Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells

Herein we reported an efficient dual DNMT and HDAC inhibitor 208 with great antiproliferative activity against U937 cells. Further studies revealed 208 affected the whole proteome profile and could induce G1 cell cycle arrest and apoptosis in U937 cells through upregulating CDK inhibitor p16 and downregulating cyclin-dependent kinases and their activators.     

Role of p21CIP1 as a determinant of SC-560 response in human HCT116 colon carcinoma cells

SC-560, a structural analogue of celecoxib, induces growth inhibition in a wide range of human cancer cells in a cyclooxygenase (COX)-independent manner. Since SC-560 suppresses the growth of cancer cells mainly by inducing cell cycle arrest, we sought to examine the role of p21CIP1, a cell cycle regulator protein, in the cellular response against SC-560 by using p21(+/+) and p21(-/-) isogenic HCT116 colon carcinoma cells. In HCT116 (p21(+/+)) cells, SC-560 dose-dependently induced growth inhibition and cell cycle arrest at the G1 phase without significant apoptosis induction. SC-560-induced cell cycle arrest was accompanied by upregulation of p21CIP1. However, the extent of SC-560-induced accumulation at the G1 phase was approximately equal in the p21(+/+) and the p21(-/-) cells. Nonetheless, the growth inhibition by SC-560 was increased in p21(-/-) cells than p21(+/+)cells. SC-560-induced reactive oxygen species (ROS) generation did not differ between p21(+/+) and p21(-/-) cells but the subsequent activation of apoptotic caspase cascade was more pronounced in p21(-/-) cells compared with p21(+/+) cells. These results suggest that p21CIP1 blocks the SC-560-induced apoptotic response of HCT116 cells. SC-560 combined with other therapy that can block p21 CIP1 expression or function may contribute to the effective treatment of colon cancer.     

DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines

The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1-20 microM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 microM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2-3 folds at 5 microM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G(2) blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity.     

A proteome-wide CDK/CRK-specific kinase inhibitor promotes tumor cell death in the absence of cell cycle progression

The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets. Thus, RGB-286147 is a proteome-wide CDK/CRK-specific kinase inhibitor whose further study could yield new insight into molecular determinants of tumor cell survival. Our results also suggest that the [1, 3, 6]-tri-substituted-pyrazolo[3,4-d]-pyrimidine-4-one kinase inhibitor scaffold is a promising template for the rational design of kinase inhibitors with potential applications to disease indications other than cancer, such as neurodegeneration, cardiac hypertrophic growth, and AIDS.     

Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAi- transfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated β-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.     

Targeting glycosylation pathways and the cell cycle: sugar-dependent activity of butyrate-carbohydrate cancer prodrugs

Short-chain fatty acid (SCFA)-carbohydrate hybrid molecules that target both histone deacetylation and glycosylation pathways to achieve sugar-dependent activity against cancer cells are described in this article. Specifically, n-butyrate esters of N-acetyl-D-mannosamine (But4ManNAc, 1) induced apoptosis, whereas corresponding N-acetyl-D-glucosamine (But4GlcNAc, 2), D-mannose (But5Man, 3), or glycerol (tributryin, 4) derivatives only provided transient cell cycle arrest. Western blots, reporter gene assays, and cell cycle analysis established that n-butyrate, when delivered to cells via any carbohydrate scaffold, functioned as a histone deacetylase inhibitor (HDACi), upregulated p21WAF1/Cip1 expression, and inhibited proliferation. However, only 1, a compound that primed sialic acid biosynthesis and modulated the expression of a different set of genes compared to 3, ultimately killed the cells. These results demonstrate that the biological activity of butyrate can be tuned by sugars to improve its anticancer properties.     

Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors

Summary Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.     

A New Oxadiazole-Based Topsentin Derivative Modulates Cyclin-Dependent Kinase 1 Expression and Exerts Cytotoxic Effects on Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal form of cancer characterized by drug resistance, urging new therapeutic strategies. In recent years, protein kinases have emerged as promising pharmacological targets for the treatment of several solid and hematological tumors. Interestingly, cyclin-dependent kinase 1 (CDK1) is overexpressed in PDAC tissues and has been correlated to the aggressive nature of these tumors because of its key role in cell cycle progression and resistance to the induction of apoptosis. For these reasons, CDK1 is one of the main causes of chemoresistance, representing a promising pharmacological target. In this study, we report the synthesis of new 1,2,4-oxadiazole compounds and evaluate their ability to inhibit the cell growth of PATU-T, Hs766T, and HPAF-II cell lines and a primary PDAC cell culture (PDAC3). Compound 6b was the most active compound, with IC₅₀ values ranging from 5.7 to 10.7 µM. Molecular docking of 6b into the active site of CDK1 showed the ability of the compound to interact effectively with the adenosine triphosphate binding pocket. Therefore, we assessed its ability to induce apoptosis (which increased 1.5- and 2-fold in PATU-T and PDAC3 cells, respectively) and to inhibit CDK1 expression, which was reduced to 45% in Hs766T. Lastly, compound 6b passed the ADME prediction, showing good pharmacokinetic parameters. These data demonstrate that 6b displays cytotoxic activity, induces apoptosis, and targets CDK1, supporting further studies for the development of similar compounds against PDAC.     

