Introduction
Lung cancer exhibits the highest incidence and
mortality rate worldwide, with ~85% of cases being histologically
classified as non-small cell lung cancer (NSCLC), of which 55–60%
are adenocarcinomas (1). The
majority of early-stage patients with NSCLC remain undiagnosed due
to the absence of clinical manifestations; when symptoms do occur,
they primarily include cough and hemoptysis. The treatment for
patients with locally advanced NSCLC primarily includes
chemotherapy, immunotherapy, molecular targeted therapy and
radiotherapy (2,3). Despite marked improvements in patient
prognosis through advancements in targeted and immunotherapeutic
approaches, the 5-year survival rate remains below 37% (4). These challenges underscore the need
to develop innovative therapeutic strategies and identify effective
chemotherapeutic agents to improve clinical outcomes.
Curcuma zedoaria Rosc. is a Traditional
Chinese Medicine component previously recorded in ‘Chinese
Pharmacopeia’ (5) amongst others
(6). It is primarily cultivated in
Guangxi and Zhejiang provinces and its rhizome, belonging to the
Curcuma genus of the Zingiberaceae family, yields
zedoary turmeric oil (ZTO), a volatile extract (7). ZTO has been shown to exhibit
multifaceted antitumor properties against lung adenocarcinoma,
including suppression of A549 cell proliferation (8), mitigation of chemotherapy-induced
adverse effects (9) and
enhancement of radiosensitivity in advanced patients with NSCLC
(10). However, the clinical use
of ZTO is hindered by its complex chemical composition and
potential adverse reactions, such as respiratory system damage and
allergic reactions (11–13), underscoring the imperative to
isolate bioactive monomers. Network pharmacology, an emerging
paradigm in systems biology, provides a robust framework to
elucidate the multi-component, multi-target and multi-pathway
synergy of Traditional Chinese Medicine (14). In the present study, network
pharmacology was employed to identify the main bioactive
components, therapeutic targets and associated signaling pathways
of ZTO in NSCLC treatment. The core targets were further validated
through molecular docking and in vitro cellular experiments,
with the aim of delineating the pharmacodynamic basis of ZTO and
optimizing its clinical utility.
Materials and methods
Key materials
Human NSCLC cell lines A549 (cat. no. CM-0016) and
H1299 (cat. no. CM-0165) were purchased from Procell Life Science
& Technology Co., Ltd. and cultured at 37°C in 5%
CO2 in DMEM (Gibco; Thermo Fisher Scientific, Inc.) or
RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.). ZTO injection
(Zhejiang Tianrui Pharmaceutical Co., Ltd.; national medical
product approval/lot no. H20064312; cat. no. 423020101), F12K
medium (cat. no. 21127030; Thermo Fisher Scientific, Inc.), FBS
(cat. no. C04001-500; Shanghai XP Biomed Ltd.) and trypsin (cat.
no. PB180225; Procell Biotechnology Co., Ltd.) were used. The
BeyoClick™ EdU Cell Proliferation Kit with AF488 (cat. no. C0071S;
Beyotime Biotechnology), 4% paraformaldehyde (cat. no. P0099;
Beyotime Biotechnology), Acridine Orange Staining Kit (cat. no.
C0233S; Beyotime Biotechnology), Annexin V-FITC Apoptosis Detection
Kit (cat. no. BA00101; Beijing Biosynthesis Biotechnology Co.,
Ltd.), TRIzol® Reagent (cat. no. 15596026CN; Invitrogen;
Thermo Fisher Scientific, Inc.) and Reverse Transcription Kit and
Real-Time Quantitative PCR Kit (cat. nos. AG0304-B and AH0104-B;
Shandong Sparkjade Biotechnology Co., Ltd.) were used. Instruments
used included the Spark™ 10M microplate reader (Tecan Group Ltd.),
Z32HK high-speed refrigerated centrifuge (HERMLE Labortechnik GmbH)
and Mastercycler® Realplex4 Real-Time PCR System
(Eppendorf SE).
MTT assay
A549 cells and H1299 cells in the logarithmic growth
phase were seeded into 96-well plates and cultured for 24 h.
