Abstract

Purpose

   Ursolic Acid (UA), a triterpenoid extracted from Hedyotis Diffusa
   Willd. (HDW), is known for its anti-inflammatory, antioxidant, and
   antitumor effects. Nevertheless, the mechanisms underlying UA’s
   anti-colorectal cancer (CRC) effects remain insufficiently understood.
   This study aimed to identify the key target proteins of UA and
   investigate their functions in CRC development.

Methods

   The cytotoxicity of three active components of HDW (UA, oleanolic acid
   (OA), and quercetin) on CRC cells was evaluated using the CCK-8 assay.
   Tandem mass tag (TMT)-based proteomics was employed to detect
   differentially expressed proteins (DEPs) in CRC cells after UA
   treatment. Bioinformatics analysis and high-content screening were used
   to identify UA’s key protein targets. The expression and role of RPLP1
   in CRC cells were investigated, including the effects of RPLP1
   knockdown and its combination with UA treatment on cell proliferation,
   migration, invasion, and apoptosis.

Results

   UA demonstrated superior inhibitory effects on CRC cells compared to OA
   and quercetin, highlighting it as a principal active ingredient of HDW.
   TMT-based proteomic analysis identified 438 upregulated and 366
   downregulated proteins after UA intervention. Among these, RPLP1 was
   identified as a critical target. UA inhibited RPLP1 expression in CRC
   cells, resulting in decreased cell proliferation, migration, and
   invasion. Furthermore, the combination of UA treatment and RPLP1
   knockdown exhibited synergistic effects in inhibiting CRC cell growth
   and migration, as well as promoting apoptosis.

Conclusions

   UA, a bioactive triterpenoid of HDW, inhibits CRC development by
   targeting and suppressing RPLP1 expression. These findings provide
   novel insights into the therapeutic of UA for CRC and highlight RPLP1
   as a promising target for intervention.

Supplementary Information

   The online version contains supplementary material available at
   10.1007/s12672-025-03486-z.

   Keywords: Colorectal cancer, Hedyotis diffusa willd, Ursolic acid,
   RPLP1, Tandem mass tags

Introduction

   As reported by recent studies, colorectal cancer (CRC) is the second
   leading cause of cancer-related mortality worldwide, accounting for a
   significant global health burden [[30]1–[31]3]. The disease often
   begins insidiously and may progress rapidly in advanced stages.
   Patients with advanced CRC frequently experience complications such as
   intestinal obstruction, systemic symptoms including weight loss and
   anemia, and metastases to critical organs such as liver, lungs, lymph
   nodes, and bones, which eventually lead to death [[32]4]. Notably, up
   to 90% of CRC-related fatalities are attributed to distant invasion and
   metastasis, highlighting the aggressive nature of advanced disease
   [[33]5]. Despite the availability of treatment such as surgical
   resection, chemoradiotherapy, and molecular-targeted therapy, these
   approaches are far from ideal [[34]6]. They are often accompanied by
   adverse effects, drug resistance, restricted survival benefits, and
   high financial costs, which together underscore the inadequacy of
   current clinical management strategies [[35]7]. Furthermore, once
   patients develop resistance to prescribed therapies, few alternative
   options remain, creating a critical gap in CRC treatment protocols.
   Given these challenges, there is an urgent need to develop novel
   therapeutic strategies that are not only effective but also demonstrate
   minimal toxicity and cost-effectiveness, providing hope for improved
   outcomes in CRC management.

