|Ovary cancer, Apoptosis, Gentiopicroside, Flow cytometry, Cytotoxic activity.
|Cancer is one of the most deadly gynecological malignancies
throughout the world and its incidence gets increased with age.
Most of the ovarian tumors are already malignant on diagnosis
due to the limited diagnostic tools and tissue anatomy. Almost
65-70% of the ovarian cancer patients have reached to late
stages of the disease resulting in a low 5-year survival rate
[1-3]. In China, the rate of ovary cancer was 8.0/100,000 and
the age-adjusted rate was 5.35/100,000 overall during period
1999-2010. Surgery is the preliminary treatment of choice for
ovarian cancer, provided patients are medically fit. Patients
who are not fit for surgery may be given adjuvant
chemotherapy and considered for surgery later. Chemotherapy
involves use of chemical compounds of natural and synthetic
origin to target and kill tumor cells . Chemotherapeutic
agents include carboplatin and paclitaxel among others.
Despite of these agents, chemotherapy treatment protocol is
often ineffective due to serious side-effects coupled with
multidrug resistance by cancer cells which eventually leads to recurrence of the disease . Therefore, there is a pressing
need for novel, effective and relatively safer anticancer
chemotherapeutic agents especially from plant origin. The
objective of the current research work was to investigate the
anticancer and apoptotic effects of gentiopicroside (chemically
known as ((3S,4R)-4-Ethenyl-3-[(2S,3R,4S,5S,6R)-3,4,5-
dihydro-3Hpyrano[3,4-c]pyran-8-one) in OVCAR-3 ovary
cancer cells. The effects of this compound on ROS generation
and NF-kB signalling pathway were also elaborated. To the
best of our knowledge, the current study constitutes the first
such report on this molecule.
Materials and Methods
Chemicals and other reagents
|Gentiopicroside (98% purity) was purchased from Sigma
Chemical Company (St. Louis. Co), and 50 mg/ml solution
dissolved in DMSO was stored at -20°C before use. 3-[4,5- dimeth-yl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide
(MTT) was purchased from Molecular Probes (USA).
Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine
Serum (FBS), Penicillin-Streptomycin, Rhodamine 123 were
obtained from Sigma-Aldrich (Sigma Aldrich, Shanghai,
China). Primary antibodies against caspase-3, caspase-9, NF-
κB, Bax, Bcl-2, PARP-1, cytochrome c, β-actin, and secondary
antibodies (goat-anti-rabbit or goat-anti-mouse) were
purchased from Millipore Pvt Ltd.
Cell line and culture conditions
|OVCAR-3 human ovary cancer cell line was purchased from
the Shanghai Institute of Cell Resource Center of Life Science
(Shanghai, China). The cells were cultured in DMEM medium
supplemented with 10% FBS, 100 μg/ml Streptomycin, and
100 U/ml Penicillin maintained at 37°C in a humidified
atmosphere with 5% CO2.
Cell proliferation assay for cell viability
|The cytotoxic effects of gentiopicroside were evaluated by
MTT assay. Briefly, cells were placed in 96-well culture plates
(2 × 105 cells/well). After 24 hours, the cells were treated with
0, 2.5, 5, 10, 20, 40 and 100 μM gentiopicroside or 0.1%
DMSO respectively for 24 h. After that MTT (10 mg/ml) was
added to each well. The cells were incubated for another 4 h,
and 250 μL DMSO was added to each well. Absorbance was
measured on a microplate reader (ELX 800; Bio-tek
Instruments, Inc., Winooski, VT, USA) at a wavelength of 490
nm and the growth inhibition ratio was calculated. The halfmaximal
Inhibitory Concentration values (IC50) were obtained
from the MTT viability curves.
Fluorescence microscopy using acridine orange and
propidium iodide double-staining
|In this assay, the apoptotic cell death induced by
gentiopicroside was evaluated by fluorescence microscopy
using propidium iodide and acridine orange double staining
according to already reported methods . OVCAR-3 cells
were plated at a density of 2 × 105 cells/ml, and then treated
with 0, 20, 40 and 100 μM concentration of gentiopicroside for
48 hours. The cells were centrifuged at 15, 000 g for 15 min,
after which cells were washed three times with PBS. After that,
10 μL each of Acridine Orange (AO) and Propidium Iodide
(PI) were added to the cell suspension. The stained cell
suspension was put onto a glass slide and these slides were
then monitored using fluorescence microscope for 30 minutes.
Around 500 cells were chosen for determining the percentages
of apoptotic, necrotic and living cells.
