ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 35
0884-6812/20/4202-0035/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
A
RTICLES
An Official Periodical of The International Academy of Cytology and the Italian Group of Uropathology
AQCH
ANALYTICAL and
QUANTITATIVE
CYTOPATHOLOGY and
HISTOPATHOLOGY®
OBJECTIVE: To investigate the mechanism of Ginseno-
side Rh2 in inhibiting the growth of NCI-N87 cells in
vitro, and to explore the possibility of Ginsenoside Rh2
becoming a STAT3 inhibitor.
STUDY DESIGN: The western blot method was em-
ployed to observe the effects of Ginsenoside Rh2 on
the expressions of p-STAT3, p-Jak2, p-Src, p-EGFR,
p-ERK1/2, and p-Akt in NCI-N87 cells. The effects of
drugs on the expressions of p-Jak2 and p-STAT3 were
measured after Ginsenoside Rh2 intervention and IL-6
(20 ng/mL) stimulation. Plasmids of DN-Jak2, DN-
EGFR, DN-Src, and CA-STAT3 were constructed and
transiently transfected to NCI-N87 cells. After being
exposed to Ginsenoside Rh2 for 24 hours, the expres-
sions of p-STAT3, p-Jak2, p-Src, and p-EGFR were
measured. Flow cytometry was applied to detect the
effect of Ginsenoside Rh2 on the cell cycle and apopto-
sis of transfected CA-STAT3 plasmid cells.
RESULTS: After treatment with Ginsenoside Rh2, the
expressions of p-STAT3 (Tyr705) and p-Jak2 in NCI-
N87 cells decreased, and the IL-induced p-Jak2 and
p-STAT3 were also inhibited. The p-Jak2 expression in
the transfected four groups of cells was decreased after
Ginsenoside Rh2 intervention, while the p-Src and
p-EGFR expressions remained the same. The transfec-
tion of CA-STAT3 plasmid could reverse the G0/G1
phase arrest and apoptosis caused by Ginsenoside Rh2
and mitigate the inhibition of p-STAT3.
CONCLUSION: Ginsenoside Rh2 selectively inhibited
the phosphorylation of Tyr705 site in the STAT3 mol-
ecule of NCI-N87 cells, in which the inhibition was
achieved by specific downregulation of the p-Jak2 pro-
tein. (Anal Quant Cytopathol Histpathol 2020;42:
35–41)
Keywords: antibodies, monoclonal; Cal-27; car-
Analytical and Quantitative Cytopathology and Histopathology®
Effects of Ginsenoside Rh2 on STAT3 Signaling
Pathway in Human Gastric Cancer Cell Line
NCI-N87 and Mechanism
Hao Yu, M.M., Xujie Wang, B.A., Qingbin Kong, B.A., and Xin Sui, B.A.
From the Department of Gastrointestinal Surgery, Weihai Central Hospital, Weihai, Shandong, P.R. China.
Hao Yu is Surgeon.
Xujie Wang is Surgeon.
Qingbin Kong is Surgeon.
Xin Sui is Surgeon.
Address correspondence to: Hao Yu, M.M., Department of Gastrointestinal Surgery, Weihai Central Hospital, 3 West Mishandong
Road, Wendeng, Weihai, Shandong 264400, P.R. China (yuhao93666@163.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. cinoma; cell differentiation; cell line, tumor; cell
proliferation/drug effects; gastrointestinal neo-
plasms; Ginsenoside Rh2; humans; Jak2; onco-
genes; protein kinase inhibitors; RNA, messenger;
signaling pathway; STAT3; STAT3 transcription
factor.
