ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 27
OBJECTIVE: To investigate the anticancer actions of
dihydrotanshinone I (DHTS) in AGS human gastric
cancer cells.
STUDY DESIGN: Cell viability was determined using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay. Intracellular reactive oxygen spe-
cies (ROS) levels were determined using flow cytome­
try. Caspase activities were measured with fluorometric
assay.
RESULTS: Results from MTT assay showed that DHTS
significantly inhibited AGS cell viability in dose- and
time-dependent manners. Elevated intracellular ROS
levels and increased apoptotic cells were observed in
DHTS-treated AGS cells. In addition, activation of
caspase-3 and caspase-8, rather than caspase-9, was no-
ticed in DHTS-treated AGS cells. Furthermore, block-
ing ROS generation with N-acetylcysteine markedly re-
versed DHTS-induced cell apoptosis.
CONCLUSION: All the findings strongly suggest that
DHTS can initiate ROS generation and induce oxida­
tive stress and cell apoptosis in AGS human gastric
cancer cells, which deserves to be further developed as an
anticancer agent. (Anal Quant Cytopathol Histpathol
2019;41:27–32)
Keywords: alternative medicine; apoptosis; apo-
ptosis/drug effects; cell line, tumor; cell prolifera­
tion/drug effects; complementary therapies; Dan­
shen; dihydrotanshinone I; gastric cancer; humans;
mitogen-activated protein kinases/metabolism;
reactive oxygen species; Salvia miltiorrhiza.
Gastric cancer is one of the leading causes of
cancer-related death in the world, with low prog­
noses and high mortality.1 Physical surgery is still
the most effective way to treat gastric cancer.2
However, the survival rate remains low due to lim-
ited therapy and delayed diagnosis approaches
for gastric cancer. Chemotherapy is one optional
way in treating gastric cancer, especially in those
patients at the late stage with metastasis.3 Natural
products and their derivatives, such as vitamins,
curcumin, and green tea, show great potential ben­
efits to control the growth of cancers.4-6
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4101-0027/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Dihydrotanshinone I Induces Apoptosis
Through Reactive Oxygen Species–Mediated
Oxidative Stress in AGS Human Gastric
Cancer Cells
Xiongdong Zhong, M.Sc., Ni Gu, M.Sc., Lei Chen, B.Sc., and Yunlong Pan, Ph.D.
From the Departments of General Surgery, The First Affiliated Hospital of Jinan University and The People’s Hospital of Zhuhai, Guang-
zhou, Guangdong Province, P.R. China.
Dr. Zhong is Associate Chief Physician, Department of General Surgery, The First Affiliated Hospital of Jinan University, and Depart­
ment of General Surgery, The People’s Hospital of Zhuhai.
Dr. Gu is Resident Physician, Department of General Surgery, The People’s Hospital of Zhuhai.
Mr. Chen is Chief Physician, Department of General Surgery, The People’s Hospital of Zhuhai.
Dr. Pan is Professor, Department of General Surgery, The First Affiliated Hospital of Jinan University.
Address correspondence to: Yunlong Pan, Ph.D., Department of General Surgery, The First Affiliated Hospital of Jinan University, No.
613 Huangpu Road, Tianhe District, Guangzhou 510632, Guangdong Province, P.R. China (yunlongpan@21cn.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Dihydrotanshinone I (DHTS) is a natural bio­
logical tanshinone identified from the herbal med­
icine called Danshen (Salvia miltiorrhiza), which has
been widely used for treating cardiac-cerebral vas­
cular diseases and hepatitis for a thousand years
in China. DHTS has been demonstrated to have
diverse biological activities, including antibacteri-
al,7 platelet aggregation inhibition,8 antiangiogen­
esis,9 antiosteoclastogenesis,10 etc. Recently, DHTS
was shown to inhibit various cancers including
colon cancer,11,12 prostate cancer,13 liver cancer,14
breast cancer,15 and cervical cancer.16 The molec-
ular mechanisms for the anticancer action of
DHTS included induction of apoptosis,12,15,16 ac-
tivation of autophagy,12 induction of cell cycle
arrest,15 etc. However, to date, the effect of DHTS
on gastric cancer has not been comprehensively
elucidated.
