Growth
Inhibition
and
Apoptosis
Induced
by
6-fluoro-3-formylchromone
in
Hepatocellular Carcinoma
Yijie Zhang#, Kailian Zheng#, Hongli Yan, Gang Jin, Chenghao Shao, Xuyu Zhou, Yingqi Zhou,
Tianlin He*
Department of General Surgery, Changhai Hospital, Shanghai 200433, China.
#
Co first author: Yijie Zhang and Kailian Zheng contribute equally to this work.
*Corresponding author: Tianlin He
Address: Department of General Surgery, Changhai Hospital, No.168 Changhai Road, Yangpu
District, Shanghai 200433, China.
Tel: +86-021-31161628
Fax: +86-021-55663159
E-mail: tianlinhe865@hotmail.com
All authors’ email
Yijie Zhang: yijiezhang568@163.com
Kailian Zheng: kailianzheng858@163.com
Hongli Yan: hongliyy@126.com
Gang Jin: gangjin2013@yeah.net
Chenghao Shao: chenghaoshao2013@yeah.net
Xuyu Zhou: xuyuzhou698@163.com
Yingqi Zhou: yingqizhou868@163.com
Abstract
Background: Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers
in human population. The 6-fluoro-3-formylchromone (FCC) has been shown to have
anti-tumor activity against various tumor cells. However, the effects of FCC on HCC cell lines
have not yet been reported. This study aims to research the effects of FCC on HCC and
advance the understanding of the molecular mechanism.Methods: HCC cell line SMMC-7721
was treated with FCC at various concentrations (0, 2, 5, 10, and 20 μg/ml) for 24, 48 and 72 h,
respectively. The proliferations of SMMC-7721 cells were measured by MTT assays. After
cultured 24 hours, cell cycle distribution and apoptosis were determined by flow cytometry.
However, the expression levels of PCNA, Bax and Bcl-2 were measured by western blotting
after 48 hours.
Results: FCC displayed a dose- and time-dependent inhibition of the SMMC-7721 cell
proliferations in vitro. It also induced apoptosis with 45.4% and caused cell accumulation in
G0/G1 phase with 21.5%. PCNA and Bcl-2 expression was significantly suppressed by FCC in
a dose-dependent manner (P < 0.05), while Bax expression was increased. Conclusions: FCC
could significantly inhibit HCC cell growth in vitro through cell cycle arrest and inducing
apoptosis by suppressing PCNA expression and modulating the Bax/Bcl-2 ratio.
Keywords: FCC; Cell Proliferation; Apoptosis.
Background
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third common cause
of cancer death in the human population [1-4]. HCC is typically aggressive and intrinsically
resistant to conventional therapies such as radiotherapy and chemotherapy [5-7] . Therefore,
more effective therapeutic agents for treating HCC are desirable.
Previous studies have shown that chromone can induce apoptosis of tumor cells and inhibit
tumor growth both in vitro and vivo [8, 9]. The derivatives also showed promising activity
against various cancers such as pancreatic, prostate, breast, adenocarcinoma and non-small cell
lung carcinoma cancers. They can affect multiple signaling pathways, such as NF-ĸB and
PI3K/Akt pathways, which play important roles in mechanism of carcinogenesis [10-12].
In eukaryotic cells, PCNA plays a crucial role in DNA replication, repair and cell proliferation
in HCC [13]. This protein was involved in synthesis of lead- and lag- DNA strands and
provided an anchorage site [14]. Applied inhibitor to down-regulate PCNA expression may
cause the cell growth inhibition. Moreover, Bcl-2 family of proteins showed the regulation
function of mitochondrial-mediated apoptosis [15]. In addition, Bcl-2 family are classified into
two opposing subfamilies, and Bcl-2 and Bax are the most representative members,
respectively [16].
