In this paper, we investigated the isoform‐specific roles of certain protein kinase C (PKC) isoforms in the regulation of skeletal muscle growth. Here, we provide the first intriguing functional ...evidence that nPKCδ (originally described as an inhibitor of proliferation in various cells types) is a key player in promoting both in vitro and in vivo skeletal muscle growth. Recombinant overexpression of a constitutively active nPKCδ in C2C12 myoblast increased proliferation and inhibited differentiation. Conversely, overexpression of kinase‐negative mutant of nPKCδ (DN‐nPKCδ) markedly inhibited cell growth. Moreover, overexpression of nPKCδ also stimulated in vivo tumour growth and induced malignant transformation in immunodeficient (SCID) mice whereas that of DN‐nPKCδ suppressed tumour formation. The role of nPKCδ in the formation of rhabdomyosarcoma was also investigated where recombinant overexpression of nPKCδ in human rhabdomyosarcoma RD cells also increased cell proliferation and enhanced tumour formation in mouse xenografts. The other isoforms investigated (PKCα, β, ε) exerted only minor (mostly growth‐inhibitory) effects in skeletal muscle cells. Collectively, our data introduce nPKCδ as a novel growth‐promoting molecule in skeletal muscles and invite further trials to exploit its therapeutic potential in the treatment of skeletal muscle malignancies.
Even targeted chemotherapies against solid cancers show a moderate success increasing the need to novel targeting strategies. To address this problem, we designed a systems-level approach ...investigating the neighbourhood of mutated or differentially expressed cancer-related proteins in four major solid cancers (colon, breast, liver and lung). Using signalling and protein-protein interaction network resources integrated with mutational and expression datasets, we analysed the properties of the direct and indirect interactors (first and second neighbours) of cancer-related proteins, not found previously related to the given cancer type. We found that first neighbours have at least as high degree, betweenness centrality and clustering coefficient as cancer-related proteins themselves, indicating a previously unknown central network position. We identified a complementary strategy for mutated and differentially expressed proteins, where the affect of differentially expressed proteins having smaller network centrality is compensated with high centrality first neighbours. These first neighbours can be considered as key, so far hidden, components in cancer rewiring, with similar importance as mutated proteins. These observations strikingly suggest targeting first neighbours as a novel strategy for disrupting cancer-specific networks. Remarkably, our survey revealed 223 marketed drugs already targeting first neighbour proteins but applied mostly outside oncology, providing a potential list for drug repurposing against solid cancers. For the very central first neighbours, whose direct targeting would cause several side effects, we suggest a cancer-mimicking strategy by targeting their interactors (second neighbours of cancer-related proteins, having a central protein affecting position, similarly to the cancer-related proteins). Hence, we propose to include first neighbours to network medicine based approaches for (but not limited to) anticancer therapies.
The vanilloid receptor VR1 is a polymodal nociceptor sensitive to capsaicin, protons, and heat. Because VR1 represents an attractive therapeutic target for conditions ranging from long-term pain to ...bladder hyperreflexia, we and other groups have sought to develop novel ligands with enhanced potencies and novel pharmacological properties. Here, we characterize two compounds, N-2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl-N'-4-(methylsulfonylamino)benzylthiourea (JYL827) and N-(4-tert-butylbenzyl)-N'-3-methoxy-4-(methylsulfonylamino)benzylthiourea (JYL1511), that function as partial agonists for rat VR1 heterologously expressed in Chinese hamster ovary cells. Both compounds showed substantially enhanced potency, inhibiting 3H resiniferatoxin binding with Ki values of 29.3 +/- 7.6 and 50.4 +/- 16.5 nM, respectively, compared with 1810 +/- 270 nM for capsaicin. The compounds showed different extents of partial agonism, 6.8 +/- 0.7% and 17.4 +/- 0.6%, respectively, and the expected corresponding degrees of partial antagonism (93.9 +/- 0.9 and 84.1 +/- 3.2%, respectively). Their IC50 values for antagonism of 45Ca2+ uptake in response to capsaicin were 67.3 +/- 24.9 nM and 3.4 +/- 0.5 nM, respectively. Protons, temperature, and protein kinase C all function as coactivators/modulators of rVR1. All enhanced the extent of partial agonism of JYL827 and JYL1511. Thus, at pH 5.5, for example, the extents of partial agonism increased to 54.9 +/- 2.5% and to 90.7 +/- 1.7%, respectively, relative to the response elicited by 300 nM capsaicin. The extents of partial antagonism decreased correspondingly. Compounds such as JYL827 and JYL1511 now permit exploration of the potential utility of partial agonists of rVR1 in animal models. Our results emphasize, moreover, the strong dependence of such partial agonists on other modulators of rVR1 and predict that their biological behavior will depend strongly on biological context.
