Cyclin-dependent Protein Kinase Research Articles

Establishment of high-throughput screening HTRF assay for identification small molecule inhibitors of Skp2-Cks1

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclin-dependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks1). Skp2 is overexpressed in esophageal carcinoma tissues and closely related with tumor poor prognosis, and perturbation of the Skp2-Cks1 interaction by inhibitors or RNAi could inhibit the proliferation and metastasis of tumor cells. Therefore, inhibition of Skp2 function by small-molecule compounds targeting Skp2-Cks1 interaction is emerging as a promising and novel anti-cancer strategy. In this study, we establish an improved high-throughput screening platform to screen Skp2 inhibitors targeting Skp2-Cks1interaction, which may provide a new therapeutic approach for the clinic.

Overexpression of cyclin-dependent kinase 1 in esophageal squamous cell carcinoma and its clinical significance.

Cyclin‐dependent kinase 1 (CDK1) plays a significant role in certain malignancies. However, it remains unclear whether CDK1 plays a role in esophageal squamous cell carcinoma (ESCC). The aim of this study was to analyze the expression and clinical value of CDK1 in ESCC. CDK1 protein in 151 ESCC tissues and 138 normal esophageal tissues was detected by immunohistochemistry. RNA‐seq of eight pairs of ESCC and adjacent esophageal specimens was performed to evaluate the levels of CDK1 mRNA. Microarray and external RNA‐seq data from 664 cases of ESCC and 1733 cases of control tissues were used to verify the difference in CDK1 expression between the two groups. A comprehensive analysis of all data was performed to evaluate the difference in CDK1 between ESCC tissues and control tissues. Further, functional enrichment analyses were performed based on differentially expressed genes (DEGs) of ESCC and co‐expressed genes (CEGs) of CDK1. In addition, a lncRNA‐miRNA‐CDK1 network was constructed. The expression of CDK1 protein was obviously increased in ESCC tissues (3.540 ± 2.923 vs. 1.040 ± 1.632, P < 0.001). RNA‐seq indicated that the mRNA level of CDK1 was also highly expressed in ESCC tissues (5.261 ± 0.703 vs. 2.229 ± 1.161, P < 0.0001). Comprehensive analysis revealed consistent up‐regulation of CDK1 (SMD = 1.41; 95% CI 1.00–1.83). Further, functional enrichment analyses revealed that the functions of these genes were mainly concentrated in the cell cycle. A triple regulatory network of PVT1‐hsa‐miR‐145‐5p/hsa‐miR‐30c‐5p‐CDK1 was constructed using in silico analysis. In summary, overexpression of CDK1 is closely related to ESCC tumorigenesis.

Cdc42-Specific GTPase-Activating Protein Rga1 Squelches Crosstalk between the High-Osmolarity Glycerol (HOG) and Mating Pheromone Response MAPK Pathways.

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of “kinetic proofreading”. Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

Increased LINC00922 in preeclampsia regulates the proliferation, invasion, and migration of placental trophoblast cells.

BackgroundRecent studies have shown that the abnormal expression of long-chain non-coding RNAs (lncRNAs) can significantly affect the biological function of trophoblast cells and lead to the occurrence of preeclampsia (PE). This study explores the expression of lncRNA LINC00922 in PE and its effect on the function of placental trophoblast cells, along with the corresponding molecular mechanism, providing a theoretical basis and molecular target for understanding the occurrence, early diagnosis, and targeted therapy of PE.MethodsFluorescence quantitative PCR was used to detect the expression of LINC00922 in 30 cases of PE tissues and normal tissues. The CCK-8 assay, clone formation experiment, and flow cytometry were used to detect the effects of LINC00922 knockdown or overexpression on the proliferation, colony formation, and cell cycle of HTR-8/SVneo placental trophoblast cells. The Transwell assay was used to detect the effects of LINC00922 knockdown or overexpression on the invasion and migration of HTR 8/SVneo cells, and western blot was used to detect the expression of cell cycle-related proteins and invasion and migration-related proteins.ResultsLINC00922 was highly expressed in PE tissues. Knockdown of LINC00922 significantly inhibited the proliferation, invasion, and migration of HTR-8/SVneo cells, along with colony formation and the ability to induce cell cycle arrest in the G0/G1 phase. However, overexpression of LINC00922 had the opposite effect. Knockdown or overexpression of LINC00922 significantly affected the expression of cell cycle-related proteins cyclin-dependent kinase 2 (CDK2), G1/S-specific cyclin-D1 (Cyclin D1), p21, proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 (MMP-9), vimentin, and E-cadherin, but had no significant effect on the expression of matrix metallopeptidase 2 (MMP-2).ConclusionsLINC00922 was highly expressed in PE, and functional experiments showed that LINC00922 could significantly affect the proliferation and invasion abilities of placental trophoblast cells, suggesting that LINC00922 may play an important role in the occurrence, early diagnosis, and treatment of PE.

