# Jaceosidin Induces Apoptosis in Human Ovary Cancer Cells through Mitochondrial Pathway

Authors: Wen Lv; Xia Sheng; Ting Chen; Qiang Xu; Xing Xie
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

We examined the antiproliferation effect of Jaceosidin (4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone) isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV-3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP), while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells. Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.


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## Body

## 1. Introduction

The normal cell function and tissue
homeostasis are maintained by a balance between proliferation and apoptosis.
Cancer is a typical disorder in which clones of malignant cells escape such
balance and proliferate inappropriately without compensatory apoptosis [1]. Generally, the growth rate of
preneoplastic or malignant cells outpaces that of normal cells as a consequence
of malfunctioning or deregulation of their cell growth and cell death
machineries [2]. The success of cancer therapies,
therefore, greatly relies on the extent to which they preferentially induce
tumour cell death while allowing survival of normal tissue. Blockade of proliferation
or induction of apoptosis has been recognized as a rational approach to eliminate
genetically damaged or preneoplastic cells before any malignancy manifests [3–6].As an important resource of anticancer
agents, many plant-derived substances have been shown to have various
bioactivities. For example, some of the Artemisia plants widely used in
traditional oriental medicine have been reported to show antimutagenic and
anti-inflammatory effects [7–9]. Our previous
study has also observed that the ethanol extract from Artemisia
vestita Wall showed an inhibitory effect on endotoxin-induced sepsis through suppressing MAPKs and NF-κB signaling in macrophages [10]. From the plant,
we further found an anticancer principle, a flavone jaceosidin. So far,
there are only a few reports on the pharmacological activities of this compound, such as inhibition of COX-2 and MMP-9 in
human mammary epithelial cells [11], suppression of E6 and E7
oncoproteins of HPV 16 [12], and induction
of apoptosis in ras-transformed human breast epithelial cells [13].
Based on the antiproliferation and apoptosis-inducing effects of jaceosidin, thus, the present study aims at demonstrating the mechanisms underlying the effects against
cancer cells from the aspect of apoptosis pathway.


## 2. Materials and Methods

### 2.1. Cell Lines

Human ovary
cancer cells CAOV-3, SKOV-3, PC3, and HeLa cells were obtained from ATCC. All the
cells were maintained in DMEM (Life Technologies Inc., Grand Island, NY, USA) supplemented with 10% FBS (Life
Technologies Inc.), 100 U/mL penicillin, and 100μg/mL streptomycin, and incubated at
37°C in a humidified atmosphere containing 5% CO2 in the air.


### 2.2. Drugs and Reagents

Jaceosidin,4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone (see Figure 1(a)), was
isolated from Artemisia vestita Wall, a traditional Tibetan medicine.
The following reagents were purchased as follows. 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium
bromide (MTT) (Sunshine Biotechnology, Nanjing, China); antibodies to poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-9, cytochrome c oxidase subunit IV (COX-IV), and cytochrome c (Cell signaling Technology,
Beverly, Mass, USA); antibody
to tubulin (Stanta Cruz Biotechnology, Stanta Cruz, Calif, USA); peroxidase-labeled antirabbit
antibody, peroxidase-labeled antimouse antibody (KPL, Gaithersburg, Md, USA); 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-benzimidazolcarbocyanine
iodide (JC-1) (Molecular Probes, Eugene, Ore, USA). Z-DEVD-FMK (BD biosciences, San Jose, Calif, USA).(a) Structure of jaceosidin. (b) Jaceosidin inhibits
proliferation of CAOV-3, SKOV-3, PC3, and HeLa cells in a concentration-dependent
manner. Cells were cultured in 96-well-plate for first 24 hours. Then, they
were treated with different concentrations of jaceosidin for further 48 hours. (c)
Jaceosidin inhibits proliferation in CAOV-3 cells in a time-dependent manner.
The CAOV-3 cells were incubated with jaceosidin for 24, 48, and 72 hours,
respectively. MTT assay was used to evaluate the proliferation rate. Each datum
indicated mean ± s.d. of three independent experiments.*P<.05, **P<.01 versus control (Dunnett’s test).
(a)(b)(c)


### 2.3. MTT Assay

Five thousands of cells per well were cultured
in a 96-well-plate
for first 24 hours. Then, they were incubated with different concentrations
of jaceosidin (10, 20, 40, and 80μM) for further 48 hours. For the time course
assay, the incubation time with jaceosidin was 24, 48, and 72 hours,
respectively. After incubation, MTT was dissolved in PBS at 5 mg/mL and added
to culture media at a final concentration of 0.5 mg/mL. After incubation at 37°C for 4 hours, the media were removed, 100 μL DMSO were
added to each well to dissolve purple crystals of formazan. The plate was shaken
for 10 minutes to allow complete solubilization. Spectrophotometric absorbance at
540 nm was read on a 96-well plate reader.


