Apoptosis and DNA intercalating activities of novel emodin derivatives

Article abstract of DOI:10.1039/c3ra23149f

Emodin belongs to the anthraquinone class of compounds and is the major bioactive compound of several herb species. A library of novel emodin derivatives was synthesized and their antiproliferative activities were evaluated against HepG2, PC-3, DU-145, MC

Full text of DOI:10.1039/c3ra23149f

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Apoptosis and DNA intercalating activities of novel
emodin derivatives3
Cite this: RSC Advances, 2013, 3,
6123
T. Narender,*a P. Sukanya,a Komal Sharmab and Surendar Reddy Bathula*b
Emodin belongs to the anthraquinone class of compounds and is the major bioactive compound of several
herb species. A library of novel emodin derivatives was synthesized and their antiproliferative activities
were evaluated against HepG2, PC-3, DU-145, MCF-7, and HEK-293 cell lines. Among these derivatives, one
showed a higher order of in vitro anticancer activity profile similar to the market drug epirubicin. The
derivative strongly inhibited the proliferation of HepG2, PC-3, DU-145 and MCF-7 cancer cell lines with IC50
values of 3.5, 5.6, 7.5, and 4.5 mM respectively. The derivative was also capable of inducing caspase-3-
mediated apoptosis in HepG2 cells by arresting the cell cycle in the S phase. Furthermore, the derivative
exhibited DNA intercalating ability comparable to doxorubicin, thus making this compound a promising
potential antitumor drug.
Received 3rd December 2012,
Accepted 6th February 2013
DOI: 10.1039/c3ra23149f
www.rsc.org/advances
properties using semi-synthetic approaches. For instance Tan
et al. have reported DNA binding pyrazole emodins,19 Eger
1. Introduction
Anthraquinones are widely distributed secondary metabolites
of plants (aloe, cascara sagrada, Senna and rhubarb), microbes,
lichens and insects, which possess various biological activ-
ities.1–3 Anthraquinones isolated from microbes such as
daunorubicin (I), doxorubicin (II), epirubicin (III) and several
synthetic analogues such as mitoxantrone (IV) and pixantrone
(V) (Fig. 1) are in clinical use for the treatment of various
cancers. Emodin (1) belongs to the anthraquinone class of
compounds and is the major bioactive compound of numer-
ous herb species such as Rheum, Polygonum (Polygonaceae),
Rhamnus (Rhamnaceae) and Cassieae (Senna)4–6 and possesses
immunosuppressive, anticancer, anti-inflammatory, anti-
atherosclerotic, and vasorelaxant effects.7–10 Several reports
have shown that emodin (1) has antiproliferative effects on
many kinds of cancer cell lines such as HER-2/neu over-
expressing breast cancer,11,12 leukemia,13,14 hepatoma,15 and
lung16 cancer cells. Also, emodin-triggered apoptosis is
mediated through the caspase and mitochondria dependent
pathways in proximal tubular epithelial HK-2 cells.17 Emodin
enhanced gefitinib induced cytotoxicity via Rad51 down
regulation and ERK1/2 inactivation in human breast cancer
B Cap-37 cells.18 Various research groups have derivatized
emodin with enhanced antitumor activities and DNA binding
et al. reported annulated tetrahydropyrazine derivatives of
emodins,20 Song et al. developed anthracene L-rhamnopyrano-
sides21 and Wang et al. synthesized emodin quaternary
ammonium salt derivatives.22
As a part of our drug discovery program on anticancer
agents from Indian medicinal plants, we isolated large
quantities of emodin (1) from the roots of Rheum emodi and
planned to carry out chemical transformations to improve its
therapeutic applications. Chemical transformation of the
bioactive compounds of medicinal herbs is one of the most
common approaches in drug discovery to improve therapeutic
properties. For example the anticancer drugs teniposide and
etoposide are derivatives of podophyllotoxin and topotecan
and irinotecan are analogues of camptothecin, which have
better therapeutic benefits than the parent natural products.