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis.

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

The modulatory properties of Astragalus membranaceus treatment on endometrial cancer: an integrated pharmacological method.

Astragalus membranaceus is a traditional Chinese medicine and has been used for adjuvant clinical therapy for a variety of cancers. However, the mechanism of its action on endometrial carcinoma is unclear. Based on the Gene Expression Omnibus (GEO) database, the Cancer Genome Atlas (TCGA) database, and the Traditional Chinese Medicine System Pharmacology Database (TCMSP™), the drug and target compounds were initially screened to construct a common network module. Twenty active compounds in Astragalus membranaceus were successfully identified, which hit by 463 potential targets related to endometrial cancer. Eight of the more highly predictive compounds (such as Jaranol, Bifendate, Isorhamnetin, Calycosin, 7-O-methylisomucronulatol, Formononetin, Kaempferol, Quercetin) were involved in DNA integrity checkpoint, cyclin-dependent protein kinase holoenzyme complex, and histone kinase activity. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway confirmed that Astragalus membranaceus might play a role in the treatment of endometrial cancer through p53 signalling pathway, transcriptional misregulation in cancer, and endometrial cancer signalling pathway. Drug-target-pathway networks were constructed using Cytoscape to provide a visual perspective. In addition, we verified that formononetin inhibited the proliferation of endometrial cancer cells through cell viability tests and clone formation tests. And qPCR and western blot found that formononetin exerts anti-cancer effects by promoting the expression of estrogen receptor beta (ERβ) and p53. Based on a systematic network pharmacology approach, our works successfully predict the active ingredients and potential targets of Astragalus membranaceus for application to endometrial cancer and helps to illustrate mechanism of action on a comprehensive level.

Fruit size control by a zinc finger protein regulating pericarp cell size in tomato

Fruit size is largely defined by the number and size of cells in the fruit. Endoreduplication – a specialized cell cycle – is highly associated with cell expansion during tomato fruit growth. However, how endoreduplication coupled with cell size is regulated remains poorly understood. In this study, we identified a zinc finger gene SlPZF1 (SolanumlycopersicumPERICARP-ASSOCIATEDZINCFINGER PROTEIN 1) that was highly expressed in the pericarp of developing fruits. Plants with altered SlPZF1 expression produced smaller fruits due to the reduction in cell size associated with weakened endoreduplication. Overexpressing SlPZF1 delayed cell division phase by enhancing early expression of several key cell cycle regulators including SlCYCD3;1 and two plant specific mitotic cyclin-dependent protein kinase (SlCDKB1 and SlCDKB2) in the pericarp tissue. Furthermore, we identified 14 putative SlPZF1 interacting proteins (PZFIs) via yeast two hybrid screening. Several PZFIs, including Pre-mRNA-splicing factor (SlSMP1/PZFI4), PAPA-1-like conserved region family protein (PZFI6), Fanconi anemia complex components (PZFI3 and PZFI10) and bHLH transcription factor LONESOME HIGHWAY (SlLHW/PZFI14), are putatively involved in cell cycle regulation. Our results demonstrate that fruit growth in tomato requires balanced expression of the novel cell size regulator SlPZF1.

