SAMPLE FULL PAPER
1”
Determination of growth inhibitory activities of several bioactive compounds of
Andrographis paniculata against a panel of human tumor cell lines: 14-deoxy-11, 12didehydroandrographolide induces a non-apoptotic programmed cell death in T-47D, a
breast carcinoma cell line
Tan Mei Lan1, Tengku Sifzizul Tengku Muhammad1, Masanori Kuroyanagi2, Shaida Fariza
Sulaiman1, Nazalan Najimudin1
1
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. 2School of
Bioresources, Hiroshima Prefectural University, Shobara-shi, Hiroshima 727 Japan.
1.25”
Introduction
Andrographis paniculata (Acanthaceae) or
widely known as Kalmegh in India was used as
a bitter ingredient in many of the traditional
formulations in the practice of Ayurveda. There
were about 26 polyherbal formulations of this
plant mentioned in Ayurveda as a popular
remedy for the treatment of various liver
disorders (Handa et al., 1986). Andrographis
paniculata was also widely known for its
usefulness and beneficial effects in general
debility, dysentery, dyspepsia, malaria, asthma,
bronchitis, filariasis and hepatitis (Kapil et al.,
1993; Jain et al., 2000). Andrographolide and
related compounds were also investigated for
their pharmacological properties and all showed
at least some degree of antipyretic, anti-malarial
and anti-inflammatory activity (Jain et al.,
2000).
However, there were no apparent data on the
cytotoxic activities of these compounds against
human tumor cell lines. Thus, the aim of this
study was to determine the growth inhibitory
activities of several bioactive compounds of
Andrographis paniculata against a panel of
human tumor cell lines and to determine the
mode of action of compounds that exhibited
potent cytotoxic activities.
Materials and methods
Cell lines and culture medium
Five different human tumor cell lines were
used; Caov-3 (human ovarian carcinoma), T47D (human breast carcinoma), Hs-578T
(human breast carcinoma), Hep G2 (human
hepatocellular carcinoma) and NCI-H23 (human
non-small cell lung carcinoma) were all
2.98”
purchased from American Type Culture
Collection (ATCC), USA. Caov-3 and Hs-578T
were cultured in DMEM, T-47D and NCI-H23
in RPMI 1640 and Hep G2 in MEM/EBSS. All
media was supplemented with 10% Fetal
Bovine Serum (FBS), 100U/ml and 100mg/ml
Penicillin-Streptomycin solution and 2mM LGlutamine (4mM L-glutamine in Hs-578T cell
medium). Additional additives such as
0.01mg/ml bovine insulin was added into T-47D
and Hs-578T cell medium, 0.1mM non-essential
amino acids into Hep G2, 10mM HEPES and
1mM sodium pyruvate in T-47D and NCI-H23
cell lines, as recommended by ATCC (USA).
1”
In vitro cytotoxicity assay
Cellular growth in the presence or absence of
experimental agents was determined using MTS
assay (CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay, Promega, USA),
according to the manufacturer’s protocol. Cell
viability was routinely determined using trypan
blue exclusion test and to make sure cell
viability was always in excess of 90%. Briefly,
near confluent cells in 96-well plates were
treated with different concentration of the
bioactive compounds. Control cells were
cultured in 0.5% (v/v) FCS-containing medium
alone. 99.9% (v/v) DMSO was used to dissolve
and dilute the compounds and the final
concentration of DMSO used was adjusted to
1% (v/v), the concentration used in control cells.
After treatment, the plates were incubated for 72
hours. Vincristine sulphate and etoposide were
used as positive controls. After 72 hours
incubation, 20 µl/well of combined MTS/PMS
solution was added and the plates were
incubated for a further 1 – 4 hours in the
0.3”
2.98”
1”
humidified 5% CO2 incubator at 370C.
Absorbance was then read at 490nm using
Vmax Kinetic Microplate Reader (Molecular
Devices, USA). Wells with complete medium
and MTS/PMS solution but without cells were
used as blanks. EC50 values were expressed as
microgram of compound concentration per
millimetre that cause a 50% growth inhibition as
compared to controls.
Detection of DNA fragmentation (apoptosis) in
T-47D cells
Cells were then subcultured into Labtek®
Chamber Slides and then incubated for 24 – 48
hours. When the cells reached between 80-90%
confluency, the medium was removed and
replaced with medium containing only 0.5%
(v/v) FBS. The cells were then incubated for a
further 4 hours and subsequently treated with
14-deoxy-11, 12-didehydroandrographolide at
concentration of EC50. Control cells were
treated with the same percentage of DMSO.
Positive control cells were treated with DNase I
and vincristine sulphate. The slides were
subsequently incubated for 24 hours. After
treatment, the cells in the chamber slides were
washed with PBS twice and subsequently
processed according to the DeadendTM
Colometric Apoptosis Detection System
(Promega, USA) protocol as described by the
manufacturer’s manual. The slides were
observed using the light microscope.