Towards this goal, we have synthesized a series of novel
O-alkylated derivatives 2–12 (Scheme 1), ester derivatives 13–16
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute,
Lucknow-226 001, U.P., India. E-mail: t_narendra@cdri.res.in (Chemistry); Fax: +91
522 2623405; Tel: +91 522 2612411
bPharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow-226 001,
U.P., India. E-mail: bsreddy@cdri.res.in (Biology)
Electronic supplementary information (ESI) available: Spectra (1H NMR, 13C
3
Fig. 1 Anticancer drugs with the anthraquinone skeleton: daunorubicin (I),
doxorubicin (II), epirubicin (III), mitoxantrone (IV), and pixantrone (V).
NMR and mass) of all compounds associated with this article. See DOI: 10.1039/
c3ra23149f
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Scheme 1 Synthesis of O-alkylated derivatives 2–12 of emodin (1). Reagents
and conditions: (a) RX, K2CO3, DMF, 60 uC, 4 h, yield: 62–80%.
Scheme 3 Synthesis of amide derivatives 19 and 20 from emodin. Reagents and
conditions: (a) Ac2O, pyridine, 60–70 uC, 4 h, yield: 98%; (b) glacial AcOH : Ac2O
(1 : 1), CrO3, 70 uC, 10 h, yield: 80%; (c) benzene, SOCl2, 70 uC, 4 h; (d) RNH2,
DCM, Et3N, 0 uC, 1 h, yield: 85%.
(Scheme 2) and amide derivatives 19, 20 (Scheme 3) from 1
and evaluated their anticancer activity against HepG2 (human
hepatocellular carcinoma), MCF-7 (breast adenocarcinoma),
DU-145 and PC-3 (human prostate cancer cells), and HEK-293
(human embryonic kidney) cell lines (Table 1). Further, we
have studied the apoptosis inducing ability, effect on cell cycle
and DNA binding properties of the most active emodin
derivative 11.
2.1.2. Collection of medicinal plant. Rheum emodi Wall.
(bark) was purchased from the local market of Lucknow, India
and the authentication was carried out by the Botany Division
of Central Drug Research Institute, Lucknow.
2.1.3. Extraction. Powdered bark of Rheum emodi Wall.
(bark) (3 kg) was placed in a glass percolator with 95% ethanol
(10 L) and allowed to stand for 24 h at room temperature. The
percolate was collected and these processes were repeated four
times. The combined percolate was evaporated under reduced
pressure at 50 uC to afford the ethanol extract. The weight of
the extract was found to be 200 g.
2. Materials and methods
2.1. General chemistry
Melting points were recorded on a Buchi-530 capillary melting
point apparatus and are uncorrected. IR spectra were recorded
on a Perkin-Elmer RX-1 spectrometer using either KBr pellets
or neat. 1H-NMR, 13C-NMR, DEPT-90 and DEPT-135 spectra
were run on Bruker Advance DPX 300 MHz and 200 MHz
spectrometers in CDCl3, DMSO-d6 and CD3OD. Chemical
shifts are reported as values in ppm relative to CHCl3(7.26)
in CDCl3 and TMS was used as an internal standard. ESI mass
2.1.4. Isolation and purification of emodin. The alcoholic
extract (200 g) was chromatographed on a column of silica gel
(60–120 mesh), eluted with hexane and chloroform (70 : 30)
and recrystallized from methanol to afford emodin (3 g).
Compound visualization was carried out under UV light, also
showing an orange spot by spraying with 10% sulfuric acid in
methanol.
spectra were recorded on
a
JEOL SX 102/DA-6000.
2.2. Preparation of emodin derivatives
Chromatography was executed with silica gel (60–120 mesh)
using mixtures of chloroform, ethyl acetate and hexane as
eluants. Visualization was done under UV light and spraying
with 10% sulfuric acid in methanol.
2.1.1. Plant background. Rheum emodi Wall. (Family:
Polygonaceae, commonly known as Revand chini and English
name rhubarb) is a stout herb, distributed in the alpine and
sub-alpine zones of the Himalayas.23,24 The roots of this
species are used widely in ayurvedic medicine. Roots of the
Indian rhubarb are inferior in aroma, and are a well known
stomachic, bitter and cathartic and used all over the world.
