Tris DBA palladium is an orally available inhibitor of GNAQ mutant uveal melanoma in vivo.

Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

Quercetin regulates ß-catenin signaling and reduces the migration of triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a lack in estrogen, progesterone, and epidermal growth factor 2 receptors. TNBC exhibits most of the characteristics of basal-like and claudin-low breast cancer subtypes. The main contributor in the mortality of TNBC is due to the higher invasive and migratory ability of these tumor cells. Some plant flavonoids inhibit the epithelial mesenchymal transition (EMT) of tumor cells and suppress cancer metastasis. In this study, we aimed to determine whether the flavonoid quercetin is effective in modulating the molecular signaling associated with EMT in TNBC. Our data indicated that quercetin can induce the expression of E-cadherin and also downregulate vimentin levels in TNBC. The ability of quercetin to modulate these EMT markers resulted in a mesenchymal-to-epithelial transition (MET). Quercetin-induced MET was linked with the alteration of nuclear localization of ß-catenin and modulation of ß-catenin target genes such as cyclin D1 and c-Myc. Furthermore, we observed that quercetin induced the anti-tumor activity of doxorubicin by inhibiting the migratory ability of TNBC cells. These results suggested that quercetin may inhibit TNBC metastasis and also improve the therapeutic efficacy of existing chemotherapeutic drugs.

Epigenetic silencing of Na,K-ATPase ß 1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma.

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na(+) and uptake of K(+) across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-ß 1 (NaK-ß) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-ß in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-ß gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients' tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-ß. Furthermore, treatment with 5-Aza-2'-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-ß protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-ß expression, which might contribute to RCC initiation and/or disease progression.

3,5-Bis(benzylidene)-1-[4-2-(morpholin-4-yl)ethoxyphenylcarbonyl]-4-piperidone hydrochloride: a lead tumor-specific cytotoxin which induces apoptosis and autophagy.

A number of N-4-(2-aminoethoxy)phenylcarbonyl derivatives of various 3,5-bis(benzylidene)-4-piperidones 2-5 demonstrated noteworthy cytotoxic potencies towards human HL-60 leukemic cells as well as human HSC-2 and HSC-4 squamous cell carcinomas. In general, toxicity towards HGF, HPC, and HPLF normal cells was substantially lower. The highest selective toxicity was noted when the terminal base is morpholine. Lead optimization was based on finding compounds which had (i) high cytotoxic potencies, (ii) a greater toxicity to neoplasms than normal cells, and (iii) drug-likeness based on the rule of five. From the biodata generated, 5a evolved as a promising lead compound for further development. The mode of action of 5a included the induction of apoptosis in HL-60 cells in which internucleosomal DNA fragmentation and activation of caspase-3 was noted. In addition, 5a caused autophagy in HSC-2 cells.

Molecular cloning and biochemical characterization of bovine retina calcineurin.

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15-21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni(2+) was found to be the strongest stimulator, while Zn(2+) was found to inhibit CaN phosphatase activity. Mn(2+) was a relatively less effective stimulator compared to Ni(2+). Fe(2+) was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe(2+) slightly inhibited CaN activity from bovine brain. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.

Expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs.

The role of N-myristoyltransferase and calcineurin is well established in signaling pathways. However, there are no data on their expression and activities in normal and inflamed lungs. The mechanisms of lung inflammation induced following administration of lipopolysaccharides (LPS) or exposure to swine barn air remain unclear. Therefore, we examined expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs of rats. Histopathology showed acute inflammation in the lungs of rats exposed to barn air or LPS but not of control rats. There was no difference in the activities of N-myristoyltransferase and calcineurin among the control, barn-exposed, and LPS-treated rat lungs. Although N-myristoyltransferase and calcineurin were localized in airway epithelium, blood vessel walls, alveolar macrophages, and septa in the lungs of rats from all the groups, the staining intensity was increased in the lungs from rats exposed to intravenous LPS or barn air. Densitometric analyses of Western blots of 55- and 60-kDa polypeptide bands corresponding to N-myristoyltransferase and calcineurin, respectively, in the lung homogenates revealed no differences among the groups. These results show that expression of myristoyltransferase and calcineurin in lung epithelium and endothelium and a cell-specific increase in immunohistochemical expression.

3,5-Bis(benzylidene)-4-oxo-1-phosphonopiperidines and related diethyl esters: Potent cytotoxins with multi-drug-resistance reverting properties.

