Deletion of glutamate carboxypeptidase II (GCPII), but not GCPIII, provided long-term benefits in mice with traumatic brain injury.

MAIN PROBLEM: N-acetylaspartylglutamate (NAAG) has neuroprotective effects in traumatic brain injury (TBI) by activating metabotropic glutamate receptor 3 (mGluR3) and reducing glutamate release. Glutamate carboxypeptidase II (GCPII) is the primary enzyme responsible for the hydrolysis of NAAG. It remains unclear whether glutamate carboxypeptidase III (GCPIII), a homolog of GCPII, can partially compensate for GCPII's function. METHODS: GCPII-/- , GCPIII-/- , and GCPII/III-/- mice were generated using CRISPR/Cas9 technology. Mice brain injury model was established through moderate controlled cortical impact (CCI). The relationship between GCPII and GCPIII was explored by analyzing injury response signals in the hippocampus and cortex of mice with different genotypes at the acute (1 day) and subacute (7 day) phase after TBI. RESULTS: In this study, we found that deletion of GCPII reduced glutamate production, excitotoxicity, and neuronal damage and improved cognitive function, but GCPIII deletion had no significant neuroprotective effect. Additionally, there was no significant difference in the neuroprotective effect between the combination of GCPII and GCPIII deletion and GCPII deletion alone. CONCLUSION: These results suggest that GCPII inhibition may be a therapeutic option for TBI, and that GCPIII may not act as a complementary enzyme to GCPII in this context.

Synthesis and evaluation of tumor-homing peptides for targeting prostate cancer.

High toxicity caused by chemotherapeutic drugs and the acquisition of drug resistance by cancer cells are the major drawbacks in cancer therapy. A promising approach to overcome the posed barriers is conjugating tumor-homing peptides to drugs or nanocarriers. Such high-affinity peptides can specifically target surface markers overexpressed by cancer cells, ensuring a rapid and cancer-specific uptake of the drugs. Since prostate-specific membrane antigen (PSMA) is overexpressed by aggressive prostate cancer cells, targeting this surface protein with peptide conjugates can lead to the development of effective strategies against prostate cancer. In this study, we aimed to determine which PSMA-binding peptide among peptides 563, 562 and 9-mer, show the highest selectivity towards PSMA using 22Rv1 prostate cancer cells, a cell line with moderate PSMA levels. Tumor-homing peptides were synthesized by fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (Fmoc-SPPS) strategy, and evaluated for their prostate cancer cell-specific targeting efficiencies by flow cytometry. Our results showed that the PSMA-binding capacity of peptide 563 was superior to those of 562, 9-mer, and 5-mer; therefore, can be utilized as a potent-targeting agent not only in the treatment of high PSMA positive but also moderate PSMA positive prostate cancer tumors.

Antihormone treatment differentially regulates PSA secretion, PSMA expression and 68Ga-PSMA uptake in LNCaP cells.

BACKGROUND: In recent years, a variety of innovative therapeutics for castration-resistant prostate cancer have been developed, including novel anti-androgenic drugs, such as abiraterone or VPC-13566. Therapeutic monitoring of these pharmaceuticals is performed either by measuring PSA levels in serum or by imaging. PET using PSMA ligands labeled with Fluor-18 or Gallium-68 is the most sensitive and specific imaging modality for detection of metastases in advanced prostate cancer. To date, it remains unclear how PSMA expression is modulated by anti-hormonal treatment and how it correlates with PSA secretion. METHODS: We analyzed modulation of PSMA-mRNA and protein expression, 68Ga-PSMA uptake and regulation of PSA secretion by abiraterone or VPC-13566 in LNCaP cells in vitro. RESULTS: We found that abiraterone and VPC-13566 upregulate PSMA protein and mRNA expression but block PSA secretion in LNCaP cells. Both anti-androgens also enhanced 68Ga-PSMA uptake normalized by the number of cells, whereas abiraterone and VPC-13566 reduced 68Ga-PSMA uptake in total LNCaP monolayers treated due to cell death. CONCLUSION: Our data indicate that PSA secretion and PSMA expression are differentially regulated upon anti-androgen treatment. This finding might be important for the interpretation of 68Ga-PSMA PET images in monitoring therapies with abiraterone and VPC-13566 in prostate cancer patients, but needs to be validated in vivo.

