chir-99021 and 6-bromoindirubin-3--oxime

Topics: Animals; Biomarkers; Buffaloes; Cell Differentiation; Embryonic Stem Cells; Female; Fertilization in Vitro; Gene Expression Regulation, Developmental; Glycogen Synthase Kinase 3; Indoles; Oximes; Pluripotent Stem Cells; Pyridines; Pyrimidines; Up-Regulation; Wnt Signaling Pathway

Cholangiocarcinoma (CCA) is the most common type of malignant tumor of the bile duct and is characterized by high morbidity and mortality; it is difficult to diagnose in the early stages and responds poorly to current conventional radiotherapy and chemotherapy. The present study investigated the role of GSK‑3β signaling on the anticancer effects of doxorubicin in human CCA cells. Blocking GSK‑3β enhanced the sensitivity of human CCA cells to doxorubicin (Dox)‑induced apoptosis, which was accompanied by decreased AKT and focal adhesion kinase (FAK) activity. Moreover, inhibiting GSK‑3β using 6‑bromoindirubin‑3'‑oxime, CHIR99021 or small interfering RNA decreased phosphorylation of FAK and AKT, and promoted apoptosis of Dox‑induced human CCA cells. Moreover, FAK inhibition suppressed AKT activity independently of phosphoinositide 3‑kinase activity. These results indicated that GSK‑3β protects human CCA cells against Dox‑induced apoptosis via sustaining FAK/AKT activity.

Topics: Bile Duct Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cholangiocarcinoma; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Oximes; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; RNA, Small Interfering

Wnt/β-catenin signalling plays a prominent role in maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). microRNAs (miRNAs) have critical roles in maintaining pluripotency and directing reprogramming. To investigate the effect of GSK3 inhibitors on miRNA expression, we analysed the miRNA expression profile of J1 mESCs in the absence or presence of CHIR99021 (CHIR) or 6-bromoindirubin-3'-oxime (BIO) by small RNA deep-sequencing. The results demonstrate that CHIR and BIO decrease mature miRNAs of most miRNA species, 90.4% and 98.1% of the differentially expressed miRNAs in BIO and CHIR treated cells were downregulated respectively. CHIR and BIO treatment leads to a slight upregulation of the primary transcripts of the miR-302-367 cluster and miR-181 family of miRNAs, these miRNAs are activated by Wnt/β-catenin signalling. However, the precursor and mature form of the miR-302-367 cluster and miR-181 family of miRNAs are downregulated by CHIR, suggesting CHIR inhibits maturation of primary miRNA. Western blot analysis shows that BIO and CHIR treatment leads to a reduction of the RNase III enzyme Drosha in the nucleus. These data suggest that BIO and CHIR inhibit miRNA maturation by disturbing nuclear localisation of Drosha. Results also show that BIO and CHIR induce miR-211 expression in J1 mESCs.

Topics: Animals; beta Catenin; Cell Cycle; Cell Line; Cell Self Renewal; Cell Shape; Colony-Forming Units Assay; Epigenesis, Genetic; Glycogen Synthase Kinase 3; High-Throughput Nucleotide Sequencing; Indoles; Mice; MicroRNAs; Mouse Embryonic Stem Cells; Multigene Family; Oximes; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Ribonuclease III; RNA Processing, Post-Transcriptional; Sequence Analysis, RNA; Transcription Factors; Wnt Signaling Pathway

Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/β-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/β-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3β (GSK3β) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the β-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3β inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a β-catenin-independent manner. In conclusion, GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.

Topics: Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Cell Line, Tumor; Cells, Cultured; Cytochrome P-450 Enzyme Inducers; Cytochrome P-450 Enzyme System; Enzyme Induction; Female; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hepatocytes; Humans; Indoles; Male; Molecular Docking Simulation; Organometallic Compounds; Oximes; Pregnane X Receptor; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Receptors, Aryl Hydrocarbon; Receptors, Steroid; Recombinant Fusion Proteins; RNA Interference; Wnt Signaling Pathway

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

Topics: Acute Radiation Syndrome; Aminophenols; Animals; Apoptosis; Bone Marrow; Cells, Cultured; Colony-Forming Units Assay; Drug Evaluation, Preclinical; Glycogen Synthase Kinase 3; Hematopoiesis; Hematopoietic Stem Cells; Indoles; Injections, Subcutaneous; Maleimides; Mice; Mice, Inbred C57BL; Oximes; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Radiation Injuries, Experimental; Whole-Body Irradiation

There is considerable interest in differentiating human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and pancreatic cells for in vitro disease modeling and cell replacement therapy. Numerous protocols use fetal bovine serum, which contains poorly defined factors to induce DE formation. Here, we compared Wnt and BMP in their ability to cooperate with Activin signaling to promote DE formation in a chemically defined medium. Varying concentrations of WNT3A, glycogen synthase kinase (GSK)-3 inhibitors CHIR99021 and 6-bromoindirubin-3'-oxime (BIO), and BMP4 could independently co-operate with Activin to effectively induce DE formation even in the absence of serum. Overall, CHIR99021 is favored due to its cost effectiveness. Surprisingly, WNT3A was ineffective in suppressing E-CADHERIN/CDH1 and pluripotency factor gene expression unlike GSK-3 inhibitors or BMP4. Our findings indicate that both Wnt and BMP effectively synergize with Activin signaling to generate DE from hPSCs, although WNT3A requires additional factors to suppress the pluripotency program inherent in hPSCs.

Topics: Activins; Blotting, Western; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Line; Endoderm; Flow Cytometry; Glycogen Synthase Kinase 3; Humans; Indoles; Oximes; Pluripotent Stem Cells; Pyridines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Serum; Wnt Signaling Pathway