Docetaxel Resistance in Prostate Cancer: Taking It Up a Notch.

Notch signaling is implicated in prostate cancer progression and docetaxel resistance. Cui and colleagues describe the additive efficacy and mechanisms of a γ-secretase inhibitor, PF-03084014, and docetaxel in preclinical models of prostate cancer, suggesting the need for further clinical development of Notch pathway modulators in men with metastatic prostate cancer.

Cytokine gene polymorphisms in ischaemic heart disease:Investigation using family based tests of association

Atherosclerosis has an inflammatory basis, with cytokines, cellular adhesion molecules and pro-inflammatory cells having important roles in the initiation and progression of this process. Interleukin (IL) 6, IL-10 and transforming growth factor (TGF) β have been proposed as important modulators of the atherosclerotic process, with IL-6 having a pro-inflammatory, atherogenic effect and IL-10 and TGF-β having anti-inflammatory, protective roles. The possible role of functional polymorphisms in the promoter regions of the IL-6, IL-10 and TGF-β genes in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population using two recently described family-based tests of association. We genotyped 1,012 individuals from 386 families with at least one member prematurely affected with IHD. Using the combined transmission disequilibrium test (TDT)/sib-TDT and the pedigree disequilibrium test, no association between any of the IL-6 -174G/C, IL-10 -1082G/A and TGF-β -509C/T polymorphisms and IHD was found. Our data demonstrate that, in an Irish population, these polymorphisms are not associated with IHD. © Springer-Verlag 2004.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer

Background BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors.

Methods We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) ( the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA ( siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided.

Results Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors ( 95% confidence interval [CI]=2.6-fold to 40.1-fold, P =.0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector ( T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: > 10(-5) versus 8.0 x 10(-9) M [ 95% CI=3.1x10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha : > 10(-5) versus 4.9 x 10(-8) M [ 95% CI=2.0 x 10(-9) to 3.9 x 10(-6) M]).

Conclusions BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Investigation of DNA polymorphisms in SMAD genes for genetic predisposition to diabetic nephropathy in patients with type 1 diabetes mellitus

Aims/hypothesis: SMAD proteins are involved in multiple signalling pathways and are key modulators of gene expression. We hypothesised that genetic variation in selected SMAD genes contributes to susceptibility to diabetic nephropathy. Methods: We selected 13 haplotype tag (ht) single nucleotide polymorphisms (SNPs) from 67 variants identified by resequencing the SMAD2 and SMAD3 genes. For SMAD1, SMAD4 and SMAD5 genes, genotype data were downloaded for 217 SNPs from Phase II of the International HapMap project. Of these, 85 SNPs met our inclusion criteria, resulting in the selection of 13 tag SNPs for further investigation. A case-control approach was employed, using 267 nephropathic patients and 442 controls with type 1 diabetes from Ireland. Two further populations (totalling 1,407 patients, 2,238 controls) were genotyped to validate initial findings. Genotyping was conducted using iPLEX, TaqMan and gel electrophoresis.
Results: The distribution of genotypes was in Hardy-Weinberg equilibrium. Analysis by the ? 2 test of genotype and allele frequencies in patients versus controls in the Irish population (n?=?709) revealed evidence for the association of one allele at 5% level of significance (rs10515478, p uncorrected?=?0.006; p corrected?=?0.04). This finding represents a relatively small difference in allele frequency of 6.4% in the patient group compared with 10.7% in the control group; this difference was not supported in subsequent investigations using DNA from European individuals with similar phenotypic characteristics.
Conclusions/interpretation: We selected an appropriate subset of variants for the investigation of common genetic risk factors and assessed SMAD1 to SMAD5 genes for association with diabetic nephropathy. We conclude that common polymorphisms in these genes do not strongly influence genetic susceptibility to diabetic nephropathy in white individuals with type 1 diabetes mellitus.

