CK2 activity is crucial for proper glucagon expression.

AIMS/HYPOTHESIS: Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells. METHODS: We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition. RESULTS: We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans. CONCLUSIONS/INTERPRETATION: These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs.

Protein Kinase CK2α', More than a Backup of CK2α.

The serine/threonine protein kinase CK2 is implicated in the regulation of fundamental processes in eukaryotic cells. CK2 consists of two catalytic α or α' isoforms and two regulatory CK2ß subunits. These three proteins exist in a free form, bound to other cellular proteins, as tetrameric holoenzymes composed of CK2α2/ß2, CK2αα'/ß2, or CK2α'2/ß2 as well as in higher molecular forms of the tetramers. The catalytic domains of CK2α and CK2α' share a 90% identity. As CK2α contains a unique C-terminal sequence. Both proteins function as protein kinases. These properties raised the question of whether both isoforms are just backups of each other or whether they are regulated differently and may then function in an isoform-specific manner. The present review provides observations that the regulation of both CK2α isoforms is partly different concerning the subcellular localization, post-translational modifications, and aggregation. Up to now, there are only a few isoform-specific cellular binding partners. The expression of both CK2α isoforms seems to vary in different cell lines, in tissues, in the cell cycle, and with differentiation. There are different reports about the expression and the functions of the CK2α isoforms in tumor cells and tissues. In many cases, a cell-type-specific expression and function is known, which raises the question about cell-specific regulators of both isoforms. Another future challenge is the identification or design of CK2α'-specific inhibitors.

Protein Kinase CK2 and Epstein-Barr Virus.

Protein kinase CK2 is a pleiotropic protein kinase, which phosphorylates a number of cellular and viral proteins. Thereby, this kinase is implicated in the regulation of cellular signaling, controlling of cell proliferation, apoptosis, angiogenesis, immune response, migration and invasion. In general, viruses use host signaling mechanisms for the replication of their genome as well as for cell transformation leading to cancer. Therefore, it is not surprising that CK2 also plays a role in controlling viral infection and the generation of cancer cells. Epstein-Barr virus (EBV) lytically infects epithelial cells of the oropharynx and B cells. These latently infected B cells subsequently become resting memory B cells when passing the germinal center. Importantly, EBV is responsible for the generation of tumors such as Burkitt's lymphoma. EBV was one of the first human viruses, which was connected to CK2 in the early nineties of the last century. The present review shows that protein kinase CK2 phosphorylates EBV encoded proteins as well as cellular proteins, which are implicated in the lytic and persistent infection and in EBV-induced neoplastic transformation. EBV-encoded and CK2-phosphorylated proteins together with CK2-phosphorylated cellular signaling proteins have the potential to provide efficient virus replication and cell transformation. Since there are powerful inhibitors known for CK2 kinase activity, CK2 might become an attractive target for the inhibition of EBV replication and cell transformation.

Inhibition of CK2 Reduces NG2 Expression in Juvenile Angiofibroma.

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve-glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-2'-deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA.

Protein Kinase CK2 Contributes to Glucose Homeostasis by Targeting Fructose-1,6-Bisphosphatase 1.

Glucose homeostasis is of critical importance for the survival of organisms. It is under hormonal control and often coordinated by the action of kinases and phosphatases. We have previously shown that CK2 regulates insulin production and secretion in pancreatic ß-cells. In order to shed more light on the CK2-regulated network of glucose homeostasis, in the present study, a qRT-PCR array was carried out with 84 diabetes-associated genes. After inhibition of CK2, fructose-1,6-bisphosphatase 1 (FBP1) showed a significant lower gene expression. Moreover, FBP1 activity was down-regulated. Being a central enzyme of gluconeogenesis, the secretion of glucose was decreased as well. Thus, FBP1 is a new factor in the CK2-regulated network implicated in carbohydrate metabolism control.

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic ß-Cells of the Line INS-1.

In pancreatic ß-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 ß-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 ß-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 ß-cells.

SGC-CK2-1 Is an Efficient Inducer of Insulin Production and Secretion in Pancreatic ß-Cells.

