Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia.

Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann's area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12.

Regulation of colorectal cancer cell epithelial to mesenchymal transition by the renin angiotensin system.

BACKGROUND AND AIM: Epithelial to mesenchymal transition (EMT) is implicated in tumor progression. We aimed to determine if the renin angiotensin system has a role in colorectal cancer (CRC) cell EMT. METHODS: Human CRC cell lines DLD-1 and LIM2405 were used in wound scratch migration assays where they were treated with renin angiotensin system peptide ANG II alone or with blockers of ANG II type 1 or 2 receptors (AT1R and AT2R). Levels of epithelial (E-cadherin), mesenchymal (ZEB1, Vimentin) markers, inducible nitric oxide synthase (iNOS), and MMP9 were determined by flow cytometry. Mice bearing CRC liver metastases and treated with blockers for AT1R or AT2R were examined for ZEB1 and iNOS by immunohistochemistry. RESULTS: ANG II increased in-vitro CRC cell migration in both cell lines, this was inhibited by AT1R (IRB) or AT2R blockade (PD123319). DLD-1 cells treated with AT1R blocker resulted in increased E-cadherin, reduced ZEB1, and Vimentin expression compared with ANG II-treated cells. Treatment with AT2R blocker decreased E-cadherin, no change in ZEB1 or Vimentin expression. AT1R blockade increased iNOS and decreased MMP9 expression in DLD-1 and LIM2405 cells. AT2R blockade decreased iNOS and MMP9 expression in both cell lines. In vivo, ZEB1 staining was higher in ANG II-treated animals compared with control and AT1R blockade treated animals, while activation of the AT2R led to an increase in iNOS compared with control and AT1R blockade. CONCLUSIONS: ANG II-induced migration of CRC cells via both AT1 and AT2 receptors; the AT1R-mediated effects were associated with changes typical of EMT.

The renin angiotensin system regulates Kupffer cells in colorectal liver metastases.

Blockade of the renin angiotensin system (RAS) can inhibit tumor growth and this may be mediated via undefined immunomodulatory actions. This study investigated the effects of RAS blockade on liver macrophages (Kupffer cells; KCs) in an orthotopic murine model of colorectal cancer (CRC) liver metastases. Here we showed that pharmacological targeting of the RAS [ANG II (31.25 µg/kg/h i.p.), ANG-(1-7) (24 µg/kg/h i.p.) or the ACE inhibitor; captopril (750 mg/kg/d i.p.)] altered endogenous KC numbers in the tumor-bearing liver throughout metastatic growth. Captopril, and to a lesser extent ANG-(1-7), increased KC numbers in the liver but not tumor. KCs were found to express the key RAS components: ACE and AT1R. Treatment with captopril and ANG II increased the number of AT1R-expressing KCs, although total KC numbers were not affected by ANG II. Captopril (0.1 µM) also increased macrophage invasion in vitro. Additionally, captopril was administered with KC depletion before tumor induction (day 0) or at established metastatic growth (day 18) using gadolinium chloride (GdCl 3; 20 mg/kg). Livers were collected at day 21 and quantitative stereology used as a measure of tumor burden. Captopril reduced growth of CRC liver metastases. However, when captopril was combined with early KC depletion (day 0) tumor growth was significantly increased compared with captopril alone. In contrast, late KC depletion (day 18) failed to influence the anti-tumor effects of captopril. The result of these studies suggests that manipulation of the RAS can alter KC numbers and may subsequently influence progression of CRC liver metastases.

Cholinergic connectivity: it's implications for psychiatric disorders.