In another set of experiments, cells were
plated onto 12-well plates at similar cell
densities and then treated accordingly with the
compound. After 24 hours and 72 hours
respectively, the treated cells were rinsed with
PBS, stained with 0.4% (w/v) trypan blue and
left for about 5 minutes. The sample was then
viewed using an inverted light microscope.
Detection of phosphatidylserine externalization
(programmed cell death) in T-47D cells
T-47D cells were prepared and stimulated
with 14-deoxy-11,12-didehydroandrographolide
as described in previous section. After 24 hours
of incubation, medium, chambers and silicon
borders of cells grown on chamber slides were
removed and the treated cells were incubated
with the Annexin-V-FLUOS labeling solution
(combination of annexin V and propidium
iodide solution) (100 µl/chamber) with
coverslips for 10 – 15 minutes at 15 – 250C as
described in the manufacturer’s protocol.
Subsequently, the slides were immediately
analyzed using a fluorescence microscope using
an excitation wavelength in the range of 450 –
500 nm and detection wavelength in the range
of 515 – 565 nm (green). Annexin V and
propidium iodide positive cells were stained in
green and red, respectively.
Calculations and statistical analysis
EC50 values for growth inhibition was
derived from a nonlinear regression model
(curvefit) based on sigmoidal dose response
curve (variable) and computed using
GraphPadPrism (Graphpad, USA). EC50 from
non-dose response curves were derived by 50%
interpolation on fit spline point-to point plots.
Data were given as mean + standard error mean
(SEM).
Results
Growth inhibitory effects of the bioactive
compounds on growth of human tumor cell
lines
The seven bioactive compounds of
Andrographis paniculata were evaluated in a
panel of human tumor cell lines that included
ovarian, breast, liver and lung carcinoma cell
lines. Andrographolide was growth inhibitory in
the range of concentration used to test all the
cell lines following a continuous incubation
under the standard conditions of the MTS assay
(Table 1). However, the compound appeared to
be non-cytotoxic against all the cell lines, as
judged by the criterion set by the National
Cancer Institute, USA (EC50 was more than 4
µg/ml) (Geran et al., 1972). As for 14deoxyandrographolide, the cytotoxic activity
was only limited to T-47D cell line (EC50 at
2.800 µg/ml). This compound appeared to be
non-cytotoxic to the rest of the cell lines.
Similarly
for
andrographiside,
deoxyandrographiside
and
TABLE 1
Growth inhibitory activities (EC50) of bioactive compounds of Andrographis paniculata [(A)
Andrographolide; (B) 14-Deoxy-andrographolide; (C) Andrographiside; (D) 14-Deoxy-11,12-didehydroandrographolide;
(E)
Neoandrographolide;
(F)
Deoxyandrographiside;
(G)
14-Deoxy-12methoxyandrographolide]; and positive controls [(H) Vincristine sulphate; (I) Etoposide] to different cell lines
at 72 hours. ND, not determined
Compounds
Cell lines
A
B
C
D
E
F
G
H
I
Caov-3
23.6
ND
ND
ND
ND
ND
ND
3.5
76.2
T-47D
13.3
2.8
ND
1.5
18.1
ND
9.9
21.7
1.9
Hs-578T
17.3
ND
ND
34.9
ND
ND
26.7
0.001
139.4
Hep G2
29.4
28.22
ND
11.5
ND
ND
ND
0.3
ND
NCI-H23
9.9
26.4
ND
41.8
ND
ND
19.5
0.002
0.1
14-deoxy-12-methoxyandrographolide,
EC50
was either above 4 µg/ml or undetermined at the
range of concentration used to test the cells,
indicating the non-cytotoxic properties of the
compounds. However, 14-Deoxy-11, 12didehydroandrographo lide cytotoxicity effects
were demonstrated in T-47D cells (EC50 at
1.520 µg/ml) but non-cytotoxic against the rest
of the cell lines.
As principle compounds of Andrographis
paniculata, andro grapholide and 14-Deoxy11,12-didehydroandrographolide appeared to be
more active as compared to the rest of the
compounds.
However,
14-Deoxy-11,12didehydroandrographolide appeared to show
more potent activity (in terms of EC50) as
compared to vincristine sulphate and etoposide,
especially against T-47D cells (EC50 at 21.700
µg/ml and 1.930 µg/ml, respectively) (Table 1).
Within the panel of cell lines, T-47D cells were
the most sensitive to 14-deoxy-11,12didehydroandrographolide, followed by 14deoxyandrographolide,
14-deoxy-12methoxyandrographolide and andro grapholide.
Although the plant was well known for its
hepatoprotective effects on liver cells (Kapil et
al., 1993, Roy Choudhury et al., 1987, Rana et
al., 1991), the results showed that none of the
compounds exhibited remarkable activity
against the Hep G2 cells. The effects of 14-
deoxy-11, 12-didehydroandrographolide on T47D cells was further evaluated.
