The anti-migratory effect of curcumin in colorectal cancer involves IKKβ/NF-κB/Snail-mediated EMT inhibition

Introduction

Colorectal cancer (CRC) is among the most commonly diagnosed malignancies on a global scale; according to recent estimates, roughly 1.9 million new cases and 935,000 deaths were attributed to this disease in 2022 alone (1). Despite meaningful therapeutic advances encompassing surgery, molecularly targeted agents, and immune checkpoint blockade, metastatic dissemination to distant organs persists as the chief contributor to CRC-associated mortality, accounting for nearly half of all deaths (2). Of the cellular processes that underpin metastasis, epithelial-mesenchymal transition (EMT) is widely regarded as a fundamental mechanism by which carcinoma cells gain the motile and invasive capabilities required for systemic spread (3,4).

EMT is a dynamic cellular reprogramming event in which epithelial cells progressively shed their cell-cell contacts and apical-basal organization while simultaneously acquiring mesenchymal attributes, including heightened motility and tissue-penetrating ability (5). At the molecular level, a group of transcription factors—among them Snail, Slug, ZEB1/2, and Twist—orchestrate this phenotypic conversion by repressing epithelial adhesion molecules such as E-cadherin and inducing mesenchymal constituents including N-cadherin, vimentin, and matrix metalloproteinases (MMPs) (6,7). Growing experimental evidence implicates the nuclear factor-kappa B (NF-κB) signaling network as a pivotal upstream driver of EMT in CRC, with IκB kinase β (IKKβ) functioning as a critical activating kinase within this cascade (8,9).

The IKKβ/NF-κB signaling module sits at the nexus of chronic inflammatory stimuli and malignant transformation (10). In brief, IKKβ-mediated phosphorylation of IκBα marks this inhibitory protein for ubiquitin-dependent proteolysis, thereby releasing NF-κB heterodimers to translocate into the nucleus and activate target genes, including the EMT-driving transcription factor Snail (11,12). Complementary studies have revealed that NF-κB-dependent transcriptional networks involving Twist1, ZEB1, and MMP-9 play integral roles in mesenchymal conversion and distant dissemination (13,14). Within the specific context of CRC, aberrant NF-κB activity has been linked to sustained proliferative signaling, apoptosis resistance, neovascularization, and metastatic organ colonization (15,16).

Curcumin (diferuloylmethane), a polyphenolic compound isolated from the rhizome of Curcuma longa L., has garnered considerable attention as a multi-targeted anticancer agent because of its broad pharmacological repertoire and well-documented tolerability (17,18). Preclinical data across diverse tumor models show that curcumin interferes with multiple cancer hallmarks, spanning aberrant cell cycle control, apoptotic evasion, angiogenic activation, and metastatic spread (19,20). Pertinently, curcumin has been reported to counteract EMT in several malignancy types by reinstating epithelial marker expression while dampening mesenchymal characteristics (21,22). In addition to direct tumor-cell effects, curcumin reshapes the stromal compartment—for instance, by redirecting M2-polarized tumor-associated macrophages toward an anti-tumor phenotype and modulating cancer-associated fibroblast behavior (23,24). Of broader relevance, plant-derived bioactive molecules have attracted growing interest as CRC-preventive agents, and emerging data further indicate that plant-origin microRNAs may contribute to anti-tumorigenic functions within the gastrointestinal milieu, reinforcing the therapeutic value of phytochemical-based strategies (25,26).

Despite accumulating evidence of curcumin’s anti-metastatic properties across various tumor types, the precise contribution of the IKKβ/NF-κB/Snail cascade to curcumin-driven migration suppression in CRC has not been systematically delineated. An additional methodological shortcoming in the published literature is the inconsistent application of drug doses across different experimental readouts within individual studies, which impedes direct correlation between molecular alterations and functional phenotypic changes. To overcome these deficiencies, the current investigation adopted a uniform, half maximal inhibitory concentration (IC₅₀)-anchored dosing strategy that was maintained across every functional and molecular assay in two genetically divergent CRC cell lines (HCT116 and SW620). Through concurrent evaluation of cell viability, cell cycle distribution, clonogenic capacity, migratory behavior, and EMT/signaling pathway status under identical treatment conditions, we aimed to construct a coherent mechanistic link between curcumin-mediated suppression of the IKKβ/NF-κB/Snail axis and inhibition of EMT-driven migration. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0236/rc).