Acetylcholine has been implicated in both the pathophysiology and treatment of a number of psychiatric disorders, with most of the data related to its role and therapeutic potential focusing on schizophrenia. However, there is little thought given to the consequences of the documented changes in the cholinergic system and how they may affect the functioning of the brain. This review looks at the cholinergic system and its interactions with the intrinsic neurotransmitters glutamate and gamma-amino butyric acid as well as those with the projection neurotransmitters most implicated in the pathophysiologies of psychiatric disorders; dopamine and serotonin. In addition, with the recent focus on the role of factors normally associated with inflammation in the pathophysiologies of psychiatric disorders, links between the cholinergic system and these factors will also be examined. These interfaces are put into context, primarily for schizophrenia, by looking at the changes in each of these systems in the disorder and exploring, theoretically, whether the changes are interconnected with those seen in the cholinergic system. Thus, this review will provide a comprehensive overview of the connectivity between the cholinergic system and some of the major areas of research into the pathophysiologies of psychiatric disorders, resulting in a critical appraisal of the potential outcomes of a dysregulated central cholinergic system.

Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study.

BACKGROUND: Mu opioid receptors have previously been shown to be altered in people with affective disorders who died as a result of suicide. We wished to determine whether these changes were more widespread and independent of psychiatric diagnoses. METHODS: Mu receptor levels were determined using [3 H]DAMGO binding in BA24 from 51 control subjects; 38 people with schizophrenia (12 suicides); 20 people with major depressive disorder (15 suicides); 13 people with bipolar disorder (5 suicides) and 9 people who had no history of psychiatric disorders but who died as a result of suicide. Mu receptor levels were further determined in BA9 and caudate-putamen from 38 people with schizophrenia and 20 control subjects using [3 H]DAMGO binding and, in all three regions, using Western blots. Data was analysed using one-way ANOVAs with Bonferroni's Multiple Comparison Test or, where data either didn't approximate to a binomial distribution or the sample size was too small to determine distribution, a Kruskal-Wallis test with Dunn's Multiple Comparison Test. RESULTS: [3 H]DAMGO binding density was lower in people who had died as a result of suicide (p<0.01). People with schizophrenia who had died as a result of suicide had lower binding than control subjects (p<0.001), whilst people with bipolar disorder (non- suicide) had higher levels of binding (p<0.05). [3 H]DAMGO binding densities, but not mu protein levels, were significantly decreased in BA9 from people with schizophrenia who died as a result of suicide (p<0.01). CONCLUSIONS: Overall these data suggest that mu opioid receptor availability is decreased in the brains of people with schizophrenia who died as a result of suicide, which would be consistent with increased levels of endogenous ligands occupying these receptors.

Altered efficacy of AT1R-targeted treatment after spontaneous cancer cell-AT1R upregulation.

BACKGROUND: Targeting of the renin angiotensin system (RAS) reduces tumour growth in experimental models of cancer. We aimed to establish if combined targeting of the 'classical' and 'alternative' arms of the RAS could result in synergistic inhibition of colorectal cancer (CRC) liver metastases. METHODS: Immediately following induction of CRC liver metastases through intrasplenic injection of murine CRC cells, treatment with irbesartan (AT1R blocker; 50 mg/kg/day s.c.), captopril (ACE inhibitor; 750 mg/kg/day i.p.), CGP42112A (AT2R agonist; 0.6 µg/kg/hr i.p.), and/or ANG-(1-7) (24 µg/kg/hr i.p.) began and continued for 21 days. Liver to body weight ratio and/or stereology were used as a measure of tumour burden. Immunohistochemistry was used to determine AT1R and VEGF expression as well as proliferation (Ki67), apoptosis (active caspase 3) and angiogenesis (CD34). RESULTS: Combined RAS therapies failed to improve upon single arm therapies. However, while irbesartan previously inhibited tumour growth in this model, in the current experiments irbesartan failed to affect tumour burden. Subsequent analysis showed a cancer-cell specific upregulation of the angiotensin II type I receptor (AT1R) in irbesartan-insensitive compared to irbesartan-sensitive tumours. The upregulation of AT1R was associated with an increase in proliferation and VEGF expression by cancer cells. While animals bearing irbesartan-sensitive tumours showed a marked decrease in the number of proliferating cells in the liver and VEGF-expressing infiltrating cells in the tumour following AT1R treatment, these were unchanged by treatment in animals bearing irbesartan-insensitive (high AT1R expressing) tumours. CONCLUSIONS: Although the results do not support increased efficacy of combined treatment, they provide intriguing evidence of the importance of RAS expression in determining patient response and tumour growth potential and suggest that components of the RAS could be used as biomarkers to aid in patient selection.