Gastric cancer is the second most common cancer
in the world. Its mortality rate is second highest
among all cancers. It is one of the most serious
malignant diseases threatening human health.1,2
The signal transducers and activators of transcrip-
tion (STATs) family is a class of proteins that play
an important or even crucial role in tumorigene-
sis and progression.3 Under normal physiological
conditions, the activated STAT3 will deactivate
the dephosphorylated monomer into the cyto-
plasm in a short time. The activation of STAT3
characterizes sustained high expression among
the main malignant tumor cells of the human
body. The sustained activation of STAT3 can pro-
mote the growth of tumor cells, inhibit the apop-
tosis of tumor cells, and stimulate the growth of
blood vessels in tumor tissues, which will dete-
riorate the condition of cancer patients.4,5 In con-
trast, targeted blocking STAT3 activation could
reverse the effect of STAT3, thereby inhibiting
tumor growth.6
Ginsenoside Rh2 is extracted from a traditional
Chinese medicine called Ginseng, with a strong
inhibitory effect on pathogenic bacteria such as he­
molytic streptococcus and staphylococcus aureus,
as well as some upper respiratory tract infection
pathogenic viruses. It is also immunity-enhancing,
liver-protective, anti-tumor, anti-inflammatory, an-
tipyretic, and hemostatic. However, the inhibitory
effect on NCI-N87 cells and its effect on apoptotic
and upstream cell cycle signaling pathway is still
unclear. Therefore, the current study has some clin-
ical significance.
Materials and Methods
MTT Assay to Detect Cell Viability
The NCI-N87 cells in logarithmic growth phase
were obtained and digested. Then the single cell
suspension was collected. The cells were counted
and seeded in 96-well culture plates at a density
of 1×104/200 μL per well, 5 compound holes for
each group. After cell adhesion, the cells were
added with Ginsenoside Rh2 at doses of 0, 10, 20,
and 40 μM, respectively, while the control group
was given the same dose of saline, and the blank
control hole was set. After 24 hours, incubation,
MTT (5 mg/mL) solution was added to each well,
and the absorbance values of each well were mea-
sured at OD 490 nm with an enzyme immuno-
assay detector.
Western Blot to Detect the Expressions of p-STAT3,
p-Jak2, p-Src, p-EGFR, p-ERK1/2, and p-Akt Proteins
NCI-N87 cells from each group were collected
and pretreated with IL-6 for 1 hour, followed
by the administration of Ginsenoside Rh2 for 24
hours. The cells were washed with PBS twice. A
total of 400 μL lysate was added to each flask,
and then 10 mmol/L PMSF 40 μL was added. The
flasks were gently agitated and then put on ice
for 10 minutes for sufficient lysis. The cells were
sucked with sterile syringe repeatedly. The lysate
product was added to the evoked potential (EP)
tube. The tube was ice-bathed for 30 minutes and
centrifuged at 12,000 g for 15 minutes. The su-
pernatant was decanted to a new EP tube. Using
protein labeled BC assay (bicinchoninic acid), the
protein concentration was measured by a micro-
plate reader. Following this, each tube was added
with 20 μL protein sample 6×buffer per 100 μL
and boiled for 5 minutes. The solution was mixed
and stored under −80°C. The samples were ob-
tained to conduct 12% SDS-PAGE electrophore-
sis for the separation of proteins. The protein
bands were transferred to the PVDF membrane
and closed under room temperature for 1 hour.
The cells were added with primary antibody
(P-STAT3, p-Jak2, p-Src, p-EGFR, p-ERK1/2, and
p-Akt, at a concentration of 1:1000). The cells were
incubated at 4°C overnight and then washed with
PBST thrice. The secondary antibody (concentra­
tion 1:1,000) was added for 1 hour incubation.
The cells were washed with PBST 3 times. After
chemiluminescence color development and fixa-
tion, the expressions of the above proteins were
measured.
Recombinant Plasmids Were Transiently Transfected
into NCI-N87 Cells
The NCI-N87 cells in logarithmic growth phase
were obtained and then digested. The single cell
suspension was collected. The cells were counted
and seeded into a 6-well culture plate at a density
of 4.5×105/2 mL per well. According to the pro­
tocols, a total of 3.5 μg of Src-RNAi, EGFR-RNAi,
Jak2-RNAi, CA-STAT3, and empty plasmids were
added to the serum-free Opti-MEM medium (In-
36 Analytical and Quantitative Cytopathology and Histopathology®
Yu et al

3. vitrogen, USA). Simultaneously, a host of 9 µL
Lipofectamine 2000 solution (Invitrogen, USA), was
added into the Opti-MEM. The reaction was car-
ried out at room temperature for 5 minutes. The
former solution containing the plasmid was add-
ed to the latter one containing Lipofectamine 2000.
