Nm23-H1/nucleoside diphosphate kinase as a key molecule in breast tumor angiogenesis

Background: Neo-angiogenesis seems to be a critical feature of breast tumor growth, migration and metastasis. Inhibition of angiogenesis may provide information regarding treatment. Since angiogenesis is the result of complex processes, controlled by several angiogenic (pro- and/or -anti) factors and their receptors, multiple ways to prevent or retrogress tumor-induced angiogenesis have been proposed. The clinically significant activity of bevacizumab and other antiangiogenic treatments have attracted a great deal of interest. Objective/methods: We discuss biological aspects of breast cancer angiogenesis and nucleoside diphosphate kinase (NDPK) as a key molecule in this process. Results/conclusions: In clinical and experimental trials, it was reported that NDPK is inversely related to breast cancer metastasis and angiogenesis. To inhibit the metastatic potential of cancer cells, Nm23-H1/NDP kinase appears to interact with many proteins involved in cellular signal transduction in angiogenesis and tumorigenesis, and therefore reduces the activation of the extracellular signal-regulated kinase (ERK)/MAPK in response to those signals.     

Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue

Nm23-H1 has been identified as a metastatic suppressor gene in murine melanoma cell lines. Several functions have been attributed to its activity in cancer, including a histidine kinase activity, DNA repair, and regulation of other proteins involved in metastatic formation. While in breast cancer, NM23-H1 overexpression indicates a benign status through impairing progression of disease, its function is opposite in other cancers; e.g., neuroblastoma. To further understand this dichotomy of function in cancer, we have analyzed its function in prostate cancer, in which the relationship between NM23-H1 expression and prognostic state is today controversial. In vitro, overexpression of NM23-H1 in PC3 cells inhibited their cell motility, while downregulation of NM23-H1 expression in these cells by RNA interference showed enhanced cell motility. Immunohistochemistry analysis performed on 346 prostate cancer tissue samples showed a relationship between high levels of NM23-H1 expression in the nuclei of these tumorigenic cells and elevated Gleason score, with high levels of NM23-H1 cytoplasmic staining related to metastatic stage. This retrospective survival study demonstrates that high levels of NM23-H1 expression in the cytoplasm determine recurrence of prostate-specific antigen levels only in those patients with metastatic disease. Our findings suggest a correlation between high levels of NM23-H1 protein in the cytoplasm of the cells and progression of prostate cancer to metastasis, thus definitively identifying NM23-H1 as a new negative prognostic marker in prostate cancer.     

Plasma membrane-associated nucleoside diphosphate kinase (nm23) in the heart is regulated by beta-adrenergic signaling

1. Receptor-independent activation of heterotrimeric G proteins by plasma membrane-associated nucleoside diphosphate kinase (NDPK) has been demonstrated in vivo, and elevated levels of NDPK were found in purified sarcolemmal membranes of patients with end-stage heart failure. 2. Among 22 consecutive patients with chronic heart failure who underwent cardiac transplantation, those treated with a beta-blocker (n=8) had a 65% lower NDPK content and activity in the cardiac sarcolemma, compared to patients with similar base line characteristics who had no beta-blocker therapy (n=14). 3. The lower NDPK was associated with a reduced NDPK-dependent, Gi-mediated inhibition of adenylyl cyclase activity, as assessed by in vitro measurement of adenylyl cyclase activity in the presence of GDP or its kinase-resistant analog guanosine 5'-O-(2-thio)diphosphate (GDPbetaS). 4. We further tested whether treatment with a beta-adrenergic agonist would induce an increase in sarcolemmal NDPK. Rats treated with isoproterenol developed myocardial hypertrophy, and NDPK in the sarcolemma rose by 60% during 14 days of treatment. The beta-blocker propranolol prevented both effects. When hypertrophy was induced with thyroid hormone, NDPK did not increase. 5. In conclusion, chronic activation of beta-adrenergic receptors increases the binding of NDPK to cardiac sarcolemma, where it may activate heterotrimeric G proteins.     