Changes in the renin angiotensin system during the development of colorectal cancer liver metastases.

BACKGROUND: Blockade of the renin angiotensin system (RAS) via angiotensin I converting enzyme (ACE) inhibition reduces growth of colorectal cancer (CRC) liver metastases in a mouse model. In this work we defined the expression of the various components of the RAS in both tumor and liver during the progression of this disease. METHODS: Immunohistochemistry and quantitative RT-PCR was used to examine RAS expression in a mouse CRC liver metastases model. CRC metastases and liver tissue was assessed separately at key stages of CRC liver metastases development in untreated (control) mice and in mice treated with the ACE inhibitor captopril (750 mg/kg/day). Non-tumor induced (sham) mice indicated the effect of tumors on normal liver RAS. The statistical significance of multiple comparisons was determined using one-way analysis of variance followed by Bonferroni adjustment with SAS/STAT software. RESULTS: Reduced volume of CRC liver metastases with captopril treatment was evident. Local RAS of CRC metastases differed from the surrounding liver, with lower angiotensin II type 1 receptor (AT1R) expression but increased ANG-(1-7) receptor (MasR) compared to the liver. The AT1R localised to cancer and stromal infiltrating cells, while other RAS receptors were detected in cancer cells only. Tumor induction led to an initial increase in AT1R and ACE expression while captopril treatment significantly increased ACE expression in the final stages of tumor growth. Conversely, captopril treatment decreased expression of AT1R and angiotensinogen. CONCLUSIONS: These results demonstrate significant changes in RAS expression in the tumor-bearing captopril treated liver and in CRC metastases. The data suggests the existence of a tumor-specific RAS that can be independently targeted by RAS blockade.

The renin-angiotensin system and malignancy.

The renin-angiotensin system (RAS) is usually associated with its systemic action on cardiovascular homoeostasis. However, recent studies suggest that at a local tissue level, the RAS influences tumour growth. The potential of the RAS as a target for cancer treatment and the suggested underlying mechanisms of its paracrine effects are reviewed here. These include modulation of angiogenesis, cellular proliferation, immune responses and extracellular matrix formation. Knowledge of the RAS has increased dramatically in recent years with the discovery of new enzymes, peptides and feedback mechanisms. The local RAS appears to influence tumour growth and metastases and there is evidence of tissue- and tumour-specific differences. Recent experimental studies provide strong evidence that drugs that inhibit the RAS have the potential to reduce cancer risk or retard tumour growth and metastases. Manipulation of the RAS may, therefore, provide a safe and inexpensive anticancer strategy.

Effect of ACE inhibitors and angiotensin II receptor antagonists in a mouse model of colorectal cancer liver metastases.

BACKGROUND AND AIMS: Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II type I receptor (AT1R) antagonists are commonly used as a treatment for hypertension. Recent experimental and population studies have suggested that these agents may exert an inhibitory effect on malignancy, possibly through anti-angiogenic pathways. The aim of this study was to investigate the effect of an ACE-I (captopril) and an AT1R antagonist (irbesartan) in colorectal cancer liver metastases. METHODS: The effect of captopril and irbesartan on tumor growth was investigated in a mouse model using quantitative stereological and histological analysis. Tumor microcirculation was assessed by microvascular resin casting. A survival study was also carried out. RESULTS: Both captopril and irbesartan markedly decreased tumor growth when compared to control (P = 0.003 and P = 0.004, respectively). However, there was no significant difference in survival or tumor necrosis for either of the drugs. Tumor microvasculature exhibited a reduction in central microvascular density, with constriction and tapering of vessels. CONCLUSION: Captopril and irbesartan caused a marked reduction in volume of colorectal cancer liver metastases and caused changes in tumor microvasculature. However, there was no difference in percentage tumor necrosis or improvements in survival. Further investigation is needed to identify the mode of action of these agents.