Am J Cancer Res 2023;13(11):5047-5064
www.ajcr.us /ISSN:2156-6976/ajcr0151648
Original Article
Brucea Javanica Oil Emulsion
Injection inhibits proliferation of pancreatic
cancer via regulating apoptosis-related genes
You Wu1,2, Ziqiao Liu1,2, Panling Xu1,2, Xin Sun1,2, Ting Wang1,2, Ping Li1,2
1
Oncology Department of Integrated Traditional Chinese and Western Medicine, The First Affiliated Hospital
of Anhui Medical University, Hefei 230022, Anhui, China; 2Department of Integrated Traditional Chinese and
Western Medicine, Anhui Medical University, Hefei 230022, Anhui, China
Received June 8, 2023; Accepted October 4, 2023; Epub November 15, 2023; Published November 30, 2023
Abstract: Brucea Javanica Oil Emulsion Injection (BJOEI) has been proven to have extensive anti-tumor effects.
But the anti-cancer mechanisms need further exploration. So, the aim of this study was to investigate the role and
mechanisms of BJOEI on pancreatic cancer using network pharmacology and experimental validation. Disease
targets were obtained from the GSE101448 dataset in the Gene Expression Omnibus (GEO) database. Eight active
ingredients were identified following a comprehensive literature search. The target genes of BJOEI were obtained
from the SwissTarget Prediction database. The core targets of BJOEI and the involved signaling pathways were
determined using the compound-target network, protein-protein interaction (PPI) network, Gene Ontology (GO), and
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. GO and KEGG enrichment analy
ses of 50 potential overlapping genes indicated that BJOEI exerted therapeutic effects on pancreatic cancer through
the apoptotic pathway. In vitro experiments further revealed that BJOEI could suppress cell growth and invasion,
arrest cells at the S stage, and cause cell apoptosis in three pancreatic cell lines. Additionally, BJOEI inhibited tumor
growth in vivo. Among the 15 key genes regulating apoptosis, 11 were upregulated, while 4 were downregulated.
PPARG emerged as a core target in bioinformatics analysis. The ability of PPARG to regulate apoptosis was validated
by Western Blot. Our findings verified that BJOEI could regulate apoptosis-related genes, especially PPARG, thereby
inducing apoptosis and inhibiting proliferation in pancreatic cancer cells. BJOEI can impede pancreatic cancer pro
gression and induce cell apoptosis. The underlying mechanism appears to be closely associated with the regulation
of apoptosis-related genes.
Keywords: Pancreatic cancer, Brucea Javanica Oil Emulsion Injection, network pharmacology, apoptosis, PPARG
Introduction
Despite notable advances, pancreatic cancer
exhibits the highest fatality rate among can
cers, with an overall five-year survival rate of
around 7% [1]. Pancreatic ductal adenocarci
noma (PDAC), the predominant subtype, exhib
its a 5-year survival rate of only 6-8%, primarily
attributed to limited treatment modalities [2].
Surgical resection is the only potentially cura
tive treatment; however, up to 80% of patients
are in advanced stage or complicated with met
astatic disease at initial diagnosis, precluding
surgical intervention. Therefore, it is important
to identify novel anti-tumor drugs and thera
peutic targets for pancreatic cancer.
As an essential part of complementary and
alternative medicinal systems, traditional
Chinese medicine has emerged as a pivotal
domain of investigation and clinical application
in cancer management [3, 4]. Chinese medicine
compound preparations have multi-target and
multi-level characteristics. Brucea Javanica Oil
Emulsion Injection (BJOEI), derived from the
Brucea javanica after petroleum ether desulfur
ization and ethyl acetate extraction, is a novel
anti-cancer drug. Its principal components
include unsaturated fatty acids, demonstrating
a distinct affinity for tumor cells [5]. BJOEI have
been proven to inhibit cell proliferation [6],
induce tumor cell apoptosis [7], and mitigate
resistance to chemotherapy drugs [8]. In China, Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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BJOEI is widely applied as an adjuvant therapy
drug for lung and gastrointestinal cancers [9,
10]. However, the precise mechanisms of BJOEI
on pancreatic cancer remains unclear.
Network pharmacology studies can provide a
novel understanding of the complex relation
ship between drugs and disease-related target
pathways. The traditional single-target, single
drug research model is gradually evolving into a
network-targeted multi-component treatment
model, which is also in line with the current
multi-target, multi-level trajectory of anti-tumor
strategies. Moreover, the pharmacological dis
ease-gene-target-drug survey pattern can
more effectively reveal the intricate connec
tions between drug constituents and diseases,
providing a more convenient and effective
learning platform for understanding the me
chanism of action of drugs. Surveillance,
Epidemiology, and End Results (SEER) data
base analysis identified race as one of the fac
tors influencing clinical features and survival
outcomes in patients with pancreatic neuroen
docrine tumors [11].
In this study, we found that BJOEI could regu
late apoptosis-related genes, especially peroxi
some proliferator-activated receptor-gamma
(PPARG), thereby inducing apoptosis and inhib
iting proliferation in pancreatic cancer cells.
