ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 93
OBJECTIVE: Sini decoction is a traditional Chinese med-
icine used for treatment of cardiovascular diseases. This
study aimed to investigate the growth and apoptosis-
regulating effects of Sini decoction on human lung
adenocarcinoma cell line A549.
STUDY DESIGN: The growth and viability of A549
cells treated with Sini decoction were analyzed by
observation under microscopy and MTT method. The
apoptosis rate of A549 cells was detected by Annexin
V-FITC/PI double staining and flow cytometry. Protein
levels of Bcl-2, Bax, and caspase-9 were determined by
western blotting.
RESULTS: Sini decoction significantly inhibited the
growth of human lung adenocarcinoma cell A549 and
caused significant morphological alterations in A549
cells in a dosage- and duration-dependent manner. The
apoptosis rate of A549 cells was significantly increased
by Sini decoction treatment and was also positively
correlated with rises in Sini decoction concentration
and duration. Sini decoction treatment promoted Bax
expression in A549 cells but inhibited the expression
of Bcl-2.
CONCLUSION: Sini decoction inhibits the growth
of lung cancer A549 cell growth by promoting cell
apoptosis, which might be mediated by regulation of
the Bcl-2 protein family. (Anal Quant Cytopathol
Histpathol 2019;41:93–101)
Keywords: A549; adenocarcinoma; cell apoptosis;
drugs, Chinese herbal; lung, pathology; lung ade-
nocarcinoma; lung cancer; lung neoplasms; med­
icine; Chinese traditional; Sini decoction.
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4103-0093/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Sini Decoction Inhibits Lung Adenocarcinoma
A549 Cell Growth by Promoting Cell
Apoptosis
Qian Wang, M.M., Chengjie Lu, M.M., Xintian Chen, M.M., Mengmeng Cai, M.M.,
Yujie Wu, M.M., Qi Yang, M.M., Liqin Yin, M.M., and Jingdong Gao, M.D.
From Suzhou Yongding Hospital, Suzhou City, Jiangsu Province; Suzhou Ninth People’s Hospital, Jiangsu Province; China Nanjing
University of Traditional Chinese Medicine, Nanjing City, Jiangsu Province; Suzhou Industrial Park, Medical and Health Management
Center, Suzhou City, Jiangsu Province; and the Department of Oncology, Suzhou Affiliated Hospital of Nanjing University of Traditional
Chinese Medicine, Suzhou City, Jiangsu Province, China.
Qian Wang, M.M., and Chengjie Lu, M.M., contributed as co–first authors.
Dr. Wang is Resident, Suzhou Yongding Hospital.
Dr. Lu is Physician, Suzhou Ninth People’s Hospital.
Drs. Chen, Cai, Wu, and Yin are Residents, China Nanjing University of Traditional Chinese Medicine.
Dr. Yang is Resident, Suzhou Industrial Park, Medical and Health Management Center.
Dr. Gao is Senior Physician, Department of Oncology, Suzhou Affiliated Hospital of Nanjing University of Traditional Chinese Medicine.
This study was supported by Suzhou Science and Technology Development–Special Industrial Technology Innovation (science and
technology for people’s livelihood–basic research on medical and health applications), Grant No. SYSD2016130.
Address correspondence to: Jingdong Gao, M.D., Department of Oncology, Suzhou Traditional Chinese Medicine Hospital, No. 18,
Yangsu Road, Gusu District, Suzhou, Jiangsu Province 215009, China (jdguo18@126.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Sini decoction, a typical traditional Chinese med­
icine consisting of the herbs Aconitum carmi­
chaelii, Glycyrrhiza uralensis, and Zingiber officinale
(Z. officinale), has been long applied for the
treatment of cardiovascular disease, such as
myocardial infarction, in Chinese medicine.1 By
high-performance liquid chromatography, a large
number of potential bioactive compounds were
identified in Sini decoction, including flavonoids,
alkaloids, and other types of chemicals,2 show-
ing its great pharmacological value and possibility
of application for treatments of various disorders.