A series of 3,5-bis(benzylidene)-4-piperidones 3 were converted into the corresponding 3,5-bis(benzylidene)-1-phosphono-4-piperidones 5 via diethyl esters 4. The analogues in series 4 and 5 displayed marked growth inhibitory properties toward human Molt 4/C8 and CEM T-lymphocytes as well as murine leukemia L1210 cells. In general, the N-phosphono compounds 5, which are more hydrophilic than the analogues in series 3 and 4, were the most potent cluster of cytotoxins, and, in particular, 3,5-bis-(2-nitrobenzylidene)-1-phosphono-4-piperidone 5 g had an average IC(50) value of 34 nM toward the two T-lymphocyte cell lines. Four of the compounds displayed potent cytotoxicity toward a panel of nearly 60 human tumor cell lines, and nanomolar IC(50) values were observed in a number of cases. The mode of action of 5 g includes the induction of apoptosis and inhibition of cellular respiration. Most of the members of series 4 as well as several analogues in series 5 are potent multi-drug resistance (MDR) reverting compounds. Various correlations were noted between certain molecular features of series 4 and 5 and cytotoxic properties, affording some guidelines in expanding this study.

Biochemical characterization of bovine brain myristoyl-CoA:protein N-myristoyltransferase type 2.

Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) from Bos tarus brain. The open reading frame codes for a 410-amino-acid protein and overexpressed in Escherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ion Ca(2+) had stimulatory effects on NMT2 activity while Mn(2+) and Zn(2+) inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

NC2213: a novel methionine aminopeptidase 2 inhibitor in human colon cancer HT29 cells.

Methionine aminopeptidase 2 (MetAP2) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. MetAP2 is overexpressed in human colon cancer. In this report we screened various MetAP2 inhibitors and treated HT29 cells with various concentrations of compounds. We evaluated the expression of MetAP2 and pp60c-src expressions in HT29 cells. In addition we also carried out the cell proliferation and cell cycle analysis in the MetAP2 inhibitor-treated HT29 cells. The cell cycle analysis of HT29 treated with 1.0 microM of NC2213 showed an arrest in the G2 phase followed by an induction in the percentage of cells undergoing apoptosis in the sub-G1 phase. Western blot analysis revealed that the MetAP2 expression was dose-dependently decreased when the HT29 cells were treated with the 3,5-bis(benzylidene)-4-piperidone derivative (NC2213). In addition, phosphorylation of Src, a myristoylated oncoprotein was significantly decreased by 1.0 microM of NC2213 as revealed by Western blot analysis. Furthermore, NC2213 also inhibits the expression of pp60c-src in HT29 cells. Interestingly, this compound also inhibits the phosphorylation at Tyr416 of pp60c-src while increasing the phosphorylation at Tyr527 of pp60c-src. NC2213 inhibits the growth of HT29 cells by inducing apoptosis and might be useful for the treatment of human colon cancer.

Tris (dibenzylideneacetone) dipalladium, a N-myristoyltransferase-1 inhibitor, is effective against melanoma growth in vitro and in vivo.

PURPOSE: Melanoma is a solid tumor that is notoriously resistant to chemotherapy, and its incidence is rapidly increasing. Recently, several signaling pathways have been shown to contribute to melanoma tumorigenesis, including constitutive activation of mitogen-activated protein kinase, Akt, and Stat-3. The activation of multiple pathways may account in part for the difficulty in treatment of melanoma. In a recent screen of compounds, we found that an organopalladium compound, Tris (dibenzylideneacetone) dipalladium (Tris DBA), showed significant antiproliferative activity against melanoma cells. Studies were carried out to determine the mechanism of action of Tris DBA. EXPERIMENTAL DESIGN: Tris DBA was tested on efficacy on proliferation of human and murine melanoma cells. To find the mechanism of action of Tris DBA, we did Western blot and gene array analyses. The ability of Tris DBA to block tumor growth in vivo was assessed. RESULTS: Tris DBA has activity against B16 murine and A375 human melanoma in vivo. Tris DBA inhibits several signaling pathways including activation of mitogen-activated protein kinase, Akt, Stat-3, and S6 kinase activation, suggesting an upstream target. Tris DBA was found to be a potent inhibitor of N-myristoyltransferase-1, which is required for optimal activity of membrane-based signaling molecules. Tris DBA showed potent antitumor activity in vivo against melanoma. CONCLUSION: Tris DBA is thus a novel inhibitor of N-myristoyltransferase-1 with significant antitumor activity and is well tolerated in vivo. Further preclinical evaluation of Tris DBA and related complexes is warranted.

