Introduction
Colorectal cancer (CRC) is the most prevalent
malignant tumor among gastrointestinal malignancies in China,
ranking fifth in both incidence and mortality among all tumors
(1–4). Notably, over the past 30 years, the
incidence and mortality rates of CRC in patients <50 years of
age have steadily increased, gradually showing a younger trend
(5). Unlike in Western countries
where CRC is often diagnosed at earlier stages due to
well-established population-based screening programs, universal
health insurance coverage for preventive services and higher public
awareness of early warning signs, CRC in China is often diagnosed
at an advanced stage, with the majority of patients presenting with
local progression or distant metastasis. Consequently, the 5-year
survival rate for late-stage patients remains at ~14% (6–9). For
patients with metastatic or recurrent CRC, chemotherapy remains a
main treatment strategy. Among platinum-based drugs, such as
oxaliplatin and cisplatin, oxaliplatin is more commonly used in
clinical practice for CRC, whereas cisplatin retains clinical
relevance, particularly in cases of oxaliplatin resistance or
intolerance (10–13). In the present study, cisplatin was
used to investigate the chemoresistance mechanisms in CRC, with the
aim of identifying novel strategies to overcome platinum-based
chemotherapy resistance.
Annexin A3 (ANXA3), a member of the membrane protein
family, is a calcium-dependent phospholipid-binding protein
(14). Alterations in the
expression level or subcellular localization of membrane-associated
proteins can trigger various pathological processes, including
tumorigenesis (14). ANXA3 has been
reported to serve important roles in numerous biological processes,
such as angiogenesis, adipocyte differentiation, tumorigenesis and
chemotherapy resistance (15). For
example, ANXA3 has been reported to be upregulated in ovarian
cancer and to serve as a potential biomarker for chemotherapy
resistance (16). It may also
enhance platinum resistance in ovarian cancer cells via the
regulation of p53 (16).
Additionally, ANXA3 has been identified as being upregulated in
lung cancer, and positively associated with clinical staging and
lymph node metastasis (17). The
relationship between ANXA3 and the development of liver cancer has
also been reported, yet its specific mechanism remains unclear
(18).
Notably, the precise mechanism by which ANXA3
mediates cisplatin resistance in CRC, especially whether it acts by
regulating key cellular detoxification pathways, remains elusive.
Glutathione S-transferase P1 (GSTP1), an important member of the
GST family, has a key role in cellular detoxification and drug
resistance (19). Specifically,
GSTP1 catalyzes the binding of glutathione with various
electrophilic compounds, including platinum-based chemotherapeutic
drugs, thereby promoting their metabolism and excretion, and
reducing the cytotoxicity of these drugs to cancer cells (20). Previous studies have demonstrated
that GSTP1 upregulation is closely associated with chemotherapeutic
resistance in multiple tumors, including CRC (21–23).
Based on the role of ANXA3 in chemotherapeutic resistance and the
key function of GSTP1, it is hypothesized that ANXA3 may mediate
cisplatin resistance in CRC through GSTP1. Moreover, exosomes, key
mediators of intercellular communication, can transfer drug
resistance between cancer cells (24,25).
Membrane-associated proteins, such as ANXA2, are routinely detected
in exosomes (26,27), which prompts the further hypothesis
that ANXA3 may also be transmitted between cells via the exosome
pathway and amplify this GSTP1-mediated drug resistance.
The present study aimed to investigate whether ANXA3
modulates cisplatin resistance in CRC through the regulation of
GSTP1 expression and to explore the availability of potential
inhibitors targeting this pathway. By addressing these issues, the
present study aimed to provide novel insights into the molecular
mechanisms underlying cisplatin resistance in CRC and to identify a
promising therapeutic strategy for overcoming chemotherapy
resistance.
Materials and methods
Cells, tissues and chemicals
CRC cell lines HCT116, LoVo, RKO, SW480 and SW620
were obtained from American Type Culture Collection. The 293T cell
line was obtained from the Shanghai Branch, Cell Bank, Chinese
Academy of Sciences and maintained in our laboratory. All cell
lines were cultured in Dulbecco's Modified Eagle Medium (HyClone™;
Cytiva) with 10% fetal bovine serum (cat. no. 10099141; Gibco;
Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml
streptomycin (cat. no. C0222; Beyotime Biotechnology) in a
humidified atmosphere of 5% CO2 at 37°C. Primary CRC
tumors (from 91 male patients and 78 female patients) were
collected from Shanghai East Hospital (Shanghai, China) between
March 2024 and February 2026, with ages ranging from 36 to 82 years
and a median age of 62 years. The present study was approved by the
Ethics Committee of Shanghai East Hospital (approval no. 2024-220).