The pyrazolopyrimidine based compound SGC-CK2-1 is a potent and highly specific CK2 inhibitor and a new tool to study the biological functions of protein kinase CK2 irrespective from off-target effects. We used this compound in comparison with the well-established CK2 inhibitor CX-4945 to analyze the importance of CK2 for insulin production and secretion from pancreatic ß-cells. Both inhibitors affected the proliferation and viability of MIN6 cells only marginally and downregulated the endogenous CK2 activity to a similar level. Furthermore, both inhibitors increased the message for insulin and boosted the secretion of insulin from storage vesicles. Thus, regarding the high specificity of SGC-CK2-1, we can clearly attribute the observed effects to biological functions of protein kinase CK2.

Protein kinase CK2 and ion channels (Review).

Protein kinase CK2 appears as a tetramer or higher molecular weight oligomer composed of catalytic CK2α, CK2α' subunits and non-catalytic regulatory CK2ß subunits or as individual subunits. It is implicated in a variety of different regulatory processes, such as Akt signalling, splicing and DNA repair within eukaryotic cells. The present review evaluates the influence of CK2 on ion channels in the plasma membrane. CK2 phosphorylates platform proteins such as calmodulin and ankyrin G, which bind to channel proteins for a physiological transport to and positioning into the membrane. In addition, CK2 directly phosphorylates a variety of channel proteins directly to regulate opening and closing of the channels. Thus, modulation of CK2 activities by specific inhibitors, by siRNA technology or by CRISPR/Cas technology has an influence on intracellular ion concentrations and thereby on cellular signalling. The physiological regulation of the intracellular ion concentration is important for cell survival and correct intracellular signalling. Disturbance of this regulation results in a variety of different diseases including epilepsy, heart failure, cystic fibrosis and diabetes. Therefore, these effects should be considered when using CK2 inhibition as a treatment option for cancer.

Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic ß-Cells.

The regulation of insulin biosynthesis and secretion in pancreatic ß-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic ß-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.

The stability of CREB3/Luman is regulated by protein kinase CK2 phosphorylation.

CREB3 (Luman) is a family member of ER resident transcription factors, which are cleaved upon the induction of ER stress. Their N-terminal fragments shuttle into the nucleus where they regulate the transcription of target genes. Here, we found that human CREB3 is phosphorylated within its transcription activation domain on serine 46 by protein kinase CK2. Further analyses revealed that the phosphorylation of this site does neither affect the cleavage by S1P/S2P proteases, nor the nuclear localisation nor the transcriptional activity of CREB3. However, phosphorylation at serine 46 reduced the stability of CREB3.

Protein Kinase CK2-A Putative Target for the Therapy of Diabetes Mellitus?

Since diabetes is a global epidemic, the development of novel therapeutic strategies for the treatment of this disease is of major clinical interest. Diabetes is differentiated in two types: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM arises from an autoimmune destruction of insulin-producing ß-cells whereas T2DM is characterized by an insulin resistance, an impaired insulin reaction of the target cells, and/or dysregulated insulin secretion. In the past, a growing number of studies have reported on the important role of the protein kinase CK2 in the regulation of the survival and endocrine function of pancreatic ß-cells. In fact, inhibition of CK2 is capable of reducing cytokine-induced loss of ß-cells and increases insulin expression as well as secretion by various pathways that are regulated by reversible phosphorylation of proteins. Moreover, CK2 inhibition modulates pathways that are involved in the development of diabetes and prevents signal transduction, leading to late complications such as diabetic retinopathy. Hence, targeting CK2 may represent a novel therapeutic strategy for the treatment of diabetes.

Influence of cryopreservation on the CATSPER2 and TEKT2 expression levels and protein levels in human spermatozoa.