3-formylchrome, one of chromone derivatives, shows significant tumor-specific cytotoxicity,
dose-dependently induced apoptosis in human oral squamous cell carcinoma cell line (HSC)
and human promyelocytic leukemia cell line (HL-60) [17]. It is also reported that some
3-formylchromone derivatives represent more potent cytotoxic activities against some tumor
cells but low cytotoxicity against normal cells [18, 19]. Among these 3-formylchromone
derivatives, 6-fluoro-3-formylchromone (FCC) was proved to be a modifier of multidrug
resistance in mouse lymphoma cells and in human Colo320 colon cancer cells [18]. Moreover,
Kawase et al. have confirmed that FCC is one of the most cytotoxic 3-formylchromone
derivatives against tumor cell lines, such as HSC-2, HSC-3, HL-60 and human submandibular
gland carcinoma cell line, through the experiments which described the effects produced by
3-formaychromones replaced at the C-6 position and evaluated the cytotoxicity against various
human cell lines [19]. Meanwhile, normal cells of human gingival fibroblast (HGF), human
pulp cell (HPC) and human periodontal ligament fibroblast (HPLF) exhibit a higher immunity
to FCC as compared with the tumor cell lines [19]. FCC is a promising approach for tumor
treatments. Nevertheless, to the best of our knowledge, there is little report describing the effect
of FCC on HCC cell lines.
In the present study, the effect of FCC on proliferation and apoptosis of HCC cell line
SMMC-7721 were investigated. In addition, to further investigate the molecular mechanisms of
FCC on SMMC-7721 cells, we also studied the expression levels of proliferation marker
PCNA and the apoptosis related proteins Bax and Bcl-2 by western blotting with FCC
treatment. Results
Effect of FCC on cell viability of SMMC-7721 cells
Chemical structure of FCC (MW=192) was shown in Figure 1A. The compound, is the
derivative of 3-formylchromone, formed when the hydrogen of the sixth carbon atom have
been replaced with fluorine. In order to determine the effects of FCC on HCC, human HCC
cell line SMMC-7721 was treated by FCC with different doses for 24 h (Figure 1B). The
proliferation of SMMC-7721 cells was inhibited by FCC in a dose-dependent manner. Further
experiments showed that FCC treatment inhibited the proliferation of SMMC-7721 cells in a
time-dependent manner (Figure 1B).
Effects of FCC on cell cycle and apoptosis of SMMC-7721 cells
The effect of FCC on cell cycle and apoptosis was observed with flow cytometry. SMMC-7721
cells were treated by FCC with various concentrations for 48 h. SMMC-7721 cells exhibited
increased cell percentages in G0/G1 phase (Table 1) with an increase from 63.80% to 85.30%.
The percentages of apoptotic cells were significantly increased in the treated group compared
to control group (p<0.05) (Table 1) in a dose-dependent manner. The apoptotic cells increased
from a total of 2.8% to 48.2% for SMMC-7721 cells. Taken together, FCC treatment could
induce HCC cell apoptosis and G0/G1 phase arrest.
Effects of FCC on PNCA, Bax and Bcl-2 expression
To further illustrate the molecular basis of the apoptosis induction by FCC, we investigated the
effect of FCC on PCNA, Bax and Bcl-2 expression in SMMC. FCC significantly suppressed
PCNA expression in a dose-dependent manner (Figure 2A and 2B). The total Bax expression
level was increased with FCC treatment in a dose-dependent manner, while the total Bcl-2
expression level was decreased, resulting in a higher Bax/Bcl-2 ratio (Figure 2A and 2C).
Discussion
To our knowledge, this is the first report describing the anti-tumor effect of FCC on HCC cell
line SMMC-7721. Our data demonstrated that FCC could inhibit the proliferation of
SMMC-7721 cells in vitro. It could down-regulate PCNA and Bcl-2 expression and up-regulate
Bax expression. The results indicated that FCC could be developed as a novel anti-tumor agent
for treating HCC.
The imbalance between cell proliferation and death is considered to be an important event in
cancer progression [19, 20]. Among the effects of anti-tumor reagents, apoptosis and growth
inhibition are the most common responses on cancer cells [21, 22]. In the present study, we
observed that the viability of SMMC-7721 cells could be significantly decreased by FCC
treatment for 24 h at various concentrations (ranging from 2 to 20 μg/ml) according to the
MTT assay. The proliferation inhibition effect of FCC on SMMC-7721 cells in vitro is
consistent with previous studies with other 3-formylchromone derivatives [13-15]. It is
suggested that the potential of FCC treatment on the proliferation of human cancer cells in
vitro.