Cisplatin and the NF-κB inhibitor JSH-23 were purchased from
Selleck Chemicals. K201 was purchased from TargetMol Chemicals
Inc.
Gene expression analysis
ANXA3 expression in colon adenocarcinoma (COAD) and
rectum adenocarcinoma (READ) was analyzed using Gene Expression
Profiling Interactive Analysis (GEPIA), an online tool that
integrates tumor tissue data from The Cancer Genome Atlas (TCGA)
and normal tissue data from the Genotype-Tissue Expression (GTEx)
project. The COAD cohort comprised 275 tumor samples and 349 normal
samples, while the READ cohort included 92 tumor samples and 318
normal samples (http://gepia2.cancer-pku.cn). Pearson correlation
analyses among ANXA3 expression and excision repair
cross-complementing 1 (ERCC1), GSTP1 or ATP7B in CRC were also
performed using the GEPIA database. The online public tumor
database UALCAN, which integrates protein expression data from the
Clinical Proteomic Tumor Analysis Consortium (CPTAC), was used to
analyze the protein expression of ANXA3 in colon cancer (https://ualcan.path.uab.edu/).
To evaluate the mRNA levels, reverse
transcription-quantitative PCR (RT-qPCR) was performed. Briefly,
total RNA was extracted from cultured cells using RNAiso Plus
(Takara Bio, Inc.), and cDNA was reverse-transcribed with the
PrimeScript™ RT reagent Kit (Takara Bio, Inc.) according to the
manufacturer's protocol. Subsequently, qPCR was carried out using
SYBR Green qPCR Master Mix (Clontech; Takara Bio, Inc.) with Roche
LightCycler® 480II real-time PCR system (Roche
Diagnostics). GAPDH was used as the reference gene, and relative
expression levels were quantified using the 2−ΔΔCt
method as described previously (28). Thermal cycling conditions included
initial denaturation at 95°C for 30 sec, followed by 40 cycles of
95°C for 5 sec and 60°C for 30 sec. Primer sequences used for qPCR
are shown in Table SI.
Immunohistochemistry (IHC)
analysis
Tissue arrays containing 169 pairs of CRC tissues
and corresponding paracancerous tissues were prepared for IHC, and
the patients' information has been described in our previous study
(29). Tissues were formalin-fixed
at room temperature for 24 h, paraffin-embedded and cut into 4-µm
sections. Sections were permeabilized with 0.1% Triton X-100,
blocked with 5% BSA at room temperature for 1 h, and incubated with
an anti-ANXA3 antibody (cat. no. ab127924; Abcam; 1:250) at 4°C
overnight. HRP-conjugated goat anti-rabbit IgG (cat. no. ab205718;
Abcam; 1:500) was applied for 1 h at room temperature, followed by
DAB chromogen detection. Staining was visualized using a light
microscope. The staining of ANXA3 was scored using a
semi-quantitative scale, as follows: 0, negative; 1, weak; 2,
moderate; 3, strong. The scoring was performed in a blinded manner.
For survival analysis, the ANXA3 score was categorized as follows:
Scores of 0–1 indicated low ANXA3 expression, while scores of 2–3
indicated high ANXA3 expression.
Western blot analysis
Western blot analysis was carried out as described
previously (30). Briefly, the
total protein from cells or exosomes was extracted using RIPA lysis
buffer (Beyotime Biotechnology) supplemented with protease and
phosphatase inhibitors. The total protein concentration was
detected using the BCA Protein Assay Kit (cat. no. P0009; Beyotime
Biotechnology), and 30 µg protein per lane was separated by 10%
SDS-PAGE. Subsequently, the proteins were transferred onto
polyvinylidene fluoride membranes (MilliporeSigma), and membranes
were blocked with 5% (w/v) non-fat milk at room temperature for 2
h. After blocking, the membranes were incubated with the indicated
primary antibodies overnight at 4°C. The anti-ANXA3 antibody (cat.