Adenoviral-mediated overexpression of human equilibrative nucleoside transporter 1 (hENT1) enhances gemcitabine response in human pancreatic cancer

Nucleoside-derived anticancer agents must be transported across the plasma membrane as a preliminary step to their conversion into active drugs. Hence, modulation of a specific nucleoside transporter may affect bioavailability and contribute significantly to sensitizing tumor cells to these anticancer agents. We have generated and functionally characterized a new recombinant adenovirus (Ad-hENT1) that has allowed us to overexpress the equilibrative nucleoside transporter hENT1 and to analyze its effects in human pancreatic tumor cells. Overexpression of hENT1 is associated with changes in cell cycle profile, in a variable manner depending on the particular cell type, thus suggesting a metabolic link between hENT1-mediated transport processes and the enzymatic machinery responsible for intracellular nucleoside metabolism. When assayed in vivo in a human pancreatic adenocarcinoma xenograft, intratumoral Ad-hENT1 injection improved the therapeutic response to gemcitabine. In summary, hENT1 overexpression is associated with alterations in nucleoside enzymatic machinery and cell cycle progression in cultured cells and enhances gemcitabine action in vivo.     

Functional characterisation of the S512Y mutant vanilloid human TRPV1 receptor

1 Mammalian transient receptor potential (TRP) channels include the nonselective cation channel TRPV1, which is activated by a range of stimuli including low pH, vanilloids and heat. Previously, selective mutagenesis experiments identified an intracellular residue (S512Y) critical to discriminating between pH and vanilloid (capsaicin) gating of the rat TRPV1 receptor. 2 In this study, switching the equivalent residue in the human TRPV1 (which has some significant differences with the rat TRPV1) also rendered this channel relatively insensitive to activation by capsaicin and proved critical in determining the receptor's sensitivity to the putative endovanilloid N-arachidonoyl-dopamine (NADA), suggesting a similar mode of activation for these two agonists. 3 Potency of pH gating was reduced; however, voltage-dependent outward rectification properties of the pH-dependent current and gating by heat and pH sensitisation of the S512Y heat response remained unaffected. 4 Surprisingly, residual capsaicin gating was detected and could be sensitised by pH even in the presence of a competitive antagonist. Taken together, these findings indicate that effective functional interaction of capsaicin with the S512Y channel still occurred, although the vanilloid-dependent gating per se was severely compromised. 5 This observation provides additional evidence for capsaicin interacting at multiple sites, distinct from the S512 residue located close to the intracellular face of the pore.     

Thirty novel genetic variations in the SLC29A1 gene encoding human equilibrative nucleoside transporter 1 (hENT1)

Thirty-nine genetic variations, including thirty novel ones, were found in the human SLC29A1 gene, which encodes equilibrative nucleoside transporter 1, from 256 Japanese cancer patients administered gemcitabine. The found novel variations included -8,166G>A, -81,10A>G, -7,947G>A, -7,789T>C, -5,595G>A, -3,803_-3,783delTCGGGGAGGTGGCAGTGGGCG, -3,548G>C, -3,414G>A, -1355T>C, -34C>G, IVS1+141G>A, IVS1+260C>T, IVS1-82C>T, 177C>G, IVS3-6C>T, 564C>T, IVS8+44T>C, IVS8+90T>C, IVS8+97T>C, IVS8+131C>T, IVS8+169G>A, 933T>C, 954C>T, IVS11-52G>C, IVS11-46G>A, 1,288G>A, 1,641C>G, 1,703_1,704delGT, 1812C>T, and 1861C>T. The frequencies were 0.051 for IVS8+169G>A, 0.012 for -7,947G>A, 0.006 for IVS1+141G>A and 1,703_1,704delGT, 0.004 for -8,166G>A, -8,110A>G, -3,548G>C, -1,355T>C, -34C>G, IVS8+44T>C, and 1,812C>T, and 0.002 for the other 19 variations. Among them, 177C>G and 1,288G>A resulted in amino acid substitutions Asp59Glu and Ala430Thr, respectively. Using the detected polymorphisms, linkage disequilibrium analysis was performed, and 28 haplotypes were identified or inferred. Our findings would provide fundamental and useful information for genotyping SLC29A1 in the Japanese and probably other Asian populations.     