Cell cycle regulation is one of the most important bioprocesses. Traditionally, the cell cycle is
divided into four phases of G1-S-G2-M. DNA replication occurs during S phase, and
chromosome segregation occurs during M phase. The S and M phases are separated by the
so-called gap phases, G1 (before DNA replication) and G2 (before mitosis) [23]. It has been
generally accepted that induction of cell cycle arrest and apoptosis are the important
bio-reactions to anti-tumor reagents [24-26]. FCM cell cycle analysis in this study confirmed
that FCC could induce a cell cycle arrest in G0/G1 phase, even the apoptosis when the damage
couldn’t be repaired on time. PCNA is a protein which is the compound of cyclin D and
cyclin-dependent kinases, involved in the proliferation cells, and it is specifically expressed in
proliferating cell nuclei [27]. It has been shown that the levels of PCNA expression are higher
in cancer tissues, including gastric, lung and breast cancer [28-30]. Several studies have
emphasized the association of PCNA with tumor malignancy grade and prognostic significance
in a number of malignancies [31, 32]. Furthermore, it is reported that non-steroidal
anti-inflammatory drugs (NSAIDs) could effectively postpone or hinder lung carcinogenesis
through down-regulating PCNA expression in rat model [33]. Our results demonstrated that
FCC could inhibit PCNA expression in SMMC-7721 cells with a dose-dependent manner.
Therefore, the proliferation inhibition in SMMC-7721 cells by FCC might be ascribed to the
suppressive effect on PCNA expression.
Members of the Bcl-2 family have been identified as key regulators of apoptosis including two
opposing subfamilies [34]. Bcl-2 proteins often form heterodimer complexes with Bax proteins,
which result in the release of cytochrome c from the mitochondria and subsequent induction of
cell death [35]. Therefore, an increase in the ratio of Bax/Bcl-2 is considered as one of the
major markers of pre-apoptosis. Several anti-tumor reagents have been confirmed to inhibit
tumor growth by influencing the Bax/Bcl-2 ratio [36, 37]. In the present study, our results also
suggest that FCC effectively induces apoptosis in SMMC-7721 cells through up-regulation of
the Bax/Bcl-2 ratio.
Taken together, our results suggested that FCC could induce G0/G1 cell cycle arrest and
apoptosis in SMMC-7721 cells through suppressing PCNA expression and increasing
Bax/Bcl-2 ratio, which advances our understanding on the molecular mechanisms of FCC in
hepatocarcinoma management. However, the effects of FCC on SMMC-7721 were not
investigated in animal models. More extensive research involving animal studies are needed in
the future.
Conclusions
FCC could significantly inhibit HCC cell growth in vitro through cell cycle arrest and inducing
apoptosis by suppressing PCNA expression and modulating the Bax/Bcl-2 ratio.
Methods
Cell culture
Human HCC cell line SMMC-7721 was purchased from Cell Bank, Chinese Academy of
Sciences (Shanghai, China). Cells were maintained in RPMI1640 medium (Gibco, Grand
Island, NY) supplemented with 10% heatinactivated fetal bovine serum (Gibco, Grand Island,
NY) at 37°C in a humidified atmosphere containing 5% CO2. In order to inhibit aminoxidase,
aminoguanidine was added into RPMI1640 medium at a final concentration of 1 mmol/L.
Antibodies and Reagents
Antibodies against PCNA, Bax and Bcl-2 were purchased from Santa Cruz (Santa Cruz
Biotechnology, Santa Cruz, CA, USA). FCC, trypsin and MTT were obtained from sigma
(Sigma, St. Louis, MO, USA). Cell Cycle Detection Kit was purchased from Thermo (Thermo
Fisher Scientific Inc, NH, USA).
Cell Viability Assay
The effect of FCC on cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazoliumbromide (MTT) assay. The cells were plated at a density of 1×104 per well in
96-well plates overnight and then treated by FCC with different concentrations of cell solution
(0, 2, 5, 10, and 20 μg/ml). After incubation for 24 h, 48 h and 72 h at 37°C in a humidified
incubator, MTT (5 mg/ml in phosphate buffered saline (PBS)) was added to each well and
incubated for 4 h; then the medium was totally removed, 0.15 ml of buffered DMSO was added
to each well. The absorbance was recorded on a microplate reader at the wavelength of 490 nm.