Considering the complexity of active compounds
in Sini decoction, further investigations showed
that Sini decoction could affect a panel of in
vivo targets and modulate pleiotropic biological
processes and signaling pathways.3 Among them,
the tumor necrosis factor–α (TNF-α) acts as one
of the major targets of Sini decoction, and hy-
paconitine, mesaconitine, and other bioactive
compounds in Sini decoction could directly bind
with TNF-α and prevent cell apoptosis during
heart failure.3 The regulation of cell death and
apoptosis by Sini decoction in cardiovascular dis­
order models indicated that Sini decoction might
also modulate pathological processes asso­
ciated
with apoptosis of other cell types such as cancers.
However, the effects of Sini decoction on cancer-
ous cells have not been previously addressed.
Lung cancer is one of the major malignant tu-
mors worldwide with significantly high incidence
and mortality.4 Lung cancer has also been the
leading cause of cancer-related human deaths in
China and many other regions.5 It has been wide­
ly established that lung cancer development was
associated with smoking, carcinogen exposure,
chronic inflammation, and genetic factors.6 By
pathologic property, lung cancers could be cate­
gorized mainly into 2 major subclasses: small-cell
lung carcinoma and non–small cell lung carcino­
ma, the latter accounting for over 80% of lung
cancer cases in clinics.7 Lung adenocarcinoma is
a common type of non–small cell lung carcino-
ma that usually originates from bronchial epi­
thelium and bronchial mucous glands.8 The
incidence of lung adenocarcinoma has been rising
steadily in recent years, accounting for more than
40% of primary lung cancers. Even worse, lung
carcinoma treatment remains a clinical challenge
due to the lack of significant symptoms at early
stages, and the majority of lung carcinoma pa-
tients are diagnosed at late stages.8 Clinical man­
agement of lung adenocarcinoma depends main-
ly on application of surgery and chemotherapy,
but the related drug resistance and high recur-
rence rates have been reported to be the main
reasons for the high mortality of lung adeno­
carcinoma.8 Novel therapeutic agents are urgent­
ly needed to effec­
tively repress lung adenocarci-
noma progression.
Medicinal herbs and related therapeutic mate­
rials long used in traditional Chinese medicines
have recently been revealed to be potential sources
for novel anti-cancer drug development.9,10 For
instance, the traditional Chinese medicine ka-sai-
ping (KSP) prevented the growth of gastric cancer
cells by activation of autophagy pathways with
lysosome and mitochondrial damage and may
be explored as a novel drug for gastric cancer
therapy.11 In a recent report the traditional Chi­
nese medicine PHY906, produced from a group
of medical herbs including Glycyrrhiza uralensis
(G. uralensis) and Paeonia lactiflora (P. lactiflora), was
used for intervention of colon cancer, exhibiting
significant anti-cancer effects by maintaining the
epithelial barrier and inducing cancer cell death
and apoptosis.12 Moreover, traditional Chinese
medicines were tested as candidates of mainte­
nance therapy for non–small cell lung carcinoma
patients who underwent treatment with first-line
chemotherapy in a number of clinical studies, which
showed the potential of prolonging progression-
free survival and improving quality of life for lung
cancer patients.13,14 Furthermore, the combination
of conventional radiation or chemical therapies
with traditional Chinese medicines might pro­
duce greatly improved therapeutic outcomes in
cancer patients.15 The combined administration of
the traditional Chinese medicine Sun-Bai-Pi ex-
tract remarkably reduced the dosage of cisplatin
needed to induce lung cancer cell death.15 Accu­
mulating studies showed that application of tra­
ditional medicines, especially the combination of
conventional and traditional medicines, might
serve as a new method for cancer treatment. How­
ever, as a traditional medicine, the effects of Sini
decoction on cancer progression and cancer cell
biology remains largely unknown.
In this study we aimed to investigate the ef-
fects of Sini decoction on lung adenocarcinoma
cell line A549—adenocarcinomic epithelial cells in
human pulmonary alveoli.16 The growth and apo­
ptosis of A549 cells under Sini decoction treat-
ment with distinct concentrations were evaluated,
94 Analytical and Quantitative Cytopathology and Histopathology®
Wang et al

3. as well as the preliminary molecular mechanisms,
which provided a basis for Sini decoction in lung
adenocarcinoma treatment.