no. ab127924; 1:1,000), anti-CD63 antibody (cat. no. ab68418;
1:500) and anti-CD81 antibody (cat. no. ab79559; 1:500) were
obtained from Abcam. The anti-Flag-tag antibody (cat. no. M185-3;
1:2,000) was purchased from Medical & Biological Laboratories
Co., Ltd. The anti-GAPDH antibody (cat. no. 97166; 1:1,000),
anti-phosphorylated (p)-NF-κB p65 (Ser536) antibody (cat. no. 3033;
1:1,000) and anti-NF-κB p65 antibody (cat. no. 8242; 1:1,000) were
purchased from Cell Signaling Technology, Inc. The anti-GSTP1
antibody (cat. no. F0781; 1:500) was obtained from Selleck
Chemicals. After washing with TBS-Tween (0.1% v/v), the membranes
were incubated with appropriate HRP-conjugated secondary antibodies
(cat. no. A0208 or A0216; Beyotime Biotechnology; 1:500) for 1 h at
room temperature. Finally, the immunoreactive bands were visualized
using an ECL substrate (Beyotime Biotechnology) and captured with a
chemiluminescence imaging system (Tanon Science and Technology Co.,
Ltd.). Semi-quantitative analysis of western blot band intensities
was performed using ImageJ software (version 2.9.0, National
Institutes of Health).
Measurement of cell viability
Cell viability was detected using the Cell Counting
Kit-8 (CCK-8) assay (cat. no. C0042; Beyotime Biotechnology) as
described previously (31,32). Briefly, CRC cells (5,000 cells/well)
were seeded in 96-well plates and allowed to adhere overnight. For
the cisplatin sensitivity assay, various CRC cell lines,
lentivirus-infected cells, or siRNA-transfected cells were
incubated with increasing concentrations of cisplatin (0, 10, 20,
40, 80 and 160 µM) for 24 h at 37°C. For combination treatment, the
cells were pretreated with 10 µM K201 or vehicle (0.1% DMSO in cell
culture medium) for 1 h at 37°C, followed by co-exposure to
increasing concentrations of cisplatin (0, 10, 20, 40, 80 and 160
µM) for 24 h at 37°C in the continued presence of K201 or vehicle.
Subsequently, 10 µl CCK-8 reagent was added to each well and the
cells were cultured for a further 1 h at 37°C. After incubation
with CCK-8 reagent, OD values were measured. Wells were assigned
randomly and the investigator performing the OD measurement was
blinded to the treatment groups. Experiments were repeated
independently three times.
Plasmid construction, lentivirus
preparation and small interfering RNA (siRNA) transfection
The human full-length ANXA3 cDNA sequence was
amplified by PCR and cloned into the lentivirus vector PLVX
(Addgene, Inc.) with a Flag tag (PLVX-Flag-ANXA3). An empty PLVX
vector (PLVX-NC) was used as the negative control. The
ANXA3-overexpressing plasmids and packaging plasmids were
co-transfected into 293T cells to produce the lentivirus and the
viral particles were prepared as described previously (33). In detail, lentiviral particles were
produced by using a second-generation lentiviral packaging system
in the present study. When 293T cells reached 70–80% confluence,
they were co-transfected with the lentiviral expression plasmid,
psPAX2 and pMD2.G at a mass ratio of 4:3:1 by using Lipofectamine
3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the
manufacturer's instruction. The total amount of plasmid DNA used
for each 10-cm dish was 20 µg. The culture supernatant containing
lentiviral particles was collected at 48 and 72 h after
transfection. LoVo and RKO cells were infected with the indicated
lentiviral particles at a multiplicity of infection of 20 and 30,
respectively, in the presence of 5 µg/ml polybrene (cat. no. H9268;
Sigma-Aldrich; Merck KGaA). Following 48 h infection, stable cells
were selected using 2 µg/ml puromycin (cat. no. P9620;
Sigma-Aldrich; Merck KGaA) for 7 days and maintained in medium
containing 1 µg/ml puromycin.
The siRNA against ANXA3 (siANXA3), the siRNA against
GSTP1 (siGSTP1) or the negative control siRNA (siNC) were
transfected into CRC cells at a final concentration of 10 nM using
Lipofectamine® RNAiMAX (Invitrogen; Thermo Fisher
Scientific, Inc.) according to the manufacturer's instructions.
Transfections were carried out at 37°C in a humidified incubator
with 5% CO2, and the transfection medium was replaced
with complete culture medium after 6 h. These cells were prepared
for subsequent experiments 24 or 48 h post-transfection. The
sequences were as follows: siANXA3 sense strand,
5′-GGAAUAUCAAGCAGCAUAUdTdT-3′; antisense strand,
5′-AUAUGCUGCUUGAUAUUCCdTdT-3′; siGSTP1 sense strand,
5′-CAUCAAUGGCAACGGGAAAdTdT-3′; antisense strand,
5′-UUUCCCGUUGCCAUUGAUGdTdT-3′; and siNC sense strand,
5′-UUCUCCGAACGUGUCACGUdTdT-3′; antisense strand,
5′-ACGUGACACGUUCGGAGAAdTdT-3′ (all synthesized by Shanghai
GenePharma Co., Ltd.).