MRI Assessment of Prostate-Specific Membrane Antigen (PSMA) Targeting by a PSMA-Targeted Magnetic Nanoparticle: Potential for Image-Guided Therapy.

Magnetic nanoparticle (MNP)-induced hyperthermia is currently being evaluated for localized prostate cancer. We evaluated the feasibility of tumor-selective delivery of prostate-specific membrane antigen (PSMA)-targeted MNPs in a murine model with high-resolution magnetic resonance imaging (MRI) after intravenous administration of MNPs at a concentration necessary for hyperthermia. A PSMA-targeted MNP was synthesized and evaluated using T2-weighted MRI, after intravenous administration of 50 mg/kg of the MNP. Significant contrast enhancement ( P < 0.0002, n = 5) was observed in PSMA(+) tumors compared to PSMA(-) tumors 24 h and 48 h after contrast agent administration. Mice were also imaged with near-infrared fluorescence imaging, to validate the MRI results. Two-photon microscopy revealed higher vascular density at the tumor periphery, which resulted in higher  peripheral accumulation of PSMA-targeted MNPs. These results suggest that the delivery of PSMA-targeted MNPs to PSMA(+) tumors is both actively targeted and passively mediated.

The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity.

Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+ -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca2+ -binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca2+ -binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca2+ for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.

Survivin and NAIP in Human Benign Prostatic Hyperplasia: Protective Role of the Association of Serenoa repens, Lycopene and Selenium from the Randomized Clinical Study.

Benign prostatic hyperplasia (BPH) treatment includes the apoptosis machinery modulation through the direct inhibition of caspase cascade. We previously demonstrated that Serenoa repens (Ser) with lycopene (Ly) and selenium (Se) reawakened apoptosis by reducing survivin and neuronal apoptosis inhibitory protein (NAIP) levels in rats. The aim of this study was to evaluate the effectiveness of Ser-Se-Ly association on survivin and NAIP expression in BPH patients. Ninety patients with lower urinary tract symptoms (LUTS) due to clinical BPH were included in this randomized, double-blind, placebo-controlled trial. Participants were randomly assigned to receive placebo (Group BPH + placebo, n = 45) or Ser-Se-Ly association (Group BPH + Ser-Se-Ly; n = 45) for 3 months. At time 0, all patients underwent prostatic biopsies. After 3 months of treatment, they underwent prostatic re-biopsy and specimens were collected for molecular, morphological, and immunohistochemical analysis. After 3 months, survivin and NAIP were significantly decreased, while caspase-3 was significantly increased in BPH patients treated with Ser-Se-Ly when compared with the other group. In BPH patients treated with Ser-Se-Ly for 3 months, the glandular epithelium was formed by a single layer of cuboidal cells. PSA showed high immunoexpression in all BPH patients and a focal positivity in Ser-Se-Ly treated patients after 3 months. Evident prostate specific membrane antigen (PSMA) immunoexpression was shown in all BPH patients, while no positivity was present after Ser-Se-Ly administration. Ser-Se-Ly proved to be effective in promoting apoptosis in BPH patients.

Focal MRI-Guided Salvage High-Dose-Rate Brachytherapy in Patients With Radiorecurrent Prostate Cancer.