### 2.4. Assay for Cell Apoptosis

Jaceosidin or quercetin was added into 12-well-plate at
indicated concentrations. In the inhibitory assay, 100μL Z-DEVD-FMK, a
capase-3 inhibitor, was added to each well along with the compounds. Twenty
four-hours later, the cells were collected, washed, and resuspended in PBS.
Apoptotic cell death was identified by double supravital staining with
recombinant FITC (fluorescein isothiocyanate)-conjugated Annexin V and PI,
using the Annexin V-FITC Apoptosis Detection kit (Becton Dickinson, Frankly
Lakes, NJ, USA)
according to the manufacturer's instructions. Flow cytometric analysis was
performed immediately after supravital staining. Data acquisition and analysis
were performed in a Becton Dickinson FACSCalibur flow cytometer using CellQuest
software.


### 2.5. Assay for Cell Mitochondrial Membrane Potential

The JC-1 dye assay was used for
determination of reduction in mitochondrial membrane potential during apoptosis.
Briefly, jaceosidin or
quercetin was added to 12-well-plate at indicated concentration. Twenty-four
hours later, cells were harvested and fixed in 0.5% PFA for 30 minutes. Then, cells
were stained with 1μL JC-1
at 1 mg/mL in DMSO. After incubation
with JC-1 for 15 minutes at 37°C,
cells were analyzed for the decrease in red-orange fluorescence. Data acquisition and analysis were
performed in a Becton Dickinson FACSCalibur flow cytometer using CellQuest
software.


### 2.6. Gel Electrophoresis and Western Blot Analysis

Cells were incubated for 24 hours in the presence of
indicated concentrations of jaceosidin or quercetin. After the incubation, the cells
were harvested and lysed. Mitochondrial protein and cytosolic protein were
isolated using Mitochondrial Fractionation Kit (Active Motif, Carlsbad, Calif, USA) according to the manufacturer’s
instructions. Proteins were quantified using a BCA protein assay kit (Pierce, Rockford, Ill, USA) according
to the manufacturer’s specifications. For western blot analysis, the proteins
were electrophoresed on a 7.5 to 10% SDS-PAGE, followed by immunoblotting on
PVDF membrane. Then, the membrane was blocked with 5% nonfat dry milk in TBS
with 0.1% Tween 20 for 1 hour at room temperature. Finally, proteins were
visualized using the enhanced chemiluminescence detection system (Cell
Signaling Technology, Beverly, Mass, USA) after incubation with antibodies to
PARP, caspase-3, caspase-9, cytochromec,
COX-IV, or tubulin overnight at 4°C. Then, they were incubated with
peroxidase-labeled antirabbit antibody or peroxidase-labeled antimouse antibody
for 1 hour at room temperature. Peroxidase activity was visualized on X-ray
film in darkroom.


### 2.7. Statistical Analysis

Results were expressed as mean ± s.d. of
three independent experiments and each experiment included triplicate sets.
Data were statistically evaluated by one-way ANOVA followed by Dunnett’s test
between control group and multiple dose groups, with the level of significance
chosen asP<.05.


## 2.1. Cell Lines

Human ovary
cancer cells CAOV-3, SKOV-3, PC3, and HeLa cells were obtained from ATCC. All the
cells were maintained in DMEM (Life Technologies Inc., Grand Island, NY, USA) supplemented with 10% FBS (Life
Technologies Inc.), 100 U/mL penicillin, and 100μg/mL streptomycin, and incubated at
37°C in a humidified atmosphere containing 5% CO2 in the air.