Materials and Methods
Cell Line and Culture
The non–small cell lung cancer cell line A549 used
in this study was purchased from American Type
Culture Collection (ATCC) (Catalog No. CCL-185)
and cultured in Dulbecco’s Modified Eagle Me-
dium (DMEM) containing 10% fetal bovine serum
at 37°C in a humidified culture cabinet with a
supply of 5% CO2, at Suzhou Hospital of Tradition­
al Chinese Medicine.
Preparation of Sini Decoction
The Sini decoction used in this study was pre-
pared as previously described with minor modi­
fications.3 The 3 medicinal herbs Aconitum car­
michaelii (30 g), Glycyrrhiza uralensis (20 g), and
Zingiber officinale (20 g) were mixed and immersed
in 12 volumes of ddH2O for 30 minutes, then
boiled and simmered on a gentle heat for anoth-
er 30 minutes. The final medicinal soup was
condensed to 1 g/mL-1 by boiling. The medicinal
soup was then cleaned by filtering and cooled
to room temperature. After being centrifuged at
13,000 rpm for 10 minutes, the medicinal soup
was filtered using a 0.45 m filter, following by a
second filtering using a 0.22 m filter. The Sini
decoction used for treating lung adenocarcinoma
A549 cells in this study was freshly prepared right
before cell culture. The Sini decoction was then
added into the DMEM for A549 cell treatment,
and distinct Sini decoction concentrations (10, 20,
30, 40, 50, 60, and 80 mg/mL-1) were tested in this
study.
Cell Morphology and Growth Observation
To analyze the influences of Sini decoction on
cancer cell growth and morphological properties,
A549 cells grown in DMEM at the logarithmic
growth phase were collected and diluted to 2×105
cells/mL using fresh DMEM, then seeded onto
6-well cell culture plates, 2 mL cell suspension
per well. Approximately 24 hours later, A549
cells were added with Sini decoction at specified
concentrations and cultured for another 48 hours,
then cell growth and morphological features were
observed using a microscope. As positive control,
the A549 cells cultured in DMEM containing 100
µg/mL-1 cisplatin was added as the positive con-
trol group. At least 3 biological and technical
replicates were performed for the reliable and
repeatable observations.
Cell Growth Inhibition Analysis by MTT Method
The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2-H-tetrazolium bromide] method using
the MTT Cell Proliferation and Cytotoxicity Assay
Kit (#C0009; Beyotime, Beijing) was performed to
determine cell growth inhibition degree accord-
ing to the manufacturer’s instructions. Briefly,
A549 cells grown in DMEM at logarithmic growth
phase were collected and the cell density was
adjusted to 6×104 cells/mL using fresh cell cul-
ture medium. Approximately 100 µL cell suspen-
sion was then added into each well of a 96-
well plate and cultured under conventional
conditions for 24 hours, and then added with
Sini decoction or cisplatin at specified concentra­
tions. Seven different Sini decoction concentra-
tions, forming a concentration gradient, were
tried in the experiment. A549 cells treated with
100 µg/mL-1 cisplatin were included as the posi-
tive control group, and cells without special
treatment were used as the negative control.
After culturing with Sini decoction for 24 hours,
48 hours, or 72 hours, 10 µL MTT solution was
added into each well, and cells were then cultured
at the same conditions for another 4 hours and
then mixed with 150 mL dimethyl sulfoxide. The
optical density (OD) values were then measured
after cells were vortexed for 1 minute, and cell
growth inhibition rates were calculated according
to the OD values. At least 3 biological repeats were
carried out.
Cell Apoptosis Analysis
The apoptosis of lung adenocarcinoma cells was
analyzed using the Annexin V-FITC/propidium
iodide (PI) double staining kit (KeyGEN BioTech,
Nanjing, China) as described by the manufac-
turer. A549 cells were collected and diluted to
4×105 cells/mL, and 2 mL cell suspensions were
then seeded into 6-well plates, followed by add-
ing fresh DMEM medium containing Sini decoc­
tion at specified concentrations. Cells treated with
cisplatin were applied as the positive control.