Subsequently, the A549 cells were treated with ZTO at final
concentrations of 0, 100, 200, 300 and 400 µg/ml at 37°C for 48 h
and the H1299 cells were treated with ZTO at final concentrations
of 0, 20, 40, 60, 80, 100, 120 and 140 µM at 37°C for 48 h. After
incubation, 10 µl MTT solution (0.5 µg/µl) was added to each well
and incubated at 37°C for 4 h. Subsequently, 150 µl DMSO was added
to each well and allowed to stand for 10 min. The absorbance was
measured at 490 nm using a multimode microplate reader.
Clonogenic survival assay
After A549 and H1299 cells were treated with 100
µg/ml and 50 µM ZTO for 48 h, A549 and H1299 cells were resuspended
at a density of 100 cells/ml and 1 ml cell suspension was seeded
into 12-well plates. Cells were cultured in a 37°C, 5%
CO2 incubator for 7–10 days until ~50 cell colonies
formed per well. The cultures were then terminated, washed twice
with PBS and fixed with methanol at room temperature for 30 min.
The cells were then stained with 0.1% crystal violet at room
temperature for 1 h, gently rinsed under running water, air-dried
and photographed. The number of colonies was quantified using
ImageJ software (version 1.54k; National Institutes of Health). A
colony was defined as a cluster of ≥50 cells; ImageJ counts were
manually verified to ensure consistency with this definition.
EdU assay
A549 and H1299 cells were seeded in 6-well plates at
a density of 5×105 cells/ml (2 ml/well) and cultured for 12 h. A549
cells were then treated with 100 µg/ml ZTO and H1299 cells were
treated with 50 µM ZTO for 48 h at 37°C. Cell proliferation was
assessed using the EdU Cell Proliferation Detection Kit (Beyotime
Biotechnology), according to the manufacturer's instructions. The
percentage of EdU-positive cells was calculated from five randomly
selected images using ImageJ software (version 1.54k).
Wound-healing assay
The A549 and H1299 cells were cultured until they
reached 80–90% confluence, after which, they were treated with 100
µg/ml and 50 µM ZTO for 48 h at 37°C, respectively. Subsequently,
the A549 and H1299 cells were scratched using a sterile 200-µl
pipette tip. The detached cells were gently washed away with PBS,
and serum-free medium was added. Wound closure was monitored and
images were captured at 0, 6 and 24 h post-scratching. The
migration rate was quantified by measuring the wound area at each
time point using ImageJ software (version 1.54k).
Transwell migration assay
After A549 and H1299 cells were treated with 100
µg/ml and 50 µM ZTO for 48 h at 37°C, respectively, they were
trypsinized, resuspended in serum-free medium and adjusted to
1×105 cells/ml. Then, a 300-µl cell suspension was added
to the upper chamber of a Transwell insert (12-well plate; 8-µm
pore size; Corning, Inc.), and 500 µl medium supplemented with 20%
FBS was added to the lower chamber as a chemoattractant. After 24 h
of incubation at 37°C with 5% CO2, the cells that
migrated to the lower membrane surface were fixed with 4%
paraformaldehyde for 0.5 h, stained with 0.1% crystal violet for 1
h at room temperature and rinsed with distilled water. The inserts
were air-dried, mounted on glass slides with neutral resin, and
images were captured under a light microscope (Olympus IX72;
Olympus Corporation). Migrated cells were counted in five randomly
selected fields per well using ImageJ software (version 1.54k).
Cell morphological analysis
A549 and H1299 cells were seeded onto glass
coverslips in 6-well plates and cultured for 12 h. A549 cells were
then treated with 100 µg/ml ZTO and H1299 cells were treated with
50 µM ZTO for 48 h. The culture medium was removed and the cells
were washed twice with PBS. Then, cells were fixed with methanol
for 30 min at room temperature. After two additional PBS washes,
cells were stained with acridine orange for 5 min at room
temperature in the dark, then rinsed three times with PBS to remove
excess dye. The coverslips were carefully inverted onto microscope
slides freshly coated with anti-fade mounting medium (applied 1–2
min before mounting) at room temperature and images were
immediately captured using a laser scanning confocal microscope
(LSM 880; Zeiss AG) with appropriate filter sets for acridine
orange detection (excitation/emission: 488/525 nm).
