ABBS 2008,40(11): Nidus vespae protein inhibiting proliferation of HepG2 hepatoma cells through extracellular signal-regulated kinase signaling pathways and inducing G1 cell cycle arrest

A protein named NVP(1) was
isolated from Nidus vespae. The aim of the present study was to
elucidate whether and how NVP(1) modulates the proliferation of HepG2 cells.
NVP(1) at a concentration of 6.6 mg/ml could arrest the cell cycle at stage G1 and inhibit
the mRNA expression of cyclinB, cyclinD1 and cyclinE. NVP(1) suppressed cdk2
protein expression, but increased p27 and p21 protein expression. However,
NVP(1) did not alter p16 protein expression levels. NVP(1) promoted apoptosis
in HepG2 cells as indicated by nuclear chromatin condensation, and in addition,
the extracellular signal-regulated kinase (ERK) signaling pathway was
activated. Moreover, the p-ERK protein expression level was attenuated when the
HepG2 cells were pretreated with ERK inhibitor PD98059. These results demonstrate
that NVP(1) inhibits proliferation of HepG2 through ERK signaling pathway.
NVP(1) could be a potential drug for liver cancer.

Keywords       Nidus vespae; NVP(1); ERK; cell
proliferation

Liver cancer is the most
frequent cause of cancer-related death in The cell cycle is
regulated through the sequential activation­ and inactivation of
cyclin-dependent protein kinases­ (Cdk) that control specific steps of the
cycle progression, such as G1-S and G2-M transitions [6,7]. Cdk activation requires
binding to different cyclins, such as cyclinA, cyclinB, cyclinE, and cyclinD,
which are expressed­ during the course of the cell cycle. Cdk activity is also
regulated by a diverse family of proteins, termed Cdk inhibitors (Cdki), that
bind and inactivate Cdk-cyclin complexes [8–13]. Two classes of Cdki are known, one is the CIP/KIP
family, which includes p21, p27, and p57 [13–15], and the other is the INK family, which includes p15,
p16, p18, and p19 [16–21]. A
central role in the cell cycle progression is played by the retinoblastoma
family of proteins, which are critical target substrates, and are
phosphorylated by the Cdk-cyclin complexes. The phosphorylation­ of
retinoblastoma proteins controls gene expression through the release of the EThe mitogen-activated protein
kinases (MAPK) family is made up of serine/threonine kinases that comprise
three major subgroups, namely extracellular regulated kinase (ERK), p38 MAPK
(p38), and C-Jun NH2-terminal kinase (JNK) [23]. Of these, ERK is shown to be
crucial in cell proliferation and differentiation. Other members of the MAPK
family, including p38 and JNK, were originally thought to mediate the cellular
stress response and apoptosis [24]. In the present research, we isolated a
protein from Nidus vespae, named NVP(1), whose apparent molecular mass
was 6.6 kDa, and investigated the mechanism of its inhibition of HepG2
cells proliferation. We found that p21, p27 and cdk2 affected the cell cycle in
HepG2 cells treated with NVP(1). Our results indicate that the ERK pathway is
required for NVP(1) induced growth arrest­ and antiproliferation response.
Therefore, NVP(1) could be a potential drug for liver cancer.

Materials and Methods

Materials

Nidus vespae was obtained
from the wild in

Extraction and purification
of Nidus vespae protein

Nidus vespae (

Cell culture

HepG2 hepatoma cell
lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% Gibco fetal bovine serum (FBS; Invitrogen,

Cell counting and cell
cycle analysis

Culture wells (6-well
cluster dishes, Costar, Sigma Aldrich, St Louis, USA) were inoculated with 3 ml
growth medium­ containing 5´104 cells and
allowed to attach overnight. Cell proliferation in response to NVP(1) (6.6 mg/ml) was determined. Cell counts were determined on days 1, 2, 3, 4 and
5. Cells from each well were removed by trypsinization and the resultant
suspension was counted using a Hemocytometer (Hausser Scientific,

