Study on the Antitumor Activity and Nephrotoxicity of Bufotalin Against Non-small Cell Lung Cancer

OBJECTIVE To evaluate the anticancer effects of bufotalin on non-small cell lung cancer(NSCLC) cells and in a nude mouse model, and to investigate the nephrotoxic potential of bufotalin and elucidate its underlying mechanisms.

METHODS ① In vitro assays: A549 NSCLC cells were exposed to varying concentrations of bufotalin(0, 100, 150, 200, 250, 300, 350, 400, 450 nmol·L⁻¹) for 24, 48, 72 h. Cell proliferation was quantified using the CCK-8 assay. Cell migration was assessed via wound healing assays following treatment with 100, 200 and 300 nmol·L⁻¹ bufotalin. ② In vivo studies: A nude mouse xenograft model was established by subcutaneous injection of A549 cells into the right axillary region. Mice were randomly assigned to control, low-, medium-, and high-dose bufotalin groups(n=6). Body weight and tumor inhibition rates were monitored. Histopathological examination of tumor and kidney tissues was performed using hematoxylin and eosin(HE) staining. Serum biomarkers indicative of renal injury were measured. Western blotting analysis was conducted to determine the expression levels of EGFR, Bax, and Bcl-2 in tumor tissues. ③ Network toxicology and molecular docking: Potential nephrotoxicity-related targets of bufotalin were predicted utilizing the Comparative Toxicogenomics Database(CTD). Nephrotoxicity-associated targets were retrieved from GeneCards and OMIM databases, and overlapping targets were identified. A protein-protein interaction network was constructed for these intersecting targets, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. Key targets were subjected to molecular docking and visualization studies. ④ Quantitative real-time PCR(qRT-PCR) was employed to validate the nephrotoxicity-related targets predicted by network toxicology.

RESULTS ① The CCK-8 assay demonstrated that bufotalin inhibited proliferation of A549 cells in a dose- and time-dependent manner. Wound healing assays revealed a significant reduction in migration rates across all bufotalin-treated groups compared to controls. ② In vivo, bufotalin administration resulted in a dose-dependent suppression of tumor growth in nude mice. HE staining of tumors from the high-dose group exhibited decreased cellular density and increased apoptotic morphological characteristics, including cell shrinkage. Western blotting analysis showed significant downregulation of EGFR expression and a decreased Bcl-2/Bax protein ratio in bufotalin-treated groups relative to controls. ③ Network toxicology analysis identified 201 potential bufotalin-associated nephrotoxicity targets. Molecular docking revealed strong binding affinities between bufotalin and key targets such as TP53, TNF, AKT1, IL-6 and STAT3. ④qRT-PCR results indicated significant upregulation of TP53, TNF and IL-6 mRNA expression in all bufotalin-treated groups compared to controls, and significant upregulation of STAT3 and AKT1 mRNA expression were observed in the medium- and high-dose bufotalin groups.

CONCLUSION  Bufotalin may inhibit the proliferation and migration of non-small cell lung cancer by suppressing EGFR expression. Additionally, it may induce certain nephrotoxic effects through the regulation of TP53, TNF, AKT1, IL-6 and STAT3.