MTT Assay In Vitro Susceptibility Testing To Optimize The Treatment Of Acute Myeloid Leukemia In The Application Program

Objective: Multi-resistant is one of the most important treatment failure factor of acute myeloid leukemia(AML), overcome and reverse of drug resistance is the key to successful treatthe recurrent refractory AML. Abnormal DNA methylation is closely related to the occurrence of leukemia development, so the DNA methylation has become to a good antitumor drug targets. Asan effective inhibitor of DNA methyltransferase, decitabine has dual effects on tumor cells, high doses has an obvious cytotoxic effect and can accelerate cell apoptosis, low doses can change the gene expression pattern and promote cell differentiation. Now it has been successfully used for clinical treatment of high risk MDS and elderly AML. Decitabine can be usefull for the refractory AML due to certain sensitization and reversal of drug resistance. This study was designed to explore the relationship between in vitro drug sensitivity test of chemotherapy drugs commonly used for AML and clinical efficacy by application of MTT. We used MTT assay to identify the optimal treatment programs of drug-resistant myeloid leukemia cells, so as to guide clinical treatment of patients with relapse or refractory AML.Methods: During 2014 to 2015, 48 cases of AML patients’ bone marrow(43 cases of newly diagnosed, five cases of relapsed or refractory) were collected in the Second Hospital of Hebei Medical University. All patients were diagnosed according to the clinical manifestation,morphological examination of the bone marrow cells, immunophenotyping and chromosome karyotyping.MTT method was used to explore the relationship between in vitro drug sensitivity test and clinical efficacy.The chemotherapy drugs were DNR+Ara-C,THP+Ara-C, MIT+Ara-C, IDA+ Ara-C, HHT+ Ara-C.Combined with the leukemia prognostic stratification index,we could make a prediction of therapeutic and prognosis evaluation. Decitabine, daunorubicin, cytarabine or combined drugs acted on K562/A02 resistant cell lines, and next we used the primary leukemia cells isolated from patients with relapsed or refractory AML for verification. The descriptive statistical data are expressed as the mean±S.D. For continuous variables with a normal distribution, the analysis of treatment effects among different groups were performed using a one-way analysis of variance analysis. All data were analyzed using Graph Pad Prism Version 5.0(San Diego, CA, USA). P<0.05 was considered statistically significant.Results: 1 The inhibition rate of combined therapy for newly diagnosed AML primary cells and the relationship between in vitro drug sensitivity test and clinical efficacy1.1 IC50 values of six single chemotherapy drugs were as follows: homoharringtonine(0.45μg/ml,mitoxantrone(0.88μg/ml),daunorubicin(0.49μg/ml),pirarubicin(0.36μg/ml),idarubicin(0.25μg/ml),and cytarabine(1.2μg/ml).1.2 The inhibition rates of sensitive group were:HA(56.69±9.45)%, MA(54.21±1.05)%,DA(53.59±11.21)%,TA(53.78±9.57)%,IA(60.78±11.28)%.The inhibition rates of resistent group were: HA(22.92±2.32)%, MA(21.38±1.40)%,DA(22.83±1.85)%,TA(21.68±1.66)%,and IA(21.16±1.68) %.1.3 Among 43 cases of newly diagnosed AML patients,35 cases were sensitivity,8 cases were drug resistance in vitro.The relationship between in vitro and in vivo efficacy of susceptibility results were: 32 cases for S/S,3 cases for S/R,1 case for R/S,7 cases for R/R.The total coincidence rate was 90.70%.The positive rate was 91.43% compared with 87.50% of the negative coincidence rate,which indicated that in vitro susceptibility testing of chemotherapy drugs had a good predictive value on in vivo efficacy.1.4 Trypan blue exclusion test was used to explore the cell vitality of primary fresh cells and recovery cells.The fresh cells had a higher average living rate than recovery cells [(96.69±1.39)% vs.(79.09±6.02)%],and the difference was statistically significant(P=0.000).Using combination drugs on recovery cells,the inhibition rate of sensitive group were HA(60.84±8.51)%, MA(59.23±7.94)%,DA(59.76±9.12)%,TA(58.60±8.92)%,IA(68.38±9.82)%.The inhibition rates of resistent group were: HA(27.53± 1.22)%,MA(26.53±3.86)%,DA(27.38±1.31)%,TA(28.52±1.49)%,IA(27.21±0.98)%. The inhibition rates of fresh primary cells were higher than recovery cells,and the difference was statistically significant. The relationship of recovery cells between in vitro and in vivo efficacy of susceptibility results were: 32 cases for S/S,10 cases for S/R,1 case for R/S,0 cases for R/R.The total coincidence rate was 74.42%. The difference of total coincidence between fresh cells and recovery cells was statistically significant.So we used fresh primary cells on the test other than the recovery cells.1.5 According to the results of the