Effect of MiR-142-3p Targeting HOXA5 on Proliferation, Cycle Arrest and Apoptosis of Acute B Lymphocytic Leukemia Cells

To investigate the effect and molecular mechanism of miR-142-3p to the proliferation, cycle and apoptosis of acute B lymphocytic leukemia (B-ALL) cells by regulating the homeobox gene 5 (HOXA5) expression. Real-time fluorescence quantitative PCR was used to detect the expression levels of miR-142-3p and HOXA5 in human B-ALL cell Nalm6 cell line and human B lymphoblast Hmy2-cir cells. Nalm6 was transfected by using liposome transfection technology, miR-142-3p mimic, pcDNA-HOXA5 overexpression plasmid, miR-142-3p mimic+pcDNA-HOXA5 overexpression plasmid, and control. The binding site of HOXA5 and miR-142-3p was predicted according to microRNA.org, and the targeting relationship between miR-142-3p and HOXA5 gene was detected by double luciferase reporter gene experiment. The effect of miR-142-3p to the proliferation of Nalm6 cells was detected using the Cell Counting Box-8 (CCK-8) method and cell clone formation experiments. Flow cytometry was used to detect the effects of miR-142-3p to cell cycle distribution and apoptosis of Nalm6 cells. The expression levels of cell cycle-related proteins, including G1 /S-specific cyclin-D1 (CyclinD1), Cyclin-dependent kinase 4 (CDK4) and B-cell lymphoma/ leukemia-2 protein (BCL-2), BCL-2 related X protein (Bax), cysteine-aspartate-specific protease (Caspase-3) were detected by Western blot. Compared with Hmy2-cir cells, miR-142-3p showed low expression in Nalm6 cells and HOXA5 showed high expression (P<0.05). MiR-142-3p and HOXA5 3'-UTR showed complementary binding regions, the luciferase activity of miR-142-3p mimic and wild-type HOXA5 3'-UTR was significantly lower than that of miR-142-3p negative control and wild-type HOXA5 3'-UTR (P<0.05). The proliferation of Nalm6 cells and the number of cell clones could be inhibited by miR-142-3p mimic after 48 and 72 hours of transfection (P<0.05), which causing G1 phase arrest of Nalm6 cells and inhibiting the expression of CyclinD1 and CDK4 protein (P<0.01), promoting the apoptosis of Nalm6 cells, and inhibiting the expression of BCL-2 protein, moreover, promoting the expression of Bax and Caspase-3 protein (P<0.05). MiR-142-3p can inhibit the proliferation of Nalm6 cells by targeting down-regulation the expression of HOXA5, arrest the G1 phase of cells, and promote apoptosis of the cells.

TP53 and CDKN2A mutations in patients with early-stage lung squamous cell carcinoma: an analysis of the correlations and prognostic outcomes.

BackgroundLung squamous cell carcinoma (LUSC) is characterized by frequent mutations of tumor protein p53 (TP53) and cyclin dependent kinase inhibitor 2A (CDKN2A). However, to date, the impact of TP53/CDKN2A status on the clinical outcome of patients with early-stage LUSC is unclear.MethodsTissue samples from 16 early-stage, surgically resected LUSCs were analyzed by next-generation sequencing (NGS). Information regarding TP53 and CDKN2A alterations and patient survival time was downloaded from The Cancer Genome Atlas (TCGA) database. The associations between TP53 and CDKN2A status and tumor characteristics, outcomes including overall survival (OS) and disease-free survival (DFS), and mutation counts were investigated.ResultsTP53 and CDKN2A exhibited a high frequency of somatic mutations in early-stage LUSC in our center. Data for 1,176 samples were collected from TCGA. CDKN2A mutation status was associated with TP53 mutation status (P=0.040). TP53 mutation was a favorable prognostic factor for early-stage LUSC. The OS times of patients with wild-type and mutated TP53 were 28.94 and 60.48 months, respectively (P=0.002). In contrast, CDKN2A mutations were significantly associated with a shorter survival time in early-stage LUSC. The OS times for wild-type and mutated CDKN2A patients were 62.81 and 37.55 months, respectively (P=0.026). Patients with TP53 mutations had higher total mutation counts compared to patients with wild-type TP53. Furthermore, OS was significantly shorter in patients with a low mutation count compared to patients with a median or high mutation count.ConclusionsEarly-stage LUSC patients with TP53 mutations had a longer OS, while those with CDKN2A mutations had a shorter OS. Furthermore, patients with TP53 mutation/CDKN2A wild-type status had a longer OS. CDKN2A mutation is a vital indicator for prognostic assessment according to TP53 status. The prolonged survival of patients with TP53 mutations may be due to their high mutation counts. Larger datasets are required to validate these observations.

Tear Film Proteome of Healthy Domestic Cats.