   Growing evidence has demonstrated that traditional Chinese medicine
   (TCM), a valuable resource for anti-CRC medications, has been utilized
   for the prevention and treatment of colorectal cancer (CRC) for
   centuries [[36]8]. TCM offers a unique therapeutic approach by
   targeting multiple pathways and factors involved in CRC progression,
   while simultaneously alleviating the adverse effects associated with
   conventional treatments such as radiotherapy, chemotherapy, and
   targeted therapy, thereby prolonging patient survival [[37]9, [38]10].
   Among the many TCM-based treatments, Hedyotis Diffusa Willd. (HDW),
   stands out as a widely used Chinese herbal medicine in anticancer
   formulations, effectively combating various human malignancies
   [[39]11]. HDW contains three primary active ingredients-ursolic acid
   (UA), oleanolic acid (OA), and quercetin-which exhibit diverse
   pharmacological properties, including antioxidant, anti-inflammatory,
   and immunomodulatory effects [[40]12]. Notably, mounting studies have
   highlighted the significant inhibitory effects of HDW on CRC
   development [[41]13–[42]15]. Among these active ingredients, UA is a
   bioactive triterpenoid with broad antitumor potential. It can be
   isolated from multiple plants species, including Oldenlandia diffusa,
   Rosmarinus officinalis, and Glechoma hederaceae [[43]16]. UA has been
   shown to effectively prevent various types of malignancies, including
   CRC [[44]17, [45]18]. Moreover, UA has the potential to enhance the
   antitumor efficacy of oxaliplatin in CRC treatment [[46]19]. Although
   these findings are promising, the precise molecular mechanisms and
   targets underlying UA’s anti-CRC effects remain insufficiently
   understood. Further research is needed to unravel the mechanistic basis
   of UA’s activity against CRC, paving the way for its potential clinical
   application.

   Quantitative proteomic analysis has emerged as a powerful tool for
   elucidating the pharmacological effects, identifying protein targets,
   and uncovering the mechanisms of action of traditional Chinese medicine
   (TCM) remedies in cancer treatment [[47]20]. With advancements in
   proteomics, particularly the use of stable isotope labeling and tandem
   mass tag (TMT)-based techniques, it is now possible to detect and
   analyze changes in thousands of proteins within minimal sample amounts,
   offering high sensitivity and accuracy [[48]21].

   In this study, we applied TMT-based quantitative proteomics to
   investigate the differential protein expression profiles following
   ursolic acid (UA) intervention in colorectal cancer (CRC) cells.
   Bioinformatics analysis and high-content screening identified the 60 S
   acidic ribosomal protein P1 (RPLP1) as a key target of UA. Subsequent
   functional assays further evaluated the role of RPLP1 in CRC, examining
   both its individual functions and the combined effects of RPLP1
   knockdown and UA treatment. By integrating proteomics with molecular
   biology techniques, this study provides a comprehensive understanding
   of the mechanisms underlying UA’s anti-CRC effects. These findings, in
   conjunction with previous research, offer valuable insights into the
   therapeutic potential of UA and lay a foundation for further
   exploration of its molecular targets.

Materials and methods

Cell culture and treatment

   Human CRC cell lines SW480 and HCT116 (Cell Bank, Chinese Academy of
   Sciences) were cultured in DMEM (Shanghai Yuanpei Biotechnology Co.,
   LTD, China) and L15 media supplemented with 10% fetal bovine serum
   (FBS, Thermo Fisher, USA). The cells were maintained at 37 °C and in a
   5% CO2 atmosphere.

Cytotoxicity assay in vitro

   To investigate the cytotoxicity of the three active ingredients (UA,
   OA, and quercetin) of HDW, SW480 cells were exposed to varying
   concentrations (0, 0.1, 1, 2, 5, 10, 20, 40, and 70 µM) of UA (Shanghai
   Taosu Biochemical Technology Co., Ltd., China), quercetin (Sigma, USA),
   and OA (Shanghai Yuanye Biotechnology Co., Ltd., China). The
   suppressive effect on proliferation of SW480 cells by these three
   active ingredients was assessed using CCK-8 assay, and their IC50
   values were determined.

CCK-8 assay

   Logarithmically growing SW480 and HCT116 cells (5 × 10^4 cells/mL) were
   seeded into each well of a 96-well plate and cultured overnight in an
   incubator. After treatments with different concentrations of UA,
   quercetin, and OA, cells were allowed to incubate for 72 h. Then, 10 µL
   CCK-8 reagent was introduced into each well for 3 h. Absorbance was
   recorded at 450 nm with a microplate reader (Model 680; Bio-199 Rad,
   Hercules, CA, USA).