These findings provide valuable insights into
the clinical management of patients with pan
creatic cancer. Significantly, genes enriched in
the apoptosis pathway may serve as a potential
target for the treatment of pancreatic cancer.
The schematic representation of our research
is depicted in Figure 1.
Materials and methods
BJOEI acquisition
BJOEI, provided by Guangzhou Baiyunshan
Mingxing Pharmaceutical Co., Ltd (approval
number: Z44021325), was obtained from
Brucea Javanica after petroleum ether deestifi
cation, followed by extraction with ethyl acetate
or chloroform.
Active components and predicted targets of
BJOEI
Through a comprehensive literature survey
[12-18], 8 active components of BJOEI were
screened. All compounds were fed into the
PubChem database (https://pubchem.ncbi.
nlm.nih.gov) to obtain their 2D chemical struc
tures. These structures were inputted into The
SwissTargetPrediction database (http://www.
swisstargetprediction.ch/) for target genes
prediction, with a probability threshold > 0.
Disease-related targets screening
The keywords “pancreatic cancer, human” were
entered into the Gene Expression Omnibus
(GEO) database (http://www.ncbi.nlm.nih.gov/
geo/). The entry criteria were set as follows:
(1) tissue samples taken from the human pan
creas; (2) a sample size ≥ 30; (3) data collect
ed within the past 5 years; and (4) both tumor
and non-tumor pancreatic tissue samples avail
able. GSE101448 complied with all criteria.
Disease-related target genes were screened
using the “GEO2R” function in the GEO data
base with selection criteria of P < 0.05 and
|log2FC| > 1.
Drug-disease-related target screening
The BJOEI target genes and pancreatic cancer
target genes were matched to obtain the over
lapping targets, and the intersecting genes
were displayed using Venn 2.1.0 (https://bio
infogp.cnb.csic.es/tools/venny/index.html) to
determine the effective targets.
Construction of BJOEI-active ingredients-tar
gets network
Multiple active compounds and relevant prima
ry genes were introduced into the Cytoscape
software 3.9.1 for the construction of a BJOEI
active elements-objects network. Nodes in
this network represented components and tar
gets, while edges symbolized the associations
between drugs with active elements and ingre
dients with target genes. This network collec
tively illustrates the intricate interplay between
BJOEI and its target genes.
Protein-protein interaction (PPI) analysis
The protein-protein interaction (PPI) network
was generated using the STRING11.5 data
base (https://cn.string-db.org/) based on over
lapping genes to elucidate the association
between the functional proteins. The screeningApoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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Figure 1. Graphical abstract.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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criteria were set as “Homo sapiens” and “medi
um confidence”, and the disconnected nodes
were concealed in the network to obtain the PPI
network. The key topological parameter and
degree described the most important nodes in
the network. Higher quantified values of topo
logical parameters indicate greater importance
of nodes [19].
Potential pathways of BJOEI in pancreatic can
cer
Gene Ontology Biological Processes (GOBP)
and Kyoto Encyclopedia of Genes and Genom
es (KEGG) pathway enrichment analysis were
performed using Metascape online tool
(https://metascape.org/) to elucidate gene
function and signaling pathways.
Cell lines
Three cell lines, Mia PaCa-2, Panc02, and
Capan-1, were procured from the American
Type Culture Collection (ATCC, USA). Panc02
and Mia PaCa-2 cells were cultured in Dul
becco’s modified Eagle’s medium (Gibco, USA)
supplemented with 10% fetal bovine serum
(FBS; Gibco), and 100 μg/ml of penicillin and
streptomycin (Gibco) each in a humid environ
ment containing 5% CO2. Capan-1 cells were
cultured under identical conditions described
above, except that the base medium was
Iscove’s Modified Dulbecco’s Medium (Gibco).
Cell viability assay
Enhanced Cell viability was quantified using
Cell Counting Kit-8 (Beyotime, China), following
the manufacturer’s instructions [20]. Briefly,
cells (1×104/well) were seeded into 96-well
plates for 24 h, and subsequently exposed to
escalating concentrations (0, 2.5, 5, 10, 20,
40, 80, and 160 μl/mL) of BJOEI. After 48 h of
incubation, a sterile CCK-8 solution was added
to each well and incubated for 30 min at 37°C.
The absorbance was measured through a
microplate reader (SpectraMax ABS plus, USA)
at 450 nm. Linear regression was applied to
determine the IC50 values.
Clonogenic assay
A clonogenic assay was carried out to assess
cell sensitivity to BJOEI [21]. Pancreatic cancer
cells were seeded (1×103/well) into 12-well
plates for one day, followed by exposure to
increasing concentrations (0, 3, 6 μL/mL) of
BJOEI for 10 days. After fixation, cells were
stained with 0.1% crystal violet (Beyotime,
China) for 30 mins, and colonies with over 50
cells were calculated using an Olympus CKX53
microscope (Olympus, Japan).