experiments,we divided patients into sensitive group(S/S)and resistent group(R/R). Collecting the information of 43 cases,statistical comparisons between sensitive group and resistent group were conducted using Chi-aquare.We found that white blood cell count,risk stratificetion were closely related with sensitivity of chemotherapy and disease recurrence(P < 0.05).Risk stratificetion was independent risk factor for sensitivity of chemotherapy and disease recurrence by using Logistic regression analysis. And the sensitive group had a higher response rate and a lower recurrence rate than the resistent group.2 The effect of single drug or combination drugs on K562/A02 resistant cell lines2.1 The IC50 of using MTT assay on K562/A02 cells and K562 cells was: 12.38μg/ml vs 0.14μg/ml.According to two-sample T-test,the sensitivity of K562/A02 cells was less than K562 cells.The multiple drug-resistant of K562/A02 cells was 87 times compared to K562 cells.So the experiments could be continued.2.2 The inhibition rates for each group of different concentrations of decitabine in K562/A02 cell line were(19.94±0.12)%,(34.05±0.11)%,(52.23 ±0.10)%,(60.10±0.07)%.The results showed that K562/A02 cell proliferation was inhibited by different concentrations of decitabine in a dose-dependent manner.As to the increased, the inhibition rate gradually increased.The difference was statistically significant(P<0.05).The inhibition rate of different concentrations of daunorubicin in K562/A02 cells were(6.97±2.32)%,(15.10 ±1.93)%,(25.11±2.11)%,(30.52±1.76)%.The inhibition rate of different concentrations of cytarabine in K562/A02 cells were(9.20±2.68)%,(19.08± 2.11)%,(25.39±1.75)%, and(36.15±3.14)%.2.3 The IC50 value of decitabine,daunorubicin and cytarabine respectively was 0.77μg/ml,7.57μg/ml,and 5.48μg/ml.The inhibition rate of each single drug of decitabine, daunorubicin, cytarabine on K562/A02 cells was(45.22± 2.07)%,(26.82±1.94)%, and(22.29±3.01)%.The inhibition rate of decitabine with daunorubicin on K562/A02 cells was(76.32±1.75)%.The inhibition rate of decitabine with cytarabine on K562/A02 cells was(79.02±2.30)%.The inhibition rate of daunorubicin with cytarabine on K562/A02 cells was(28.05±1.20)%. The inhibition rate of decitabine with daunorubicin with daunorubicin and decitabine monotherapy treatment groups were significantly different(P=0.000). The difference between decitabine with cytarabine and decitabine with cytarabine monotherapy treatment groups was statistically significant(P=0.000). The difference between decitabine with cytarabine and decitabine with cytarabine was statistically significant(P=0.000). Cytarabine with daunorubicin compared with another two groups were significantly different(P =0.000).3 Monotherapy or combined therapy for primary refractory AML cells3.1 The inhibition rates of decitabine on 5 patients with refractory AML primary cells were:(40.95±3.42)%,(42.12±2.34)%,(40.91±3.26)%,(40.14±3.20)%,(42.56±3.55)%.The inhibition rates of daunorubicin were(20.25±2.84)%,(20.80±3.06)%,(21.19±3.57)%,(19.68±3.24)%,(20.87±4.18)%.The inhibition rates of cytarabine were(17.19±2.97)%,(17.30±3.20)%,(15.30±2.25)%,(17.36±1.72)%,(17.95±2.23)%.3.2 The inhibition rate of decitabine with daunorubicin were:(73.08±1.69)%,(72.80±1.07)%,(73.88±1.71)%,(73.98±1.45)%,(73.15±2.12)%.The inhibition rate of decitabine with cytarabine were:(75.78±3.06)%,(75.56±2.29)%,(74.55±2.87)%,(76.23±2.21)%,(75.66±2.52) %.The inhibition rate of daunorubicin with cytarabine were:(27.06±1.82)%、(27.72±1.88)%、(27.70±1.63)%、(26.39±1.27)%、(25.18±2.24)%.The group of decitabine with daunorubicin compared with decitabine or daunorubicin treatment groups were significantly different(P=0.000).The group of decitabine with cytarabine compared with decitabine or daunorubicin were significantly different(P=0.000).The group of daunorubicin with cytarabine compared with another two group were significantly different( P=0.000).According to the comparation of K562/A02 cells with refractory primary cells,we found that there were significant differences(P<0.01).We concluded that the sensitivity of K562/A02 cells to drugs was higher than that of the refractory primary cells.Conclusions: 1 In vitro susceptibility testing had a good correlation with clinical efficacy.It can be used to choose the optimal chemotherapy regimens and guide treatment.2 Sensitive group had a higher response rate and lower relapse rate than the resistant group. Risk stratificetion was independent risk factor for sensitivity of chemotherapy and disease recurrence.3 Decitabine could reverse the resistant cell line K562/A02 of cytarabine or daunorubicin.And it could also reverse the patients of relapse and refractory of cytarabine or daunorubicin.The chemotherapy regiments based on decitabine had a good prospect on relapsed or refractory AML patients.