Feasibility of dietary folic acid reduction intervention for men on active surveillance for prostate cancer.

BACKGROUND & AIMS: Fortification of the US food supply has increased folic acid intake and resulted in a concomitant decrease in neural tube defects in women. However, a body evidence supports the hypothesis that increased circulating folate levels due to excessive dietary or supplemental folic acid may be harmful for men with prostate cancer. Therefore, this pilot study aimed to investigate the feasibility of a reduced folic acid dietary intervention in men on an active surveillance monitoring program for prostate cancer. METHODS: Men with low-grade prostate cancer enrolled into a 12-week dietary folic acid reduction diet. Primary outcome was red blood cell (RBC) folate reduction at 12 weeks. Other outcomes include serum folate, homocysteine, and vitamin B12 levels. The number of patients who complete the trial and reasons for disenrollment or dropout were also assessed. RESULTS: Twenty-eight participants were enrolled into the dietary intervention study. Six participants withdrew from the study and a total of 21 participants completed all baseline and week 12 biochemical assessments. Only 18 participants completed all dietary questionnaires. Participants withdrew from the study due to difficulty with the diet or personal reasons. A substantial reduction was noted in serum folate (p < 0.007), RBC folate (p < 0.001) and dietary consumption of folic acid from foods (p = 0.003) and supplements (p = 0.003) without reduction in serum homocysteine or vitamin B12. Although an overall decrease in PSA from baseline to twelve weeks was found, the reduction was not significant (-3.55 ng/mL, p = 0.197). CONCLUSIONS: This phase 1 feasibility study reduced dietary folic acid intake from food and supplements and successfully lowered serum and RBC folate without resulting harmful effects. Data from this study supports future intervention trials with a larger prostate cancer active surveillance population and has the potential to reduce prostate cancer progression. There are no interventions to reduce progression of prostate cancer in man on active surveillance.

A Perspective on the Evolving Story of PSMA Biology, PSMA-Based Imaging, and Endoradiotherapeutic Strategies.

In this review, we cover the evolution of knowledge on the biology of prostate-specific membrane antigen (PSMA) and its translation to therapy. The usual key to discovery is a realistic model for experimentation and for testing a hypothesis. A realistic model is especially needed in the case of the human prostate, which differs significantly from the prostate of species often used as research models. We will emphasize the genetic characterization of PSMA, the nature of the PSMA protein, and its role as a carboxypeptidase, with differing important substrates and products in different tissues. We give special prominence to the importance of PSMA as a target for imaging and therapy in prostate cancer and its underdeveloped role for imaging and targeting the neovasculature of tumors other than prostate cancer. Lastly, we bring attention to its importance in other nonprostatic tissues.

Contrasting roles of the ABCG2 Q141K variant in prostate cancer.

ABCG2 is a membrane transport protein that effluxes growth-promoting molecules, such as folates and dihydrotestosterone, as well as chemotherapeutic agents. Therefore it is important to determine how variants of ABCG2 affect the transporter function in order to determine whether modified treatment regimens may be necessary for patients harboring ABCG2 variants. Previous studies have demonstrated an association between the ABCG2 Q141K variant and overall survival after a prostate cancer diagnosis. We report here that in patients with recurrent prostate cancer, those who carry the ABCG2 Q141K variant had a significantly shorter time to PSA recurrence post-prostatectomy than patients homozygous for wild-type ABCG2 (P=0.01). Transport studies showed that wild-type ABCG2 was able to efflux more folic acid than the Q141K variant (P<0.002), suggesting that retained tumoral folate contributes to the decreased time to PSA recurrence in the Q141K variant patients. In a seemingly conflicting study, it was previously reported that docetaxel-treated Q141K variant prostate cancer patients have a longer survival time. We found this may be due to less efficient docetaxel efflux in cells with the Q141K variant versus wild-type ABCG2. In human prostate cancer tissues, confocal microscopy revealed that all genotypes had a mixture of cytoplasmic and plasma membrane staining, with noticeably less staining in the two homozygous KK patients. In conclusion, the Q141K variant plays contrasting roles in prostate cancer: 1) by decreasing folate efflux, increased intracellular folate levels result in enhanced tumor cell proliferation and therefore time to recurrence decreases; and 2) in patients treated with docetaxel, by decreasing its efflux, intratumoral docetaxel levels and tumor cell drug sensitivity increase and therefore patient survival time increases. Taken together, these data suggest that a patient's ABCG2 genotype may be important when determining a personalized treatment plan.

