| ObjectiveThe continued complete remission (CCR) of acute leukemia has been raised greatly because of individualized therapy. There had close relationship between the drug's metabolism and leukemic cells. Vitro study showed that daunorubicin(DNR) could induce apoptosis in leukemic line accompanied with changes of reactive oxygen species (ROS)levels. This study aimed to get the role of ROS in the cellular sensitivity to DNR in acute lymphocyte leukemia (ALL) and provide evidence for individualized therapy.MethodsLeukemic cells were isolated from children with leukemia using ficoll liquid. The ROS levels were detected using 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) by flow cytometry in the control group, groups with DNR alone (50ng/ml,100ng/ml,500ng/ml), group with emodin(E)10uM/L only and group incubated with E(10uM/L ) and DNR(50ng/ml) for 24 hours. The ROS levels of U937 cells were taken as control every time. Cell viabilities of groups above and nacetylcysteine( NAC)10mM/L combined with DNR100ng/ml were determined by MTT assay after incubated for 48 hours. The morphology of apoptosis was observed both by Annexinⅴ/PI reactivity using FCM analysis and the inverted microscope after the cells were stained by hoechest33342/PI.Results1 There was an inverse correlation between basal ROS contents and IC50 values of DNR in 11 ALL and 14 AL(11 ALL,3 AML)cells(P<0.05).2 The individual basal ROS levels had significant difference in ALL cells(p<0.05). After incubated with DNR(50,100,500ng/ml) for 24 hours, the relative ROS level of U937 were 0.2908±0.1315 in 11 ALL cells,0.2849±0.1254 in 9 ALL cells and 0.2565±0.0987 in 7 ALL cells. The level increased compared with basal ROS levels(0.2602±0.1178) when the DNR was 50 and 100ng/ml(P<0.05).However, ROS levels decreased when incubated with DNR 500ng/ml(P<0.05).The same phenomenon was observed in AL cells (11 ALL, 4 AML).3 After incubated with DNR(50,100,500ng/ml) for 48 hours, the cell viabilities were 73.38±15.87%,55.51±16.41% and 39.55±18.87%(p<0.05)。The cell viabilities also decreased when concentration increased in AL cells(11 ALL,3 AML).4 Cell viabilities were 56.33±17.46% and 104.63±22.19% when treated with DNR 100ng/ml alone and combined with NAC(10mM/L ) for 48 hours(p<0.05).NAC greatly attenuated the cytotoxicity of DNR.5 An increase of apoptotic signal and morphologic changes have been detected in ALL cells When the DNR-treated cells were analyzed for reactivity with Annexinⅴand stained by hoechst33342-PI separately. Moreover, it seemed more significant when concentration increased.6 The relative basal ROS levels of U937 cells were 0.2587±0.1294 in 10 ALL patients. The ROS values were 0.2941±0.1447,0.3159±0.1522 and 0.3421±0.1706 when treated with DNR50ng/ml , E10uM/L alone and combined. They could cause an increase of cellular ROS ( p<0.05 ) .Moreover, the combination of both compounds had a higher levels than DNR alone (p>0.05). The same phenomenon was observed in AL cells (11 ALL,4 AML)but the combined group didn't have higher ROS levels than DNR alone.7 E (10uM/L ) didn't have cytotoxicity to cells after incubated for 48 hours in 11 ALL cells(P>0.05). Addition of E(10uM/L ) to DNR significantly decreased the cell viability from 75.45±18.01% to 58.22±19.16%( p<0.05). Combination index (CI) < 1 indicated synergistic activity. The inhibition to cells could reach to the inhibition ratio when DNR was 100ng/ml.The same results came out in 14 AL cells (11 ALL, 3 AML).8 The DNR-resistant ( IC50>225.77ng/ml ) cells showed significantly lower basal cellular ROS contents ( 0.1811±0.0765) than the DNR-sensitive (IC50<225.77ng/ml)cells (0.3065±0.0594 , P < 0.01). The viability of the DNR-resistant cells decreased from 88.82±11.68% to 73.18±13.12% when addition of E(10uM/L )(P<0.05). However E didn't have a sensitization to DNR-sensitive cells. The same results came out in 14 AL cells (11 ALL, 3 AML). 9 In combination with E in ALL cells,apoptotic morphologic enhanced.10 In 19 ALL patients, basal ROS levels of low risk cells were higher than high risk cells(P<0.05).However, there were no significant correlation between basal cellular ROS contents with sex, age, immunity grouping, the number of primary leukemia cells in peripheral blood, therapy,the prednisone responder and the proportion of juvenile cell in bone marrow after treated 19 days. The basal cellular ROS levels were higher in AML than that in ALL(P<0.05).Conclusion1 The basal ROS levels of ALL and AL cells had direct correlation with the cytotoxicity to DNR.2 The individual basal cellular ROS of ALL cells had significant difference. The ROS levels of ALL and AL cells increased after incubated with DNR. ROS took part in the cytotoxicity of DNR. The cytotoxicity of DNR relied on its concentration.3 E caused an increase of cellular ROS and the cytotoxic activity to DNR in ALL and AL cells. They had synergistic activity.4 E increased the cytotoxic effect of DNR in ALL and AL cells that were resistant to DNR in vitro. While, there was no sensitization to DNR-sensitive cells.5 Apoptosis was one of the mechanisms in the cytotoxic effect of DNR in ALL cells.6 Basal ROS levels of low risk cells were higher than high risk cells in ALL .The basal cellular ROS of AML was higher than that of ALL.
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