wogonin and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

The 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.. mRNA 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.. We 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. Our 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. Video Abstract.

Topics: Animals; Apoptosis; Cell Proliferation; Cyclin-Dependent Kinase 9; Flavanones; GATA1 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Molecular Targeted Therapy; Multiprotein Complexes; Nuclear Proteins; Phosphorylation; Positive Transcriptional Elongation Factor B; Transcription Factors; Xenograft Model Antitumor Assays

Constitutive NF-E2-related factor 2 (Nrf2, NFE2L2) activation has been recently reported to play a pivotal role in enhancing cell survival and resistance to anticancer drugs in many tumors. Wogonin had strong reversal potency via reduction of Nrf2 mRNA in Adriamycin (ADR)-induced resistant human chronic myelogenous leukemia (CML) K562/A02, but the mechanism of reduction of Nrf2 mRNA was still unclear. In this study, we aimed to delineate the mechanism by which Wogonin suppressed transcription of Nrf2 in resistant CML cells and further evaluate the reversal effects of Wogonin on the established animal models. Data indicated that Wogonin suppressed transcription of Nrf2 by NF-κB inactivation. Wogonin inhibited the binding of p65 to Nrf2 by suppression of the κB-binding activity. Further research revealed the κB2 site was responsible for the decreased Nrf2 by Wogonin in resistant K562 cells. Furthermore, reduction of pY705-Stat3 was involved in inhibition of the binding of p65 to Nrf2 by Wogonin. In vivo, Wogonin potentiated the inhibitory effect of ADR on leukemia development by suppressing pY705-Stat3 and Nrf2 signaling. In summary, these results demonstrated Wogonin could combat chemoresistance effectively through inhibiting Nrf2 via Stat3/NF-κB signaling, and supported that Wogonin can be developed into an efficient natural sensitizer for resistant human myelogenous leukemia.

Topics: Animals; Apoptosis; Doxorubicin; Drug Resistance, Neoplasm; Flavanones; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Inbred NOD; Mice, SCID; NF-E2-Related Factor 2; NF-kappa B; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transcription Factor RelA; Transcription, Genetic

Constitutive Stat5 activation enhanced cell survival and resistance to imatinib (IM) in chronic myelogenous leukemia (CML) cells. However, the mechanism of Stat5 activation in mediating resistance to IM in bone marrow (BM) microenvironment has not been evaluated precisely. In this study, we reported HS-5-derived conditioned medium (CM) significantly enhanced IM resistance in K562 and KU812. Interestingly, upregulation of the proportion of CD34+ subpopulation was found in CML cells. Subsequently, the BCR/ABL-independent activation of Stat5 increased P-glycoprotein (P-gp) activity in CM-mediated protection of CML stem cells (LSCs) from IM. Further research revealed Stat5 activation increased the DNA binding activity of NF-κB though binding of p-Stat5 and p-RelA in nucleus. Moreover, highly acetylated RelA was required for Stat5-mediated RelA nuclear binding. The study further confirmed that Wogonin potentiated the inhibitory effects of IM on leukemia development by suppressing Stat5 pathway both in CM model and the K562 xenograft model. In summary, results clearly demonstrated BCR/ABL-independent Stat5 survival pathway could contribute to resistance of CML LSCs to IM in BM microenvironment and suggested that natural durgs effectively inhibiting Stat5 may be an attractive approach to overcome resistance to BCR/ABL kinase inhibitors.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Bone Marrow; Cell Line; Cell Line, Tumor; Culture Media, Conditioned; Drug Resistance, Neoplasm; Drug Synergism; Flavanones; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice, Inbred NOD; Mice, SCID; Microscopy, Confocal; NF-kappa B; Signal Transduction; STAT5 Transcription Factor; Tumor Microenvironment; Xenograft Model Antitumor Assays

Wogonin, a flavonoid derived from Scutellaria baicalensis Georgi, has been demonstrated to be highly effective in treating hematologic malignancies. In this study, we investigated the anticancer effects of wogonin on K562 cells, K562 imatinib-resistant cells, and primary patient-derived CML cells. Wogonin up-regulated transcription factor GATA-1 and enhanced binding between GATA-1 and FOG-1, thereby increasing expression of erythroid-differentiation genes. Wogonin also up-regulated the expression of p21 and induced cell cycle arrest. Studies employing benzidine staining and analyses of cell surface markers glycophorin A (GPA) and CD71 indicated that wogonin promoted differentiation of K562, imatinib-resistant K562, and primary patient-derived CML cells. Wogonin also enhanced binding between GATA-1 and MEK, resulting in inhibition of the growth of CML cells. Additionally, in vivo studies showed that wogonin decreased the number of CML cells and prolonged survival of NOD/SCID mice injected with K562 and imatinib-resistant K562 cells. These data suggested that wogonin induces cycle arrest and erythroid differentiation in vitro and inhibits proliferation in vivo.

Topics: Animals; Antineoplastic Agents; Benzamides; Cell Cycle Checkpoints; Cell Differentiation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Erythroid Cells; Extracellular Signal-Regulated MAP Kinases; Female; Flavanones; GATA1 Transcription Factor; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Kinase Kinases; Mice, Inbred NOD; Mice, SCID; Nuclear Proteins; Piperazines; Protein Kinase Inhibitors; Pyrimidines; RNA Interference; Signal Transduction; Time Factors; Transcription Factors; Transfection; Tumor Cells, Cultured; Xenograft Model Antitumor Assays