Phase contrast microscopic evaluation of OVCAR-3
cancer cells after treatment
|Phase contrast microscopic evaluation of OVCAR-3 cancer
cells after drug treatment was done using inverted light
microscope (Olympus, PA, USA). In brief, OVCAR-3 cells
were seeded in 6-well plate at a density of 2 × 105 cells/well The cells were treated with 0, 20, 40 and 100 μM of
gentiopicroside for 48 hours. The morphological changes were
monitored and the same spot of cells was photographed. The
images were captured at a magnification of 400X.
Measurement of mitochondrial membrane potential
|The effect of gentiopicroside on the loss of mitochondrial
transmembrane potential was measured by flow cytometry
using rhodamine-123 fluorescent probe. The OVCAR-3 cells
(2 × 105 cells/well) were treated with 0, 20, 40 and 100 μM of
gentiopicroside for 48 h. Rhodamine-123 dye was added 2 h
before termination of the experiment. The cells were harvested,
washed twice with PBS and then incubated in PI for 10 min.
The decrease in fluorescence intensity, due to loss of ΛΨm,
was analyzed using flow cytometry (FACSCalibur™; BD
Biosciences). The mean fluorescence intensity was detected
using the FL1 channel of the BD FACSCalibur™.
Western blot analysis
|OVCAR-3 cells were treated with 0, 20, 40 and 100 μM dose
of gentiopicroside and then incubated for 48 h. The adherent
and floating cells were harvested and then washed three times
with PBS and then lysed in RIPA buffer and protease inhibitor
for 10 min. After centrifugation, the protein content was
determined by for Western blotting analysis. The protein
lysates (20 μg/lane) were separated by 10% SDS-PAGE and
blotted onto nitrocellulose membranes (Millipore, Bedford,
MA, USA). Each membrane was blocked with 6% skim milk,
and then incubated with the designated primary antibodies
against caspase-9, caspase-3, Bcl-2, NF-κB, PARP-1,
cytochrome c, and β-actin overnight at 4°C. Subsequently, the
membrane was incubated with the secondary antibodies (HRPconjugated
goat anti-rabbit or goat anti-mouse IgG) for 1 h at
room temperature and the formed immunocomplex was
visualized by western blotting detection reagents (Trans Gene,
|Data are presented as the mean ± SEM. All experiments were
repeated at least three times. The differences between groups
were analyzed by one-way ANOVA, significance of difference
was indicated as *P<0.05, **P<0.01.
Cytotoxic activity of gentiopicroside in OVCAR-3
|The chemical structure of gentiopicroside is shown in Figure 1.
The results of the current study indicated that gentiopicroside
exerted potent cytotoxic effects in a time-dependent as well as
dose-dependent manner. The potency of a chemical compound
is expressed in terms of IC50 (half-maximal Inhibitory
Concentration) value which is the concentration which causes
50% growth inhibition. The IC50 values at 24 and 48 h time
intervals was found to be 22.4 and 8.2 μM respectively (Figure
Apoptosis quantification using Acridine Orange (AO)
and propidium iodide staining
|Fluorescence microscopy can be used to examine the apoptotic
morphological changes in cancer cells after drug treatment.
The present study indicated that gentiopicroside induced potent
apoptotic morphological changes in a dose-dependent manner
|As can be seen from Figure 3, untreated control cells (Figure 3A) revealed green colour indicating undamaged nuclear
structure. However, after treatment with 20 and 40 μM dose of
gentiopicroside, signs of mild apoptosis could be seen from the
induced morphological changes including nuclear
fragmentation and membrane blebbing (Figures 3B and 3C).
However, at higher dose (100 μM) dose of gentiopicroside,
extensive apoptosis could be observed characterized by
reddish-orange fluorescence which arises due to the binding of
AO dye to the fragmented DNA (Figure 3D). Around 500
cells were chosen for quantification of apoptosis.
Phase contrast microscopic evaluation of the
gentiopicroside-induced cell cytotoxicity
|Exposure of OVCAR-3 cells to 0, 20, 40 and 100 μM of
gentiopicroside for 48 h resulted in a substantial decrease in
cell count and, furthermore, induced morphological changes
that were characteristic of cytotoxicity in OVCAR-3 cells
under phase-contrasted microscopy following exposure to the
drug (Figure 4). The results indicated that the number of cells
with a vacuolated cytoplasm was markedly higher in the
gentiopicroside-treated group compared with the control group.
The appearance of vacuoles in the cytoplasm is an indication of
Gentiopicroside induced mitochondrial membrane
potential loss (Δψm) in OVCAR-3 cancer cells
|Further experiments studied the effect of gentiopicroside on the
loss of mitochondrial membrane potential in OVACR-3 cells
using flow cytometry using Rh-123 as a fluorescent probe. Figure 5 A-D show the effect of the compound on Δψm
indicating that gentiopicroside exerts potent depolarizing
effects on the mitochondrial transmembrane potential. The
percentage of OVACR-3 cells with depolarized mitochondria
increased from 5.2% in untreated cells to 16.7%, 44.5% and
67.3% in cells treated with 20, 40 and 100 μM dose of
gentiopicroside respectively (Figure 6).
