Adrenomedullin prevents apoptosis in prostate cancer cells

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents

Entropy Measures of Electroencephalograms towards the Diagnosis of Psychogenic Non-Epileptic Seizures

Psychogenic non-epileptic seizures (PNES) may resemble epileptic seizures but are not caused by epileptic activity. However, the analysis of electroencephalogram (EEG) signals with entropy algorithms could help identify patterns that differentiate PNES and epilepsy. Furthermore, the use of machine learning could reduce the current diagnosis costs by automating classification. The current study extracted the approximate sample, spectral, singular value decomposition, and Renyi entropies from interictal EEGs and electrocardiograms (ECG)s of 48 PNES and 29 epilepsy subjects in the broad, delta, theta, alpha, beta, and gamma frequency bands. Each feature-band pair was classified by a support vector machine (SVM), k-nearest neighbour (kNN), random forest (RF), and gradient boosting machine (GBM). In most cases, the broad band returned higher accuracy, gamma returned the lowest, and combining the six bands together improved classifier performance. The Renyi entropy was the best feature and returned high accuracy in every band. The highest balanced accuracy, 95.03%, was obtained by the kNN with Renyi entropy and combining all bands except broad. This analysis showed that entropy measures can differentiate between interictal PNES and epilepsy with high accuracy, and improved performances indicate that combining bands is an effective improvement for diagnosing PNES from EEGs and ECGs

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation

Investigation of Alzheimer’s Disease EEG Frequency Components with Lempel-Ziv Complexity

. This pilot study applied Lempel-Ziv Complexity (LZC) to 22 resting EEG signals, collected using the 10-20 international system, from 11 patients with Alzheimer’s disease (AD) and 11 age-matched controls. This allowed for frequency band analysis as the EEG signals were first prefiltered with a third order Hamming window in the ranges F to F+WHz with both F and W equal to 1-30Hz respectively. Control subjects were found to have a greater signal complexity than AD patients with statistically significant bands seen at various ranges in all 16 electrodes. The maximum statistical significance (Student’s t test, p<0.01) was increased over the findings with traditional signal filtering techniques allowing the whole range, with a maximum significance of p=3.50e-6 at electrode T4 between 7-18Hz. Electrode F4 also showed significantly high statistically significant differences. The maximum accuracy, both controls and AD patients correctly identified, found with Receiver Operating Characteristic Curves was 95.45% (21 of 22 subjects correctly classified) at T4 (7-18Hz and 7-20Hz), Fp2 (8-32Hz) and F4 (6-21Hz), which is significantly more accurate than the most accurate methods previously applied to this dataset. The beta band (13-30Hz) was found to be most influential in separating the two test groups in this study with the best range suggested to be 5-26Hz, combining traditional theta, alpha and beta bands. These findings suggest pre-filtering has a significant effect on method outcomes and can be successfully tailored to improve the statistical effectiveness of LZC at distinguishing between these two EEG groups. However, more testing is required to investigate the effectiveness at distinguishing other signal dynamics

Adrenomedullin and proadrenomedullin N-terminal 20 peptide in the normal prostate and in prostate carcinoma

There is increasing evidence for the important role played by regulatory peptides in the physiology of the normal and neoplastic prostate. Adrenomedullin (AM) and pro-adrenomedullin N-terminal 20 peptide (PAMP) are recently discovered regulatory peptides widely expressed in the normal prostate and in prostate carcinoma. AM is produced in secretory, stroma, and endothelial cells and in neurons of the prostate ganglia. PAMP is only produced by neuroendocrine cells. The expression of AM mRNA is regulated by androgens in the rat prostate. The number of neuroendocrine cells expressing PAMP is increased in prostate carcinoma after androgen deprivation, which shows that this peptide could regulate androgen-independent prostate tumor growth. However, the roles of AM and PAMP in the normal prostate and in prostate carcinoma are yet to be elucidated

2-Methyl-3-(n-octylsulfan­yl)quinoxaline

All the non-H atoms of the title compound, C17H24N2S, lie almost in a common plane (r.m.s. deviation = 0.049 Å). The octyl chain adopts an all-trans conformation

1-Benzyl-3-methyl­quinoxalin-2(1H)-one

The asymmetric unit of the title compound, C16H14N2O, contains three independent mol­ecules. The dihedral angles between the quinoxaline and phenyl planes in the three mol­ecules are 82.58 (8), 85.66 (9) and 85.36 (9)°. The crystal packing is stabilized by C—H⋯O and C—H⋯N hydrogen bonds

Androgen-independent expression of adrenomedullin and peptidylglycine alpha-amidating monooxygenase in human prostatic carcinoma

Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues

Overexpression of adrenomedullin gene markedly inhibits proliferation of PC3 prostate cancer cells in vitro and in vivo

The expression of the gene encoding adrenomedullin (AM), a multifunctional peptide hormone, in the prostate is localized to the epithelial cells. Prostate cancer cells are derived from prostatic epithelial cells. To elucidate the potential role of the AM gene in prostate cancer progression, we have stably-transfected the PC3 human prostate cancer cell line with an AM gene expression vector. The AM-transfected PC3 sublines were studied along with parental and empty vector transfected PC3 cells as controls. The average level of AM in the conditioned media of AM-transfected cells was 0.959+/-0.113 nM, a physiologically relevant concentration. The ectopic expression of AM gene inhibited the proliferation of PC3 cells in culture dishes. In addition, anchorage-independent growth of the transfected sublines was virtually abolished in soft agar assays. Flow cytometry studies showed that overexpression of AM gene caused a very significant G(1)/G(0) cell cycle arrest. In vivo experiments demonstrated that AM gene expression markedly inhibited the growth of xenograft tumors in nude mice. Our in vivo and in vitro studies suggest that AM could strongly suppress the malignancy of prostate cancer cells, via autocrine and/or paracrine mechanisms

Gene expression profiling identifies IL-13 receptor alpha 2 chain as a therapeutic target in prostate tumor cells overexpressing adrenomedullin

Human adrenomedullin (AM) is a 52 amino acid peptide, which shares homology with the calcitonin gene-related peptide. Overexpression of AM in the prostate carcinoma cell line PC-3 results in growth inhibition with a 20% (for human AM) and 35% (for rat AM) increase in doubling time compared to parental or mock-transfected cells. We demonstrate by gene expression profiling that AM overexpression results in the dysregulation of approximately 100 genes. Examples of such genes include many involved in the formation of the cytoskeleton, cell adhesion and the extracellular matrix, as well as regulators of the cell cycle and apoptosis, cytokines and transcription factors. Several genes related to cell growth arrest, such as GADD45, IGF-BP6 and RUNX-3, are upregulated by AM. Interestingly, interleukin-13 receptor alpha 2 (IL-13R alpha 2) transcripts were significantly increased in clones overexpressing AM, which was confirmed by semiquantitative RT-PCR analysis. In addition, PC-3 cells treated with AM showed an overexpression of IL-13R alpha 2, which was abolished when cells were preincubated with an anti-AM blocking antibody. When PC-3 cells overexpressing AM and the IL-13R alpha 2 were treated with the highly specific IL13-PE38 cytotoxin, which binds to this receptor, a concentration-dependent inhibition of protein synthesis was observed. The IC(50) (concentration of cytotoxin inhibiting protein synthesis by 50%) ranged from 1 to 4 ng/ml. This cytotoxicity was specific as it was neutralized by the excess of IL-13 and confirmed by clonogenic assays. This study describes a novel AM-induced mechanism of tumor sensitization through the upregulation of functional IL-13R alpha 2 chain, an ideal target for the highly specific recombinant chimeric cytotoxin IL13-PE38