Mechanism of Cucurbitacin B Targeting <scp>ZNF70</scp> / <scp>NLRP3</scp> Axis Against Inflammation‐Associated Colorectal Cancer

Cucurbitacin B is a naturally occurring tetracyclic triterpenoid extracted from plants in the Cucumis melo L. It demonstrates various pharmacological activities, such as hepatoprotective, anti-inflammatory, and anti-tumor effects. However, its therapeutic effect on inflammation-associated colorectal cancer (CRC) and its mechanism of action have not been elucidated. This study is aimed to investigate the effects of cucurbitacin B on inflammation-associated CRC and the mechanism of action. In vitro, the effect of cucurbitacin B on the inflammatory response and the related ZNF70/NLRP3 pathway was examined using western blotting, CCK-8, ELISA, and immunofluorescence assays. In the HCT116/THP-1 supernatant co-culture system, EdU, colony formation, and wound healing assays were performed to evaluate the effects of cucurbitacin B on the proliferation, migration, and epithelial-mesenchymal transition (EMT) progression of HCT116 cells in response to inflammation. In vivo, a mouse model of inflammatory CRC was constructed by administering azoxymethane (AOM) and dextran sodium sulfate (DSS). To study the role of ZNF70, an adenoviral vector AAV was used to knock out ZNF70 in mice. High-performance liquid chromatography (HPLC) was utilized to assess the toxic impact of cucurbitacin B on mice. Our results indicate that cucurbitacin B reduces the expression of NLRP3 inflammasome-associated proteins by downregulating the production of ZNF70. The co-culture experiment showed that cucurbitacin B inhibited the proliferation, migration, and EMT of HCT116 cells in the inflammatory microenvironment. In vivo studies demonstrated that the knockdown of ZNF70 restored body weight, improved colon length, reduced tumor burden, and increased survival rate in AOM/DSS model mice. This effect was further enhanced by the addition of cucurbitacin B. The HPLC results showed that cucurbitacin B was non-toxic to the heart, liver, spleen, lungs, and kidneys of mice. In conclusion, the downregulation of ZNF70 by cucurbitacin B inhibits the activation of NLRP3 inflammasomes and reduces the promoting effect of the inflammatory microenvironment on CRC proliferation, migration, and the EMT process. Our findings may provide new insights for the development of treatments for inflammation-associated CRC.