5
018
N. Kishore et al. / Bioorg. Med. Chem. 22 (2014) 5013–5019
Table 3
Activation (%) of caspase 3/7
more cytotoxic and specific against human cervical epithelial car-
cinoma (HeLa), prostate epithelial carcinoma (DU-145) and breast
adenocarcinoma (MCF-7) cancer cell lines as confirmed in cell
cycle, apoptosis and caspase 3/7 activation as compared to the
other derivatives. The derivative 2,5-dihydroxy-7-methyl-1,4-
naphthoquinone (19) was found to be the most potent among
all; though not inducing apoptosis up to 48 h suggested that may
be slow induction occurred, which had to be tested for longer time.
The derivative 5 (8-chloro-5-hydroxy-6-methyl-1,4-naphthoqui-
none) and 6 (5-hydroxy-6-methyl-1,4-naphtho-quinone) could be
further modified to reduce the toxicity.
Compounds
Caspase 3/7 activity (% above control)
Cisplatin
5.89
Compound 4
Compound 6
Compound 19
ꢀ5.21
ꢀ5.14
7.39
4
4
. Material and methods
.1. Cell culture
Four human cancer cell lines, breast adenocarcinoma (MCF-7),
cervical epithelial carcinoma (HeLa), oesophageal carcinoma
SNO) and prostate epithelial carcinoma (DU-145) (Highveld Bio-
(
logical, SA) were maintained in culture flasks in complete Mini-
mum Essential Medium, Eagle supplemented with 10% fetal
bovine serum (Highveld Biological, SA), in a humidified 5% CO
2
incubator at 37 °C. Upon reaching confluence, the cells were tryp-
sinized (0.25% trypsin containing 0.01% EDTA) for 10 min at 37 °C
Figure 5. Activation (%) of caspase 3/7.
and then stopped by the addition of complete medium. About
5
1
ꢁ 10 of the viable cells were then re-suspended in complete
medium. U937 cells were maintained in culture flasks in complete
RPMI 1640 medium (Sigma, Germany) supplemented with 10%
fetal bovine serum (Delta Bioproducts, SA), containing 25 mM
2
4 h, suggesting that apoptosis occurred very soon after exposure
and shorter incubation time may have been better. This is sup-
ported by the very low percentage of viable cells seen after only
HEPES and 2 mM glutamine, in a humidified 5% CO
7 °C.
2
incubator at
2
4 h. The compounds 2–5 and 19 significantly inhibited the growth
of HeLa and DU-145 cells in vitro, possibly by either down-regula-
tion of antiapoptotic Bcl-X and up-regulation of proapoptotic Bax
3
L
waf1/cip1
4.2. Cytotoxicity assay
or by increasing the p21
cycle arrest in G or G
phase.36 Copeland et al. has reported that
,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone exhibits cytotox-
icity on androgen-dependent and -independent prostate cancer
protein, which is involved in cell
1
2
Cytotoxicity of the adherent cells was measured by the XTT
2
0
(
Sodium 3 -[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-
3
7
methoxy-6-nitro) benzene sulfonic acid hydrate) method using
1
cell lines inducing apoptosis by arresting cell cycle at G phase.
the Cell Proliferation Kit II (Roche Diagnostics GmbH). The cancer
The cell cycle alterations indicate that cell cycle arrest is one of
the primary mechanisms responsible for the anticancer activity
of synthesized 7-MJ derivatives in HeLa and DU-145 cells.
5
cells (100
l
l) were seeded at 1 ꢁ 10 per ml onto a microtiter plate
and incubated for 24 h to allow the cells to attach to the bottom of
the plate. A dilution series was made for the compounds (0.1–
1
00
for 48 h. The XTT reagent was added to a final concentration of
.3 mg/ml and incubated for 1–2 h. After incubation the absor-
lg/ml), which was added to the microtiter plate and incubated
2
.6. Caspase 3/7 activity
0
Based on the cell cycle analysis, compounds 4, 6 and 19 were
bance of the colour complex was quantified at 490 nm using an
ELISA plate reader with a reference wavelength set at 690 nm.
The 50% cell survival (IC50) was analysed by using GraphPad Prism
further tested for induction of apoptosis. The induction of apopto-
sis was confirmed by induction of caspases. It is clear from Table 3
that after incubation cisplatin and compound 19 activated caspase
(
version 4) from the concentration-effect relationship.
3
6
/7 activity by 6.6% and 7.4%, respectively, while compounds 4 and
(Fig. 5) inhibited caspase 3/7 activity by ꢀ4.1% and ꢀ3.2%,
respectively. No activation of caspase 3/7 by compounds 4 and 6
suggests that they induce apoptosis through a different pathway.
Surprisingly, compound 19 activated caspase 3/7, although no
apoptosis was seen during cell cycle analysis. It is possible that
the induction and execution of apoptosis by 19 is slower than
the other compounds (may be more time is required for inducing
4.3. Peripheral blood mononuclear cells (PBMCs)
Blood was obtained from healthy adult volunteers. PBMCs were
separated with BD Vacutainer™ CPT™ cell preparation tubes con-
taining sodium heparin. The method was followed as published38
but with different final concentrations tested (1, 10 and 100 lg/
apoptosis). The increase in G
3.1% might be the first indication of cell cycle arrest that could
lead to apoptosis induction.
0
/G
1
observed at 24 h from 38.0% to
ml) using a fluorescence read at 560Ex/590Em using a Fluoroskan
Ascent FL fluorometer (ThermoLabsystems, Finland).
5
4
.4. Cell cycle analysis
3
. Conclusions
Compounds with the lowest IC50 values were further investi-
The compounds, 2,5-dihydroxy-7-methyl-1,4-naphthoquinone
19), 8-Chloro-5-hydroxy-6-methyl-1,4-naphthoquinone (5), 8-
fluoro-5-hydroxy-7-methyl-1,4-naphthoquinone (4) and 5-
hydroxy-6-methyl-1,4-naphtho-quinone (6) were found to be
gated for the mechanism of action. U937 cells were seeded in com-
6
(
plete RPMI 1640 medium at a density of 2 ꢁ 10 cells per millilitre
in flasks. After a recovery period of 24 h, the selected compounds
(1–6) were dissolved in DMSO (2 lg/ml) and added to the cells.