Study On The Reversal Function Of As₂O₃ On P-gp-mediated Multidrug Resistance In Leukemia Cells And Its Mechanism

Objective:The main cause for the failure of clinical cancer chemotherapy is the formation of multidrug resistance（MDR）of tumor cells,which means tumor cells once is exposed to a cytotoxic agent they will develop cross-resistance to a range of structurally and functionally unrelated compounds.There is a variety of mechanisms on MDR in resistant cell and overexpression of P-glycoprotein（P-gp）is one of the main mechanisms involved in the development of MDR.P-gp is encoded by the MDR1 gene,also referred to as ABCB1,which is one of ATP-binding cassette（ABC）transporters family.ABCB1efficiently removes cytotoxic drugs from the intracellular environment by drug-efflux pump through an ATP-dependent mechanism.Therefore it is possible to counteract some cases of MDR by inhibiting P-gp in cancer.Leukemia treatment depends mainly on the chemotherapy and bone marrow transplantation.Clinically,60-80%of acute myeloid leukemia（AML）patientsexperiencecompleteremissionfollowingroutine chemotherapy treatment,however,numerous patients relapse.Tumor cell resistance to chemotherapy is therefore a more common phenomena in leukemia treatment.P-gp expression was significantly higher in leukemia cells of relapsed or refractory patients than that in treatment-sensitive patients of AML.The P-gp overexpression is also considered as the main reason on acute myeloid leukemia cell to MDR.It is reported that the proportion of relapsed/refractory patients increases with age.During the differentiation of leukemia cells,CD34 is expressed in AML malignant cells,while the expression P-gp is decreased.Whether CD34 levels correlate with the expression or function of P-gp remains unclear.The competition inhibitors to P-gp has been used as the adjuvant treatment for clinical AML to reversal drug resistance,but the results are not very good.Arsenic trioxide（As₂O₃）has been used over the last centuries as a form of traditional herbal medicine.applied to the treatment of acute promyelocytic leukemia（APL）and some solid tumor,the treatment has achieved some effect.Recently it was found that arsenic trioxide inhibits P-gp expression of leukemia cells.Up to date,the mechanism on which As₂O₃ reverses P-gp-mediated MDR is not clear.The upstream region of the MDR1 gene promoter lacks the TATA box commonly found in many protein-coding genes;however,it does contain a reverse CCAAT and GC-rich region that can bind with NF-Y and Sp family transcription factors,which recruit histone acetyltransferase to initiate histone acetylation,chromatin remodeling,thus,activating the MDR1 promoter.Induction of apoptosis is considered to be the mechanism by which As₂O₃ acts as a chemotherapeutic agent.Mathas et al found that As₂O₃ inhibits the activity of nuclear factor-κB（NF-κB）and induces apoptosis.NF-κB also induces the expression of P-gp in addition to inducing the expression of antiapoptotic genes.Bentires-Alj et al demonstrated that the promoter of MDR1contained an NF-κB binding sequence that activates transcription of an MDR1promoter-driven reporter gene.These findings support the notion that As₂O₃ has an effect on the reversal of drug resistance by inhibition of NF-κB,consequently inducing apoptosis and expression of MDR1.In this research,we used As₂O₃ as a treatment of reversal MDR and studied the effect of As₂O₃ on P-gp expression in human leukemia cells.We identified that the influence of NF-κB signal passway on P-gp promoter activity may be one of the mechanisms by which As₂O₃ regulates P-gp expression.Furthermore,we probed the role of As₂O₃ in the regulation of P-gp by NF-κB.Methods:In this research,85 cases of acute myeloid leukemia,human leukemia cell lines,sensitive cell line K562/S and the resistant cell line K562/D were used as research objects.Immuno cell chemistry was used to test P-gp and CD₃₄4 expression in leukemic cells from peripheral blood or bone marrow of leukemia patients.Flow cytometry was performed to test P-gp