Contribution of hypothermia and CB1 receptor activation to protective effects of TAK-937, a cannabinoid receptor agonist, in rat transient MCAO model

Background

Cannabinoid (CB) receptor agonists are expected to alleviate ischemic brain damage by modulating neurotransmission and neuroinflammatory responses via CB₁ and CB₂ receptors, respectively. In a previous study, TAK-937, a novel potent and selective CB₁ and CB₂ receptor agonist, was shown to exert significant cerebroprotective effects accompanied by hypothermia after transient middle cerebral artery occlusion (MCAO) in rats. Sustained hypothermia itself induces significant neuroprotective effects. In the present studies, we examined the relative contribution of hypothermia and CB₁ receptor activation to the cerebroprotective effects of TAK-937.

Methodology/Principal Findings

Using a multichannel brain temperature controlling system we developed, the brain temperature of freely moving rats was telemetrically monitored and maintained between 37 and 38°C during intravenous infusion of TAK-937 (100 µg/kg/h) or vehicle for 24 h after 2 h MCAO. AM251, a selective CB₁ receptor antagonist, was administered intraperitoneally at 30 mg/kg 30 min before starting intravenous infusion of TAK-937 (100 µg/kg/h) for 24 h. Rats were sacrificed and their brains were isolated 26 h after MCAO in both experiments. When the hypothermic effect of TAK-937 was completely reversed by a brain temperature controlling system, the infarct-reducing effect of TAK-937 was attenuated in part, but remained significant. On the other hand, concomitant AM251 treatment with TAK-937 completely abolished the hypothermic and infarct-reducing effects of TAK-937.

Conclusions/Significance

We conclude that the cerebroprotective effects of TAK-937 were at least in part mediated by induction of hypothermia, and mainly mediated by CB₁ receptor activation.     

Effect of Immune Reconstitution on the Incidence of HIV-Related Hodgkin Lymphoma

Background

The incidence of Hodgkin lymphoma (HL) has increased since introduction of combined antiretroviral therapy (cART). While HIV-related HL is highly associated with EBV, the causes underlying the rising incidence remain unclear. The aim of this study was to evaluate the effect of immune reconstitution on HL incidence among a cohort of HIV-infected male veterans ever receiving cART.

Methods

We performed a retrospective cohort study utilizing data from the Veterans Affairs HIV Clinical Case Registry from 1985-2010. HL cases were identified using ICD-9 codes (201.4-9). Poisson regression was conducted to evaluate relationships between cART-related immunologic measures (e.g., nadir CD4 before cART, time-updated CD4, % time undetectable HIV RNA) and HL incidence. Additionally, we examined CD4 change after cART initiation.

Results

31,056 cART users contributed 287,256 person-years and 196 HL cases (IR=6.8/10,000 person-years). Rate of CD4 increase after cART was worse among HL cases than non-cases (p<0.05). In multivariate regression, HL risk was elevated among veterans with recent CD4 200-350 cells/µL (IRR=1.67, 95%CI=1.16-2.40) and <200 cells/µL (IRR=1.61, 95%CI=1.09-2.39), compared to >350 cells/µL. HL risk was lower among veterans with >80% time undetectable HIV RNA (IRR=0.57, 95%CI=0.35-0.92) and 40-80% undetectable (IRR=0.68, 95%CI=0.47-0.99), compared to <40% undetectable. HL risk was higher in the first 12 months (IRR=2.02, 95%CI=1.32-3.10) and 12-24 months (IRR=1.75, 95%CI=1.16-2.64) after cART initiation, compared to >36 months.

Conclusion

These data highlight immunosuppression and poor viral control may increase HL risk, specifically during immune reconstitution in the interval post cART initiation. Findings suggest an immune reconstitution type mechanism in HIV-related HL development.     

Cannabinoids Alleviate Experimentally Induced Intestinal Inflammation by Acting at Central and Peripheral Receptors

Background and Aims

In an attempt to further investigate the role of cannabinoid (CB) system in the pathogenesis of inflammatory bowel diseases, we employed two recently developed ligands, AM841 (a covalently acting CB agonist) and CB13 (a peripherally-restricted CB agonist) to establish whether central and peripheral CB sites are involved in the anti-inflammatory action in the intestine.

Methods and Results

AM841 (0.01, 0.1 and 1 mg/kg, i.p.) significantly decreased inflammation scores in dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated mice when administered before induction of colitis or as a treatment of existing intestinal inflammation. The effect was absent in CB₁, CB₂ and CB_1/2-deficient mice. A peripherally-restricted agonist CB13 did not alleviate colitis when given i.p. (0.1 mg/kg), but significantly decreased inflammation score after central administration (0.1 µg/animal).