The aim of this study was to investigate the proteins found in tear film of healthy domestic cats. Schirmer tear test strips were used to collect tear samples of twelve healthy cats, which were mixed, centrifuged, and placed in a single 1.5 mL microtube that was frozen at −20°C, until analysis by two-dimensional polyacrylamide gel and mass spectrometry associated with high-performance liquid chromatography. The resulting spectra were analyzed and compared with the Swiss-Prot search tool. Forty peptides were detected in the analyzed protein fragments of 90 spots, with 16 proteins identified. Of these, the authors confirmed what has been already found in other studies: lactotransferrin, serum albumin, allergenic lipocalins, and neutrophil gelatinase-associated lipocalin. Others were considered novel in tear film samples of all species: cyclin-dependent protein kinase, serine/arginine repetitive matrix protein, apelin receptor, secretory protein related to C1q/TNF, Wee1, α-1,4 glucan phosphorylase, and WD repeat domain 1. The network was divided into 11 clusters, and a biological function was assigned. Most of the proteins have functions in the defense and maintenance of feline ocular surface homeostasis. Serum albumin is a bottleneck protein, with a high betweenness value. This paper is a pioneer in reporting, in-depth, the tear film proteome of domestic cats.

IL-6/STAT3 signaling activation exacerbates high fructose-induced podocyte hypertrophy by ketohexokinase-A-mediated tristetraprolin down-regulation

Glomerular hypertrophy is a crucial factor of severe podocyte damage and proteinuria. Our previous study showed that high fructose induced podocyte injury. The current study aimed to explore a novel molecular mechanism underlying podocyte hypertrophy induced by high fructose. Here we demonstrated for the first time that high fructose significantly initiated the hypertrophy in rat glomeruli and differentiated human podocytes (HPCs). Consistently, it induced inflammatory response with the down-regulation of anti-inflammatory factor zinc-finger protein tristetraprolin (TTP) and the activation of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling in these animal and cell models. Subsequently, high-expression of microRNA-92a-3p (miR-92a-3p) and its target protein cyclin-dependent kinase inhibitor p57 (P57) down-regulation, representing abnormal proliferation and apoptosis, were observed in vivo and in vitro. Moreover, high fructose increased ketohexokinase-A (KHK-A) expression in rat glomeruli and differentiated HPCs. Exogenous IL-6 stimulation up-regulated IL-6/STAT3 signaling and miR-92a-3p, reduced P57 expression and promoted podocyte proliferation, apoptosis and hypertrophy in vitro. The data from anti-inflammatory agent maslinic acid treatment or TTP siRNA transfection showed that high fructose may decrease TTP to activate IL-6/STAT3 signaling in podocyte overproliferation and apoptosis, causing podocyte hypertrophy. Whereas, KHK-A siRNA transfection remarkably restored high fructose-induced TTP down-regulation, IL-6/STAT3 signaling activation, podocyte overproliferation, apoptosis and hypertrophy in differentiated HPCs. Taken together, these results suggested that high fructose possibly increased KHK-A expression to down-regulate TTP, subsequently activated IL-6/STAT3 signaling to interfere with podocyte proliferation and apoptosis by up-regulating miR-92a-3p to suppress P57 expression, causing podocyte hypertrophy. Therefore, the inactivation of IL-6/STAT3 to relieve podocyte hypertrophy mediated by inhibiting KHK-A to increase TTP may be a novel strategy for high fructose diet-associated podocyte injury and proteinuria.

P28-10 Cisplatin induces DNA damage associated with cell cycle arrest and apoptosis in PC9 cells

Cisplatin is an effective anticancer drug for the treatment of various tumors, including non-small-cell lung cancer (NSCLC). Here, we investigated the cytotoxicity and action mechanism of cisplatin in PC9 cells. PC9 cells were treated with cisplatin and then evaluated with cell viability assay, Giemsa staining, DAPI staining, flow cytometry, SA-beta-gal staining, and western blot analysis. Our results revealed that cytotoxic activity was associated with a DNA damage response signaling pathway. It exhibited a significant anti-proliferative effect on PC9 cells, and this effect was concentration-dependent. Cells undergoing cisplatin treatment showed morphological signs of apoptosis. Cell cycle arrest was associated with inhibition of E2F-1 activity by the cyclin-dependent protein kinase inhibitor p21WAF1/CIP1. Cisplatin-induced PC9 cells apoptosis through upregulation of Fas-L, Fas, Bak, tBID as well as the proteolysis of PARP. Cisplatin also contributed to the loss of mitochondrial membrane potential and activation of a caspase cascade. Moreover, the apoptotic effect of cisplatin was ROS dependent, as evidenced by ROS generation. Taken together, cisplatin showed anticancer effects by regulating cell cycle, inducing apoptosis through caspase activation, and generating ROS, suggesting that cisplatin has a considerable therapeutic potential against PC9 cells.

