Daunorubicin In Cells Research Articles

MiR-181a modulates acute myeloid leukemia susceptibility to natural killer cells

Although daunorubicin (DNR) is the most widely used anthracycline to treat acute myeloid leukemia (AML), resistance to this drug remains a critical problem. The aim of this study was to investigate the relationship between AML resistance to daunorubicin and susceptibility to natural killer (NK) cell-mediated cell lysis, and the putative expression of miRs. For this purpose, we used the parental AML cell lines U-937 and KG-1 and their equivalent resistant U937(R) and KG-1(R) cell lines. We demonstrate for the first time that the acquisition of resistance to DNR by the parental cell lines resulted in the acquisition of cross-resistance to NK cell-mediated cytotoxicity. miR microarray analysis revealed that this cross-resistance was associated with miR-181a downregulation and the subsequent regulation of MAP3K10 and MAP2K1 tyrosine kinases and the BCL-2 (BCL-2 and MCL-1) family. Overexpression of miR-181a in AML blasts resulted in the attenuation of their resistance to DNR and to NK-cell-mediated killing. These data point to a determinant role of miR-181a in the sensitization of leukemic resistant cells to DNR and NK cells and suggest that miR-181a may provide a promising option for the treatment of immuno- and chemo-resistant blasts.

Identification of Genomic Classifiers That Distinguish Induction Failure in T-Lineage Acute Lymphoblastic Leukemia in COG Study 9404.

The clinical features of age, white count, and presence of extramedullary disease cannot predict risk for induction failure (IF) in patients who present with T-cell acute lymphoblastic leukemia (T-ALL). On the basis of recent observations that gene expression profiles can distinguish clinicopathologic cohorts of patients with acute leukemia, we hypothesized that microarray analyses performed on diagnostic T-ALL bone marrow samples might identify a genomic classifier for IF patients. Using a case-control study design for children and young adults treated for T-ALL on Children's Oncology Group Study 9404, we analyzed 50 cryopreserved T-ALL samples using Affymetrix U133A Plus 2 genechips, which have 54,000 genes, ESTs and genomic classifiers. Following RMA normalization, we used Prognostic Multi-array Analysis (PAM) to identify a 116-member genomic classifier that could accurately identify all 6 IF cases from the 44 patients who achieved remission. Within the IF cohort, 37 genes were up-regulated and 79 were down-regulated in comparison to other outcome groups. To further investigate the genetic mechanisms governing IF, we developed four cell lines with acquired drug resistance: Jurkat and Sup T1; each having resistance to daunorubicin (DNR) and asparaginase (ASP). Using a comparative analysis for fold-change in gene expression among 6 IF patients and the T-ALL DNR and ASP-resistant cell lines, we identified seven genes that were up-regulated, and another set of seven genes that were commonly down-regulated. To validate the potential use of our 116-member gene set in predicting IF in T-ALL, we tested our genomic classifier in 42 cases which were treated on COG study 8704 and hybridized to the Affymetrix U133Av.2 chip. Because only 85 probes were shared between U133A Plus 2 and U133Av. 2 chips, we employed shrunken class centroids to constrain our classifier to 25 rank-ordered probes. This smaller classifier correctly identified the single IF case in 8704, as well as another patient who was an early treatment failure, indicating that similar genomic classifiers may identify IF patients in different clinical trials. These results indicate that genetic profiling may be useful in prospectively identifying IF patients in T-ALL. In addition, we identified genes that were commonly upregulated in IF patients and T-ALL cell lines with intrinsic drug resistance.