Reactive oxygen species (ROS) are produced
by various redox metabolic reactions, which play
important roles in numerous physiological and
pathological processes, including arteriosclerosis
and carcinogenesis.17 ROS, especially H2O2, are key
elements for inflammatory cell recruitment.18 Fur­
thermore, ROS work as second messengers to in-
duce diverse redox-sensitive signaling molecules,
for example, the stress-activated p38 MAP kinases.19
The activation of p38 MAPK by ROS will eventual­
ly induce cell death, including apoptosis, necrosis,
and autophagy.20
In the current study the anticancer action of
DHTS was investigated in human gastric cancer
cell line AGS and human gastric epithelial cell line
GES-1. The underlying mechanisms for the pro-
apoptotic effects of DHTS were also evaluated
using numerous methods.
Materials and Methods
All experiments were approved by the Research
Committee of The First Affiliated Hospital of Jinan
University and carried out in accordance with the
approved guidelines.
Reagents
Dihydrotanshinone I (DHTS, purity >98%) was
obtained from TianJin Zhongxin Pharmaceutical
Group Co. Ltd. (Tianjin, China). RPMI culture
medium, N-acetylcysteine (NAC), 2’7’-dichlorodi­
hydrofluorescein diacetate (H2DCFDA), annexin
V/PI (propidium iodide) apoptosis kit, and di-
methylsulfoxide (DMSO) were purchased from
Sigma (St. Louis, Missouri, USA). Fetal bovine
serum and penicillin-streptomycin were purchased
from Tianjin Hao Yang Biological Manufacture
Co. Ltd. (Tianjin, China). Unless stated otherwise,
other reagents were purchased from Sigma. DHTS
was dissolved in DMSO and stocked at −20°C,
which was diluted to the desired concentrations
with fresh culture medium before usage. The final
concentration of DMSO was lower than 0.5% in
fresh medium, which showed no obvious effect on
cell viability.
Cell Culture
Human gastric cancer cell line AGS and human
gastric epithelial cell line GES-1 were obtained
from the Cell Bank of the Committee on Type
Culture Collection of the Chinese Academy of
Sciences (Shanghai, China) and cultured in RPMI
1640 medium supplemented with 10% fetal bovine
serum and 1% penicillin-streptomycin at 37°C
under 5% CO2.
Cell Viability Assay
The effects of DHTS on cell viability of AGS cells
and GES-1 cells were measured with 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay. Briefly, the cells were seed­
ed in 96-well plates (4×103 cells/well) and then
incubated with DHTS (0, 5, 10, 20 μM) for 48
hours. The medium was then replaced with fresh
culture medium containing 20 μL of 5 mg/mL MTT
solution. After further incubation for 4 hours, the
supernatants were replaced with 100 μL DMSO
to dissolve the formed blue formazan crystal.
Absorbances at a wavelength of 570 nm were
collected using a multiple fluorescent plate reader
(Bio-Rad Laboratories, USA). The data were shown
as the percentage of cell viability compared with
vehicle control group.
Measurement of Intracellular Reactive Oxygen
Species (ROS) Level
The intracellular ROS levels were determined
using a fluorogenic probe H2DCFDA. In brief,
AGS cells were treated with different concentra­
tions of DHTS with or without NAC (500 μM) for
2 or 4 hours. Then, the cells were washed twice
with phosphate buffered saline (PBS) and incu-
bated with H2DCFDA (30 μM) at 37°C for 30 min­
utes. After incubation the cells were then washed
twice and resuspended in fresh PBS. The fluores­
cence of 10,000 individual cells from each group
were collected and analyzed using a FACStar flow
28 Analytical and Quantitative Cytopathology and Histopathology®
Zhong et al

3. cytometer (Becton Dickinson, Franklin Lakes, New
Jersey, USA).
Oxidative Stress Detection
Reduced glutathione (GSH)/oxidized disulfide
(GSSG) ratio was measured to determine oxida­
tive stress in AGS cells with DHTS treatment. In
this study the concentrations of total glutathione
(T-GSH), GSH, and GSSG were measured using
enzymatic methods. T-GSH was measured by
testing 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB)-
GSSG reductase recycling. GSSG was determined
using 5-thio-2-nitrobenzoic acid (TNB) generated
from reduced GSH reaction with DTNB. The sig­
nal measured at 410 nm was used to assay the TNB
formation. The reduced GSH level was calculated
by subtracting GSSG from T-GSH.