The mixture was gently agitated and evenly mixed
to react at room temperature for 20 minutes. Then,
the mixture was added dropwise to a 6-well plate,
and the plate was gently shaken for sufficient mix
of plasmid complex with the medium. After being
cultured in an incubator for 48 hours, the green
fluorescence expression was measured with a fluo-
rescence inverted microscope.
Western Blot to Detect the Jak2/STAT3 Signaling
Pathway in NCI-N87 Cells
The NCI-N87 cells and the transfected DN-EGFR,
DN-Src, DN-Jak2, and CA-STAT3 were treated
with 40 μM Ginsenoside Rh2 for 24 hours. Sub­
sequently, the western blot method was applied
to detect the expression of P-Jak2, p-Src, and p-Src
and p-STAT3 proteins using the same procedure
described above.
Flow Cytometry to Detect Cell Cycle
The NCI-N87 cells at logarithmic growth phase
were washed twice with PBS and then digested
with 0.25% trypsin for 3 minutes. The cells were
collected to make a single cell suspension, and
then the numbers were counted. The cells were
seeded into 6-well plates at the density of 4×105/
well, 2 mL each. After cell adhesion, a host of
2 mL Ginsenoside Rh2 at 0 and 40 μM was
added, respectively. After 24 hours of culture,
the cells were collected and centrifuged. The den­
sity was adjusted to 1×106/mL, and then 70%
ethanol was added for 4°C overnight. The sec-
ond day, the cells were washed twice with PBS
and incubated with RNAse (8 μg/mL) and pro­
pidium iodide (PI) (10 μg/mL) for 30 minutes. The
cell cycle was detected by flow cytometry.
Annexin V-FITC to Detect Apoptosis
Cells were treated with flow cytometry. Cells were
collected according to the Annexin V-FITC assay
kit (Becton Dickinson, USA). Cells were harvested
and digested by trypsin without EDTA. The cell
concentration was adjusted to 1×106/mL by cen-
trifugation and then washed twice with PBS. The
cells were centrifuged at 1,000 rpm for 5 minutes,
the supernatant was discarded, and the process
was repeated 3 times. The cells were resuspended
with 400 μL of 1× binding buffer, and then 5 μL
of Annexin V-FITC and 10 μL of PI were added
and gently mixed. The cells were incubated at
room temperature under darkness for 15 minutes.
The apoptosis was detected after 1 hour.
Statistical Methods
All data are expressed as mean±standard devia­
tion (SD). Comparison between the two groups
was conducted using t test, and comparison be-
tween multiple sets of data (>2) was through the
one-way ANOVA, with p<0.05 indicating statis-
tical significance. The data were analyzed with
Graphpad Prism 5.0 (Graphpad Software, San
Diego, California, USA).
Results
MTT Assay to Detect Cell Viability
The results of MTT showed that, after drug ad-
ministration, the proliferation of cells was inhib-
ited in a positively proportional way. The inhibi-
tion rate of the high concentration group was the
highest, indicating that different concentrations of
Ginsenoside Rh2 can effectively inhibit the prolif­
eration of NCI-N87 (Figure 1).
The Associated Plasmid Transfection of NCI-N87
Cells
The NCI-N87 cells were successfully transfected
with EGFR-RNAi, Src-RNAi, Jak2-RNAi, and emp­
ty plasmids, and the transfected cells were iden-
tified by RT-PCR. The experiment showed that
Volume 42, Number 2/April 2020 37
Ginsenoside Rh2 in Gastric Cancer
Figure 1 Inhibitory effect of Ginsenoside Rh2 on NCI-N87 cells.