NF-κB inhibitor treatment
For pharmacological inhibition of NF-κB signaling,
LoVo cells were seeded in 6-well plates at 5×105
cells/well and allowed to adhere overnight at 37°C with 5%
CO2. And then, cells were incubated with 5 µM NF-κB
inhibitor JSH-23 (Selleck Chemicals) or vehicle control (DMSO) for
48 h at 37°C with 5% CO2, followed by RNA and protein
extraction for further analysis.
Isolation of exosomes
Exosomes were isolated as described previously
(34). Briefly, LoVo cells infected
with PLVX-NC or PLVX-Flag-ANXA3-derived lentiviruses were incubated
for 72 h at 37°C. Cell culture supernatants were then collected and
subjected to sequential centrifugation: 300 × g for 10 min at 4°C
to remove cells, 2,000 × g for 20 min at 4°C to remove debris and
10,000 × g for 30 min at 4°C to remove large vesicles. Exosomes
were then isolated from the resulting supernatant using the
Hieff® Quick Exosome Isolation Kit Plus (cat. no.
41205ES10; Shanghai Yeasen Biotechnology Co., Ltd.) according to
the manufacturer's instructions. The total protein concentration of
the isolated exosomes was detected using BCA Protein Assay Kit
according to the manufacturer's protocol. The expression levels of
exosomal biomarkers CD63 and CD81 were then detected by western
blot analysis as aforementioned. For exosome treatment, LoVo cells
were incubated with 50 µg/ml OE-ANXA3-exosomes or NC-exosomes at
37°C with 5% CO2 for 24 h.
Luciferase assay
The GSTP1 luciferase reporter gene construct (−336
to +74) was generated as reported previously (35). For the luciferase assay, LoVo cells
(5×104 cells/well) were seeded in 24-well plates and
cultured overnight. The cells were then co-transfected with 400 ng
GSTP1 luciferase reporter plasmid (constructed by inserting GSTP1
promoter into PGL4 vector, and 40 ng pRL-TK Renilla internal
control vector (Promega Corporation) using Lipofectamine 3,000
according to the manufacturer's instructions. After 24 h, the cells
were infected with the PLVX-Flag-ANXA3-derived lentiviruses for 48
h or incubated with the ANXA3-overexpressing exosomes for 24 h,
followed by luciferase assay using the Dual-Luciferase Reporter
Assay System (Promega Corporation) according to the manufacturer's
instructions. Firefly luciferase activity was normalized to the
corresponding Renilla luciferase activity for each well. All
transfections and treatments were performed in three independent
replicates.
Virtual screening
Virtual screening was performed using molecular
docking as described previously (36). ANXA3 structure (UniProt ID: P12429;
http://www.uniprot.org/uniprotkb/P12429/entry) was
obtained from AlphaFold database (version 2.3.2; http://alphafold.ebi.ac.uk/entry/P12429)
and the scoring of the molecular docking was completed using the
Schrödinger Suite (HTVS/SP/XP modes, Schrödinger LLC), followed by
Sybyl clustering conducted in Sybyl-X 2.0 (Certara L.P.). Finally,
the candidate compound K201 was screened out based on the scores
and PubMed literature search (https://pubmed.ncbi.nlm.nih.gov). The docking score of
K201 was −5.378 kcal/mol and Molecular Mechanics/Generalized Born
Surface Area of K201 was −54.204 kcal/mol.
Statistical analysis
In vitro experiments were independently
repeated at least three times unless otherwise indicated. The
software GraphPad Prism 5 (Dotmatics) was used to generate figures
and perform statistical analysis. Continuous data are presented as
the mean ± SD, and were compared between two independent groups
using unpaired Student's t-test. When data were paired or matched,
paired Student's t-test was used. Categorical data are presented as
median with interquartile range (IQR); the Wilcoxon signed-rank
test was applied for non-continuous data (such as IHC scores).
One-way or two-way ANOVA was used to compare three or more groups,
followed by Tukey's post hoc test for multiple comparisons. The
Kaplan-Meier method and log-rank test were used for survival
analysis. For IHC score distributions (scores 0, 1, 2 and 3),
Bowker's test was used to compare CRC and NT groups, and Fisher's
exact test was used for comparisons across clinical stages I–IV.
P<0.05 was considered to indicate a statistically significant
difference.
Results
Elevated ANXA3 is a negative index for
patients with CRC
In our previous study, ANXA3 was identified as being
upregulated in both primary and liver metastatic CRC (37). To further validate this finding, the
publicly available cancer data from the GEPIA database were
analyzed, which integrates TCGA normal and GTEx datasets, and the
results revealed significantly upregulated mRNA expression levels
of ANXA3 in COAD and READ tissues (Fig.
