Exploration of the mechanism of curcumin in the regulation of apoptosis for the treatment of colorectal cancer

Abstract

Background

The incidence of colorectal cancer (CRC) is steadily increasing, and its standard treatment regimen improves the survival rate of tumor patients, but metastatic CRC is the main cause of death in CRC patients. As a low-toxicity natural compound, curcumin, a traditional Chinese medicine, can effectively inhibit the growth of tumor cells by mediating various biological processes. This study aimed to investigate the molecular mechanism underlying curcumin in the treatment of CRC using a combination of network pharmacology analysis and experimental validation.

Methods

The GeneCards database was used to identify potential targets associated with CRC and apoptosis. Target concentrations for curcumin and apoptosis were identified from the Search Tool for Interacting Chemicals (STITCH) and GeneCards databases, respectively. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using the ‘clusterprofile’ package in R software. Furthermore, to examine the impact of curcumin on the viability and apoptosis of colon cancer cell lines, Cell Counting Kit-8 (CCK-8) assays and flow cytometry analyses were performed. Lastly, Western blot analysis was conducted to validate curcumin’s effects on proapoptotic protein.

Results

A total of 25 essential genes were identified for protein-protein interaction (PPI) network construction and enrichment analysis. The results of the CCK-8 assay indicated that curcumin exerted inhibitory effects on in vitro proliferation. Moreover, the results of flow cytometry demonstrated that curcumin triggered apoptosis in SW480 cells and HCT116 cells. Finally, western blot analysis revealed that curcumin down-regulated the expression of MDM2 and COX-2.

Conclusions

This study suggests a possible therapeutic approach for CRC by modulating key genes associated with apoptosis, such as MDM2 and COX-2, offering a novel therapeutic strategy for CRC.

Highlight box.

Key findings

• Curcumin inhibits the progression of colorectal cancer (CRC) by promoting apoptosis in CRC cells.

• Curcumin down-regulates the expression of MDM-2 and COX2 and concurrently promotes apoptosis in CRC cells.

• In this study, the vital role of multiple targets in the treatment of CRC was elucidated through network pharmacology and bioinformatics analysis.

What is known and what is new?

• Curcumin exerts therapeutic effects in the treatment of CRC.

• Curcumin can promote apoptosis and down-regulate key protein targets, thereby exerting therapeutic effects in CRC.

What is the implication, and what should change now?

• The potential mechanisms of curcumin for these targets may be an important direction for future research.

Introduction

According to the International Agency for Research on Cancer (1), colorectal cancer (CRC) ranks as the third most prevalent cancer and the second leading cause of cancer-related mortality. Despite advances in endoscopic technology, which have increased the detection rate of precancerous lesions and early-stage CRC, some patients are diagnosed at advanced stages, whilst others experience tumor recurrence or metastasis postoperatively. For patients with advanced CRC, treatment typically involves a comprehensive strategy that combines chemotherapeutic agents and molecular targeted drugs (2). Meanwhile, in some cases, treatment aims to convert unresectable lesions into resectable ones, thus achieving cure or prolonging the survival time of patients. At present, the therapeutic outcomes for metastatic CRC remain limited, highlighting the urgent need for developing novel therapeutic agents. Traditional Chinese medicines (TCMs), which offer multi-component, multi-target benefits, are associated with low drug-resistance rates and have garnered extensive attention for the treatment of CRC. However, the mechanism of action underlying TCM in CRC is complex and elusive, warranting further in-depth studies to improve its anti-tumorigenic efficacy. Thus, the present study aimed to explore the role and mechanism of curcumin in the treatment of CRC.

