Colorectal cancer cell line SW480 (Procell, Wuhan, China), galloflavin (Topscience, Shanghai, China, 98% purity), lipopolysaccharide (LPS), and adenosine triphosphate (ATP) (Sigma, United Sates), Cell Counting Kit-8 (CCK-8) (MCE, Shanghai, China), monoclonal antibodies against NLRP3, ASC, C-Myc, and P21 (Abcam, United Sates), NLRP3 overexpression plasmid pCDH-NLRP3 (Invitrogen, United Sates), BCA protein quantitation kit (Beyotime Biotechnology Company, Shanghai, China), DAB immunohistochemical staining kit (Abcam, United Sates), Hematoxylin and Eosin (H&E) staining kit (Beyotime Biotechnology, Shanghai, China), DMEM high glucose medium (Gibco, United Sates) and ELISA kits of IL-1β, IL-18, and TNF-α (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China) were purchased. Balb/c nude mice were maintained at Jiaxing University.

SW480 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. SW480 cells were passaged after 3–5 days, followed by cell viability detection using trypan blue. Cells were divided into different groups: the DMSO group was the control cell; cells in the L/A group were treated with lipopolysaccharide (LPS) (0.1 g/L), and adenosine triphosphate (ATP) (1 μM) mimics the inflammatory microenvironment; and cells in the Gal groups were pretreated with 5 and 15 μM Gal for 6 h, followed by L/A intervention for 6 h.

SW480 cells of the logarithmic phase were inoculated into 96-well plates and cultured overnight. After cell adherence, cells were pretreated with Gal and further treated with LPS and ATP. The cell viability was measured at 6, 12, 24, and 48 h. In brief, 10 μL of the CCK-8 reagent was added to each well for 4 h, and fresh serum-free medium was replaced (100μL/well). The absorbance was measured at 450 nm by using a microplate reader. The absorbance of blank medium was removed, and the absorbance at 0 h was used as the control to calculate cell viability. Results were shown as %, and three replicates were set in each group.

The cell culture medium was changed to serum-free medium 12 h before the experiment. In brief, 40 μL Matrigel was coated into the Transwell chamber. Cells were digested, washed with 1 μL PBS twice, and 500 μL serum-free medium was added to the 24-well plate. A total of 5×10⁵ cells were resuspended, and 200–250 μL of cell suspension was added to the Transwell chamber to ensure that there were no bubbles between the lower complete medium and the Transwell chamber. After cell adherence, cells were treated with Gal and further treated with LPS + ATP, followed by incubation for 24 h. Afterward, cells were stained with 500 μL of 0.1% crystal violet staining solution (prepared with methanol and diluted with PBS) at room temperature in dark for 15 min, rinsed with PBS, wiped with a cotton swab, and dried. Cells were observed under an inverted fluorescence microscope (200x) to count and photograph the number of cells penetrating through the membrane. Five fields of view were randomly selected for cell counting, followed by calculation of the average value. Matrigel was not added for the migration assay. The number of migrated cells was directly counted, and three replicates were set in each group.

SW480 cells were inoculated in the 24-well plate at a density of 5×10⁴ cells/well. After cell adherence overnight, a sterile 10 μL pipette tip was used to scratch a straight line, and 100 μL PBS was subsequently used to discard cell debris, followed by incubation in a serum-free culture medium at 37°C with 5% CO₂. Cells were treated with Gal and further treated with LPS + ATP. The cell migration and the width of the scratch were observed under an inverted microscope at 0 and 24 h. The image analysis software ImageJ was used to measure the width of the cell scratch. The migration rate was calculated according to the formula: cell migration rate = (cell scratch width_24h − cell scratch width_0h)/cell scratch width_0h × 100%, and three replicates were set in each group.

Cells were inoculated into a 6-well plate at a density of 5×10³ cells/well and further incubated. Fresh culture medium was changed every other week, and cells were subjected to Coomassie blue staining after 2 weeks. A cell clone with over 30 cells was counted as a colony. A cell colony was observed and photographed under a light microscope at 100x magnification. Five fields of view were randomly selected to record the number of colonies, followed by calculation of the average value. Three samples were set in each group, and the experiment was performed three times.