Methods

Cell culture and reagents

Two human CRC cell lines, HCT116 (wild-type p53) and SW620 (mutant p53, R273H), were sourced from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Routine culture was carried out in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco), under standard humidified conditions (37 ℃, 5% CO₂). Curcumin (purity ≥98%; MedChemExpress, Monmouth Junction, NJ, USA, CAS 458-37-7) was reconstituted in dimethyl sulfoxide (DMSO) at a stock concentration of 100 mM and stored light-protected at −20 ℃. Working DMSO levels in culture media were maintained at or below 0.1%.

MTT cell viability assay and concentration selection

Cytotoxicity was quantified with the MTT colorimetric method to obtain IC₅₀ values and to define working concentrations for downstream experiments. In brief, cells were dispensed into 96-well plates (5×10³ per well) and left to attach overnight. Graded curcumin concentrations (HCT116: 0–30 µM in eight increments; SW620: 0–45 µM in ten increments) were applied for 48 h. At the endpoint, 20 µL MTT reagent (5 mg/mL) was introduced, and the plates were returned to the incubator for 4 h. After careful aspiration of the medium, 150 µL DMSO was dispensed to solubilize the formazan product, and optical density was recorded at 490 nm on a Tecan microplate reader (Tecan US, Inc., Morrisville, NC, USA). Viability was expressed as a percentage of untreated controls. IC₅₀ values were computed by nonlinear curve fitting [log(inhibitor) vs. normalized response] in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Two working concentrations, corresponding approximately to IC₃₀ and IC_50–60, were then selected for all subsequent assays: 6 and 12 µM (HCT116) and 10 and 20 µM (SW620).

Flow cytometric cell cycle analysis

Cell cycle phase distribution was assessed by propidium iodide (PI) staining. Cells seeded in 6-well plates were exposed to curcumin at the designated concentrations for 48 h, after which they were harvested by trypsinization and washed twice with cold phosphate-buffered saline (PBS). Nuclear DNA was labeled with PI (50 µg/mL) for 15 min at room temperature in darkness. Fluorescence data were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA; 4 lasers, 13 colors), collecting ≥10,000 events per sample. Phase proportions (G0/G1, S, and G2/M) were determined with FlowJo v10.8, and the combined G1 + S fraction served as a surrogate index of proliferative activity.

Colony formation assay

Long-term clonogenic capacity was evaluated by plating cells at a density of 1×10³ per well in 6-well dishes with 2 mL DMEM. After overnight adherence, the medium was replaced with curcumin-containing medium at the specified concentrations. Following a 48-h drug challenge, cultures were switched to drug-free complete medium, which was renewed every three days. On day 14, colonies were immobilized with 4% paraformaldehyde (15 min) and stained with 0.1% crystal violet (20 min) at ambient temperature. After gentle rinsing and air-drying, plates were photographed, and clusters exceeding 50 cells were counted with ImageJ (NIH, USA). Clonogenic efficiency was calculated as (colony number / seeded cell number) × 100%.

Wound healing assay

Cell migration was evaluated via a scratch wound approach. Monolayers grown to 90–95% confluence in 6-well plates were scored with a sterile 200 µL pipette tip to generate a uniform cell-free gap. Floating cells were removed by two PBS rinses, and cultures were maintained in serum-free medium supplemented with curcumin at the indicated concentrations. The omission of serum was intended to minimize confounding contributions of cell proliferation to gap closure. Phase-contrast images of the wound region were recorded at 0, 24, and 48 h using an inverted microscope (Olympus, Tokyo, Japan) equipped with a digital camera. Gap closure was measured with ImageJ and expressed as [(Area_oh − Area_th) / Area_oh] × 100%.