Curcuma longa, also referred to as turmeric, is a popular TCM with outstanding therapeutic efficacy. As a crucial polyphenol isolated from turmeric rhizomes, it regulates multiple biological processes (3). Furthermore, curcumin exerts anti-oxidants, anti-inflammatory, anti-microbial (4), and anti-cancer effects (5,6). Notably, it can modulate the expression of anti-apoptotic genes, activate caspases, and upregulate the expression of the P53 protein. According to earlier studies, curcumin inhibits tumor invasion by modulating the activities of matrix metalloproteases (MMPs) and downregulating the expression of chemokines and growth factors such as HER-2 and epidermal growth factor receptor (EGFR) (7). Furthermore, it suppresses angiogenesis by inhibiting the activity of angiogenic cytokines such as IL-6, IL-23, and IL-1β (8). Curcumin has been shown to inhibit tumor proliferation by reducing the levels of pro-inflammatory molecules, including COX-2, lipoxygenase-2, inducible nitric oxide synthase (iNOS), and related cytokines (9). Lastly, it down-regulates the expression of serine/threonine-dependent cell cycle protein kinases (CDKs), thereby inhibiting cell cycle progression in tumor cells and suppressing their growth (10).

Over the past few decades, compelling evidence suggests that apoptosis serves as a natural barrier against tumorigenesis (11). Apoptosis is defined as a regulated form of programmed cell death (12). Curcumin has been established to promote apoptosis and inflammation-associated programmed cell death pathways. Caspases participate in the regulation of the apoptotic process (13). Specifically, caspase-3 catalyzes the cleavage of key cellular components, contributing to chromatin condensation and DNA fragmentation during apoptosis (14). In addition, caspases-8 and -9 can initiate a series of proteolytic events involving effector caspases, which leads to the progressive disassembly and phagocytic clearance of cancer cells. A previous study described that the activation of caspases plays a vital role in triggering apoptosis in kaempferol-induced CRC (13). Furthermore, the inactivation of caspase-8, -9, -7 and -3 leads to resistance to 5-fluorouracil (5-FU) in Caco-2 cells harboring mutant p53. The activation of caspases can induce apoptosis in CRC. In the present study, protein-protein interaction (PPI) analysis revealed that curcumin targets MDM2 and COX-2, highlighting their relevance in the treatment of CRC.

MDM2 acts as an E3 ubiquitin ligase and exerts oncogenic effects by primarily ubiquitinating p53 degradation (15). In CRC, MDM2 overexpression inactivates p53 and drives cell proliferation and resistance to apoptosis (16). In certain types of CRCs harboring p53 mutations, MDM2 may act through non-classical pathways (modulation of the NF-κB or Rb pathway). WDR43 has been reported to enhance the MDM2-mediated ubiquitination of p53 by binding to RPL11, thereby decreasing the stability of the p53 protein and ultimately promoting proliferation and chemoresistance in CRC cells (17). The combination of chidamide and oxaliplatin modulates the RPS27A-MDM2-P53 axis in CRC, thereby inhibiting tumor progression (18). COX-2 mediates the synthesis of prostaglandins (PGE2) and promotes inflammation, angiogenesis, and cell survival. It is overexpressed in cancerous tissues and is correlated with CRC progression and poor prognosis (19). In contrast, COX-2 inhibitors, such as aspirin, reduce the risk of CRC. The COX-2 inhibitor celecoxib inhibits tumor progression by altering the expression of MDM2 and COX-2 in A549 cells. Nevertheless, this process also confers drug resistance (20). Therefore, there is a pressing need to explore the role of MDM2 and COX-2 in CRC.

This study utilized network pharmacology and experimental validation to comprehensively investigate the mechanism by which curcumin regulates apoptosis in the treatment of CRC (Figure 1). The results initially revealed that curcumin regulates apoptosis by binding to MDM2 and COX-2, providing valuable insights into CRC therapy. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-359/rc).

Figure 1.

Flow chart illustrating the survey strategies used in this study. CRC, colorectal cancer; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction.

Methods

Potential targets of curcumin

The Search Tool for Interacting Chemicals (STITCH) database, a database commonly used to identify potential interactions between drugs and proteins, was used to identify proteins that interact with curcumin (http://stitch.embl.de/). A high confidence cut-off value of 0.700 was set, and the homo sapiens species was selected.