Effect of gentiopicroside on the apoptosis-related
protein expressions including NF-kB, Bcl-2,
caspase-3, caspase-9 and PARP
|Western blot analysis was employed to investigate the effect of
gentiopicroside on the expression levels of various apoptosisrelated
proteins and β-actin was used as a loading control.
Gentiopicroside treatment at various doses (0, 20, 40 and 100
μM) for 48 h led to up-regulation of cleaved PARP-1,
cytochrome c, caspase-3 and caspase-9 while as it was found
that the gentiopicroside treatment resulted in down-regulation
of NF-kB and Bcl-2 in a dose dependent manner. All these
proteins play key roles in the induction of apoptosis in cells
|Apoptosis, which involves programmed cell death, is a highly
organized biochemical process essential for the development of
most of the organisms. Two main signalling pathways have
been identified for the process of apoptosis, one is the extrinsic
pathway and the other is the intrinsic pathway. Any
deregulation of the apoptotic process leads to serious disorders
including cancer [7,8]. Apoptosis process is characterized by
several morphological changes in the cells including
membrane blebbing, cell shrinkage, nuclear and DNA
fragmentation and formation of apoptotic bodies. Currently,
there is an increasing demand for searching chemotherapeutic
agents which can act as apoptotic stimuli for the induction of
apoptosis in cancer cells [9,10].
|Mitochondria has also been reported to play crucial roles in
many biochemical processes including the apoptosis process.
Mitochondria is involved in many key biochemical processes
including release of caspase activators, loss of mitochondrial
membrane potential and involvement of pro- and antiapoptotic
proteins . NF-kB, which is a pro-survival transcription
factor, plays crucial role in cell proliferation and cell survival
and it makes cells less prone to the process of apoptosis. This
transcription factor is involved in inhibiting the apoptosis
process and as such its down-regulation leads to induction of
apoptosis . NF-κB activation has also been witnessed in
many solid tumors. NF-κB activation results from primary
inflammation or the consequence of formation of an
inflammatory microenvironment during cancer development.
|NF-κB activation in cancer may be the result of either exposure
to proinflammatory stimuli in the tumor microenvironment or
mutational activation of upstream components in IKK–NF-κB
signaling pathways . Alterations in mitochondrial
membrane potential has been shown to trigger induction of
apoptosis and has even been suggested to be essential to the
apoptotic pathway. Opening of the mitochondrial permeability
transition pore has been shown to induce depolarization of the
transmembrane potential, release of apoptogenic factors and
loss of oxidative phosphorylation .
|Gentiopicroside, also known as gentiopicrin, is a plant
secoiridoid constituent of many plant species of Gentiana
genus including Gentiana macrophylla, Gentiana gelida,
Gentiana lutea etc. It has been reported to exhibit smooth
muscle relaxing activity in guinea pig ileum .
Gentiopicroside has also been reported to suppress the
chemically and immunologically induced liver injuries in mice
. Gentiopicroside has been reported to exhibit antitumor
activity in mice bearing the experimental tumor leukemia P
388. It has also shown activity in human hepatoma Hep3B
cells [17,18]. In this study, we evaluated the effect of
gentiopicroside on the proliferation and apoptosis in OVACR-3
ovary cancer cells.
|The results indicated that gentiopicroside is a potent cytotoxic
agent inhibiting the proliferation of OVCAR-3 cells in a dose
and time-dependent manners. Fluorescence and phase contrast
microscopic results indicated that gentiopicroside induces
characteristic morphological features of apoptosis marked by
reddish-orange fluorescence in treated cells. Gentiopicroside
also led to the loss of mitochondrial membrane potential in
OVCAR-3 cells. The percentage of cells with depolarized
mitochondria increased from 5.2% in untreated cells to 16.7%,
44.5% and 67.3% in cells treated with 20, 40 and 100 μM dose
of gentiopicroside respectively.
|The current study also examined the effect of gentiopicroside
on the expression levels of various apoptosis-related proteins.
It was observed that 0, 20, 40 and 100 μM dose of
gentiopicroside treatment for 48 h led to up-regulation of
cleaved PARP-1, cytochrome c, caspase-3 and caspase-9 while
as this treatment resulted in down-regulation of NF-kB and
Bcl-2 in a dose dependent manner.
|The current findings reveal that gentiopicroside induces
antiproliferative and apoptotic effects in OVCAR-3 ovary
cancer cells through the induction of mitochondrial membrane
potential loss and by altering the expression levels of various
apoptosis-related proteins including NF-kB, Bcl-2, cleaved
PARP-1, cytochrome c, caspase-3 and caspase-9.
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