Conclusions

This is the first evidence that central and peripheral CB receptors are responsible for the protective and therapeutic action of cannabinoids in mouse models of colitis. Our observations provide new insight to CB pharmacology and validate the use of novel ligands AM841 and CB13 as potent tools in CB-related research.     

Increased expression of cannabinoid CB₁ receptors in Achilles tendinosis

Background

The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB₁) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis.

Methodology

Cannabinoid CB₁ receptor immunoreactivity (CB₁IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons.

Principal Findings

CB₁IR was seen as a granular pattern in the tenocytes. CB₁IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB₁ receptor expression in tendinosis tissue compared to control tissue.

Conclusion

Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder.     

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

Background

Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB₁ and CB₂ receptors mediate this therapeutic effect is unclear.

Principal Findings

We generated astrocytoma subclones that express set levels of CB₁ and CB₂, and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB₁, CB₂ and AKT, but still through a mechanism involving ERK1/2.

Significance

The high expression level of CB₁ and CB₂ receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB₁ and CB₂ receptors, yet still activate ERK1/2.     

The Suppressive Role and Aberrent Promoter Methylation of BTG3 in the Progression of Hepatocellular Carcinoma

Background

BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.

Methodology

The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.

Results

BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

Conclusion

Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.     

Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

Background

Severe asthma is associated with T helper (T_H) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.

Objective

To determine the anti-inflammatory potential of anthraquinones in-vivo.

Methods

BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.

Results

Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of T_H2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.

Conclusion

Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.     

Knockdown of FRAT1 Expression by RNA Interference Inhibits Human Glioblastoma Cell Growth,Migration and Invasion

Background

FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades.

Methods

In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi.

Results

FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice.

Conclusions

Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.     

Resistance to Bleomycin in Cancer Cell Lines Is Characterized by Prolonged Doubling Time,Reduced DNA Damage and Evasion of G2/M Arrest and Apoptosis

Background

To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC₅₀ values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.

Results

Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC₅₀ and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC₅₀ and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05).

Conclusion

Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.     

Hepatitis C Virus Induces the Cannabinoid Receptor 1

Background

Activation of hepatic CB₁ receptors (CB₁) is associated with steatosis and fibrosis in experimental forms of liver disease. However, CB₁ expression has not been assessed in patients with chronic hepatitis C (CHC), a disease associated with insulin resistance, steatosis and metabolic disturbance. We aimed to determine the importance and explore the associations of CB₁ expression in CHC.

Methods

CB₁ receptor mRNA was measured by real time quantitative PCR on extracted liver tissue from 88 patients with CHC (genotypes 1 and 3), 12 controls and 10 patients with chronic hepatitis B (CHB). The Huh7/JFH1 Hepatitis C virus (HCV) cell culture model was used to validate results.

Principal Findings

CB₁ was expressed in all patients with CHC and levels were 6-fold higher than in controls (P<0.001). CB₁ expression increased with fibrosis stage, with cirrhotics having up to a 2 fold up-regulation compared to those with low fibrosis stage (p<0.05). Even in mild CHC with no steatosis (F0-1), CB₁ levels remained substantially greater than in controls (p<0.001) and in those with mild CHB (F0-1; p<0.001). Huh7 cells infected with JFH-1 HCV showed an 8-fold upregulation of CB₁, and CB₁ expression directly correlated with the percentage of cells infected over time, suggesting that CB₁ is an HCV inducible gene. While HCV structural proteins appear essential for CB₁ induction, there was no core genotype specific difference in CB₁ expression. CB₁ significantly increased with steatosis grade, primarily driven by patients with genotype 3 CHC. In genotype 3 patients, CB₁ correlated with SREBP-1c and its downstream target FASN (SREBP-1c; R = 0.37, FASN; R = 0.39, p<0.05 for both).

Conclusions/Significance

CB₁ is up-regulated in CHC and is associated with increased steatosis in genotype 3. It is induced by the hepatitis C virus.     

Association between Cannabinoid CB1 Receptor Expression and Akt Signalling in Prostate Cancer

Background

In prostate cancer, tumour expression of cannabinoid CB₁ receptors is associated with a poor prognosis. One explanation for this association comes from experiments with transfected astrocytoma cells, where a high CB receptor expression recruits the Akt signalling survival pathway. In the present study, we have investigated the association between CB₁ receptor expression and the Akt pathway in a well-characterised prostate cancer tissue microarray.

Methodology/Principal Findings

Phosphorylated Akt immunoreactivity (pAkt-IR) scores were available in the database. CB₁ receptor immunoreactivity (CB₁IR) was rescored from previously published data using the same scale as pAkt-IR. There was a highly significant correlation between CB₁IR and pAkt-IR. Further, cases with high expression levels of both biomarkers were much more likely to have a more severe form of the disease at diagnosis than those with low expression levels. The two biomarkers had additive effects, rather than an interaction, upon disease-specific survival.

