B.-Y. Lin, et al.
EuropeanJournalofPharmacology877(2020)173093
performed using intermediates 2 and 5. Intermediate 5 (5 mmol,
0.903 g) was slowly added to a solution of 2 (5 mmol, 0.685 g) in 50 ml
of isopropanol. After reacting at room temperature for 8–10 h, iso-
propanol was removed under a vacuum. The pH of the reaction system
was adjusted to 9 with NaHCO3 and then extracted with EtOAc. The
organic phase was concentrated to obtain the crude product and then
2.2.7. Colchicine competitive binding inhibition assay
An assay of the competitive inhibition of colchicine binding was
conducted using protocol reported in the literature (Saeedian
a
tained 0.1 mg/ml tubulin, 1.0 M monosodium glutamate, 1.0 mM
MgCl2, 0.1 M glucose-1-phosphate, 1.0 mM GTP, 0.5 mg/ml BSA,
5.0 μM [3H]colchicine, and different concentrations of the tested
compound) was warmed to 37 °C and incubated for 30 min. After in-
cubation, the reaction mixtures were quenched with ice-cold water. The
mixtures were then filtered with 0.45-μm nitrocellulose Millipore filter
and washed three times with ice-cold water. Finally, 3 ml of scintilla-
tion liquid solution was added to each sample. After vortexing, the
radioactivity of the samples was recorded with a liquid scintillation
counter. The obtained values represent the averages of three in-
dependent experiments and three independent samples.
purified
by
column
chromatography
(EtOAc:
petroleum
ether = 20:1–3:1) to afford the target compound, AQ-4.
2.2.2. Structural identification and purity testing of the target compound,
AQ-4
The chemical structure of the novel compound, AQ-4, was identified
by 1H NMR and 13C NMR spectra at 400 and 101 MHz, respectively. The
spectra were recorded using TMS as the internal standard on Bruker
BioSpin GmbH spectrometer higher solution mass spectra (HR-MS). The
purity of AQ-4 was detected by HPLC with
a C18 column
2.2.8. Immunofluorescence microscopy
(4.6 mm × 150 mm, 5 μm, Phenomenex, Torrance, CA, USA). Five
microliters of sample were injected and separated in isogradient chro-
matography condition which was consist of 50% H2O (A) and 50%
acetonitrile (B). The mobile phase flow rate was set to 0.5 ml/min.
After plating in a confocal culture dish, 1 × 105 cells were grown
for 24 h before incubation with or without AQ-4 (1, 10, or 20 nM) for
12 h. After washing with phosphate-buffered saline (PBS), the cells
were fixed with 4% paraformaldehyde for 15 min, permeabilized with
0.01% Triton X-100 for 15 min, and incubated with 10% goat serum for
30 min at room temperature. Subsequently, the cells were incubated
with tubulin antibody at 4 °C overnight. The nuclei were stained with
Hoechst 33342 (10 μg/ml) in the dark at room temperature for 30 min
after incubated with the Alexa-Fluor 488-conjugated antibody for 1 h.
Images of intracellular microtubule were captured using a Zeiss LSM
570 laser scanning confocal microscope (Carl Zeiss, Germany).
2.2.3. Cell culture
The human cancer cell lines used in this study were purchased from
Shanghai Institute of Cell Resource Center of Life Science (Shanghai,
China). Cells were cultured in DMEM or RPMI 1640 containing 10% (v/
v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and
100 μg/ml streptomycin. Cells were maintained at 37 °C in an atmo-
sphere with 5% CO2 and 90% relative humidity.
2.2.9. Flow cytometry of cell cycle analysis
A549 cells in the logarithmic phase of growth were seeded in 6-well
plates at a density of 2 × 105 cells/well and grown for 24 h.