Cells were inoculated into 6-well plates at a density of 1×10⁶ cells/mL. Cells were treated with Gal, subsequently treated with LPS + ATP and digested. The collected cells were lysed within 500 μL of the RIPA protein lysate on ice for 30 min and centrifuged at 12,000 r/min at 4°C for 10 min. The supernatant was collected and subjected to protein concentration by using the BCA kit. The protein sample was mixed with the loading buffer and boiled at 100°C for 5 min. The prepared protein sample (25 μL/well) was subjected to SDS-PAGE gel (5% concentration gel and 10% separation gel) at 60 V and subsequently at 120 V, transferred to the membrane at 4°C for 1.5 h. The PVDF membrane was blocked with 5% skimmed milk powder for 2 h, incubated with monoclonal antibody at 4°C overnight, washed with TBST, incubated with HRP-labeled goat anti-rabbit IgG at 37°C for 2 h, and visualized with ECL, followed by image acquisition by an automatic gel imaging system. GAPDH was used as an internal control to analyze the protein expression level.

The cell culture supernatant was collected to determine the levels of IL-6, TNF-α, and IL-1β in cell experiments. In brief, the supernatant was collected and centrifuged at 3000 g, followed by detection of inflammatory factors (including IL-1β, IL-18, and TNF-α) by ELISA kits. The absorbance value was measured at 450 nm by using a microplate reader (BioTek, United Sates) according to the manufacturer’s instructions, and the results were expressed in pg/ml.

For the animal experiment, after the mice were sacrificed by carbon dioxide asphyxiation, the tumor tissue was ground with liquid nitrogen, lysed within 1.0 ml of RIPA lysate on ice for 30 min, and centrifuged at 10000 g for 15 min, followed by protein quantification of the supernatant. The levels of inflammatory factors were measured according to the manufacturer’s instructions.

The animal experiment is approved by Jiaxing University ethics approval. Nude mice, 4–5 weeks old, 19–23 g of weight, were raised in the SPF environment. SW480 cells were cultured to the logarithmic phase and digested, followed by the adjustment of cell concentration to 3 × 10⁷/ml. Afterward, 0.2 ml cell suspension (6 × 10⁶ cells) was injected into the right hind limb after disinfection. Nodules appeared at the injection site within 3–4 days. Mice were subsequently divided into control and galloflavin groups. Mice of the galloflavin group were administered with 5 mg/kg and 15 mg/kg galloflavin once daily. Mice were kept for 15 days and sacrificed by carbon dioxide asphyxiation, followed by tumor resection and measurement of tumor volume.

The tumor tissues of mice were embedded in paraffin, serially cut into 4 μm-thick sections, deparaffinized with xylene, dehydrated with gradient ethanol, rinsed with tap water for 2 min, stained with hematoxylin for 3 min, rinsed with tap water for 2 min, treated with 1% acid alcohol for 2 s, rinsed with tap water for 2 min, treated with 1% ammonia water for 20 s, treated with 0.5% eosin alcohol for 10 s, dehydrated with gradient alcohol, transparent with xylene, and sealed with neutral gum. Finally, the pathological change of the liver tissue was observed under a light microscope.

Tumor tissue sections were deparaffinized in xylene three times, soaked in absolute ethanol for 5 min, and immersed in 95–85% ethanol twice. The slices were immersed in 0.01 mol/L citrate buffer (PH = 6.0), subjected to antigen retrieval using microwave at 98°C for 20 min, cooled down at room temperature for 30 min, and rinsed with distilled water. The slices were incubated within 3% hydrogen peroxide at room temperature for 10 min to eliminate endogenous peroxidase, blocked with 2% BSA, incubated with the monoclonal antibody (dilution 1:300) at 4°C, incubated with peroxidase-labeled streptomycin for 15 min, rinsed with PBS three times (5 min each time), and visualized with freshly prepared DAB. After observing the visualization reaction under a microscope, the slices were counterstained with hematoxylin and mounted, followed by observing under an upright Olympus-BX51 microscope.

We performed overexpression of NLRP3 in SW480 cells, followed by LPS and ATP treatment to induce the inflammatory microenvironment. As a result, L/A induction could promote the viability and promote cell proliferation of SW480 cells; the overexpression of NLRP3 (NLRP3-OE) further upregulated the cell viability, which was significantly higher than that of L/A (Figure 1A). Colony formation assay also showed that the number of colonies of L/A was significantly higher than that of control. NLRP3-OE could further promote colony formation in the inflammatory environment, with a significantly higher number of colonies than that of L/A (Figures 1B,C). Cell migration and invasion assays also showed that L/A promoted the migration and invasion of SW480 cells, with more cells than that in the control group. NLRP3-OE further upregulated cell migration and invasion abilities, which were significantly higher than those of L/A (Figures 1D–F). Wound healing assay also demonstrated that the migration rate of NLRP3-OE cells was significantly upregulated, which was higher than that of L/A and control (Figures 1G,H).