2.2.1. General method for O-alkylation (Method A). To a
magnetically stirred solution of compound 1 (100 mg, 0.37
mmol) in DMF (5 mL) at room temperature was added the
respective aliphatic halide (RX) (0.33 mmol) and K2CO3 (0.92
mmol). The whole reaction mixture was stirred for 4 h at 60–70
uC. It was then extracted with ethyl acetate (3 6 25 mL). The
organic layer was washed with water, dried over anhydrous
Na2SO4 and evaporated under reduced pressure. Then the
crude product was chromatographed on silica gel to afford the
desired compound.
2.2.2. General method for esterification (Method B). To a
magnetically stirred solution of the appropriate acid (0.44
mmol) and compound 1 (100 mg, 0.37 mmol) in dry DCM (5
mL), was added DIPC (0.55 mmol) and DIEA (0.37 mmol). The
whole reaction mixture was stirred for
6 h at room
temperature. It was then extracted with ethyl acetate (3 6 25
mL). The organic layer was washed with water, dried over
anhydrous Na2SO4 and evaporated under reduced pressure.
Then the crude product was chromatographed on silica gel to
afford the desired compound.
Scheme 2 Synthesis of ester derivatives 13–16 from emodin (1). Reagents and
conditions: (a) acid, dry DCM, DIPC, DIEA, 0 uC–rt, 6 h, yield: 60–65%.
2.2.3. General method for preparation of amide derivatives
(Method C). Compound 1 (100 mg, 0.37 mmol) was magne-
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Table 1 Chemical structures and in vitro anticancer activities (IC50 in mM) of emodin (1) and its derivatives (2–20)
IC50 (mM)
Comp. No.
Chemical structure
HepG2
PC-3
145 ¡ 0.02
DU-145
MCF-7
HEK-293
1
119 ¡ 0.16
114 ¡ 0.06
162 ¡ 0.09
150 ¡ 0.04
2
3
127 ¡ 0.14
48 ¡ 0.12
137 ¡ 0.01
76 ¡ 0.13
124 ¡ 0.024
140 ¡ 0.054
162 ¡ 0.011
49 ¡ 0.024
78 ¡ 0.21
69 ¡ 0.32
4
5
127 ¡ 0.11
130 ¡ 0.06
114 ¡ 0.03
125 ¡ 0.87
122 ¡ 0.9
92 ¡ 0.029
56 ¡ 0.012
110 ¡ 0.9
87 ¡ 0.1
75 ¡ 0.43
6
7
8
9
100 ¡ 0.069
103 ¡ 0.022
36 ¡ 0.149
15 ¡ 0.032
97 ¡ 0.024
120 ¡ 0.017
37 ¡ 0.07
82 ¡ 0.03
125 ¡ 0.07
34 ¡ 0.033
11 ¡ 0.099
89 ¡ 0.09
114 ¡ 0.04
39 ¡ 0.012
10 ¡ 0.078
90 ¡ 0.02
140 ¡ 0.09
42 ¡ 0.067
17 ¡ 0.056
12 ¡ 0.08
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Table 1 (Continued)
IC50 (mM)
Comp. No.
Chemical structure
HepG2
PC-3
22 ¡ 0.02
DU-145
MCF-7
20 ¡ 0.04
HEK-293
10
25 ¡ 0.055
3.5 ¡ 0.11
98 ¡ 0.036
29 ¡ 0.043
27 ¡ 0.08
11
12
5.6 ¡ 0.01
7.5 ¡ 0.013
4.5 ¡ 0.022
12 ¡ 0.9
89 ¡ 0.02
86 ¡ 0.012
94 ¡ 0.011
84 ¡ 0.09
13
53 ¡ 0.13
49 ¡ 0.03
51 ¡ 0.045
45 ¡ 0.023
57 ¡ 0.043
14
15
74 ¡ 0.16
80 ¡ 0.011
86 ¡ 0.077
78 ¡ 0.05
84 ¡ 0.06
130 ¡ 0.01
112 ¡ 0.013
129 ¡ 0.013
124 ¡ 0.034
119 ¡ 0.07
16
25 ¡ 0.05
32 ¡ 0.08
29 ¡ 0.014
22 ¡ 0.09
34 ¡ 0.3
17
18
34 ¡ 0.13
42 ¡ 0.12
36 ¡ 0.02
24 ¡ 0.07
32 ¡ 0.04
27 ¡ 0.09
46 ¡ 0.05
218 ¡ 0.14
41 ¡ 0.019
40 ¡ 0.9
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Table 1 (Continued)
IC50 (mM)
Comp. No.
