Experimental And Clinical Study On Drug-resistant Mechanisms And Management Strategies Of Myeloid Leukemia Cells Induced By Anthracyclines

Section 1: Response and mechanisms of K562 cells treated with doxorubicinObjective To understand the drug-resistant mechanisms of the K562 cells treated with doxorubicin in terms of cell cycle arrest, apoptosis, telomerase activity, expression of mdr1 gene and Pgp. Methods cell cycle analysis, apoptosis and Pgp expression using flow cytometry, determination of telomerase activity using photometric enzyme immunoassay for the detection of telomerase activity utilizing the Telomeric Repeat Amplification Protocal (TRAP), expression of MDR1 gene and hTERT gene using real time PCR. Results The response features of K562 cells treated with doxorubicin at 0.01mg/l,0.03 mg/1,0.05 mg/1, 0.1 mg/1 respectively, and at 24h,36h,48h,60h,72h, 4.5d,respectively,were observed. With the concentrations of doxorubicin and the time increased, the inhibition of cell proliferative capacity was elevated. At the time of 36^th hours after treated with any concentration of doxorubicin ( 0.01mg/l,0.03 mg/L,0.05 mg/l,0.1 mg/l), the K562 cell proliferative capacity was the highest, and the cells were arrested predominantly in the G2/M phase, the rate of cell apoptosis was 0%. At the 24^th hours, with the concentrations of doxorubicin increased, the cell apoptosis was decreased. When treated with 0.01mg/L doxorubicin, the telomerase activity in K562 cells was higher than that in control (K562 cellswithout treated) at the time of 36^th hours, but at the other any time, the results were reversed. When treated with 0.03mg/l,0.05mg/l and 0.1mg/l doxorubicin, the telomerase activity in K562 cells was reduced before the time of 36^th hours, and it was at the time of 36^th hours that the telomerase activity began to be higher than that in control and lasted. In terms of hTERT gene expression in K562 cells treated with doxorubicin, there was no change compared with that in control cells. At the 4.5^th day after treated with 0.05mg/l doxorubicin, K562 cell proliferative capacity was the lowest, at the 9^th day, cell proliferative capacity began to increase, and at the 31^th day, the cell survival rate reached more than 80% and lasted. At the 12^th hour after treated with 0.05mg/l doxorubicin, the expression of mdrlgene began to increase, and at the 40^th day, the increased mdr1 gene was 14636 times than that in control (K562 cells without treated). The expression of Pgp began to increase at the 32^th after treated with 0.05mg/l doxorubicin, and it was at the 38^th day that the expression of Pgp reached the level of K562/A02. Conclusions Treated with doxorubicin, K562 cells were arrested predominantly in the G2/M phase, the cell apoptosis was inhibited, and the telomerase activity was elevated. Doxorubicin can elevate the expression of the mdrlgene and the Pgp protein in K562 cells at the different time.Section 2: Response and mechanisms of K562 cells treated with doxorubicin and homoharringtonineObjective To further understand the drug-resistant mechanisms of the K562 cells treated with doxorubicin, the K562 cells were treated with doxorubicin togather with homoharringtonine, through observing the changes in terms of cell cycle arrest, apoptosis, telomerase activity, expression of mdr1 gene and Pgp. Methods cell cycle analysis, apoptosis and Pgp expression using flow cytometry, determination of telomerase activity using photometric enzyme immunoassay for the detection of telomerase activity utilizing the Telomeric Repeat Amplification Protocal (TRAP), expression of mdr1 gene using real time PCR. Results With the concentrations of doxorubicin (0.01mg/l, 0.03mg/l,0.05mg/l,respectively) in combination withhomoharringtonine, and the time increased, the inhibition of cell proliferative capacity was elevated. For 10μg/l homoharringtonine, with the time increased, the inhibition of cell proliferative capacity was also elevated. As to the inhibition of K562 cell proliferative capacity, 10μg/l homoharringtonine was between 0.01mg/l and 0.03mg/l doxorubicin. Treated with 10μg/1 homoharringtonine, only 4.79% of the K562 cells were arrested in the G2/M phase compared with 0% in control K562 cells. At the time of 36^th hours after treated with homoharringtonine in combinatin with doxorubicin (0.03mg/l,0.05mg/l,respectively), the K562 cells in the G2/M phase increased, but the degree of the increasing was less than that in terms of the K562 cells treated with doxorubicin (0.03mg/l, 0.05mg/l, respectively) only. Treated with 10μg/l homoharringtonine, the apoptosis rate of K562 cells decreased from 24.68% at the time of 24^th hours to 0% at the time of 36^th hours, and at the time of 24^th hours, the apoptosis rate elevated again. For those K562 cells treated with 10μg/l homoharringtonine in combination with 0.01mg/l , 0.03mg/l ,0.05mg/l doxorubicin, respectively, at the time of 36^th hours, all of the apoptosis rate were 0%, and at the time of 24^th hours, the apoptosis rates were increased compared with those in terms of doxorubicin only (0.01mg/l , 0.03mg/l ,0.05mg/l,respectively). 10μg/l homoharringtonine could reduce the telomerase activity in K562 cells with that at the time of 36^th hours, which was increased, excepted. 10μg/l homoharringtonine could also reduced the telomerase activity induced by doxorubicin in K562 cells, and could also make the time when telomerase activity begin to increase from at the time of 24^th hours to at the time of 12^th hours. At the 5.5^th day after treated with 10μg/l homoharringtonine, K562 cell proliferative capacity was the lowest, at the 11^th day, cell proliferative capacity increase to be 70% less and more and lasted. At the 86^th hour after treated with 10μg/l homoharringtonine, the expression of mdr1 gene began to increase, and at the 48^th day, the increased mdrlgene was 120 times than that in control (K562 cells without treated). The expression of Pgp began to increase at the 19^th after treated with 10μg/l homoharringtonine, and at the 48^th day, increased to 27.46%. Conclusions Homoharringtonine could inhibit the cell cycle arrest in G2/M phase, increase the rate of cell apoptosis, reduce the higher telomerase activitycaused by doxorubicin in K562 cells with some degree. Homoharringtonine could also elevate the expression of the mdr1 gene and the Pgp protein in K562 cells at the different time.Section 3: Homoharringtonine in combination with cytarabine and aclarubicin in the treat ment of patients with de novo acute myeloid leukemiaObjective To assess the efficacy and toxicity of HAA regimen (homoharritonine 4 mg/m² /day, day 1-3; cytarabine 150 mg/m² /day, day 1-7; aclarubicin 12 mg/m² /day, day 1-7) as an induction therapy in the treatment of de novo acute myeloid leukemia (AML). Methods 48 patients with newly diagnosed AML, aged 35 (14-57) years, were entered into this clinical study. Results The median follow-up was 26 months. Eighty three per cent of patients achieved CR, and the first single course of induction HAA regimen resulted in CR rate of 79 %. The CR rate of 100%, 82% and 33% were achieved in patients with favorable, intermediate and unfavorable cytogenetics, respectively. For all patients who achieved CR, the median time from the initiation of the induction therapy to the evaluation of the remission status was 32 days. For all patients, the estimated 3 years OS rate was 53%, while for patients with M5, the estimated OS rate at 3 years was 75%. The toxicities associated with HAA regimen were acceptable, and the most common toxicity was infection. Conclusions This study suggested that HAA regimen might be a well-tolerable, effective induction regimen in young adult patients with AML.