Conclusions/Significance

The present study provides data that is consistent with the hypothesis that at a high CB₁ receptor expression, the Akt signalling pathway becomes operative.     

Human Omental-Derived Adipose Stem Cells Increase Ovarian Cancer Proliferation,Migration, and Chemoresistance

Objectives

Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination.

Materials and Methods

We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment.

Results

O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries.

Conclusions

ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro.     

Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

Purpose

The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling.

Methods

Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-β₁ integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined.

Principal Findings

We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-β₁ integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632.

Conclusions/Significance

The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow.     

Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis

Aims/Hypothesis

To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function.

Methods

Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6) and rat (INS-1E) beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1). Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS). Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests.

Results

24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P<0.05). Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P<0.05) and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein). Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P<0.05) and reactive oxygen species increased by more than twofold (P<0.05) following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively.

Conclusions/Interpretation

Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell.     

Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies

Background

We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents.

Design and Methods

Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG).

Results

Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses.

Conclusions

Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.     

Ulcerative Colitis Induces Changes on the Expression of the Endocannabinoid System in the Human Colonic Tissue

Background

Recent studies suggest potential roles of the endocannabinoid system in gastrointestinal inflammation. Although cannabinoid CB₂ receptor expression is increased in inflammatory disorders, the presence and function of the remaining proteins of the endocannabinoid system in the colonic tissue is not well characterized.

Methodology

Cannabinoid CB₁ and CB₂ receptors, the enzymes for endocannabinoid biosynthesis DAGLα, DAGLβ and NAPE-PLD, and the endocannabinoid-degradating enzymes FAAH and MAGL were analysed in both acute untreated active ulcerative pancolitis and treated quiescent patients in comparison with healthy human colonic tissue by immunocytochemistry. Analyses were carried out according to clinical criteria, taking into account the severity at onset and treatment received.

Principal Findings

Western blot and immunocytochemistry indicated that the endocannabinoid system is present in the colonic tissue, but it shows a differential distribution in epithelium, lamina propria, smooth muscle and enteric plexi. Quantification of epithelial immunoreactivity showed an increase of CB₂ receptor, DAGLα and MAGL expression, mainly in mild and moderate pancolitis patients. In contrast, NAPE-PLD expression decreased in moderate and severe pancolitis patients. During quiescent pancolitis, CB₁, CB₂ and DAGLα expression dropped, while NAPE-PLD expression rose, mainly in patients treated with 5-ASA or 5-ASA+corticosteroids. The number of immune cells containing MAGL and FAAH in the lamina propria increased in acute pancolitis patients, but dropped after treatment.

Conclusions

Endocannabinoids signaling pathway, through CB₂ receptor, may reduce colitis-associated inflammation suggesting a potential drugable target for the treatment of inflammatory bowel diseases.     

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy

Background aims

EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1.

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation

Background

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction.

Methodology/Principal Findings

Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing.

Conclusions/Significance

Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.     

Pharmacodynamic Consequences of Administration of VLA-4 Antagonist CDP323 to Multiple Sclerosis Subjects: A Randomized,Double-Blind Phase 1/2 Study

Background

Lymphocyte inhibition by antagonism of α4 integrins is a validated therapeutic approach for relapsing multiple sclerosis (RMS).

Objective

Investigate the effect of CDP323, an oral α₄-integrin inhibitor, on lymphocyte biomarkers in RMS.

Methods

Seventy-one RMS subjects aged 18–65 years with Expanded Disability Status Scale scores ≤6.5 were randomized to 28-day treatment with CDP323 100 mg twice daily (bid), 500 mg bid, 1000 mg once daily (qd), 1000 mg bid, or placebo.

Results

Relative to placebo, all dosages of CDP323 significantly decreased the capacity of lymphocytes to bind vascular adhesion molecule-1 (VCAM-1) and the expression of α₄-integrin on VCAM-1–binding cells. All but the 100-mg bid dosage significantly increased total lymphocytes and naive B cells, memory B cells, and T cells in peripheral blood compared with placebo, and the dose-response relationship was shown to be linear. Marked increases were also observed in natural killer cells and hematopoietic progenitor cells, but only with the 500-mg bid and 1000-mg bid dosages. There were no significant changes in monocytes. The number of samples for regulator and inflammatory T cells was too small to draw any definitive conclusions.

Conclusions

CDP323 at daily doses of 1000 or 2000 mg induced significant increases in total lymphocyte count and suppressed VCAM-1 binding by reducing unbound very late antigen-4 expression on lymphocytes.

Trial Registration

ClinicalTrials.gov NCT00726648.