Divergence between dietary folate intake and concentrations in the serum and red blood cells of aging males in the United States.

BACKGROUND & AIMS: As part of a broader study examining the relationship between serum folate concentrations and prostate cancer progression, we determined if there are age related changes in serum folate concentration compared to folate intake in the U.S. male population. METHODS: Weighted data from the 2007-2008 and 2009-2010 NHANES databases was analyzed. A subpopulation of male participants was selected who were older than one year of age, had completed two days of dietary recall including supplement usage, and had fasted for at least 4 h prior to having their serum folate measured. Total dietary folate equivalent (DFE) intake (mcg) represented the combination of all natural food folate and folic acid from fortification and dietary supplements. Geometric means of serum folate (nM), red blood cell (RBC) folate (nM), and DFE intake were calculated for nine consecutive age groups, with each group generally representing a 10 year span. Analysis was then focused on males older than 20 years of age. RESULTS: A total of 19,142 subjects were in the initial NHANES population, which represented over 294 million people within the United States. Applying our inclusion criteria created a final subpopulation size of 3775. Subsequent analysis of the age groups for all males older than 20 years found the following: The mean serum folate (nM) with 95% CI levels ranged from 28.2 (26.6, 29.9) to 55.1 (47.5, 63.9). RBC folate (nM) concentrations with 95% CI levels without any fasting exclusions ranged from 795.6 (741.5, 853.7) to 1038.4 (910.7, 1184.2). Serum and RBC folate concentrations were significantly higher with age across these age groups (p < 0.001). However, the mean total daily DFE intake did not significantly differ ranging from 640.4 (574.7, 713.7) to 720.2 (665, 780) mcg, (p = 0.373). Serum folate concentrations in men with total daily DFE intake of at least 1000 mcg increased more significantly with increasing age than serum folate concentrations in men with less than 400 mcg of total daily DFE intake (p < 0.001). There was a similar trend with the RBC folate concentrations (p = 0.054). CONCLUSIONS: We observed higher serum and RBC folate concentrations and a divergence between dietary folate intake and these folate concentrations in older males. This phenomenon was evident at total DFE intakes that were significantly less than the 1000 mcg tolerable upper intake level currently recommended by the Institute of Medicine.

Novel nuclear localization of fatty acid synthase correlates with prostate cancer aggressiveness.

Fatty acid synthase is up-regulated in a variety of cancers, including prostate cancer. Up-regulation of fatty acid synthase not only increases production of fatty acids in tumors but also contributes to the transformed phenotype by conferring growth and survival advantages. In addition, increased fatty acid synthase expression in prostate cancer correlates with poor prognosis, although the mechanism(s) by which this occurs are not completely understood. Because fatty acid synthase is expressed at low levels in normal cells, it is currently a major target for anticancer drug design. Fatty acid synthase is normally found in the cytosol; however, we have discovered that it also localizes to the nucleus in a subset of prostate cancer cells. Analysis of the fatty acid synthase protein sequence indicated the presence of a nuclear localization signal, and subcellular fractionation of LNCaP prostate cancer cells, as well as immunofluorescent confocal microscopy of patient prostate tumor tissue and LNCaPs confirmed nuclear localization of this protein. Finally, immunohistochemical analysis of prostate cancer tissue indicated that nuclear localization of fatty acid synthase correlates with Gleason grade, implicating a potentially novel role in prostate cancer progression. Possible clinical implications include improving the accuracy of prostate biopsies in the diagnosis of low- versus intermediate-risk prostate cancer and the uncovering of novel metabolic pathways for the therapeutic targeting of androgen-independent prostate cancer.

The prostate-specific membrane antigen: lessons and current clinical implications from 20 years of research.