INTRODUCTION: Whole-gland salvage treatment of radiorecurrent prostate cancer has a high rate of severe toxicity. The standard of care in case of a biochemical recurrence is androgen deprivation treatment, which is associated with morbidity and negative effects on quality of life. A salvage treatment with acceptable toxicity might postpone the start of androgen deprivation treatment, might have a positive influence on the patients' quality of life, and might even be curative. Here, toxicity and biochemical outcome are described after magnetic resonance imaging-guided focal salvage high-dose-rate brachytherapy in patients with radiorecurrent prostate cancer. MATERIALS AND METHODS: Seventeen patients with pathologically proven locally recurrent prostate cancer were treated with focal high-dose-rate brachytherapy in a single 19-Gy fraction using magnetic resonance imaging for treatment guidance. Primary radiotherapy consisted of external beam radiotherapy or low-dose-rate brachytherapy. Tumors were delineated with Ga-68-prostate-specific membrane antigen or F18-choline positron emission tomography in combination with multiparametric magnetic resonance imaging. All patients had a prostate-specific antigen level of less than 10 ng/mL at the time of recurrence and a prostate-specific antigen doubling time of ≥12 months. Toxicity was measured by using the Common Terminology Criteria for Adverse Events version 4. RESULTS: Eight of 17 patients had follow-up interval of at least 1 year. At a median follow-up interval of 10 months (range 3-40 months), 1 patient experienced a biochemical recurrence according to the Phoenix criteria, and prostate-specific membrane antigen testing revealed that this was due to a distant nodal metastasis. One patient had a grade 3 urethral stricture at 2 years after treatment. CONCLUSION: Focal salvage high-dose-rate brachytherapy in patients with radiorecurrent prostate cancer showed grade 3 toxicity in 1 of 17 patients and a distant nodal metastasis in another patient. Whether this treatment option leads to cure in a subset of patients or whether it can successfully postpone androgen deprivation treatment needs further investigation.

PSMA Expression in Tumor Neovasculature Endothelial Cells of Follicular Thyroid Adenoma as Identified by Molecular Imaging Using 68Ga-PSMA Ligand PET/CT.

The prostate-specific membrane antigen (PSMA) is expressed by both prostate cancer and other neoplasms. We report the case of a 65-year-old man with castration-resistant metastatic prostate cancer who underwent Ga-PSMA ligand PET/CT for restaging of disease. Ga-PSMA ligand accumulation was noted in a thyroid lesion, suspicious for thyroid malignancy on complementary ultrasound. Subsequent resection and histopathological analysis showed follicular thyroid adenoma with PSMA expression in tumor neovasculature endothelial cells, but not in thyroid epithelial cells. It is important to be aware that both malignant and benign thyroid neoplasms may show PSMA expression to avoid misinterpretation.

Folate Acts in E. coli to Accelerate C. elegans Aging Independently of Bacterial Biosynthesis.

Folates are cofactors for biosynthetic enzymes in all eukaryotic and prokaryotic cells. Animals cannot synthesize folate and must acquire it from their diet or microbiota. Previously, we showed that inhibiting E. coli folate synthesis increases C. elegans lifespan. Here, we show that restriction or supplementation of C. elegans folate does not influence lifespan. Thus, folate is required in E. coli to shorten worm lifespan. Bacterial proliferation in the intestine has been proposed as a mechanism for the life-shortening influence of E. coli. However, we found no correlation between C. elegans survival and bacterial growth in a screen of 1,000+ E. coli deletion mutants. Nine mutants increased worm lifespan robustly, suggesting specific gene regulation is required for the life-shortening activity of E. coli. Disrupting the biosynthetic folate cycle did not increase lifespan. Thus, folate acts through a growth-independent route in E. coli to accelerate animal aging.

Development of a complementary PET/MR dual-modal imaging probe for targeting prostate-specific membrane antigen (PSMA).