**p<0.05 vs. 0 μm, &&p<0.01 vs. 10 μm, &p<0.05 vs. 10 μm,
#p<0.05 vs. 20 μm.

4. EGFR-RNAi (7992-1, DN-EGFR), Src-RNAi (18911-
1, DN-Src), and Jak2-RNAi (19019-1, DN-Jak2)
plasmids have the most significant depleting ef-
fects on the corresponding EGFR, Src, and Jak2
proteins (Figure 2).
Detecting the Expressions of p-STAT3, p-Jak2, p-Src,
p-EGFR, p-ERK1/2, and p-Akt by Western Blotting
Western blot results showed that Ginsenoside Rh2
decreased the expressions of p-STAT3, p-Jak2,
p-Src, p-EGFR, and p-ERK1/2 proteins but had
little effect on p-Akt protein (Figure 3).
A dose of 40 μM Ginsenoside Rh2 was admin-
istered to NCI-N87 cells for 24 hours, and recom­
binant IL-6 was added to intervene for 1 hour.
Western blot results revealed that IL-6 could
increase the expressions of p-Jak2 and p-STAT3
proteins, while the intervention of Ginsenoside
Rh2 inhibited the expression of both proteins,
but with minor changes in the Jak2 and STAT3
expressions in the control group (Figure 4).
38 Analytical and Quantitative Cytopathology and Histopathology®
Yu et al
Figure 2 Results of associated plasmid transfection of NCI-N87
cells. Figure 3 The effect of Ginsenoside Rh2 on the expressions of
p-STAT3, p-Jak2, p-Src, p-EGFR, p-ERK1/2, and p-Akt proteins.
Figure 4 The effect of Ginsenoside Rh2 on the expressions of
p-STAT3 and p-Jak2.

5. Ginsenoside Rh2 Inhibited the Jak2/STAT3 Signaling
Pathway of Transfection-Associated Plasmid
NCI-N87 Cells
As shown in Figure 4, Ginsenoside Rh2 inhibited
the expressions of p-Jak2 and p-STAT3 in empty
plasmid cells, with the similar mechanism as
DN-Jak2 depleting p-Jak2. The expressions of p-
EGFR and p-Src proteins, which transfected DN-
EGFR and DN-Src plasmid cells, were inhibited.
However, the two proteins that transfected empty
plasmid cells were not affected by Ginsenoside
Rh2. Transfection of CA-STAT3 plasmid cells at­
tenuated the inhibitory effect of DHA on p-STAT3,
and the CA-STAT3 empty plasmid p-STAT3 was
inhibited by Ginsenoside Rh2 (Fig­
ure 5).
Flow Cytometry to Detect Cell Cycles
The results showed that the proportion of apop-
totic cells increased in transfected empty plas-
mid NCI-N87 cells after being administrated with
Ginsenoside Rh2, whereas the cells in G0/G1
phase decreased after adding Ginsenoside Rh2
to NCI-N87 cells that were transfected with CA-
STAT3 plasmid, and the apoptosis reduced as
well. It was suggested that after treatment with
Ginsenoside Rh2, apoptosis and G0/G1 cycle ar-
rest of cells in transfected empty plasmids were
significantly increased, while CA-STAT3 could at­
tenuate or partially reverse the effects of Ginseno-
side Rh2 (Figures 6–7).
Discussion
In the pathogenesis and treatment of tumors, mem-
bers of the STATs family as targets are so impor-
tant that the famous scholar Hua Yu called it the
advent of a new molecular target era.7 In addition
to being activated by proteins such as Jak2, Src,
FGF, VEGFRs, and c-Met, many signaling path-
ways such as mTOR, MAPK, and p38 can activate
Volume 42, Number 2/April 2020 39
Ginsenoside Rh2 in Gastric Cancer
Figure 5 The effect of Ginsenoside Rh2 on Jak2/STAT3 signaling
pathway in transfection-associated plasmid NCI-N87 cells.
