Thymoquinone Induces Downregulation of BCR-ABL/JAK/STAT Pathway and Apoptosis in K562 Leukemia Cells

Abstract

Objective: BCR ABL oncogene encodes the BCR-ABL chimeric protein, which is a constitutively activated non-receptor tyrosine kinase. The BCR-ABL oncoprotein is a key molecular basis for the pathogenesis of chronic myeloid leukemia (CML) via activation of several downstream signaling pathways including JAK/STAT pathway. Development of leukemia involves constitutive activation of signaling molecules including, JAK2, STAT3, STAT5A and STAT5B. Thymoquinone (TQ) is a bioactive constituent of Nigella sativa that has shown anticancer properties in various cancers. The present study aimed to evaluate the effect of TQ on the expression of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes and their consequences on the cell proliferation and apoptosis in K562 CML cells.

Methods: BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis.

Results: TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001).

Conclusion: our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.

Keywords: Apoptosis; BCR-ABL; CML; STAT; thymoquinone.

Figure 1

Dose-Dependent Inhibitory Effects of TQ on the Proliferation of K562 Cells. Trypan blue exclusion assay was used to assess the growth inhibitory effects of different concentrations of TQ (0 μM, 3 μM, 6 μM, 9 μM, 12 μM, 15 μM, 18 μM, 21 μM, 24 μM, 27 μM and 30 μM) on K562 cells after 24 hours of treatment, the IC50 value was 23 ± 4.3 μM. Data were stated as percentages of viable cells relative to the untreated cells. The values were presented as mean ± SD of three independent experiments. * p < 0.05, **p < 0.01, ***p < 0.00

Figure 2

Dose-Course Flow Cytometry Analysis of K562 Cells after TQ Treatment. (A) Cells were treated with 12 μM, 18 μM, 24 μM, 27 μM and 30 μM of TQ for 24 hours. The apoptosis events in K562 cells were assessed using Annexin V-FITC and PI- double stain. (B) The bar graph is the percentages of cells relative to the untreated cells after 24 hours of treatment. Values were presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 3

Effect of TQ on the mRNA expression of BCRABL, JAK2, STAT3, STAT5A and STAT5B in K562 cells. RT-qPCR analysis were performed in triplicate before and after treatment with 15 μM of TQ for 48 hours. The graph showed the down-regulation of BCRABL, JAK2, STAT3, STAT5A and STAT5B. Wilcoxon signed rank test was conducted and values were stated as median (interquartile range); ***p < 0.001

Figure 4.

Effect of TQ on JAK2 and STAT5 Protein Levels in K562 Cells. Jess simple western analysis was performed in triplicate. The cells were treated with 15 μM of TQ for 48 hours. (A) Representative image of JAK2 and STAT5 protein levels from Jess simple western blotting. (B) Bar graph shows the reduction of JAK2 and STAT5 protein levels in TQ-treated K562 cells. Wilcoxon signed rank test was conducted and values were stated as median (interquartile range); ***p < 0.001