Targeted blockage of signal transducer and activator of transcription 5 signaling pathway with decoy oligodeoxynucleotides suppresses leukemic K562 cell growth

Abstract

The protein signal transducer and activator of transcription 5 (STAT5) of the JAK/STAT pathway is constitutively activated because of its phosphorylation by tyrosine kinase activity of fusion protein BCR-ABL in chronic myelogenous leukemia (CML) cells. This study investigated the potential therapeutic effect of STAT5 decoy oligodeoxynucleotides (ODN) using leukemia K562 cells as a model. Our results showed that transfection of 21-mer-long STAT5 decoy ODN into K562 cells effectively inhibited cell proliferation and induced cell apoptosis. Further, STAT5 decoy ODN downregulated STAT5 targets bcl-xL, cyclinD1, and c-myc at both mRNA and protein levels in a sequence-specific manner. Collectively, these data demonstrate the therapeutic effect of blocking the STAT5 signal pathway by cis-element decoy for cancer characterized by constitutive STAT5 activation. Thus, our study provides support for STAT5 as a potential target downstream of BCR-ABL for CML treatment and helps establish the concept of targeting STAT5 by decoy ODN as a novel therapy approach for imatinib-resistant CML.

FIG. 1.

Incorporation and subcellular localization of FAM-labeled ODN in K562 cells. Dynamic change of intracellular FAM-labeled ODN in K562 cells on day 1 (A), day 3 (B), and day 5 (C) after transfection (original × 400). (D) Confocal laser scanning microscopy demonstrating internalization of the STAT5 decoy ODN in K562 cells. Internalized ODN (light gray, i) and nuclei (dark gray, ii) were overlaid with the corresponding differential interface contrast image iii. Intracellular distribution of FAM-labeled ODN was acquired by confocal laser scanning microscopy (i, FAM-labeled ODN, light gray; ii, nuclei, dark gray; iii, color overlay). The scale bar is 20 μm. STAT5, signal transducer and activator of transcription 5; ODN, oligodeoxynucleotides.

FIG. 2.

Effect of the STAT5 decoy ODN on K562 cell proliferation. K562 cells treated with 0–25 μM STAT5 decoy ODN demonstrated a dose-dependent cell proliferation inhibition (A). K562 cells (B) and HL-60 cells (C) were treated with the STAT5 decoy ODN or mutant ODN at a concentration of 25 μM. Viable cells were counted by trypan blue dye exclusion at indicated time points after treatment.

FIG. 3.

Effect of STAT5 decoy ODN on cell cycle and apoptosis of K562 cells. Cell cycle was assayed by the PI staining method (upper panel), and cell apoptosis was examined by the Annexin-V/PI double staining method (lower panel) as described in the Materials and Methods section. The numbers indicate the percentage of cells in each quadrant (lower left: FITC−/PI−, intact cells; lower right: FITC+/PI−, apoptotic cells; upper left: FITC−/PI+, necrotic cells; upper right: FITC+/PI+, late apoptotic or necrotic cells). PI, propidium iodide.

FIG. 4.

Ultrastructural change of K562 cells at 48 h after transfection with STAT5 decoy ODN. Untreated K562 cells (A) exhibited “normal” morphology, whereas ODN-treated cells (B–D) exhibited morphologic changes of apoptosis (indicated by white arrow), such as karyopyknosis and nuclear fragmentation (B), increased electron density (C), and apoptotic body (D).

FIG. 5.

Effect of STAT5 decoy ODN on STAT5 promoter activity. K562 cells were transiently cotransfected with luciferase reporter plasmid and STAT5 decoy or mutant ODNs. Cells were harvested and luciferase activity was determined at 48 h after transfection. Luciferase activity was represented as arbitrary units and as mean ± standard deviation from three independent experiments. There was significant difference between the decoy ODN-treated group, the mutant ODN-treated group, and untreated groups; *p < 0.05.

FIG. 6.

Specific binding of STAT5 to STAT5 decoy ODN. Electrophoretic mobility shift assay was performed using the STAT5 binding sequences and nuclear protein extracts of K562 cells. Competitions were performed with a 100-fold molar excess of unlabeled probe or a 100-fold molar excess of mutated probe. For supershift experiments, nuclear protein extracts were preincubated with STAT5a, STAT5b, or STAT3 monoclonal antibody, respectively. Arrows indicate migrational location of supershift band, STAT5-DNA complex, or free probe.

FIG. 7.

Effect of STAT5 decoy ODN on transcription of c-myc, bcl-xL, and cyclinD1. (A) Semiquantitative reverse transcription (RT)–polymerase chain reaction results for c-myc, bcl-xL, and cyclinD1 in K562 and HL-60 cells at 48 h after STAT5 decoy or mutant ODN treatment. M: DNA marker; lanes 1, 2: c-myc; lanes 3, 4: GAPDH; lanes 5, 6: bcl-xL; lanes 7, 8: cyclinD1; lanes 1, 3, 5, 7: mutant ODN group; lanes 2, 4, 6, 8: decoy ODN group. (B) Gel intensity was analyzed using the Bio-Rad software (Gel Doc 1000). Values are represented as mean ± standard deviation of three independent experiments. Statistical significance was determined as *p < 0.05 compared with the mutant ODN control.

FIG. 8.

Effect of STAT5 decoy ODN on protein expression of c-myc, bcl-xL, and cyclinD1. Lanes 1, 3, 5: mutant ODN group; lanes 2, 4, 6: decoy ODN group. (A) Western blot analysis of c-myc, bcl-xL, and cyclinD1 in lysates of K562 and HL-60 cells at 48 h after STAT5 decoy or mutant ODN treatment. β-Actin was used as a loading control. (B) Figures were representative of three independent experiments with similar results. Statistical significance was determined as *p < 0.05 compared with the mutant ODN control.