N3,N7-diaminophenothiazinium derivatives as antagonists of α7-nicotinic acetylcholine receptors expressed in Xenopus oocytes

Derivatization of phenothiazine (PTZ, 1) has been a commonly used method to develop drugs with various pharmacological properties. In the present study, a series of PTZ derivatives 1-11 were investigated on the inhibition of the cloned α7 subunit of the human nicotinic acetylcholine receptor (α7-nAChR) expressed in Xenopus oocytes by using the two-electrode voltage-clamp technique. In the first series of experiments, the effect of unsubstituted phenothiazine 1 on α7-nAChRs was compared with that of the N3,N7-diaminophenothiazin-5-ium derivative 2, and of sequentially methylated derivatives 3-6. In the second set of experiments, the effects of N3,N7-tetra-ethyl- to n-hexylphenothiazin-5-ium derivatives 7-11 were tested. Despite the lack of activity found for 1, a reversible inhibition of α7-nAChRs, ranging from moderate to potent, was observed as a result of a sequential amine- and methylamine substitution of 1. The inhibition of ACh (100 μM)-induced currents was concentration-dependent with IC(50) values ranging from 0.4 to 16.8 μM. However, an optimal inhibitory activity was achieved by prolongation of alkyl chains up to propyl size, as found in PTZ derivative 8, whereas further lengthening of alkyl chains to n-butyl-, n-pentyl-, or n-hexyl groups resulted in inactive derivatives 9-11. The results evidently suggest the presence of a lipophilic binding pocket of narrow tolerability on the receptor protein. These results emphasize the importance of amine and/or alkylamine moieties for the inhibitory effect of PTZ derivatives and provide further insights for the development of novel antagonists targeting α7-nAChRs.     

PTEN deletion and heme oxygenase-1 overexpression cooperate in prostate cancer progression and are associated with adverse clinical outcome

Overexpression of the pro-survival protein heme oxygenase-1 (HO-1) and loss of the pro-apoptotic tumour suppressor PTEN are common events in prostate cancer (PCA). We assessed the occurrence of both HO-1 expression and PTEN deletion in two cohorts of men with localized and castration-resistant prostate cancer (CRPC). The phenotypic cooperation of these markers was examined in preclinical and clinical models. Overall, there was a statistically significant difference in HO-1 epithelial expression between benign, high-grade prostatic intraepithelial neoplasia (HGPIN), localized PCA, and CRPC (p < 0.0001). The highest epithelial HO-1 expression was noted in CRPC (2.00 ± 0.89), followed by benign prostate tissue (1.49 ± 1.03) (p = 0.0003), localized PCA (1.20 ± 0.95), and HGPIN (1.07 ± 0.87) (p < 0.0001). However, the difference between HGPIN and PCA was not statistically significant (p = 0.21). PTEN deletions were observed in 35/55 (63.6%) versus 68/183 (37.1%) cases of CRPC and localized PCA, respectively. Although neither HO-1 overexpression nor PTEN deletions alone in localized PCA showed a statistically significant association with PSA relapse, the combined status of both markers correlated with disease progression (log-rank test, p = 0.01). In a preclinical model, inhibition of HO-1 by shRNA in PTEN-deficient PC3M cell line and their matched cells where PTEN is restored strongly reduced cell growth and invasion in vitro and inhibited tumour growth and lung metastasis formation in mice compared to cells where only HO-1 is inhibited or PTEN is restored. In summary, we provide clinical and experimental evidence for cooperation between epithelial HO-1 expression and PTEN deletions in relation to the PCA patient's outcome. These findings could potentially lead to the discovery of novel therapeutic modalities for advanced PCA.     

Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype

Particulate vaccines are emerging promising technologies for the creation of tunable prophylactics against a wide variety of conditions. Vesicular and solid biodegradable polymer platforms, exemplified by liposomes and polyesters, respectively, are two of the most ubiquitous platforms in vaccine delivery studies. Here we directly compared the efficacy of each in a long-term immunization study and in protection against a model bacterial antigen. Immunization with poly(lactide-co-glycolide) (PLGA) nanoparticles elicited prolonged antibody titers compared to liposomes and alum. The magnitude of the cellular immune response was also highest in mice vaccinated with PLGA, which also showed a higher frequency of effector-like memory T cell phenotype, leading to an effective clearance of intracellular bacteria. The difference in performance of these two common particulate platforms is shown not to be due to material differences but appears to be connected to the kinetics of antigen delivery. Thus, this study highlights the importance of sustained antigen release mediated by particulate platforms and its role in the long-term appearance of effector memory cellular response.     

Age-related decline in white matter tract integrity and cognitive performance: A DTI tractography and structural equation modeling study

Age-related decline in microstructural integrity of certain white matter tracts may explain cognitive decline associated with normal aging. Whole brain tractography and a clustering segmentation in 48 healthy individuals across the adult lifespan were used to examine: interhemispheric (corpus callosum), intrahemispheric association (cingulum, uncinate, arcuate, inferior longitudinal, inferior occipitofrontal), and projection (corticospinal) fibers. Principal components analysis reduced cognitive tests into 6 meaningful factors: (1) memory and executive function; (2) visuomotor dexterity; (3) motor speed; (4) attention and working memory; (5) set-shifting/flexibility; and (6) visuospatial construction. Using theory-based structural equation modeling, relationships among age, white matter tract integrity, and cognitive performance were investigated. Parsimonious model fit demonstrated relationships where decline in white matter integrity may explain age-related decline in cognitive performance: inferior longitudinal fasciculus (ILF) with visuomotor dexterity; the inferior occipitofrontal fasciculus with visuospatial construction; and posterior fibers (i.e., splenium) of the corpus callosum with memory and executive function. Our findings suggest that decline in the microstructural integrity of white matter fibers can account for cognitive decline in normal aging.     

Interactions and relationships of PTEN, ERG, SPINK1 and AR in castration-resistant prostate cancer

Bismar T A, Yoshimoto M, Duan Q, Liu S, Sircar K & Squire J A
(2012) Histopathology  60, 645–652
Interactions and relationships of PTEN, ERG, SPINK1 and AR in castration‐resistant prostate cancer Aims: Recently, ETS‐related gene (ERG) gene rearrangements, phosphatase tensin homologue (PTEN) deletions and serine protease inhibitor Kazal type 1 (SPINK1) overexpression were investigated as potential markers for molecularly subtyping prostate cancer (PCA). However, their incidence and co‐association in castration‐resistant PCA (CRPC) has not been characterized fully. Methods and results: A cohort of 59 CRPC patients was investigated for ERG rearrangements, PTEN deletions and androgen receptor (AR) amplification by fluorescence in‐situ hybridization. SPINK1 overexpression was assessed by immunohistochemistry. ERG rearrangements and PTEN deletions were detected in 22 of 53 (41.5%) and 35 of 55 (63.6%) of cases, with 15 of 22 (68.1%) of ERG rearrangements occurring through deletions. SPINK1 overexpression occurred in three of 51 (5.8%) of cases exclusively in non‐ERG rearranged and AR amplification was detected in 12 of 49 (24.4%) of cases. Only PTEN deletions showed intrafocal heterogeneity occurring in nine of 35 (25.7%) of cases. PTEN deletions were significantly associated with each of ERG rearrangements occurring by deletions only (P = 0.001), AR amplification (P = 0.002) and SPINK1 overexpression (P = 0.002). None of the SPINK1 overexpressing tumours showed AR amplification (P = 0.005) and all occurred in PTEN deleted foci (P = 0.002). Conclusion: The study supports the heterogeneous nature of CRPC and confirms a significant association between PTEN, ERG, AR and SPINK1. Characterizing combined markers will aid in defining PCA subgroups relevant to prognosis contributing to the design of improved therapeutic approaches for CRPC.