Annexin V-FITC
After A549 and H1299 cells were treated with 100
µg/ml and 50 µM ZTO for 48 h, respectively, they were trypsinized
(using EDTA-free trypsin), transferred to centrifuge tubes and
pelleted by centrifugation at 1,000 × g for 5 min at 4°C. The
supernatant was discarded and cells were resuspended in PBS, then
centrifuged (1,000 × g, 5 min, 4°C). Cell pellets were stained with
5 µl Annexin V-FITC and 10 µl PI solution, followed by incubation
at 37°C in the dark for 30 min with gentle mixing. The cell
suspensions were then filtered through a 400-mesh nylon sieve and
analyzed using a Cytomics FC500 Flow Cytometer (Beckman Coulter,
Inc.) within 1 h. All samples were analyzed with CXP Software
ver.2.3 (Beckman Coulter, Inc.). Apoptotic populations were
quantified using Annexin V-FITC+/PI-(early apoptosis), Annexin
V-FITC+/PI+ (late apoptosis/necrosis) and Annexin
V-FITC-/PI-(viable cells).
Platforms, software and databases used
for network pharmacology
Specific platforms, software and databases used for
network pharmacology analysis are shown in Table I.
 | Table I.Technical specifications: Platforms,
software and databases. |
Table I.
Technical specifications: Platforms,
software and databases.
Target prediction of active
constituents in ZTO
Primary chemical constituents of ZTO were
systematically retrieved from the Traditional Chinese Medicine
Systems Pharmacology database (TCMSP) and supplemented with
evidence from the literature (7,11,12).
The chemical structures of the identified compounds were analyzed
using the PubChem database, while putative therapeutic targets were
predicted using the Swiss Target Prediction database. Targets with
a probability >0 were selected as the target proteins.
Probability indicates the confidence level of the predicted
interaction between the compound and the target.
Screening of ZTO targets for
NSCLC
Potential therapeutic targets associated with NSCLC
were identified by searching the Online Mendelian Inheritance in
Man (OMIM) database, GeneCards database and Therapeutic Target
Database (TTD) using the keyword ‘non-small cell lung cancer’. The
retrieved targets were combined and duplicate entries were
eliminated. High-confidence targets were then prioritized based on
relevance scores and functional annotations.
Construction of the ZTO-NSCLC
compound-target network
Overlapping targets between ZTO active components
and NSCLC were identified and the ‘ZTO-NSCLC Compound-Target
Network’ was constructed using Cytoscape (version 3.10.1) (15). Node degree values were analyzed and
the top five active components with the highest degree values were
selected to visualize their pharmacological associations.
Construction of protein-protein
interaction (PPI) network and screening of key targets
Standard targets of ZTO for lung cancer treatment
were imported into the Search Tool for the Retrieval of Interacting
Genes/Proteins (STRING) database (version 12.0), with the species
limited to Homo sapiens and a confidence score threshold of
>0.900. Disconnected nodes were excluded to generate the PPI
network. Network topology analysis was conducted using Cytoscape
(version 3.10.1) and the degree values of each target were
calculated to identify hub genes.
Gene ontology (GO) and Kyoto
encyclopedia of genes and genomes (KEGG) enrichment analysis
Overlapping target genes between ZTO and NSCLC were
imported into the DAVID database for GO and KEGG pathway enrichment
analysis. Visualization of the enrichment results, including bubble
plots and bar charts, was performed using the Bioinformatics Online
Analysis Platform (https://www.bioinformatics.com.cn/).
Molecular docking analysis
SDF files of the primary active components of ZTO
were retrieved from the PubChem database, while the 3D structures
of key target proteins were collected from the Protein Data Bank
(PDB; http://www.rcsb.org/). The protein
structures were optimized by removing water molecules using PyMOL
(version 2.1.0; Schrödinger, Inc.), followed by hydrogenation and
format conversion to PDBQT using AutoDock Tools (version 1.5.6;
Molecular Graphics Laboratory, The Scripps Research Institute).
Molecular docking was performed using the AutoDock Vina (version
2.0) module embedded in PyRx software (version 0.8; http://pyrx.sourceforge.io/) to calculate binding
energies and generate output files.
Reverse-transcription quantitative PCR
(RT-qPCR)
After treatment with 100 µg/ml ZTO for 48 h, the
mRNA expression levels of ESR1 and EGFR in A549 cells were analyzed
using RT-qPCR. Total RNA was extracted and reverse-transcribed into
cDNA using the ReverTra Ace™ qPCR RT Master Mix with gDNA Remover
(Toyobo Co., Ltd.), as described by Zhang et al (16). qPCR was performed with TB Green
Premix Ex Taq (Tli RNaseH Plus; cat. no. RR420L; Takara Bio, Inc.)
by cycling at 95°C for 2 min, followed by 40 cycles of 95°C for 15
sec and 60°C for 30 sec. All reactions were run in triplicate and
relative gene expression was calculated using the 2−ΔΔCq
method (17). The primer sequences
used, including that of the reference gene β-actin, are listed in
Table II.
| Table II.Primers used for reverse
transcription-quantitative PCR. |
Table II.