Persistent reversal of P-glycoprotein-mediated daunorubicin resistance by tetrandrine in multidrug-resistant human T lymphoblastoid leukemia MOLT-4 cells

Multidrug resistance (MDR) represents a major problem in cancer chemotherapy. P-glycoprotein (P-gp), the drug efflux pump that mediates this resistance, can be inhibited by compounds with a variety of pharmacological functions, thus circumventing the MDR phenotype. The present study was performed to evaluate a unique MDR-reversal feature of a bisbenzylisoquinoline alkaloid tetrandrine (TET) in a P-gp expressing MOLT-4 MDR line (MOLT-4/DNR) established in our laboratory. Cell viability was determined by an MTT assay. P-gp function was characterized by determining the Rh123 accumulation/efflux capacity. P-gp overexpression in resistant MOLT-4/DNR cells was confirmed by flow cytometry analysis after staining with phycoerythrin-conjugated anti-P-gp monoclonal antibody 17F9. Compared to ciclosporin A (CsA), TET exhibited stronger activity to reverse drug resistance to daunorubicin (DNR), vinblastine (VLB) and doxorubicin (DOX) in MOLT-4/DNR cells. TET showed no cytotoxic effects on parental MOLT-4 cells lacking P-gp expression or on the resistant MOLT-4/DNR cells. TET modulated DNR cytotoxicity even after it was washed with the medium for 24 h, while CsA almost completely lost its reversal capability 24 h after washing. TET and CsA similarly increased the accumulation of Rh123 in resistant MOLT-4/DNR cells. However, TET inhibited Rh123 efflux from resistant cells even after washing with the medium, while CsA rapidly lost its ability to inhibit Rh123 efflux after washing. The current study suggests that TET enhances the cytotoxicity of anticancer drugs in the P-gp expressing MDR cell line by modulating P-gp in a different manner to the well-known P-gp inhibitor CsA.

Characterization and modulation of drug transport kinetics in K562 c1.6 daunorubicin-resistant cell line.

The effects of cyclosporin A (CSA) and cellular energy depletion on daunorubicin (DAU) transport kinetics were investigated in a human erythroid leukaemia cell line K562 c1.6 selected for resistance to daunorubicin. K562 c1.6/DAU resistant cells displayed high levels of P-glycoprotein and a high level of multidrug resistance against several antitumour drugs. The resistance factors of K562 c1.6/DAU cells to DAU, doxorubicin, vinblastine and etoposide were 106, 114, 85 and 13 respectively. A 1.6-fold decrease (P < 0.01, n = 8) in DAU accumulation and a 4-fold increase (P < 0.001, n = 8) in DAU efflux were shown in the resistant cells when compared to K562 c1.6 drug-sensitive parental cells. K562 c1.6/DAU cells were also shown to reach a DAU saturation level (SL) 8-fold faster (P < 0.001, n = 8) than the parental cells. Addition of CSA to the resistant cells led to a dose-dependent increase in cellular DAU retention, while no such effect was observed in the sensitive cells by the introduction of CSA. Resistance to the antitumour drugs could be reduced to various extents by CSA. The patterns of changes and modulations of DAU transport kinetics, as well as chemosensitivity in K562 c1.6/DAU cells were found to be similar to a vinblastine-resistant leukaemia cell line CEM/VLB100. However, K562 c1.6/DAU cells were more resistant to DAU, doxorubicin and etoposide than the CEM/VLB100 cells. An increase in DAU accumulation, intracellular SL and the time to reach 90% saturation level (SL90), and a decrease in DAU efflux in the resistant but not the sensitive cells were found in response to ATP depletion by sodium azide. These effects could be completely reversed by addition of glucose. Our results suggest that the presence of an energy-dependent effluxing mechanism responsible for the decreased drug accumulation and enhanced drug efflux may make a major contribution to the mechanism of resistance in K562 c1.6/DAU resistant cells.

Cellular pharmacokinetics of daunorubicin: uptake by leukaemic cells in vivo and fate.

In 6 patients with acute myeloblastic leukaemia, daunorubicin was assayed in leukaemic cells from peripheral blood or bone marrow. The cells were separated from red blood cells and granulocytes by centrifugation on a Ficoll-Isopac gradient. The assay was performed by high performance liquid chromatography. Daunorubicin concentrations in peripheral leukaemic cells, 2 hours after the end of the infusion, were much higher than in plasma, the cell/plasma concentration ratio reaching about 350 and rising to almost 700 at 24 h. At that time, drug concentrations were even higher in the bone marrow leukaemic cells. The value of the assay of daunorubicin in cells as method for monitoring therapy is discussed.