Investigating CRC and apoptosis-associated proteins

The GeneCards database was used to identify proteins related to CRC and apoptosis.

Identification of crucial targets and construction of PPI network

Key proteins strongly correlated with curcumin, CRC, and apoptosis were identified using the Venn diagram (21) following which a PPI network was constructed using the STRING database (https://string-db.org/) (22).

Pathway enrichment analysis

A total of 25 proteins identified through the PPI network were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in R software.

Cell culture

Human CRC cells SW480 and HCT116 were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium (Gibco, Thermo Fisher Scientific, Inc., Grand Island, NY, USA) and McCoy’s5A (Wuhan Pricella Biotechnology Co., Ltd., Wuhan, China) medium, respectively. Both media were supplemented with 10% fetal bovine serum (FBS) (Sangon Biotech, Shanghai, China) and 1% penicillin-streptomycin (Cytiva, Shanghai, China). ATCC-derived human cell lines were used in this study. Given that the content and purpose of the study were within the authorization granted by the provider, the Ethics Review Committee of Dalian University Affiliated Xinhua Hospital waived the requirement for ethical approval for this study.

Cell viability assays

Curcumin was procured from RHAWN. Briefly, 0.36838 g of curcumin powder was dissolved in 10 mL of dimethyl sulfoxide (DMSO) and filtered through a 0.22-µm filter to prepare a reserve solution (0.1 mol/L) with a final concentration of DMSO <0.1%. Curcumin stocks were diluted to different concentrations in DMEM (or McCoy’s5A) medium supplemented with 10% FBS for the treatment of CRC cells. SW480 and HCT116 cells were seeded into a 96-well plate at a density of 4×10³ and 5×10³ cells/well, respectively, and incubated for 24 h at 37 ℃ in an incubator under a 5% CO₂ atmosphere. Subsequently, CRC cells were treated with varying concentrations of curcumin for the pre-defined duration. Next, 10 µL of Cell Counting Kit-8 (CCK-8) cell proliferation assay reagent (KeyGEN BioTECH, Nanjing, China) was introduced into each well to assess cell viability. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Statistical analysis was carried out, and a growth curve was plotted. The cell viability ratio was calculated as follows: cell viability ratio (%) = A450 (study group)/A450 (control group) ×100%. The half maximal inhibitory concentration (IC50) value is the curcumin concentration at which cell growth inhibition is 50%. Two concentrations before and after IC50 were selected for subsequent experiments. SW480 and HCT116 were seeded into 96-well plates with 4×10³ and 5×10³ cells per well, respectively, and 10 µL of CCK-8 reagent was added to detect the absorbance of different concentrations of curcumin action at different time points (0, 12, 24, 36 and 48 h).

Flow cytometry analysis

SW480 cells treated with 20 and 40 µmol/L curcumin, as well as HCT116 cells exposed to 12.5 and 25 µmol/L curcumin for 24 h, were collected using ethylene diamine tetraacetic acid (EDTA)-free trypsin (KeyGEN BioTECH, Nanjing, China). After centrifugation with phosphate buffered saline (PBS), the cell pellet was incubated for 30 min with 2.5 µL propidium iodide (PI) and stained with annexin V fluorescein isothiocyanate (annexin V-FITC). The stained cells were analyzed using a flow cytometer (BECKMAN, Brea, CA, USA), and data were processed using FlowJo software.