We tried to develop a dual-modal PET/MR imaging probe using a straightforward one-pot method by encapsulation with specific amphiphiles. In this study, iron oxide (IO) nanoparticles were encapsulated with three amphiphiles containing PEG, DOTA and the prostate-specific membrane antigen (PSMA)-targeting ligand in aqueous medium. The diameter of the prepared nanoparticle DOTA-IO-GUL was 11.01±1.54nm. DOTA-IO-GUL was labeled with (68)Ga in high efficiency. The DOTA-IO-GUL showed a dose-dependent binding to LNCaP (PSMA positive) cells via a competitive binding study against (125)I-labeled MIP-1072 (PSMA-targeting agent). Additionally, PET and MR imaging results showed PSMA selective uptake by only 22Rv1 (PSMA positive) but not PC-3 (PSMA negative) in dual-tumor xenograft mouse model study. MR imaging showed high resolution, and PET imaging enabled quantification and confirmation of the specificity. In conclusion, we have successfully developed the specific PSMA-targeting IO nanoparticle, DOTA-IO-GUL, as a dual-modality probe for complementary PET/MR imaging. FROM THE CLINICAL EDITOR: The combination of using Positron Emission Tomography (PET) and computed tomography (CT) in clinical practice is now the norm. With advances in technology, the next step would be to develop combined PET and Magnetic Resonance (MR) dual-imaging. In this article, the authors described their positive study on the development of a dual-modal PET/MR imaging probe using a prostate cancer model.

Randomized multicenter investigation of folate plus vitamin B12 supplementation in schizophrenia.

IMPORTANCE: More effective treatments are needed for negative symptoms of schizophrenia, which are typically chronic, disabling, and costly. Negative symptoms have previously been associated with reduced blood folate levels, especially among patients with low-functioning variants in genes that regulate folate metabolism, suggesting the potential utility of folate supplementation. OBJECTIVES: To determine whether folic acid plus vitamin B12 supplementation reduces negative symptoms of schizophrenia and whether functional variants in folate-related genes influence treatment response. DESIGN: Parallel-group, randomized, double-blind, placebo-controlled clinical trial of 16 weeks of treatment with 2 mg of folic acid and 400 µg of vitamin B12. SETTING: Three community mental health centers affiliated with academic medical centers in the United States. PARTICIPANTS: Outpatients with chronic schizophrenia who were psychiatrically stable but displayed persistent symptoms despite antipsychotic treatment. Eligible patients were 18 to 68 years old, were treated with an antipsychotic agent for 6 months or more at a stable dose for 6 weeks or more, and scored 60 or more on the Positive and Negative Syndrome Scale. INTERVENTION: One hundred forty subjects were randomized to receive daily oral folic acid plus vitamin B12 or placebo. MAIN OUTCOME MEASURES: Change in negative symptoms (Scale for the Assessment of Negative Symptoms [SANS]), as well as positive and total symptoms (Positive and Negative Syndrome Scale). RESULTS: Folate plus vitamin B12 improved negative symptoms significantly compared with placebo (group difference, -0.33 change in SANS score per week; 95% CI, -0.62 to -0.05) when genotype was taken into account but not when genotype was excluded. An interaction of the 484C>T variant of FOLH1 (rs202676) with treatment was observed (P = .02), where only patients homozygous for the 484T allele demonstrated significantly greater benefit with active treatment (-0.59 change in SANS score per week; 95% CI, -0.99 to -0.18). In parallel, we observed an inverse relationship between red blood cell folate concentration at baseline and 484C allele load (P = .03), which persisted until 8 weeks of treatment. Change in positive and total symptoms did not differ between treatment groups. CONCLUSIONS: Folate plus vitamin B12 supplementation can improve negative symptoms of schizophrenia, but treatment response is influenced by genetic variation in folate absorption. These findings support a personalized medicine approach for the treatment of negative symptoms. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00611806.

Genetic variation throughout the folate metabolic pathway influences negative symptom severity in schizophrenia.