Wound healing assay
A conventional wound healing assay was
utilized [22]. Approximately 1×106 cells were
seeded in a well. The next day, a scratch was
created on the cell monolayer using a pipette
tip while being held vertically and not tilted.
Subsequently, the cells were washed with PBS
thrice, the scratched cells were removed, and
serum-free culture medium was added. The
cells were then incubated in a 37°C, 5% CO2
incubator. Finally, images were collected at 0 h,
24 h, and 48 h.
Cell apoptosis assay
Cell apoptosis was measured using an apopto
sis reagent (Beyotime, China) [23]. Briefly, can
cer cells (1×105/well) were seeded in a 6-
well plate for one day and then treated with
varying doses of BJOEI (0, 3, 6 μL/mL) for 2
days. Then, the cells were treated with Annexin
V-FITC and propidium iodide solution for 20
mins and analyzed using a Cyto FLEXS flow
cytometer (Beckman COULTER, USA).
Cell cycle analysis
Cell Cycle reagent (Beyotime, Chia) was utilized
as described previously [24]. Briefly, cells were
treated with BJOEI at different concentrations
for 48 h, followed by fixation with chilled 70%
ethanol for one day. Subsequently, the cells
were dyed with propidium iodide for 30 min in
the dark and analyzed using ModfitLT 5 soft
ware (Verity Software House, USA).
Real-time PCR
Total RNA was extracted from experimental
cells using an RNA Isolation Kit with Spin
Column (Beyotime, China). The BeyoFastTM
SYBR Green One-Step qRT-PCR Kit (Beyotime,
China) was employed for continuous reverse Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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transcription and fluorescence quantitative
PCR using LightCycler 96 Real-Time PCR sys
tem (Roche, Switzerland). All experiments were
repeated thrice. The sequences of gene prim
ers are provided in Table S1.
Immunoblot
Total protein extraction from cells was conduct
ed using the Radio Immunoprecipitation Assay
(RIPA) lysis buffer and BCA kit (Beyotime).
Following SDS-PAGE separation, protein bands
were electro-transferred onto polyvinylidene
difluoride (PVDF) membranes (0.45 μm, Beyo
time). Samples were then incubated in 5%
skimmed milk in 1×TBST (1 h), followed by over
night incubation at 4°C with primary antibod
ies, including β-actin (Beyotime, China), BAX
rabbit (BBI, China), BCL2 (BBI, China), and
CASP3 (BBI, China), PPARG (Beyotime, China),
and GW9662 (Sigma-Aldrich, USA). Samples
were then washed and incubated with HRP
labeled goat anti-rabbit IgG (H+L) (Beyotime,
China) for 1 h at room temperature. The bands
were visualized using an ECL Kit (Beyotime,
China), while ImageJ.JS (http://cnij.imjoy.io/)
was utilized for quantification.
Animal models
Female C57BL/6 mice, aged 4 weeks, were
sourced from HFK Bio-Technology (China). The
animals were housed in a pathogen-free envi
ronment with a temperature of 22 ± 1°C, a
humidity of 50 ± 1%, and a 12 h light/dark
cycle. The mice had ad libitum access to water
and food. A 7-day acclimatization period was
provided for all animals. Assays involving ani
mals were approved by the Experimental
Animal Ethics Committee of Anhui Medical
University (approval number LLSC20221106).
To establish subcutaneous tumor models,
1×106 Panc02 cells were injected subcutane
ously into the right flank of each animal. After
one week, tumor-harboring animals were divid
ed into three groups (n = 6 mice in each group):
a control and two BJOEI treatment groups. The
BJOEI-treated mice received an intraperitoneal
injection of BJOEI (25 mL/kg or 50 mL/kg)
every day, while the control group received
equivalent volumes of PBS once daily. Mouse
weights and tumor sizes were measured at
3-day intervals. Vernier caliper was used to
measure the tumor size, which was calculated
with the following formula: 1/2 a×b2, where a
and b are the long and short diameters, respec
tively. After 18 days of treatment, the animals
were euthanized by CO2 inhalation [25], fol
lowed by tumor extraction and weighing.
Immunohistochemistry
Tumor tissues were subjected to formalin fixa
tion, dehydration, paraffin-embedding, and
then sectioned at a thickness of 4 μm. Follow
ing deparaffinization, hydration, and micro
wave-based antigen retrieval, the sections
were incubated with 3% H2O2 for 1 h and then
blocked with 5% bovine serum albumin
(Gentihold, China) for 20 min. The sections
were subsequently incubated with primary
antibody anti-Ki-67 (Beyotime, China; overnight
at 4°C) and secondary antibody IgG H&L
(HRP) (Beyotime, China; 60 min at room tem
perature). Diaminobenzidine and hematoxylin
were utilized for development and counter
staining, respectively. An Olympus inverted flu
orescence microscope (Olympus) was used to
image five randomly selected high-power fields
(400×) per specimen. Finally, ImageJ was used
for quantification.
Statistical analysis
GraphPad Prism (version 9.0, USA) software
was used to perform statistical analyses.
