.84 (1H, dd, J = 13.6, 3.8 Hz, H-18), 2.33–2.55 (2H, m, H-2).
2.8. Cell apoptosis analysis
2.3. 2-Morpholinomethyl-3-oxo-olean-12-en-28-oic acid (SZC015)
To determine whether apoptosis involves in the inhibition effect of
SZC015 on cell viability in H322 cells, the Annexin V-FITC Apoptosis De-
tection kit was used according to the manufacturer's instructions. Brief-
ly, H322 cells were seeded in 6-well plates and further cultured at 37 °C
for 24 h. Then cells were treated with different concentrations of
SZC015 for 24 h. H322 cells were washed, harvested, and collected. Fi-
nally, the samples were stained with 5 μl of Annexin-V FITC and
propidium iodide for 30 min in the dark at room temperature. These
samples were then analyzed by flow cytometry immediately (BD
FACSAria II; BD Co; America).
Morpholine hydrochloride 1.23 g (10.0 mmol), SnCl2 0.38 g
(2.0 mmol) and paraformaldehyde 0.3 g were added to a solution of
3-oxo-OA (0.91 g, 2.0 mmol) in 40 ml ethanol. The reaction mixture
was heated at reflux for 20 h and then filtrated. The filtrate was concen-
trated to dryness under reduced pressure. The residue was dissolved in
200 ml ethyl acetate, then washed with saturated brine (50 ml × 3). The
organic layer was dried with anhydrous sodium sulfate and concentrat-
ed in vacuo. The crude product was purified by silica gel column chro-
matography eluting with chloroform/ethanol/water (15:1:0.1, V:V:V)
to give compound SZC015 as a pale yellow solid (0.17 g, 15.3% yield).
mp 162–164 °C. MS (API-ES Negative)m/z:588.5 (M + Cl)−;(API-ES
2.9. Propidium iodide staining
Positive)m/z 554.5(M + H)+ 1H NMR (CDCl3, 400 MHz), 0.83, 0.90,
.
0.93, 1.07, 1.10, 1.11, 1.22 (each s, 3H, 7 × CH3), 2.59 (t, 4H,
N(CH2CH2)2O), 2.85 (dd, 1H, J = 3.4, 13.0 Hz, H-18), 3.77 (brs, 4H, N
(CH2CH2)2O), 5.29 (brs, 1H, J = 3.3 Hz, H-12). 13C NMR (100 MHz,
CDCl3), δ: 207.4, 183.0, 143.7, 122.2, 66.4, 58.2, 54.1, 53.4, 52.8, 48.5,
47.9, 47.1, 47.0, 46.6, 45.4, 41.9, 41.9, 39.5, 39.1, 38.6, 36.7, 33.9, 32.5,
30.7, 29.7, 28.2, 25.8, 25.8, 25.4, 23.7, 20.2, 18.0, 17.3. The derivatives
were dissolved in DMSO and stored at 4 °C.
To determine whether cell cycle arrest involves in the inhibition ef-
fect of SZC015 on cell viability in H322 cells. Cells were plated in 6-well
plates, incubated at 37 °C for 24 h then cells were treated with different
concentrations of SZC015 for 24 h, subsequently collected, and fixed in
70% cold ethanol overnight at 4 °C. The samples were resuspended
with the cell cycle analysis solution that contains 50 mg/ml PI and
1 mg/ml RNAse in 1 ml of sodium citrate buffer, and then incubated in