Comparison of the Profile of the AML Drug Midostaurin (Rydapt®) As a Kinase Inhibitor with Those of Its Predominant Primary Human Metabolites

Background: The multi-targeted kinase inhibitor midostaurin (M) has single agent activity in internal tandem duplication and tyrosine kinase domain mutant FLT3 acute myeloid leukemia (AML). When added to standard chemotherapy M (50 mg, BID) significantly improved both event-free and overall survival in AML patients harboring activating FLT3 mutations (Stone RM, NEJM 2017). In humans, M is converted into 3 primary metabolites: the 10-O-demethylated compound CGP62221 and two epimeric C3-hydroxylated compounds, e1 and e2 (Fig 1). Following the administration of a single 50 mg dose of [14C]-M, the plasma AUC0-96hr of M, CGP62221, e1 and e2 were 18.0, 22.5, 4.12 and 25.8 µg Eq*h/mL respectively (He H, Drug Metab Dispos 2017) and following 50 mg BID the steady-state plasma trough levels of M, CGP62221 and e2 were 0.82, 1.48 and 6.73 µM (Wang Y, J Clin Pharmacol 2008), indicating substantial accumulation of the latter metabolite. Here we compare the kinase profile of M with those of its predominant metabolites.Methods: Reaction of M with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone afforded a mixture of oxidation products. After purification by chromatography (silica gel) and recrysallisation, X-ray crystallography of the more lipophilic epimer (VE12.094; m.p. 298-301°C) confirmed it to have (S)-stereochemistry (Fig 1). Comparison of this epimer with the products from cryopreserved human hepatocyte (BioreclamationIVT, Baltimore, MD, USA)-mediated metabolism of M showed it to be identical (ultra high performance liquid chromatography - mass spectrometry) to e1; the corresponding 3-(R)-epimer (e2) could not be purified. CGP58546 (Hoehn P, J Antibiot 1995) was treated with benzoic anhydride to give CGP62221 (purified by chromatography; m.p. 246°C). The profiles of these compounds as kinase inhibitors were determined using recombinant enzymes (13 lipid and 320 protein kinases) in radiometric transphosphorylation assays (ProQinase GmbH, Freiburg, Germany) and where >50% inhibition was observed at 10 µM, concentrations required to inhibit activity by 50% (IC50 value) were calculated from the dose-response curves.Results: All four compounds showed inhibition of both serine-threonine and tyrosine protein kinases, consistent with their binding in a type-1 ATP-competitive binding mode. M and CGP62221 showed both a similar pattern of selectivity against 320 protein kinases, as well as similar degrees of potency, although CGP62221 (the most potent inhibitor of PRKG2) was less active against LCK (Tables 1 and 2). The presence of a hydroxyl-group in the pyrrole-ring (e1 and e2) generally reduced potency resulting in increased selectivity towards VEGFR2 (none of the compounds evaluated inhibited VEGFR1 by >50% at 10 µM). PDK1 was the only kinase substantially more sensitive to e2 compared with e1 (>3-fold). None of the compounds substantially (>50%) inhibited the activity of any lipid kinase, nor of AKT, nor of any cyclin-dependent protein kinase at 10 µM.Conclusion: The disparate effects of FLT3-targeting drugs in AML clinical trials has been commented upon (Stone RM, Blood 2017). However, the efficacy and side-effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent, but often comprise of complex cooperative effects between the properties of the parent and active metabolites. AML is a heterogeneous malignancy and activating mutations in the FLT3 and KIT receptor kinases are particularly implicated among the mutations driving the malignant phenotype (Patel JP, NEJM 2012). In the case of M, the major circulating metabolites are multi-targeted protein kinase inhibitors with activity against mutant forms of FLT3 and KIT, as well as several kinases involved in their signaling pathways or implicated in AML via stromal support, such as IGF1R (Chapuis N, Haematologica 2010), JAK (Furqan M, Biomarker Res 2013), LYN (Robinson LJ, Exp Hematol 2005), PDK1 (Zabkiewicz J, Haematologica 2014), RET (Rudat S, ASH 2016), SRC (Leischner H, Blood 2012) and SYK (Puissant A, Cancer Cell 2014). Notably, CGP62221, e1 and e2, which are major circulating components following chronic dosing of M, exhibited substantial kinase activity at physiologically relevant doses. A complex interplay between the kinase activities of M and its metabolites may contribute to the efficacy of M in AML and help engender the distinctive effects of M compared to other FLT3 inhibitors in this malignancy. [Display omitted] DisclosuresManley:Novartis International: Employment. Caravatti:Novartis International: Employment. Furet:Novartis International: Employment. Roesel:Novartis International AG: Employment. Tran:Novartis International: Employment. Wagner:Novartis Pharmaceuticals: Employment.