OBJECTIVE: Despite a multitude of detection and treatment advances in the past 2 decades, prostate cancer remains the second leading cause of deaths due to cancer among men in the United States. Technological evolution and expanding knowledge of tumor biomarkers have invigorated exploration in prostate cancer therapeutics. Prostate-specific membrane antigen (PSMA) was one of the first prostate cancer biomarkers successfully cloned. Since then, it has been characterized as the prototypical cell-surface marker for prostate cancer and has been the subject of intense clinical inquiry. In this article, we review the relevant research in PSMA on the 20th anniversary of its cloning. METHODS AND MATERIALS: A PubMed search using the keywords "prostate-specific membrane antigen" or "glutamate carboxypeptidase II" provided 1019 results. An additional 3 abstracts were included from scientific meetings. Articles were vetted by title and abstract with emphasis placed on those with clinically relevant findings. RESULTS: Sixty articles were selected for inclusion. PSMA was discovered and cloned in 1993. Its structure and function were further delineated in the ensuing decade. Consensus sites of expression in normal physiology are prostate, kidney, nervous system, and small intestine. PSMA has been implicated in the neovasculature of several tumors including urothelial and renal cell carcinomas. In prostate cancer, expression of PSMA is directly related to the Gleason grade. PSMA has been tested both in imaging and therapeutics in a number of prostate cancer clinical trials. Several recent approaches to target PSMA include the use of small molecule inhibitors, PSMA-based immunotherapy, RNA aptamer conjugates, and PSMA-targeted prodrug therapy. Future study of PSMA in prostate cancer might focus on its intracellular functions and possible role in tumor neurogenesis. CONCLUSIONS: Twenty years from its discovery, PSMA represents a viable biomarker and treatment target in prostate cancer. Research to delineate its precise role in prostate carcinogenesis and within the therapeutic armamentarium for patients with prostate cancer remains encouraging.

Opposing roles of folate in prostate cancer.

The US diet has been fortified with folic acid to prevent neural tube defects since 1998. The Physician Data Queries from the National Cancer Institute describe folate as protective against prostate cancer, whereas its synthetic analog, folic acid, is considered to increase prostate cancer risk when taken at levels easily achievable by eating fortified food or taking over-the-counter supplements. We review the present literature to examine the effects of folate and folic acid on prostate cancer, help interpret previous epidemiologic data, and provide clarification regarding the apparently opposing roles of folate for patients with prostate cancer. A literature search was conducted in Medline to identify studies investigating the effect of nutrition and specifically folate and folic acid on prostate carcinogenesis and progression. In addition, the National Health and Nutrition Examination Survey database was analyzed for trends in serum folate levels before and after mandatory fortification. Folate likely plays a dual role in prostate carcinogenesis. There remains conflicting epidemiologic evidence regarding folate and prostate cancer risk; however, there is growing experimental evidence that higher circulating folate levels can contribute to prostate cancer progression. Further research is needed to clarify these complex relationships.

NAAG peptidase inhibitors and deletion of NAAG peptidase gene enhance memory in novel object recognition test.

The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is inactivated by the extracellular enzyme glutamate carboxypeptidase II. Inhibitors of this enzyme reverse dizocilpine (MK-801)-induced impairment of short-term memory in the novel object recognition test. The objective of this study was to test the hypothesis that NAAG peptidase inhibition enhances long-term (24h delay) memory of C57BL mice. These mice and mice in which glutamate carboxypeptidase II had been knocked out were presented with two identical objects to explore for 10min on day 1 and tested with one of these familiar objects and one novel object on day 2. Memory was assessed as the degree to which the mice recalled the familiar object and explored the novel object to a greater extent on day 2. Uninjected mice or mice injected with saline prior to the acquisition session on day 1 demonstrated a lack of memory of the acquisition experience by exploring the familiar and novel objects to the same extent on day 2. Mice treated with glutamate carboxypeptidase II inhibitors ZJ43 or 2-PMPA prior to the acquisition trial explored the novel object significantly more time than the familiar object on day 2. Consistent with these results, mice in which glutamate carboxypeptidase II had been knocked out distinguished the novel from the familiar object on day 2 while their heterozygous colony mates did not. Inhibition of glutamate carboxypeptidase II enhances recognition memory, a therapeutic action that might be useful in treatment of memory deficits related to age and neurological disorders.

Deficiency of DNA repair nuclease ERCC1-XPF promotes prostate cancer progression in a tissue recombination model.