Measurement data were demonstrated as
mean ± standard deviation based on three
independent assays. Shapiro-Wilk test and QQ
plot were used to assess the data distribution.
Data conforming to a normal distribution were
analyzed using t-tests or one-way ANOVA fol
lowed by Dunnett’s multiple comparison test
[19]. P < 0.05 was considered statically
significant.
Results
Chemical components and potential targets of
BJOEI
Through a literature review, we identified eight
compounds of BJOEI, including oleic acid, lin
oleic acid, olein, palmitic acid, sesquiterpenes,
stearic acid, α-Linolenic acid, and anthraqui
none. Comprehensive information and 2D
structure of these 8 mixtures were retrieved
from the PubChem database, and the results
are summarized in Table S2. Furthermore, theApoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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target prediction of these 8 active components
was carried out in the SwissTarget Prediction
database, yielding 250 predicted target genes
after deduplication. The drug components and
action targets were visualized using the
Cytoscape software (Figure 2).
Identification of potential targets for BJOEI
treatment in pancreatic cancer
Total RNA expression data from 24 normal tis
sue samples and 19 pancreatic tumor tissue
samples were obtained from GSE101448 in
the GEO database. A total of 2573 differential
genes were obtained from GSE101448 from
both normal and pancreatic tumor tissues. The
obtained differential genes and 250 drug com
ponent prediction target genes were then incor
porated into Venn 2.1.0 software for plotting
and analysis. After intersecting the two, we
obtained 50 disease-drug component common
targets. Details about these 50 overlapping
target genes are listed in Table S3. The 50 tar
get genes and 8 active ingredients were used
to form an “overlapping target gene-corre
sponding compound of BJOEI” network dia
gram (Figure 3A).
PPI network construction and analysis
We subjected the 50 overlapping genes to the
STRING database to obtain the PPI data. We
Figure 2. The “Compounds-targets” network.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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Figure 3. Screening key targets and functional analysis. A. Overlapping target gene-corresponding compound network of BJOEI; B. PPI network diagram of candidate
genes; C. KEGG pathway of BJOEI and GO biological processes, GO cell components, GO molecular functions.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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then constructed and visualized the PPI net
work, comprising 50 nodes and 105 edges,
using Cytoscape (Figure 3B). A higher degree
indicates a larger node size, with red indicating
the highest degree and yellow indicating the
lowest degree. The top node based on degree
values was PPARG, suggesting a pivotal role of
PPARG within the PPI network pertaining to
pancreatic cancer.
Potential pathways influenced by BJOEI in pan
creatic cancer
Fifty overlapping genes were entered into the
Metascape platform for the GO and KEGG
pathway analyses. The top 2 pathways in the
KEGG pathway analysis included pathways in
cancer and apoptosis. Additionally, GO analysis
revealed targets related to biological process
es, particularly the positive regulation process
es encompassing cell death and the apoptotic
process, both of which emerged as top hits
based on P-value < 0.05. The targets and rank
ings of cell components and molecular func
tions are shown in Figure 3C.
Cytotoxic impact of BJOEI on pancreatic cancer
cells
The CCK8 test demonstrated a dose-depen
dent suppression of Capan-1, Panc02, and Mia
PaCa-2 cells by BJOEI compared with that in
the control cells (Figure 4A). The IC50 values of
BJOEI on Panc02, Capan-1, and Mia Paca-2
cells were 7.7 μl/mL, 7.3 μL/mL, and 6.243
μL/mL, respectively, at 48 h. Furthermore,
increasing BJOEI levels inhibited colony forma
tion (P < 0.05 vs. control; Figure 4B).
Next, we examined the effect of BJOEI on pan
creatic cancer cell migration using wound heal
ing assays. BJOEI was able to inhibit the migra
tory abilities of cancer cells compared with the
control group (P < 0.05; Figure 5).
BJOEI induced cell cycle arrest and cell apop
tosis
Cells administered with BJOEI exhibited S
phase arrest (Figure 6A). Moreover, the apop
totic rate was significantly correlated with BJOEI
concentration, suggesting BJOEI-induced pan
creatic cancer cell apoptosis (P < 0.05 vs. con
trol; Figure 6B). Apoptosis-related proteins,
such as BAX, BCL2, and cleaved caspase3,
were confirmed using immunoblotting. Our
results showed that BJOEI upregulated BAX
and cleaved caspase3 expression and down
regulated BCL2 expression (Figure 7).
BJOEI inhibited tumor growth in vivo
To examine BJOEI’s anticancer properties in
pancreatic cancer in vivo, we established a
Panc02 cell tumor model in female C57BL/6
mice. Following tumor formation after 7 days of
cell injection, animals were randomized into
respective groups. No toxicity was observed
throughout the study. Remarkably, the aver
age tumor size exhibited slower growth after
BJOEI treatment compared to that in the con
trol group (Figure 8A and 8B). Histological
examination revealed distinctive architectural
alterations, including poor gland generation,
predominant solid tumor growth, and substan
tial neutrophil infiltration in the control group
compared to the BJOEI-treated mice, highlight
ing BJOEI’s potential to restrain neoplastic pro
gression. Consistent with this, immunohisto
chemistry analysis demonstrated a reduction
in the number of tumor cells expressing the
proliferation marker Ki-67 after BJOEI treat
ment compared with controls (Figure 8C).