RNA extraction and
semiquantitative reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted
(20 mg), and DNase was used
to eliminate the contamination of genomic DNA. The PCR primers pairs used for genes
amplification were: actin (NM_001100) (432 bp) actin 1, 5‘-caggtcatcactatcggcaa-3‘;
actin 2, 5‘-caaagaaagggtgtaaaacgc-3‘; cyclinB(NM_031966) (385 bp)
cyclinB 1, 5‘-tcgcctgagcctattttggt-3‘; cyclinB 2, 5‘-gcatcttcttgggcacacaa-3‘;
cyclinE(NM_001238) (366 bp) cyclinE 1, 5‘-aaaat­cgacag­gac­g­g­c­gag-3‘;
cyclinE 2, 5‘-tgccaagtaaaaggtctccc-3‘; cyclin D1(NM_053056) (310
bp) cyclin D1 1, 5‘-aatgtgtgcag­aagg­a­ggtc-3‘; cyclin D1 2, 5‘-ttgagcttgttcaccaggag-3‘;
proliferating­ cell nuclear antigen (PCNA) (NM_002592) (410 bp) PCNA 1, 5‘-gcactcaaggacctcatcaa-3‘;
and PCNA 2, 5‘-atatggctgagatctcggca-3‘. After denaturation at 94 ºC
for 2 min, PCR was carried out in a DNA thermal cycler (Biometra Thermocycler,
Göttingen, Germany)
for 30 cycles. Each cycle includes denaturation at 94 ºC
for 40 s, annealing at 54 ºC for 40 s, and extension at 72 ºC
for 60 s, followed by a final extension at 72 ºC
for 5 min. The PCR products were separated on 2% agarose gel in TAE buffer (

Western blot analysis

HepG2 cells were
collected by rubber policeman, washed in PBS, and lysed in ice-cold PBS buffer
[0.l M Tris-HCl, pH 8.0,

Statistical analysis of
the data

All data are presented
as mean±standard deviation. Significant­ differences among
the groups were determined using the unpaired Student’s t-test. A value
of P<0.05 was accepted as an indication of statistical significance. All the figures shown in this article were obtained from at least three independent experiments.

Results

Detection of molecular
weight of NVP(1)

Molecular weight of
NVP(1) was 6600 Da detected by mass spectrometry (data not shown). The result
was then confirmed by 15% SDS-PAGE assay [Fig. 1(A)].

Inhibition of HepG2 proliferation
by NVP(1) in vitro

In the absence of
NVP(1), 10% fetal bovine serum caused a time dependent increase in cell
numbers, whereas the increase in cell number was obviously reduced in the
presence­ of 6.6 mg/ml NVP(1) [Fig.
1(B)].

Comparing with serum-treated
control cultures, cells treated with 6.6 mg/ml NVP(1) for 48 h showed unique morphological changes.
The cells were much smaller in their size with a change in morphology to
spherical shape [Fig. The IC50 of NVP(1)
on the assembly of HepG2 cells was assessed by quantifying the cell count in
the presence of different concentrations of NVP(1) between 3.4710–4 mg/ml and 65 mg/ml for 3 d. The percentage of inhibition induced by
NVP(1) was normalized against controls and plotted against different
concentrations of NVP(1) used in the experiment. The IC50 was calculated to
be 6.6 mg/ml [Fig.
1(E)].

The role of NVP(1) in
regulating the HepG2 cell cycle

To gain insight into the
mechanism of growth inhibitory effects of NVP(1), we assessed cell cycle
distribution of HepG2 cells by flow cytometry. Treatment of HepG2 cells with
6.6 mg/ml of NVP(1) for 24h
resulted in the increased accumulation of the cells in G1 phase [Fig.
1(D)]. Cultured­ HepG2 cells were first synchronized and then treated with
NVP(1) for 24 h. In controls, 35.4%2.7% of HepG2 cells mainly progressed into S
phase after serum stimulation for 24 h, however, in the presence of NVP(1)
treatment, 89.0%3.5% of HepG2 cells remained in the G1 phases, with only
10.95% of cells entering S phase [Fig. 1(D)]. On average, the G1 phase cells
treated with NVP(1) was 90.6%2.0% (n=4). This indicates that the growth
suppressive effect of NVP(1) is to arrest the cell cycle at the G1 phase.  