## 2.2. Drugs and Reagents

Jaceosidin,4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone (see Figure 1(a)), was
isolated from Artemisia vestita Wall, a traditional Tibetan medicine.
The following reagents were purchased as follows. 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium
bromide (MTT) (Sunshine Biotechnology, Nanjing, China); antibodies to poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-9, cytochrome c oxidase subunit IV (COX-IV), and cytochrome c (Cell signaling Technology,
Beverly, Mass, USA); antibody
to tubulin (Stanta Cruz Biotechnology, Stanta Cruz, Calif, USA); peroxidase-labeled antirabbit
antibody, peroxidase-labeled antimouse antibody (KPL, Gaithersburg, Md, USA); 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-benzimidazolcarbocyanine
iodide (JC-1) (Molecular Probes, Eugene, Ore, USA). Z-DEVD-FMK (BD biosciences, San Jose, Calif, USA).(a) Structure of jaceosidin. (b) Jaceosidin inhibits
proliferation of CAOV-3, SKOV-3, PC3, and HeLa cells in a concentration-dependent
manner. Cells were cultured in 96-well-plate for first 24 hours. Then, they
were treated with different concentrations of jaceosidin for further 48 hours. (c)
Jaceosidin inhibits proliferation in CAOV-3 cells in a time-dependent manner.
The CAOV-3 cells were incubated with jaceosidin for 24, 48, and 72 hours,
respectively. MTT assay was used to evaluate the proliferation rate. Each datum
indicated mean ± s.d. of three independent experiments.*P<.05, **P<.01 versus control (Dunnett’s test).
(a)(b)(c)


## 2.3. MTT Assay

Five thousands of cells per well were cultured
in a 96-well-plate
for first 24 hours. Then, they were incubated with different concentrations
of jaceosidin (10, 20, 40, and 80μM) for further 48 hours. For the time course
assay, the incubation time with jaceosidin was 24, 48, and 72 hours,
respectively. After incubation, MTT was dissolved in PBS at 5 mg/mL and added
to culture media at a final concentration of 0.5 mg/mL. After incubation at 37°C for 4 hours, the media were removed, 100 μL DMSO were
added to each well to dissolve purple crystals of formazan. The plate was shaken
for 10 minutes to allow complete solubilization. Spectrophotometric absorbance at
540 nm was read on a 96-well plate reader.


## 2.4. Assay for Cell Apoptosis

Jaceosidin or quercetin was added into 12-well-plate at
indicated concentrations. In the inhibitory assay, 100μL Z-DEVD-FMK, a
capase-3 inhibitor, was added to each well along with the compounds. Twenty
four-hours later, the cells were collected, washed, and resuspended in PBS.
Apoptotic cell death was identified by double supravital staining with
recombinant FITC (fluorescein isothiocyanate)-conjugated Annexin V and PI,
using the Annexin V-FITC Apoptosis Detection kit (Becton Dickinson, Frankly
Lakes, NJ, USA)
according to the manufacturer's instructions. Flow cytometric analysis was
performed immediately after supravital staining. Data acquisition and analysis
were performed in a Becton Dickinson FACSCalibur flow cytometer using CellQuest
software.


## 2.5. Assay for Cell Mitochondrial Membrane Potential

The JC-1 dye assay was used for
determination of reduction in mitochondrial membrane potential during apoptosis.
Briefly, jaceosidin or
quercetin was added to 12-well-plate at indicated concentration. Twenty-four
hours later, cells were harvested and fixed in 0.5% PFA for 30 minutes. Then, cells
were stained with 1μL JC-1
at 1 mg/mL in DMSO. After incubation
with JC-1 for 15 minutes at 37°C,
cells were analyzed for the decrease in red-orange fluorescence. Data acquisition and analysis were
performed in a Becton Dickinson FACSCalibur flow cytometer using CellQuest
software.


## 2.6. Gel Electrophoresis and Western Blot Analysis

Cells were incubated for 24 hours in the presence of
indicated concentrations of jaceosidin or quercetin. After the incubation, the cells
were harvested and lysed. Mitochondrial protein and cytosolic protein were
isolated using Mitochondrial Fractionation Kit (Active Motif, Carlsbad, Calif, USA) according to the manufacturer’s
instructions. Proteins were quantified using a BCA protein assay kit (Pierce, Rockford, Ill, USA) according
to the manufacturer’s specifications. For western blot analysis, the proteins
were electrophoresed on a 7.5 to 10% SDS-PAGE, followed by immunoblotting on
PVDF membrane. Then, the membrane was blocked with 5% nonfat dry milk in TBS
with 0.1% Tween 20 for 1 hour at room temperature. Finally, proteins were
visualized using the enhanced chemiluminescence detection system (Cell
Signaling Technology, Beverly, Mass, USA) after incubation with antibodies to
PARP, caspase-3, caspase-9, cytochromec,
COX-IV, or tubulin overnight at 4°C. Then, they were incubated with
peroxidase-labeled antirabbit antibody or peroxidase-labeled antimouse antibody
for 1 hour at room temperature. Peroxidase activity was visualized on X-ray
film in darkroom.


## 2.7. Statistical Analysis

Results were expressed as mean ± s.d. of
three independent experiments and each experiment included triplicate sets.
Data were statistically evaluated by one-way ANOVA followed by Dunnett’s test
between control group and multiple dose groups, with the level of significance
chosen asP<.05.