We investigated the effect of thapsigargin, a well-known sarcoplasmic reticulum ATPase (SERCA) inhibitor, on the non-specific Ca
2+ channel vanilloid receptor-1 (VR1) in CHO-VR1 cells. We found that ...thapsigargin inhibited the VR-1 mediated
45
Ca
2+
uptake of CHO-VR1 cells
(
IC
50=6.4±1.9
μM)
and the
3
H
RTX binding to VR1
(
IC
50=4.0±1.3
μM)
. Further analysis revealed that thapsigargin is a mixed-type inhibitor, suggesting both direct and indirect interactions between thapsigargin and the capsaicin binding site of VR1. Thapsigargin alone transiently elevated the Ca
2+
i
in CHO-VR1 cells
(
EC
50=44
nM)
. However,
45
Ca
2+
uptake was not detected after thapsigargin treatment, indicating that the emptying of the thapsigargin sensitive intracellular pools of Ca
2+ was responsible for the elevated Ca
2+
i
level rather than the activation of VR-1. We conclude that thapsigargin represents a new prototype of a VR1 inhibitor and that caution should be exercised in interpreting the effects of thapsigargin, especially when it is used in the micromolar range to inhibit SERCA activity.
Adrenocortical cancer is a rare tumor and its prognosis is poor. Although numerous tumor-associated genetic and signal transduction alterations have been described to date, its pathogenesis is still ...unclear. Hybridization-based DNA microarray approaches may reveal significant gene expression alterations and may thus contribute to a better understanding of tumorigenesis and may identify molecular markers applicable for the distinction of benign and malignant lesions. Beside gene expression patterns, studies on microRNAs seem to be useful, as well. Novel therapeutical targets might be established by these approaches. In this review, the authors attempt to summarize the main findings of mRNA and microRNA expression microarray studies performed to date in adrenocortical cancer including a recent meta-analysis of gene expression data and present novel pathogenic pathways.
The role of oxidative stress (OXS) due to myocardial nitric oxide synthase (NOS) uncoupling related to oxidative depletion of its cofactor tetrahydrobiopterin (BH4) emerged in the pathogenesis of ...heart failure with preserved ejection fraction. We determined the prevalence of six single nucleotide polymorphisms (SNPs) of genes encoding enzymes related to OXS, BH4 metabolism, and NOS function in ≥60-year-old 94 patients with hypertension and 18 age-matched controls with normal ejection fraction. Using echocardiography, 56/94 (60%) patients with hypertension had left ventricular (LV) diastolic dysfunction (HTDD+ group) and 38/94 (40%) patients had normal LV diastolic function (HTDD- group). Four SNPs (rs841, rs3783641, rs10483639, and rs807267) of guanosine triphosphate cyclohydrolase-1, the rate-limiting enzyme in BH4 synthesis, one (rs4880) of manganese superoxide dismutase, and one (rs1799983) of endothelial NOS genes were genotyped using real-time polymerase chain reaction method and Taqman probes. Protein carbonylation, BH4, and total biopterin levels were measured from plasma samples. No between-groups difference in minor allele frequency of SNPs was found. We calculated a genetic score indicating risk for OXS based on the minor allele frequencies of the SNPs. A high genetic risk for OXS was significantly associated with HTDD+ even after adjustment for confounding variables (odds ratio 95% confidence interval:4.79 1.12-20.54; P = .035). In both patient groups protein carbonylation (P < .05 for both), plasma BH4 (P < .01 for both) and in the HTDD+ group total biopterin (P < .05) increased versus controls. In conclusion, in patients with hypertension and normal ejection fraction, a potential precursor of heart failure with preserved ejection fraction, a partly genetically determined increased OXS, seems to be associated with the presence of LV diastolic dysfunction.
PTEN phosphatase, a product of PTEN tumor suppressor gene, exists in cells in phosphorylated and unphosphorylated form and has a central role in regulation of PI3K/Akt signalling which is involved in ...non-genomic action of estradiol. The purpose of this study was to analyze the level of total PTEN and phosphoPTEN parallel to phosphoAkt in leiomyoma and adjacent myometrium during menstrual cycle and at menopause. The expression of total PTEN in leiomyoma and myometrium did not change throughout the experiments. However, the level of phosphoPTEN was increased in leiomyoma during menstrual cycle. The phosphorylation of PTEN in myometrium was lower during secretory phase than that of proliferative phase. The phosphoAkt was abundant in leiomyoma, and its expression was higher during menstrual cycle than in myometrium. The phosphorylation of PTEN was directly related to phosphoAkt, suggesting a direct link between the inactivation of PTEN and activation of Akt. At the decline of sexual steroids, at menopause, no differences were observed in the expression of studied proteins between the two types of tissues. Our results suggest that the altered phosphorylation of PTEN protein and the consequent activation of survival signals may contribute to the pathomechanism of leiomyoma.