Transwell migration assay

Directional motility was further examined with Transwell chambers (6.5 mm, 8.0 µm pore PET membrane; Corning, Corning, NY, USA). After initial seeding and overnight attachment in the upper compartment, the medium was exchanged for serum-free medium containing curcumin at the designated concentrations. A suspension of 2×10⁴ cells in 200 µL serum-free curcumin-supplemented medium was loaded into the upper insert, while 600 µL of complete medium with 10% FBS was placed below as a chemoattractant. After 48 h at 37 ℃, non-migrated cells on the upper membrane surface were removed with a cotton swab. Transmigrated cells on the lower surface were fixed (4% paraformaldehyde, 20 min) and stained (0.1% crystal violet, 15 min). Five random fields per insert were captured at 200× magnification, and migrated cells were enumerated using ImageJ. Results are presented as a percentage of the control value.

Western blot analysis

After 48 h of curcumin exposure at the specified concentrations, cells were collected and lysed in RIPA buffer (Beyotime, Shanghai, China) fortified with protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Total protein content was measured with a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal protein loads (30 µg per lane) were resolved on 10% SDS-polyacrylamide gels and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were blocked for 1 h at room temperature in 5% bovine serum albumin (BSA) dissolved in Tris-buffered saline with 0.1% Tween-20 (TBST), then probed overnight at 4 ℃ with the following primary antibodies from Cell Signaling Technology (Danvers, MA, USA): E-cadherin (#3195), MMP-2 (#40994), N-cadherin (#13116), Snail (#3879), NF-κB p65 (#8242), phospho-NF-κB p65 (#3033), and GAPDH (#8884). Anti-VEGF (ab150375) was from Abcam (Cambridge, UK), and anti-IKKβ (sc-34673) from Santa Cruz Biotechnology (Dallas, TX, USA). After three TBST washes, membranes were incubated with HRP-conjugated secondary antibodies (1:5,000; Cell Signaling Technology) for 1 h at room temperature. Bands were visualized with enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) on a ChemiDoc system (Bio-Rad, Hercules, CA, USA). Densitometric analysis was performed in ImageJ: target-protein band intensities were first normalized to the corresponding GAPDH signal and subsequently expressed relative to the untreated control (set to 1.0). All quantifications represent three independent biological replicates.

Statistical analysis

Each experiment was conducted in triplicate, and results are reported as mean ± standard deviation (SD). Statistical computations were carried out in GraphPad Prism 9.0. Multi-group comparisons were made by one-way analysis of variance (ANOVA) with Tukey’s post hoc correction. Groups sharing the same letter are not significantly different from each other (P≥0.05). Different letters indicate statistically significant differences between groups (P<0.05). Detailed statistical significance levels are reported in the corresponding figure legends.

Results

Curcumin inhibits CRC cell viability and determination of IC50 values

MTT-based viability measurements were first performed to characterize curcumin’s concentration-dependent cytotoxic profile and to identify appropriate dosing regimens for subsequent experiments. As shown in Figure 1A,1B, increasing curcumin concentrations produced a progressive decline in cell viability in both HCT116 and SW620 lines. Nonlinear regression of the resulting dose-response data yielded IC₅₀ estimates of 11.8 µM for HCT116 and 19.5 µM for SW620 cells (Figure 1C,1D). The comparatively greater tolerance of SW620 cells is in keeping with their origin from a lymph node metastasis and their inherently more aggressive biological behavior.

Figure 1 Curcumin inhibits CRC cell viability in a concentration-dependent manner. (A,B) Cell viability bar charts of HCT116 (A) and SW620 (B) cells following treatment with a series of curcumin concentrations for 48 hours. (C,D) Dose-response curves of curcumin in HCT116 (C) and SW620 (D) cells after 48-hour treatment. IC₅₀ values: 11.8 µM (HCT116) and 19.5 µM (SW620). Data are mean ± SD (n=3). Bars not sharing a common letter differ significantly (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; CRC, colorectal cancer; IC₅₀, half maximal inhibitory concentration; SD, standard deviation.

Guided by these IC₅₀ values, two standardized treatment concentrations were established for all downstream functional assays: 6 and 12 µM for HCT116 (approximating IC₃₀ and IC_50–60, respectively) and 10 and 20 µM for SW620 (analogous range). These doses were chosen to span a sublethal-to-moderate cytotoxicity window, thereby retaining enough viable cells for reliable functional readouts while permitting detection of concentration-dependent biological effects.