Western blotting

The expression levels of COX-2 and MDM2 in CRC cells were evaluated by treating cells with curcumin for 24 hours, the curcumin concentration corresponding to a statistically significant total apoptosis rate was chosen. SW480 and HCT116 cells were lysed using RIPA lysis buffer (strong) containing protease inhibitors, phosphatase inhibitors, and phenylmethanesulfonyl fluoride (PMSF) on ice (4 ℃). After centrifugation, the supernatant was collected, and protein concentration was quantified using a BCA kit (KeyGEN BioTECH). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% skimmed milk for 2 h. Next, the membranes were incubated overnight with primary antibodies, comprising MDM2 (SC965, dilution 1:800, Santa Cruz, Dallas, TX, USA), COX-2 (SC19999, dilution 1:100, Santa Cruz), and GAPDH (ET1601-4, dilution 1:5,000, HUABIO, Hangzhou, China), at 4 ℃. Thereafter, the membranes were incubated with secondary antibodies for 2h. The following horseradish peroxidase (HRP)-conjugated secondary antibodies were used in this study: goat anti-mouse immunoglobulin G (IgG) (#31430, dilution 1:5,000, Thermo Fisher Scientific) and goat anti-rabbit IgG (#31460, dilution 1:5,000, Thermo Fisher Scientific). The gel was developed using an ECL luminescent solution (Biodragon, Suzhou, China) in a chemiluminescence gel imager (Jena, Germany), and the gray values of the bands were quantified using ImageJ software.

Statistical analysis

The statistical analysis of data was conducted using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). The results of both measurement and ranked data in this study were presented as mean ± standard deviation. Student’s t-test was employed for comparing two groups, while one-way analysis of variance (ANOVA) and Bonferroni post hoc tests were utilized for evaluating differences among three or more groups. Statistical significance was defined as P<0.05.

Results

Potential targets associated with curcumin, apoptosis and CRC

Curcumin screening through the STITCH database identified 60 protein targets with high confidence (0.7) for the ensuing analysis. Apoptosis-associated and CRC-associated genes were screened using the GeneCards database.

Identification of critical genes and construction of a PPI network

A Venn diagram was plotted to identify critical genes (Figure 2). The results revealed 25 crucial genes, namely TP53, ATM, CHEK2, PTEN, EGFR, CDKN2A, RB1, AKT1, CCND1, CDKN1B, MTOR, PPARG, STAT3, CDKN1A, SRC, MDM2, PTGS2, BCL2, CDK2, CASP3, GSK3B, MAPK8, XIAP, SIRT1 and FOXO3. We summarized the role of 25 genes in apoptosis and CRC (Table 1). Afterward, these genes were used to construct a PPI network (Figure 3).

Figure 2.

Venn diagram. CRC, colorectal cancer.

Table 1. 25 key genes in apoptosis and colorectal cancer.

