Journal List > Korean J Hematol > v.40(3) > 1032620

Lee, Jin, Kim, and Kim: The Effect of Auranofin on Thrombomodulin Expression in Acute Promyelocytic Leukemia Cell

Abstract

Background

Acute promyelocytic leukemia (APL) is distinguished from other forms of leukemia by its association with bleeding diatheses. All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), which have been effectively used in the treatment of the APL, promptly improve coagulation/bleeding syndromes by regulating the expressions of tissue factor (TF) and thrombomodulin (TM). We have previously shown a novel activity of auranofin (AF), in that it induced apoptosis and differentiation of NB4 cells. To study whether AF also possesses similar anticoagulant effects to those of ATRA and As2O3, its effects on the expressions of TM and TF were investigated.

Methods

NB4 cells derived from APL were incubated with 1μM of AF. After incubation for 12, 24 and 48 hours, the AF-regulated expressions of TM and TF were analyzed by RT-PCR, Northern blot and Western blot. The assay for the TM antigen on the cell surface was performed using a flow cytometry.

Results

The expression of the TM gene was increased for upto 12 hours after the AF treatment, but no change was observed in the expression of the TF gene. Western blot analysis also demonstrated that AF increased the level of TM proteinin a time-dependent manner. FACS data showed the TM antigen on the cell surface to gradually increase for upto 48 hours in AF-treated cells.

Conclusion

The results of this study indicate that AF can have an antithrombotic function via the upregulation of the expression of TM, which suggests it may partially contribute to the improvement of coagulopathies in APL.

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Fig. 1.
The structure of AF.
kjh-40-135f1.tif
Fig. 2.
The effect of AF on TM and TF gene expressions. NB4 cells were treated with 1μM of AF for 12 and 24 hours, and then total cellular RNA was isolated using RNAzol B solution. (A) To analyze TM and TF gene expressions, RT-PCR was performed as described in materials and methods. Equal amounts of RNA used in each sample were evaluated by detecting β-actin expression. (B) Northern blot analysis. 20μg of total RNA per lane was loaded and electrophoresed on a 1.2% agarose/formaldehyde gel. DIG-labeled PCR product of human TM gene was used as a probe. 28 S and 18 S ribosomal RNA shown in the EtBr-stained gel indicate the equal amount of loaded RNA samples. The results shown (A and B) are representative of at least two independent experiments.
kjh-40-135f2.tif
Fig. 3.
The enhancement of TM antigen level by AF. After treatment with 1μM of AF for the indicated time, 50μg of cellular proteins were subjected to SDS-polyacrylamide gel electrophoresis and the TM protein was analyzed by Western blot by using anti-TM antibody. The blot shown is representative of three independent experiments.
kjh-40-135f3.tif
Fig. 4.
The flow cytometric analysis of surface TM antigen. The NB4 cells were treated with 1μM of AF for 0, 12, 24 and 48 hours. After that, the cells were sequentially incubated with anti-TM antibody and FITC -conjugated anti-rabbit IgG, and analyzed by a flow cytometer. Note that the population of cells with FITC-fluorescence was elevated in a time-dependent manner. Dashed line is for isotype control by using rabbit IgG instead of anti-TM antibody. The data shown represent a typical result obtained from two independent experiments.
kjh-40-135f4.tif
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