PKB binding proteins: Getting in on the akt

Protein kinase B (PKB) has emerged as the focal point for many signal transduction pathways, regulating multiple cellular processes such as glucose metabolism, transcription, apoptosis, cell proliferation, angiogenesis, and cell motility. In addition to acting as a kinase toward many substrates involved in these processes, PKB forms complexes with other proteins that are not substrates, but rather act as modulators of PKB activity and function. In this review, we discuss the implications of these data in understanding the multitude of functions predicted for PKB in cells.

KCNQ Currents and Their Contribution to Resting Membrane Potential and the Excitability of Interstitial Cells of Cajal From the Guinea Pig Bladder

PURPOSE: The presence of novel KCNQ currents was investigated in guinea pig bladder interstitial cells of Cajal and their contribution to the maintenance of the resting membrane potential was assessed. MATERIALS AND METHODS: Enzymatically dispersed interstitial cells of Cajal were patch clamped with K(+) filled pipettes in voltage clamp and current clamp modes. Pharmacological modulators of KCNQ channels were tested on membrane currents and the resting membrane potential. RESULTS: Cells were stepped from -60 to 40 mV to evoke voltage dependent currents using a modified K(+) pipette solution containing ethylene glycol tetraacetic acid (5 mM) and adenosine triphosphate (3 mM) to eliminate large conductance Ca activated K channel and K(adenosine triphosphate) currents. Application of the KCNQ blockers XE991, linopirdine (Tocris Bioscience, Ellisville, Missouri) and chromanol 293B (Sigma) decreased the outward current in concentration dependent fashion. The current-voltage relationship of XE991 sensitive current revealed a voltage dependent, outwardly rectifying current that activated positive to -60 mV and showed little inactivation. The KCNQ openers flupirtine and meclofenamic acid (Sigma) increased outward currents across the voltage range. In current clamp mode XE991 or chromanol 293B decreased interstitial cell of Cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells. Flupirtine or meclofenamic acid hyperpolarized interstitial cells of Cajal and inhibited any spontaneous electrical activity. CONCLUSIONS: This study provides electrophysiological evidence that bladder interstitial cells of Cajal have KCNQ currents with a role in the regulation of interstitial cell of Cajal resting membrane potential and excitability. These novel findings provide key information on the ion channels present in bladder interstitial cells of Cajal and they may indicate relevant targets for the development of new therapies for bladder instability.

Thrombopoietin, flt3-ligand and c-kit-ligand modulate HOX gene expression in expanding cord blood CD133(+) cells

Haemopoietic stem/progenitor cell (HSPC) development is regulated by extrinsic and intrinsic stimuli. Extrinsic modulators include growth factors and cell adhesion molecules, whereas intrinsic regulation is achieved with many transcription factor families, of which the HOX gene products are known to be important in haemopoiesis. Umbilical cord blood CD133(+) HSPC proliferation potential was tested in liquid culture with 'TPOFLK' (thrombopoietin, flt-3 ligand and c-kit ligand, promoting HSPC survival and self-renewal), in comparison to 'K36EG' (c-kit-ligand, interleukins-3 and -6, erythropoietin and granulocyte colony-stimulating factor, inducing haemopoietic differentiation). TPOFLK induced a higher CD133(+) HSPC proliferation (up to 60-fold more, at week 8) and maintained a higher frequency of the primitive colony-forming cells than K36EG. Quantitative polymerase chain reaction analysis revealed opposite expression patterns for specific HOX genes in expanding cord blood CD133(+) HSPC. After 8 weeks in liquid culture, TPOFLK increased the expression of HOX B3, B4 and A9 (associated with uncommitted HSPC) and reduced the expression of HOX B8 and A10 (expressed in committed myeloid cells) when compared to K36EG. These results suggest that TPOFLK induces CD133(+) HSPC proliferation, self-renewal and maintenance, up-regulation of HOX B3, B4 and A9 and down-regulation of HOX B8 and A10 gene expression.