Myristoyltransferase and calcineurin: novel molecular therapeutic target for epilepsy.

N-myristoylation is a co-translational, irreversible addition of a fatty acyl moiety to the amino terminus of many eukaryotic cellular proteins. These myristoylated proteins in the cell have diverse biological functions such as signal transduction, cellular transformation and oncogensis. Known myristoylated proteins [Src family kinases, the catalytic subunit of cAMP-dependent protein kinase and calcineurin (CaN)] are either protein kinases or a protein phosphatases which modulate various cellular metabolic processes. Myristoylation is catalyzed by N-myristoyltransferase (NMT) and is recognized to be a widespread and functionally important modification of proteins. The main objective of this review is to focus on the potential role of NMT and CaN in epileptic brain and its involvement in neuronal apoptosis. The findings on the interaction of NMT and CaN with various signaling molecules in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis. Understanding the regulation of NMT by specific inhibitors may help us to control the action of this enzyme on its specific substrates and may lead to improvements in the management of various neurological disorders like Alzheimer's disease, ischemia and epilepsy.

N-acyl-3,5-bis(arylidene)-4-piperidones and related compounds which stimulate fyn kinase.

This study is part of a long term project designed to explore the hypothesis that stimulation of cancer cells followed by treatment with one or more cytotoxic agents may create greater damage to tumours than to the corresponding normal tissues. The aim of the present investigation was to discover various compounds which stimulate a protein tyrosine kinase, namely fyn kinase. The N-acyl-3,5-bis(arylidene)-4-piperidones and related analogues activated this enzyme using concentrations of 25 microM while representative molecules achieved this result at 0.1 microM. Molecular modelling suggested that the compounds interact transiently with the ATP binding site of fyn kinase thereby enhancing the catalytic phosphorylation of proteins. In the future, candidate antineoplastic agents will be designed which incorporate the structural features of these enzyme stimulators with the goal of their being formed in vitro and in vivo prior to the release of cytotoxins.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) splice variants from bovine cardiac muscle.

Calmodulin-dependent cyclic nucleotide phopshodiesterase (PDE1) has been extensively characterized and is a key enzyme involved in the complex interaction between cyclic nucleotide and Ca(2+) second-messenger systems. It is well established that PDE1 exists in different isozymes. For example, bovine brain tissue has two PDE1 isozymes (PDE1A2 and PDE1B1) whereas only one form (PDE1A1) is reported in bovine cardiac tissue. In this study, we report the cloning of two cDNA splice variants of PDE1: PDE1-small and PDE1-large, from bovine cardiac tissue. Their amino acid sequence similarity to PDE1 sequences from other mammalian species showed that all are very conserved, suggesting their importance in cellular functions. Interestingly, compared to other mammalian species, bovine PDE1A, PDE-small and PDE-large show a deletion at the C-terminal end of the catalytic domain of the gene. Although the significance of this deletion at this crucial location of the gene is not known, we have successfully over-expressed both PDE1-small and PDE1-large splice variants in E. coli and these splice variants are characterized in terms of Western blot, biotinylated calmodulin overlay and peptide mass fingerprinting. Results from these studies suggested that these two splice variants belong to the PDE1 superfamily. To our knowledge, this is the first report on cloning and characterization of these cDNA variants from bovine cardiac tissue. Since there are at least two isoforms of PDE1 in bovine heart tissue, this merits further in-depth study.

Role of calpain and caspase system in the regulation of N-myristoyltransferase in human colon cancer (Review).

A number of viral and eukaryotic proteins which undergo a lipophilic modification by the enzyme N-myristoyltransferase (NMT: NMT1 and NMT2) are required for signal transduction and regulatory functions. We reported a higher expression of NMT2 in most of the cases of cancerous tissues compared to normal tissues by Western blot analysis. Furthermore, protein-protein interaction of NMTs revealed that m-calpain interacts with NMT1 while caspase-3 interacts with NMT2. Our findings provide the first evidence of higher expression of NMT2 in human colorectal adenocarcinomas and the interaction of both forms of NMT with various signaling molecules. In this review, we summarize the recent findings on NMT2 in human colon cancer in our laboratory.

Potential role of N-myristoyltransferase in cancer.

Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. The treatment of colonic cancer remains difficult because of the lack of effective chemotherapeutic agents; therefore it is important to continue to search for cellular functions that can be disrupted by chemotherapeutic drugs resulting in the inhibition of the development and progression of cancer. The current knowledge of the modification of proteins by myristoylation involving myristoyl-CoA: protein N-myristoyltransferase (NMT) is in its infancy. This process is involved in the pathogenesis of cancer. We have reported for the first time that NMT activity and protein expression were higher in human colorectal cancer, gallbladder carcinoma and brain tumors. In addition, an increase in NMT activity appeared at an early stage in colonic carcinogenesis. It is conceivable therefore that NMT can be used as a potential marker for the early detection of cancer. These observations lead to the possibility of developing NMT specific inhibitors, which may be therapeutically useful. We proposed that HSC70 and/or enolase could be used as an anticancer therapeutic target. This review summarized the status of NMT in cancer which has been carried in our laboratory.

Phosphorylation and dephosphorylation of human myristoyltransferase type 1.

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational and (or) post-translational transfer of myristate to the amino terminal glycine residue of a number of important proteins, especially the non-receptor tyrosine kinases whose activity is important for tumorigenesis. Human NMT was found to be phosphorylated by non-receptor tyrosine kinase family members of Lyn, Fyn, and Lck and dephosphorylated by the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In this review, we discuss the cross-talk that exists between NMT and their N-myristoylated protein substrates. The cross-talk among NMT, tyrosine kinases, and phosphatases may be determined by their subcellular localization and by the physiological state of the cell.

Regulation of calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1): review.

The response of living cells to change in cell environment depends on the action of second messenger molecules. The two second messenger molecules cAMP and Ca2+ regulate a large number of eukaryotic cellular events. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1) is one of the key enzymes involved in the complex interaction between cAMP and Ca2+ second messenger systems. Some PDE1 isozymes have similar kinetic and immunological properties but are differentially regulated by Ca2+ and calmodulin. Accumulating evidence suggests that the activity of PDE1 is selectively regulated by cross-talk between Ca2+ and cAMP signalling pathways. These isozymes are also further distinguished by various pharmacological agents. We have demonstrated a potentially novel regulation of PDE1 by calpain. This study suggests that limited proteolysis by calpain could be an alternative mechanism for the activation of PDE1. We have also shown PDE1 activity, expression and effect of calpain in the rat model in vitro of cardiac ischemia-reperfusion.

N-myristoyltransferase 2 expression in human colon cancer: cross-talk between the calpain and caspase system.

A number of viral and eukaryotic proteins which undergo a lipophilic modification by the enzyme N-myristoyltransferase (NMT: NMT1 and NMT2) are required for signal transduction and regulatory functions. To investigate whether NMT2 contributes to the pathogenesis of colorectal carcinoma, we observed a higher expression of NMT2 in most of the cases of cancerous tissues compared to normal tissues (84.6% of cases; P < 0.05) by Western blot analysis. Furthermore, protein-protein interaction of NMTs revealed that m-calpain interacts with NMT1 while caspase-3 interacts with NMT2. Our findings provide the first evidence of higher expression of NMT2 in human colorectal adenocarcinomas and the interaction of both forms of NMT with various signaling molecules.

Interaction between heat shock protein 70 kDa and calcineurin in cardiovascular systems (Review).

Cells have the capability of defending themselves from various stressors by activating a genetic program with the production of substances known as heat shock proteins (Hsps) and their regulatory partners, the heat shock transcription factors. Hsps play a major role in systemic hypertension, coronary artery disease, carotid atherosclerosis, myocardial infarction and myocardial ischemia. In this review we discuss the interaction between Hsp70 and CaN which was carried out in our laboratory. We demonstrated that the cardiac Hsp70 stimulated a 2-fold increase in calcineurin (CaN) activity. In addition, the pull-down assay revealed that Hsp70 directly interacts with CaN. Furthermore, expressed cardiac specific Hsp70 was phosphorylated in vitro by cAMP-dependent protein kinase. The phosphorylated Hsp70 was unable to activate the phosphatase activity of CaN. For the first time we demonstrated that Hsp70 is phosphorylated by cAMP-dependent protein kinase and provides an on/off switch for the regulation of CaN signaling by Hsp70. This will lead to therapeutic benefit in human diseases such as atherosclerosis, cardiomyopathy, congestive heart failure, and ischemia.