Primers used for reverse
transcription-quantitative PCR.
| Primer | Direction | Sequence
(5′-3′) |
|---|
| ESR1 | Forward |
GCTTACTGACCAACCTGGCAGA |
|
| Reverse |
GGATCTCTAGCCAGGCACATTC |
| EGFR | Forward |
AACACCCTGGTCTGGAAGTAC |
|
| Reverse |
TCGTTGGACAGCCTTCAAGACC |
| β-actin | Forward |
AGCGAGCATCCCCCAAAGTT |
|
| Reverse |
GGGCACGAAGGCTCATCATT |
Western blotting
Whole-cell lysate was prepared using RIPA lysis
buffer (cat. no. P0013B; Beyotime Biotechnology). The lysate was
centrifuged at 13,000 × g at 4°C for 15 min. The supernatant was
collected and protein concentration was measured with a BCA protein
assay kit (Beyotime Biotechnology). Equal amounts of protein (12 µg
per lane) were loaded onto 10% SDS-PAGE gels, separated and then
transferred to PVDF membranes. The membranes were blocked with
BeyoFC™ Fc Receptor Blocking Solution (Human) (Beyotime
Biotechnology) at room temperature for 15 min, then incubated
overnight at 4°C with primary antibodies against ESR1 (1:300; cat.
no. WL00940; Shenyang Wanli Biotechnology Co., Ltd.) and EGFR
(1:300; cat. no. CY5064; Shanghai Abways Biotechnology Co., Ltd.).
Afterward, the membranes were incubated with IRDye®
800CW goat anti-rabbit IgG secondary antibody (1:15,000; cat. no.
926–32211; LICORbio) or IRDye® 800CW goat anti-mouse
secondary antibody (1:15,000; cat. no. 926-32210; LICORbio) at room
temperature for 1 h, then washed with 1X PBS-0.1% (v/v) Tween-20
(pH 7.4). The western blots were visualized using the
Odyssey® Infrared imaging system (LI-COR Biosciences)
and the bands were analyzed and normalized to mouse β-actin
(1:5,000; cat. no. ab8227; Abcam). using ImageJ software.
Mycoplasma testing
A 2–3 day-old cell culture medium was used as the
sample for Mycoplasma testing. A 30 µl PCR reaction was
assembled by adding 15 µl TransDetect® PCR Mycoplasma
SuperMix II (2×; TransGen Biotech Co., Ltd.), 2 µl Myco Primer Mix
II, 8 µl MycoFree Water and 5 µl template in that order. Both a
negative control (MycoFree Water) and a positive control
(MycoPositive Control Template II) were included. The following
program was then run: initial denaturation at 95°C for 4 min; 35
cycles of 95°C for 10 sec, 62°C for 25 sec and 72°C for 10 sec;
final extension at 72°C for 5 min. A total of 10 µl product was
loaded on a 1.5% agarose gel. Following electrophoresis, the gel
was stained with ethidium bromide and visualized under UV
transillumination; a ~254 bp band indicated a positive result. The
samples were compared with controls to determine Mycoplasma
contamination.
Statistical analysis
Statistical analyses were performed using SPSS
(version 25.0; IBM Corp.) and GraphPad Prism (version 8.0;
Dotmatics) software. Intergroup differences were evaluated using
unpaired Student's t-test. Data are expressed as the mean ± SD.
P<0.05 was considered to indicate a statistically significant
difference.
Results
ZTO inhibits proliferation of lung
cancer A549 cells and H1299 cells
To investigate the effects of different
concentrations of ZTO on the proliferation of human lung cancer
cell lines A549 and H1299, the inhibitory effects were measured
using an MTT assay. The results demonstrated that ZTO inhibited the
proliferation of both cell lines in a concentration-dependent
manner (Fig. 1A). The
IC50 values were determined to be 96.73 µg/ml for A549
cells and 46.82 µM for H1299 cells. Therefore, ZTO concentrations
of 100 µg/ml and 50 µM were selected for subsequent experiments.