Effect of red clover-derived isoflavone supplementation on insulin-like growth factor, lipid and antioxidant status in healthy female volunteers: a pilot study

Background: Isoflavones are estrogen-like plant compounds that may protect against cardiovascular disease and endocrine-responsive cancer. Isoflavones may, because of their ability to act as selective estrogen receptor modulators, alter insulin-like growth factor (IGF) status.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Synthesis and SAR of novel, non-MPEP chemotype mGluR5 NAMs identified by functional HTS

This Letter describes the discovery and SAR of three novel series of mGluR5 non-competitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype identified by a functional HTS approach. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and further examples of a mode of pharmacology 'switch' to provide PAMs with a non-MPEP scaffold. (C) 2009 Elsevier Ltd. All rights reserved.

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?), cultures were treated with the PPAR? ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPAR? antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPAR? ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

Discovery and SAR of a novel series of non-MPEP site mGlu(5) PAMs based on an aryl glycine sulfonammide scaffold

Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu(5)) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site mGlu(5) PAM chemotype.

Good news-bad news:the Yin and Yang of immune privilege in the eye

The eye and the brain are prototypical tissues manifesting immune privilege (IP) in which immune responses to foreign antigens, particularly alloantigens are suppressed, and even completely inhibited. Explanations for this phenomenon are numerous and mostly reflect our evolving understanding of the molecular and cellular processes underpinning immunological responses generally. IP is now viewed as a property of many tissues and the level of expression of IP varies not only with the tissue but with the nature of the foreign antigen and changes in the limited conditions under which privilege can operate as a mechanism of immunological tolerance. As a result, IP functions normally as a homeostatic mechanism preserving normal function in tissues, particularly those with highly specialized function and limited capacity for renewal such as the eye and brain. However, IP is relatively easily bypassed in the face of a sufficiently strong immunological response, and the privileged tissues may be at greater risk of collateral damage because its natural defenses are more easily breached than in a fully immunocompetent tissue which rapidly rejects foreign antigen and restores integrity. This two-edged sword cuts its swathe through the eye: under most circumstances, IP mechanisms such as blood-ocular barriers, intraocular immune modulators, induction of T regulatory cells, lack of lymphatics, and other properties maintain tissue integrity; however, when these are breached, various degrees of tissue damage occur from severe tissue destruction in retinal viral infections and other forms of uveoretinal inflammation, to less severe inflammatory responses in conditions such as macular degeneration. Conversely, ocular IP and tumor-related IP can combine to permit extensive tumor growth and increased risk of metastasis thus threatening the survival of the host.

Structure-based design of HSPA5 inhibitors: From peptide to small molecule inhibitors

We identified nine small-molecule hit compounds of Heat shock 70 kDa protein 5 (HSPA5) from cascade in silico screening based on the binding modes of the tetrapeptides derived from the peptide substrate or inhibitors of Escherichia coli HSP70. Two compounds exhibit promising inhibition activities from cancer cell viability and tumor inhibition assays. The binding modes of the hit compounds provide a platform for development of selective small molecule inhibitors of HSPA5. (C) 2013 Elsevier Ltd. All rights reserved.

Immune modulation in advanced radiotherapies: Targeting out-of-field effects

By virtue of being a localized treatment modality, radiotherapy is unable to deliver a tumoricidal radiation dose to tissues outside of the irradiated field. Nevertheless, ionizing radiation may result in radiation damage mediated by a bystander like effect away from the irradiated field, but this response is likely to be modest when radiotherapy is the sole treatment modality. Over the last decade there has been a re-emergence of immune modulating therapies as anti-cancer treatment modalities. Clinical trials on vaccines have on the whole been largely disappointing, but greater response rates have been observed from the immune checkpoint modulators. A clinical benefit of using such agents has been shown in disease sites such as melanoma and non-small cell lung cancer. There is growing pre-clinical data and a number of case reports which suggest the presence of abscopal effects when radiotherapy is co-administered with immune checkpoint inhibitors, suggesting that this combination may lead to an enhanced tumour response outside of the primary treatment field. In this review, the mechanisms of such an enhanced out-of-field tumour response, the potential clinical utilities, the optimal radiotherapy delivery and considerations for clinical follow-up following treatment are discussed.