Abstract
BackgroundThe positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. Cyclin-dependent kinase 9 (CDK9), a core component of P-TEFb, regulates the process of transcription elongation, which is associated with differentiation and apoptosis in many cancer types. Wogonin, a natural CDK9 inhibitor isolated from Scutellaria baicalensis. This study aimed to investigate the involved molecular mechanisms of wogonin on anti- chronic myeloid leukemia (CML) cells.Materials and methodsmRNA and protein levels were analysed by RT-qPCR and western blot. Flow cytometry was used to assess cell differentiation and apoptosis. Cell transfection, immunofluorescence analysis and co-immunoprecipitation (co-IP) assays were applied to address the potential regulatory mechanism of wogonin. KU-812 cells xenograft NOD/SCID mice model was used to assess and verify the mechanism in vivo.ResultsWe reported that the anti-CML effects in K562, KU-812 and primary CML cells induced by wogonin were regulated by P-TEFb complex. We also confirmed the relationship between CDK9 and erythroid differentiation via knockdown the expression of CDK9. For further study the mechanism of erythroid differentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA-1 and FOG-1 but decreased the binding between GATA-1 and RUNX1, which were depended on P-TEFb. Also, wogonin induced apoptosis and decreased the mRNA and protein levels of MCL-1 in KU-812 cells, which is the downstream of P-TEFb. In vivo studies showed wogonin had good anti-tumor effects in KU-812 xenografts NOD/ SCID mice model and decreased the proportion of human CD45+ cells in spleens of mice. We also verified that wogonin exhibited anti-CML effects through modulating P-TEFb activity in vivo.ConclusionsOur study indicated a special mechanism involving the regulation of P-TEFb kinase activity in CML cells, providing evidences for further application of wogonin in CML clinical treatment.8cwZMBLp1zasMdEPaNEt3gVideo
Highlights
The positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription
We reported that the anti-chronic myeloid leukemia (CML) effects in K562, KU-812 and primary CML cells induced by wogonin were regulated by P-TEFb complex
For further study the mechanism of erythroid differentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA binding protein 1 (GATA-1) and friend of GATA-1 (FOG-1) but decreased the binding between GATA-1 and Runt-related transcription factor 1 (RUNX1), which were depended on P-TEFb
Summary
The positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. CML is a malignant clonal disease that originates in hematopoietic stem cells [1]. It is divided into three phases including chronic phase, accelerated phase and blast phase [2]. Tyrosine kinase inhibitors (TKIs) are currently the preferred drug in clinical CML patients and Imatinib is the first generation of TKIs used for the treatment of CML [5]. TKIs therapy can alleviate the condition of most CML patients, long-term use cannot eliminate the existence of factors such as leukemia stem cells and high price [6, 7].
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