The colony-forming efficiency and percentage of EdU-positive cells
in both A549 and H1299 cells were found to be significantly
decreased compared with the control group (Fig. 1B and C).
ZTO inhibits the migration of lung
cancer A549 and H1299 cells
Effects of ZTO on the migratory capacity of A549 and
H1299 cells were evaluated by wound-healing and Transwell assays.
Compared with the control group, the wound healing rate and number
of migrated cells were markedly reduced in the ZTO-treated groups
(Fig. 2). These results indicated
that ZTO suppressed the migration of lung adenocarcinoma cells.
ZTO induces apoptosis in lung cancer
A549 cells and H1299 cells
As shown in Fig.
3A, acridine orange staining revealed that untreated A549 and
H1299 cells exhibited high density, intact cellular structure and
nearly round nuclei. By contrast, ZTO-treated cells displayed
marked morphological changes, including cell shrinkage, chromatin
condensation in the nuclei and early apoptotic alterations. The
Annexin V-FITC/PI dual staining results demonstrated that, compared
with the control group, ZTO treatment significantly increased the
early apoptosis rate of A549 and H1299 cells (Fig. 3B). These findings collectively
indicated that ZTO promoted apoptosis in A549 cells.
Screening of ZTO components and target
prediction results
Through the TCMSP database and supplementary
literature review, >100 components from ZTO were identified.
Based on preliminary experiments and screening criteria from the
literature, 27 major active components of ZTO were identified
(Table III). Subsequently,
target prediction was performed for these active components. After
entering the components into the database and applying filtering
parameters (probability >0), 301 target genes were obtained for
further analysis.
| Table III.Basic information of chemical
composition of zedoary turmeric oil. |
Table III.
Basic information of chemical
composition of zedoary turmeric oil.
| First author,
year | PubChem no. | Chemical
component | Simplified
molecular input line entry system | (Refs.) |
|---|
| Zhong et al,
2025 | CID14543198 |
Isoprocurcumenol |
CC(=C1CC2C(CCC2(C)O)C(=C)CC1=O)C | (7) |
|
| CID24836956 | Neocurdione |
CC1CCC=C(CC(=O)C(CC1=O)C(C)C)C |
|
|
| CID6441391 | Curdione |
CC1CCC=C(CC(=O)C(CC1=O)C(C)C)C |
|
|
| CID14240392 | Curcumol |
CC1CCC2C13CC(C(O3)(CC2=C)O)C(C)C |
|
|
| CID167812 | Curcumenol |
CC1CCC2C13CC(=C(C)C)C(O3)(C=C2C)O |
|
|
| CID10399139 | Isocurcumenol |
CC1CCC2C13CC(=C(C)C)C(O3)(CC2=C)O |
|
|
| CID3081930 | Curzerenone |
CC1=COC2=C1C(=O)C(C(C2)(C)=C)C(=C)C |
|
|
| CID6436348 | Germacrone |
CC1=CCC(=C(C)C)C(=O)CC(=CCC1)C |
|
|
| CID572766 | Curzerene |
CC1=COC2=C1CC(C(C2)(C)C=C)C(=C)C |
|
|
| CID6918391 | β-Elemene |
CC(=C)C1CCC(C(C1)C(=C)C)(C)C=C |
|
|
| CID6442617 |
Dehydrocurdione |
CC1CCC=C(CC(=O)C(=C(C)C)CC1=O)C |
|
|
| CID24834047 | Zedoarondiol |
CC(=C1CC2C(CCC2(C)O)C(CC1=O)(C)O)C |
|
|
| CID6654 | α-Pinene |
CC1=CCC2CC1C2(C)C |
|
|
| CID6616 | Camphene |
CC1(C2CCC(C2)C1=C)C |
|
|
| CID6552009 | Camphor |
CC1(C2CCC1(C(C2)O)C)C |
|
|
| CID64685 | Borneol |
CC1(C2CCC1(C(C2)O)C)C |
|
| Yang et al,
2022 | CID91753231 | Germacrone-4,
5-epoxide |
CC1=CCCC2(C(O2)CC(=C(C)C)C(=O)C1)C | (12) |
|
| CID12309452 | γ-Elemene |
CC(=C1CCC(C(C1)C(=C)C)(C)C=C)C |
|
|
| CID12309449 | δ-Elemene |
CC(C)C1=CC(C(CC1)(C)C=C)C(=C)C |
|
|
| CID10399140 |
13-Hydroxygermacrone |
CC1=CCC(=C(C)CO)C(=O)CC(=CCC1)C |
|
|
| CID14191393 | Curcumanolide
A |
CC1CCC(C12CC(=C(C)C)C(=O)O2)C(=C)C |
|
|
| CID5315469 | Bisacumol |
CC1=CC=C(C=C1)C(C)CC(C=C(C)C)O |
|
|
| CID10263440 |
