Treatment with tiludronic acid helps reduce the development of experimental osteoarthritis lesions in dogs with anterior cruciate ligament transection followed by reconstructive surgery: a 1-year study with quantitative magnetic resonance imaging.

OBJECTIVE: to investigate over a 1-year period in dogs that underwent extracapsular stabilization surgery (ECS) following anterior cruciate ligament (ACL) transection: whether reconstructive surgery could prevent osteoarthritis (OA) progression and whether treatment with the bisphosphonate tiludronic acid (TA) could improve the chronic evolution of OA structural changes. METHODS: ACL transection was performed on dogs on Day 0 and ECS on Day 28. Dogs were randomly divided into 2 groups: 15 received placebo and 16 were treated with TA (2 mg/kg subcutaneous injection) on Days 14, 28, 56, and 84. Magnetic resonance images were acquired on Days -10, 26, 91, 210, and 357, and cartilage volume was quantified. At sacrifice (Day 364), cartilage from femoral condyles and tibial plateaus was macroscopically and histologically evaluated. Expression levels of MMP-1, -3, -13, ADAMTS-4, -5, BMP-2, FGF-2, IGF-1, TGF-ß1, collagen type II, and aggrecan were determined using real-time RT-PCR. RESULTS: the loss of cartilage volume observed after ACL transection stabilized following ECS. Thereafter, a gradual gain occurred, with the cartilage volume loss on the tibial plateaus reduced at Day 91 (p < 0.02) and Day 210 (p < 0.001) in the TA-treated dogs. At sacrifice, TA-treated dogs presented a reduction in the severity of macroscopic (p = 0.03 for plateaus) and histologic (p = 0.07 for plateaus) cartilage lesions, had a better preserved collagen network, and showed decreased MMP-13 (p = 0.04), MMP-1 and MMP-3 levels. CONCLUSION: our findings indicate that in dogs with ACL transection, ECS greatly prevents development of cartilage volume loss. Treatment with TA provided an additional benefit of reducing the development of OA lesions.

1,2-Diamines as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1).

We have identified the N(1)-benzyl-N(2)-methylethane-1,2-diamine unit as a substitute for the (S)-alanine benzylamide moiety for the design of co-activator associated arginine methyltransferase 1 (CARM1) inhibitors. The potency of these inhibitors is in the same order of magnitude as their predecessors and their clearance, volume of distribution, and half lives were greatly improved.

N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1).

A series of N-benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides targeting co-activator associated arginine methyltransferase 1 (CARM1) have been designed and synthesized. The potency of these inhibitors was influenced by the nature of the heteroaryl fragment with the thiophene analogues being superior to thiazole, pyridine, isoindoline and benzofuran based inhibitors.

The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells.

Accumulating evidence indicates that elevated levels of prostaglandin E(2) (PGE(2)) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE(2) exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE(2) to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE(2)-induced ERK phosphorylation and cell proliferation of HCA-7 cells. In order to identify downstream target genes of ERK1/2 signaling, we found that PGE(2) induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE(2) treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE(2) driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE(2) in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer.

Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.

Profiling of selenomethionine responsive genes in colon cancer by microarray analysis.

High-selenium containing yeast is being evaluated in clinical trials against colon polyp recurrence. However, the molecular targets for the anticancer effects of selenium remain unclear. Previous studies by our group demonstrated that selenomethionine-induced growth arrest appears to be mediated by activation of ERK and subsequent phosphorylation of RSK and histone H3. These results suggest that selenomethionine can alter gene expression. In the present study, we have used cDNA microarrays to determine whether gene expression differences exist in HCT116 colon cancer cells treated with selenomethionine. These experiments reveal statistically significant expression changes for 50 genes. Genes we found to increase with selenomethionine treatment include KLK6, ATOX1, SGK, GJB2, DAP-1, PLAU, VIM, DPYSL2, STC2 and PXN. Conversely, genes downregulated by selenomethionine include PRKACB, LIM, DEPP, MYC, CDH5, ELF3, VSNL1, SAT and EGLN3. Further analysis of those genes using chromatin immunoprecipitation experiments showed that phosphorylated histone H3 on serine 10 bound to the GJB2 promoter (connexin 26) or the serum glucocorticoid kinase promoter is increased with selenomethionine treatment. Cells overexpressing CX26 or DAP-1 displayed a reduced number of colonies which suggests that these two genes could play a functional role in the growth inhibitory effects of selenomethionine. These data support the notion that selenomethionine-induced growth inhibition is associated with global changes in gene expression. They also demonstrate that selenomethionine can modify chromatin state to alter gene transcription. Finally, our studies provide a practical foundation for the further development of biomarkers to monitor the efficacy of selenomethionine in clinical trials.