After co-culture with Sini decoction–containing
medium for 24 hours, 48 hours, or 72 hours, A549
cells were collected by centrifuge at 1,500 r/min-1
for 5 minutes at 4°C, followed by 2 washes with
PBS solution. For analysis by flow cytometry,
Volume 41, Number 3/June 2019 95
Sini Decoction Inhibits Lung Adenocarcinoma

4. cells were first fixed with 70% ethanol for 30
minutes, washed twice with PBS solution again,
incubated with 1,000 μL Annexin V-FITC solution
for 10 min in the dark, then mixed with 5 μL PI
solution followed by incubation for 5 minutes in
the dark. The percentages of apoptotic A549 cells
were finally determined by a flow cytometry sys­
tem (BD Biosciences).
Western Blotting
After being treated with Sini decoction for 48
hours, cells were collected by centrifuge at 1,500
r/min-1 for 5 minutes at 4°C, washed twice with
1 mL PBS solution, mixed, and incubated with
150 L cell lysis buffer (#160811; Beyotime) on ice
for 30 minutes. After being centrifuged at 12,000
r/min-1 for 5 minutes at 4°C, the supernatant
was collected into a new Eppendorf tube, fol­
lowed by protein concentration determination.
Appropriate total proteins were mixed with 5×
loading buffer and boiled at 100°C for 5 min-
utes, separated by 12% SDS-PAGE gels, and
transferred onto PVDF membranes. The PVDF
membranes were then blocked with 5% lipid-
free milk solution for 1 hour at room tempera­
ture, incubated with primary antibodies dilated
in TBST buffer for 1–2 hours at room tempera­
ture, washed with TBST 3 times, and incubated
with secondary antibodies diluted with TBST
buffer for 1 hour at room temperature. After be-
ing washed 3 times with TBST for 5 minutes,
the enhanced chemilumines­
cence kit was finally
used to detect the immune complexes accord-
ing to the manufacturer’s instructions (Thermo
Scien­
tific Pierce). The antibodies used in this
study were listed as follows: anti-Bcl-2 anti-
bodies (#ab32124; Abcam), anti-Bax antibodies
(#ab32503; Abcam), anti-Caspase-9 antibodies
(#ab52298; Abcam) and anti-beta-actin antibodies
(#ab8227; Abcam). At least 3 biological replicates
were carried out for quantitative comparison of
protein abundances.
Statistical Analysis
Data in this study were presented as mean±
standard deviation and analyzed with the SPSS
17.0 software package. Significant differences
between groups were statistically evaluated by
Student’s t test, analysis of variance (ANOVA),
and homogeneity test of variance, as appropri-
ate. Significant differences were defined by a
p value of <0.05.
Results
Sini Decoction Induces Significant Morphological
Alterations in A549 Cells
For preliminary analysis of the effects of Sini de-
coction on lung adenocarcinoma cell functions, the
A549 cells were treated with 20, 40, and 80 mg/
mL-1 Sini decoction for 48 hours, and the mor­
phological properties of A549 cells were closely
observed. The A549 cells treated with cisplatin
(100 µg/mL-1) for 48 hours were applied as the
positive control, and A549 cells with no specific
treatment were used as the negative control. As
expected, the cell number and growth rate of A549
cells treated with cisplatin were greatly suppressed
as compared with the negative control (Figure
1A–B). Under microscopy, we also observed that
cell number and growth rate of A549 cells treat­
ed with different concentrations of Sini decoction
were markedly lower than those of the negative
control group, showing growth alteration similar
to that of the cisplatin group (Figure 1A–E).
Moreover, Sini decoction treatment induced visible
shrinkage, rounding, and suspension phenotypes
in A549 cells, which demonstrated Sini decoction
dosage-dependent tendencies. These preliminary
observations suggested that Sini decoction might
prohibit A549 cell growth in a dosage-dependent
manner.