Epiprocurcumenol |
CC1=CC(=O)C(=C(C)C)CC2C1CCC2(C)O |
|
|
| CID636458 | Furanodiene |
CC1=CCC2=C(CC(=CCC1)C)OC=C2C |
|
|
| CID14543200 |
Neoprocurcumenol |
CC1=C2CCC(C2CC(=C(C)C)C(=O)C1)(C)O |
|
| Wu et al,
2022 | CID91457 | β-Eudesmol |
CC12CCCC(=C)C1CC(CC2)C(C)(C)O | (11) |
|
| CID153845 | Curcumenone |
CC(=C1CC2C(C2(CC1=O)C)CCC(=O)C)C |
|
Network pharmacological analysis of
ZTO-NSCLC interactions
Using ‘non-small cell lung cancer’ as the keyword,
156 targets were retrieved from the OMIM database, 1,317 targets
(with relevance score >4 times the median value) from the
GeneCards database and 100 targets from the TTD database. After
merging and deduplication, 1,483 NSCLC-associated targets were
identified. A cross-analysis of 301 ZTO targets and 1,483 NSCLC
targets identified 96 common drug-disease targets (Table SI). A ZTO-NSCLC compound-target
interaction network diagram was then constructed (Fig. 4A), whereby the number of edges
emitted by active compounds reflects their potential regulatory
scope. The top five active compounds with the highest target
connectivity were curcumenone, zedoarondiol, 13-hydroxygermacrone,
neocurdione and curcumol, indicating their key roles in NSCLC
intervention.
The 96 overlapping targets were further analyzed
using the STRING database to build a PPI network (Fig. 4B), comprising 96 nodes and 202
edges. Visualization and topological analysis using Cytoscape
(Fig. 4C and Table IV) revealed that node size and
color intensity were associated with degree values. The top five
core targets ranked by degree values were ESR1, EGFR, PIK3CA,
PIK3CB and PTPN11, indicating their central roles in mediating the
anti-NSCLC effects of ZTO.
| Table IV.Key targets. |
Table IV.
Key targets.
| Target | Degree value | Betweenness
centrality | Closeness
centrality |
|---|
| ESR1 | 15 | 0.230 | 0.430 |
| EGFR | 15 | 0.130 | 0.410 |
| PIK3CA | 15 | 0.060 | 0.380 |
| PIK3CB | 15 | 0.060 | 0.367 |
| PTPN11 | 15 | 0.050 | 0.367 |
GO and KEGG pathway enrichment
analysis
GO functional enrichment analysis of the 96
overlapping targets was performed using the DAVID database,
yielding 526 significant entries (Fig.
5A). These included 391 biological process terms primarily
associated with ‘Signal transduction’, ‘Phosphorylation’ and
‘Response to xenobiotic stimulus’, 52 cellular component terms
enriched in the ‘Cytoplasm’, ‘Nucleus’ and ‘Membrane raft’ as well
as 83 molecular function terms mainly associated with ‘Protein
tyrosine kinase activity’, ‘Transcription coactivator binding’ and
‘Protein kinase binding’. KEGG pathway enrichment analysis
identified 526 significant pathways (Fig. 5B), predominantly associated with
‘EGFR tyrosine kinase inhibitor resistance’, ‘PI3K-Akt signaling
pathway’, ‘Pathways in cancer’, ‘HIF-1 signaling pathway’ and
‘Proteoglycans in cancer’. These results suggested that ZTO exerted
therapeutic effects on NSCLC through multi-target modulation of
oncogenic signaling pathways and regulation of cellular
proliferation.
Molecular docking validation
Molecular docking was performed to validate the
interactions between the top five core components of ZTO
(curcumenone, curcumadiol, 13-hydroxygermacrone, neocurdione and
curcumol) and the key targets (ESR1, EGFR, PIK3CA, PIK3CB and
PTPN11) identified for NSCLC treatment. The results demonstrated
that the binding energies of all active components with the core
targets were <-5.0 kcal/mol, indicating spontaneous and stable
binding (Table V). The binding
mode analyses, focusing on ESR1 and EGFR as representative cases,
are illustrated in Fig. 6.
Notably, ESR1 exhibited powerful interactions with the core
components, with binding energies consistently <-7.0 kcal/mol,
suggesting high structural stability (18).
| Table V.Molecular docking results
(kcal/mol). |
Table V.
Molecular docking results
(kcal/mol).
| Compound | Curcumenone | Zedoarondiol |
13-Hydroxygermacrone | Neocurdione | Curcumol |
|---|
| ESR1 | −7.9 | −8.5 | −7.8 | −7.8 | −7.4 |
| EGFR | −6.2 | −6.4 | −6.3 | −6.5 | −6.6 |
| PIK3CA | −6.8 | −7.8 | −7.3 | −7.2 | −7.7 |
| PIK3CB | −6.7 | −7.0 | −7.0 | −7.5 | −6.5 |
| PIPN11 | −6.2 | −6.5 | −6.5 | −6.2 | −6.4 |
Effects of ZTO on ESR1 and EGFR
expression levels in A549 lung cancer cells
To determine whether ZTO exerts its effects through
ESR1 and EGFR, RT-qPCR and western blotting analyses were performed
to assess changes in the expression levels of these two genes
following ZTO treatment. The results demonstrated that, compared
with the control group, ZTO significantly downregulated ESR1 and
EGFR mRNA levels by 46.56 and 32.61%, respectively (Fig. 7A). At the protein level, ESR1 and
EGFR expression decreased by 35.77 and 28.99%, respectively
(Fig. 7B). These findings
suggested that ZTO inhibited proliferation and induces apoptosis in
A549 lung cancer cells by downregulating ESR1 and EGFR
expression.
Discussion
A continuous rise in newly diagnosed lung cancer
cases in China has been observed in recent years, posing a marked
threat to human health (19). The
poor prognosis of patients with locally advanced NSCLC may be
attributed to the toxic side effects of conventional
chemotherapeutic agents on normal cells while targeting tumor cells
(20). Traditional Chinese
medicine preparations, characterized by multi-target effects and
fewer adverse reactions, hold unique advantages. ZTO, primarily
composed of volatile oils extracted from Curcuma zedoaria,
is a natural product containing a number of bioactive components.
Clinical and experimental studies have demonstrated its
antimicrobial (21),
anti-inflammatory (22) and
anticancer (23) properties. In
the present study, ZTO treatment significantly reduced the
viability, colony-forming ability and number of EdU-positive A549
and H1299 cells, as well as inhibited cell migration and increased
the early apoptosis rate. These findings indicated that ZTO
inhibited proliferation and induced apoptosis in A549 and H1299
human lung cancer cells.
However, the chemical composition of ZTO is complex.
To clarify the pharmacologically active components responsible for
its therapeutic effects, network pharmacology analysis was
performed to identify the primary bioactive constituents of ZTO,
with results showing that these included curcumenone, zedoarondiol,
13-hydroxygermacrone, neocurdione and curcumol, exhibit extensive
potential targets, consistent with findings reported in prior
studies (24–30). Curcumol has been identified as an
effective inducer of apoptosis in a number of cancer cell types by
targeting key signaling pathways commonly dysregulated in
malignancies, such as the MAPK/ERK, PI3K/Akt and NF-κB pathways
(24–26). Zedoarondiol suppresses the
proliferation of human bronchial smooth muscle cells (27), where germacrone inhibited A549 cell
proliferation and induced apoptosis (28). Curcumenone and its associated
bioactive components regulate vascular contraction and inflammatory
responses (29). Neocurdione has
also demonstrated notable tumor growth inhibitory activity
(30), collectively indicating
that these components likely represent the central constituents
underlying the efficacy of ZTO against NSCLC.
GO and KEGG analyses were performed to elucidate the
biological processes and signaling pathways underlying the
inhibitory effects of ZTO on proliferation and induction of
apoptosis in human lung cancer A549 cells. The results revealed
that ZTO exerted antitumor effects by modulating the EGFR pathway,
PI3K-Akt signaling pathway and other key pathways.
Studies have indicated that EGFR-mediated signaling
pathways serve an important role in regulating physiological
processes, including cell proliferation and differentiation
(31,32). The MAPK and PI3K/Akt pathways,
downstream of EGFR, serve a key role in these effects. The PI3K/Akt
pathway regulates diverse cellular processes and is critically
involved in cancer progression (33). In combination with celecoxib,
curcumol has been shown to markedly reduce the phosphorylation of
PI3K, Akt, ERK and p38MAPK of A549 and H1299 cells, thereby
inducing apoptosis. Notably, the mitogenic PI3K pathway and its
downstream NF-κB signaling are known for their ability to suppress
apoptosis and promote proliferation (34). Curcumol may also inhibit
proliferation and invasion while promoting apoptosis in lung cancer
cells by activating the JAK1/STAT3 signaling pathway (35). Zedoarondiol suppresses human
bronchial smooth muscle cell proliferation through the
caveolin-1/platelet-derived growth factor signaling pathway,
potentially by inhibiting the activation of the MAPK and PI3K/AKT
pathways (27). Germacrone likely
inhibits cell proliferation by modulating the Akt/mouse double
minute 2 homolog/p53 signaling axis (28).
PPI network analysis was conducted to identify the
core targets underlying the inhibitory effects of ZTO on the
proliferation and induction of apoptosis in human lung cancer A549
cells. The PPI network revealed that ESR1, EGFR, PIK3CA, PIK3CB and
PTPN11 were the core therapeutic targets of ZTO in NSCLC. ESR1, a
key target in the estrogen signaling pathway, has been shown to
suppress A549 cell proliferation, migration and xenograft tumor
formation in nude mice when knocked down (36). Notably, PIK3CA expression has been
associated with EGFR mutations, whereas positive expression of
PIK3CB and mTOR increases the risk of lymph node metastasis in
patients with lung adenocarcinoma (37). EGFR, a transmembrane protein with
cytoplasmic kinase activity, transduces growth factor signals and
is a primary therapeutic target in NSCLC. Although EGFR tyrosine
kinase inhibitors have been clinically developed to effectively
treat NSCLC, drug resistance remains a challenge (38). RT-qPCR and western blotting results
demonstrated that ZTO significantly downregulated the mRNA and
protein expression of EGFR and ESR1 in A549 cells, leading to
reduced proliferative capacity. These findings suggested that ZTO
may provide a novel opportunity for synergistic antitumor activity
in combination with targeted therapies for NSCLC. Although the
present study demonstrated that ZTO downregulated EGFR and ESR1,
which are upstream regulators of the PI3K/Akt pathway, whether each
core monomeric component individually contributes to this effect
and whether there exist synergistic or antagonistic interactions
among these components, remains to be elucidated. Furthermore, the
present study lacks in vivo animal experimental data. The
antitumor activity, bioavailability and systemic toxicity of ZTO
and its monomeric components in animal models of non-small cell
lung cancer therefore await further validation.
In summary, the present study employed network
pharmacology to identify potential core components and key targets
of ZTO in the treatment of NSCLC. Molecular docking analysis
revealed favorable binding interactions between these core
components and their targets, indicating their robust
pharmacological activity. In vitro experiments determined
that ZTO inhibits the proliferation of A549 cells and downregulates
the expression of ESR1 and EGFR, which are pivotal regulators of
oncogenic signaling pathways. These findings indicated that ZTO
exerts its therapeutic effects on NSCLC through a complex network
mechanism involving multicomponent synergy, multi-target modulation
and multi-pathway regulation. Further in-depth investigations
should be pursued in subsequent studies. The present study
therefore provides marked theoretical support for the clinical
translation of ZTO and highlights its potential as a complementary
agent in targeted NSCLC therapies.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the Natural Science
Foundation of Heilongjiang Province (grant no. LH2023H104) and the
Traditional Chinese Medicine Scientific Research Project of
Heilongjiang Province (grant no. ZHY2025-299).
Availability of data and materials
The data generated in the present study are included
in the figures and/or tables of this article.
Authors' contributions
HL and WZ conceptualized the present study. QY
performed the confocal laser microscope imaging and analysis. HL,
LL, YL and YS performed the bioinformatics analyses, including
molecular docking and network pharmacology analysis. JJ, ZW, WZ and
BL performed data analysis. HL, LL and WZ wrote, reviewed and
edited the original manuscript. HL provided supervision and
acquired funding for the present study. HL and BL confirm the
authenticity of all the raw data. All authors read and approved the
final manuscript.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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