| Objective: Drug resistance of leukemia cells is a main obstacle forthe success of chemotherapy. Especially for the patients with refractory or relapsed ALL, the result of are usually unsatisfactory.If we could detect the drug sensitivity before chemotherapy, and optimize the treatmentoptions according to the results,which will effectively improve the success rate of chemotherapy, prolonged the survival time. For acute lymph-oblastic leukemia(ALL), the previous studies suggested that the resistanceto conventional chemotherapy drugs is very common,Therefore, seekingsome drug sensitizers to reduce drug resistance will greatly improve theprognosis of patients.At present, preclinical tumor model test in vivo co-nfirmed that proteasome inhibitor(bortezomib) could significantly inhibitvarious tumor cells growth, including multiple myeloma and ALL, indu-cetumor cell apoptosis and suppress the angiogenesis.In addition, in vitrostudy have also shown that bortezomib could increase the sensitivity of chemotherapy drugs, overcome drug resistance of myeloma cells on dex-amethasone, doxorubicin, melphalan and other drugs.But so far, the relevant reports is lacked about whether bortezomib could effectively reducedrug resistance of ALL cells on chemotherapy drugs in vitro.This study was designed to test the sensitivity of chemotherapy dr-ugs to ALL cells in vitro,and to investigate the correlation with efficacyof clinical treatment, which will provide some strong evidences for app-lying clinical drug sensitivity detection as a promising prognostic factors.Combined with the others prognostic indicator,make an overal prognosisprediction for patients with ALL. In addition, for relapsed ALL, we selected the Bortezomib as a potential chemotherapy sensitizer(On the one hand, increasing the killing effect of conventional chemotherapy drugs, on the other hand, reversing drug resistance of leukemia cells on chemotherapy drugs). In this study, we compared the effects among treatment groups(bortezomib monotherapy; bortezomib+fludarabine+cytarabine; bortezomib+daunorubicin) on drug resistant leukemia cells.And the unterlyingmechanisms of bortezomib to increase anti-tumor effect of chemotherapydrug were explored, so as to provide more alternative combined chemot-herapy schemes for clinical recurrent refractory ALL, even provideinga theoretical basis for developing the new therapeutic strategies.Methods:1 Firstly, using MTT method to detect the sensitivity of single drug and chemotherapy drugs for 36 cases of newly diagnosed ALL patients, to observe the effect of proliferation inhibition. Combined with the others prognostic indicator, predicting the efficacy and prognosis of ALL patients.2 Selected CEM/C1 cells(T-ALL cell line)as the study object, appl-ying different concentration of Bortezomib to treat CEM/C1 cells, and measure the effect of proliferation inhibition.Also, the effect of Bortezo-mibwith Fludarabine and Cytarabine+Bortezomib+Daunorubicin on CEM/C1 cells were detected.3 Selected CEM/C1 cells(T-ALL cell line) as study object,applying①Bortezomib(0.36ug/ml)②Fludarabine(158.5ug/ml)③Cytarabine(19.5ug/ml)④Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml)⑤Bortezomib(0.36ug/ml)+Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml) to conduct the proliferat-ion inhibiting test for CEM/C1 cells.4 Selected the fresh primary leukemia cells obstaining from refract-ory or relapsed ALL patients, and applied different concentration of Bortezomib, Bortezomib, Fludarabine, Cytarabine, Fludarabine+Cytarabine, B-ortezomib+Fludarabine+Cytarabine,Daunorubicin,Bortezomib+Daunorubicinto conduct proliferation inhibiting test.The effect of different drugs andtra-ditional chemotherapy have been compared.5 This paper adopts SPSS22.0 statistical software for data analysis of the samples, which greatly improves the efficiency and scientificity of research. Statistical comparisons among groups as well as two or more rates were conducted using Chi-square test. Logistic regression was used to evaluate the multivariate analysis.Results: 1 The correlation between the inhibitory effect of combination drugs oninitial treatment primary cells and the clinical efficacy. 