Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

The differential effects of chlorpromazine and haloperidol on latent inhibition in healthy volunteers.

Latent inhibition (LI) is a measure of reduced learning about a stimulus to which there has been prior exposure without any consequence. It therefore requires a comparison between a pre-exposed (PE) and a non-pre-exposed (NPE) condition. Since, in animals, LI is disrupted by amphetamines and enhanced by antipsychotics, LI disruption has been proposed as a measure of the characteristic attentional deficit in schizophrenia: the inability to ignore irrelevant stimuli. The findings in humans are, however, inconsistent. In particular, a recent investigation suggested that since haloperidol disrupted LI in healthy volunteers, and LI was normal in non-medicated patients with schizophrenia, the previous findings in schizophrenic patients were entirely due to the negative effects of their medication on LI (Williams et al., 1998). We conducted two studies of antipsychotic drug effects on auditory LI using a within-subject, parallel group design in healthy volunteers. In the first of these, single doses of haloperidol (1 mg. i.v.) were compared with paroxetine (20 mg p.o.) and placebo, and in the second, chlorpromazine (100 mg p.o.) was compared with lorazepam (2 mg. p.o.) and placebo. Eye movements, neuropsychological test performance (spatial working memory (SWM), Tower of London and intra/extra dimensional shift, from the CANTAB test battery) and visual analogue rating scales, were also included as other measures of attention and frontal lobe function. Haloperidol was associated with a non-significant reduction in LI scores, and dysphoria/akathisia (Barnes Akathisia Rating Scale) in three-quarters of the subjects. The LI finding may be explained by increased distractibility which was indicated by an increase in antisaccade directional errors in this group. In contrast, LI was significantly increased by chlorpromazine but not by an equally sedative dose of lorazepam (both drugs causing marked decreases in peak saccadic velocity). Paroxetine had no effect on LI, eye movements or CANTAB neuropsychological test performance. Haloperidol was associated with impaired SWM, which correlated with the degree of dysphoria/akathisia, but no other drug effects on CANTAB measures were detected. We conclude that the effect of antipsychotics on LI is both modality and pharmacologically dependent and that further research using a wider range of antipsychotic compounds is necessary to clarify the cognitive effects of these drugs, and to determine whether there are important differences between them.

Effects of amisulpride, risperidone and chlorpromazine on auditory and visual latent inhibition, prepulse inhibition, executive function and eye movements in healthy volunteers.

In view of the evidence that cognitive deficits in schizophrenia are critically important for long-term outcome, it is essential to establish the effects that the various antipsychotic compounds have on cognition, particularly second-generation drugs. This parallel group, placebo-controlled study aimed to compare the effects in healthy volunteers (n = 128) of acute doses of the atypical antipsychotics amisulpride (300 mg) and risperidone (3 mg) to those of chlorpromazine (100 mg) on tests thought relevant to the schizophrenic process: auditory and visual latent inhibition, prepulse inhibition of the acoustic startle response, executive function and eye movements. The drugs tested were not found to affect auditory latent inhibition, prepulse inhibition or executive functioning as measured by the Cambridge Neuropsychological Test Battery and the FAS test of verbal fluency. However, risperidone disrupted and amisulpride showed a trend to disrupt visual latent inhibition. Although amisulpride did not affect eye movements, both risperidone and chlorpromazine decreased peak saccadic velocity and increased antisaccade error rates, which, in the risperidone group, correlated with drug-induced akathisia. It was concluded that single doses of these drugs appear to have little effect on cognition, but may affect eye movement parameters in accordance with the amount of sedation and akathisia they produce. The effect risperidone had on latent inhibition is likely to relate to its serotonergic properties. Furthermore, as the trend for disrupted visual latent inhibition following amisulpride was similar in nature to that which would be expected with amphetamine, it was concluded that its behaviour in this model is consistent with its preferential presynaptic dopamine antagonistic activity in low dose and its efficacy in the negative symptoms of schizophrenia.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p<or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p<or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p<or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy

The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-alpha can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-alpha-/- and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-alpha-/- and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNFalpha-/- and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-alpha significantly, improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy.

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

Effect of the metabolic inhibitor, methimazole on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered in the presence of a metabolic inhibitor. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-sensitive isolates Were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 mu M). Flukes were then incubated for I further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); MTZ+NADPH+TCBZ (15 mu g/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 mu g/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed Using scanning electron microscopy'. After treatment with either TCBZ or TCBZ.SO alone, there was greater surface disruption to the triclabendazole-susceptible than -resistant isolate. However, co-incubation with MTZ and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant isolate than with each drug oil its own; this was not seen for the TCBZ-susceptible Cullompton isolate. Results of this study support the concept of altered drug metabolism in TCBZ-Resistant flukes and this process may play a role in the development of drug resistance.

Inhibition of cytochrome P450-mediated metabolism enhances ex vivo susceptibility of Fasciola hepatica to triclabendazole

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP P450) system was inhibited using piperonyl butoxide (PB). The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP P450 system was inhibited by a 2 h pre-incubation in PB (100 mu M). Flukes were then incubated for a further 22 h in NCTC medium containing either PB; PB + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); PB + NADPH + TCBZ (15 mu g/ml); or PB + NADPH + TCBZ.SO (15 mu g/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible than the resistant isolate. However, co-incubation with PB and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant Oberon isolate than with each drug on its own. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action, and only with TCBZ.SO. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.

The inhibition of human cytomegalovirus (hCMV) protease by hydroxylamine derivatives.

Aryl hydroxylamine derivs. have been synthesized that are some of the most potent inhibitors of hCMV protease prepd. to date (IC50 14-60 nM). Mass spectrometry studies indicate that oxazinone derived hydroxylamines inhibit the enzyme by acylation of Ser132 whereas non-oxazinone derived hydroxylamines appear to inhibit via formation of a sulfinanilide at Cys138.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Multisubstrate adduct inhibitors: Drug design and biological tools

In drug discovery, different methods exist to create new inhibitors possessing satisfactory biological activity. The multisubstrate adduct inhibitor (MAI) approach is one of these methods, which consists of a covalent combination between analogs of the substrate and the cofactor or of the multiple substrates used by the target enzyme. Adopted as the first line of investigation for many enzymes, this method has brought insights into the enzymatic mechanism, structure, and inhibitory requirements. In this review, the MAI approach, applied to different classes of enzyme, is reported from the point of view of biological activity.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 mu M), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ+NADPH+TCBZ (15 mu g/ml), or KTZ+NADPH+triclabendazole sulphoxide (TCBZ. SO; 15 mu g/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ. SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ+TCBZ, but more particularly KTZ+TCBZ. SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.