Measurement of Apoptotic Cells by Flow Cytometry
The apoptotic cells in DHTS-treated AGS cells
were determined using flow cytometry with
Annexin V/PI apoptosis detection kit under the
manufacturer’s instructions. In brief, AGS cells
were treated with DHTS with or without NAC
(500 μM) for 48 hours and washed 3 times with
ice-cold PBS. Annexin V-FITC (5 μL) and PI (1 mg/
mL) were then added into the cells and further
incubated for another 30 minutes. The stained
cells were washed twice with PBS, and data from
20,000 cells of each group were collected and
analyzed by a BD flow cytometer.
Detection of Caspase Activity
Caspase-3, caspase-8, and caspase-9 activities were
measured with fluorometric assay kits following
the manufacturer’s instructions (Abcam). Briefly,
AGS cells were collected and lysed with lysis buf­
fer. Proteins (20 μg) were incubated with caspase-
3 substrate Ac-DEVD-AMC, caspase-8 substrate
Ac-LEDH-AMC, or caspase-9 substrate Ac-IETD-
AMC (50 μg) at 37°C for 2 hours, respectively.
The mixtures were then transferred into a 96-well
microplate with black bottom. Fluorescences at
400 nm and 505 nm for noncleaved substrate and
cleaved substrate, respectively, were recorded in a
fluorescent plate reader (Millipore, USA).
Statistical Analysis
The results were analyzed by the analysis of vari­
ance (ANOVA) followed by Student’s t test using
GraphPad Prism version 5.0 (GraphPad Co., USA).
All data were shown as means±standard devia­
tion (SD) from 3 independent experiments. In all
comparisons, p<0.05 was considered statistically
significant.
Results
DHTS Inhibited Cell Proliferation in AGS Cells
MTT assay was used to access the effects of DHTS
on cell proliferation in human gastric cancer cell
AGS and human gastric epithelial cell GES-1. As
shown in Figure 1A, treatment with DHTS (5, 10,
and 20 μM) markedly inhibited cell proliferation
in AGS cells in a dose-dependent manner. In the
meantime, DHTS showed no obvious effects in
GES-1 cell treatment with the same concentrations
(Figure 1B).
DHTS Induced Oxidative Stress in AGS Cells
The intracellular ROS level and the GSH/GSSG
ratio were measured to determine oxidative stress
in AGS cells under DHTS treatment. The results
showed that DHTS treatment (10 μM) significant-
ly increased the intracellular ROS level after incu­
bation for 2 or 4 hours. NAC could remarkably
reverse the induction of intracellular ROS levels
in AGS with DHTS treatment (Figure 2A). In ad-
dition, DHTS (10 μM) significantly inhibited the
ratio of GSH/GSSG in AGS cells, while NAC could
recover the inhibition of the ratio of GSH/GSSG in
AGS cells (Figure 2B).
DHTS Induced Cell Apoptosis in AGS Cells
The apoptotic cells in DHTS-treated AGS cells were
determined using flow cytometry with Annexin
V/PI double staining. As shown in Figure 3, the
apoptotic cells were significantly increased by
DHTS (10 μM) treatment. In addition, the pro­
apoptotic effect of DHTS was inhibited by NAC
(500 μM) pretreatment.
DHTS Activated Caspase-3 and -8 Activities in AGS
Cells
As shown in Figure 4, treatment of AGS cells with
DHTS (10 μM) resulted in remarkable elevations of
caspase-3 and -8 activities, rather than caspase-9.
The activations of caspase-3 and -8 by DHTS in
AGS cells were further inhibited by NAC, a ROS
inhibitor.
Discussion
Gastric cancer is one leading malignancy in the
world, with an increased mortality rate. Although
chemoresistance becomes a big concern, chemo­
Volume 41, Number 1/February 2019 29
Dihydrotanshinone I in Gastric Cancer Cells

4. therapy remains one of the effective options for
treating gastric cancer. It is urgent to develop nov-
el chemotherapeutic agents. Due to the diversity
of molecular structures, natural products are con­
sidered as a valuable source of novel chemothera­
peutic agents. DHTS, a tanshinone from Danshen
(Salvia miltiorrhiza), exhibits various biological ac-
tivities.8,12 In the present study the results indicated
that DHTS exerted promising antitumor effects by
inducing cell apoptosis through generation of ROS
and activation of p38 MAPK pathway in human
gastric cancer cells.
Apoptosis, known as programmed cell death,
causes diverse cell changes including cell shrink­
age, cell membrane blebbing, nuclear and DNA
fragmentation, and consequently cellular dysfunc­
tion.21 Apoptosis is controlled by a broad range
of cell signals originating intracellularly or extra­
30 Analytical and Quantitative Cytopathology and Histopathology®
Zhong et al
Figure 1 Effects of DHTS on cell viability of (A) AGS and (B) GES-1 cells. Data are expressed as means±SD. **p<0.01, ***p<0.001
when compared with vehicle control group.
