Effect Of Dihydromyricetin On The Proliferation And Apoptosis Of Acute T-Cell Lymphoblastic Leukemia Jurkat Cells And The Mechanism

Objectives:To investigate the effect of dihydromyriceti(DMY) on the proliferation of acute T-cell lymphoblastic leukemia Jurkat cells and to explore the possible mechanism from the perspective of autophagy.Methods : The acute T-cell lymphoblastic leukemia Jurkat cells were cultivated in RPMI1640 medium. Jurkat cells were incubated by the RPMI1640 complete culture medium included different concentrations of DMY(20, 40, 60, 80 and 100 mg/L) and 5% fetal calf serum for 12, 24 and 48 h. Jurkat cells were incubated with 60 mg/L DMY and 10 mmol/L autophagy inhibitor 3-methyladenine(3-MA) for 24 h. MTT assay was used to detect the cell viability and the cell proliferation inhibition rate was calculated. Cell apoptosis was tested by flow cytometry and cell apoptosis rate was calculated. The ultrastructural of Jurkat cells was observated by transmission electron microscope. The expressions of autophagy related gene Beclin 1, LC3 and p62/SQSTM1 in Jurkat cells were measured by Western Blot. The m RNA and protein expressions of Mammalian target of rapamycin(m TOR) were measured by Real-time PCR and Western Blot respectively. Results:1. The proliferation of Jurkat cells was inbibited by DMY in the dose and time dependent manner(P?0.05). 50% inhibition concentrations of DMY at 12 h, 24 h and 48 h time point were 217, 155 and 92 mg/L respectively. The optimal concentration and time of DMY was treatment with 60 mg/L DMY for 24 h.2. The apoptosis of Jurkat cells was inbibited by DMY in the dose and time dependent manner(P?0.05). The optimal concentration and time of DMY was treatment with 60 mg/L DMY for 24 h.3. Cell morphology of Jurkat cells in the control group was normal. Cell morphology was irregular. The nucleus was round or oval. Chromatin was distributed evenly. The nuclear membrane and nucleolus of Jurkat cells were clear. A large number of mitochondria, endoplasmic reticulum, ribosomes and other organelles were distributed in the cytoplasm. The autophagic vacuole and autophagy in the cytoplasm of Jurkat cells were occasionally observated in the control group. In DMY treatment for 24 h group, cell morphology was irregular. The nucleus of Jurkat cells was sparse into reticular. Chromatin was partly condensated in the nucleus. In the cytoplasm, the mitochondria was partially vacuole. Cell organelle was swelling. Partial cell membrane was ruptured. The cells were swelling. The autophagy with double membrane structure and autolysosome with single layer structure in the cytoplasm of Jurkat cells were obviously observated, which ytoplasmic components and damaged organelles in different degradation stages were ncluded in them. The number of autophagy and autolysosome in the cytoplasm of Jurkat cells was significantly increased compared with the control group in the DMY treatment for 24 h group. Compared with the control group, the percentage of autophagic vacuole area and total area of cytoplasm in the DMY treatment for 24 h group was significantly increased(P?0.05).4. Compared with the control group, the expressions of Beclin 1 and LC3-? and the ratio of LC3-?/LC3-?were significantly increased, and the expressions of p62 in Jurkat cellswere significantly decreased in the DMY treatment for 24 h group(P?0.05).5. Compared with the control group, the m RNA and protein expressions of m TOR in Jurkat cells were significantly decreased in the DMY treatment for 24 h group(P?0.05).6. The role of DMY which inhibited the proliferation of Jurkat cells and induced the apoptosis of Jurkat cells was partly abolished by autophagy inhibitor 3-methyladenine(3-MA)(P?0.05). Conclusions : Dihydromyricetin inhibits the proliferation of Jurkat cells and induces the apoptosis of Jurkat cells, which the mechanism may be related with down regulation of m TOR exoression and induction of autophagy.