Insights into susceptibility of antiviral drugs against the E119G mutant of 2009 influenza A (H1N1) neuraminidase by molecular dynamics simulations and free energy calculations

Neuraminidase inhibitors (NAIs) play vital roles in controlling human influenza epidemics and pandemics. However, the emergence of new human influenza virus mutant strains resistant to existing antiviral drugs has been becoming a major challenge. Therefore, it is critical to uncover the mechanisms of drug resistance and seek alternative treatments to combat drug resistance. In this study, molecular dynamics (MD) simulations and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) were applied to investigate the different sensitivities of oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV) against the E119G mutant of 2009 A/H1N1 neuraminidase. The predicted binding free energies indicate that the E119G mutation in NA confers resistance to all of the three studied inhibitors. The ordering of the level of drug resistance predicted by the binding free energies for the three inhibitors is ZNV > PRV > OTV, which agrees well with the experimental data. Drug resistance arises primarily from the unfavorable shifts of the polar interactions between NA and the inhibitors. It comes as a surprise that the mutation of Glu119 that can form strong H-bonds with the inhibitors in the wild-type protein does not have direct impact on the binding affinities of both OTV and PRV due to the regulation of the strong unfavorable polar desolvation energies. The indirectly conformational variations of the inhibitors, which caused by the E119G mutation, are responsible for the loss of the binding free energies. However, for ZNV, the E119G mutation has both direct and indirect influences on the drug binding. The structural and quantitative viewpoint obtained from this study provides valuable information for the rational design of novel and effective drugs to combat drug resistance.     

Induction of G0/G1 arrest and apoptosis by 3-hydroxycinnamic acid in human cervix epithelial carcinoma (HeLa) cells

The present studies were undertaken to analyze the factors regulating 3-hydroxycinnamic acid-induced apoptosis and cell cycle arrest. Treatment of human cervix HeLa cells with 3-hydroxycinnamic acid induced apoptosis and G0/G1-phase arrest. The percentage of apoptosis induced by 3-hydroxycinnamic acid in HeLa cells was increased with incubation time. The results also demonstrated that 3-hydroxycinnamic acid increased the expression of p53, caspase-3, Bax and cyclin B. These results demonstrated that 3-hydroxycinnamic acid induced apoptosis through p53- and caspase-3-dependent pathways.     

人甲状旁腺激素(1-34)突变蛋白的设计与活性验证

通过对NCBI数据库中不同物种同源序列进行比对protein-protein BLAST,并利用计算机软件DiscoveryStudio3.1进行分子对接和分子动力学模拟,对天然人甲状旁腺激素(1 34)的3个氨基酸R25K26K27进行Q25E26L27置换,并对突变体的生物活性进行了评价。结果显示:突变后PTH(1 34)-(RKK-QEL)和突变前PTH(1 34)多肽主链叠合后均方根偏差RMSD值为2.509 3,说明两者主链结构构象差异不大;PTH(1 34)-(RKK-QEL)与受体蛋白PTH1R的相互作用能为599.253 kcal.mol 1,较天然PTH(1 34)的554.083 kcal.mol 1增强7.5%;氢键数目由32对增加至38对;PTH(1 34)-(RKK-QEL)能显著刺激UAMS-32P细胞RANKL基因的表达(P<0.01),并抑制OPG基因的表达(P<0.01);PTH(1 34)-(RKK-QEL)作用于UAMS-32P和小鼠原代股骨骨髓细胞共培养体系时,能显著刺激破骨细胞的形成(P<0.01),并且活性高于PTH(1 34)标准品。     

The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.     

Estrogen desensitizes 5-HT(1A) receptors and reduces levels of G(z), G(i1) and G(i3) proteins in the hypothalamus

The present study investigated whether estrogen would desensitize hypothalamic serotonin(1A) (5-HT(1A)) receptors by examining the neuroendocrine response to 8-OH-DPAT, a 5-HT(1A) agonist. Rats were ovariectomized, allowed to recover for 5 days, then given 2 daily injections of estradiol benzoate or vehicle (10 microg/day, s.c.). Twenty-four hours after the second injection, rats were challenged with a sub-maximal dose of 8-OH-DPAT (50 microg/kg, sc) or saline 15 min prior to sacrifice. 8-OH-DPAT produced a significant increase in plasma oxytocin, ACTH and corticosterone levels in ovariectomized rats. While estrogen treatment for 2 days did not alter basal hormone levels, it did significantly reduce the magnitude of oxytocin, ACTH and corticosterone responses to 8-OH-DPAT. The reduction in hormone responses was accompanied by a significant reduction in hypothalamic levels of G(z), G(i1) and G(i3) proteins (by 50%, 30% and 50%, respectively). These findings suggest that a reduction in these G proteins may contribute to the mechanisms underlying estrogen-induced desensitization of 5-HT(1A) receptors. The desensitization of 5-HT(1A) receptors has been suggested to underlie the therapeutic effects of antidepressant 5-HT uptake inhibitors (SSRIs). Thus, the present results suggest that estrogen or estrogen-like substances in combination with SSRIs may prove effective in developing novel therapeutic strategies for neuropsychiatric disorders in women.     