Figure 2 Effects of DHTS on ROS and GSH/GSSG levels in AGS cells. (A) AGS cells were treated with DHTS (10 μM) with or without
NAC (500 μM) for 2 or 4 h. The intracellular ROS level was determined using a fluorogenic probe H2DCFDA. (B) AGS cells were treated
with DHTS (10 μM) with or without NAC (500 μM) for 4 h. Reduced glutathione (GSH)/oxidized disulfide (GSSG) ratio was measured
to determine oxidative stress in AGS cells. Data are expressed as means±SD. **p<0.01 when compared with vehicle control group;
#p<0.05 when compared with DHTS treatment group.

5. cellularly, including caspases and the proteins of
the bcl-2 family. Mitochondria plays a crucial role
in mediating apoptosis through various apoptotic
proteins, for example cytochrome c, whose major
functions are the control of cellular metabolism
and apoptosis and induction of the caspase cas­
cade as well.22 The caspases are a group of enzymes
called cysteine proteases, and caspases working
as the major executors of apoptotic processes. In
our current study we demonstrated that DHTS
induced remarkable elevations of caspase-3 and
-8 activities, rather than caspase-9 (Figure 4). The
activations of caspase-3 and -8 by DHTS in AGS
cells were partially inhibited by NAC, a ROS in-
hibitor, suggesting that the proapoptotic effects of
DHTS are ROS dependent.
Recently, various reports demonstrated that ROS
served as central modulators in the apoptosis.23,24
In addition, ROS were also shown to induce apo­
ptosis through regulating phosphorylation and ac-
tivation of the MAPK pathways, resulting in de-
creased antiapoptotic protein levels and increased
proapoptotic protein expression, and followed by
cell death.23,24 Our present results showed that
ROS generation was involved in DHTS-induced
apoptosis in gastric cancer.
Gastric cancer is a common malignant tumor
affecting approximately 760,000 people worldwide
each year. Although surgical removal is the most
effective treatment, chemotherapy and radiation
therapy are still used as palliative treatment for
gastric cancer. DHTS firstly identified from Salvia
miltiorrhiza exhibited potent anticancer activities
through diverse mechanisms containing inhibi­
tion of cancer cell growth, induction of cell-cycle
arrest, and reduction of tumor progression, etc.11-16
In the present study dihydrotanshinone I induced
apoptosis through ROS-mediated oxidative stress
in AGS human gastric cancer cells. In the future,
it is warranted to verify the anticancer action of
Volume 41, Number 1/February 2019 31
Dihydrotanshinone I in Gastric Cancer Cells
Figure 3 Apoptosis induced by DHTS in AGS cells. AGS cells
were treated with DHTS with or without NAC (500 μM) for
48 h. Annexin V-FITC (5 μL) and propidium iodide (1 mg/mL)
were then added into the cells and further incubated for another
30 min. Data of 20,000 cells from each group were collected
and analyzed by a BD flow cytometer. Data are expressed as
means±SD. **p<0.01 when compared with vehicle control
group; #p<0.05 when compared with DHTS treatment group.
Figure 4 DHTS induced caspase activity in AGS cells. Caspase-3, -8, and -9 activity were monitored after DHTS treatment in AGS cells.
Data are expressed as means±SD. **p<0.01 when compared with vehicle control group; #p<0.05 when compared with DHTS treatment
group.

6. dihydrotanshinone I in a gastric cancer mouse mod­
el before applying to gastric cancer patients.
Limitation of application of herbal medicines as
drugs for cancer patients is probably due to the
following reasons: (1) various secret components
are added into highly complex personalized pre­
scriptions in some traditional medicines, (2) lack of
sound proof of efficacy in treating specific cancer
patients, and (3) long-term and short-term safety
considerations, such as specific usage, minimal ef-
fective dosage, and tolerable maximal dosage, etc.
It is also important to note that there is still a long
way to go before active ingredients from herbal
medicines are accepted as treatment candidates in
standard care for cancer patients.
Taken together, the obtained results demon-
strated that DHTS was able to inhibit cell prolif­
eration and induce ROS-mediated cell apoptosis.
These results strongly suggest that DHTS is a po-
tential candidate for treating gastric cancers and
deserves to be further developed as a therapeutic
agent for human cancers.
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