1.1 Trypan blue exclusion found that the living fresh primary ALL cellsrate is from 95% to 99%, average living cell rate is(97.16±1.31)%, theliving cell rate after cryopreservation and resuscitation is from 67% to 89.99%,which shows that the cell activities decreased, average living cellrate is(82.85±5.62)%, the results were statistically significant(P<0.05). 1.2 The correlation between the results of chemosensitivity test in vitroand sensitivity in vivo, 31 in vitro drug-sensitivity patients, 5 drug resi-stance in vitro patients, 31 in vivo drug-sensitivity patients, 5 drug resi-stance in vivo patients.The comparison of chemosensitivity test in vitro and chemical therapeutic efficacy in vivo, 30 S/S cases, 1 S/R case, 1 R/Scases, 4 R/R cases, the total coincidence rate was 96.77%,the rateforpositive and negative was respectively 97.06% and 80%. A comparisonbe-tween proliferation of refractory ALL primary cells and proliferationof living cell after cryopreservation and resuscitationhad statistical sign-ificance(P<0.05). 1.3 Through comparison of the inhibition rates of cells between sensitiv-ity group and resistance group with different combination schemes ofmultiagent chemotherapy, the average inhibition rate of sensitivity grou-p under the VCTD scheme was(61.32±7.88)% and that of resistancegroup was(28.65±1.81)%.The figures were(62.34±7.97)% and(27.24±2.24)% under the VCID scheme,(63.07±7.39)% and(28.69±0.99)% underthe VCILD scheme for sensitivity group and resistance group, respect-ively. 1.4 The complete remission rates of drug sensitive group and drug resi-stant group were compared with Chi-square test. The results between thetwo groups have no statistically significant difference(P=0.000). The rec-urrence rates of drug sensitive group and drug resistant group were co-mpared with Chi-square test. The results between the two groups have-statistically significant difference(P=0.011). 1.5 The seven factors were processed single factor analysis as foll-ows: age, white cell count of newly diagnosed patients(T-ALL WBC≥100×109/L, B-ALL WBC≥30×109/L)extra-medullary diseases, complex karyotypic, prognostic stratification,remission rate and recurrence rate. Results of si-ngle factor analysis shows that there is ststistical relation between age,white cell count of newly diagnosed patients,prognostic stratification, re-mission rate and recurrence rate(P<0.05).The indexes of single factora-nalysis which have a statistical significance were assigned, and then theindexes were analyzed with multivariate analysis by Logistic regressionanalysis.The results suggest that age,white cell count of newly diagnosedALL patients and prognostic stratification are independent risk factors ofrecurrence. 2 The influence of single-agent therapy and combination therapy on pr-oliferation of ALLcell Line CEM/C1 Cells(Tepotecan- drug fast). 2.1 Tepotecan(24.5ug/ml) was selected to test the drug resistance of CEM/C1 cell, the result showed that the inhibition rate is(15.02±2.46) %. 2.2 Bortezomib with different concentrations(0.025ug/ml, 0.25ug/mland 2.5ug/ml) was selected to act on leukemia CEM/C1 cells for 48 h with the inhibition rates of different groups of(24.51±1.98)%,(44.17±1.16)% and(55.02±2.46)%, respectively. The experimental results indicatedthat bortezomib had inhibitory effect on proliferation of CEM/C1 cells and the effect was dose dependant.With the increase of the action con-centration,the inhibition rate was rose. Ithad statistical significance(P=0.00). 2.3 ①Bortezomib(0.36ug/ml),②Daunorubicin(0.49ug/ml),and③Bortezomib(0.36ug/ml)+Daunorubicin(0.49ug/ml) were selected to act on CEM/C1 c-ells for 48 h with the inhibition rates of different groups of(44.46±1.6to effect on proliferation of refractory ALL primary cells(A,B,C,D,E,F)for 48 h, the inhibition rates had statistical significance. 