TMT-based proteomics analysis

   When SW480 cells treated with UA and DMSO (negative control) reached a
   concentration of 4 × 10^3 cells/mL, they were lysed with RIPA buffer to
   extract total protein. After determining the protein concentration and
   purity, 200 µg protein extract was subjected to Filter-Aided Sample
   Preparation (FASP) digestion. UA-treated and DMSO-treated (negative
   control) SW480 cells were lysed with RIPA buffer to extract total
   protein. After determining the protein concentration and purity, 200 µg
   protein extract was subjected to Filter-Aided Sample Preparation (FASP)
   digestion. The solutions were collected and lyophilized. TMT labeling
   was then conducted utilizing a TMT labeling kit (Thermo Fisher
   Scientific). Subsequently, high-performance liquid chromatography
   fractionation (HPLC) and liquid chromatography–mass spectrometry/mass
   spectrometry (LC–MS/MS) analysis were performed. Each experimental
   sample underwent separation using an Easy nLC system at nanoflow rates.
   After equilibrating the column with 100% buffer A (0.1% formic acid
   aqueous solution), samples were introduced into an analytical column
   (Thermo Fisher Scientific) for separation at a flow rate of 300 nL/min.
   Following chromatographic separation, analysis was conducted using a Q
   Exactive Plus mass spectrometer for 90 min, operating in positive ion
   mode with a mass scan range of 350–1800 m/z. The first-level mass
   resolution was 70,000, with an AGC target of 3e6 and a first-level
   maximum IT of 50 ms. Peptide and peptide fragment mass-to-charge ratios
   were determined through a series of ten MS2 scans following each full
   scan. Using the HCD method, the MS2 activation was conducted, with the
   isolation window set to 2 m/z. The second-level mass resolution was
   35,000 with 1 microscan, a maximum injection time of 45 ms, and a
   normalized collision energy of 30 eV.TMT-based proteomics was repeated
   four times.

Identification of deps and functional enrichment analysis

   Protein identification and quantification were executed using Mascot2.6
   and Proteome Discoverer 2.1 (Thermo Fisher Scientific), based on the
   UniProt human database (Uniprot_HomoSapiens_20386_20180905). DEPs were
   identified by Student’s t-test with a threshold value of |fold change
   (FC)| >1.2 and P < 0.05. Subsequently, functional enrichment analysis
   for DEPs was conducted, and significantly enriched GO terms and KEGG
   pathways were selected using Fisher’s Exact Test.

High-content screening

   To screen for key DEPs in CRC, high-content screening was employed to
   evaluate the impact of 9 DEPs on HCT116 cell proliferation. Briefly,
   HCT116 cells were collected after being infected with shRNAs (shPC
   (Myc, positive control), shCtrl (negative control), shSTK26, shPKN1,
   shZC3H8, shRPLP1, shDDX20, shFOXK2, shFXR1, shPCGF2, and shTRIM24) for
   72 h. See Supplementary Table [49]1 for detailed target information.
   Cells (1,000 cells/well) were plated into 96-well plates and
   photographed daily for 5 days utilizing a Celigo image cytometer
   (Nexcelom Bioscience, Lawrence, MA, USA). On the 5th day, the
   fold-change (FC) value of the cell count in the shCtrl group (negative
   control group) was compared with that in the sh-target gene groups.
   When FC ≥ 2, cell proliferation in the sh-target gene group exhibited
   significant inhibition in comparison to the shCtrl group, and the
   target gene was considered a proliferation-related positive regulator.

Plasmids construction and transfection

   To evaluate the functional roles of RPLP1 in CRC, shRNAs targeting
   RPLP1 (shRNA-57068, shRNA-57069, and shRNA-57070) were designed and
   synthesized for RPLP1 knockdown. An shRNA non-target sequence was
   utilized as the negative control (NC). Using Lipofectamine 2000
   (Invitrogen Life Technologies, USA), 293T packaging cells were
   transfected with lentiviruses coexpressing the shRNA sequences psPAX2
   (Addgene, USA), and pMD2.G (Addgene). Subsequently, the cells were
   harvested 48 h post-transfection for further experiments.

Quantitative real-time PCR

   Total RNA was isolated from SW480 and HCT116 cells using TRIzol reagent
   (Invitrogen Life Technologies, USA). Reverse transcription into cDNA
   was conducted using iScript cDNA synthesis kit (Bio-Rad, USA). qRT-PCR
   was employed to detect target gene expression using SYBR Green qPCR
   Master Mix (Servicebio). GAPDH was used as the internal control.
   Relative quantification was performed using the 2^−ΔΔCT method.