These findings demonstrated that BJOEI effec
tively inhibited Panc02-derived tumor growth in
mice.
Apoptosis genes played an essential role in
BJOEI inhibiting pancreatic cancer
The genes enriched to apoptosis pathway
and positive regulation of apoptotic processes
were validated by RT-PCR (Figure 9). These 15
genes are listed in Table S4. Following BJOEI
treatment, the mRNA expression of PPARG,
CTSB, PARP3, PLA2G1B, FAP, ADORA1, PTPRC,
VDR, PARP4, IDO1, and CTSK were found to be
upregulated, while the expression of MMP2,
PTGS2, BACE1, and TOP2A were downregulat
ed.
Based on the PPI network and the pathway
enrichment analyses, the top target PPARG was
selected for subsequent experimental verifica
tion. Immunoblot further demonstrated that
BJOEI could upregulate PPARG (Figure 10A).
We used PPARG inhibitor, GW9662, to study
the effect of BJOEI on pancreatic cell apopto
sis. Compared with the control group, the addi-Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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tion of GW9662 partially rescued BJOEI’s pro
apoptotic effect on these three BJOEI cell lines
(Figure 10B). These findings were consistent
with the results from bioinformatics analysis.
Figure 4. BJOEI suppressed pancreatic cancer cell growth in vitro. A. BJOEI inhibited pancreatic cancer viability in
a dose-dependent manner; B. BJOEI significantly inhibited the migration of Capan-1, Mia Paca-2 and Panc02 cells.
N = 3; ns, not significant; **P < 0.01, ***P < 0.001, vs. 0 group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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Discussion
Traditional Chinese medicine has been utilized
in China for thousands of years to prevent and
treat malignant tumors at diverse stages,
including pancreatic cancer. In traditional
Chinese medicine, the etiology and pathogen
esis of pancreatic cancer are often related to
Figure 5. BJOEI suppressed pancreatic cancer cell growth in vitro. BJOEI significantly inhibited migration of Capan-1,
Panc02, and Mia Paca-2 cells. N = 3; ns, not significant; **P < 0.01, vs. 0 group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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Figure 6. BJOEI induced cell cycle arrest in the S phase and caused apoptosis. A. BJOEI arrested Panc02, Capan-1, and Mia PaCa-2 cells in the S phase; B. BJOEI
induced cell apoptosis. N = 3; *P < 0.05, **P < 0.01, ***P < 0.001, vs. 0 group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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“dampness, heat, phlegm, and blood stasis”,
whereby the disease emergence is attributed
to the gradual accumulation of pathogens and
toxins over time [26]. BJOEI can guard the
zhengqi and warmth, disperse the knots, and
contribute to the flow of qi and blood circula
tion, threby treating pancreatic cancer. Modern
pharmacological research has also highlighted
the anti-tumor effects of BJOEI [27, 28].
Multiple active ingredients in BJOEI exhibit a
synergistic anti-tumor effect [29]. BJOEI con
sists of 85% triglycerides and 10% oleic acids,
interlaced with saturated and unsaturated
fatty acids along with triterpene alcohols.
Notably, unsaturated fatty acid components,
such as oleic acid and linoleic acid, exhibit a
specific affinity for tumor cell membranes, with
a significant anti-tumor activity [8]. Triglycerid
es have an indirect anti-cancer activity and can
be hydrolyzed into oleic acid, subsequently
exerting anti-tumor activity. Oleic acid can
decrease insulin secretion and enhance
sensitivity, thereby potentially curbing PDAC
through attenuation of hyperinsulinemia, lead
ing to DNA damage and, in turn, tumor growth
[30]. In contrast, Palmitic acid, the most com
mon saturated free fatty acid, causes lipotoxic
ity and apoptosis of non-fat cells when at full
capacity [31]. Additionally, accumulation of pal
mitic acid can conduce pancreatic β-cell dys
function and cardiac myocyte apoptosis [31].
Sesquiterpenes, such as alantolactone, can
sensitize human pancreatic cancer cells to
EGFR inhibitors by inhibiting STAT3 signaling
[32]. Emodin, a natural anthraquinone deriva
tive, can inhibit pancreatic cancer epithelial
mesenchymal transition [33]. As a composite
formulation of bioactive constituents, BJOEI
would potentially attract widespread attention
in the years ahead. However, the mechanism of
BJOEI in pancreatic cancer is still not complete
ly understood.