BACKGROUND: The excision repair cross complementing (ERCC1) gene product plays a vital role in the nucleotide excision repair (NER) and DNA interstrand crosslink repair pathways, which protect the genome from mutations and chromosomal aberrations, respectively. Genetic deletion of Ercc1 in the mouse causes dramatically accelerated aging. We examined the effect of Ercc1 deletion in the development of prostate cancer in a prostate recapitulation model as Ercc1 deficient mice die within four weeks of birth. METHODS: Prostate tissues from Ercc1(-/-) mice or wild-type littermates were combined with embryonic rat urogenital mesenchyme and grown as renal grafts for a total of 8, 16, and 24 weeks before histological, expression and proliferative evaluation. RESULTS: Invasive adenocarcinoma was observed in Ercc1(-/-) tissue recombinants but not wild-type as early as 8 weeks post-grafting. PIN-like lesions in Ercc1(-/-) tissue recombinants had more cytologic and architectural atypia than wild-type (P = 0.02, P = 0.0065, and P = 0.0003 at the 8, 16, and 24 weeks, respectively), as well as more proliferative cells (P = 0.022 and P = 0.033 at 8 and 16 weeks, respectively). With serial grafting, Ercc1(-/-) tissue recombinants progressed to a more severe histopathological phenotype more rapidly than wild-type (P = 0.011). CONCLUSIONS: Results show that ERCC1 and by implication the NER and/or interstrand crosslink repair mechanisms protect against prostate carcinogenesis and mutations or polymorphisms affecting these DNA repair pathways may predispose prostate epithelial cells to transformation.

False negative results from using common PCR reagents.

BACKGROUND: The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG), and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results. FINDINGS: We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA. CONCLUSIONS: These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is.

Increased cancer cell proliferation in prostate cancer patients with high levels of serum folate.

BACKGROUND: A recent clinical trial revealed that folic acid supplementation is associated with an increased incidence of prostate cancer (Figueiredo et al., J Natl Cancer Inst 2009; 101(6): 432-435). As tumor cells in culture proliferate directly in response to available folic acid, the goal of our study was to determine if there is a similar relationship between patient folate status, and the proliferative capacity of tumors in men with prostate cancer. METHODS: Serum folate and/or prostate tissue folate was determined in 87 randomly selected patients undergoing surgery for prostate cancer, and compared to tumor proliferation in a subset. RESULTS: Fasting serum folate levels were positively correlated with prostate tumor tissue folate content (n = 15; r = 0.577, P < 0.03). Mean serum folate was 62.6 nM (7.5-145.2 nM), 39.5% of patients used supplements containing folic acid (n = 86). The top quartile of patients had serum folates above 82 nM, six times the level considered adequate. Of these, 48% reported no supplement use. Among 50 patients with Gleason 7 disease, the mean proliferation index as determined by Ki67 staining was 6.17 ± 3.2% and 0.86 ± 0.92% in the tumors from patients in the highest (117 ± 15 nM) and lowest (18 ± 9 nM) quintiles for serum folate, respectively (P < 0.0001). CONCLUSIONS: Increased cancer cell proliferation in men with higher serum folate concentrations is consistent with an increase in prostate cancer incidence observed with folate supplementation. Unexpectedly, more than 25% of patients had serum folate levels greater than sixfold adequate. Nearly half of these men reported no supplement use, suggesting either altered folate metabolism and/or sustained consumption of folic acid from fortified foods.

Expression of prostate-specific membrane antigen (PSMA), increases cell folate uptake and proliferation and suggests a novel role for PSMA in the uptake of the non-polyglutamated folate, folic acid.

BACKGROUND: Prostate specific membrane antigen (PSMA) is a unique folate hydrolase that is significantly upregulated in prostate cancer. In a mouse model, PSMA is able to facilitate prostate carcinogenesis, however, little is known about the mechanism by which this occurs. As PSMA is able to hydrolyze polyglutamated folates, and cancer cells proliferate directly in response to available folate, we examined if expression of human PSMA in PC-3 cells confers a proliferative advantage in a microenvironment with physiologically relevant folate levels. METHODS: Proliferation and folate uptake of PC-3 prostate cancer cells expressing human-PSMA or vector alone was assessed in media containing low (LF; 1 nM), physiological (PF; 25 nM), or high (HF; 2.3 microM) folate with or without poly-gamma-glutamated folate (Pte-Glu(5)) or folic acid, and a specific inhibitor of the enzymatic activity of PSMA, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). Folic acid was tested for its ability to competitively inhibit the enzymatic activity of PSMA. RESULTS: Proliferation of PC-3-PSMA cells grown in the presence of poly-gamma-glutamated folate, was significantly higher than that of PC-3-vector cells, an advantage which was attenuated by the addition of 2-PMPA. In media containing physiologic levels of folate, PSMA expression increased folic acid uptake approximately twofold over non-expressing cells. Folic acid was able to inhibit hydrolysis of N-[4-(phenylazo)-benzoyl]-glutamyl-gamma-glutamic acid (PABGgG) by PSMA in a competitive inhibition assay. CONCLUSION: These findings implicate PSMA in both the metabolism of polyglutamated folates, and in the uptake of monoglutamated folates. Under conditions of LF or PF levels, PSMA gives cells expressing it a proliferative advantage.