Gene	Effect on apoptosis in CRC cells	Mutations/abnormalities in colorectal cancer
TP53	Tumor suppressor protein that activates pro-apoptotic genes (e.g., BAX, PUMA) in response to DNA damage, inducing apoptosis	High-frequency mutations (loss of function), leading to apoptosis inhibition and tumor progression
ATM	DNA double-strand break repair key kinase, activates downstream CHEK2 and TP53, triggers apoptosis or repair	Mutations or reduced expression leads to defective DNA repair and genomic instability that may impair apoptotic signaling
CHEK2	Activation by ATM phosphorylates TP53, enhancing its stability and promoting cell cycle arrest or apoptosis	Mutations lead to inactivation of the TP53 pathway and apoptosis is blocked
PTEN	Antagonizes the PI3K/AKT pathway, inhibits the pro-survival signaling of AKT, and promotes the activation of pro-apoptotic proteins (e.g., BAD)	Deletion or mutation leads to overactivation of the PI3K/AKT pathway and inhibits apoptosis
EGFR	Activates downstream RAS/MAPK and PI3K/AKT pathways to promote cell proliferation and inhibit apoptosis	Overexpression or sustained activation results in inhibition of apoptosis, tumor growth and metastasis
CDKN2A	Encodes p16INK4a (inhibits CDK4/6, blocks cell cycle) and p14ARF (stabilizes TP53, pro-apoptotic) proteins	High-frequency methylation or deletion, resulting in uncontrolled cell cycle and impaired TP53 function
RB1	Regulates G1/S phase transition and binds E2F to inhibit proliferation; releases pro-apoptotic factors upon inactivation	Mutations are rare, but pathway inactivation (e.g., CDKN2A deletion, CCND1 overexpression) is common
AKT1	Core kinase of the PI3K/AKT/mTOR pathway that phosphorylates pro-apoptotic proteins (e.g., BAD, Caspase-9) and inhibits their activity to promote cell survival	Amplification or activation mutations (e.g., PIK3CA mutation, PTEN deletion) lead to AKT overactivation
CCND1	Cell cycle protein D1, binds to CDK4/6 to drive the G1/S phase transition; overexpression accelerates proliferation and may inhibit pro-apoptotic signaling	Amplification or overexpression results in cell cycle dysregulation and is associated with poor prognosis
CDKN1B	Encodes a p27 protein that inhibits the CDK2/4 complex and blocks the cell cycle; low expression leads to accelerated proliferation and reduced apoptosis	Low expression or accelerated degradation (e.g., SKP2 overexpression) is associated with an aggressive phenotype
MTOR	PI3K/AKT/mTOR signaling pathway core kinase that promotes cell growth and metabolism and inhibits autophagy and apoptosis	Activating mutations or abnormal upstream signaling (PI3K/AKT) lead to overactivation
PPARG	Nuclear receptor transcription factor that regulates lipid metabolism and cell differentiation; activation may induce differentiation or pro-apoptosis	Down-regulated expression or loss of function results in uncontrolled proliferation and inhibition of apoptosis
STAT3	Transcription factor that promotes expression of anti-apoptotic genes (BCL2, MCL1) and inhibits pro-apoptotic signaling when activated by IL-6/JAK	Persistent phosphorylation activation, associated with inflammatory microenvironment and chemoresistance
CDKN1A	Cell cycle inhibitor, regulated by TP53; inhibits proliferation at high expression, but may inhibit apoptosis	Dysfunctional when TP53 is mutated, or aberrantly expressed independently of TP53
SRC	Non-receptor tyrosine kinase that activates proliferation/survival pathways (e.g., MAPK, FAK) and inhibits pro-apoptotic signaling	Overexpressed or aberrantly activated, promotes invasive metastasis and apoptosis resistance
MDM2	E3 ubiquitin ligase that degrades TP53; inhibits TP53-mediated apoptosis when overexpressed	Amplification or overexpression, leading to TP53 inactivation and genomic instability
PTGS2	Prostaglandin synthase, promotes production of inflammatory mediators (e.g. PGE2) and activates pro-survival pathways such as PI3K/AKT	Overexpression is associated with inflammation-associated carcinogenesis and chemoresistance
BCL2	Anti-apoptotic protein that inhibits the mitochondrial apoptotic pathway (blocks BAX/BAK activation and cytochrome C release)	Overexpression results in blockage of the mitochondrial apoptotic pathway and is associated with poor prognosis
CDK2	Cell cycle-dependent kinase that drives G1/S and S-phase processes; inhibits apoptotic signaling associated with cell cycle arrest	Overexpression or activating mutations lead to uncontrolled proliferation and inhibition of apoptosis
CASP3	Apoptosis executes cysteine asparaginase, which cleaves downstream substrates (e.g. PARP) to trigger programmed cell death	Activity is inhibited (e.g., XIAP overexpression), resulting in blocked apoptotic execution
GSK3B	Glycogen synthase kinase 3β, involved in the Wnt/β-catenin, PI3K/AKT pathway; phosphorylates pro-apoptotic proteins and is highly dependent on the signaling environment for function	Activity is often inhibited (e.g., AKT hyperactivation), leading to accumulation of pro-survival proteins
MAPK8	Stress-activated kinase that phosphorylates pro-apoptotic proteins (e.g. c-JUN, BCL2 family members) in response to DNA damage or oxidative stress	Continued activation may promote survival, or inhibition of activity may lead to attenuated apoptotic signaling
XIAP	X-linked apoptosis inhibitory protein that directly binds and inhibits caspase-3/7/9 activity, blocking apoptosis execution	Overexpression leads to apoptosis resistance and is associated with chemo-/radiotherapy tolerance
SIRT1	NAD+-dependent deacetylase that regulates stress response and metabolism; deacetylates pro-apoptotic proteins (e.g., p53, FOXO3) with a dual function	Overexpression inhibits p53-dependent apoptosis but may activate FOXO3-dependent pro-apoptotic pathways
FOXO3	Transcription factor that activates pro-apoptotic genes (e.g., BIM, PUMA); regulated by the PI3K/AKT pathway	Often inactivated by AKT overactivation, with reduced intranuclear localization and decreased expression of pro-apoptotic genes

CRC, colorectal cancer.