Colony formation assay

   Logarithmic growth-phased SW480 and HCT116 cells (1000 cells/well) were
   inoculated into a 6-well plate. After incubation for 2–3 weeks, the
   formed cell clones were immobilized with 4% paraformaldehyde and
   stained with Giemsa (DingGuo Biotechnology) for 10–30 min. The number
   of clones was counted under a microscope.

Edu assay

   BeyoClick^TM null EdU-488 proliferation assay (Beyotime) was conducted
   to detect cell proliferation. Briefly, cells (1 × 10^6 cells/mL) at
   logarithmic growth phase were seeded into a 6-well plate and incubated
   overnight. The cells were then incubated with EdU (10 µM) for 2 h.
   After being fixed and permeated, the cells were incubated with
   BeyoClick™ EdU-488. After washing thrice, the cell nuclei were labeled
   with Hoechst 33342 (RiboBio, Shanghai, China) in the dark for 10 min.
   Treated cells were observed under a fluorescence microscope (CKX41;
   Olympus, Tokyo, Japan).

Wound healing assay for assessing cell migration

   SW480 cells (2 × 10^5 cells/well) at logarithmic growth phase were
   plated into 6-well plates. Upon reaching 80 to 90% confluence, a
   scratch was created in the center of the cell monolayer using a 100 µL
   pipette tip, which was aligned vertically to the well’s base to ensure
   a straight scratch. After washing with PBS to remove debris three
   times, complete culture medium was added, and cells were incubated in a
   5% CO[2] incubator at 37 °C. Wound healing along the scratch was
   observed at 0 and 24 h.

Transwell assay for detecting cell invasion and migration

   Cells (5 × 10^4 cells/mL) were starved under serum-free conditions for
   12 h, harvested, and suspended in serum-free culture medium. The
   transwell chambers were coated with Matrigel. Then, cell suspension
   (200 µL) and culture medium supplemented with 10% serum (500 µL) were
   introduced into the upper and lower chamber, respectively. After 72 h,
   the chambers underwent fixation with 4% paraformaldehyde for 30 min.
   After washing, chambers were stained with 0.1% crystal violet for
   5 min. Cell invasion to the lower chamber was observed using a
   fluorescence microscope (CKX41).

   For quantitative analysis, images were captured at 1 and 24 h
   post-scratch for each well, selecting five representative fields per
   well. The number of invaded cells was quantified using GraphPad Prism
   7.0 software. The relative invasion was calculated as the number of
   cells that migrated through the Matrigel-coated membrane divided by the
   total number of cells seeded in the upper chamber. The migration rate
   was calculated as the number of cells that migrated to the lower
   surface of the membrane divided by the total number of cells seeded in
   the upper chamber.​.

Apoptosis analysis

   Logarithmically growing cells (1 × 10^6 cells/well) were trypsinized,
   suspended in complete medium, and plated into a 6-well plate. After
   different treatments, cell apoptosis was detected using an Annexin
   V-FITC kit (Biosea Biotechnology Co., Beijing, China). Briefly, cells
   (5 × 10^5 cells/mL) were resuspended in 1 × binding buffer and
   incubated with Annexin V-FITC (5 µL) and propidium iodide (PI, 10 µL)
   in the dark for 15 min. A flow cytometer (FACSCalibur,
   Becton-Dickinson, San Jose, CA, USA) was used to detect cell apoptosis,
   and CellQuest software (Becton, Franklin Lakes, NJ, USA) was used for
   data analysis.

Statistical analysis

   All experiments were repeated thrice. All data were depicted as
   mean ± standard deviation (SD). Statistical analysis was completed
   using SPSS 17.0 software, with statistical significance defined as
   P < 0.05.

Results

Identification of active ingredients of HDW

   To screen the active ingredients of HDW, the cytotoxicity of three
   active ingredients of HDW was evaluated. The results revealed that UA
   (Fig. [50]1A) and quercetin (Fig. [51]1B) showed strong inhibitory
   effects on SW480 cell proliferation, whereas OA (Fig. [52]1C) had no
   obvious inhibitory effect on SW480 cells. Among the three ingredients,
   UA exhibited the lowest IC50 value and was thus regarded as the active
   ingredient for subsequent experiments.

Fig. 1.