KEGG analysis revealed that cancer and apop
tosis pathways exhibited the highest number of
genes enriched in tumor-associated signaling
pathways, underscoring their pivotal role in
mediating the effects of BJOEI on pancreatic
cancer. Subsequent in vitro experiments
revealed that BJOEI suppressed pancreatic
cancer cell growth and aggression, increased
cell apoptosis, and induced S-phase cell cycle
arrest. Immunoblotting further corroborated
BJOEI’s pro-apoptotic influence, as evidenced
by modulations in BAX, BCL2, and caspase3
expressions, thereby confirming the regulation
of apoptosis. BJOEI reduced cell apoptosis by
Figure 7. Protein expression in Panc02, Mia paca-2 and Capan-1 cells after BJOEI treatment. BJOEI upregulated BAX
and cleaved caspase 3 but downregulated BCL2 expression. N = 3; **P < 0.01, vs. 0 group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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regulating apoptosis-related proteins, therefore
suppressing tumor growth. Furthermore, qPCR
results demonstrated that BJOEI could upregu
late VDR, CTSK, PARP4, FAP, CTSB, ADORA1,
PARP3, PTPRC, IDO1, PPARG, and PLA2G1B
while downregulating TOP2A, PTGS2, BACE1,
and MMP2. These results indicate that BJOEI
exerts therapeutic effects on pancreatic can
cer by modulating apoptosis-related genes.
The induction of tumor cell death is an effec
tive strategy to control tumor development.
Apoptosis involves the activation, expression,
and regulation of a series of genes. The Bcl-2
family, encompassing anti-apoptotic (e.g., Bcl-
2) and proapoptotic (e.g., Bax) proteins, plays
a crucial role in the process of cell apoptosis
[34, 35], regulating and activating cysteine
protease caspases, as well as leading to apop
tosis [35]. Multiple genes and pathways can
regulate the expression of Bcl-2 family genes.
Our RT-PCR results demonstrated that the
expression of apoptosis-related genes, includ
ing PPARG, CTSB, PARP3, PLA2G1B, FAP,
ADORA1, PTPRC, VDR, PARP4, IDO1, CTSK,
MMP2, PTGS2, BACE1, and TOP2A, which play
crucial roles in modulating the Bcl-2 family
genes, were regulated in pancreatic cell lines
upon BJOEI treatment. Among them, the PPI
results suggested that PPARG may play a key
Figure 8. BJOEI suppressed tumor growth in mice. A. BJOEI suppressed panc02 mouse xenograft tumor growth; B.
BJOEI did not affect mouse weight; C. BJOEI suppressed Ki-67 expression in tumor tissues. Magnification: 50×. N =
3; *P < 0.05, **P < 0.01, vs. control group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
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role in the anti-pancreatic cancer process,
prompting further experimental verification.
Immunoblot analysis showed an increase in
PPARG expression after BJOEI treatment. We
then used a PPARG inhibitor, GW9662, to study
the effect of BJOEI on pancreatic cell apopto
sis. It was found that GW9662 partially rescued
the pro-apoptotic effect of BJOEI on these three
Figure 9. BJOEI modulated apoptotic pathway genes. The mRNA expression of PPARG, CTSB, PARP3, PLA2G1B, FAP,
ADORA1, PTPRC, VDR, PARP4, IDO1, CTSK, were upregulated, while those of MMP2, PTGS2, BACE1 and TOP2A were
downregulated. N = 3; *P < 0.05, **P < 0.01, vs. control group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
5061
Am J Cancer Res 2023;13(11):5047-5064
BJOEI cell lines compared with the control
group [36]. PPARG, a transcriptional factor, has
been reported to be a key regulator of cellular
energy metabolism. Our finding aligns with pre
vious reports showing that PPARG inhibition
could promote proliferation and inhibit apopto
sis through the Bcl-2/Caspase3 pathways [37,
38]. These findings verify that BJOEI can
regulate apoptosis-related genes, especially
PPARG, thereby inducing apoptosis and inhibit
ing proliferation of pancreatic cancer cells.
However, this study has several limitations.
First, the in vivo effect of BJOEI on pancreatic
cancer needs to be further explored. Second,
we haven’t verified all the genes that related to
apoptosis. Finally, a comprehensive examina
tion of the potential side effects of BJOIE (e.g.,
the effect on the digestive system) needs to be
explored. Future research should shed light on
BJOEI’s role in pancreatic cancer while illumi
nating its mechanism of action.
Conclusions
Incorporating network pharmacology and ex
perimental methods, this study has explored
the mechanism underlying pancreatic cancer
attenuation through BJOEI intervention. Our
findings suggest that BJOEI can suppress the
Figure 10. BJOEI inhibited the apoptosis of pancreatic cancer by activating PPARG, a key gene in the apoptotic path
way. Pancreatic cancer cells were pre-treated with 2 μM GW9662 for 6 h, followed by 6 μL/mL BJOEI treatment for
48 h, then the protein expression was assessed by Western blotting. A. BJOEI upregulated the expression of PPARG;
B. Compared with the control group, the addition of GW9662 partially rescued BJOEI’s pro-apoptotic effect on these
three BJOEI cell lines. N = 3; **P < 0.01, vs. 0 group.Apoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
5062
Am J Cancer Res 2023;13(11):5047-5064
progression of pancreatic cancer and cause
cell apoptosis. The mechanism might be asso
ciated with the modulation of apoptosis-related
genes.