Exploring multi-target inhibitors using in silico approach targeting cell cycle dysregulator–CDK proteins

Cyclin-dependent kinases (CDKs) belong to a family of multifunctional enzymes that control cell cycle modifications, transcription, and cell proliferation. Their dysfunctions result in different diseases like cancer making them an important drug target in oncology and beyond. The present study aims at identifying the selective inhibitors for ATP binding site in CDK proteins (CDK1, CDK2, CDK4, and CDK5) following a multi-target drug designing approach. Significant challenges lie in identifying the selective inhibitor for the ATP binding site as this region is highly conserved in all protein kinases. Molecular docking coupled with molecular dynamics simulation and free energy of binding calculations (MMPBSA/MMGBSA) were used to identify the potent competitive ATP binding site inhibitors. All the four proteins were docked against the library of drug-like compounds and the outcomes of the docking study were further analyzed by Molecular dynamics (total of 6μs) and MMPB/GBSA techniques. Five different inhibitors for structurally distant protein kinases, i.e. CDK1, CDK2, CDK4, and CDK5 are identified with the binding energy (ΔG bind-PB) in the range −18.24 to −28.43Kcal/mol. Mechanistic complexities associated with the binding of the inhibitor are unraveled by carefully analyzing the MD trajectories. It is observed that certain residues (Lys33, Asp127, Asp145, Tyr15, Gly16, Asn144) and regions are critical for the retention of inhibitors in active pocket, and significant conformational changes take place in the active site region as well as its neighbor following the entry of the ligand inside active pocket as inferred by RMSD and RMSF. It is observed that LIG3 and LIG4 are the best possible inhibitors as reflected from their high binding energy, interaction pattern, and their retention inside the active pocket. This study will facilitate the process of multi-target drug designing against CDK proteins and can be used in the development of potential therapeutics against different diseases.

Proteomic and phosphoproteomic profiles of Sertoli cells in buffalo

Sertoli cells provide nutrients and support for germ cell differentiation and maintain a stable microenvironment for spermatogenesis. Comprehensive identification of Sertoli cellular proteins is important in understanding spermatogenesis. In this study, we performed an integrative analysis of the proteome and phosphoproteome to explore the role of Sertoli cells in spermatogenesis. A total of 2912 and 753 proteins were identified from the proteome and phosphoproteome in Sertoli cells, respectively; 438 proteins were common to the proteome and phosphoproteome. Data are available via ProteomeXchange with identifier PXD024984. In the proteome, ACTG1, ACTB, ACTA2, MYH9 were the most abundant proteins. Gene Ontology (GO) analysis indicated that most of the proteins were involved in the processes of localization, biosynthesis, gene expression, and transport. In addition, some of the proteins related to Sertoli cell functions were also enriched. In the phosphoproteome, most of the proteins were involved in gene expression and the RNA metabolic process; the pathways mainly involved the spliceosome, mitogen-activated protein kinase signaling pathway, focal adhesion, and tight junctions. The pleckstrin homology-like domain is the most highly enriched protein domain in phosphoproteins. Cyclin-dependent kinases and protein kinases C were found to be highly active kinases in the kinase-substrate network analysis. Ten proteins most closely related to network stability were found in the analysis of the network interactions of proteins identified jointly in the phosphoproteome and proteome. Through immunohistochemistry and immunofluorescence verification of vimentin, it was found that there were localization differences between phosphorylated and non-phosphorylated vimentin in testicular tissue. This study is the first in-depth proteomic and phosphoproteomic analysis of buffalo testicular Sertoli cells. The results provide insight into the role of Sertoli cells in spermatogenesis and provide clues for further study of male reproduction.