Figure 3.

PPI network for crucial proteins. PPI, protein-protein interaction.

GO and KEGG enrichment analysis

The 25 crucial genes were subjected to enrichment analysis. The three most enriched biological processes identified by GO analysis were regulation of G1/S transition of mitotic cell cycle, regulation of cell cycle G1/S phase transition, and G1/S transition of mitotic cell cycle (Figure 4A). KEGG metabolic pathway enrichment analysis uncovered the three most enriched metabolic pathways to be the prostate cancer pathway, the endocrine resistance pathway, and the cellular senescence pathway (Figure 4B).

Figure 4.

GO and KEGG analysis of the potential therapeutic targets. (A) GO enrichment analysis of potential targets for the treatment of CRC by curcumin; (B) KEGG enrichment analysis of potential targets for CRC treatment using curcumin. BP, biological process; CC, cellular component; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; PML, promyelocytic leukemia nuclear.

Curcumin exerts an anti-proliferative effects on CRC cells

Treatment of SW480 cells (10, 20, 30, 40, 60 and 80 µmol/L) and HCT116 cells (5, 10, 15, 20, 25 and 30 µmol/L) were treated with concentration-graded curcumin for 24 h, and the results indicated that the proliferative rate of CRC cells in the curcumin group was significantly lower compared to the control group. More importantly, curcumin inhibited the cell proliferation in a concentration-dependent manner. The IC50 values were 32.15 µmol/L for SW480 cells and 14.90 µmol/L for HCT116 cells (Figure 5A,5B).

Figure 5.

Curcumin has anti-tumor proliferative activity on colorectal cancer cells in vitro. (A,B) SW480 and HCT116 cell activity after 24 h of curcumin incubation at different concentrations, in a concentration-dependent manner; one-way ANOVA. (C,D) Effect of curcumin at different concentrations at different time points of 0, 12, 24, 36, and 48 h on SW480 and HCT116 cell activity, in a time-dependent manner. Values are mean ± standard deviation of at least three independent determinations; n=6, one-way ANOVA, *, P<0.05; **, P<0.01, compared with the control group. ANOVA, analysis of variance; Con, control; Cur[12.5], curcumin 12.5 µmol/L; Cur[20], curcumin 20 µmol/L; Cur[25], curcumin 25 µmol/L; Cur[40], curcumin 40 µmol/L.

The viability of CRC cells treated with different concentrations of curcumin at different time points (0, 12, 24, 36 and 48 h) was examined. As anticipated, the results demonstrated that cell viability decreased with the prolonged exposure to curcumin (Figure 5C,5D) and that curcumin inhibited tumor cell proliferation in a time-dependent manner.

Curcumin promotes human colon cancer cells apoptosis

To determine whether curcumin inhibits CRC cells by promoting apoptosis, flow cytometry was employed to assess apoptosis in CRC cells following treatment with varying concentrations of curcumin for 24 h. Interestingly, the total apoptosis rate of CRC cells was significantly higher in the curcumin-treated groups compared with the control group, and the pro-apoptotic effects of curcumin were concentration-dependent (P<0.05) (Figure 6).

Figure 6.