   [53]Fig. 1
   [54]Open in a new tab

   CCK8 assay evaluated the inhibitory effects of three effective
   ingredients of HDW on CRC cells. A: UA. B: Oleanolic acid. C:
   Quercetin. UA: ursolic acid

DEPs screening and functional enrichment analysis

   TMT labeling-based proteomic analysis was conducted on SW480 cells
   treated with 12 µM UA and DMSO for 72 h, respectively. In this
   analysis, the labeling efficiency was 99.4%. The quality control
   results showed that the quality deviation of ≥ 90% peptide segments was
   small, indicating that the data were reliable. A total of 804 DEPs (438
   upregulated and 366 downregulated) were screened in UA-treated SW480
   and control cells. A volcano plot of the DEPs is shown in Fig. [55]2A.
   Moreover, the heatmap showed that UA-treated SW480 cells and control
   cells could be distinguished based on the expression of DEPs
   (Fig. [56]2B). RPLP1 and STK26 exhibited marked differential expression
   in UA-treated SW480 cells compared to control cells (Supplementary
   Table [57]1). Furthermore, GO enrichment analysis of the DEPs was
   performed (Fig. [58]2C). With respect to biological process (BP), DEPs
   were mainly enriched in the negative regulation of endopeptidase
   activity. Regarding molecular function (MF), DEPs were markedly
   involved in heparin binding. With respect to cellular components (CC),
   DEPs were primarily localized in the integral component of the plasma
   membrane. Moreover, these DEPs were implicated in KEGG pathways such as
   complement and coagulation cascades, cell adhesion molecules, and
   steroid biosynthesis (Fig. [59]2D).

Fig. 2.

   [60]Fig. 2
   [61]Open in a new tab

   Identification of DEPs after UA treatment based on TMT-based proteomic
   analysis and functional enrichment analysis. A: Volcano plot of DEPs.
   The horizontal axis is the fold change of protein expression after log2
   conversion, and the vertical axis is the P value of significant
   difference test after -log10 conversion. B: Heatmap showed the
   expression of DEPs in all samples. A1, A2, A3, A4: negative control
   group (SW480 + DMSO); B1, B2, B3, B4: UA group (SW480 + UA). C: Gene
   Ontology annotation results. D: KEGG pathway enrichment results. DEPs:
   differentially expressed proteins; UA: ursolic acid; TMT: tandem mass
   tag

RPLP1 was identified as a gene related to CRC cell proliferation

   High-content screening with a Celigo image cytometer was used to record
   HCT116 cell proliferation for 5 days following treatment with shRNAs
   targeting key DEPs (Supplementary Fig. 1). Based on FC on day 5, HCT116
   cells treated with shSTK26 and shRPLP1 showed significant proliferation
   inhibition (Fig. [62]3). The high-content screening results showed that
   after HCT116 cells were treated with shRNA targeting RPLP1, their
   proliferation was significantly inhibited. RPLP1 was identified as a
   key gene related to CRC cell proliferation.

Fig. 3.

   [63]Fig. 3
   [64]Open in a new tab

   High-content screening showed HCT116 cell proliferation for 5 days
   after treating with shSTK26 and shRPLP1. ShPC was the positive control
   and shCtrl was the negative control

UA inhibited RPLP1 expression in CRC cells

   The effect of UA on RPLP1 mRNA expression was detected by qPCR.
   Different concentrations (10 and 20 µM) of UA significantly inhibited
   RPLP1 mRNA expression in CRC cells (Fig. [65]4).

Fig. 4.

   [66]Fig. 4
   [67]Open in a new tab

   qPCR showed RPLP1 mRNA expression in HCT116 and SW480 cells after
   treatment with different concentrations (10 and 20 µM) of UA. **
   P < 0.01 and *** P < 0.001 compared to control group. UA: ursolic acid

