Cinobufagin Promotes Cell Cycle Arrest and Apoptosis to Block Human Esophageal Squamous Cell Carcinoma Cells Growth <i>via</i> the p73 Signalling Pathway

Abstract

Cinobufagin isolated from traditional Chinese herbs has antitumour, anaesthetic, analgesic and antiinflammatory effects. Recently, the antitumour activity of cinobufagin has attracted increasing attention from researchers. However, the anticancer activity of this drug on esophageal cancer cells and the precise mechanism are unclear. In this study, we determined the inhibitory effect of cinobufagin on the growth of three esophageal squamous cell carcinoma cell lines and explored its underlying mechanism. EC-109, Kyse-150, and Kyse-520 cells were treated with different concentrations of cinobufagin. The results of the Cell Counting Kit-8 (CCK-8) and clone formation assays showed that cinobufagin significantly reduced cell proliferation in a dose-and time-dependent manner. Also, flow cytometry and Hoechst 33342 staining indicated that the inhibition of growth induced by cinobufagin was mediated by G2/M cell cycle arrest and apoptosis. In addition, the expression of proteins related to cell cycle arrest and apoptosis was assessed by realtime quantitative (q)RT-PCR and Western blot. The results showed that cinobufagin caused G2/M arrest via upregulation of p21 and Wee1 and downregulation of cyclin B1 and Cdc2 at the mRNA and protein levels and induced apoptosis via upregulation of cleaved caspase-3, Puma and Noxa expression and an increased Bax/Bcl-2 ratio. Other data further showed that cinobufagin increased p73 expression and decreased Mdm2 expression, whereas p53 expression was not significantly changed. Taken together, these results suggest that growth inhibition of cinobufagin in esophageal cancer cells might act through the p73 pathway and its downstream molecules.

Cinobufagin Suppresses Melanoma Cell Growth by Inhibiting LEF1

“…Although cytotoxic side effects in cardiac myocytes by the administration of a high dose of cinobufagin contained in Chinese Medicine has been reported [37], emerging evidence has shown that cinobufagin effectively suppresses cancer growth and progression by inducing cell cycle arrest and apoptosis and inhibiting angiogenesis [38]. For example, cinobufagin was found to promote cell cycle arrest at the G2/M phase and apoptosis in hepatocellular carcinoma and esophageal squamous cell carcinoma by increasing the expression levels of pro-apoptotic p73 and BAX [39,40]. Cinobufagin was found to induce apoptosis in osteosarcoma cancer cells by suppressing cancer-promoting signaling pathways such as STAT3, Notch and NF-kB, and activating mitochondria-mediated apoptosis pathways [41][42][43][44].…”

miR-200b regulates cellular senescence and inflammatory responses by targeting ZEB2 in pulmonary emphysema

“…For instance, P16 is a good marker for acute injury, such as acute kidney injury, in which p16 plays a critical role not only in cellular senescence but also in inflammation, apoptosis, and the excessive accumulation of reactive oxygen species (ROS). A recent study indicated that cinobufagin could indirectly increase the expression of p21 to increase G2/M arrest [24], inducing cell apoptosis, which indicates that p21 functions in apoptosis as well as in cellular senescence. Consequently, here, we chose SA-b-GAL staining to define cellular senescence, which is widely accepted in senescent research as the gold standard.…”

Function of p21 and its therapeutic effects in esophageal cancer (Review)

“…These drugs cause cell cycle arrest and induce apoptosis by increasing the expression of p21. Furthermore, some substances obtained from Chinese medicine have been shown to exert therapeutic effects on EC, which also proves the value of Chinese medicine in antitumor therapy (57).…”

“…Cinobufagin, isolated from traditional Chinese herbs, exerts antitumor, anesthetic, analgesic and anti-inflammatory effects. A previous study by Deng et al (57) indicated that cinobufagin induced ESCC cell cycle arrest at the G 2 /M phase and promoted apoptosis by regulating the expression of p21, indicating that traditional Chinese medicine may prove useful for the treatment of EC.…”