1A). Concurrently, data from the UALCAN database (matched with
CPTAC samples) revealed that the protein levels of ANXA3 were
elevated in the primary tumor tissues of colon cancer (Fig. 1B). To validate these findings, a
tissue array was prepared and subsequently evaluated by IHC
staining. These results confirmed that ANXA3 was significantly
upregulated in CRC tumor tissues (Fig.
1C and 1D; Table SII).
Furthermore, Kaplan-Meier survival analysis revealed that high
expression of ANXA3 was associated with poor prognosis in patients
with CRC (Fig. 1E). Taken together,
these preliminary data indicated that ANXA3 was upregulated in CRC
tumors and elevated ANXA3 was associated with a poor prognosis for
patients with CRC.
 | Figure 1.Elevated ANXA3 is a negative index
for patients with CRC. (A) Online public tumor database Gene
Expression Profiling Interactive Analysis was used to analyze the
mRNA expression of ANXA3 in COAD and READ. Data were analyzed using
unpaired Student's t-test and are presented as the mean ± SD. (B)
Online public tumor database UALCAN was used to analyze the protein
expression of ANXA3 in colon cancer. Data were analyzed using
unpaired Student's t-test and are presented as the mean ± SD. IHC
staining was performed to detect the expression of ANXA3 in a
tissue microarray containing 169 pairs of CRC cancerous tissues and
corresponding paracancerous tissues, and the (C) representative
images and (D) statistical analysis are shown. ‘-’ represents a
score of 0, ‘+’ represents a score of 1, ‘++’ represents a score of
2, ‘+++’ represents a score of 3. Data are presented as box plots
showing the median and interquartile range, and statistical
analysis was performed using the Wilcoxon signed-rank test. (E)
Overall survival of patients with CRC with low or high ANXA3
(n=169) was analyzed by Kaplan-Meier curve analysis. *P<0.05,
****P<0.0001. ANXA3, annexin A3; T, tumor tissues; N, normal
tissues; COAD, colon adenocarcinoma; CRC, colorectal cancer; IHC,
immunohistochemistry; READ, rectum adenocarcinoma. |
ANXA3 mediates cisplatin sensitivity
and regulates GSTP1 expression in CRC cells
First, the expression levels of ANXA3 in five CRC
cell lines were detected, including HCT116, LoVo, RKO, SW480 and
SW620. As shown in Fig. 2A, ANXA3
was markedly downregulated in LoVo and RKO cell lines compared with
in the other cell lines. Subsequently, these five CRC cell lines
were incubated with cisplatin and CCK-8 assays were performed to
assess drug sensitivity. The results showed that CRC cell lines
with high ANXA3 expression exhibited reduced sensitivity to
cisplatin compared with those with low ANXA3 (Fig. 2B). To further validate this
relationship, ANXA3 was overexpressed by lentiviral plasmid
transduction in LoVo and RKO cells, which exhibited low endogenous
ANXA3 (Fig. 2C), and the results
showed that overexpression of ANXA3 significantly reduced the
sensitivity of cisplatin in CRC cells (Fig. 2D and E). By contrast, when ANXA3 was
silenced in HCT116 and SW480 cells with high endogenous ANXA3
(Fig. 2F and G), these CRC cells
became more sensitive to cisplatin (Fig. 2H and I). Taken together, the
aforementioned results suggested that ANXA3 mediates cisplatin
sensitivity in CRC cells.
 | Figure 2.Elevated ANXA3 reduces the
sensitivity of cisplatin in CRC cells. (A) CRC cell lines,
including HCT116, LoVo, RKO, SW480 and SW620, were lysed for
western blot analysis to detect the protein levels of ANXA3. (B)
CRC cell lines were incubated with increasing concentrations of
cisplatin for 24 h, followed by CCK-8 assay. Data are presented as
the mean ± SD and P-values were calculated via two-way ANOVA.