RPLP1 knockdown inhibited the malignant behaviors of CRC cells

   To elucidate the role of RPLP1 in CRC, RPLP1 expression was suppressed
   in HCT116 and SW480 cells by stable transfection with shRPLP1, which
   included three specific sequences: shRNA-57,068, shRNA-57,069, and
   shRNA-57,070. In both cell lines, only shRNA-57,068 transfection
   significantly inhibited RPLP1 expression compared to sh-NC (P < 0.05,
   Fig. [68]5A). The impact of shRPLP1 transfection on CRC cell viability
   was examined using CCK-8 assays. The results revealed a notable
   reduction in cell viability of HCT116 and SW480 cells following
   transfection with shRNA-57068 and shRNA-57069 (P < 0.001), with the
   suppressive effect of shRNA-57068 being the strongest (Fig. [69]5B).
   Consequently, shRNA-57068 was selected for subsequent experiments to
   knock down RPLP1 expression in HCT116 and SW480 cells. Colony formation
   (Fig. [70]5C) and EdU (Fig. [71]5D) assays showed a significant
   reduction in the colony-forming ability and proliferative rate of both
   cell types upon RPLP1 knockdown (P < 0.001, Fig. [72]5C-D). Moreover,
   the transwell assay showed that RPLP1 knockdown dramatically suppressed
   the invasion of both cell lines (P < 0.001, Fig. [73]5E). The wound
   healing assay demonstrated a remarkable decrease in the migration of
   HCT116 and SW480 cells (P < 0.001, Fig. [74]5F). Collectively, RPLP1
   knockdown attenuated the malignant behaviors of CRC cells.

Fig. 5.

   [75]Fig. 5
   [76]Open in a new tab

   RPLP1 knockdown inhibited the malignant behaviors of CRC cells. A: qPCR
   showed RPLP1 mRNA expression in HCT116 and SW480 cells after
   transfection of shRPLP1 (shRNA-57068, shRNA-57069, and shRNA-57070). B:
   CCK-8 assays revealed the viability of HCT116 and SW480 cells after
   transfection of shRPLP1 (shRNA-57068, shRNA-57069, and shRNA-57070). C:
   Colony formation assay showed the effects of RPLP1 knockdown on
   colony-forming abilities of HCT116 and SW480 cells. D: EdU assay
   demonstrated the effects of RPLP1 knockdown on the proliferation of
   HCT116 and SW480 cells. E: Transwell assay showed the effects of RPLP1
   knockdown on HCT116 and SW480 cell invasion. F: Wound healing assay
   indicated the effects of RPLP1 knockdown on HCT116 and SW480 cell
   migration. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to NC
   group. UA: ursolic acid; NC: negative control

RPLP1 knockdown and UA synergistically suppressed the malignant behaviors of
CRC cells

   We investigated the combined effects of RPLP1 knockdown and UA
   treatment on HCT116 and SW480 cells. The CCK-8 assay results revealed
   that both RPLP1 knockdown and UA individually reduced the viability of
   both cell lines (P < 0.001, Fig. [77]6A). In addition, the combination
   of RPLP1 knockdown and UA treatment showed stronger inhibition than
   single treatment alone in both cell lines (P < 0.001, Fig. [78]6A).
   Moreover, both colony formation (Fig. [79]6B) and EdU (Fig. [80]6C)
   assays showed that RPLP1 knockdown and UA synergistically decreased the
   colony-forming ability and proliferation of both cell lines
   (P < 0.001), corroborating the results of the CCK-8 assay. Moreover,
   the wound-healing assay revealed that the combination of RPLP1
   knockdown and UA dramatically inhibited cell migration (P < 0.001).
   Compared with the UA group, the combined treatment exhibited a more
   pronounced impact in suppressing the migration of both cell types
   (P < 0.001, Fig. [81]6D). Furthermore, RPLP1 knockdown, UA treatment,
   and their combination significantly promoted apoptosis in both cell
   lines (P < 0.001, Fig. [82]6E). The combined treatment resulted in
   significantly higher levels of apoptosis than UA treatment alone
   (P < 0.001, Fig. [83]6E). Taken together, RPLP1 knockdown and UA
   treatment demonstrated synergistic effects on the inhibition of CRC
   cell proliferation and migration and promoting apoptosis.

Fig. 6.

   Fig. 6
   [84]Open in a new tab

   RPLP1 knockdown and UA synergistically inhibited the malignant
   behaviors of CRC cells. HCT116 and SW480 cells were treated with
   shRPLP1, UA and their combination. A: CCK-8 assays revealed the
   viability of HCT116 and SW480 cells after different treatments. B:
   Colony formation assay showed the colony-forming abilities of HCT116
   and SW480 cells after different treatments. C: EdU assay demonstrated
   the proliferation of HCT116 and SW480 cells after different treatments.
   D: Wound healing assay displayed the migratory activities of HCT116 and
   SW480 cells after different treatments. E: Flow cytometry analysis
   detected apoptosis of HCT116 and SW480 cells after different
   treatments. *** P < 0.001 compared to NC group. # P < 0.05 and ##
   P < 0.01 compared to UA groups. UA: ursolic acid; NC: negative control

