Abstract
Background: In this study, we mainly aimed to explore the correlation between galloflavin and NLRP3 and its effect on colorectal cancer. Methods: NLRP3 was overexpressed in SW480 cells; LPS + ATP was used to mimic the inflammatory microenvironment. Wound healing assay and Transwell assay were utilized to detect cell migration and invasion abilities; CCK-8 assay was performed to detect cell viability alterations; colony formation assay was conducted to detect colony formation ability; Western blot was used to detect the levels of NLRP3, ASC, C-Myc, and P21. SW480 cells were pretreated with high-dose and low-dose galloflavin, followed by observation of their effects on cell metastasis and invasion. NLRP3 was knocked out in SW480 to construct the SW480-NLRP3−/− cell line, followed by high-dose galloflavin treatment and subsequent observation of cell metastasis and invasion abilities. Small molecule–protein docking and pull-down assay were performed to confirm the targeting relationship between galloflavin and NLRP3. After constructing a tumor-bearing mice model, galloflavin was intragastrically administered, followed by detection of tumor growth, expression of NLRP3 and ASC by immunohistochemistry, and tumor histopathology by H&E staining. Results: After NLRP3 overexpression and LPS/ATP induction in SW480, the cell migration and invasion abilities were significantly enhanced, and cell viability was also enhanced. The activation of NLRP3 could promote the malignant behavior of colorectal cancer cells in the inflammatory microenvironment. Galloflavin treatment could significantly attenuate the malignant behavior of SW480 in the inflammatory microenvironment and inhibit the migration and invasion capabilities of SW480. The knockout of NLRP3 inhibited the effect of galloflavin, which did not significantly change the migration and invasion abilities. Molecular docking and pull-down assay revealed a targeted binding relationship between galloflavin and NLRP3 and that galloflavin is bound to NLRP3 not ASC protein. Moreover, galloflavin could inhibit tumor growth and decrease the expression of NLRP in tumor-bearing mice. Conclusion: In this study, we found that NLRP3 could promote the migration and invasion of colorectal cancer cells in the inflammatory microenvironment. Galloflavin could inhibit the malignant behavior of colorectal cancer cells by targeting NLRP3.
Highlights
In this study, we mainly aimed to explore the correlation between galloflavin and NLRP3 and its effect on colorectal cancer
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, associated speck-like protein-containing CARD (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
We found that the LPS/ATP-induced inflammatory microenvironment could promote cell viability, colony formation, metastasis, and invasion in SW480 cells, indicating that the inflammatory microenvironment could promote the malignant behavior of colorectal cancer
Summary
We mainly aimed to explore the correlation between galloflavin and NLRP3 and its effect on colorectal cancer. Inflammatory TME is defined as the inflammatory internal environment during tumorigenesis and tumor progression, which is mainly composed of interstitial fibroblasts, blood vessels and lymphatic network, extracellular matrix components, massive inflammatory cells, and inflammatory factors (Bauer et al, 2020). This special “biological system” involves the mutual regulation and influence of various cells, cytokines, and chemical factors, rendering the complicated and dynamic inflammatory TME (He et al, 2021; Zhou et al, 2021). NLRP3 is expected as a novel target for tumor and microenvironment regulation [10]
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