6
S. Xu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
activity with the IC50 values from 0.093 to 1.101
the pharmacophore (3-OH/4-OCH3) are also found in other tubulin
inhibitors such as colchicin and combretastatin A4.
l
Μ. Interesting,
ploid peacks was still obvious at 50 nΜ (38.7%). However, when
the cells were exposed at relatively high concentration of 17
(100 or 500 nΜ), most of the cells died and the G2/M phase almost
disappeared; cell cycle arrest at the sub-G1 peack (a characteristic
hypodiploid peack) was found to be the main peak, which indi-
cated most cells underwent DNA fragment. These results are con-
sistent with the performance of most tubulin inhibitors.
To study the morphological alterations of cancer cells caused by
compound 17, human cervical carcinoma (Hela) cells were exposed
to different concentrations of 17 (0.05, 0.10 and 0.50 lΜ) for 24 h
To elucidate whether these compounds target the tubulin-
microtubule system, compounds 10 and 17, which showed better
anti-proliferative activity in the initial cytotoxicity screening, was
chosen to test the inhibitory effect on microtubule assembly
in vitro, with MPC-6827 as the reference compound. As shown in
Fig. 2, the increased fluorescence intensity with time of the purified
and unpolymerized tubulin control samples indicated that tubulin
polymerization had occurred. The IC50 of compounds 10, 17 and
and then observed under an LSM 570 laser confocal microscope. As
shown in Fig. 5, the integrity of the mitotic spindle and cellular
microtubule network of the Hela cells was obvious in the control
MPC-6827 were 6.11 0.12, 5.809 0.14, and 2.74 0.10
respectively.
lM,
Compound 17 displayed good antiproliferative activity and
effective tubulin polymerization inhibition, therefore we decided
to perform a molecular docking study to investigate the potential
binding site of compound 17 to colchicine binding site of tubu-
lin-microtubule system. Docking studies with the suite AutoDock
Vina predicted 17 to bind in an orientation analogous to colchicine.
As shown in Fig. 3A, colchicine could be re-docked in the colchicine
binding site in a similar conformation to the X-ray structure of the
colchicine (PDB code: 1SA0), which indicated the reliability of our
docking method. Then we applied this docking method to 17 (Fig.
3B). The docking results showed that 17 occupied the binding cav-
ity of tubulin. Fig. 3C and D provided further evidence that com-
pound 17 could bind the same site as MPC-6827. The binding
free energy of compound 17 and MPC-6827 inclusion complexes
with protein obtained from AutoDock-Vina are ꢀ8.6 and ꢀ7.9
kcal molꢀ1, respectively, which confirmed that 17-protein complex
is more stable than that of MPC-6827-protein.
Then the flow cytometry analysis was performed to study its
effect on the cell cycle using human cervical carcinoma (Hela) cell.
As indicated in Fig. 4, comparing with control, the tetraploid
peacks (28.3%) increased when Hela cells were treated with 17 at
50 nM for 24 h, indicating 17 induced cell cycle arrest at the G2/
M phase. The percentage of cells at the G2/M phase increased to
39.6% and 55.6% with the increase of 17 to 100, and 500 nM,
respectively. When incubation time was extended to 48 h, tetra-
group. After treatment with 17 at the concentration of 0.050
the spindle microtubule organization was significantly deranged.
At 0.50 Μ, the nucleus of the cells narrowed sharply and spindle
lΜ,
l
microtubules were disorganized completely.
Subsequently, apoptosis was measured using propidium iodide
(PI) and fluorescent immunolabeling of the protein annexin-V (V-
FITC) by flow cytometry. When Hela cells were treated for 48 h
with 17 at indicated concentrations, the Hela cells were harvested,
stained with Annexin V-FITC and PI, and analyzed by flow cytom-
etry. The results showed 10.44%, 23.21%, 69.90% and 0.18% of the
early and late apoptosis cells at the concentrations of 0.05, 0.1
and 0.5 lΜ or DMSO (0.01%), respectively (Fig. 6A). When the incu-
bation time was extended to 72 h (Fig. 6B), the early and late apop-
tosis cells increased to 17.22%, 32.72%, 84.69% and 0.18%,
respectively. These results indicated that 17 induced cell apoptosis
in a concentration- and time-dependent manner.
Mitochondria are highly dynamic organelles which play an
important role in regulating the life and death of cells, and its dys-
function is closely related to apoptosis. To study the possible
involvement of mitochondrial dysfunction in 17-induced apoptosis
of the cells, we performed the quantitative MMPs (mitochondrial
transmembrane potential) assay with JC-1 staining. The change
of MMP could be detected by flow cytometry analysis or a laser
scanning confocal microscope. As shown in Fig. 7, when the Hela
cells were exposed to the indicated concentrations (50, 100, and
Fig. 7. The Hela cells were treated with 17 at the indicated concentration for 24 h, followed by incubation with the fluorescence probe JC-1 for 30 min. Then, the cells were
analyzed by flow cytometry (A) or fluorescence microscopy (B). The experiments were performed at least three times, and the results of the representative experiments are
shown.
Xu, Shaoyu