Discussion

   In the present study, we identified UA, the major active ingredient of
   Polygonum cuspidatum, as having significant inhibitory effects on the
   malignant behaviors of CRC cells. UA markedly suppressed CRC cell
   proliferation, migration, and invasion, while also inducing apoptosis.
   To explore the active components, we screened three major ingredients
   of Polygonum cuspidatum (UA, quercetin, and OA) and found that UA
   exhibited the lowest IC50 value in SW480 cells, indicating the
   strongest anti-proliferative effect. Consequently, UA was selected for
   further investigation. Subsequently, we employed TMT-based quantitative
   proteomic analysis to investigate global protein expression changes and
   uncover the potential mechanisms of UA treatment. A total of 804
   differentially expressed proteins (DEPs) were identified, comprising
   438 upregulated and 366 downregulated proteins. These DEPs were
   enriched in biological processes such as the negative regulation of
   endopeptidase activity and molecular functions like heparin binding,
   and were localized within cellular structures such as the integral
   component of the plasma membrane. Pathway enrichment analysis revealed
   significant associations with KEGG pathways, including complement and
   coagulation cascades, cell adhesion molecules, and steroid
   biosynthesis. Mechanistically, high-content screening identified RPLP1,
   a ribosomal protein-related gene, as a key factor closely associated
   with CRC cell proliferation. qPCR analysis demonstrated that UA
   treatment significantly downregulated the expression of RPLP1 mRNA in
   CRC cells. Functional assays further confirmed that shRNA-mediated
   knockdown of RPLP1 suppressed CRC cell proliferation, colony formation,
   migration, and invasion, while promoting apoptosis. Importantly, the
   combination of UA treatment and RPLP1 knockdown exhibited synergistic
   effects on CRC cells. Compared with either treatment alone, the
   combined approach significantly enhanced the inhibition of cell
   proliferation and migration and further promoted apoptosis. These
   findings highlight the potential of RPLP1 as a therapeutic target for
   UA in the treatment of CRC.

   HDW is a renowned heat-clearing and detoxifying herb frequently
   utilized in TCM formulations, with its inclusion reported in up to 5.1%
   of anticancer TCM prescriptions [[85]22]. Among its bioactive
   components, UA has demonstrated anti-inflammatory, antioxidant, and
   antitumor properties [[86]23]. Previous studies have shown that UA
   possesses a wide range of biological functions, including improving
   hepatic lipid deposition and inhibiting liver fibrosis [[87]24].
   scavenging free radicals and exhibiting potent anti-inflammatory and
   antiviral effects [[88]25, [89]26]; protecting lung tissues from heat
   stress-induced lung injury [[90]27]; enhancing glucose tolerance and
   preventing hyperlipidemia [[91]28]; anti-depressant effects [[92]29],
   inhibiting angiogenesis [[93]30], and antitumor effects [[94]31,
   [95]32]. The findings of this study further strengthen the growing body
   of evidence supporting the therapeutic potential of UA in colorectal
   cancer (CRC) treatment. Additionally, UA exerts its anti-CRC effects
   through various mechanisms. For instance, Lu et al. indicated that UA
   suppresses CRC cell growth by regulating SNTB1 to modulate the
   PKN2/Akt/ERK pathway [[96]33]. Zhang et al. demonstrated that UA
   attenuates the invasive potential of CRC cells via modulating the
   TGF‑β1/ZEB1/miR‑200c pathway [[97]34]. Chen et al. revealed that UA
   alleviates CRC by engaging the non-canonical Hedgehog pathway in a
   smoothened-independent fashion, which is contingent upon AKT signaling
   activation [[98]35]. Several studies have confirmed that UA exerts the
   inhibitory effect on CRC via modulation of multiple signaling pathways
   [[99]18, [100]36].