Curcumin induces cell cycle arrest in CRC cells

To determine whether curcumin interferes with cell cycle progression, flow cytometric analysis was carried out after PI staining. As illustrated in Figure 2, curcumin produced marked shifts in cell cycle phase distribution across both cell lines. HCT116 cells exhibited a concentration-dependent decrease in the combined G1 + S compartment, pointing to compromised replicative transit.

Figure 2 Curcumin induces cell cycle arrest in colorectal cancer cells. (A,C) Representative flow cytometry histograms of cell cycle distribution in HCT116 (A) and SW620 (C) cells treated with curcumin for 48 hours. (B,D) Quantification of cell cycle phase distribution in HCT116 (B) and SW620 (D) cells. Data are mean ± SD (n=3). Bars not sharing a common letter differ significantly (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; SD, standard deviation.

SW620 cells showed an analogous response, with a statistically significant contraction of the G1 + S fraction at both treatment doses. Together, these observations demonstrate that curcumin restricts CRC cell proliferation, at least partly, by perturbing orderly cell cycle progression and thereby limiting the replicative capacity of tumor cells.

Curcumin suppresses colony formation of CRC cells

Clonogenic assays were employed to assess the sustained proliferative capacity of curcumin-treated cells. As presented in Figure 3, curcumin elicited a pronounced, dose-responsive reduction in colony numbers in both HCT116 and SW620 lines. These data confirm that curcumin substantially compromises the long-term clonogenic potential of CRC cells at doses that retain sufficient viability for parallel functional analyses.

Figure 3 Curcumin suppresses colony formation capacity of CRC cells. (A,C) Representative images of colony formation in HCT116 (A) and SW620 (C) cells fixed with 4% paraformaldehyde and stained with 0.1% crystal violet after 14 days of curcumin treatment. (B,D) Quantification of colony formation efficiency. Data are mean ± SD (n=3). Groups labeled with different letters are significantly different (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; CRC, colorectal cancer; SD, standard deviation.

Curcumin suppresses migration of CRC cells

Scratch wound assays, performed in serum-free medium to limit proliferative interference, were used to gauge curcumin’s effect on cell motility. As presented in Figure 4, gap closure was substantially impeded by curcumin in both HCT116 and SW620 cells, with inhibition increasing in a concentration- and time-dependent fashion.

Figure 4 Curcumin inhibits CRC cell migration in wound healing assay. (A,C) Representative images of wound healing in HCT116 (A) and SW620 (C) cells at 0, 24, and 48 hours (scale bar: 100 µm). (B,D) Quantification of wound closure rates. Data are mean ± SD (n=3). Groups labeled with different letters are significantly different (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; CRC, colorectal cancer; SD, standard deviation.

Transwell migration experiments under chemotactic gradient conditions yielded concordant results. As depicted in Figure 5, curcumin dose-dependently diminished the number of cells traversing the membrane in both lines, collectively establishing that curcumin effectively hinders CRC cell migration under in vitro conditions.

Figure 5 Curcumin suppresses CRC cell migration in Transwell assay. (A,C) Representative images of migrated HCT116 (A) and SW620 (C) cells fixed with 4% paraformaldehyde and stained with 0.1% crystal violet after 48-hour incubation (scale bar: 100 µm). (B,D) Quantification of migrated cells in HCT116 (B) and SW620 (D). Data are mean ± SD (n=3). Groups labeled with different letters are significantly different (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; CRC, colorectal cancer; SD, standard deviation.

Curcumin reverses EMT by modulating epithelial and mesenchymal markers

Because EMT is tightly linked to migratory and invasive competence, we next asked whether curcumin’s anti-migratory action was accompanied by alterations in EMT-related protein expression. Immunoblotting revealed that curcumin elicited a dose-responsive elevation of the epithelial adhesion molecule E-cadherin in both HCT116 and SW620 cells (Figure 6A,6C,6F,6H). In parallel, the mesenchymal-associated proteins N-cadherin (Figure 6A,6B,6F,6G), MMP-2 (Figure 6A,6D,6F,6I), and VEGF (Figure 6A,6E,6F,6J) were all markedly down-regulated following curcumin treatment. These data demonstrate that curcumin reverses the EMT phenotype in CRC cells, restoring epithelial features while suppressing mesenchymal characteristics.