The detection of inflammatory factors found that L/A could promote the expression of inflammatory factors. To be specific, the expression levels of IL-6, TNF-α, and IL-1β were significantly higher than those in the control group. NLRP3-OE could further improve the levels of IL-6, TNF-α, and IL-1β in the inflammation environment, which were higher than those in the L/A group (Figures 2A–C). When detecting the protein expression, we found that LPS/ATP could promote the expression of NLRP3, c-Myc, and P21 and promote cell proliferation. The inflammatory microenvironment could promote the malignant behavior of SW480. The expression of c-Myc and P21 was further upregulated in NLRP3-OE, indicating that NLRP3 could promote the proliferation of SW480 (Figures 2D,E). Taken together, NLRP3 could promote the malignant behavior of SW480 cells in the inflammatory microenvironment.

LPS/ATP was used to simulate the inflammatory environment. Gal intervention could inhibit the upregulation of cell viability in the inflammatory microenvironment. Cell viability in Gal was significantly lower than that in the L/A group, with an obvious effect of high-dose Gal than low-dose (Figure 3A). Colony formation showed that Gal could inhibit colony formation of SW480 cells in the inflammatory environment, with a significantly lower number of colonies than L/A (Figures 3B,C). Cell invasion and migration assay showed that Gal inhibited the migration and invasion of SW480 in the inflammatory environment, with a significantly lower number of migrated and invaded cells than that of the L/A group (Figures 3D–F). Wound healing assay also showed that Gal inhibited the migration ability of cells (Figures 3G,H).

Detection of inflammatory factors showed that Gal could inhibit the release of inflammatory factors. The levels of IL-6, TNF-α, and IL-1β were significantly downregulated, which was lower than that of L/A. High-dose Gal had a higher ability to inhibit inflammatory factors than that of low-dose (Figures 4A–C). Protein detection revealed that Gal could inhibit the expression of P21 and c-Myc. Most importantly, Gal could also inhibit the level of NLRP3 (Figures 4D,E).

To investigate whether Gal acted through NLRP3, SW480-NLRP3^−/− cells were intervened with 15 μM Gal in, showing Gal could not further inhibit the malignant behavior in SW480-NLRP3^−/− cells. Cell viability, colony formation, migration, or invasion ability was not significantly changed between the groups. Meanwhile, Gal had no effect on the level of inflammatory factors (Figure 5).

The small molecule–protein docking model showed that the amino acid residue Thr80 formed a hydrogen bond with the ligand small molecule Gal, and the amino acid residues Arg79, Tyr220, Cys178, Gln179, Lys77, Ser78, and Thr227 form a hydrophobic bond with the ligand small molecule. Gal could bind to the hydrophobic pocket of NLRP3 and affect the assembly and formation of the NLRP3 inflammasome (Figures 6A–C).

Meanwhile, biotin-labeled Gal was used for protein binding in the pull-down assay. As a result, we found that Gal and NLRP3 had a binding relationship, rather than ASC, which further confirmed the targeted-binding relationship between Gal and NLRP3 (Figure 6D).

We performed Gal intervention in tumor-bearing mice and found that Gal could significantly inhibit tumor growth. From the tumor growth curve, Gal inhibited tumor growth and tumor volume in a time-dependent pattern, and high-dose Gal had a more obvious inhibitory effect on tumor growth (Figure 7C). H&E staining showed that Gal could cause tumor tissue damage, which was associated with the inhibitory effect of Gal on SW480 growth. IHC also revealed a high expression of NLRP3 and ASC in tumors. Gal could inhibit the levels of NLRP3 and ASC, which was consistent with the results of cell experiments (Figures 7A,B). Protein detection also found that Gal could inhibit the expression of NLRP3 and suppress the expression of c-Myc and P21 (Figure 7G). Gal inhibited the level of inflammatory factors in the tumor, which was significantly lower than Control and relieved the inflammatory microenvironment.(Figures 7D–F).