Chemical structure
HepG2
PC-3
20 ¡ 0.09
DU-145
39 ¡ 0.01
MCF-7
24 ¡ 0.07
HEK-293
19
19 ¡ 0.003
20 ¡ 0.019
4.6 ¡ 0.9
42 ¡ 0.06
20
III
19 ¡ 0.06
25 ¡ 0.014
21 ¡ 0.05
29 ¡ 0.06
Epirubicin (marketed drug)
9.9 ¡ 0.03
5.7 ¡ 0.01
3.7 ¡ 0.08
4.2 ¡ 0.09
tically stirred in a solution of pyridine (2 mL) and acetic
anhydride (1.85 mmol) at 60–70 uC for 4 h. The reaction
mixture was put into cold water for crystallization, then
filtered and dried. To the resultant crude acetate was gradually
added 10 mL of a mixture of acetic anhydride and glacial acetic
acid (1 : 1) and CrO3 at 45 uC and the resulting mixture stirred
for 10 h at 70 uC. The acetic anhydride and glacial acetic acid
mixture was removed by vacuum. The residue was then
extracted with ethyl acetate (3 6 25 mL). The organic layer
was washed with water, dried over anhydrous Na2SO4 and
evaporated under reduced pressure. Then the crude product
was chromatographed on silica gel to afford the desired
compound. The acid was magnetically stirred in a solution of
benzene (2 mL) and SOCl2 (5 mL) at 70 uC for 4 h. Benzene was
removed by vacuum. To the residue was then added dry DCM,
amine (0.25 mmol) and triethylamine (0.38 mmol) and the
mixture maintained at 0 uC for 1 h. DCM was removed by
vacuum. The residue was then extracted with ethyl acetate (3
6 25 mL), and the organic layer was washed with water, dried
over anhydrous Na2SO4 and evaporated under reduced
pressure. Then the crude product was chromatographed on
silica gel to afford the desired compound.
purchased from Calbiochem. MTT dye was procured from
Sigma-Aldrich. All the flow cytometry experiments were
performed using a FACScan (Becton Dickinson, Mountain
View, CA) flow cytometer, equipped with a single 488 nm argon
laser. All the experiments with live animals were done under
the animal protocol approved by the CDRI Institutional
Animal Ethics Committee.
2.3.2. MTT assay for cell viability. Cell viability was assessed
by MTT assay, which is based on the reduction of MTT by
mitochondrial dehydrogenases of viable cells to form a purple
formazan product. Briefly, cells (5 6 103/well) were plated in
96 well plates. After incubating overnight, the cells were
treated with six different concentrations (100, 50, 20, 10, 5, 2.5
mM) as triplicates of emodin derivatives for 48 h. Subsequently,
10 mL of MTT (10 mg mL21) was added to each well and
incubated for 3 h. The MTT formazan formed by viable cells
was dissolved in 100 mL of DMSO and shaken for 10 min. The
absorbance was measured on an ELISA reader. Each test was
repeated at least three times. The concentration of the
compound which gives the 50% growth inhibition value
corresponds to IC50, calculated using GraphPad prism 5.
2.3.3. Apoptosis studies. Quantitation of apoptotic cells by
annexin V staining was carried out according to the manufac-
turer’s instructions. Briefly, cells (5 6 105 cells/well) were
seeded in 6 well plates and treated with 5, 10 and 20 mM of
derivative 11 for 24 h. After incubation, cells were washed with
PBS stained with 1.25 mL of annexin V-FITC and 10 mL of media
binding reagent and incubated for 20 min. After that 10 mL of
propidium iodide (PI) was added and samples were analysed
using a flow cytometer. Annexin V-FITC was analyzed using
excitation and emission settings of 488 nm and 535 nm (FL-1
channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and
clumps were gated out using forward and orthogonal light
scatter. Each experiment was repeated two times indepen-
dently.
