Abstract: BACKGROUND: Toosendan, a bitter and cold traditional Chinese medicine, has little toxicity and does not require high growth environment. It can significantly inhibit the proliferation of tumor cells in solid tumors, but it is rarely reported in blood tumors. Based on the previous research results, combined with the theory of “combating poison with poison” of traditional Chinese medicine, this paper discusses the mechanism of its main component toosendanin against acute lymphocyte leukemia.  
OBJECTIVE: To explore the mechanism of ethanol extract of toosendan inhibiting proliferation of leukemia CEM cells.
METHODS:  Human acute T lymphoblastic leukemia cell line (CEM cells) was cultured to logarithmic growth stage. After being treated with ethanol extracts of toosendan at five concentration gradients for 24, 48, and 72 hours, their inhibition rates were obtained by MTT assay. Considering the influence of osmotic pressure of cells and ensuring the development of subsequent experiments, the suitable ethanol extracts of toosendan with low, medium, and high concentrations (16, 80, and 400 mg/L) were screened out. The ethanol extract of toosendan had been exposed to peripheral blood lymphocytes of rats for 24, 48, and 72 hours. MTT assay and Giemsa-Reich staining were used to explore its toxic effects. Hoechst 33258 staining was used to observe the apoptotic bodies of CEM cells. RT-qPCR assay was used to detect the transcription of p53, Bcl-2, Bax, Cyt-C, Caspase-9, and Caspase-3 genes in CEM cells after treatment for 24 and 48 hours. Western blot assay was used to detect the expression of p53, Bcl-2, and Bax protein in CEM cells after treatment for 24 and 48 hours.  
RESULTS AND CONCLUSION: (1) Low, medium and high doses (16, 80, and 400 mg/L) of ethanol extract of toosendan had no significant inhibitory effect on normal lymphocytes. (2) After treatment with 16, 80, and 400 mg/L of ethanol extract of toosendan in CEM cells, the apoptotic bodies with blue light could be seen by Hoechst 33258 staining. (3) Compared with the control group, the 80 and 400 mg/L ethanol extract of toosendan increased the transcription of p53, Bax, Cyt-C, Caspase-9, and Caspase-3 genes (P < 0.05 or P < 0.01), and decreased the transcription level of Bcl-2 gene (P < 0.05 or P < 0.01), especially at 48 hours. (4) After administration for 24 hours, compared with the control group, the expression of Bcl-2 protein decreased, the expression of Bax protein increased in the 16, 80, and 400 mg/L extract groups (P < 0.05 or P < 0.01), and the expression of p53 increased in the 400 mg/L extract group (P < 0.05). After treatment for 48 hours, compared with the control group, the p53 protein expression of CEM cells treated with 80 and 400 mg/L ethanol extract of toosendan was significantly increased (P < 0.01), but Bcl-2 protein expression was decreased (P < 0.05 or P < 0.01), and Bax protein expression was significantly increased (P < 0.05 or P < 0.01). (5) The ethanol extract of toosendan can significantly inhibit the proliferation of leukemia CEM cells in a concentration and time dependence and its mechanism may be realized by inducing apoptosis mediated by mitochondria.

Key words: ethanol extract of toosendan, traditional Chinese medicine, combat poison with poison, leukemia, CEM cells, mitochondrial pathway, apoptosis, molecular mechanism