Figure 6 Curcumin reverses EMT by modulating epithelial and mesenchymal markers. (A,F) Western blot analysis of EMT markers (N-cadherin, E-cadherin, MMP-2, VEGF) in curcumin-treated HCT116 and SW620 cells. (B-E,G-J) Quantification of N-cadherin (B,G), E-cadherin (C,H), MMP-2 (D,I), and VEGF (E,J) expression. Band intensities were normalized to GAPDH and expressed relative to the untreated control. Data are mean ± SD (n=3). Groups labeled with different letters are significantly different (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; EMT, epithelial-mesenchymal transition; SD, standard deviation.

Curcumin suppresses the IKKβ/NF-κB/Snail signaling axis

We next interrogated the upstream signaling events responsible for curcumin-induced EMT reversal by focusing on the IKKβ/NF-κB/Snail pathway. Immunoblot analysis showed that curcumin markedly reduced IKKβ protein levels (Figure 7A,7B,7F,7G) and phosphorylated NF-κB p65 (p-NF-κB) abundance (Figure 7A,7D,7F,7I) in both cell lines. Of note, total NF-κB p65 protein remained largely unaltered (Figure 7A,7C,7F,7H), whereas its phosphorylated, transcriptionally active form declined in a concentration-dependent manner.

Figure 7 Curcumin suppresses the IKKβ/NF-κB/Snail signaling axis. (A,F) Western blot analysis of IKKβ, NF-κB p65, p-NF-κB p65, and Snail in curcumin-treated HCT116 and SW620 cells. (B-E,G-J) Quantification of IKKβ (B,G), total NF-κB p65 (C,H), p-NF-κB p65 (D,I), and Snail (E,J) expression. Band intensities were normalized to GAPDH and expressed relative to the untreated control. Data are mean ± SD (n=3). Groups labeled with different letters are significantly different (P<0.05; one-way ANOVA with Tukey’s post hoc test). ANOVA, analysis of variance; SD, standard deviation.

Concomitantly, Snail—a pivotal EMT-inducing transcription factor and established NF-κB target—was substantially down-regulated by curcumin (Figure 7A,7E,7F,7J). In HCT116 cells, IKKβ, p-NF-κB, and Snail were all significantly diminished (Figure 7A-7E), and a parallel pattern was observed in SW620 cells (Figure 7F-7J). These findings collectively indicate that curcumin restrains CRC cell migration by suppressing the IKKβ/NF-κB/Snail signaling cascade, thereby blocking EMT-driven acquisition of metastatic potential.

Discussion

Curcumin, a turmeric-derived polyphenol, has drawn sustained research attention as a candidate anticancer compound because of its favorable safety record and ability to simultaneously modulate multiple oncogenic pathways. While its antiproliferative actions in CRC have been documented by numerous groups, the specific signaling architecture through which curcumin limits cell motility has not been fully resolved. The present investigation was therefore undertaken to determine whether curcumin suppresses CRC cell migration by targeting the IKKβ/NF-κB/Snail axis and consequently reversing EMT. Our results show that curcumin robustly attenuates the migratory behavior of both HCT116 and SW620 cells, as demonstrated by complementary wound healing and Transwell assays. In parallel, curcumin exhibited concentration-dependent cytotoxicity, yielding IC₅₀ values of 11.8 µM (HCT116) and 19.5 µM (SW620) at the 48-h time point. Flow cytometric data revealed curcumin-induced cell cycle disturbance, reflected in a shrinkage of the G1 + S compartment, and clonogenic survival was markedly compromised. These findings align with prior reports describing curcumin’s growth-inhibitory activity in CRC (27) and provide a platform for dissecting the molecular basis of its anti-migratory effects.