Analysis of apoptosis by annexin V-FITC/PI. (A,B) SW480 cells after different concentrations of curcumin were stained with PI, and apoptosis was detected by flow cytometry and statistically analyzed. (C,D) HCT116 cells after different concentrations of curcumin were stained with PI, and apoptosis was detected by flow cytometry and statistically analyzed. Apoptosis rate was calculated as the total cells with early (red) and late (blue) apoptosis on the right side. Values are mean ± standard deviation of three independent determinations; n=3, one-way ANOVA. ns, P>0.05; **, P<0.01, compared with the control group. ANOVA, analysis of variance; Con, control; Cur[12.5], curcumin 12.5 µmol/L; Cur[20], curcumin 20 µmol/L; Cur[25], curcumin 25 µmol/L; Cur[40], curcumin 40 µmol/L; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PI, propidium iodide.

Curcumin down-regulates MDM2 and COX-2 protein expression

Furthermore, Western blot analysis was conducted to explore the effects of curcumin on CRC cells. Through network pharmacological analysis, two target proteins, MDM2 and COX-2, were selected. The results indicated that the expression levels of MDM2 and COX-2 significantly decreased with increasing curcumin concentrations in both SW480 and HCT116 CRC cells (Figure 7) (P<0.05). Therefore, we speculate that the down-regulation of MDM2 and COX-2 might inhibit CRC progression.

Figure 7.

Curcumin down-regulates tumor cell action targets to exert anti-tumor effects. (A) SW480 cells and HCT116 cells were treated with 40 and 25 µmol/L curcumin for 24 h, respectively. The expression of cellular COX-2 and MDM2 was detected by protein blotting, and GAPDH was used as a control. (B) Statistical analysis of COX-2 and MDM2 expression in SW480 cells. (C) Statistical analysis of COX-2 and MDM2 expression in HCT116 cells. Values are mean ± standard deviation from three independent determinations; n=3, Student’s t-test. *, P<0.05; **, P<0.01, compared with the control group. Con, Control; Cur[25], curcumin 25 µmol/L; Cur[40], curcumin 40 µmol/L.

Discussion

As is well documented, the incidence of CRC is steadily rising, presenting a significant threat to human health (23). Although standard treatment protocols for CRC have improved the survival rate of tumor patients, approximately 20% of CRC patients are diagnosed at stage IV, which is the chief cause of death in CRC patients (24). In recent years, the TCM curcumin has gained widespread attention owing to its remarkable anticancer potential. As a low-toxicity natural compound, it can effectively inhibit the growth of tumor cells by mediating various biological processes. Therefore, further investigation into the effects of curcumin on the malignant biological behavior of CRC cells and its underlying mechanisms of action may offer a theoretical reference for the development of novel anti-CRC therapies.

Noteworthily, a literature review signaled that curcumin may regulate apoptosis in human malignancies (25). Apoptosis is a type of programmed cell death that could influence the development of CRC. While numerous studies have investigated the effects of curcumin, the relationship between curcumin and apoptosis in CRC requires further elucidation. Calibasi-Kocal et al. reported that curcumin inhibited the migratory and invasive abilities of CRC cells (26). At the same time, Huang et al. concluded that curcumin induced apoptosis in CRC stem cells (27). To the best of our knowledge, this is the first study to explore the potential mechanisms by which curcumin modulates apoptosis in CRC, potentially offering a novel therapeutic strategy.

Network pharmacology analysis was employed to identify key cross-talk proteins associated with curcumin, apoptosis, and CRC, including TP53, MDM2, PTGS2, etc. Next, a PPI network was constructed using the STRING database and further enriched to identify biological processes such as cell cycle regulation and related signaling pathways. The results implied that these selected proteins are involved in various critical biological processes and pathways. Furthermore, the results of the CCK-8 assay showed that curcumin inhibited tumor cell growth in a concentration-dependent and time-dependent manner. Flow cytometry results suggested that curcumin, at specific concentrations, induced apoptosis in CRC cells. Finally, the results of western blot analysis indicated that curcumin modulated apoptosis by regulating the expression of MDM2 and COX-2, consistent with the observations of previous studies.