****P<0.0001. (C) Western blotting was used to detect the
effectiveness of the PLVX-derived ANXA3-overexpressing lentivirus
system in LoVo and RKO cells. (D) LoVo and (E) RKO cells infected
with PLVX-NC or PLVX-Flag-ANXA3-derived lentiviruses were incubated
with increasing concentrations of cisplatin for 24 h, followed by
CCK-8 assay. Data are presented as the mean ± SD and P-values were
calculated via two-way ANOVA. *P<0.05, **P<0.01,
****P<0.0001 vs. PLVX-NC group at that concentration. (F) HCT116
and (G) SW480 cells were transfected with siNC or siANXA3 for 24 h,
followed by reverse transcription-quantitative PCR analyses to
assess the mRNA levels of ANXA3. GAPDH was used as an internal
control. Data were analyzed using unpaired Student's t-test and are
presented as the mean ± SD. ***P<0.001. (H) HCT116 and (I) SW480
cells transfected with siNC or siANXA3 were incubated with
increasing concentrations of cisplatin for 24 h, followed by CCK-8
assay. Data are presented as the mean ± SD and P-values were
calculated via two-way ANOVA. **P<0.01, ***P<0.001,
****P<0.0001 vs. siNC at that concentration. All experiments
were repeated independently three times. ANXA3, annexin A3; CCK-8,
Cell Counting Kit-8; CRC, colorectal cancer; NC, negative control;
si, small interfering. |
Next, the public tumor database GEPIA was analyzed
and it was found that ANXA3 expression in CRC was positively
correlated with the expression of several important genes
associated with chemoresistance, including ERCC1 (r=0.24;
P<0.05), GSTP1 (r=0.45; P<0.05) and ATP7B (r=0.27; P<0.05)
(Fig. 3A). The expression levels of
these genes were also confirmed using RT-qPCR and GSTP1 was
revealed to be the most prominently upregulated following ANXA3
overexpression in CRC cells (Fig. 3B
and C). Western blot analysis further revealed that ANXA3
overexpression markedly increased GSTP1 protein levels in CRC cells
(Fig. 3D). Combined with the mRNA
levels, it was hypothesized that ANXA3 may regulate the
transcriptional expression of GSTP1. To assess this, a luciferase
reporter system driven by the GSTP1 promoter was used, and the
luciferase assay showed that ANXA3 significantly increased the
activity of the GSTP1 promoter (Fig.
3E). By contrast, silencing ANXA3 decreased GSTP1 expression
(Fig. 3F). The aforementioned
results indicate that ANXA3 promoted the transcriptional expression
of GSTP1 in CRC cells. Given that NF-κB has been reported to be an
important transcription factor mediating the expression of GSTP1
(38), whether ANXA3 exerted its
function by activating NF-κB was investigated. As shown in Fig. 3G, overexpression of ANXA3
upregulated the expression levels of p-NF-κB p65. Moreover, the
regulatory effect of ANXA3 on GSTP1 expression was also blocked by
the NF-κB inhibitor JSH-23 (Fig. 3H and
I), which indicated that ANXA3 regulated GSTP1 expression in
CRC cells in an NF-κB-dependent manner. In addition, as
demonstrated in Fig. S1, knockdown
of GSTP1 significantly suppressed ANXA3-mediated cisplatin
resistance, which further suggested that GSTP1 may serve as a key
downstream molecule through which ANXA3 exerts its effect on drug
sensitivity.
 | Figure 3.ANXA3 regulates GSTP1 expression in
colorectal cancer cells in an NF-κB-dependent manner. (A) ANXA3
gene co-expression analyses were conducted using the publicly
available cancer database Gene Expression Profiling Interactive
Analysis, including ERCC1, GSTP1 and ATP7B. LoVo cells were
infected with PLVX-NC or PLVX-Flag-ANXA3 lentiviruses, and cultured
for 48 h. (B) Then, western blotting was performed to assess the
expression of Flag-ANXA3. (C) RT-qPCR was performed to assess the
mRNA levels of ERCC1, GSTP1 and ATP7B. GAPDH was used as an
internal control. (D) Western blotting was performed to assess the
expression of GSTP1. (E) Luciferase assay was conducted to assess
the effects of ANXA3 on the activity of the GSTP1 promoter (−336 to
+74). (F) HCT116 cells were transfected with siNC or siANXA3 for 48
h, followed by western blotting to assess the protein levels of
GSTP1 and ANXA3. GAPDH was used as a loading control. (G) LoVo
cells were infected with PLVX-NC or PLVX-Flag-ANXA3 for 48 h,
followed by western blotting to assess the expression levels of
p-NF-κB p65, NF-κB p65 and GAPDH. LoVo cells were infected with
PLVX-Flag-ANXA3-derived lentivirus or incubated with 5 µM NF-κB
inhibitor JSH-23 for 48 h at 37°C, followed by (H) RT-qPCR analyses
to assess the mRNA levels of GSTP1 or (I) western blotting to
assess the protein expression of GSTP1. GAPDH was used as an
internal control. Data are presented as the mean ± SD and P-values
were calculated by unpaired Student's t-test or one-way ANOVA. All
experiments were repeated independently three times. *P<0.05,
***P<0.001, ****P<0.0001. ANXA3, annexin A3; ERCC1, excision
repair cross-complementing 1; GSTP1, glutathione S-transferase P1;
NC, negative control; p-, phosphorylated; RT-qPCR, reverse
transcription-quantitative PCR; si, small interfering. |
ANXA3-overexpressing exosomes
(OE-ANXA3-exosomes) upregulate GSTP1 expression and reduce
cisplatin sensitivity in CRC cells
For the present study, OE-ANXA3-exosomes were also
generated. As shown in Fig. 4A and
B, ANXA3-overexpressing CRC cells were successfully
established, and exosomes isolated from the culture supernatants
were subsequently characterized by western blot analysis, which
confirmed the expression of exosomal biomarkers CD63 and CD81.