Subsequently, the cells were treated with or without AQ-4 (1, 10,
20 nM) for indicated times. The harvested cells were pre-incubated with
RNAse A (Keygen Biotech, China) at 37 °C for 30 min, and then stained
with the PI solution (Keygen Biotech) for another 30 min. Finally, ap-
proximately 15,000 events were collected for detection by flow cyto-
metry (Beckman Coulter, Epics XL). The percentages of cells in each
phase of the cell cycle were analyzed using the EXPO32 ADC analysis
software.
2.2.4. Antiproliferative activity evaluation
Cells in the logarithmic phase of growth were treated with trypsin.
Afterwards, approximately 5 × 103 cells were seeded in 96-well plates
and incubation for additional 24 h. After the cells were incubated with
or without different concentrations of the test compounds (from
100 μM to 1 nM) for 48 h, 10 μl of CCK-8 solution was added. After
2–4 h incubation, the absorbance was recorded at 450 nm using a
multifunction microplate reader (Multiskan™ FC, ThermoFisher
Scientific). Each experiment was independently repeated at least three
times. IC50 values (the drug concentrations required to inhibit cell
growth by 50%) were calculated with GraphPad Prism Software version
5.02 (GraphPad Inc., San Diego, USA).
2.2.10. Flow cytometry of apoptosis analysis
The A549 cell sample was prepared using the method described for
the cell cycle analysis. Cells harvested by centrifugation were treated
with 5 μL of Annexin-V/FITC binding buffer (10 mM HEPES, 140 mM
NaCl, and 2.5 mM CaCl2 at pH 7.4, Keygen Biotech, China) for 15 min,
and subsequently incubated in the dark with PI solution for another
15 min. Finally, approximately 15,000 events from each sample were
analyzed by flow cytometry (Beckman Coulter, Epics XL). Apoptotic
cells in each stage were calculated using EXPO32 ADC Analysis soft-
ware.
2.2.5. Metabolic stability study
The in vitro metabolic stability of AQ-4, CA-4 and Verubulin was
analyzed using a method reported in the literature. Briefly, compounds
were incubated with commercially available human or rat liver mi-
crosomes in a NADPH regenerating system. The reaction system volume
was set at 500 μl, and the final concentration of microsomes was 1 mg/
ml. After the mixture was shaken incubated at 37 °C for indicated times,
500 μl of ice-cold acetonitrile was added to terminate the reaction. The
supernatants were harvested through centrifuging at 12,500 g for
10 min at 4 °C and then directly analyzed using liquid chromatography-
mass spectrometry (LC-MS/MS).
2.2.11. Caspase-3 activity evaluation
A549 cells were seeded in a 96-well plate and grown for 24 h. Then
the cells were treated with or without different concentrations of AQ-4
(1, 10, 20 nM) for 24 h. After replacing the medium with a fresh
medium containing 5 μM GreenNuc™ Caspase-3 substrate solution, cells
were incubated in the dark for another 30 min. Caspase-3 activity was
measured with a multifunction microplate reader (Multiskan™ FC,
Thermo Fisher Scientific) at an emission wavelength of 410 nm and
excitation wavelength of 340 nm.
2.2.6. In vitro tubulin polymerization inhibition assay
A cell-free purified tubulin polymerization assay was performed as
(0.4 mg, > 97% purity) was pre-mixed with the test compounds at
37 °C for 1 min in 100 μl general tubulin buffer solution (80 mM PIPES,
2.0 mM MgCl2, 0.5 mM EGTA, and 1 mM GTP, and 10.2% glycerol, pH
6.9). After the addition of 55 μl of the tubulin solution, the reaction was
initiated and fluorescence intensity increment was monitored every
60 s at a wavelength of 340 nm for 80 min using a multifunction mi-
croplate reader (Multiskan™ FC, ThermoFisher Scientific).
2.2.12. Mitochondrial membrane potential (MMP) detection
JC-1 (Beyotime, China), a lipophilic cationic dye, was applied to
monitor the state of intracellular MMP. In the normal state, when MMP
is high, JC-1 aggregates to display bright red fluorescence.
3