Low serum folate levels previously have been associated with negative symptom risk in schizophrenia, as has the hypofunctional 677C>T variant of the MTHFR gene. This study examined whether other missense polymorphisms in folate-regulating enzymes, in concert with MTHFR, influence negative symptoms in schizophrenia, and whether total risk allele load interacts with serum folate status to further stratify negative symptom risk. Medicated outpatients with schizophrenia (n = 219), all of European origin and some included in a previous report, were rated with the Positive and Negative Syndrome Scale. A subset of 82 patients also underwent nonfasting serum folate testing. Patients were genotyped for the MTHFR 677C>T (rs1801133), MTHFR 1298A>C (rs1801131), MTR 2756A>G (rs1805087), MTRR 203A>G (rs1801394), FOLH1 484T>C (rs202676), RFC 80A>G (rs1051266), and COMT 675G>A (rs4680) polymorphisms. All genotypes were entered into a linear regression model to determine significant predictors of negative symptoms, and risk scores were calculated based on total risk allele dose. Four variants, MTHFR 677T, MTR 2756A, FOLH1 484C, and COMT 675A, emerged as significant independent predictors of negative symptom severity, accounting for significantly greater variance in negative symptoms than MTHFR 677C>T alone. Total allele dose across the 4 variants predicted negative symptom severity only among patients with low folate levels. These findings indicate that multiple genetic variants within the folate metabolic pathway contribute to negative symptoms of schizophrenia. A relationship between folate level and negative symptom severity among patients with greater genetic vulnerability is biologically plausible and suggests the utility of folate supplementation in these patients.

Bioavailability of polyglutamyl folic acid relative to that of monoglutamyl folic acid in subjects with different genotypes of the glutamate carboxypeptidase II gene.

BACKGROUND: Before dietary folate is absorbed, polyglutamate folates are deconjugated to monoglutamates by folylpoly-gamma-glutamyl carboxypeptidase in the small intestine. The 1561T allele of the glutamate carboxypeptidase II gene (GCPII), which codes for folylpoly-gamma-glutamyl carboxypeptidase, may impair intestinal absorption of dietary folates. OBJECTIVE: Our aim was to study the bioavailability of polyglutamyl folic acid relative to that of monoglutamyl folic acid across GCPII 1561 genotypes. DESIGN: In a randomized study, 180 healthy adults aged 50-75 y received 323 nmol monoglutamyl folic acid/d (n = 59), 262 nmol heptaglutamyl folic acid/d (n = 61), or placebo (n = 60) for 12 wk. Genotypes were assessed after the intervention. The bioavailability of heptaglutamyl folic acid relative to that of monoglutamyl folic acid was calculated by using the changes in serum folate concentration in the treatment groups, after correction for changes in the placebo group and for the administered dose. RESULTS: No subjects with the TT genotype were encountered. At baseline, serum and erythrocyte folate concentrations were higher (P < 0.05) in subjects with the CT genotype [16.3 nmol/L (geometric x; 95% CI: 13.7, 19.3 nmol/L) and 863 nmol/L (735, 1012 nmol/L), respectively; n = 19] than in subjects with the CC genotype [13.7 (13.1, 14.3) and 685 (652, 721) nmol/L, respectively; n = 161]. Baseline homocysteine concentrations were not significantly different between genotypes. The bioavailability of heptaglutamyl folic acid relative to that of monoglutamyl folic acid was not significantly different between subjects with the CC (64%; 52%, 76%) and CT genotypes (70%; 49%, 91%). CONCLUSIONS: The 1561T allele of the GCPII gene does not impair the bioavailability of polyglutamyl folic acid. However, the allele is associated with higher folate status. This association may be explained by yet unidentified factors controlling the expression of the GCPII gene.

Early embryonic death of glutamate carboxypeptidase II (NAALADase) homozygous mutants.

Glutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 microM) of the neuropeptide N-acetylaspartylglutamate to yield N-acetylaspartate and glutamate and also serves as a high-affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N-acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N-acetylaspartylglutamate. The PGK-Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild-type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild-type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis.