Keywords TMT proteomics - Weichang'an - gastric cancer - differentially expressed protein
Introduction
On February 1, 2024, the International Agency for Research on Cancer (IARC) released
the latest global cancer statistics. By 2022, there were an estimated 20 million new
cancer cases and nearly 10 million cancer-related deaths worldwide. Among these, there
were 970,000 new cases of gastric cancer and 660,000 deaths, ranking fifth in both
incidence and mortality rates.[1 ] In China, the detection rate of early-stage gastric cancer remains low, and most
patients are diagnosed at an advanced stage, losing the opportunity for curative resection,
which results in poor prognosis. Traditional chemotherapy and radiation therapies
have limited efficacy. Due to the high spatiotemporal heterogeneity of gastric cancer,
progress in precise targeted therapies has been slow.[2 ] Traditional Chinese medicine (TCM) plays an important role in the prevention and
treatment of gastric cancer. Our research team has developed a TCM compound, Weichang'an,
which primarily strengthens the spleen, targeting the major pathogenic factor of spleen
and stomach deficiency. This formulation has been used clinically for nearly 30 years
as an in-hospital preparation and has shown definitive efficacy in improving the spleen
deficiency syndrome in gastric cancer patients, extending survival, and reducing recurrence
and metastasis after radical surgery.[3 ]
[4 ]
[5 ]
With advancements in medical technology, research in TCM has also deepened, with the
ultimate goal of enhancing clinical efficacy, improving patient quality of life, and
prolonging survival. High-throughput screening technologies are increasingly used
in scientific research, and proteomics has become an indispensable tool in functional
genomics. It not only provides potential biomarkers for diagnosis but also offers
insights for better understanding the mechanisms of TCM in treating diseases. Tandem
mass tags (TMT) technology, developed by Thermo Fisher Scientific in the United States,
is a high-throughput proteomics technique that labels peptide amino groups with 2
to 10 stable isotopes for mass spectrometry analysis.[6 ] This study aims to identify key proteins involved in the treatment of human gastric
cancer in nude mice with the TCM compound Weichang'an, using proteomics to explore
the biological basis of its effect on gastric cancer and provide a reference for further
understanding its therapeutic mechanisms.
Materials
Experimental Animals and Cells
Sixteen 7- to 8-week-old female BALB/C nude mice, weighing 16 to 20 g, were provided
by the Animal Facility of the Shanghai Cancer Institute, with animal production license
number SCXK (Hu) 2002–0001. The animals were housed and experiments were conducted
in strict accordance with specific pathogen-free (SPF) standards. The animal facility
had the license number SYXK (Hu) 2002–0009. The animal experiment was approved by
the Ethics Committee of Experimental Animals at Shanghai University of Traditional
Chinese Medicine, with ethical approval number LHERAW-20003. The human gastric cancer
cell line MKN45 was kindly provided by the Department of Pharmacy, East China University
of Science and Technology.
Reagents
The reagents used in the study were the following: Triton X-100 (MedChemExpress, USA,
CAS number: 9002–93–1); DL-dithiothreitol (DTT), trifluoroacetic acid (TFA), iodoacetamide
(IAA), ammonium bicarbonate (NH4 HCO3 ), N,N,N',N'-tetramethylethylenediamine (TEMED), urea (Sigma-Aldrich, USA, product
numbers: 43819, T6508, V900335, A6141, T8133, U5128); TMT labeling kit, Pierce Dilution-Free
Rapid Gold BCA Protein Assay, Thermo Fisher Scientific, USA, catalog number: A58332,
A55861); trypsin (Promega, USA, catalog number: V5113); acetonitrile (Merck, Germany,
product number: 1.00030); formic acid (FA; Millipore, USA, product number: 5.33002);
BCA protein concentration assay kit (Shanghai Beyotime Biotechnology Co., Ltd., China,
product number: P0012); tetraethylammonium bromide [TEAB; Qi Yi Biotechnology (Shanghai)
Co., Ltd., China, catalog number: 71–91–0].
Instruments
The instruments used were the following: Q Exactive HF-X quadrupole-Orbitrap liquid
chromatography-mass spectrometry system, EASY-nLC1200 chromatography system, Multiskan
FC microplate reader (Thermo Fisher Scientific, USA); JY96-IIN ultrasonic cell crusher
(Ningbo XinZhi Biotechnology Co., Ltd., China); and PowerPac electrophoresis system
(BIO-RAD, USA).
Methods
Preparation of Weichang'an
The TCM compound formula Weichang'an consists of 12 g of Taizishen (Pseudostellariae
Radix), 12 g of fried Baizhu (Atractylodis Macrocephalae Rhizoma), 15 g of Fuling
(Poria), 9 g of Xiakucao (Prunellae Spica), 9 g of Jiang Banxia (Pinelliae Rhizoma
Praeparatum cum Zingibere et Alumine), 9 g of Chenpi (Citri Reticulatae Pericarpium),
30 g of Hongteng (Sargentodoxae Caulis), 30 g of wild Putaoteng (Vitis quinquangularis
Rehd), 3 Bihu (Gekko japonicus Dumeril et Bibron), 30 g of Sheng Muli (Ostreae Concha), and 9 g of Lyu Emei (Prunus mume Sieb. et Zucc). The TCM decoction pieces were provided and identified by the TCM
Pharmacy at Longhua Hospital, Shanghai University of Traditional Chinese Medicine.
Weichang'an water extract was prepared using the decoction method, with the drug concentration
adjusted to 240 g/L (per liter of crude drug), sterilized at high temperature, and
stored at 4°C for later use. High-performance liquid chromatography (HPLC) was used
to quantitatively measure the active ingredient hesperidin in the Weichang'an decoction
to assess its quality.[7 ]
Establishment of Subcutaneous Tumor Model in Nude Mice and Grouping
The MKN45 cell line was expanded in culture, and 1 × 107 cells were suspended in 0.2mL serum-free culture medium. The cell suspension was
injected subcutaneously into the axillae of nude mice (0.1 mL per side). On the seventh
day postinjection, noticeable tumor nodules appeared at the injection sites, indicating
successful establishment of the tumor model. Mice with successful tumor formation
were randomly divided into two groups: the Weichang'an group and the model group,
with eight mice each group. Mice in the Weichang'an group were orally administered
0.5 mL of Weichang'an decoction, while those in the model group were given 0.5 mL
of normal saline, once daily for 21 days. On the 28th day of the experiment, the mice
were euthanized by cervical dislocation, the tumors were excised, and then immediately
frozen.
Protein Sample Preparation
Mouse tumor samples were taken from the –80°C freezer and ground into powder using
liquid nitrogen. Each group of samples was mixed with four times the volume of lysis
buffer (containing 8 mol/L urea and 1% protease inhibitor) and subjected to ultrasonic
lysis. The samples were then centrifuged at 12,000× g for 10 min at 4°C, and the supernatant was collected. Protein concentration was determined
using a bicinchoninic acid (BCA) protein assay kit. A protein solution of 80 µg was
mixed with DTT to achieve a final concentration of 5 mmol/L and reduced at 56°C for
30 min. IAA was then added to a final concentration of 11 mmol/L, and the mixture
was incubated at room temperature for 15 min in the dark. Finally, the samples were
diluted to a concentration lower than 2 mol/L. Trypsin was added at a mass ratio of
1:50 (trypsin:protein) and incubated at 37°C overnight for digestion. Afterward, 1%
trypsin was added for a further 4 h of digestion. The trypsin-digested peptides were
desalinated using a Strata X C18 column (Phenomenex), and the desalinated peptides
were vacuum freeze-dried. The peptides were then dissolved in 0.5 mol/L TEAB and labeled
using the TMT reagent kit according to the manufacturer's instructions.
HPLC Fractionation
Peptides were fractionated using high pH reverse-phase liquid chromatography with
an Agilent 300Extend C18 column (particle size of 5 µm, inner diameter of 4.6 mm,
length of 250 mm). The labeled peptides from each group were mixed and fractionated
using an Agilent 1260 infinity II HPLC system. The mobile phase A consisted of 10 mmol/L
HCOONH4 , 5% CAN, pH 10.0, and phase B was 10 mmol/L HCOONH4 , 85% CAN, pH 10.0. The column was equilibrated with mobile phase A, and the sample
was injected using an autosampler for separation on the column at a flow rate of 1 mL/min.
The gradient elution conditions were as follows: 0 to 25th minutes, 0% B; 25th to 30th min, linear gradient 0 to 7% B; 30th to 65th min, linear gradient 7% to 40% B; and 65th to 70th min, linear gradient 40% to 100% B; 70th to 85th min, 100% B. The elution was monitored at 214 nm, and fractions were collected every
minute, yielding approximately 40 fractions. After freeze-drying, the samples were
redissolved in 0.1% FA and combined into different fractions.
Liquid Chromatography-Mass Spectrometry Analysis
The combined peptide fractions were dissolved in mobile phase A (0.1% FA aqueous solution)
and separated using an EASY-nLC 1200 ultra-HPLC system. The mobile phase B was 0.1%
FA acetonitrile solution. The liquid chromatography gradient was as follows: 0 to
60th min, 6% to 20% B; 60th to 80th min, 20% to 30% B; 80th to 86th min, 30% to 80% B; and 86th to 90th min, 80% B, with a flow rate maintained at 350 nL/min. After separation by ultra-HPLC,
peptides were ionized using an NSI ion source and analyzed using an Orbitrap Fusion
Lumos mass spectrometer. The ion source voltage was set to 2.4 kV, and both the parent
ions and their fragment ions were analyzed by high-resolution Orbitrap. Data were
collected using data-dependent acquisition (DDA) mode, with automatic gain control
(AGC) set to 5 × 104 , a signal threshold of 5,000 ions/s, and a maximum injection time of 100 ms. The
dynamic exclusion time for tandem mass spectrometry scans was set to 30 s to prevent
repetitive scanning of parent ions.
Data Processing
Secondary mass spectrometry data were searched using the MaxQuant database. The search
parameters were set to SwissProt Human (20,130 sequences) and SwissProt Mouse (16,839
sequences), with a reverse database included to calculate the false-positive rate
due to random matches. Common contaminant databases were also added. The enzyme digestion
method was set to trypsin/P; the maximum number of missed cleavages was set to 2;
the minimum peptide length was set to 7 amino acid residues; the maximum number of
modifications per peptide was set to 5. The mass tolerance for the primary parent
ion was set to 5 ppm for Main search and 20 ppm for First search, and the mass tolerance
for secondary fragment ions was set to 0.02 Da. Cysteine alkylation was set as a fixed
modification, while variable modifications included methionine oxidation and N-terminal
acetylation of the protein. The quantification method used was TMT-10plex, and the
false-positive rates for PSM identification and protein identification were both set
to 1%. Differential proteins were screened using the p -value and fold change parameters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes
and Genomes (KEGG) databases were used for enrichment analysis of the differential
proteins. GO analysis described the proteins from three dimensions: molecular function,
biological process, and cellular component. Fisher's exact test was used to calculate
and select significantly different protein pathways.
Statistical Analysis
Data were analyzed using SPSS 22.0 software, and results were expressed as mean ± standard
deviation (x̅ ± s). The study compared two groups, and since the data met normality tests, independent
sample T -tests were used. A p -value of less than 0.05 was considered statistically significant.
Results
Weichang'an Inhibits Subcutaneous Tumor Growth in Human Gastric Cancer Nude Mice
Compared with the model group, the tumor weight of the nude mice in the Weichang'an
group was significantly reduced, with a statistically significant difference (p = 0.000), and the tumor inhibition rate was 41.40%. This indicates that Weichang'an
can inhibit the growth of subcutaneous transplanted tumors in human gastric cancer
nude mice (see [Table 1 ]).
Table 1
Effect of Weichang'an on subcutaneous tumor growth in nude mice (x̅ ± S)
Group
n
Tumor mass (m/g)
p -value
Tumor inhibition rate/%
Model group
8
2.15 ± 0.24
0.000
41.40
Weichang'an group
8
1.26 ± 0.11
Differential Protein Identification
Proteomic analysis identified differential expression proteins in human gastric cancer
nude mice subcutaneous transplanted tumors after intervention with the compound Weichang'an.
A total of 2,856 proteins containing quantitative information were identified. Proteins
with a fold change greater than 1.2 were considered significantly upregulated, and
those with a fold change less than 0.83 were considered significantly downregulated.
Among the quantified proteins, 13 proteins were significantly downregulated and 25
proteins were significantly upregulated in the Weichang'an group (see [Fig. 1 ] and [Tables 2 ] and [3 ]).
Fig. 1 Number of differentially expressed proteins at different thresholds.Notes: Q1 (0~0.77):
fold change range from 0 to 0.77; Q2 (0.77~0.83): fold change range from 0.77 to 0.83;
Q3 (1.2~1.3): fold change range from 1.2 to 1.3; Q4 (>1.3): fold change range greater
than 1.3. The numbers on each bar represent the number of proteins in each range.
Table 2
Differential proteins with downregulated expression in the Weichang'an group
No.
Names of differential proteins
Fold change
p -value
1
Glyceraldehyde-3-phosphatedehydrogenase, testis-specific
0.761
0.006
2
Serine/threonine-protein kinase Chk1
0.572
0.010
3
Ribosomal L1 domain-containing protein 1
0.741
0.035
4
Complement factor D
0.813
0.041
5
Collagen α-1(I) chain
0.749
0.038
6
Clathrin light chain A
0.79
0.010
7
Fibrillin-1
0.77
0.003
8
Interferon-induced protein with tetratricopeptide repeats 5
0.809
0.016
9
Ribosome production factor 2 homolog
0.675
0.037
10
G-protein coupled receptor family C group 5 member C
0.781
0.018
11
1-acyl-sn-glycerol-3-phosphate acyltransferase epsilon
0.811
0.009
12
Lysosomal thioesterase PPT2
0.811
0.031
13
Feline leukemia virus subgroup C receptor-related protein 1
0.73
0.019
Table 3
Differential proteins with upregulated expression in the Weichang'an group
No.
Names of differential proteins
Fold change
p -value
1
PDZ and LIM domain protein 1
1.241
0.006
2
Tumor protein D54
1.214
0.019
3
Putative nucleoside diphosphate kinase
1.345
0.012
4
Steroid hormone receptor ERR2
2.255
0.029
5
Protein-L-isoaspartate (D-aspartate) O-methyltransferase
1.202
0.047
6
Cofilin-1
1.244
0.001
7
Tyrosine-protein phosphatase nonreceptor type 6
1.269
0.014
8
Lysosomal acid lipase/cholesteryl ester hydrolase
1.275
0.046
9
Basal cell adhesion molecule
1.261
0.029
10
Myomesin-1
1.317
0.014
11
UV excision repair protein RAD23 homolog B
1.201
0.026
12
Tumor protein D52
1.219
0.020
13
Cytochrome c oxidase assembly factor 6 homolog
1.238
0.048
14
Calmodulin-lysine N-methyltransferase
1.245
0.006
15
Plasminogen activator inhibitor 1 RNA-binding protein
1.302
0.027
16
Coiled-coil domain-containing protein 12
1.239
0.046
17
Uncharacterized protein KIAA1143
1.284
0.010
18
Carboxymethylenebutenolidase homolog
1.235
0.032
19
Transcription elongation factor A protein-like 4
1.245
0.038
20
Serine/arginine-related protein 53
1.223
0.001
21
Nonstructural maintenance of chromosomes element 3 homolog
1.247
0.024
22
Equilibrative nucleoside transporter 1
1.226
0.028
23
Zinc finger protein 106
1.217
0.027
24
Ubiquitin-like conjugating enzyme ATG3
1.228
0.002
25
Epidermal growth factor receptor substrate 15-like 1
1.236
0.044
Differentially Expressed Protein Enrichment Analysis
GO and KEGG enrichment analyses were performed on the differentially expressed proteins,
and the significance of these proteins in specific functional annotations was evaluated.
GO enrichment analysis of the differentially expressed proteins was conducted using
the GO database. The results showed that, in terms of cellular components, the differentially
expressed proteins were primarily enriched in the cell membrane, extracellular matrix,
and envelope. In terms of molecular functions, the differentially expressed proteins
were primarily enriched in cell adhesion molecule binding, extracellular matrix structural
components, signal receptor activity, and molecular sensor activity. In terms of biological
processes, the differentially expressed proteins were mainly enriched in the negative
regulation of cell adhesion, hematopoiesis, development of the skeletal system, and
cellular responses to stimuli such as peptide hormones, epidermal growth factor, and
insulin-like growth factor (see [Fig. 2 ]). KEGG enrichment analysis of the differentially expressed proteins was performed
using the KEGG database to predict the signaling pathways involved in these proteins,
thereby revealing the mechanism of action of the Weichang'an formula in the treatment
of gastric cancer. Pathway annotation of the differentially expressed protein genes
based on the KEGG database identified all the pathways associated with the differentially
expressed proteins, and pathways significantly related to the differentially expressed
proteins were selected with p < 0.05, with lysosome signaling pathway being identified as a significant pathway
(see [Fig. 3 ]).
Fig. 2 Gene Ontology enrichment analysis of differential proteins.
Fig. 3 KEGG signaling pathway enrichment analysis of differential proteins.
Discussion
TCM treatment for advanced gastric cancer not only acts synergistically with chemotherapy
drugs to enhance efficacy and reduce toxicity but also plays a role in prolonging
survival and improving quality of life. Through long-term clinical practice and basic
research, under the guidance of TCM theory, Prof. Qiu proposed the academic concept
that “spleen deficiency is key in gastric cancer” and that “tonifying the spleen is
essential throughout the treatment process,” which led to the formulation of the TCM
compound Weichang'an. In this formula, herbs like Taizishen (Pseudostellariae Radix),
Baibiandou (Lablab Semen Album), Baizhu (Atractylodis Macrocephalae Rhizoma), and
Fuling (Poria) serve as the main ingredients, primarily exerting effects on tonifying
the spleen and regulating qi. Herbs such as Hongteng (Sargentodoxae Caulis) and Baqia
(Smilacis Chinae Rhizoma) act as adjuncts for clearing heat and detoxifying; raw Muli
(Ostreae Concha) and Xiakucao (Prunellae Spica) help soften masses and resolve phlegm,
while Jiangbanxia (Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine), Qingpi
(Citri Reticulatae Pericarpium Viride), and Chenpi (Citri Reticulatae Pericarpium)
work to harmonize the stomach and regulate qi. Although the efficacy of Weichang'an
in treating gastric cancer is significant, its underlying mechanism is not yet fully
understood. With the continuous advancement of high-throughput screening technologies,
proteomics has made important contributions to the modernization of TCM research,
particularly in exploring disease-related biomarkers and targets, with quantitative
proteomics being widely used for this purpose.[8 ]
[9 ]
TMT is a high-throughput proteomics technology developed by Thermo Fisher Scientific
in the United States. It involves mass spectrometry analysis of peptides labeled with
2 to 10 stable isotopes. This method offers several advantages, including short run
times, simple operation steps, simultaneous analysis of multiple samples, high protein
identification, and good result reproducibility.[10 ] In this study, TMT labeling, HPLC fractionation, and quantitative proteomics techniques
were used to analyze the impact of gastrointestinal agents on protein expression in
gastric cancer subcutaneous xenograft tumors, exploring the mechanism of gastrointestinal
treatment in gastric cancer at the protein level. A total of 3,385 proteins were identified
based on TMT quantitative proteomics, of which 2,856 proteins contained quantitative
information. Among the quantified proteins, 13 proteins were downregulated and 25
proteins were upregulated in the gastrointestinal agent–treated group. Further literature
analysis of the differential proteins revealed that these proteins were mainly related
to the cytoskeleton, cell cycle, DNA damage repair, and autophagy.
PDZ and LIM domain protein 1 (PDLIM1), also known as CLP-36, is a member of the PDZ-LIM
family of adaptor proteins. It is associated with the cytoskeleton, neuronal signal
transduction, organ development, and tumorigenesis.[11 ] Studies have shown that PDLIM1 is involved in the invasion, migration, and metastasis
of tumor cells in breast cancer, colon cancer, and gliomas.[12 ]
[13 ]
[14 ] Serine/threonine-protein kinase Chk1 (CHEK1), also known as checkpoint kinase 1,
is a key protein that controls the cell cycle and DNA stability. It is essential for
checkpoint-mediated cell cycle arrest in response to DNA damage or unreplicated DNA,
regulating cell cycle checkpoints and coordinating DNA repair.[15 ]
[16 ] Research has found that high expression of CHEK1 is associated with poor prognosis
in patients with non-small-cell lung carcinoma (NSCLC) and also has a similar correlation
in ovarian cancer.[17 ]
[18 ] Fibrillin-1 (FBN1) is a structural component of microfibrils in the extracellular
matrix, serving as an important and complex lattice protein that regulates the microenvironment.[19 ]
[20 ]
[21 ] Recent studies have shown that FBN1 is closely related to tumorigenesis and progression.
Silencing FBN1 expression can reduce proliferation and induce apoptosis in thyroid
papillary carcinoma cells. High expression of FBN1 may induce early recurrence of
ovarian cancer and sensitivity to platinum-based chemotherapy.[22 ]
[23 ] Ribosomal L1 domain-containing protein 1 (RSL1D1), also known as cellular senescence-inhibited
gene protein (CSIG), is involved in regulating multiple biological processes such
as the cell cycle, cellular senescence, apoptosis, and tumor metastasis. Studies have
shown that RSL1D1 is highly expressed in liver cancer and prostate cancer tissues,
and its expression is associated with poor prognosis in patients.[24 ]
[25 ] Collagen α-1 (I) chain (COL1A1), also known as α-1 type I collagen, is a major component
of the extracellular matrix, primarily maintaining the integrity of the cytoskeletal
structure. Its degradation is an important process in the invasion and metastasis
of tumor cells.[26 ]
[27 ] COL1A1 is highly expressed in gastric cancer tissues and is positively correlated
with tumor cell invasion and metastasis.[28 ] Silencing COL1A1 in gastric cancer BGC-823 cells can inhibit tumor cell proliferation
and migration ability.[29 ] Cofilin-1 (CFL1), a low-molecular-weight actin-regulating protein,[30 ] plays an important role in cell proliferation and migration and is a key regulator
in tumor cell invasion and metastasis.[31 ] Recent studies have found that CFL1 is highly expressed in various tumor tissues,
including renal cell carcinoma, ovarian cancer, oral squamous cell carcinoma, pancreatic
cancer, and breast cancer.[32 ]
[33 ]
[34 ] Clinical studies show that CFL1 is closely associated with pathological differentiation,
tumor size, lymph node metastasis, and clinical staging in gastric cancer, and plays
an important role in tumor cell invasion and metastasis.[35 ] Ultraviolet (UV) excision repair protein RAD23 homolog B (RAD23B) is involved in
nucleotide excision repair. Increasing evidence suggests that tumorigenesis is associated
with the expression levels of DNA repair genes. The expression level of HR23B, the
protein encoded by the RAD23B gene, is closely related to tumor invasion and prognosis. Genome-wide association
studies have demonstrated that single nucleotide polymorphism (SNP) in RAD23B are associated with esophageal cancer and bladder cancer.[36 ]
[37 ] Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1) plays a central role
in fibrinolysis, angiogenesis, wound healing, and tumor cell invasion and metastasis.[38 ]
[39 ] Recent studies have found that SERBP1 is significantly overexpressed in ovarian
cancer epithelial cells and is strongly correlated with advanced tumor staging.[40 ] In contrast, overexpression of SERBP1 is positively correlated with a good prognosis
in human breast cancer, suggesting that SERBP1 may have a potential protective role
in breast cancer progression.[41 ] Equilibrative nucleoside transporter 1 (SLC29A1), a member of the solute carrier
(SLC) transporter family, is a type of membrane transporter.[42 ] The SLC superfamily plays an important role in the homeostasis of endogenous compounds,
drug delivery, and tumor drug resistance.[43 ]
[44 ] The study found that the expression of SLC29A1 is positively correlated with the
chemotherapy efficacy in Asian pancreatic cancer patients;[44 ] significant upregulation of SLC29A1 in colorectal cancer, astrocytoma, and breast
cancer cells helps reduce cisplatin resistance and enhances cell viability;[45 ] silencing the expression of SLC29A1 reduces the drug sensitivity in leukemia and
lung cancer cells.[46 ] The ubiquitin-like conjugating enzyme ATG3, also known as autophagy-related gene
3, plays a key role in the formation of autophagosomes.[47 ] During the autophagic process, microtubule-associated protein 1 light chain 3-I
(LC3-I) is converted into the ubiquitin-like conjugating enzyme ATG3, and with the
help of ATG5-ATG12-ATG16, it is lipidated into LC3-II. Studies have shown that silencing
ATG3 expression reduces autophagic activity and inhibits the migration ability of
NSCLC, possibly by inhibiting the activation of the Notch/snail signaling pathway,
thereby affecting the process of epithelial–mesenchymal transition (EMT).[48 ]
GO enrichment analysis revealed that the differentially expressed proteins were mainly
enriched in cellular components such as the cell membrane, extracellular matrix, and
envelope. These proteins participate in biological processes such as the negative
regulation of cell adhesion, hematopoiesis, skeletal system development, and cellular
responses to stimuli such as peptide hormones, epidermal growth factor, and insulin-like
growth factor. They play roles in functions such as cell adhesion molecule binding,
extracellular matrix structural component, receptor activity, and molecular sensor
activity. KEGG enrichment analysis identified the lysosome pathway as a key signaling
pathway. Studies have shown that autophagic dysfunction is closely related to tumor
occurrence and malignant transformation.[49 ] Depending on the cellular conditions and the duration and intensity of stress stimuli,
autophagy can either suppress or promote cancer cell death. Moreover, inhibiting autophagy
can increase the sensitivity of tumor cells to chemotherapy drugs and radiotherapy.[50 ]
Conclusion
This study, through proteomics, found that cytoskeleton-related proteins, cell cycle–related
proteins, and autophagy-related proteins may be involved in the inhibitory effect
of Weichang'an on human gastric cancer cells, and elucidates the biological basis
of Weichang'an in the treatment of gastric cancer. In the future, we will further
conduct experimental studies on the treatment of gastric cancer with Weichang'an,
focusing on cytoskeleton proteins that inhibit invasion and metastasis, as well as
autophagy-lysosome-related pathways. We aim to explore the molecular mechanisms underlying
Weichang'an's treatment of gastric cancer and identify key intervention targets in
gastric cancer treatment so as to provide further evidence for the prevention and
treatment of gastric cancer.