Combined Treatment with PI3K Inhibitors BYL-719 and CAL-101 Is a Promising Antiproliferative Strategy in Human Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer arising from skeletal muscle myogenic progenitors. Recent studies have shown an important role for AKT signaling in RMS progression. Aberrant activation of the PI3K/AKT axis is one of the most frequent events occurring in human cancers and serves to disconnect the control of cell growth, survival, and metabolism from exogenous growth stimuli. In the study reported here, a panel of five compounds targeting the catalytic subunits of the four class I PI3K isoforms (p110α, BYL-719 inhibitor; p110β, TGX-221 inhibitor; p110γ, {"type":"entrez-protein","attrs":{"text":"CZC24832","term_id":"994587862","term_text":"CZC24832"}}CZC24832; p110δ, CAL-101 inhibitor) and the dual p110α/p110δ, AZD8835 inhibitor, were tested on the RMS cell lines RD, A204, and SJCRH30. Cytotoxicity, cell cycle, apoptosis, and the activation of downstream targets were analyzed. Of the individual inhibitors, BYL-719 demonstrated the most anti-tumorgenic properties. BYL-719 treatment resulted in G1/G0 phase cell cycle arrest and apoptosis. When combined with CAL-101, BYL-719 decreased cell viability and induced apoptosis in a synergistic manner, equaling or surpassing results achieved with AZD8835. In conclusion, our findings indicate that BYL-719, either alone or in combination with the p110δ inhibitor, CAL-101, could represent an efficient treatment for human rhabdomyosarcoma presenting with aberrant upregulation of the PI3K signaling pathway.     

Heterogeneous nuclear ribonuclear protein C is increased in the celecoxib-induced growth inhibition of human oral squamous cell carcinoma

Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) that is a critical factor in carcinogenesis, but precise mechanism of its action remains to be elucidated. Here we evaluated the inhibitory effect of celecoxib on cell growth of human oral squamous cell carcinoma (OSCC) YD-10(B), which was established to be used as in vitro OSCC model, and identified celecoxib-regulated protein by proteomics techniques. Celecoxib (IC(50)=37 microM) inhibited the growth of YD-10(B) cells with the decrease of COX-2 protein expression. Its inhibition could be linked in the arrest of G(1) phase with increased levels of p(27) protein, a specific CDK inhibitor. Using proteomics, the 10- to 20-fold increase of heterogeneous nuclear ribonuclear protein C (hnRNP C), which has been suggested to be related with the translation of p(27) mRNA, was observed in celecoxib-treated YD-10(B) cells. In summary, celecoxib has a potential to induce the protein expression of hnRNP C and its increase subsequently induce the translation of p(27) mRNA, which trigger the inhibition of cell growth via p(27)-regulated cell cycle arrest in YD-10(B) cells. In addition, YD-10(B) cells could be useful to study the pathological mechanism of OSCC.     

Effects of the cyclin-dependent kinase 10 (CDK10) on the tamoxifen sensitivity of keloid samples

Cyclin-dependent kinase 10 (CDK10) is a cell cycle regulating protein kinase, which has just been discriminated in recent years. In this paper, mRNA and protein expression of CDK10 were first investigated by a comparative study between 23 human keloid tissue samples and their adjacent normal skin. To further address its potential as a therapeutic target in the treatment of keloid, a plasmid expressing the CDK10 gene was transfected into keloid fibroblast. The effects on tamoxifen-induced apoptosis were then investigated using Western blot assay and flow cytometry. Results showed that there is a generally down-regulated expression of CDK10 in keloid compared to normal skin samples. Transfection with the recombinant CDK10 plasmid significantly decreased the viability of cells and increased the apoptosis rates. Tamoxifen sensitivity in keloid fibroblasts was observed after treatment with the recombinant CDK10 plasmid. The results suggested that CDK10 may play an important role in enhancement of tamoxifen efficiency, and its expression may have a synergistic effect on keloid treatments.     

Differential modulation of zinc-stimulated p21(Cip/WAF1) and cyclin D1 induction by inhibition of PI3 kinase in HT-29 colorectal cancer cells

Activation of the extra cellular signal regulated kinase (ERK) pathway is involved in both proliferation and growth arrest of cells depending on intensity and duration of stimuli. In this study, we have elucidated differential regulation of the zinc-stimulated p21(CiP/WAF1) and cyclin D1 activation by inhibition of phosphoinositide 3-kinase (PI3K). In HT-29 colorectal cancer cells, the ERK activities were increased by zinc, which was accompanied by the induction of p21(Cip/WAF1) and cyclin D1. However, in the HT-29 cells pre-treated with PI3K inhibitor, LY294002, zinc induced further the p21(CiP/WAF) induction whereas abrogated cyclin D1 induction. In addition, the induction of p21(Cip/WAF1) expression completely inhibited the incorporation of bromodeoxyuridine (BrdU) into the nucleus, indicating that p21(CiP/WAF1) is an important indicator for ERK-dependent growth arrest. These studies suggest presence of an inter-related regulatory mechanism of cell proliferation by ERK and PI3K pathways.     

Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21(Waf1), p27(Kip1) and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21(Waf1) was increased, while p27(Kip1) and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21(Waf1) over-expression.