2.3. Biological studies
2.3.1. Cell lines and culture. The human cancer cell lines e.g.
HepG2 (hepatocellular carcinoma), DU-145 and PC-3 (human
prostate cancer cells), MCF-7 (breast adenocarcinoma), and
normal non-transformed cell type, human embryonic kidney
(HEK-293), used in the present study were obtained from the
ATCC (American Type Culture Collection, USA). Mouse skin
fibroblasts were isolated from mice maintained in RPMI 1640
(Roswell Park Memorial Institute, Merck) with 10% FBS
(Merck), supplemented with 1% antibiotic and antimycotic
solution (Gibco, USA) at 37 uC in a humidified incubator with
5% CO2. All stock solutions of the compounds were prepared
in cell culture grade DMSO (dimethyl sulfoxide) and stored at
220 uC. Compounds were diluted in culture media prior to use
in experiments. Annexin V FITC apoptosis detection kits were
2.3.4. Cell cycle analysis. For cell cycle distribution studies
post-treatment, cells were fixed in 70% ethanol overnight,
rehydrated in PBS with ribonuclease A (100 mg mL21) and
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Triton-X (1%) for 30 min at room temperature then PI (50 mg
ml21) added before incubation in the dark for 30 min and
analysis with a flow cytometer. Experiments were repeated two
times independently.
acid derivative 18 (Scheme 3).27 The resultant acid 18 was
converted to its acid chloride by using SOCl2 in benzene at 70
uC, then further reacted with alkylamine in the presence of
triethylamine in situ at 0 uC to obtain amides 19 and 20
(Scheme 3).
2.3.5. Caspase-3 activation assay. The activity of caspase-3
was determined using a caspase-3 colorimetric assay kit
(Sigma Aldrich) according to the manufacturer’s protocol.
Briefly, HepG2 cells (1 6 106 cells/well) were treated with 5, 10
and 20 mM of derivative 11 for 24 h. The cells were harvested
and lysed by addition of lysis buffer. Cell lysates were mixed
with colorimetric substrate (Ac-DEVD-pNA) and incubated at
37 uC in the dark for 4 h. The absorbance was measured at 405
nm in an ELISA reader. Caspase-3 activity was expressed as the
change of the activity compared to the control.
2.3.6. DNA intercalation assay. Calf thymus DNA (8.8 mM)
was loaded with Tris buffer containing ethidium bromide (4.4
mM). To each well were added compounds 9, 11, 19, etoposide
and DOX at 5, 10 and 20 mM. After incubation for 30 min at 25
uC fluorescence was measured by using a fluorimeter in
duplicate experiments with two control wells (no compound =
3.2. Biology
3.2.1. Anti-proliferative activity. All the emodin derivatives
(2–20) were evaluated for in vitro anticancer activity using an
MTT assay across different molar concentrations (2.5, 5, 10,
20, 50, 100 mM) as triplicates. The growth-inhibitory effects
were studied in four human cancer cell lines, HepG2
(hepatocellular carcinoma), DU-145 and PC-3 (human prostate
cancer cells), MCF-7 (breast adenocarcinoma) and one non-
cancer cell line, HEK-293 (human embryonic kidney). The
parent compound, emodin (1) has an IC50 of 119, 145, 114, 162
and 150 mM against HepG2, PC-3, DU-145, MCF-7 and HEK-
293 respectively (Table 1). All the derivatives 2–20 prepared
from emodin (1) exhibited improved in vitro anticancer activity
against HepG2, PC-3, and MCF-7 cell lines compared to parent
compound 1 (Table 1). Except 2, 7, 19, all other derivatives also
exhibited improved activity against the DU-145 cell line.
