Cinobufagin

Cardiotoxicity of traditional Chinese medicines (TCMs) is an important factor in pharmacogenetic cardiovascular diseases, which directly affects the clinical use of TCM. Recently, cinobufagin (CBG), the active substance of Toad venom, has shown as a potential anti-tumor natural product. However, its cardiotoxicity limits the clinical application and the intrinsic mechanism is unclear. Here, we found that CBG induced oxidative stress and cell apoptosis in rat cardiomyocytes (H9c2), which caused cardiomyocyte injury. SET and MYND domain containing 1 (SMYD1) is a histone methyltransferase containing a SET-MYND domain, which is specifically expressed only in cardiomyocytes and skeletal muscle cells. RNA-Seq analysis revealed that the expression of SMYD1 was significantly decreased in the CBG treated H9c2 cells. Overexpression of SMYD1 alleviated CBG-induced myocardial injury, while knockdown of SMYD1 did the opposite. To summary, our study indicated that CBG induced cell apoptosis and oxidative stress via suppressing the expression of SMYD1 and ultimately leaded to myocardial toxicity. This research revealed the side-effects of CBG in myocardial toxicity and provided mechanism basis in specific disease conditions for CBG therapy.

This study aims to investigate the therapeutic efficacy and molecular mechanism of cinobufagin in non‐small cell lung cancer (NSCLC) via pyroptosis induction. Bronchial epithelial cells and NSCLC cell lines were treated with gradient concentrations of cinobufagin. Cell viability was evaluated using Cell Counting Kit‐8 (CCK‐8) assay. RNA‐sequencing was performed to identify differentially expressed genes. Lactate dehydrogenase (LDH) release was measured via cytotoxicity detection kit. Pyroptotic morphological changes were observed by transmission electron microscopy. Western blotting analysed expression levels of pyroptosis‐related proteins. In vivo efficacy was validated in nude mouse xenograft models. Immunohistochemistry evaluated tumour pyroptosis markers, whilst flow cytometry analysed tumour‐infiltrating CD8+ T cells and natural killer (NK) cells. Cinobufagin demonstrated selective cytotoxicity against NSCLC cells with minimal toxicity to normal bronchial epithelium. RNA‐seq analysis revealed significant enrichment of pyroptosis‐related pathways. Functional experiments confirmed cinobufagin‐induced LDH release, characteristic pyroptotic morphological changes and upregulation of cleaved caspase‐3 and Gasdermin E (GSDME)‐NT in NSCLC cells. In xenograft models, cinobufagin treatment reduced tumour volume compared to controls. Mechanistically, this was associated with enhanced caspase‐3 activation and GSDME‐NT accumulation in tumour tissues. Notably, cinobufagin treatment significantly increased NK cell infiltration and activity. Cinobufagin exerts antitumor effects in NSCLC through caspase‐3/GSDME‐mediated pyroptosis induction, accompanied by immune microenvironment modulation. These findings provide preclinical evidence for cinobufagin as a potential therapeutic agent targeting pyroptosis in NSCLC.