The effect on cell proliferation was assessed as the percent cell viability wherein
vehicle-treated cells were taken as 100% viable.
Cell Cycle Analysis
After FCC treatment, the DNA content and cell cycle distribution of SMMC-7721 cells were
determined by flow cytometry. Cells plated at a density of 1×106 per well in 6-well plates,
treated with FCC and then harvested at 24 h. The cells were washed twice with PBS. They
were then fixed in cold 70% ethanol and stored at 4°C for 30 min. Then ethanol was removed
and the cells were resuspended in PBS. The fixed cells were then washed with PBS, treated
with RNase (100 mg/ml), and stained with Propidium Iodide (PI, 50 mg/ml) in the dark for 30
min at 37°C. Cell cycle was analyzed by flow cytometry.
Western Blot Analysis
SMMC-7721 cells were treated with FCC (0, 2, 5, 10, and 20 μg/ml) for 48 h in complete cell
medium. After 48 h of treatment, cells were harvested and cell lysates were prepared and stored
at -80°C for later use. The protein content in the lysates was determined using a modified
Lowry assay [38]. For Western blot analysis, 50 μg of protein from each sample were subjected
to separate on a SDS-PAGE gel. After electrophoresis, proteins were electroblotted to
polyvinylidene difluoride (PVDF) membranes, and subsequently incubated in blocking buffer
(5% nonfat dry milk) for 12 h at 4°C. The blots were incubated with appropriate primary
antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary
antibody. The blots were detected with chemiluminescence.
Statistical Analysis
All data represents at least three independent experiments and results were shown as mean ±
SD. Statistical differences between two groups were determined by Student’s t-test. A
significant difference was considered as p< 0.05.
List of abbreviations
HCC: Hepatocellular carcinoma; FCC: 6-fluoro-3-formylchromone; NSAIDs: non-steroidal
anti-inflammatory drugs; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide;
PBS: phosphate buffered saline; PVDF: polyvinylidene difluoride; HRP: horseradish
peroxidase.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Yijie Zhang and Kailian Zheng designed this study. Jing Wei performed the statistical analysis.
Chenghao Shao and Gang Jin carried out the study. Yingqi Zhou collected important
background information, Xuyu Zhou and Tianlin He drafted the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
None.
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Figure Legends
Figure 1 FCC Inhibited Cell Proliferation of SMMC-7721 cells.
(A) Chemical Structure of FCC. (B) Viability of SMMC-7721 cells treated with FCC. MTT
assay was performed to measure cell growth inhibition rate at 24 h, 48 h and 72 h after FCC
treatment. Data shown were representatives of three experiments.
Figure 2 Effects of FCC treatment on PCNA, Bax, and Bcl-2 expression.
(A) SMMC-7721 cells were treated with FCC at doses of 0, 2, 5, 10 and 20 μg/ml for 48 h. The
cell lysates were prepared and analyzed for PCNA, Bax and Bcl-2 expression by Western blot
analysis. Equal loading was confirmed by stripping immunoblots and reprobing for β-actin.
Data shown were representatives of three experiments. (B) Statistical analysis of PCNA
quantification. (C) The ratio of Bax to Bcl-2 protein. * p< 0.05, ** p< 0.01, compared with
control.
Table 1 Effects of FCC on cell cycle and apoptosis of SMMC-7721 cells.
FCC (μg/mL)
G0/G1
0
63.8±2.8
2
S
G2/M
apoptosis (%)
20.9±3.8
15.3±2.1
2.8±1.3
69.1±2.1
16.8±2.4**
14.1±1.8
9.5±1.1**
5
76.4±3.1
13.2±1.4**
10.4±0.9*
22.4±1.9**
10
80.2±1.9
11.0±3.0**
8.8±1.7**
37.5±2.1**
20
85.3±2.2
8.6±2.3**
6.1±2.0**
48.2±1.7**
Cell cycle analysis and apoptosis of SMMC-7721 following FCC treatment for 48 h by flow
cytometry. * p< 0.05, ** p< 0.01, compared with control.