A previous study reported that elevated MDM2 expression levels were associated with decreased survival rates in mesothelioma patients, indicating the potential of MDM2 as an oncogene (28). Additionally, earlier studies have identified an association between curcumin and tumor cell apoptosis. Yavuz Türel et al. validated that curcumin induces apoptosis in colon carcinoma cells by upregulating the expression of FADD, CASP3, and CASP8 (29). However, the relationship between curcumin and MDM2 in CRC remains to be elucidated. Our results demonstrated that curcumin down-regulates MDM2 expression, suggesting that curcumin may inhibit CRC development by targeting MDM2.

COX-2 has been identified in numerous tumor tissues (30). Shirali et al. demonstrated that physical activity following diagnosis can lead to improved treatment outcomes in patients with COX-2-positive tumors (31). Zhao et al. determined that COX-2 can activate inflammatory processes (32). Uncontrolled upregulation of COX-2 may promote the development of certain malignant tumors and inflammatory conditions (33). Herein, curcumin was found to bind to the COX-2 enzyme to regulate apoptosis in CRC. These findings suggest a significant association between the PTGS2 gene and curcumin in the context of CRC. Nonetheless, additional research is required to validate this relationship.

Although this study initially revealed the regulatory effects of curcumin on the target targets in an in vitro model, the following limitations still exist and need to be improved in subsequent studies. The experimental data were all based on cellular models, and the functions of the target genes were not directly verified by gene knockdown/overexpression experiments. In addition, curcumin, as a multi-targeted natural compound, may produce off-target effects through non-specific binding (e.g., modulation of broad-spectrum pathways such as NF-κB, STAT3, etc.), and the current study has not yet localized its direct targets of action through proteomics or chemical probes. Our team is currently evaluating the in vivo antitumor activity by establishing a tumor-bearing mouse model and developing structural derivatives of curcumin to improve target specificity.

In recent years, clinical trials involving curcumin nanopreparations have made significant progress in the treatment of various diseases (34). Studies have systematically explored the safety, pharmacokinetic characteristics, and therapeutic efficacy of this preparation, especially in terms of anti-tumorigenic effects, which have shown unique advantages. A large number of clinical trials have established that nanocurcumin can be used in the treatment of multiple sclerosis, chronic kidney disease, ankylosing spondylitis, metabolic syndrome, and malignant tumors (35).

The mechanism of action of curcumin is intricate, involving a network of interrelated and interactive pathways. It is worthwhile emphasizing that curcumin has a multi-targeted mechanism of action that can regulate the metabolism of tumor cells, induce apoptosis, and inhibit the abnormal proliferation of tumor cells. However, clinical observations have reported that this substance may elicit digestive disorders (manifested as bloating, diarrhea, etc.), neurological symptoms (such as persistent headache), and other adverse reactions. Besides, its clinical application faces significant pharmacokinetic challenges, largely characterized by low oral bioavailability, which is closely related to insufficient intestinal mucosal absorption, pronounced hepatic first-pass effect, and rapid systemic clearance rate. Consequently, although in vitro experimental findings have demonstrated its anticancer potential, it is vital to establish a standardized drug delivery system, improve pharmacodynamic evaluation models, and conduct multicenter randomized controlled trials to systematically validate its clinical efficacy and safety.

Conclusions

This study explored crucial CRC-associated genes and their interactions with curcumin and apoptosis. A total of 25 essential genes were identified, and their biological relevance was examined. The results indicate that curcumin may regulate apoptosis by binding to MDM2 and COX-2 in CRC. Overall, these findings may aid in developing and designing novel curcumin-based anti-CRC drugs. Our findings strongly support further in-depth investigation of curcumin for its anti-tumor activities, such as its inhibition of tumor cell viability and induction of apoptosis in CRC cells. Additionally, its tumor-suppressive effects in CRC tumor models should be evaluated. Taken together, we hypothesize that these findings could expand our understanding of the effects of curcumin on CRC treatment.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Given that the content and purpose of the study were within the authorization granted by the provider, the Ethics Review Committee of Dalian University Affiliated Xinhua Hospital waived the requirement for ethical approval for this study.

Data Sharing Statement

Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-359/dss

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DOI: 10.21037/tcr-2025-359