Sini Decoction Suppresses A549 Cell Growth and
Viability
For more insights into the effects of Sini decoction
on lung adenocarcinoma cell functions, the A549
cells were cultured in DMEM medium contain-
ing 10, 20, 30, 40, 50, 60, and 80 mg/mL-1 Sini de-
coction for 24 hours, 48 hours, or 72 hours, re-
spectively. A549 cells cultured in DMEM medium
containing 100 µg/mL-1 cisplatin for correspond-
ing times were used as the positive control. The
cell viabilities of A549 cells were then determined
by the MTT method. A549 cells treated with cis-
platin showed greatly increasing inhibition rates
with the increase of treatment duration, and 100
µg/mL-1 cisplatin for 72 hours led to a significant
cell inhibition rate of 69.53% (p<0.01), showing
that the cellular system in this study was suitable
for assessing inhibition effect (Figure 2A–B). The
viability of A549 cells was markedly suppressed
by treatment of Sini decoction at different con­
centrations compared with the negative control
group, showing a dosage-dependent A549 inhi-
bition by Sini decoction (Figure 2A–B). The inhi­
96 Analytical and Quantitative Cytopathology and Histopathology®
Wang et al

5. bition rates in cells treated with 50, 60, and 80
mg/mL-1 Sini decoction for 24 hours were 19.98%,
22.45%, and 40.3%, respectively—all significantly
higher than those of the negative control (p<0.01).
Additionally, the inhibition of A549 cell viability
showed positive correlation with the Sini decoc­
tion treatment duration, and cells treated with
Sini decoction for 72 hours showed significantly
effective growth inhibition, in comparison to those
treated with Sini decoction for 24 hours or 48
hours (Figure 2A–B). These cell viability assay re-
sults demonstrate that Sini decoction possesses
high potential in inhibiting lung adenocarcinoma
cell growth, which is comparable to the widely
applied anti-cancer chemical, cisplatin.
Sini Decoction Induces A549 Cell Apoptosis
Effective induction of malignant cell apoptosis
acts as one of the key mechanisms mediating the
therapeutic functions of the majority of widely
applied anti-cancer drugs. To fully reveal the bi-
ological functions of Sini decoction on cellular
processes of lung cancer cells, A549 cells cul­
tured in DMEM medium were treated with dis-
tinct concentrations of Sini decoction for 24 hours,
48 hours, or 72 hours as described above. Then,
the percentages of apoptotic A549 cells in each
group were quantitatively analyzed by flow cy-
tometry. We observed that, after Sini decoction
treatment for 24 hours, the percentages of apo-
ptotic A549 cells were significantly increased as
compared with the control group of cells cultured
under normal conditions (Figure 3A and 3C). By
Sini decoction treatment for 48 hours, the A549
Volume 41, Number 3/June 2019 97
Sini Decoction Inhibits Lung Adenocarcinoma
Figure 1
Morphological changes
of A549 cells induced
by Sini decoction. Lung
adenocarcinoma A549
cells were cultured with
different concentrations of
Sini decoction for 48 h and
observed under microscopy
(magnification, ×400). A549
cells treated with cisplatin
were used as the positive
control, and cells with no
treatment were applied as
negative control.
Figure 2 Suppression of A549 cell growth by Sini decoction.
(A) Detailed information of A549 cell inhibition rates induced
by Sini decoction treatment. (B) Dependence of A549 inhibition
rates with Sini decoction concentration and treatment duration.
Lung adenocarcinoma A549 cells were cultured with Sini
decoction at distinct concentrations for 24 h, 48 h, and 72 h,
respectively. The growth and viability of A549 cells were then
evaluated by MTT method. A549 cells treated with cisplatin were
used as the positive control, and cells with no treatment were
applied as negative control. *p<0.05; **p<0.01.

6. cells exhibited even higher rates of apoptosis,
in comparison with the negative control group
(Figure 3B–C). Also, the apoptosis of A549 cells
showed close positive correlation with Sini decoc­
tion concentration and duration, and 60 mg/mL-1
Sini decoction for 48 hours resulted in significant
apoptosis in over 16% of lung cancer cells (Figure
3C). These analyses by double staining and flow
cytometry demonstrated that Sini decoction could
effectively induce apoptosis in lung adenocarcino­
ma A549 cells, further suggesting that Sini decoc­
tion might act as a novel anti–lung cancer drug.