Increased gene copy number of the transcription factor E2F1 in malignant melanoma.

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.

Studies into the anticancer effects of selenomethionine against human colon cancer.

Colorectal cancer is the third most frequent fatal malignant neoplasm in the United States and is expected to cause significant morbidity and mortality. The recent recall of cyclooxygenase-2 inhibitors from clinical trials highlights the need to develop other agents for cancer chemoprevention trials. Intervention strategies with selenium compounds represent a viable option to reduce colon cancer. Here we discuss epidemiologic studies and ongoing clinical trials with selenium. In addition, we discuss preclinical mechanistic studies that provide insights into the biochemical and molecular bases for the anticancer effects of selenomethionine.

Death-signal-induced relocalization of cyclin-dependent kinase 11 to mitochondria.

Fas receptor-Fas ligand interaction appears to be important in carcinogenesis, tumour outgrowth and metastasis. Emerging evidence suggests that CDK11 (cyclin-dependent kinase 11) plays a role in apoptosis and melanoma development. Here, we show that CDK11p110 protein kinase was cleaved after induction of apoptosis by Fas. The N-terminal portion of CDK11p110, CDK11p60, was translocated from the nucleus to the mitochondria. The targeting of CDK11p60 to mitochondria occurred as early as 12 h after treatment. Overexpression of EGFP (enhanced green fluorescent protein)-tagged CDK11p60 could partially break down the mitochondrial membrane potential, induce cytochrome c release and promote apoptosis. Reduction of endogenous CDK11p110 protein levels with siRNA (small interfering RNA) resulted in the suppression of both cytochrome c release and apoptosis. In addition, subcellular fractionation studies of Fas-mediated apoptosis demonstrated that CDK11p60 was associated with the mitochondrial import motor, mitochondrial heat shock protein 70. Taken together, our data suggest that CDK11p60 can contribute to apoptosis by direct signalling at the mitochondria, thereby amplifying Fas-induced apoptosis in melanoma cells.

Selenomethionine regulates cyclooxygenase-2 (COX-2) expression through nuclear factor-kappa B (NF-kappaB) in colon cancer cells.

Previously, we showed that selenomethionine (Se-Met) inhibits growth of colon cancer cells via suppressing COX-2 expression at both mRNA and protein level. However, the molecular mechanism by which Se-Met suppresses COX-2 expression remains to be elucidated. To this end, we transiently transfected HCA-7 cells with different COX-2 promoter constructs followed by Se-Met treatment (90 microM) for 12 h. The results suggested the role of nuclear factor-kappa B (NF-kappaB) in transcriptional regulation of COX-2. We also observed complete inhibition of DNA binding activity of NF-kappaB in Se-Met (90 microM) treated HCA-7 cells as shown by electrophoretic mobility shift assay (EMSA). Supershift assays with anti-p65 antibody identified p65 subunit in the protein complex. We further demonstrate dose-dependent inhibition of nuclear translocation of NF-kappaB/p65 in Se-Met treated HCA-7 cells, which could explain the observed reduction in DNA binding of NF-kappaB/p65. These results suggest that Se-Met regulates COX-2 at transcriptional level by modulating the activity of NF-kappaB transcription factor.

Selenomethionine induces sustained ERK phosphorylation leading to cell-cycle arrest in human colon cancer cells.

Selenomethionine (SeMet) is being tested alone and in combination with other agents in cancer chemoprevention trials. However, the molecular targets and the signaling mechanism underlying the anticancer effect of this compound are not completely clear. Here, we provide evidence that SeMet can induce cell-growth arrest and that the growth inhibition is associated with S-G2/M cell-cycle arrest. Coincidentally with the cell-cycle arrest, we observed a striking increase in cyclin B as well as phosphorylation of the cyclin-dependent kinase Cdc2. Since activation of the mitogen-activated protein kinase (MAPK) cascade has been associated with cell-cycle arrest and growth inhibition, we evaluated the activation of extracellular signal-regulated kinase (ERK). We found that SeMet induced phosphorylation of the MAPK ERK in a dose-dependent manner. We also demonstrate phosphorylation of ribosomal S6 kinase (p90RSK) by SeMet. Additionally, we show phosphorylation of histone H3 in a concentration-dependent manner. Furthermore, the phosphorylation of p90RSK and histone H3 were both antagonized by the MEK inhibitor U0126, implying that SeMet-induced phosphorylation of p90RSK and histone H3 are at least in part ERK pathway dependent. Based on these results, we propose that SeMet induced growth arrest and phosphorylation of histone H3 are mediated by persistent ERK and p90RSK activation. These new data provide valuable insights into the biological effects of SeMet at clinically relevant concentrations.