Induction of programmed cell death by 14deoxy-11,12-didehydroandrogra pholide in T47D cells
14-Deoxy-11,12-didehydroandrogra-pholide
was found to be cytotoxic against the T-47D
cells as demonstrated in previous results. In
order to determine the mechanism of cell death
elicited by the bioactive compound, T-47D cells
were incubated for 24 hours with the compound
at EC50 concentration for 72 hours (1.520 g/ml,
Table 1). After incubation, the cells were then
subjected to a modified TUNEL assay using
Deadend
Apoptosis
Detection
System
(Promega, USA). Positive and negative controls
were carried out simultaneously.
Interestingly, the treated cells were not
labeled and nearly non-visible when observed
under light microscope, under all fields and
magnifications as compared to cells treated with
DNase I and vincristine sulphate (Figure 1).
This clearly indicated that no DNA
fragmentation occurred at 24 hours, thus cell
death was most probably non-apoptotic in
nature.
Trypan
blue
exclusion
assay
demonstrated that very few cells were positively
stained after 24 and 72 hours incubation with
14-deoxy-11,12-didehydroandrographolide
a
b
c
d
FIGURE 1 The effect of (a) 14-Deoxy-11,12didehydroandrographolide, (b) 1% (v/v) DMSO,
(c) 1U/ml DNase I and (d) 21.70
µg/ml
vincristine sulphate on T-47D cells after 24 hours
incubation. DNA fragmentation was detected
using
DeadendTM
Colometric
Apoptosis
Detection System (Promega, USA).
(data not shown). This simple experiment
actually ruled out necrosis as the mode of cell
death.
To investigate further, T-47D cells were
incubated for 24 hours with the compound at
EC50 concentration for 72 hours. After
incubation, the cells were subjected to the
Annexin-V-FLUOS™ (Roche, Germany) assay.
The presence of weakly scattered annexin V
positive cells were evident (Figure 2), indicating
an early phase of programmed cell death as
compared to negative control cells. A high
percentage of cells with homogeneous and high
intensity staining of propidium iodide was
observed, indicating either necrosis, late stage
apoptosis (secondary necrosis) or programmed
cells death have taken place. As apoptosis and
necrosis was ruled out as the mechanism of cell
death
elicited
by
14-deoxy-11,12didehydroandrographolide, the high percentage
of positive annexin V cells and cells that took
up the propidium iodide dye may indicate that
cell death was mostly due to programmed cell
death as indicated in Type II non-apoptotic
programmed cell death.
a
b
FIGURE 2 The cells were treated with (a) 1.50
µg/ml of the compound and (b) 1% (v) DMSO for
24 hours and stained with the Annexin-VFLUOSTM kit (Roche, Germany).
Discussion
Results of the present study revealed that the
principle compounds, andrographolide and 14deoxy-11,12-didehydroandrographolide appeared to be more active as compared to the rest of
the compounds. 14-deoxy-11,12-didehydroandrographolide also showed more potent activity
(in terms of EC50) as compared to vincristine
sulphate and etoposide, especially against T47D cells (EC50 at 1.520 µg/ml), indicating that
this compound could be a possible candidate
against breast cancer. Although vincristine
sulphate did not appear to produce a prominent
growth inhibition pattern against T-47D cells
using MTS assay, this anticancer agent
produced a high percentage of apoptotic cells
(demonstrated DNA fragmentation) in T-47D
cells (Figure 1). The cell death cause by 14deoxy-11,12-didehydroandro grapholide against
T-47D cells appeared to be non-apoptotic (as
revealed by lack of DNA fragmentation) and
non-necrotic (as revealed by trypan blue
exclusion assay) but possibly programmed cell
death (as revealed by annexin V and propidium
iodide staining). The non-apoptotic programmed
cell death was probably the Type II nonapoptotic programmed cell death (vacuolar) as
demonstrated by the high percentage of cells
that took up the propidium iodide dye. Cells
undergoing Type II death was known to take up
the propidium iodide like necrotic cells, due to
cell porosity instead of cell membrane
disruption (Prof Ivor D. Bowen, personal
communication).
The results in this study suggested that 14deoxy-11,12-didehydroandro grapholide may be
possible candidate for the treatment of breast
cancer and the compound inhibited growth of T47D cells via a non-apoptotic programmed cell
death mechanism. Although the characteristic
staining pattern by annexin V-propidium iodide
suggested that cell death may be programmed
cell death or Type II programmed cell death
(vacuolar),
further
investigations
were
warranted such as ultrastructural analysis using
transmission electron microscope and gene
expression
studies.
The
non-apoptotic
programmed cell death may be another potential
target in cancer therapeutics.
Acknowledgements
The authors wished to thank the Ministry of
Science, Technology and Innovation (MOSTI),
Malaysia for the National Science Fellowship
awarded to Tan Mei Lan and for IRPA grant
awarded to Shaida Fariza Sulaiman and Tengku
Sifzizul Tengku Muhammad. The authors were
also grateful to Prof. I.D. Bowen of Cardiff
University, Cardiff, UK for his expertise and
experience in the interpretation of the
ultrastructural analysis results.
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