Calycosin Induces Gastric Cancer Cell Apoptosis via the ROS-Mediated MAPK/STAT3/NF-κB Pathway.

BackgroundCalycosin, an active compound in plants, can promote the apoptosis of various cancer cells; however, the mechanism by which it regulates reactive oxygen species (ROS) in gastric cancer (GC) cells remains unclear.PurposeIn this study, we investigated the effects of calycosin on apoptosis, the cell cycle, and migration in GC cells under ROS regulation.ResultsThe results of the Cell Counting Kit-8 assay suggested that calycosin had significant cytotoxic effects on 12 gastric cancer cells, but no significant cytotoxic effects on normal cells. Hoechst 33342/propidium iodide (PI) double staining and flow cytometry showed that calycosin had clear pro-apoptotic effects on AGS cells. Western blotting revealed that the expression of cytochrome C and pro-apoptotic proteins B-cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad), cleaved (cle)-caspase-3, and cle-poly (ADP-ribose) polymerase gradually increased, and the expression of anti-apoptotic protein Bcl-2 gradually decreased. Calycosin also decreased the expression of extracellular signal-regulated kinase, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation levels of p38, c-Jun N-terminal kinase, and inhibitor of NF-κB. In addition, calycosin markedly increased ROS accumulation, and pretreatment with active oxygen scavenger n-acetyl-l-cysteine (NAC) clearly inhibited apoptosis. Calycosin downregulated the cell cycle proteins cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclin D1, and cyclin E; upregulated p21 and p27; and arrested cells in the G0/G1 phase. Similarly, calycosin also downregulated Snail family transcriptional repressor 1, E-cadherin, and β-catenin and inhibited cell migration. However, pretreatment with NAC inhibited the calycosin-induced effects of cycle arrest and migration.ConclusionIn summary, calycosin induces apoptosis via ROS-mediated MAPK/STAT3/NF-κB pathways, thereby exerting its anti-carcinogenic functions in GC cells.

Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy.

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.

Diabetes induces dysregulation of microRNAs associated with survival, proliferation and self-renewal in cardiac progenitor cells.

Diabetes mellitus causes a progressive loss of functional efficacy in stem cells, including cardiac progenitor cells (CPCs). The underlying molecular mechanism is still not known. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate genes at the post-transcriptional level. We aimed to determine if diabetes mellitus induces dysregulation of miRNAs in CPCs and to test if in vitro therapeutic modulation of miRNAs would improve the functions of diabetic CPCs. CPCs were isolated from a mouse model of type 2 diabetes (db/db), non-diabetic mice and human right atrial appendage heart tissue. Total RNA isolated from mouse CPCs was miRNA profiled using Nanostring analysis. Bioinformatic analysis was employed to predict the functional effects of altered miRNAs. MS analysis was applied to determine the targets, which were confirmed by western blot analysis. Finally, to assess the beneficial effects of therapeutic modulation of miRNAs in vitro and in vivo, prosurvival miR-30c-5p was overexpressed in mouse and human diabetic CPCs, and the functional consequences were determined by measuring the level of apoptotic cell death, cardiac function and mitochondrial membrane potential (MMP). Among 599 miRNAs analysed in mouse CPCs via Nanostring analysis, 16 miRNAs showed significant dysregulation in the diabetic CPCs. Using bioinformatics tools and quantitative real-time PCR (qPCR) validation, four altered miRNAs (miR-30c-5p, miR-329-3p, miR-376c-3p and miR-495-3p) were identified to play an important role in cell proliferation and survival. Diabetes mellitus significantly downregulated miR-30c-5p, while it upregulated miR-329-3p, miR-376c-3p and miR-495-3p. MS analysis revealed proapoptotic voltage-dependent anion-selective channel 1 (VDAC1) as a direct target for miR-30c-5p, and cell cycle regulator, cyclin-dependent protein kinase 6 (CDK6), as the direct target for miR-329-3p, miR-376c-3p and miR-495-3p. Western blot analyses showed a marked increase in VDAC1 expression, while CDK6 expression was downregulated in diabetic CPCs. Finally, in vitro and in vivo overexpression of miR-30c-5p markedly reduced the apoptotic cell death and preserved MMP in diabetic CPCs via inhibition of VDAC1. Our results demonstrate that diabetes mellitus induces a marked dysregulation of miRNAs associated with stem cell survival, proliferation and differentiation, and that therapeutic overexpression of prosurvival miR-30c-5p reduced diabetes-induced cell death and loss of MMP in CPCs via the newly identified target for miR-30c-5p, VDAC1.