Rational selection of drug concentrations constitutes a pivotal methodological element in migration-oriented studies. Here, working doses were anchored to IC₅₀ values—6 and 12 µM for HCT116, 10 and 20 µM for SW620—and kept constant across every assay. This design reflects the particular demands of migration experiments as compared with straightforward viability measurements. Whereas cytotoxicity endpoints primarily capture cell death, migration readouts require an intact motility apparatus and a sufficient population of viable cells. The lower dose (approximately IC₃₀) was chosen to limit cytotoxic interference while maintaining approximately 70% viability, a threshold that ensures meaningful wound closure and Transwell data. The higher dose (approximately IC_50–60) enables dose-response assessment and generates more pronounced biological effects without excessive cell loss that would confound migration endpoints. Holding concentrations uniform across migration assays, cell cycle analysis, clonogenic assays, and immunoblotting allows direct mechanistic linkage between cellular phenotypes and molecular changes. The greater curcumin tolerance displayed by SW620 cells relative to HCT116 cells is biologically noteworthy, likely reflecting the metastatic origin and inherently more aggressive nature of the SW620 line. Such differential sensitivity highlights the heterogeneity of CRC and supports the case for individualized dose calibration rather than blanket concentration choices across cell models (28).

EMT has been increasingly recognized as a core driver of metastatic progression, enabling epithelial tumor cells to acquire mesenchymal features that promote dissemination and tissue invasion. The molecular hallmarks of this transition include downregulation of E-cadherin-dependent intercellular junctions, concomitant upregulation of N-cadherin, and elevated expression of extracellular matrix-remodeling enzymes and pro-angiogenic mediators (29). E-cadherin is essential for maintaining epithelial cohesion, and its transcriptional silencing releases tumor cells from spatial constraints. N-cadherin, by contrast, facilitates dynamic interactions between migrating cells and the surrounding matrix (30). MMP-2, a basement-membrane-degrading gelatinase, supports the initial invasive steps of metastasis, whereas VEGF promotes the neovascular supply required for tumor expansion and hematogenous dissemination (31). The coordinated regulation of these molecules therefore constitutes a molecular fingerprint of metastatic competence in CRC. In agreement with this framework, our immunoblot data showed that curcumin induced a marked elevation of E-cadherin alongside dose-dependent suppression of N-cadherin, MMP-2, and VEGF in both cell lines—a profile consistent with reversal of the EMT phenotype.

The EMT marker panel selected for this study—E-cadherin, N-cadherin, MMP-2, and VEGF—was designed to capture functionally distinct facets of the epithelial-to-mesenchymal switch. MMP-2 was chosen as the representative metalloproteinase because of its established involvement in degrading type IV collagen in the basement membrane during early invasion and its transcriptional regulation by NF-κB, which directly connects it to the signaling axis under investigation. Although inclusion of additional EMT markers such as vimentin, ZEB1, Slug, and Twist would yield a more exhaustive characterization of the EMT landscape, the four markers interrogated here encompass key functional mediators spanning cell-cell adhesion (E-cadherin), mesenchymal motility (N-cadherin), matrix remodeling (MMP-2), and angiogenic support (VEGF). Their coherent, dose-dependent modulation across both cell lines constitutes strong evidence for functional EMT reversal. Expanding the marker panel to include vimentin and additional upstream transcription factors represents an important priority for subsequent studies.

The molecular changes documented here translated into clear functional readouts: wound closure rates were markedly diminished and Transwell migration was substantially suppressed upon curcumin treatment. The degree of migration inhibition tracked closely with the magnitude of EMT marker modulation, pointing to a direct mechanistic connection. It is noteworthy that the robust E-cadherin restoration observed with curcumin contrasts with findings for certain other phytochemicals. For example, 6-shogaol has been reported to produce inconsistent or even paradoxical E-cadherin responses in some CRC cell lines despite reducing migration (32), a discrepancy that may stem from compound-specific regulatory mechanisms or from E-cadherin-independent motility pathways. Zheng et al. (33) documented comparable E-cadherin recovery following curcumin treatment in gastric cancer cells, supporting cross-cancer generalizability. Furthermore, Tomeh et al. (34) have reviewed curcumin derivatives as anticancer agents, emphasizing opportunities for structural optimization to improve bioavailability and potency. The consistent E-cadherin upregulation in the present work strengthens the view that curcumin reliably reverses the EMT phenotype in CRC cells.