This study designed to assess the expression level of CATSPER2 and TEKT2 and to evaluate the levels of CatSper2 and Tektin2 proteins in human spermatozoa before and after cryopreservation. One hundred and twenty semen samples were included in this study. All the samples were subjected to qPCR and Western blot analysis. The results showed a significant reduction in the expression levels of CATSPER2 and TEKT2 in the cryopreserved compared to the fresh samples (P = 0.0039 and P = 0.0166, respectively), and the results showed down-regulation in the expression level of CATSPER2 and TEKT2 genes between the study groups. Moreover, the protein levels of the CatSper2 and Tektin2 were lower in cryopreserved samples compared to fresh samples (P = 0.0001). In conclusion, the reduction in the proteins level and expression level of the CATSPER2 and TEKT2 in cryopreserved samples could be used as an indicator of sperm motility loss.

The impact of cigarette smoking on protamines 1 and 2 transcripts in human spermatozoa.

This study was conducted to evaluate the relation between cigarette smoking, semen quality and ratios of protamine mRNAs in smokers and non-smokers. Spermatozoa from 123 men and 64 smokers and 59 non-smokers whose female partners attended an assisted reproduction and andrology laboratory were evaluated. Protamine mRNA was extracted from purified sperm, reverse-transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine 1 (PRM1) and protamine 2 (PRM2). The main outcomes showed that PRM1 mRNA levels in smokers were significantly lower (p = 0.05) than that of non-smokers. Additionally, PRM2 mRNA levels in smokers were significantly lower (p = 0.001) than that of non-smokers. PRM1/PRM2 mRNA ratios in non-smokers samples show significant differences (p = 0.001) compared with those in smokers. PRM1/PRM2 mRNA ratios were negatively and significantly correlated (p = 0.001) with semen volume, sperm count and normal sperm morphology. We concluded that sperm quality and sperm protamine mRNAs were negatively affected by smoking, and these data will serve as new evidence for the hazardous effect of smoking on male fertility. Additionally, protamine transcripts ratios may serve as a marker for male fertility.

The status of global DNA methylation in the spermatozoa of smokers and non-smokers.

RESEARCH QUESTION: Does regular smoking affect semen quality and the levels of DNA methylation in mature human spermatozoa? DESIGN: Spermatozoa from 109 men were evaluated (55 smokers and 54 non-smokers). DNA was extracted from purified spermatozoa, and DNA methylation was quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Global DNA methylation of non-smokers is significantly lower (P < 0.001) than that of smokers (4.85 ±â€¯2.72 and 7.08 ±â€¯1.77 ng/µl, respectively). Moreover, the mean global DNA methylation levels were significantly correlated (r = 0.22;P = 0.02) with non-condensed chromatin in the spermatozoa. Levels of non-condensed chromatin were significantly higher (P < 0.001) in smokers (29.75 ±â€¯9.38%) compared with non-smokers (20.96 ±â€¯11.31%). Furthermore, global sperm DNA methylation was negatively correlated with high significance (P < 0.010) with sperm: count (r = -0.27), motility (r = -0.30) and vitality (r = -0.26). CONCLUSION: Smoking interferes with DNA methylation. Also, DNA methylation is significantly correlated with sperm parameters and sperm non-condensed chromatin. These data emphasize another detrimental effect of smoking on male fertility. DNA methylation may, therefore, be considered as a fertility marker in men.

Ecto-protein kinase CK2, the neglected form of CK2.

Ecto-protein kinases, including protein kinase CK2 (former name, casein kinase 2), have been the focus of research for more than 30 years. At the beginning of the ecto-kinase research their identification was performed with substrates and inhibitors whose specificity under the current knowledge was rather limited. Since all currently known ecto-kinases, including ecto-CK2, have intracellular counterparts, one has to exclude that an ecto-localization originates from intracellular counterparts after cell damage. Protein kinase CK2 is involved in cellular key processes such as cell cycle progression, inhibition of apoptosis, DNA damage repair, differentiation and many other processes. CK2 is composed of two catalytic CK2α or CK2α' subunits and two non-catalytic CK2ß subunits. Progress in the ecto-kinase and in particular ecto-CK2 studies was made with the use of transfected tagged CK2 subunits, which allowed to follow their individual transport and localization on the cell surface after transfection. Furthermore, immunofluorescence studies with antibodies against CK2 subunits as well as affinity chromatography with a binding partner of CK2 subunits have improved ecto-kinase research. The use of new and more specific inhibitors as well as of substrates, which do not cross the plasma membrane, have further improved the specificity for ecto-CK2. From the various substrates of ecto-CK2, it can be concluded that ecto-CK2 plays a role in Alzheimer disease, cell adhesion, platelet aggregation, immune response and cellular signalling. New tools and techniques, to study ecto-CK2 activity, are required to identify new substrates and thereby new functional implications for ecto-CK2.

