Decitabine Combination With Chemotherapy Reversal Of Multidrug Resistance Mechanisms In Leukemia Cell Lines

Objective: Leukemia is a malignant tumor of the hematopoietic system,bone marrow hematopoietic tissue does not work properly due to theformation of intracellular DNA variation，Current treatments is mainly forhematopoietic stem cell transplantation and chemotherapy. Hematopoieticstem cell transplantation is the only possible way to cure leukemia, but thereare many restrictions, the majority of patients can not be operatedhematopoietic stem cell transplantation, most patients usually requirechemotherapy to treat, whereas chemotherapy is prone to drug resistance andrelapse. Multidrug resistance (MDR) is one of the main obstacles tochemotherapy for tumors. Human multidrug resistance gene product ofmembrane protein P170glycoprotein is over expressed, P170forms a channelin the cell membrane with the energy of ATP hydrolysis to form intracellulardrug pump will enter the extracellular and intracellular drug concentrationdecreased, resulting in multi-drug resistance.DNA methylation of tumor suppressor genes which inactivation ofmalignant tumor occurrence and development is one of the importantmechanisms.A variety of studies have shown that methylation can be reversedwith drugs,the demethylation treatment mechanism of action is different fromchemotherapy and hematopoietic stem cell transplantation. Decitabine(5-aza-2deoxycytidine, DCA) is a DNA methyltransferase inhibitors, toexert anti-tumor activity through inhibition of the methylation level of thetumor cells, and perform a dose related mechanism, high concentration hascell toxic effect, low concentration is to methylation role. Present domesticand foreign has been reported in the research, application decitabine andcombination chemotherapy in the treatment of refractory leukemia achievebetter clinical results, the mechanism of which is worth further study.The objective of this study is to observe the reversal effect of decitabine andAdriamycin on the multidrug resisted of leukemia cell strain K562and toexplore its feasibility to treat leukemia. In order to understand the role ofdecitabine in leukemia multidrug resistance mechanism, lay the experimentalfoundation for exploring more reasonable combination chemotherapyregimens to treat leukemia.Methods:1First of all, we used cck8assay to examine the effects of DCA or ADRwith different concentrations on the growth of k562cells.2we used flow cytometric analysis detection to examine the effects ofDCA on the growth of K562cells were examined by flow cytometric analysisdetection.3The RT-PCR to assay the mRNA expression of the MDR-1.4Then, we used flow cytometric analysis detection to examine the effectsof DCA or ADR with different concentrations and their combinations on thegrowth of K562cells were examined by flow cytometric analysis detection.5The RT-PCR to assay the mRNA expression of the MDR-1.6Synergetic effects of DCA and ADR were analyzed by median-effectprinciple.Results:1Effect of DCA alone or ADR on the proliferation of K562/ADR cellsDates from cck8assays show that1-500μg/ml DCA or0.1-50μg/ml ADRtreated cells, inhibition rates enhanced in a time-and dose-dependentlymanner.(Fig.1, Fig.2, Table1, Table2). DCA48-hour IC5035.9μg/ml；ADR48-hour IC5012.8μg/ml.2Flow cytometry results show that the role of single-agent decitabine ordoxorubicin hydrochloride to K562/ADR cell extended with the duration ofaction and concentration, the apoptotic rate was also increased (Fig.3Fig.4,Table3, Table4)3RT-PCR results showed that single-agent decitabine role in K562/ADRcells can inhibit gene expression of MDR and in a time-and dose-dependent, 1μg/ml of decitabine role in K562/ADM cells6h~48h MDR-1geneexpression:(0.71,0.69,0.60,0.53);5μg/ml of decitabine role in K562/ADMcells6h~48h MDR-1gene expression:(0.70、0.67、0.54、0.36);10μg/ml ofdecitabine role in K562/ADM cells6h~48h MDR-1gene expression:(0.70、0.66、0.41、0.27）(Table8).4We selected a concentration of9μg/ml/ml of Adriamycin and aconcentration of1μg/ml、5μg/ml、10μg/ml/ml of Decitabine, K562/ADRcells were treated with9μg/ml Adriamycin or1μg/ml、5μg/ml、10μg/ml Decitabine in combination for48h.The experiment is divided into thefollowing groups:(1) Blank control group: not a member of any drugs;(2)ADR group: K562/ADR cells were treated with9μg/ml ADR alone for48h;(3) DCA group: K562cells were sseparately treated with1μg/ml、5μg/ml、10μg/ml of DCA alone for48hours;(4) Combination therapy group a:K562/ADR cells were treated with the DCA and different concentrations ofADR in combination for48h;(4) Combination therapy group b: K562/ADRcells were treated with different concentrations of DCA pretreated for6h andthen give ADR for a total of48h; inhibition rates were （17.8±1.6）%、（30.7±1.4）%、（36.33±0.78）%、（13.4±1.25）%，（31.3±1.75）%、（47.53±1.80）%、（69.07±1.80）%、（45.53±1.20）%、（61.7±1.51）%、（80.73±2.12）%, The inhibition rates of K562/ADR cells in combination groups were higherthan those in the two single treatment groups(P<0.05),especially in thecombined treatment group in group b (table-3Fig.3).5Flow cytometry results showed that single-agent Decitabine orAdriamycin can be time-dose-dependent manner to induce apoptosis inK562/ADR cells (Fig.4). Flow cytometry results showed that: blank controlgroup, DCA group and ADR group, the combination therapy group acombination group b, apoptosis rate of the combination therapy （16.67±0.87）%、（22.67±1.55）%(32.73±2.40)%(12.17±1.64)%、（29.10±1.91）%、（44.20±1.85）%、（67.03±1.48）%、（43.37±1.49）%、（59.87±2.43）%、（78.83±1.30）%, and combined treatment group compared with themonotherapy group difference was statistically significant (P <0.05)(table-3 Fig.4). The apoptosis rates of K562/ADR cells in combination groups werehigher than those in the two single treatment groups (P<0.05), especially in thecombined treatment group in group b.６By RT-PCR results show that MDR-1gene expression changes inK562/ADR cells: RT-PCR results showed that: blank control group, the DCAgroup ADR group, the combination of a group of the combination therapygroup b of MDR-1gene expression levels were0.72±0.01、0.53±0.02、0.36±0.02、0.27±0.02、0.67±0.02、0.50±0.02、0.36±0.02、0.25±0.03、0.42±0.03、0.24±0.02、0.14±0.01. Two joint mode of administration of MDR-1expression than those in the monotherapy group, especially in the combinationtherapy group b was the lowest statistically significant (P <0.05)(table7, Fig.6.7) Test.Conclusion:1Decitabine or Adriamycin has a dose-and time-dependentant proliferation and proapoptotic effect on K562/ADR lymphoma cells invitro. The50%inhibitory concentration of Decitabine or Adriamycinrespectively on K562/ADR cells for48h is35.9μg/ml and12.8μg/ml.2DCA and ADR could reverse the multidrug resistance on leukemia cellstrain K562．3. DCA sequential ADR could reverse the multidrug resistance onleukemia cell strain K562.4. The combination therapy group b is greater than combination therapygroup a.