Subsequently, CRC cells were incubated with OE-ANXA3-exosomes or
NC-exosomes, and the results indicated that OE-ANXA3-exosomes
significantly upregulated the transcriptional expression of GSTP1
(Fig. 4C and D). Additionally,
OE-ANXA3-exosomes significantly increased the luciferase activity
of the GSTP1 promoter (Fig. 4E).
Subsequently, CRC cells were incubated with OE-ANXA3-exosomes or
cisplatin, and the results indicated that OE-ANXA3-exosomes
significantly decreased the drug sensitivity of cisplatin in CRC
cells (Fig. 4F). Moreover, the
cisplatin-triggered downregulation of GSTP1 was partially reversed
by the treatment with OE-ANXA3-exosomes (Fig. 4G and H). Based on these findings, it
is speculated that ANXA3 may be involved in the process of
exosome-mediated cisplatin resistance in CRC cells through
regulating GSTP1 expression.
Combined K201 and cisplatin treatment
significantly inhibits CRC viability
Finally, molecular docking-based virtual screening
identified K201 as a potential compound interacting with ANXA3. As
shown in Fig. 5A-C, K201 formed π-π
interactions with His8 (HIE8) and Phe206, as well as a hydrogen
bond and a salt bridge with Asp283 of ANXA3. Subsequently, CRC
cells harboring different expression levels of ANXA3 were incubated
with K201, and the results showed that K201 exhibited stronger
cytotoxicity in HCT116 cells with high ANXA3 expression than in RKO
and LoVo cells with low ANXA3 expression (Fig. S2). CRC cells with high ANXA3
expression (including HCT116 and SW480) were then incubated with
cisplatin alone or in combination with K201, and K201 was shown to
significantly enhance the cytotoxicity of cisplatin in CRC cells
(Fig. 5D and E). Critically, the
enhanced cytotoxicity observed in the combination group (K201 +
cisplatin) was significantly greater than that of K201 alone,
arguing against a simple additive toxic effect and supporting a
specific sensitizing mechanism (Fig.
5F). Moreover, ectopic overexpression of ANXA3 partially
reversed the K201-mediated cisplatin sensitization in CRC cells
(Fig. 5F), which suggested that the
level of ectopically expressed ANXA3, while sufficient to
significantly attenuate K201 activity, may not fully saturate all
available targets in the presence of the drug. These results
collectively suggested that targeting ANXA3 activity with K201 may
enhance cisplatin efficacy in CRC cells.
Discussion
ANXA3 exhibits high structural homology with other
membrane-associated proteins, particularly ANXA5 (39). It has been reported that the mRNA
levels of ANXA5 are upregulated in tumor tissues of colorectal
adenocarcinoma, and increased ANXA5 expression is associated with
higher tumor stage and poor prognosis (40). This aligns with the broader
phenomena observed within the annexin family, where some members
are dysregulated in tumors. The present study showed that ANXA3 was
significantly upregulated in CRC primary tumors, and its high
expression was associated with shorter overall survival, which
reinforces the existing literature reporting ANXA3 upregulation
across various tumor types, such as breast cancer and pancreatic
cancer (41).
Beyond its expression patterns, the role of ANXA3 in
chemoresistance has emerged as another important function. For
example, cervical cancer cells have been shown to become sensitive
to cisplatin after ANXA3 is silenced, whereas the opposite effect
has been observed when ANXA3 is overexpressed in cervical cancer
cells (42). Similarly, in breast
cancer, ANXA3 knockdown enhances the sensitivity of cells to
doxorubicin (DOX) by promoting drug uptake, and the combination of
DOX treatment and ANXA3 downregulation can synergistically suppress
tumor growth and metastasis (43).