Apoptosis Pathways Associated with Sini Decoction
Treatment
For a preliminary understanding of the molecular
mechanisms underlying A549 cell apoptosis in-
duced by Sini decoction, the protein abundances
of 3 major components of the cell apoptosis path­
way were determined by western blotting. We
found that the protein level of Bcl-2, a key onco­
gene associated with various tumors in humans,
was significantly downregulated in A549 cells
treated with Sini decoction (Figure 4A–B). On the
contrary, the expression level of Bax, an important
tumor suppressor which belongs to the same pro­
tein family as Bcl-2, was then increased by Sini
decoction treatment (Figure 4A–B). These results
showed that the Bcl-2 family proteins might play
critical roles during A549 cell apoptosis induced
by Sini decoction. Unexpectedly, the expression
level of Caspase-9, one major component of the
classical apoptosis pathway, in A549 cells under
Sini decoction treatment were greatly repressed
as compared with the control group (Figure 4).
When the Sini decoction concentration increased
to 60 mg/mL-1, the Caspase became even nonde­
tectable by western blotting in A549 cells. These
molecular alterations showed that Sini decoction
might be able to induce lung cancer cell apopto­
sis via special pathways. Our preliminary assay
showed that Bcl-2 family-mediated apoptosis
pathways might play key mediating roles during
A549 cell apoptosis caused by Sini decoction
treatment.
Discussion
Cellular models have been applied as important
systems for screening and development of novel
anti-cancer drugs.17,18 The lung adenocarcinoma
A549 cells were first isolated and characterized
from human alveolar basal epithelial cells in 1972
and have since been widely used in the study
of lung cancer pathology and also as an in vitro
cellular model for pharmaceutical studies and
novel drug screening.19 A549 cells have also been
regularly applied for the characterization of new
agents that could regulate lung cancer cell apopto­
sis, as potential anti-cancer drugs.20 For instance,
using A549 cells as a cellular model, gambogenic
acid from Garcinia hanburryi was revealed to be
a potential anti-cancer drug which could induce
cancer cell apoptosis through mitogen-activated
protein kinase (MAPK) signaling pathway.21 Al-
98 Analytical and Quantitative Cytopathology and Histopathology®
Wang et al
Figure 3
Induction of A549 cell
apoptosis by Sini decoction.
(A) A549 cell apoptosis by
flow cytometry after being
treated with Sini decoction
for 24 h. (B) A549 cell
apoptosis after Sini decoction
treatment for 48 h.
(C) Statistical analysis of A549
cell apoptosis rate following
Sini decoction treatment.
Lung adenocarcinoma A549
cells were treated with
Sini decoction at distinct
concentrations for 24 h or
48 h, respectively. The
apoptosis of A549 cells were
then measured by Annexin
V-FITC and PI double staining
and flow cytometry.

7. though far from being applied in the clinical
management of cancer patients, anti-cancer drug
candidates selected by appropriate cellular mod­
els such as A549 cells have provided the basis
for subsequent preclinical and clinical research.
Thus, in the present study we addressed the anti-
cancer functions of Sini decoction in A549 cells,
which might promote its future applications as
cancer therapy or complementary medicine for
lung cancer patients.
Sini decoction has been previously used for
treatment of cardiovascular disease in traditional
Chinese medicine and could regulate cell growth
and apoptosis associated with heart and vascular
disorders.19 The capability of Sini decoction in
regulating vascular cell apoptosis suggested that
it might also influence the cell growth and apo­
ptosis linked with tumorigenesis, which has never
been investigated in previous reports. To analyze
the effects of Sini decoction on lung cancer cell
functions, we treated A549 cell with different con­
centrations of Sini decoction for distinct durations.
We observed in this study that Sini decoction
could induce significant morphological changes
in A549 cells and also effectively inhibit A549
growth and promote A549 cell apoptosis. For
molecular mechanisms, we performed analysis of
major apoptosis-related proteins and found that
the Bcl family proteins and caspase-related signal­
ing might mediate the apoptosis-promoting role
of Sini decoction. These results clearly showed
that Sini decoction could promote lung cancer
cell apoptosis, and the remarkable increase of
cell apoptosis in A549 cells cultured in medium
containing Sini decoction persuasively showed
that Sini decoction might be explored as a novel
anti–lung cancer agent.