Inhibition of CHK1 kinase by Gö6976 converts 8-chloro-adenosine-induced G2/M arrest into S arrest in human myelocytic leukemia K562 cells

8-Chloro-cAMP (8-Cl-cAMP) and its metabolite 8-chloro-adenosine (8-Cl-Ado) inhibit cell growth by 8-Cl-Ado-converted 8-Cl-ATP that targets cell-cycle control and RNA metabolism. However, the cell-cycle checkpoint pathways remain to be identified. Recent studies have shown that 8-Cl-cAMP administration and 8-Cl-Ado exposure may damage chromosomal DNA in vivo and in vitro. In this study, we demonstrate that 8-Cl-Ado-induced DNA damage activates G2/M phase checkpoint, which is associated with ATM-activated CHK1-CDC25C-CDC2 pathway joined by BRCA1-CHK1 branch in apoptosis-resistant human myelocytic leukemia K562 (p53-null) cells. Inhibition of CHK1 kinase by Gö6976, an inhibitor of CHK1 activity, can promote DNA damage and lead to the activation of CHK2, converting G2/M checkpoint into intra-S-phase checkpoint in which two parallel branches, the ATM-CHK2-CDC25A-CDK2 and the ATM-NBS1/SMC1 cascades, are involved. These observations may provide aid in better understanding of the mechanisms of 8-Cl-cAMP and 8-Cl-Ado actions and in potential design of the combined therapy.     

Differential coupling of 5-HT(1) receptors to G proteins of the G(i) family

1: Since all 5-HT(1) receptors couple to G(i)-type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5-HT(1) receptors has been unclear. 2: In previous studies we have used transfected cells to demonstrate that 5-HT(1B) receptors can couple more efficiently than 5-HT(1A) receptors to activation of extracellular signal-regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5-HT(1) receptors differentially couple to isoforms of G(ialpha). 3: In the present study we utilized a model system in which pertussis toxin resistant forms of human G(ialpha1), G(ialpha2), and G(ialpha3) were used to directly compare the coupling of human 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptors to each G(ialpha) in transfected human HeLa cells. 4: 5-HT(1A) receptors displayed a preference for G(ialpha1) and G(ialpha2), relative to G(ialpha3). Pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5-HT in the absence of pertussis toxin. 5: In contrast, pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 32%, 118%, and 35% of 5-HT(1B) receptor-stimulated activity, respectively, indicating that 5-HT(1B) receptors coupled primarily through G(ialpha2). A similar preference for G(ialpha2) was found in studies of the 5-HT(1D) receptor, where toxin resistant G(ialpha1), G(ialpha2), and G(ialpha3) rescued 30%, 70%, and 40% of activity, respectively. 6: In conclusion, the observed differential coupling of 5-HT(1) receptors to isoforms of G(ialpha), provides additional evidence for our previous findings that the subtypes of 5-HT(1) receptors exhibit similar, but distinct, functions.     

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell-surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A(1) receptor abundance. The regional co-localization of hENT1 and A(1) receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A(1) receptors in human brain.     

Role of the equilibrative and concentrative nucleoside transporters in the intestinal absorption of the nucleoside drug, ribavirin, in wild-type and ent1(-/-) mice