Among all these derivatives (2–20), the O-alkylated derivative
with a basic amine group (11) turned out to be the most potent
antiproliferative agent with IC50 values of 3.5, 5.6, 7.5 and 4.5
mM against HepG2, PC-3, DU-145 and MCF-7 cancer cell lines
respectively, which are comparable to those of the marketed
anthraquinone class of anticancer drug (epirubicin IC50 values
of 4.6, 9.9, 5.7 and 3.7 mM). The O-geranylated derivative (9)
also showed significant antiproliferative activity against all the
cancer cell lines (IC50 values of 15, 12, 11 and 10 mM) followed
by O-propyne derivative 10 (IC50 values of 25, 22, 29 and 20
mM). Among other O-alkylated derivatives (2–8), compound 8
with a C-22 alkyl chain showed good antiproliferative activity
(IC50 values of 36, 37, 34, 39 and 42 mM) followed by derivative
3 with a C-12 alkyl chain (IC50 values of 48, 76, 78, 69 and 49
mM). Esters 13–17 prepared from 1 also exhibited improved
activity over the parent compound. The nicotinic acid
derivative (16) and triacetate (17) have better activity profiles
than other ester derivatives 13–15 with IC50 values of 25, 32,
29, 22 mM and 34, 42, 36, 24 mM against HepG2, PC-3, DU-145
and MCF-7 cancer cell lines respectively. The amides 19 and 20
prepared from acid derivative 18 exhibited good antiprolifera-
tive activities with IC50 values of 19, 20, 39 and 24 mM and 20,
19, 25 and 21 mM against HepG2, PC-3, DU-145 and MCF-7
cancer cell lines respectively. The most active compounds 9, 11
and19 were also tested against mouse skin fibroblasts (Fig. S1,
100% fluorescence and no DNA
= 0% fluorescence).
Doxorubicin was used as a positive control for the DNA
intercalation assay while etoposide was used as a negative
control. Fluorescence readings are reported as % fluorescence
readings compared to controls.
2.4. Statistical analysis
All data are expressed as mean ¡ standard deviation for three
experiments. A probability value of p , 0.05 was considered
statistically significant. All the statistical analysis was per-
formed using Graph Pad Prism version 5.00 for Windows
(Graph Pad Software, USA).
3. Results and discussion
3.1. Chemistry
3.1.1. O-Alkylation. In order to synthesize O-alkylated
derivatives emodin (1) was reacted with various long chain
alkyl halides, geranyl bromide, 3-bromopropyne, 2-bromo-
N,N-dimethylethanamine and N-(2-bromoethyl)-N-isopropyl-
propan-2-amine under basic conditions using K2CO3 and
DMF as a solvent (Scheme 1). Out of three hydroxyls of emodin
(1), two hydroxyls are involved in chelation with a carbonyl
group, therefore only meta-substituted derivatives 2–12 were
obtained. The presence of chelated hydroxyls was confirmed
1
from H-NMR spectral data (please see ESI ).
3
ESI ). These compounds did not show any significant
cytotoxicity against them.
3
3.1.2. Esterification. Since emodin (1) contains free hydroxyl
groups we planned to prepare several esters to study their
anticancer activities. Emodin (1) was reacted with the
respective aromatic acids in the presence of DIPC and DIEA
in dry DCM at 0 uC to rt (Scheme 2), which provided the meta-
substituted ester derivatives 13–16.25,26
3.1.3. Amide formation. To transform the methyl group into
an amide functionality, emodin (1) was converted to its
triacetate 17 with acetic anhydride in the presence of pyridine
and subsequently oxidized with chromium trioxide in the
presence of glacial acetic acid and acetic anhydride to provide
3.2.2. Apoptosis studies. Most of the currently used cytotoxic
drugs induce cell killing by the use of a process known as
apoptosis.28 In this study we measured the apoptosis inducing
ability of derivative 11 in HepG2 cells by flow cytometry.
Annexin V specially binds to phosphatidylserine and has been
employed in the determination of apoptotic cells. Annexin V/PI
staining was performed to determine early (annexin V positive,
PI negative), late apoptotic (annexin V positive, PI positive) and
necrotic (annexin V negative, PI positive) cells followed by 24 h
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treatment with 5, 10 and 20 mM concentration of derivative 11
in HepG2 cells.
In Fig. 2, compared to the control, compound treatment
increased the number of both annexin V and PI positive cells
for all tested concentrations. It is evident from the above data
that derivative 11 induces apoptosis in HepG2 cells.
3.2.3. Cell cycle analysis. In this study we also examined the
effect of derivative 11 on the cell cycle of HepG2 cells by flow
cytometry in PI (propidium iodide) stained cells after
treatment with 11 for 24 h. Fig. 3 shows the DNA distribution
histograms of HepG2 cells in the absence (control) and
presence (5, 10, and 20 mM) of derivative 11. The treatment
of HepG2 cells with 11 led to an obvious increase in the
percentage of cells in the S phase, accompanied by a
corresponding reduction in the percentage of cells in the G0/
G1 phase in a concentration dependent manner. Treatments
with 5, 10 and 20 mM of derivative 11 caused an increase of the
S-phase population to 28.05%, 27.45%, and 66.59% respec-
tively, compared with the S-phase population of 15.80% of
untreated HepG2 cells, indicating the induction of S-phase
arrest by derivative 11. The above results suggest that the
antiproliferative mechanism of derivative 11 on HepG2 cells is
due to the S-phase arrest of the cell cycle.