A series of
N-(3-acyloxy-2-benzylpropyl)-
N′-(4-hydroxy-3-methoxybenzyl)thiourea derivatives were investigated as vanilloid receptor ligands in an effort to discover a novel class of analgesics. The ...proposed pharmacophore model of resiniferatoxin, which includes the C
20-homovanillic moiety, the C
3-carbonyl and the orthoester phenyl ring as key pharmacophoric groups, was utilized as a guide for drug design. The compounds were synthesized after several steps from diethylmalonate and evaluated in vitro in a receptor binding assay and in a capsaicin-activated channel assay. Additional evaluation of analgesic activity, anti-inflammatory activity and pungency was conducted in animal models by the writhing test, the ear edema assay, and the eye-wiping test, respectively. Among the new compounds,
23 and
28 were found to be the most potent receptor agonists of the series with
K
i values of 19
nM and 11
nM, respectively. Their strong in vitro potencies were also reflected by an excellent analgesic profile in animal tests with ED
50 values of 0.5
μg/kg for
23 and 1.0
μg/kg for
28. Relative to capsaicin these compounds appear to be ca. 600 and 300 times more potent. Both
23 and
28 were found to be less pungent than capsaicin based on the eye-wiping test. However, the compounds did not show significant anti-inflammatory activity. A molecular modeling study comparing the energy-minimized structures of resiniferatoxin and
35 demonstrated a good correlation in the spatial disposition of the corresponding key pharmacophores. The thioureas described in this investigation, which were designed as simplified resiniferatoxin surrogates, represent a novel class of potent vanilloid receptor agonists endowed with potent analgesic activity and reduced pungency.
We recently reported that N-(4-t-butylbenzyl)-N'-4-(methylsulfonylamino)benzyl thiourea (2) was a high affinity antagonist of the vanilloid receptor with a binding affinity of K(i)=63 nM and an ...antagonism of K(i)=53.9 nM in rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells (Mol. Pharmacol. 2002, 62, 947-956). In an effort to further improve binding affinity and antagonistic potency, we have modified the C-region of the lead 4-t-butylbenzyl group with diverse surrogates, such as araalkyl, alkyl, 4-alkynylbenzyl, indanyl, 3,3-diarylpropyl, 4-alkoxybenzyl, 4-substituted piperazine and piperidine. The lipophilic surrogates, arylalkyl and alkyl, conferred modest decreases in binding affinities and antagonistic potencies; the groups having heteroatoms resulted in dramatic decreases. Our findings indicate that 4-t-butylbenzyl is one of the most favorable groups for high receptor binding and potent antagonism to VR1 in this structural series.
Objective Ecallantide is a recombinant peptide in the same class as aprotinin that inhibits plasma kallikrein, a major component of the contact coagulation and inflammatory cascades. Therefore, ...ecallantide was expected to reduce blood loss associated with cardiac surgery requiring cardiopulmonary bypass. Methods This prospective multinational, randomized, double-blind trial enrolled patients undergoing cardiac surgery using cardiopulmonary bypass for procedures associated with a high risk of bleeding. Patients were randomly assigned to ecallantide (n = 109) or tranexamic acid (high dose, n = 24; low dose, n = 85). Efficacy was assessed from the volume of packed red blood cells administered within the first 12 hours after surgery. Results The study was terminated early after the independent data safety and monitoring board observed a statistically significantly higher 30-day mortality in the ecallantide group (12%) than in the tranexamic acid groups (4%, P = .041). Patients receiving ecallantide received more packed red blood cells within 12 hours of surgery than tranexamic acid-treated patients: median = 900 mL (95% confidence interval, 600–1070) versus 300 mL (95% confidence interval, 0–523) ( P < .001). Similar differences were seen at 24 hours and at discharge. Patients treated with the higher tranexamic acid dose received less packed red blood cells, 0 mL (95% confidence interval, 280–600), than the group treated with the lower dose, 400 mL (95% confidence interval, 0–400) ( P = .008). No deaths occurred in the higher dose tranexamic acid group. Conclusions Ecallantide was less effective at reducing perioperative blood loss than tranexamic acid. High-dose tranexamic acid was more effective than the low dose in reducing blood loss.
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