Objective: To investigate the effects of Cinobufacini (Cino) injection on the proliferation of human esophageal carcinoma cells ECA109

analyze the effect of Cin combined with irradiation on cell cycle

and explore the potential mechanisms of Cino injection combined with radiotherapy. Method: ECA109 cells were divided into blank group

Cino group

radiotherapy group

and combined group (Cino+radiotherapy). After the esophageal carcinoma cells ECA109 were treated for 48 h

methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to detect the effects of Cino on proliferation of ECA109 cells

flow cytometry (FCM) was used to detect the changes in cell cycles

reverse transcription-polymerase chain reaction(RT-PCR) and Western blot were used to respectively detect the mRNA expressions and protein expressions of ring finger protein 2 (RNF2)

p16 and cyclin-dependent kinase 4 (CDK4) in ECA109 cells. Result: Cinobufacini injection significantly inhibited ECA109 cells proliferation in doses and time dependent ways. As compared with the blank group

the proportion of cells in G0/G1 phase were significant increased (P<0.05) and the proportion of cells in S phase were significant decreased (P<0.05) in various treatment groups. As compared with the blank group

the mRNA and protein expressions of RNF2 in the combined group were significantly decreased (P<0.05)

mRNA and protein expressions of p16 were higher in various treatment groups (P<0.05)

while mRNA and protein expressions of CDK were lower in the various treatment groups (P<0.05). Conclusion: Cino injection can effectively inhibit the proliferation of human esophageal carcinoma ECA109 cells and arrest cell cycle at G0/G1 phase. The mechanism might be partly related to the down-regulation of RNF2

CDK4 levels

and the up-regulation of p16 mRNA expressions.