A key interpretive consideration for any migration study is the potential confounding contribution of proliferation inhibition. To mitigate this, we performed wound healing experiments in serum-free medium, an approach that markedly reduces cell division and thereby permits a more faithful assessment of intrinsic migratory capacity. The Transwell assay, which captures active directional movement through a defined pore size over a fixed period, further complements the scratch assay as a proliferation-independent readout. Although inclusion of a mitotic inhibitor such as mitomycin C would provide an additional safeguard against proliferative confounding, the convergent outcomes from both serum-free wound healing and Transwell approaches strongly argue that curcumin exerts genuine anti-migratory effects that are separable from its antiproliferative activity. This interpretation is reinforced by the observation that the lower curcumin dose (approximately IC₃₀) preserved roughly 70% cell viability, making it unlikely that migration suppression merely reflects cytotoxic attrition. Moreover, whereas a trypan blue exclusion test would offer the most direct means of distinguishing cell death from growth inhibition, our combination of MTT data—showing graded, dose-dependent viability reductions—and flow cytometric evidence of cell cycle arrest rather than a dominant sub-G1 population collectively points to proliferation inhibition, not extensive cell killing, as the predominant effect at the lower concentration.

NF-κB occupies a central position in the transcriptional circuitry that couples inflammatory cues to malignant progression, with established contributions to tumor cell survival, expansion, and dissemination. Under resting conditions, NF-κB dimers are retained in the cytoplasm through association with inhibitory IκB proteins. Pathway activation—principally via IKKβ-dependent phosphorylation of IκBα—triggers IκBα proteolysis and subsequent nuclear entry of NF-κB, where it transactivates a spectrum of genes encoding survival factors, inflammatory mediators, and EMT-promoting transcription factors, notably Snail. By directly silencing E-cadherin transcription while simultaneously inducing mesenchymal gene expression, Snail functions as a master switch of the EMT program. The IKKβ/NF-κB/Snail cascade therefore represents a critical signaling conduit for metastatic progression in CRC (35,36), and pharmacological intervention at this node has shown promise in experimental tumor models (37).

In the present work, curcumin produced a dose-dependent decline in IKKβ, phospho-NF-κB p65, and Snail protein abundance across both cell lines. Critically, total NF-κB p65 remained essentially constant, whereas its phosphorylated (transcriptionally competent) form was substantially reduced—an observation indicating that curcumin targets the activation machinery rather than causing indiscriminate translational down-regulation. The concomitant reduction in Snail, a well-characterized direct NF-κB transcriptional target, provides functional corroboration that NF-κB-dependent gene expression was indeed attenuated. We recognize that demonstrating NF-κB nuclear translocation directly—via immunofluorescence staining or nuclear-cytoplasmic fractionation—would furnish more definitive proof of curcumin-mediated suppression of NF-κB nuclear activity. Herein, Ser536 phosphorylation of NF-κB p65 served as a widely validated surrogate for transcriptional activation and nuclear import. The pronounced, dose-dependent decrease in p-NF-κB p65, together with stable total NF-κB levels, strongly supports the conclusion that curcumin suppresses NF-κB activation rather than merely lowering overall protein abundance. Confirming the subcellular redistribution of NF-κB in curcumin-treated cells remains a high-priority objective for future mechanistic work.

We acknowledge that the current dataset establishes associative, rather than unequivocally causal, relationships between curcumin exposure and IKKβ/NF-κB/Snail pathway suppression. Formal causal validation—through IKKβ knockdown or selective pharmacological inhibition, NF-κB luciferase reporter assays, or Snail over-expression rescue experiments—would substantially fortify the mechanistic claims. Notwithstanding this limitation, several convergent lines of evidence support the proposed sequential signaling model. First, the dose-dependent correspondence between IKKβ down-regulation, reduced NF-κB phosphorylation, diminished Snail levels, and functional migration inhibition is more compatible with a linear cascade than with unrelated parallel events. Second, published biochemical data have demonstrated that curcumin physically engages and inhibits IKKβ kinase activity (38), while IKKβ blockade by independent means recapitulates the EMT reversal documented here (36). Third, the reproducibility of both molecular and functional responses in two genetically distinct CRC lines reduces the probability of cell line-specific artifacts. Definitive establishment of causality within this signaling axis through genetic and pharmacological perturbation strategies constitutes an essential next step.

