Mechanism of Cinobufotalin Regulating Intestinal Microbiota and Inhibiting the Progression of Gastric Cancer Cells

OBJECTIVE To investigate the effects of cinobufotalin on the proliferation, migration, and invasion of gastric cancer cells and in a mouse xenograft model of gastric cancer, as well as to explore the mechanisms by which cinobufotalin modulates the gut microbiota to exert anti-tumor effects.

METHODS The half-maximal inhibitory concentration(IC₅₀) of cinobufotalin on gastric cancer cells was determined using the CCK8 assay; the colony formation assay was employed to assess the inhibitory effect of cinobufotalin on gastric cancer cell proliferation; scratch wound healing and Transwell assays were used to evaluate the impact of cinobufotalin on gastric cancer cell migration and invasion; apoptosis assays were conducted to determine the pro-apoptotic effects of cinobufotalin on gastric cancer cells; Western blotting analysis was performed to examine the effects of cinobufotalin on the expression of epithelial-mesenchymal transition-related proteins. The orthotopic xenograft mouse model was established using nude mice, which were randomly divided into three groups(n=5 per group): control group, low-dose cinobufotalin group(6 mL·kg⁻¹), and high-dose cinobufotalin group(12 mL·kg⁻¹). After four weeks of oral cinobufotalin administration, the effect of cinobufotalin on tumor cell proliferation in vivo was evaluated. Additionally, fecal samples were collected for metagenomic analysis to assess changes in gut bacterial composition.

RESULTS Cinobufotalin inhibited the proliferation, migration, and invasion of gastric cancer cell lines MKN1 and HGC27 in vitro and significantly promoted gastric cancer cell apoptosis. cinobufotalin increased the expression of E-cadherin protein while reducing the expression of N-cadherin and Vimentin proteins in gastric cancer cells. In vivo, cinobufotalin suppressed the growth of xenograft tumors in nude mice and altered the composition of the gut microbiota. cinobufotalin treatment increased the abundance of Bacteroidetes while reducing the abundance of Firmicutes and Proteobacteria in the gut microbiota.

CONCLUSION Cinobufotalin effectively inhibits the proliferation, migration, and invasion of gastric cancer cells both in vitro and in vivo. The anti-tumor effects of cinobufotalin are closely associated with its modulation of the gut microbiota composition in mice.