Identification and characterization of the human Cdc2l2 gene promoter.

The CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) protein kinases are part of the large family of p34(cdc2)-related kinases and have been shown to play a role in cell cycle progression, RNA processing and apoptosis. They are encoded by two genes-cell division control like 1 (Cdc2L1) and cell division control like 2 (Cdc2L2). To date, little is known about the transcription factors controlling their expression. To understand the mechanisms underlying the regulation of CDK11 gene expression, we cloned and identified the Cdc2L2 promoter and determined its transcriptional regulatory elements. By deletion analysis, a region between nucleotides -145 and +10 was identified to be critical for basal level transcription of the Cdc2L2 gene. Sequencing analysis revealed that the proximal promoter of the Cdc2L2 gene is GC rich and does not contain TATA and CAAT boxes. However, multiple consensus and near consensus transcription factor binding sites were found to be present in this region, such as two Ets-1, one cAMP-responsive element (CRE) and one TCF11/LCR-F1/Nrf1 binding sites. Site-directed mutagenesis and transfection studies revealed that all these binding sites were necessary to achieve sustained transcriptional activity. Electrophoretic mobility shift assay confirmed that transcription factors Ets-1 and CREB bind to the Cdc2L2 promoter elements, indicating their potential role in the transcriptional regulation of Cdc2L2 gene. More importantly, Ets-1, CREB and phosphorylated CREB were found binding to the endogenous Cdc2L2 promoter using chromatin immunoprecipitation (CHIP) assay. Our results provide the foundation for further studies into the regulation of Cdc2L2 gene expression in normal homeostasis and cancer.

Chemoprevention of prostate cancer with selenium: an update on current clinical trials and preclinical findings.

Prostate cancer is the most common cancer diagnosed and the second leading cause of cancer-related deaths in men in the United States. The etiological factors that give rise to prostate cancer are not known. Therefore, it is not possible to develop primary intervention strategies to remove the causative agents from the environment. However, secondary intervention strategies with selenium (Se) compounds and other agents represent a viable option to reduce the morbidity and mortality of prostate cancer. In this review, we discuss ongoing clinical trials. In addition, we discuss preclinical mechanistic studies that provide insights into the biochemical and molecular basis for the anti-carcinogenic activity of both inorganic and organic forms of Se.

The p34cdc2-related cyclin-dependent kinase 11 interacts with the p47 subunit of eukaryotic initiation factor 3 during apoptosis.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. However, substrates of CDK11 during apoptosis have not been identified. We used a yeast two-hybrid screening strategy and identified eukaryotic initiation factor 3 p47 protein (eIF3 p47) as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11(p46)). We demonstrate that the eIF3 p47 can interact with CDK11 in vitro and in vivo, and the interaction can be strengthened by stimulation of apoptosis. EIF3 p47 contains a Mov34/JAB domain and appears to interact with CDK11(p46) through this motif. We show in vitro that the caspase-processed CDK11(p46) can phosphorylate eIF3 p47 at a specific serine residue (Ser(46)) and that eIF3 p47 is phosphorylated in vivo during apoptosis. Purified recombinant CDK11(p46) inhibited translation of a reporter gene in vitro in a dose-dependent manner. In contrast, a kinase-defective mutant CDK11(p46M) did not inhibit translation of the reporter gene. Stable expression of CDK11(p46) in vivo inhibited the synthesis of a transfected luciferase reporter protein and overall cellular protein synthesis. These data provide insight into the cellular function of CDK11 during apoptosis.

Selenomethionine inhibits growth and suppresses cyclooxygenase-2 (COX-2) protein expression in human colon cancer cell lines.

Currently, selenium (in the form of high selenium containing yeast or selenomethionine) is being evaluated for anticancer effects against both human colon polyp recurrence and human prostate cancer, respectively. Chemical speciation analysis of the high selenium containing yeast indicates that selenomethionine (SeMet) is a major constituent of selenized yeast. We tested the hypothesis that SeMet might affect colon cancer cell growth by mechanisms involving cyclooxygenases (COX). The growth of all four-colon cancer cell lines tested was inhibited by selenomethionine. Furthermore, selenomethionine decreased COX-2 protein and PGE2 levels in HCA-7 cells. Selenomethionine suppressed COX-2 RNA levels in HCA-7 cells which could account for decreased COX-2 protein levels. Finally, the addition of PGE2 protected cells from the antiproliferative effects of selenomethionine in a concentration dependent manner. Selenomethionine might regulate COX-2 at the transcriptional level. These data suggests that Se-Met-induced cell growth inhibition may be, in part, mediated by COX-2 dependent mechanisms. The results of this study support the use of selenium agents in colon cancer chemoprevention trials.