Functional interplay between the transcription factors USF1 and PDX-1 and protein kinase CK2 in pancreatic ß-cells.

Glucose homeostasis is regulated by insulin, which is produced in the ß-cells of the pancreas. The synthesis of insulin is controlled by several transcription factors including PDX-1, USF1 and USF2. Both, PDX-1 and USF1 were identified as substrates for protein kinase CK2. Here, we have analysed the interplay of PDX-1, USF1 and CK2 in the regulation of PDX-1 gene transcription. We found that the PDX-1 promoter is dose-dependently transactivated by PDX-1 and transrepressed by USF1. With increasing glucose concentrations the transrepression of the PDX-1 promoter by USF1 is successively abrogated. PDX-1 binding to its own promoter was not influenced by glucose, whereas USF1 binding to the PDX-1 promoter was reduced. The same effect was observed after inhibition of the protein kinase activity by three different inhibitors or by using a phospho-mutant of USF1. Moreover, phosphorylation of USF1 by CK2 seems to strengthen the interaction between USF1 and PDX-1. Thus, CK2 is a negative regulator of the USF1-dependent PDX-1 transcription. Moreover, upon inhibition of CK2 in primary islets, insulin expression as well as insulin secretion were enhanced without affecting the viability of the cells. Therefore, inhibition of CK2 activity may be a promising approach to stimulate insulin production in pancreatic ß-cells.

Quinalizarin inhibits adipogenesis through down-regulation of transcription factors and microRNA modulation.

BACKGROUND: Protein kinase CK2 is induced early in adipogenesis whereas later on, this kinase seems to be dispensable. Here, we have analysed how CK2 might be involved in early steps of differentiation of 3T3-L1 cells. METHODS: 3T3-L1 cells were differentiated to adipocytes in the absence or presence of quinalizarin. The expression and localization of important transcription factors was analysed by Western blot and immunofluorescence. DNA binding capacity and transactivation was analysed with pull-down assays and with luciferase reporter experiments, respectively. mRNA was detected with qRT-PCR, miRNAs with Northern hybridization and qRT-PCR. RESULTS: We show that clonal expansion was considerably repressed upon inhibition of CK2 with quinalizarin. Moreover, to prevent adipogenesis CK2 inhibition had to take place before day 4 of differentiation. Neither the expression at the protein or at the RNA level nor the subcellular localization of the transcription factors C/EBPß and C/EBPδ was affected by CK2 inhibition. There was, however, a drastic reduction in the mRNA and protein levels of C/EBPα and PPARγ2. Upon inhibition of CK2, we found a significant up-regulation of the level of the microRNAs miR-27a and miR-27b, which are known to target PPARγ mRNA. CONCLUSIONS: Time course experiments revealed that CK2 seems to be required at early time points after the induction of differentiation. One important target of CK2 was identified as PPARγ, which is down-regulated after inhibition of CK2. GENERAL SIGNIFICANCE: This is the first report about i) cellular targets of CK2 during adipogenesis and ii) a role of CK2 in microRNA regulation.

Impact of protein kinase CK2 inhibitors on proliferation and differentiation of neural stem cells.