Acknowledgements
The authors thank the Center for Scientific
Research of Anhui Medical University for their
valuable contribution to our experiment. The
study was supported by the Anhui Provincial
Key Research and Development Plan (20210-
4a07020021).
Disclosure of conflict of interest
None.
Abbreviations
BJOEI, Brucea Javanica oil emulsion injection;
GEO, Gene Expression Omnibus; GEPIA, Gene
Expression Profiling Interactive Analysis; CTSK,
Cathepsin K; PLA2G1B, Phospholipase A2
Group IB; PARP3, Poly (ADP-Ribose) Polyme
rase Family Member 3; CTSB, Cathepsin B;
IDO1, Indoleamine 2,3-Dioxygenase 1; ADO
RA1, Adenosine A1 Receptor; PPARG, Pero
xisome proliferator-activated receptor gamma;
PARP4, Poly (ADP-Ribose) Polymerase Family
Member 4; TOP2A, DNA Topoisomerase II
Alpha; VDR, Vitamin D Receptor; FAP, Poly (ADP
Ribose) Polymerase; PTGS2, Prostaglandin
Endoperoxide Synthase 2; BACE1, Beta
Secretase 1; MMP2, Matrix Metallopeptidase
2; PTRPC, Protein Tyrosine Phosphatase Re
ceptor Type C; GOBP, Gene Ontology Biologi
cal Processes; GOCC, GO cell components;
GOMF, GO molecular functions; KEGG, Kyoto
Encyclopedia of Genes and Genomes; PDB,
Protein Data Bank; ATCC, American Type
Culture Collection; CCK-8, Cell Counting Kit-8;
RT-PCR, Real-time PCR.
Address correspondence to: Dr. Ping Li, Oncology
Department of Integrated Traditional Chinese and
Western Medicine, The First Affiliated Hospital of
Anhui Medical University, No. 120, Wanshui Road,
Shushan District, Hefei 230022, Anhui, China. Tel:
+86-0551-6590-8527; E-mail: 1964liping@sina.
com
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1
Table S1. The sequences of the primers for genes
Gene
Forward
Reverse
PLA2G1B
AGACACATGACAACTGCTATGA
AGAGCACGAGTATGAATAGGTG
PTPRC
AATCCTCTTGCTCTGTGCCTCTTTG
GAACACTCACCTTCACCTGCCTTC
PARP4
TTTCGTACAATGGTGTCGACTA
GAAAAGGCGACTCATTCTCATC
PRRP3
CTCACACTTTTACACCGTCATC
CACTTGAGAAGCTGGTAGTCTC
CTSK
TTCCAGTTTTACAGCAAAGGTG
CGTTGTTCTTATTTCGAGCCAT
CTSB
ATACTCAGAGGACAGGATCACT
ATCTTTTCCCAGTACTGATCGG
VDR
AAAGGTCATTGGCTTTGCTAAG
CTTGACTTCAGCAGTACGATCT
IDO1
CTGCCTGATCTCATAGAGTCTG
TTGTGGTCTGTGAGATGATCAA
PPARG
AGATCATTTACACAATGCTGGC
TAAAGTCACCAAAAGGCTTTCG
PTGS2
TGTCAAAACCGAGGTGTATGTA
AACGTTCCAAAATCCCTTGAAG
FAP
AGCAGTGGTCGGAATGTTCAAGTG
ATGTCTCGCCTCCTCTGTCTTCTG
MMP2
ATTGTATTTGATGGCATCGCTC
ATTCATTCCCTGCAAAGAACAC
TOP2A
AAGATTCATTGAAGACGCTTCG
GCTGTAAAATGCCATTTCTTGC
ADORA1
CCTATGTTTGGCTGGAACAATC
GTACTCCATGCTGATGACCTTC
BACE1
CAGACAAGTTCTTCATCAACGG
GTCTGCTTTACCAGAGAGTCAAApoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
2
Table S2. Information about the 8 active compounds of BJOEI
PubChem CID
COMPOUND
Canonical SMILES
STRUCTURE
Oleic acid
445639
CCCCCCCCC=CCCCCCCCC(=O) O
Linoleic acid
5280450
CCCCCC=CCC=CCCCCCCCC(=O) O
Olein
45102063
CCCCCCCCC=CCCCCCCCC(=O) OCC(COC(=O) C) O
Palmitic acid
985
CCCCCCCCCCCCCCCC(=O) OApoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
3
Sesquiterpenes
667450
CC1C2CCC3(C=CC(=O) C(=C3C2OC1=O) C) C
Stearic acid
5281
CCCCCCCCCCCCCCCCCC(=O) O
α-Linolenic acid
5280934
CCC=CCC=CCC=CCCCCCCCC(=O) O
Anthraquinone
6780
C1=CC=C2C(=C1)C(=O)C3=CC=CC=C3C2=OApoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
4
Table S3. Basic information of the 50 genes
Gene names Protein names
Gene related pathways
PPARG
Peroxisome proliferator-activated receptor gamma
PIP3 activates AKT signaling
SCD
Stearoyl-CoA Desaturase
Metabolism of steroids
PTGS1
Prostaglandin-Endoperoxide Synthase 1
Arachidonic acid metabolism
PTGES
Prostaglandin E Synthase