Figure 6 CA-STAT3 reverses cell cycle arrest induced by
Ginsenoside Rh2. **p<0.05 vs. 0 μm, &p<0.05 vs. 40 μm,
##p<0.01 vs. 0 μm+CA-STAT3.

6. STAT3 as well. Of all the oncogenes, the EGFR,
Src family, and Jak2 are the three most import-
ant regulatory signaling proteins that can activate
STAT3,8 and in particular the Jak2/STAT3 signal-
ing pathway is the most classic and important. In
recent years, tumor researchers are paying more
and more attention to the relationship between
STAT3 and tumors, and the research is getting
in-depth. The STAT3 oncogene effect in animal
experiments is very significant; the existence of a
separate activation can lead to normal cell malig-
nancy and tumorigenesis, which is an important
starting factor in leading to malignant tumor cells
to obtain treatment resistance.9-12
The present experiment showed that the changes
of p-Erk1/2 and p-Akt in the two signal pathways
were not obvious after intervention of different
concentrations of Ginsenoside Rh2. Ginsenoside
Rh2 has no inhibitory effect on Ser727 activation
of site STAT3, which is likely because Ginseno-
side Rh2 has no effect on upstream p-Erk1/2,
leading to the absence of serine phosphorylation
of STAT3. The state of p-Erk1/2 to a certain ex-
tent affected the activation of the Ser727 site. The
data suggested that Ginsenoside Rh2 inhibition
of STAT3 activation is at least partially selective
and specific. Meanwhile, the study also found that
Ginsenoside Rh2 can specifically inhibit Jak2 ac-
tivation, suggesting that Ginsenoside Rh2 inhibi-
tion of STAT3 depends on the decline of p-Jak2,
indicating that Ginsenoside Rh2 played a role in
inhibiting Jak2/STAT3 signaling pathway. Inflam-
matory mediators IL-6 can bind to receptors on
the cell membrane, and the outcome complexes
can strongly activate Jak2, which can induce the
phosphorylation of Tyr705 in STAT3. Thus, IL-6
is recognized as a potent Jak2/STAT3 stimulant.
In the present study, the expressions of p-Jak2
and p-STAT3 proteins increased in all three kinds
of NCI-N87 cells treated with IL-6, and the inter-
vention of Ginsenoside Rh2 could reduce such
expressions, which suggested that Ginsenoside
Rh2 inhibited STAT3 activation by blocking Jak2
phosphorylation.
RNA interference refers to a biologically highly
conventional, double-stranded RNA-induced gene
silencing mechanism that inhibits gene expression
by hindering the translation or transcription of a
particular gene.13,14 In the present study, NCI-N87
cells were transfected with Src-RNAi, EGFR-RNAi,
Jak2-RNAi, CA-STAT3 plasmids, and correspond-
ing empty plasmids. The transfected cells were
identified and screened by RT-PCR. The optimal
interference suppression gene was examined ex-
perimentally. After transfection with a certain dose
of Ginsenoside Rh2 for 24 hours, Jak2 and STAT3
protein activation was found to be reduced. This
suggests that Ginsenoside Rh2 inhibition of NCI-
N87 cell growth is achieved by specifically block-
ing the Jak2/STAT3 signaling pathway. CA-STAT3
can partially reverse this inhibitory effect and
can partially offset the Ginsenoside Rh2-induced
G0/G1 cycle arrest and apoptosis. Therefore, the
blocking of STAT3 activation is located in the in-
hibition of upstream Jak2 protein activation, and
the state of the Jak2 protein is a key factor in in-
hibiting cell proliferation.
In summary, Ginsenoside Rh2 selectively inhib­
ited the phosphorylation of Tyr705 site in the
STAT3 molecule of NCI-N87 cells, in which the
inhibition was achieved by specific downregula-
tion of the p-Jak2 protein.
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Figure 7
CA-STAT3 reverses cell cycle
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Ginsenoside Rh2.

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