The molecular mechanisms underlying the sig­
nificantly increased A549 cell apoptosis caused
by Sini decoction need further investigation. Cell
apoptosis is a biological process mediated by pleio­
tropic signaling pathways and plays key roles in
the development of various human cancers.22,23
Our preliminary analysis showed that the expres­
sion of Bcl-2 and Bax in A549 cells were oppo­
sitely altered by treatment with Sini decoction,
indicating that Bcl-2 protein family members and
related signaling pathways might function as the
major molecular mechanisms of A549 cell apopto­
sis induced by Sini decoction. The Bcl-2 protein
family consists of more than 20 main members
and can be classified into 2 subclasses according
to their contrary roles in regulating cell apopto­
sis.24,25 Bcl-2 acts as the representative member of
anti-apoptosis subclass of Bcl-2 family proteins,
which inhibits progression of cell apoptosis by
suppression of cytochrome C release from the
mitochondria.26,27 However, members of the other
subclass of the Bcl-2 family, including Bax, serve
as critical drivers and mediators of cell apopto­
sis.28 The opposite alteration of Bcl-2 and Bax ex-
pression in A549 cells induced by Sini decoction
in the present study confirmed the effects of Sini
decoction in regulating lung cancer cell apoptosis.
For a comprehensive understanding of the roles
of Bcl-2 protein family in A459 cell apoptosis
caused by Sini decoction, the expression and
roles of other members of this family deserve
further investigation. The disclosure of molecular
networks underlying the lung cancer cell apopto-
sis induced by Sini decoction might provide a basis
Volume 41, Number 3/June 2019 99
Sini Decoction Inhibits Lung Adenocarcinoma
Figure 4 Apoptosis signaling pathways regulated by Sini
decoction in A549 cells. (A) Apoptosis signaling protein
abundances in A549 cell after Sini decoction treatment. A549
cells treated with distinct concentrations of Sini decoction for
48 h and total proteins were extracted and subjected to protein
abundance analysis by western blotting. Beta-actin protein
was used as the internal standard for comparison. (B) Statistical
analysis of apoptosis-related protein expression in A549 cells.
Bcl-2 = B-cell lymphoma–2, Bax = Bcl-2-associated X protein.
*p<0.05; **p<0.01.

8. for application of Sini decoction for lung cancer
therapy. Moreover, the demonstration of cancer
cell apoptosis-promoting capability of Sini decoc­
tion might also inspire screening of novel drugs
from traditional medicines for cancer treatment.
Besides Sini decoction, many other traditional
drugs were reported to possess regulatory func­
tions in cell apoptosis processes.19 Combined with
results shown in the present study, it is reason-
able to speculate that future studies on the com­
bination of different traditional medicines with
apoptosis-promoting effects might be an efficient
way of characterizing valuable candidates for
cancer therapy. Furthermore, the dysregulation of
cell growth and apoptosis is a common feature
of cancers originating from different human or-
gans, thus our results also suggest that Sini de-
coction and other traditional medicine might like­
ly repress the progression of other human cancers.
It is also worth mentioning that Caspase-9 protein
abundance in A549 cells treated with Sini decoc-
tion was greatly downregulated, showing the par-
ticular features of lung cancer cell apoptosis in-
duced by this traditional medicine, which also
deserves further investigation.
Conclusion
Generally, we demonstrated in this study that
Sini decoction, a traditional Chinese medicine pre­
viously used for the treatment of cardiovascular
disorders, could effectively inhibit lung adenocar­
cinoma A549 cell growth and significantly pro-
mote A549 cell apoptosis. The regulation of lung
cancer cell apoptosis by Sini decoction might
possibly be mediated by the Bcl-2 and Bax protein
family–associated signaling pathways. Our results
provided a basis for the application of Sini decoc­
tion and other traditional medicine in the clinical
management of lung and other human cancers.
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Sini Decoction Inhibits Lung Adenocarcinoma