## 3. Results

Jaceosidin Concentration-Dependently and Time-Ddependently Inhibited Proliferation of  Various Cancer Cells
To examine the effects of jaceosidin on different
kinds of tumour cells, exponentially growing cells were treated by jaceosidin for
48 hours. As shown in Figure1(b), jaceosidin significantly reduced the proliferation of CAOV-3,
SKOV-3, PC3, and HeLa cells in a dose-dependent manner. The flavone also showed
a time-dependent inhibition on the proliferation of CAOV-3 cells (see Figure 1(c)).Jaceosidin Induced Apoptosis in CAOV-3 Cells in a Caspase-3-Dependent Manner
As shown in Figures2(a) and 2(b),
Annexin-V positive cells were considered as early and late apoptosis
populations. Treatment of jaceosidin induced apoptosis of CAOV-3 cells in a dose-dependent
manner. Against the apoptosis induction, caspase-3 inhibitor Z-DEVD-FMK
significantly reversed the proapoptotic effect of jaceosidin in CAOV-3 cells (see
Figure 2(c)).Jaceosidin promotes apoptosis of CAOV-3 cells in a
caspase-3-dependent manner. CAOV-3 cells were seeded in 12-well-plate
overnight, and then were treated with different concentrations of jaceosidin or
10μM quercetin in the presence or absence of 100 μM Z-DEVD-FMK for 24 hours.
Annexin-V/PI stain was used to measure apoptosis rate. Apoptosis rate
is measured by flow cytometry. (a) The results are representative of three separate experiments. (b) Data are expressed as a
histogram of mean ± s.d. of three independent experiments. (c) Effect of
Z-DEVD-FMK on the proapoptotic activity of jaceosidin. Each datum indicated
mean ± s.d. of three independent experiments. *P<.05, **P<.01 (Dunnett’s test).
(a)(b)(c)Jaceosidin Induced Loss of Mitochondrial Membrane Potential (MMP) and Cytochromec Release from Mitochondria to Cytosol
In Figure3(a), mitochondrial
potential alteration was examined upon jaceosidin treatment. CAOV-3 cells were
incubated with jaceosidin or quercetin for 24 hours. As the result, a
dose-dependent reduction in MMP was detected in the groups treated with
jaceosidin. Quercetin also showed a strong decrease in MMP.
To confirm if jaceosidin induces apoptosis through mitochondrial pathway,
we incubated CAOV-3 cells with jaceosidin for 24 hours. Then, we collected cells
and isolated mitochondria protein. Thirtyμg of each protein were subjected to Western
blotting. Equal protein loading was confirmed by immunodetection of COX-IV for
mitochondria protein or tubulin for cytosol protein. As shown in Figure 3(b),
jaceosidin greatly increased the cytosol cytochrome c and decreased the mitochondria cytochrome c as
compared with the control group.Jaceosidin
dose-dependently (a) induces loss of mitochondrial membrane potential and (b) induces
cytochromec to release from
mitochondria to cytosol. CAOV-3
cells were seeded into 12-well-plate and cultured overnight. Jaceosidin or
quercetin was added into each well. Twenty-four hours later, cells were
harvested for measuring mitochondrial membrane potential by flow cytometry
using JC-1 staining. Cells on the lower side showed lower membrane potential. One
of three independent experiments is shown. In addition, mitochondria and
cytosol proteins were prepared from cells treated with 80 μM jaceosidin,
which were subjected to western blotting for measuring cytochrome c in mitochondria and cytosol.
(a)(b)Jaceosidin Activated Caspase-3 and its Related Proteins to Promote Apoptosis
To examine the status of the caspase-3
protein, we performed Western blot analysis by using an anticaspase-3 antibody,
which recognizes both procaspase-3 and cleaved caspase-3, the p17 large/active
subunit of caspase-3. As shown in Figure4, the p17 cleavage product appeared
in lysates of CAOV-3 cells treated with 80 μM jaceosidin and 10 μM quercetin. Jaceosidin
and quercetin also cleaved PARP to a band of 85 kDa. The amount of cleaved
caspase-9 was also increased significantly in the cells treated with quercetin,
and slightly with jaceosidin.Figure 4
Jaceosidin activates caspase-3
and its related proteins to promote apoptosis. Whole cell lysates prepared from CAOV-3
cells, which had been incubated with jaceosidin or quercetin for 24 hours. The
proteins were subjected to Western blot analysis using the respective
antibodies as indicated. Data are representative of three separate experiments.