Fig. 3 Effect of emodin derivative 11 on cell cycle progression of HepG2 cells.
Cells were treated with different concentrations of derivative 11 starting from 5,
10 and 20 mM for 24 h, washed with PBS, fixed in 70% ethanol and stained with
PBS containing PI (50 mg mL21), as described in the materials and methods
section. The percentages of cells at each cell cycle phase are shown, respectively.
3.2.4. Capase-3 activation. To find out whether the induction
of apoptosis by 11 is caspase mediated or not, we examined
the caspase-3 activation in HepG2 cells. Caspases are cysteinyl
aspartate peptidases and cleave substrate proteins at aspartate
residues upon receiving an apoptotic signal. Among these,
caspase-3 is a key protein in the apoptosis mechanism since it
executes both extrinsic and intrinsic pathways.29,30 Our studies
indicated a concentration dependent increase in caspase-3
activity with derivative 11 treatment as compared with the
untreated control. Caspase-3 activation was enhanced 2.8, 7.01
and 13.64 fold by 11 in cells treated with 5, 10 and 20 mM
respectively, when compared with the control group (Fig. 4).
These results confirmed that the apoptosis induction is due to
caspase-3 activation by derivative 11 in HepG2 cells.
3.2.5. DNA interaction. In this study we evaluated the DNA
interacting abilities of antiproliferative emodin derivatives 9,
11, and 19 using an ethidium bromide displacement assay.
The fluorescence intensity of ethidium bromide increases
upon the binding of DNA and subsequent displacement of
ethidium is apparent by a decrease in emission intensity upon
addition of a competitive inhibitor.31 We have included DOX, a
known DNA interacting anticancer drug for comparison. Calf
thymus DNA was preincubated with ethidium bromide for 30
min in buffer before newly synthesized compound addition.
Fig. 2 Analysis of apoptosis induction by compound 11 in the HepG2 cell line.
Cells were treated with different concentrations of 11 for 24 h and apoptosis
was analyzed using annexin V and propidium iodide double staining by flow
cytometry.
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inducing cell cycle arrest and caspase dependent apoptosis in
HepG2 cell lines. Along with those derivatives 9, 11 and 19
have shown comparable DNA intercalation with DOX. These
synthetic emodin derivatives may hold promise for developing
an alternative to the existing anthraquinone class of antic-
ancer agents. Further work is in progress in our laboratory to
evaluate their in vivo anticancer potentials and in-depth
mechanistic aspects.
Acknowledgements
Fig. 4 Measurement of caspase-3 activities. Dose-dependent induction of
caspase-3 in HepG2 cell lines. Colorimetric determination of general caspase
was done as described in the materials and methods section. Each data point is
the representation of triplicate treatments. (**P , 0.01 vs. negative control).
The authors are thankful to Dr T. K. Chakraborty, Director,
CSIR-CDRI for constant encouragement for the program on
natural products of biological importance. We also thank the
Ministry of Earth Sciences (MoES) and EMPOWER project
from CSIR, New Delhi for providing a research grant and
fellowship. Komal Sharma is thankful to DST for an INSPIRE
fellowship. Mr J. P. Chaturvedi, Miss Astha Shukla and Miss
Manali Agrawal are thanked for technical support, SAIF, CDRI
for spectral data. This is CSIR-C.D.R.I. Communication-8402.
Derivatives 9, 11 and 19 and etoposide (negative control) were
incubated with ethidium bromide DNA complex for 30 min
and the fluorescence measured. As shown in Fig. 5, DOX has a
very strong effect in this assay, while derivatives 9, 11 and 19
have comparable effect. Considering the nearly equipotent
DNA binding affinity of the derivative 11 with DOX, we can
conclude that the antiproliferative effect of the derivative 11 is
due to DNA intercalation.
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