NVP(1) induces apoptosis
in HepG2 cells

In the flow cytometric
analysis, the observation of sub-G1 peaks at 24 h following NVP(1) treatment has
suggested that NVP(1) could promote apoptosis in HepG2 cells following­ cycle
arrest [Fig. 1(D)]. One of the early events of apoptosis is the
condensation of nuclear chromatin. We therefore investigated the morphology of
NVP(1)-treated cells using PI staining, cells treated 20 mg/ml NVP(1) for 48 h displayed the typical
morphology of nuclear chromatin condensation [Fig.

NVP(1) decreases cyclins
(B, E, and D1) mRNA levels

In order to examine whether
NVP(1) could affect cyclins transcription, semi-quantitive RT-PCR were
performed using primer pairs specific for cyclinB, cyclinD1, cyclinE, and using
actin as a control. It was shown clearly that there were significant
decreases in the mRNA level of cyclinB, cyclinD1, and cyclinE in
HepG2 cells treated with NVP(1) (Fig. 2). Surprisingly, mRNA was almost
undetectable­ after NVP(1), treatment for 20 h. However, mRNA expressions of
PCNA (Fig. 2) and actin (Fig. 2) were not
influenced by NVP(1).

Effect of NVP(1)
treatment on the expression of Cdk and Cdki in HepG2 cells

In order to understand
the signaling pathways in NVP(1) induced HepG2 cells growth inhibition,
specific cell cycle protein mediators were monitored at different time points
following NVP(1) treatment by Western blot.

It is known that the
cyclin dependent kinase inhibitors p27 and p21 proteins bind and antagonize
cyclin/cdks complexes­ in order to halt cell cycle progression [26]. p27
protein levels remained low level 5 min following NVP(1) treatment, and then
sharply increased later (Fig. 3).The increase in p21 protein was
dramatic 15–120 min
following NVP(1) treatment, whereas the p21 protein was not detected in 0–5 min following NVP(1) treatment (Fig. 3).
No changes in p16 protein were observed in HepG2 cells treated with NVP(1) (Fig.
3).

Cdk2 proteins are
expressed during G1 phase of the cell cycle and cyclinD1/cdk2, 4
or 6 are required for G1 progression­ [27]. In HepG2 cells, Cdk2
protein levels were significantly down-regulated following NVP(1) treatment­ (Fig.
3). The actin protein levels were not changed (Fig. 3).

Inhibition of HepG2
proliferation by NVP(1) via the activation of the ERK signaling pathway

In order to explore the
signaling pathway involved in the inhibition of HepG2 proliferation by NVP(1),
we detected the activation of three distinct downstream mitogen-activated­
protein kinases, ERK, c-Jun N-terminal kinase (JNK), and p38, by measuring the
proportion of their phosphorylated­ forms. HepG2 cells cultured in the presence­
of NVP(1) were terminated at various times. The levels of total ERK protein
remained the same in both control and treated cells [Fig. 4(A)].
However, the proportion of phosphorylated ERK increased in a time- and
dose-dependent manner [Fig. 4(B)]. The total protein levels of p38 and
JNK were also measured and no changes were observed. The phosphorylated forms
of p38 and JNK were not detectable­ (data not shown). These results demonstrate
that NVP(1) inhibits HepG2 cell proliferation through the ERK signaling
pathway, whereas the p38 and JNK signaling­ pathways are not involved.

The MEK1/2 inhibitor
blocks phosphorylation of ERK

To confirm NVP(1)
inhibiting HepG2 cells proliferation through the ERK signaling pathway, we
treated HepG2 cells with PD98059, the potent inhibitor of MEK/ERK, for 1 h
before NVP(1) was added to the culture medium for 5, 15, 30, 60 and 120 min
respectively. Lysates were used for Western blot analysis. The levels of total
ERK protein and actin remained the same in both control and treated cells [Fig.
5(A)]. Fig. 5(B) shows that in the presence of PD98059 (20 mM), phosphorylated ERK was dramatically reduced.
Dimthyl sulfoxide (DMSO) alone had no effect on the level of phosphorylated ERK
protein when compared­ to the control [Fig. 5(B)]. The results
demonstrated­ that NVP(1) activated phosphorylation-ERK signal pathway.