## 4. Discussion

In the present study, we first examined
the effect of jaceosidin on some kinds of cancer cell lines (see Figure1(b)).
CAOV-3 cells were initiated from human ovary adenocarcinoma of a 54-years-old
Caucasian female [14]. SKOV-3 is also a human ovary
adenocarcinoma cell line, and it is resistant to tumor necrosis factor and to
several cytotoxic drugs including diphtheria toxin, cis-platinum and adriamycin
[15]. PC3, as an epithelial cell line,
was established from a human grade IV prostatic adenocarcinoma metastatic to
bone [16]. And HeLa cells
are human cervix adenocarcinoma cells [17]. Though the inhibition of
proliferation rate by jaceosidin was observed to different degrees against
different kinds of cancer cells, a concentration-dependent action was shown in
all 4 kinds of cell lines used. This finding suggests that jaceosidin may have
a relatively wide spectrum of antitumor growth. In CAOV-3 cells, the compound
also showed a time-dependent inhibition against the cell proliferation (see Figure
1(c)). As the ovary cancer cell line CAOV-3 showed the most significant
inhibitory rate, we chose it to do the following studies.Apoptosis, a morphologically and
biochemically defined form of cell death [18], plays a role in a wide variety
of biologic systems. As apoptosis is often caused by chemotherapeutic drugs [19], we used annexin V-PI staining
to study the apoptosis rate of cells treated with jaceosidin (see Figures 2(a)
and 2(b)). The result indicated that jaceosidin could dose-dependently induce
the apoptosis of CAOV-3 cells. The IC50 was less than 40 μM. Treatment of
caspase-3 inhibitor led to a blockade of the proapoptotic effect of jaceosidin
(see Figure 2(c)), indicating caspase-3 is involved in the underlying mechanism
of jaceosidin’s anticancer effect.Chemotherapeutic agents have been
described to induce apoptosis in two major routes: the extrinsic or death
receptor-associated route
and the intrinsic or mitochondrial route [20]. In the intrinsic route the
execution phase is initiated by release of cytochrome c and other polypeptides from the mitochondrial intermembrane space
[21]. This release is accompanied by
a dissipation of mitochondrial inner transmembrane potential [22]. It was reported that quercetin,
another widely used flavone, has strong proapoptotic effect in cancer cells [23]. In the present
study, we used it as positive control in the apoptosis assay for comparison. To
further access the antitumour mechanism of jaceosidin, we observed that mitochondrial membrane potential
decreased in a dose-dependent manner by jaceosidin (see Figure 3(a)). This
result suggests that jaceosidin probably promotes the mitochondrial pathway. As
expected, the next experiment further demonstrated that jaceosidin treatment
greatly induced the cytochrome c to release from mitochondria to
cytosol in the cells (see Figure 3(b)), indicating involvement of the intrinsic
apoptosis route via mitochondria.Once released into the cytoplasm,
cytochromec usually interacts with
Apaf-1 (apoptotic protease-activating factor-1), adenosine triphosphate-deoxyadenosine
triphosphate (ATP/dATP), and procaspase-9 to form a complex known as the
apoptosome [24]. In the
apoptosome, caspase-9 is activated which in turn activates effector caspases,
like procaspase-3 and -7 [25]. The effector caspases may cleave
a number of structural and regulatory cellular proteins including PARP and lamin protein and are
responsible for the typical morphologic and biochemical features of an
apoptotic cell [26, 27]. Figure 4 showed the effects of
jaceosidin on these events occurred in the apoptosis, where jaceosidin markedly
caused increases in the cleaved form of PARP, caspase-3, and caspase-9. This
result further confirmed that jaceosidin promotes apoptosis through capase-3
via mitochondria. Walle et al. reported that 5, 7-dimethoxyflavone and
5, 7, 4′-trimethoxyflavone
were both 10 times more potent inhibitors of cell proliferation than the
corresponding unmethylated analogs chrysin and apigenin, fully methylated
flavones appear to have great potential as cancer
chemopreventive/chemotherapeutic agents [28]. However, in our experiments,
quercetin (3, 3′, 4′, 5, 7-pentahydroxyflavone)
showed obviously better efficacy than jaceosidin (4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone). Comparison of the structure of these compounds
suggested that 3-hydroxyl group might be very important for the function of
flavone compounds. Further experiments need to be done to confirm this
hypothesis.Overall, our study has proved an antitumour effect of
jaceosidin, a flavone fromArtemisia
vestita Wall. Its mechanism underlying the effect mainly involves the
induction of apoptosis through activating caspase-3 via mitochondrial pathway.
The findings obtained here may be helpful for understanding the properties of
jaceosidin as a candidate of antitumour drug.



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*Source: 394802-2008-07-17.xml*