Influence of Rapamycin on apoptosis of acute lymphoblastic leukemia EU-4 cells induced by Methotrexate / 中华实用儿科临床杂志

ABSTRACT

Objective To explore the influence of Rapamycin (RAPA) on apoptosis of acute lymphoblastic leukemia EU-4 cells induced by Methotrexate (MTX).Methods EU-4 cells were pretreated 0.5 h with 5 pμg/L,10 μg/L,25 μg/L,50 μg/L and 100 μg/L RAPA.RAPA untreated group was set up as control group.The cells were collected and the expression of LC-3 was assayed by using Western blot.The apoptosis of EU-4 cells was detected by using flow cytometry (FCM).The effects of different concentrations of RAPA pretreatment on autophagy and apoptosis of EU-4 cells were observed,and the pretreatment concentration of RAPA was determined.EU-4 cells were divided into RAPA pretreatment group and untreated group.The cells were treated with 0.05 μmol/L,0.10 pμmol/L and 0.20 μmol/L MTX for 24 h,respectively.The apoptotic rate was detected by using FCM.Western blot was performed to test the expression of LC-3 protein,while the absorbance(A562) was measured by using microplate reader,and the protein concentration in the sample was calculated according to the standard curve.Results The apoptotic rates of EU-4 cells in the control group and the different concentrations of PAPA pretreatment group showed that the control group,5 μg/L,10 μg/L,25 μg/L,50 μg/L and 100 μg/L RAPA were (7.51 ±0.32)％,(7.33 ±0.41)％,(7.71 ± 0.51) ％,(7.63 ± 0.38) ％,(7.80 ± 0.43) ％ and (16.66 ± 0.87) ％,respectively.The apoptotic rates of EU-4 cells in the 5 μg/L,10 μg/L,25 μg/L and 50 μg/L PAPA pretreatment group had no significant difference from those in the control group(t =0.427,-0.417,-0.297,-3.561,all P ＞ 0.05).The apoptotic rate of EU-4 cells in 100 μg/L PAPA pretreatment group was significantly higher than that in control group,and the difference was significant (t =-28.815,P ＜ 0.01).Combined with the results of Western blot and FCM,50 μg/L was used as the pretreatment of PAPA.The apoptosis rates of EU-4 cells in PAPA pretreatment group were (50.23 ± 2.11) ％,(66.88 ± 2.89) ％ and (73.11 ± 2.67) ％ after treated with 0.05 μmol/L,0.10 μmol/L and 0.20 μmol/L MTX for 24 h,respectively.The apoptotic rates of EU-4 cells in PAPA unpretreatment group were (22.53 ± 1.67) ％,(42.82 ± 2.26) ％ and (53.22 ± 1.93)％ after treated with 0.05 μmol/L,0.10 μmol/L and 0.20 μmol/L MTX for 24 h,respectively.The apoptotic rates of EU-4 cells in RAPA pretreatment group were significantly higher than those in untreated group in the same concentration of MTX treatment after 24 h,and the differences were significant(t =12.693,66.148,14.429;all P ＜ 0.01).Conclusion With RAPA pretreatment,relative low dose MTX can induce a great deal of acute lymphoblastic leukemia EU-4 cells apoptosis.