Prostate specific membrane antigen (PSMA) expression gives prostate cancer cells a growth advantage in a physiologically relevant folate environment in vitro.

BACKGROUND: Prostate specific membrane antigen (PSMA) expression is correlated with stage and grade of prostate cancer suggesting that it confers a growth advantage. We studied if PSMA folate hydrolase activity provides cells a growth advantage in a low folate (LF) micro-environment by hydrolyzing extracellular poly-gamma-glutamated folate to a form that cells can import. METHODS: Proliferation of LNCaP and DU-145 cells was assessed in media containing low (LF), physiological (PF), or high (HF) folate with or without penta-gamma-glutamated folate and a PSMA specific folate hydrolase inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). RESULTS: LNCaP cells, which express PSMA, and DU-145 cells, which do not, displayed decreased proliferation when grown in LF or PF compared to HF media. This reduction in proliferation was eliminated in LNCaP cells when penta-gamma-glutamated folate was added to the media. In the presence of penta-gamma-glutamated folic acid DU-145 cells displayed increased growth but this was still significantly lower than growth in HF medium. Addition of 2-PMPA attenuated the increased growth seen in LNCaP cells but had no effect on DU-145 cell growth. CONCLUSIONS: The folate hydrolase activity of PSMA may provide a growth advantage in LF and PF environments.

Mice lacking glutamate carboxypeptidase II are protected from peripheral neuropathy and ischemic brain injury.

Excessive glutamate release is associated with neuronal damage. A new strategy for the treatment of neuronal injury involves inhibition of the neuropeptidase glutamate carboxypeptidase II (GCP II), also known as N-acetylated alpha-linked acidic dipeptidase. GCP II is believed to mediate the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to glutamate and N-acetyl-aspartate, and inhibition of NAAG peptidase activity (by GCP II and other peptidases) is neuroprotective. Mice were generated in which the Folh1 gene encoding GCP II was disrupted (Folh1-/- mice). No overt behavioral differences were apparent between Folh1-/- mice and wild-type littermates, with respect to their overall performance in locomotion, coordination, pain threshold, cognition and psychiatric behavioral paradigms. Morphological analysis of peripheral nerves, however, showed significantly smaller axons (reduced myelin sheaths and axon diameters) in sciatic nerves from Folh1-/- mice. Following sciatic nerve crush, Folh1-/- mice suffered less injury and recovered faster than wild-type littermates. In a model of ischemic injury, the Folh1-/- mice exhibited a significant reduction (p < 0.05) in infarct volume compared with their wild-type littermates when subjected to middle cerebral artery occlusion, a model of stroke. These findings support the hypothesis that GCP II inhibitors may represent a novel treatment for peripheral neuropathies as well as stroke.

Comparative analysis of prostate-specific membrane antigen (PSMA) versus a prostate-specific membrane antigen-like gene.

BACKGROUND: Currently prostate-specific membrane antigen (PSMA) is showing promise both as an imaging and therapeutic target for occult prostate cancer metastases. First generation antibodies against PSMA are used for the FDA approved Prostascint trade mark monoclonal antibody scan and second generation antibodies are being developed for therapeutic targeting as well as imaging 1. However, there have been reports describing PSMA expression in non-prostatic tissues including kidney, liver, and brain. As we had previously showed the existence of a human PSMA homolog, we set out to determine if this non-prostatic expression was due to expression of the PSMA or another gene. MATERIALS AND METHODS: The PSMA homolog (PSMA-like) cDNA was cloned by screening a liver cDNA library. mRNA expression of the PSMA and PSMA-like genes was determined via Northern blot analysis using two different probes and protein expression confirmed in some tissues via Western blot analysis. Transcriptional regulation of the two genes was examined using reporter constructs driving luciferase expression. RESULTS: The PSMA-like gene possesses 98% identity to the PSMA gene at the nucleotide level and is expressed in kidney and liver under the control of a different promoter to the PSMA gene. The PSMA gene is expressed in several human tissues and is most abundant in the nervous system and the prostate. CONCLUSION: The non-prostatic expression of PSMA should be taken into consideration when designing clinical strategies targeting PSMA.

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate.

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.