When viewed in aggregate, the molecular data converge on a coherent mechanistic model: by suppressing IKKβ and thereby preventing NF-κB phosphorylation, curcumin curtails Snail transcription, which in turn relieves E-cadherin repression and lowers the abundance of mesenchymal markers (N-cadherin, MMP-2, VEGF). This coordinated molecular reprogramming manifests functionally as diminished cell migration, an effect that was consistent across both HCT116 and SW620 cells notwithstanding their divergent genetic backgrounds and metastatic potentials.

A further noteworthy observation pertains to the distinct p53 genotypes of the two cell lines: HCT116 carries wild-type p53, whereas SW620 harbors the R273H gain-of-function mutation. Despite this fundamental genetic disparity, curcumin consistently modulated EMT markers and the IKKβ/NF-κB/Snail axis in both lines, implying that curcumin’s anti-migratory mechanism operates largely independent of p53 status. The elevated IC₅₀ in SW620 cells (19.5 µM vs. 11.8 µM) may partly reflect augmented survival signaling conferred by mutant p53 gain-of-function activities, compounded by the inherently more resilient phenotype of this metastasis-derived line. Such p53-independent efficacy carries therapeutic relevance, given that p53 mutations are detected in roughly 40–50% of CRC tumors and frequently confer resistance to standard chemotherapeutic regimens (39).

From a translational standpoint, curcumin’s ability to engage the IKKβ/NF-κB/Snail axis positions it as a candidate warranting deeper preclinical evaluation for CRC management. Nevertheless, limited systemic bioavailability remains a formidable barrier to clinical translation (40). Promising delivery innovations—including nanoparticle encapsulation, liposomal carriers, and self-micro-emulsifying drug delivery systems—have begun to address this hurdle (41,42). The differential dose sensitivity observed between HCT116 and SW620 cells additionally suggests that molecular profiling may be required to identify patient subgroups most likely to derive benefit from curcumin-based therapeutic strategies.

Several limitations of the present study merit acknowledgment. First, exclusive dependence on two-dimensional in vitro cell culture cannot fully replicate the complexity of the tumor microenvironment, host immune interactions, or pharmacokinetic variables relevant to clinical translation. Validation in xenograft or orthotopic metastasis models, with concurrent assessment of the IKKβ/NF-κB/Snail axis in tumor tissue, would markedly reinforce the translational significance of these findings. Second, as detailed above, the mechanistic evidence is correlative in nature; definitive pathway causality would require genetic or pharmacological loss-of-function and gain-of-function experiments targeting IKKβ, NF-κB, and Snail individually. Third, although functionally representative, the EMT marker panel could be broadened to include vimentin, ZEB1, and Twist for a more comprehensive depiction of the EMT program. Fourth, direct evaluation of NF-κB nuclear translocation by immunofluorescence or subcellular fractionation would add additional mechanistic resolution. Notwithstanding these caveats, the present study demonstrates that curcumin suppresses CRC migration via the IKKβ/NF-κB/Snail-mediated EMT pathway using a standardized, IC₅₀-anchored dosing approach that promotes experimental reproducibility and permits direct correlation between molecular and functional readouts.

Conclusions

In summary, the present data demonstrate that curcumin suppresses CRC cell migration by down-regulating the IKKβ/NF-κB/Snail signaling cascade and reversing the EMT phenotype. Curcumin treatment elevated E-cadherin while reducing N-cadherin, MMP-2, and VEGF levels, culminating in attenuated motility in both HCT116 and SW620 cells. These findings suggest that curcumin merits further preclinical and clinical investigation as a potential adjunctive approach to restrict CRC metastasis; however, strategies to enhance bioavailability and in vivo validation remain essential prerequisites before clinical translation can be considered.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0236/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0236/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0236/prf

Funding: This study was supported by the Science and Technology Development Fund, Macau SAR (No. 0114/2022/A, 0018/2024/RIA1).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0236/coif). The authors have no conflicts of interest to declare.

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.

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