Cinobufagin-induced DNA damage response activates G2/M checkpoint and apoptosis to cause selective cytotoxicity in cancer cells

Abstract

Background Processed extracts from toad skin and parotoid gland have long been used to treat various illnesses including cancer in many Asian countries. Recent studies have uncovered a family of bufadienolides as the responsible pharmacological compounds, and the two major molecules, cinobufagin and bufalin, have been shown to possess robust antitumor activity; however, the underlying mechanisms remain poorly understood. Methods Intracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry, and DNA damage was analyzed by immunofluorescent staining and the alkaline comet assay. Cytotoxicity was measured by MTT as well as colony formation assays, and cell cycle and apoptosis were analyzed by flow cytometry. In addition, apoptosis was further characterized by TUNEL and mitochondrial membrane potential assays. Results Here we showed that sublethal doses of cinobufagin suppressed the viability of many cancer but not noncancerous cell lines. This tumor-selective cytotoxicity was preceded by a rapid, cancer-specific increase in cellular ROS and was significantly reduced by the ROS inhibitor N-acetyl cysteine (NAC), indicating oxidative stress as the primary source of cinobufagin-induced cancer cell toxicity. Sublethal cinobufagin-induced ROS overload resulted in oxidative DNA damage and intense replication stress in cancer cells, leading to strong DNA damage response (DDR) signaling. Subsequent phosphorylation of CDC25C and stabilization of p53 downstream of DDR resulted in activation of the G2/M checkpoint followed by induction of apoptosis. These data indicate that cinobufagin suppresses cancer cell viability via DDR-mediated G2 arrest and apoptosis. Conclusion As elevated oxidative pressure is shared by most cancer cells that renders them sensitive to further oxidative insult, these studies suggest that nontoxic doses of cinobufagin can be used to exploit a cancer vulnerability for induction of cancer-specific cytotoxicity.

Screening of traditional Chinese medicine monomers as ribonucleotide reductase M2 inhibitors for tumor treatment

“…In non-small cell lung cancer, cinobufagin could suppress proliferation, migration, and invasion of cancer cells by inhibiting the expression of G9a[ 27 ]. By interfering with the cell cycle, cinobufagin also inhibits the survival of cancer cells and promotes apoptosis[ 28 ]. Moreover, many other anti-tumor pathways are involved, such as the Notch signaling pathway[ 29 ], AURKA/mTOR/eIF4E axis[ 30 ], c-Myc pathway[ 31 ], and ROS/JNK/p38 signaling pathway[ 32 ].…”

Cinobufagin inhibits proliferation and induces apoptosis of hepatocarcinoma cells by activating apoptosis, AKT, and ERK pathways

“…Previously, cinobufagin was shown to inhibit the growth of cancer cells but did not reduce the viability of noncancerous cells such as human L-O2 liver and NCM460 colon epithelial cell lines (Dai et al, 2018b;Niu et al, 2021), demonstrating that cinobufagin is selectively toxic to cancerous cells. Our study showed that the cytotoxicity of cinobufagin to HepG2 cells is concentrationand time-dependent, high concentration and long exposure generated more cytotoxicity, suggesting that cinobufagin might interact with HepG2 directly and indirectly.…”

Predictive DNA damage signaling for low‑dose ionizing radiation

“…Given the aforementioned panel of markers, the radioprotective activity of each modulator was investigated. First, cinobufagin was selected as a candidate radioprotector as it may activate the ATM-CHK2 signaling pathway, which promotes DNA repair ( 32 ). Several studies have demonstrated that cinobufagin exerts anti-inflammatory ( 33 ), anti-bacterial ( 34 ), antitumor ( 35 , 36 ) and immune-enhancing effects ( 37 , 38 ).…”