Discussion

We detected the extracts
from Nidus vespae in order to explore whether they can inhibit
proliferation of HepG2. First, we isolated one kind of protein designated
NVP(1) from Nidus vespae to investigate the molecular mechanism­ of its
inhibition on proliferation in HepG2 cells. We found that NVP(1) could suppress
HepG2 cell proliferation in both a time- and dose-dependent manner. The same
effect­ was obtained from PC3 prostate cancer cells and primary cultured
bronchial smooth muscle cells of wister rats, the results indicating that
NVP(1)-inhibited cell proliferation is not selected (data not shown).
Additionally, HepG2 treated with NVP(1) was dramatically blocked at the G1 phase. The
results of the Western blot analysis clearly demonstrate­ that NVP(1) induces
cell cycle arrest specifically­ at the G1 phase via the
up-regulation of p21 and p27 and inhibition of cdk2 protein expression.
Moreover, we found that NVP(1) enhanced the ERK activation­ by phosphorylation.

The importance of cdk2
and cdk4 activation and the subsequent phosphorylation of pRb in the G1 to S
transition­ have been emphasized in a variety of cells [28]. The
phosphorylation­ of pRb is required to release EMitogen activated
protein kinases signaling cascade pathways comprise a group of three main
pathways, including ERK 1 and 2, and two stress-activated protein kinases
designated JNK and p38 [35,36]. We analyzed whether the Mitogen activated
protein kinase pathway is involved in NVP(1) dependent HepG2 growth inhibit.
Among the Mitogen activated protein kinases family, the ERK1/2 cascade is
critical to proliferation of many types of cells [37], our data showed that NVP(1)
inhibited HepG2 proliferation and increased ERK1/2 activity in a dose- and
time-dependent manner. At the same time, we were not being able to detect p-p38
and p-JNK proteins in the HepG2 treated with NVP(1). It is suggested that
NVP(1) inhibits HepG2 mitogenesis, in part by increasing ERK1/2 signaling but
not through p38 and JNK signal pathways. Therefore, we further examined whether
specific inhibition of ERK, PD98059 can attenuate NVP(1)-induced senescence or
apopotosis in HepG2. The results [Fig. 5(B)] showed that inhibition of
p-ERK significantly suppressed NVP(1)-induced HepG2 death.

NVP(1) seems to halt the
cell cycle progression until the sub-G1 population increases-a common
indication of the presence of apoptosis cells, if this proposition is correct,
it implies that HepG2 will start to undergo apoptosis some times after the
NVP(1)-induced cell cycle arrest. Many apoptotic bodies were observed by
propidium iodide­ DNA staining when HepG2 cells were treated by NVP(1) for 24 h
or 48 h [Fig. 1(C)]. At present, NVP(1) is only detected by molecular
weight. However, the amino acid sequence and construction for protein have not
been elucidated. Moreover, the receptor associated with NVP(1) is also not
clear. The experiment presented here testified­ that NVP(1) promotes apoptosis
and inhibits cell proliferation. It is suggested that NVP(1)-induced growth
suppression contributes to synthesis effects including apoptosis, cell
proliferation, and cell cycle arrest.

In summary,
we have demonstrated that NVP(1) prevents HepG2 cell proliferation through
activating the ERK signal pathway. On the other hand, NVP(1) arrests the cell
cycle at G1. Finally, the question
of whether NVP(1) may act as a regulator of apoptosis or senescence for liver
cancer in vivo remains to be answered. Our data suggest a possible agent
in clinical therapeutic treatment for patients­ who are suffering from cancer
and provide important evidence­ for understanding the mechanism underlying­ the
inhibitory effect of NVP(1) on HepG2 cell proliferation.

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