BAF57/SMARCE1 Interacting with Splicing Factor SRSF1 Regulates Mechanical Stress-Induced Alternative Splicing of Cyclin D1.

Background: Cyclin D1 regulates cyclin-dependent protein kinase activity of the cell cycle, and cyclin D1 alternative splicing generates a cyclin D1b isoform, acting as a mediator of aberrant cellular proliferation. As alternative splicing processes are sensitive to mechanical stimuli, whether the alternative splicing of cyclin D1 is regulated by mechanical stress and what kinds of factors may act as the regulator of mechano-induced alternative splicing remain unknown. Methods: The alternative splicing of Cyclin D1 was examined using reverse transcription polymerase chain reaction (RT-PCR) in osteoblast cell lines and keratinocyte cells loaded by a cyclic stretch. The expression of splicing factors and switching defective/sucrose non-fermenting (SWI/SNF) complex subunits were detected in stretched cells using real-time quantitative PCR (RT-qPCR). The protein interaction was tested by co-immunoprecipitation assay (Co-IP). Results: Cyclin D1 expression decreased with its splice variant upregulated in stretched cells. Serine/arginine-rich splicing factor 1 (SRSF1) and SWI/SNF complex subunit Brahma-related gene-1-associated factor 57 (BAF57), also named SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1 (SMARCE1), could respond to mechanical stimuli. Overexpression and knockdown experiments indicated the BAF57/SMARCE1 is probably a critical factor regulating the alternative splicing of cyclin D1. Co-IP showed an interaction between BAF57/SMARCE1 and SRSF1, implying a possible underlying mechanism of the regulator role of BAF57/SMARCE1 in the splicing process of cyclin D1. Conclusions: The splicing factor SRSF1 and BAF57/SMARCE1 are possibly responsible for the mechanical stress-induced alternative splicing of cyclin D1.

Maintenance of cellular annexin A1 level is essential for PI3K/AKT/mTOR-mediated proliferation of pancreatic beta cells.

Annexin A1 (AnxA1, also known as lipocortin-1), is a calcium-dependent phospholipid binding protein with diverse functions. Previous studies have indicated that AnxA1 is associated with age-related β-cell dysfunction and aging, which lead to decreased β-cell proliferation capacity. However, it has been uncertain whether AnxA1 affects the proliferation of pancreatic beta (β) cells. In the present study, we reduced AnxA1 expression in the MIN6 islet β-cell line using small interfering RNA (AnxA1-siRNA), then measured cell cycle distribution and cellular proliferation. We also measured the expression levels of cell cycle-related proteins such as cyclin D1, cyclin E, and cyclin-dependent kinase 2 (CDK2) by Western blot analysis. We investigated the phosphatidylinositol 3-kinase (PI3K)/ serine/threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway to explore the potential mechanism underlying the observed effects. Knockdown of AnxA1 expression using siRNA reduced the rates of MIN6 cell proliferation. The proportions of cells in S and G2/M phases also decreased upon inhibition of AnxA1. Moreover, AnxA1 protein expression in MIN6 cells was positively related to the protein levels of cyclin D1, cyclin E, and CDK2. Activation of the PI3K/Akt/mTOR signaling pathway by AnxA1 may be involved in the signaling cascade to regulate cell proliferation. This study identified a positive correlation between AnxA1 protein and pancreatic β-cell proliferation. AnxA1 protein expression might affect the proliferation of MIN6 cells via regulation of cyclin D1, cyclin E, and CDK2 proteins, as well as the PI3K/Akt/mTOR signaling pathway.