Moreover, ANXA3 has been shown to be upregulated in
oxaliplatin-resistant lung cancer cells, indicating that
downregulation of ANXA3 may overcome such resistance (44). Consistent with these reports across
different cancer types, the present study provided CRC-specific
insights: It was demonstrated that ANXA3 expression was associated
with cisplatin resistance, and ANXA3 overexpression promoted the
expression of GSTP1, a known drug-resistance gene in CRC, through
the NF-κB signaling pathway. Collectively, these findings position
ANXA3 within the specific pathway of cisplatin response in CRC
cells. While ANXA3 can regulate GSTP1 to modulate cisplatin
sensitivity, alternative mechanisms may also contribute to
cisplatin resistance in CRC cells. For example, ANXA3 might
modulate drug influx/efflux transporters, interfere with DNA damage
repair pathways, or regulate broader oxidative stress responses
beyond GSTP1-mediated conjugation (45–47).
Future studies are needed to investigate the potential relationship
between these pathways and the GSTP1-dependent mechanism proposed
in the present study.
It is well known that exosomes can induce cancer
cells to develop resistance to chemotherapeutic drugs, a topic that
has attracted extensive attention in recent years. It has been
reported that exosomes enriched with ANXA3 can effectively abolish
ferroptosis in laryngeal squamous cell carcinoma (LSCC) cells and
induce lymphatic metastasis, thereby promoting the progression of
LSCC (48). The data in the present
study are consistent with and extend this phenomenon, demonstrating
that OE-ANXA3-exosomes could markedly decrease the sensitivity of
CRC cells to cisplatin by regulating the transcriptional expression
of GSTP1. This indicates a potential common mechanism where
ANXA3-rich exosomes mediate drug sensitivity across different tumor
types, with ANXA3 regulating GSTP1 as an important pathway worthy
of further investigation. In addition to the aforementioned
mechanism, recent studies have found other mechanisms underlying
CRC chemoresistance. For example, GIPC1 has been reported to
suppress CRC chemoresistance by regulating the TTC7B/mTOR/NF-κB
axis (49). ARL4C has also been
reported to promote oxaliplatin resistance in CRC by activating the
RAP1/PI3K-Akt-mTOR signaling axis and inhibiting its own
ubiquitination to enhance protein stability (50). Whether ANXA3 also affects these
newly discovered chemoresistance mechanisms remains to be explored
in future work.
Additionally, to overcome the insensitivity of cells
to cisplatin caused by ANXA3 overexpression, the compound K201 was
identified through virtual screening. The present study revealed
that K201 significantly reduced cisplatin resistance in
ANXA3-elevated cells, and overexpression of ANXA3 partially
reversed the K201-mediated cisplatin sensitization. While the
results from the molecular docking and functional assays indicated
that K201 interacts with ANXA3, direct binding evidence (for
example, cellular thermal shift assay or drug affinity responsive
target stability assay) is currently lacking. This will be further
investigated in future work.
Despite the findings of the present study, several
limitations should be acknowledged. First, the findings are limited
to in vitro experiments and the therapeutic relevance of the
ANXA3-GSTP1 axis and K201 remains unvalidated in vivo.
Second, elucidating the specific cell death mechanisms regulated by
ANXA3 represents another important direction for future research
and we plan to address this in subsequent studies. Third, the
characterization of exosomes in the current study remains
preliminary and a more comprehensive isolation and validation
protocol is needed. Finally, the survival analysis is limited by
insufficient clinical adjustment, such as patient comorbidities and
treatment regimens. Collectively, these limitations underscore the
need for further validation in future investigations.
In conclusion, the present study suggested that
ANXA3 may mediate cisplatin sensitivity in CRC cells via regulation
of GSTP1. These in vitro findings indicate that targeting
ANXA3 could be a potential strategy for overcoming chemoresistance
in CRC. However, these results remain preliminary and require
further validation in more complex models and clinical studies to
establish their therapeutic relevance.
Supplementary Material
Supporting Data
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the Fundamental Research
Funds for the Central Universities (grant no. 22120240280), the
Open Project of the Key Laboratory of Embedded System and Service
Computing (Ministry of Education, Tongji University; grant no.
ESSCKF 2024-08), and the High Quality Innovation Platform of
Science and Education Innovation Zone in Suzhou Industrial Park-Key
Platform Project (grant no. YZCXPT2023104).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
YY and XJ designed the study and supervised the
research process. XC, XX, XY, KH, HW, SZ, CM and RH performed the
experiments, and collected and analyzed the data. XC and XX
generated the figures. XX and YY wrote the original draft. YY and
XJ confirmed the authenticity of all the raw data. All authors read
and approved the final version of the manuscript.
Ethics approval and consent to
participate
The present study was approved by the Ethics
Committee of Shanghai East Hospital (approval no. 2024-220). All
patients provided written informed consent.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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