function.Real-time reverse transcription-polymerase chain reaction（RT-PCR）was used to test the changes of MDR1 expression leval in P-gp expression positive leukemic cells of 3 relapsed/refractory patiants and primary cultured cells of 9 AML patiants and drug resistant cell line K562/D treated with As₂O₃.Flow cytometry was used to test P-gp expression and functional changes in K562/D cells treated with As₂O₃.EMSA was carried out to identify the NF-κB responsive elements within MDR1 promoter.Western blot was performed to test the influence of As₂O₃ on NF-κB p65 subunit,IκB and phosphorylated IκB expression levels.MDR1promoter luciferase reporter gene was constructed.Luciferase assay was performed to detect the role of NF-κB element in the regulation of TNF-αinduced MDR1promoter activity by As₂O₃.After cells were transfected with wild-type,mutant construct of IκB expression plasmids and p65siRNA,luciferase assay were carried out to analyze the role of NF-κB element in the regulation of TNF-αinduced MDR1promoter activity by As₂O₃.Results:1.Immunocytochemistry results showed 36.5%and 50.0%P-gp expressions in63 patients with newly treatment group and in 22 patients with relapsed/refractory group,respectively.No significant difference（P>0.05）between the two groups was found.P-gp function positive was 28.6%and 59.1%in the two groups,respectively,and there was a significant difference between the two groups（P<0.05）.P-gp expression level and function were positive correlated（P=0.0001,r=0.579）.2.Immunocytochemistry results showed 31.7%and 59.1%CD₃₄4 expressions in 63 patients with newly diagnosed group and in 22 patients with relapsed/refractory group,respectivelly（P<0.05）.CD₃₄4 expression and P-gp expression levels were positively correlated（P=0.0001.r=0.483）;CD₃₄4 expression levels and P-gp function were positively correlated（P=0.0001,r=0.550）.3.In the 46 cases（<50 years）of initial treatment group,17.4%CD₃₄4 expression was positive,26.1%P-gp expression positive and 19.6%P-gp function positive;In the 17 patients（≥50 years）of initial treatment group,64.7%CD₃₄4 was positive,64.7%P-gp positive,52.9%function P-gp positive,which were higher than those in<50 years group（P<0.05）.4.Real-time RT-PCR results showed that As₂O₃ reduced overall MDR1 expression in leukemia cells in vivo,primary cultured acute myeloid leukemia cells and K562/D cells in vitro.5.Results from flow cytometry assy showed that As₂O₃ reduced P-gp expression and function of drug release in K562/D cells.6.EMSA result demonstrated that the position+561⁺571 of the MDR1 promoter was an NF-κB responsive element.7.Western blot results showed that As₂O₃ decreased IκB degradation,down-regulated phosphorylated IκB levels and reduced nuclear NF-κB p65 protein in K562/D cells.8.Results from luciferase assay showed that TNF-αpositively regulated the activity of MDR1 promoter and As₂O₃inhibited TNF-α-induced activity of MDR1 promoter.9.Results from luciferase assay showed that the activity of MDR1 promoter inhibited by As₂O₃,which was regulated by NF-κB response element and cited by TNF-α,significantly decreased after cell transfected with wild-type and mutant IκB expression plasmids.The decreased activity of the former was less than that transfed with mutant IκB plasmid.10.Luciferase assy resluts showed the activity of MDR1 promoter inhibited by As₂O₃ which was regulated by NF-κB response element and cited by TNF-α.was significantly decreased after cell transfection with p65siRNA.Conclusions:1.P-gp overexpression is the main reason in clinical drug resistance in acute myeloid leukemia patients.2.As₂O₃ inhibites MDR1 expression in K562/D cells and clinical acute myeloid leukemia cells.3.As₂O₃ reverses P-gp-mediated leukemia cell resistance thorough NF-κB pathway.The position+561⁺571 of the MDR1 promoter was an NF-κB responsive element.As₂O₃ inhibits IκB phosphorylation probably through inhibiting the phosphorylase activity,resulting in the inhibition of NF-κB activity and MDR1 gene expression leading to the reversal of the drug resistance in leukemia cells.