Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair

Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viability

Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation

Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation.

Bone Morphogenetic Protein 2 Is Involved in Oocyte Maturation Through an Autocrine/Paracrine Pathway in Scylla paramamosain

Ovary-secreted autocrine/paracrine factors play important roles in regulating oocyte maturation via the autocrine/paracrine pathway. This study aimed to evaluate the functions of bone morphogenetic protein 2 (BMP2) in oocyte maturation and communication between follicle cells and oocytes. In our study, we first identified BMP2 from the mud crab Scylla paramamosain. Quantitative real-time PCR showed that BMP2 was detected in diverse tissues, notably in the ovary, stomach and gill. The expression levels of BMP2 transcripts increased during vitellogenesis. Spatial expression of BMP2 and receptors in the ovary revealed that BMP2 was exclusively detected in oocytes, whereas the receptors were expressed in both follicle cells and oocytes. RNAi tests revealed that the expression of cyclin B first decreased at 2 h and then increased at 4 h after BMP2 knockdown. These combined findings suggest that BMP2 may promote oocyte maturation through an autocrine/paracrine pathway in S. paramamosain.

Quantitative phosphoproteomics reveals regulatory state dependent efficacy of chemical Cdk1 inhibition and distinct Cdk5 complexes as novel Wee1 substrates

Wee1 kinase plays a central role in the eukaryotic cell cycle via its well-known negative regulation of Cdk1 activity at the G2/M transition, preventing progression into mitosis until DNA replication and/or DNA damage repair is complete. Recent genetic evidence in yeast, flies and human cells have suggested additional functions of Wee1 in mitosis and during mitotic exit, respectively. To discover new candidate substrates of Wee1 kinase, we used SILAC-based phosphoproteomics and selective chemical inhibition to quantitatively compare phosphorylation site abundances in the presence and absence of Wee1 activity. Unexpectedly, we uncovered a role for the Wee1-dependent phosphorylation of Cdk1-cyclin B at tyrosine 15 (Y15) in facilitating chemical inhibition of Cdk1-cyclin B by the inhibitor RO3306. Thermal shift stability assays demonstrated greater binding affinity of RO3306 for Y15-phosphorylated Cdk1-cyclin B versus unphosphorylated complex, providing an additional molecular basis for the observed Wee1 inhibitor-based toxicity in human cells. In addition, our experiments identified Cdk5-CABLES and Cdk5-cyclin B as novel substrates of Wee1 during chemically induced exit from mitosis. Collectively, these experiments facilitate a greater understanding of the Wee1-Cdk1 signaling axis and uncover new candidate substrates for Wee1.

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1 activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CDK1 activating kinases (CAK) or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a tripartite complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).

Scaffold Repurposing of In-House Small Molecule Candidates Leads to Discovery of First-in-Class CDK-1/HER-2 Dual Inhibitors: In Vitro and In Silico Screening

Recently, multitargeted drugs are considered a potential approach in treating cancer. In this study, twelve in-house indole-based derivatives were preliminary evaluated for their inhibitory activities over VEGFR-2, CDK-1/cyclin B and HER-2. Compound 15l showed the most inhibitory activities among the tested derivatives over CDK-1/cyclin B and HER-2. Compound 15l was tested for its selectivity in a small kinase panel. It showed dual selectivity for CDK-1/cyclin B and HER-2. Moreover, in vitro cytotoxicity assay was assessed for the selected series against nine NCI cell lines. Compound 15l showed the most potent inhibitory activities among the tested compounds. A deep in silico molecular docking study was conducted for compound 15l to identify the possible binding modes into CDK-1/cyclin B and HER-2. The docking results revealed that compound 15l displayed interesting binding modes with the key amino acids in the binding sites of both kinases. In vitro and in silico studies demonstrate the indole-based derivative 15l as a selective dual CDK-1 and HER-2 inhibitor. This emphasizes a new challenge in drug development strategies and signals a significant milestone for further structural and molecular optimization of these indole-based derivatives in order to achieve a drug-like property.

Prenatal Glucocorticoid Administration Accelerates the Maturation of Fetal Rat Hepatocyte

Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because the liver is immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats.Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real time-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, HNF4α, HGF, Thy-1, cyclin B, and CDK1 in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production.The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration.The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in premature fetuses.