Arachidonic acid metabolism
PDE4D
Phosphodiesterase 4D
GPCR downstream signalling
RORC
RAR Related Orphan Receptor C
Gene expression (Transcription)
AKR1B10
Aldo-Keto Reductase Family 1 Member B10
Visual phototransduction
PTGER2
Prostaglandin E Receptor 2
Arachidonic acid metabolism
PTGS2
Prostaglandin-Endoperoxide Synthase 2
Arachidonic acid metabolism
SERPINA6
Serpin Family A Member 6
Metabolism of steroid hormones
G6PD
Glucose-6-Phosphate Dehydrogenase
KEAP1-NFE2L2 pathway
NPC1L1
NPC1 Like Intracellular Cholesterol Transporter 1
Digestion and absorption
PLA2G1B
Phospholipase A2 Group IB
Glycerophospholipid biosynthesis
BACE1
Beta-Secretase 1
Alzheimer’s disease and miRNA effects
TOP2A
DNA Topoisomerase II Alpha
Transport of the SLBP independent Mature
mRNA
ACHE
GeneCards Symbol: ACHE
Glycerophospholipid biosynthesis
PTGER4
Prostaglandin E Receptor 4
ADORA2B mediated anti-inflammatory
cytokines production
IDO1
Indoleamine 2,3-Dioxygenase 1
NAD de novo biosynthesis II (from trypto
phan)
FDFT1
Farnesyl-Diphosphate Farnesyltransferase 1
Superpathway of cholesterol biosynthesis
OXER1
Oxoeicosanoid Receptor 1
GPCR downstream signalling
SLC16A1
Solute Carrier Family 16 Member 1
Transport of inorganic cations/anions and
amino acids/oligopeptides
CCKBR
Cholecystokinin B Receptor
Cholecystokinin B Receptor
ENPP2
Ectonucleotide Pyrophosphatase/Phosphodiesterase
2
Metabolism of water-soluble vitamins and
cofactors
GPBAR1
G Protein-Coupled Bile Acid Receptor 1
ADORA2B mediated anti-inflammatory
cytokines production
RBP4
Retinol Binding Protein 4
Visual phototransduction
MMP2
Matrix Metallopeptidase 2
Matrix metalloproteinases
PRKCQ
Protein Kinase C Theta
Prolactin Signaling
LPAR5
Lysophosphatidic Acid Receptor 5
GPCR downstream signaling
VDR
Vitamin D Receptor
Gene expression (Transcription)
HSD17B3
Hydroxysteroid 17-Beta Dehydrogenase 3
Androstenedione and testosterone biosyn
thesis and metabolism p.1
CDC45
Cell Division Cycle 45
Activation of the pre-replicative complex
PTPRC
Protein Tyrosine Phosphatase Receptor Type C
Activation of cAMP-Dependent PKA
RARA
Retinoic Acid Receptor Alpha
RNA Polymerase I Promoter Opening
EPHX2
Epoxide Hydrolase 2
Arachidonic acid metabolism
PRSS1
Serine Protease 1
Cobalamin (Cbl, vitamin B12) transport
and metabolism
CTSK
Cathepsin K
Gene expression (Transcription)
ADORA1
Adenosine A1 Receptor
Purinergic signaling
CDC25C
Cell Division Cycle 25C
Defective Intrinsic Pathway for Apoptosis
25C
CTSS
Cathepsin S
Innate Immune SystemApoptosis-mediated inhibition of pancreatic cancer growth by BJOEI
5
CTSB
Cathepsin B
Collagen chain trimerization
PARP3
Poly (ADP-Ribose) Polymerase Family Member 3
Overview of interferons-mediated signaling
pathway
PARP4
Poly (ADP-Ribose) Polymerase Family Member 4
SARS-CoV-1 Infection
FAP
Fibroblast Activation Protein Alpha
Extracellular region
CTRC
Chymotrypsin C
Urokinase-type plasminogen activator (uPA)
and uPAR-mediated signaling
MMP3
Matrix Metallopeptidase 3
Matrix metalloproteinases
FGFR1
Fibroblast Growth Factor Receptor 1
Apoptotic Pathways in Synovial Fibroblasts
NOS1
Nitric Oxide Synthase 1
Superpathway of L-citrulline metabolism
MME
Membrane Metalloendopeptidase
Innate Immune System
MGLL
Monoglyceride Lipase
Glycerophospholipid biosynthesis
GCG
Glucagon
ADORA2B mediated anti-inflammatory
cytokines production
Table S4. Fifteen genes relate to apoptosis for pancreatic cancer treatment
PARP4
CTSB
CTSK
PARP3
ADORA1
FAP
IDO1
MMP2
PLA2G1B
PPARG
PTGS2
PTPRC
TOP2A
VDR
BACE1