3.3 The effect of tradition chemotherapy regimens and new chemot-herapy regimens on proliferation of refractory ALL primary cells. ①VCTD, ②VCID, ③VCILD, ④Bortezomib(0.36ug/ml)+Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml) and ⑤Bortezomib(0.36ug/ml)+Daunorubicin(0.49ug/ml) were selected to act on the following six groups(A,B,C,D,E,F) with relapse or refractory ALL primary cells respectively for 48 h, the inhibition rates had statistical significance.Conclusions:1 The primary cells of ALL patients in MTT chemosensitivity test in vitro should be fresh cells.After cryopreservation and resuscitation thevitality of the primary cell is poor so to affect the results of MTT test.2 MTT chemosensitivity test in vitro is consistent with the therape-utic efficacy in vivo, which could be used to choose the combination ofchemotherapy regimens for individual treatment of ALL patients.3 The scheme on the ALL patients who were sensitive to drug su-sceptibility test in vitro had a good therapeutic effect of conventional c-hemotherapy. However, the effect of traditional chemotherapy scheme onALL patients who were resistant to drug susceptibility test was not sati-sfactory, so the therapeutic scheme was needed to be adjusted in clinic-alpractice.Especially the patients with older age,high leukocyte count andhigh risk stratification for initial treatment, called for optimized chemot-herapeutic scheme because of the poor chemotherapy sensitivity.4 Acute T-lymphocyte leukemia cell strain CEM/C1 cells(topotecandrug resistance),bortezomib in combination with daunorubicin, bortezomibcombined with fludarabine and cytarabine had a sensitizing effect.5 Bortezomib in combination with chemotherapy drugs could optim-ize the chemotherapeutic scheme of ALL patients with refractory recure-nce and improve the remission rate. 1)%,(43.59±2.04)% and(54.94±1.06)%, respectively. These groups had st-atistical significance. 2.4 ①Bortezomib(0.36ug/ml),②Fludarabine(158.5ug/ml),③Cytarabine(19.5ug/ml),④Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml),and⑤Bortezomib(0.36ug/ml)+Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml) were selectedto act on leukemia CEM/C1 cells for 48 h with the inhibition rates of different groups of(44.46±1.61)%,(38.02±1.29)%,(24.59±2.89)%,(45.64±1.08)%, and(56.09±2.14)%, respectively. These groups had statistical si-gnificance. 3 The influence of single-agent therapy and combination therapy on proliferation of refractory ALL primary cells. 3.1 Bortezomib with different concentrations(0.025ug/ml, 0.25ug/mland 2.5ug/ml)was selected to act on three groups of refractory ALL pri-marycells for 48 h, the inhibition rates of different groups were(24.51±1.98)%,(44.17±1.16)% and(55.02±2.46)%, respectively. The inhibition rates were(24.78±1.84)%,(44.98±1.04)% and(55.79±3.07)% for group A,(26.26±2.91)%,(44.99±0.65)% and(55.40±3.19)% for group B, and(25.73±1.56)%,(45.34±1.28)% and(57.73±3.43)% for group C. The experimentalre sults showed that bortezomib had inhibitory effect on proliferationofr-efractory ALL primary cells,and the effect was dose dependant.With the increase of the action concentration, the inhibition rate was rose it had statistical significance(P=0.00). 3.2 The effect of Bortezomib and non-Bortezomib drugs on proliferationof refractory ALL primary cells. 3.2.1 ①Bortezomib(0.36ug/ml),②Daunorubicin(0.49ug/ml),and③Bortezomib(0.36ug/ml)+ Daunorubicin(0.49ug/ml) were selected to act on prolifera-tion of refractory ALL primary cells(A,B,C,D,E,F) for 48 h, the inhibitio-nrates had statistical significance. 3.2.2 ①Bortezomib(0.36ug/ml),②Fludarabine(158.5ug/ml),③Cytarabine(19.5ug/ml),④Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml),and⑤Bortezomib(0.36ug/ml)+Fludarabine(158.5ug/ml)+Cytarabine(19.5ug/ml) were selected... |
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