   Proteomics analysis provides a comprehensive overview of protein
   profile changes and is widely used in the investigation of molecular
   mechanisms, biomarkers, and therapeutic targets across various
   diseases. In this study, TMT-based proteomics was employed to explore
   the potential mechanisms of UA in CRC. A total of 804 DEPs were
   identified, among which RPLP1 was recognized as a pivotal target
   protein of UA. These findings suggest that DEPs regulated by UA are
   primarily enriched in the regulation of extracellular matrix functions
   and specific metabolic pathways. For instance, cell adhesion molecules,
   closely associated with CRC progression [[101]37], may be regulated by
   UA. Cell adhesion serves as a determinant of metastasis, often leading
   to tumor cell arrest and extravasation [[102]37]. Moreover, cell
   adhesion is influenced by heparin binding and integrin function
   inhibition [[103]38], as highlighted by the GO analysis showing common
   DEPs involved in heparin-binding processes. Additionally, steroid
   hormone biosynthesis can inhibit MAPK signaling pathways, reducing the
   expression and activity of endopeptidases [[104]39, [105]40]. GO
   analysis of common DEPs further revealed their involvement in processes
   impacting the integral components of the plasma membrane. An increasing
   body of evidence suggests that integral components of the plasma
   membrane, including receptor proteins, ion channels, and transport
   proteins, are not only structural constituents but also play crucial
   functional roles in CRC progression. These membrane components
   participate in pharmacological nutrient absorption and tumor
   progression by regulating extracellular signal transduction, metabolite
   transmembrane transport, and interactions with the tumor
   microenvironment. For example, receptor proteins such as EGFR and TGF-β
   receptors mediate growth factor signaling pathways, promoting CRC cell
   proliferation and invasion [[106]41, [107]42]. Ion channels, such as
   calcium channels, are critical for maintaining intracellular ion
   homeostasis and signal transduction [[108]43]. Transport proteins,
   including GLUT1 and amino acid transporters from the SLC family,
   facilitate the uptake of essential metabolites required for rapid
   cancer cell proliferation [[109]44, [110]45]. Therefore, proteomics
   analysis serves as a powerful tool for evaluating the mechanisms of UA
   in CRC.

   In various colorectal cancer (CRC) studies, apoptosis is closely linked
   to cancer cell death [[111]46]. Apoptosis removes damaged cells through
   morphological and biochemical changes, and its occurrence in CRC cells
   is associated with tumor progression, metastasis, and chemotherapy
   resistance [[112]47]. RPLP1, a ribosomal protein, is involved in
   protein synthesis and apoptosis. Changes in RPLP1 expression can affect
   apoptosis-related pathways and regulate cell survival [[113]48]. RPLP1
   is upregulated in various cancers [[114]49–[115]51], and its knockdown
   reduces cell proliferation and migration [[116]52]. In our study, UA
   intervention inhibited RPLP1 expression in CRC cells, and RPLP1
   knockdown suppressed malignant behaviors. These results suggest that
   RPLP1 may be a key regulator in CRC progression and a crucial target of
   UA in CRC.

Conclusion

   The strength of this study is the identification of RPLP1 as a protein
   target of UA against CRC using a TMT-based proteomic approach and
   experimental validation. However, this study has some limitations.
   Firstly, RPLP1 protein expression was not verified in clinical CRC
   samples. Secondly, the potential mechanisms by which RPLP1 regulates
   CRC development were not investigated. More studies are needed to
   confirm these results. In conclusion, our results indicate that UA, a
   bioactive triterpenoid in HDW, inhibits CRC development by inhibiting
   RPLP1 expression. Our findings provide new perspectives for identifying
   the protein targets of UA for CRC treatment.

   Author contributions.

   Li-Min Zhu, Zhen-Ye Xu carried out the conception and design of the
   research, Li-Min Zhu, Hai-Xia Shi participated in the acquisition of
   data. Li-Min Zhu, Hai-Bin Deng carried out the analysis and
   interpretation of data. Li-Min Zhu, Hai-Xia Shi participated in the
   design of the study and performed the statistical analysis. Li-Min Zhu
   participated in obtaining funding. Li-Min Zhu conceived of the study,
   and participated in its design and coordination and drafted the
   manuscript and revision of manuscript for important intellectual
   content. All authors read and approved the final manuscript.

Supplementary Information

   [117]Supplementary Material 1.^ (23KB, xls)
   [118]Supplementary Material 2.^ (33.6KB, xls)
   [119]Supplementary Material 3.^ (113.5KB, xlsx)
   [120]Supplementary Material 4^ (116.5KB, png)

Acknowledgements