Ribavirin is frontline treatment for hepatitis C virus infection. To determine the role of nucleoside transporters in the intestinal absorption of orally administered ribavirin, we perfused the intestines of Ent1(-/-) and wild-type mice, in situ, with [(3)H] ribavirin (20, 200, and 5000 μM) in the presence and absence of sodium. The decrease in luminal ribavirin concentration over 30 min was measured at 5 min intervals. Blood samples were collected approximately every 10 min. Ribavirin plus phosphorylated metabolite concentrations (hereafter referred to as ribavirin) were determined in tissue, blood, and plasma by HPLC fractionation and scintillation counting. There was no significant difference between wild-type and Ent1(-/-) mice in intestinal loss of ribavirin at any ribavirin concentration studied. Perfusions without sodium drastically reduced the intestinal loss of ribavirin in both wild-type and Ent1(-/-) mice. After 20 μM ribavirin perfusions, Ent1(-/-) intestinal tissue contained 8-fold greater ribavirin than wild-type mice (p < 0.01). Ribavirin concentrations in the wild-type intestinal tissue were 70-fold higher after 200 vs 20 μM perfusions (p < 0.001), indicating saturation of intestinal ribavirin efflux and possibly other processes as well. Ribavirin plasma concentrations were significantly higher in wild-type mice (2.7-fold) vs Ent1(-/-) mice at 30 min after the 20 μM perfusion (p < 0.01). These results suggest that, at lower intestinal concentrations of ribavirin, concentrative and equilibrative nucleoside transporters are important in the intestinal absorption of ribavirin. At higher intestinal concentrations, these transporters are saturated and other processes in the intestine (transport and/or metabolism) play an important role in the absorption of ribavirin.     

Fluoxetine mediates G0/G1 arrest by inducing functional inhibition of cyclin dependent kinase subunit (CKS)1

Fluoxetine, a well-known antidepressant used clinically for mental depression has gained attention in cancer research owing to its chemosensitizing potential in drug resistant cell lines. Some preliminary reports, however, suggested its independent cytotoxic potential which is not yet well characterized. Our aim in this study was to characterize its antiproliferative activity in tumor cells and to further elucidate the mechanism. We found that fluoxetine sensitized the effect of cyclophosphamide even in drug sensitive MDA MB 231 and SiHa cells. IC(50) values of 28 and 32 microM were obtained for fluoxetine mediated antiproliferative response in these cells. Further, PARP and caspase 3 cleavage analyses confirmed fluoxetine mediated apoptosis at molecular level. Cell cycle analysis showed that fluoxetine arrested cells at G0/G1 phase in a time dependent manner. The application of bioinformatics tools at this juncture predicted CKS1 as one of the possible targets of fluoxetine, which is of relevance to cell cycle biology. Fluoxetine showed the potential to disrupt skp2-CKS1 assembly required for ubiquitination and proteasomal degradation of p27 and p21. Our in vitro results were in agreement with the predictions made in silico. We found that fluoxetine treatment could accumulate p27 and p21, an immediate outcome characteristic of functional inhibition of CKS1. This was accompanied by the accumulation of cyclin E, another possible target of CKS1. We observed CKS1 downregulation also upon prolonged fluoxetine treatment. Fluoxetine had downregulated cyclin A which confirmed G0/G1 arrest at the molecular level. We conclude that fluoxetine induced cell cycle arrest is CKS1 dependent.     

The 4G/4G genotype of the 4G/5G polymorphism of the type-1 plasminogen activator inhibitor (PAI-1) gene is a determinant of penetrating behaviour in patients with Crohn's disease

BACKGROUND: Crohn's disease is a heterogeneous disorder with polygenic inheritance. AIM: To assess the effect of the 4G/5G polymorphism of the type-1 plasminogen activator inhibitor (PAI-1) gene, the major inhibitor of fibrinolysis, on Crohn's disease susceptibility and phenotype. METHODS: One hundred and fifty-seven patients with Crohn's disease and 350 controls were included prospectively. Medical records were reviewed to determine changes in the Crohn's disease phenotype. The 4G/5G polymorphism was assessed by polymerase chain reaction techniques. RESULTS: The frequencies of the 4G/4G, 4G/5G and 5G/5G genotypes were similar in patients with Crohn's disease and controls. The 4G/4G genotype (P < 0.0001; odds ratio, 4.84) and male sex (P = 0.009; odds ratio, 2.63) were independent risk factors for penetrating behaviour in Crohn's disease. Most Crohn's disease patients had a non-penetrating phenotype at diagnosis. The probability of development of a penetrating phenotype within 5 years of diagnosis was higher in patients with the 4G/4G genotype (72% vs. 19%, P < 0.0001). CONCLUSIONS: The 4G/4G genotype of the PAI-1 gene does not influence Crohn's disease susceptibility, but increases by five-fold the probability of penetrating behaviour. Most patients with the 4G/4G genotype have a non-penetrating phenotype at diagnosis, but develop a penetrating behaviour within 5 years. Genotyping the 4G/5G polymorphism may be useful for the identification of a sub-group of patients with aggressive Crohn's disease, who might benefit from specific therapy.