BACKGROUND: Protein kinases play central roles in cell and tissue development. Protein kinase CK2, an ubiquitously expressed serine/threonine kinase has severe impacts on embryo- and spermatogenesis. Since its role in neurogenesis has so far only been investigated in very few studies, we analysed the role of CK2 in neural stem cells by using two specific inhibitors. METHODS: Neural stem cells were isolated from the subventricular zone of neonatal mice, using a neurosphere approach. Proliferation of the neurospheres, as well as their differentiation was investigated with and without inhibition of CK2. Changes in proliferation were assessed by counting the number and measuring the diameter of the neurospheres. Furthermore, the absolute cell numbers within the neurospheres were estimated. Differentiation was induced by retinoic acid in single cells after dissociation of the neurospheres. CK2 was inhibited at consecutive time points after induction of the differentiation process. RESULTS: CK2 inhibition reduced the amount and size of proliferating neurospheres dose dependently. Adding the CK2 inhibitor CX-4945 at the start of differentiation we observed a dose-dependent effect of CX-4945 on cell viability and glia cell differentiation. Adding quinalizarin, a second CK2 inhibitor, at the start of differentiation led to an elevated level of apoptosis, which was accompanied by a reduced neural differentiation. Adding the CK2 inhibitors at 72 h after the start of differentiation had no effect on stem cell differentiation. Conclusion: Inhibition of CK2 influences early gliogenesis in a time point and concentration dependent manner. GENERAL SIGNIFICANCE: The use of a CK2 inhibitor significantly affects the neural stem cell niche.

The mammalian STE20-like kinase 1 (MST1) is a substrate for the apoptosis inhibiting protein kinase CK2.

Apoptosis and the response to cell stress are evolutionary highly conserved mechanisms. Both processes require strict regulation, which is often performed by protein kinases. The mammalian Sterile 20-like kinase 1 (MST1) is a pro-apoptotic protein kinase, which is activated and cleaved by caspases upon the induction of cell stress. Being a phosphoprotein itself, the activity of MST1 is regulated by phosphorylation. Protein kinase CK2 is an anti-apoptotic protein kinase which seems to be involved in the regulation of many different cellular processes including apoptosis. There is increasing evidence that the cleavage of many substrates by caspases is regulated by phosphorylation in the close vicinity of the caspase cleavage sites. One of these kinases, implicated in the phosphorylation of caspase substrates, is protein kinase CK2. Here, we report that serine 320 of the MST1 protein is a novel phosphorylation site for the anti-apoptotic protein kinase CK2. Although serine 320 is in close vicinity to the caspase 3 cleavage site, caspase 3 cleavage of MST1 is not affected by CK2 phosphorylation. Using biochemical approaches, we were able to show that MST1 co-localizes with the CK2 subunits in the pancreatic ß-cell line INS-1 and that full-length MST1 and the activated N-terminal fragment of MST1 both interacted with the CK2 subunits in vitro and in vivo. MST1 is a basophilic kinase whereas CK2 is an acidophilic kinase. Thus, binding of these two kinases in the cytosol and in the nucleus opens the door to the phosphorylation of a variety of new substrates.

Novel coumarin- and quinolinone-based polycycles as cell division cycle 25-A and -C phosphatases inhibitors induce proliferation arrest and apoptosis in cancer cells.

Cell division cycle phosphatases CDC25 A, B and C are involved in modulating cell cycle processes and are found overexpressed in a large panel of cancer typology. Here, we describe the development of two novel quinone-polycycle series of CDC25A and C inhibitors on the one hand 1a-k, coumarin-based, and on the other 2a-g, quinolinone-based, which inhibit either enzymes up to a sub-micro molar level and at single-digit micro molar concentrations, respectively. When tested in six different cancer cell lines, compound 2c displayed the highest efficacy to arrest cell viability, showing in almost all cell lines sub-micro molar IC50 values, a profile even better than the reference compound NCS95397. To investigate the putative binding mode of the inhibitors and to develop quantitative structure-activity relationships, molecular docking and 3-D QSAR studies were also carried out. Four selected inhibitors, 1a, 1d, 2a and 2c have been also tested in A431 cancer cells; among them, compound 2c was the most potent one leading to cell proliferation arrest and decreased CDC25C protein levels together with its splicing variant. Compound 2c displayed increased phosphorylation levels of histone H3, induction of PARP and caspase 3 cleavage, highlighting its contribution to cell death through pro-apoptotic effects.