α-Emitting cancer therapy using 211 At-AAMT targeting LAT1.

α-Methyl-l-tyrosine (AMT) has a high affinity for the cancer-specific l-type amino acid transporter 1 (LAT1). Therefore, we established an anti-cancer therapy, with 211 At-labeled α-methyl-l-tyrosine (211 At-AAMT) as a carrier of 211 At into tumors. 211 At-AAMT had high affinity for LAT1, inhibited tumor cell growth, and induced DNA double-stranded breaks in vitro. We evaluated the accumulation of 211 At-AAMT in vivo and the role of LAT1. Treatment with 0.4 MBq/mouse 211 At-AAMT inhibited tumor growth in the PANC-1 tumor model and 1 MBq/mouse 211 At-AAMT inhibited metastasis in the lung of the B16F10 metastasis model. Our results suggested that 211 At would be useful for anti-cancer therapy and that LAT1 is suitable as a target for radionuclide therapy.

18F-Boramino acid PET/CT in healthy volunteers and glioma patients.

PURPOSE: In this work, the safety, biodistribution, and radiation dosimetry of large neutral amino acid transporter type-1 (LAT-1) targeting PET tracer 18F-trifluorobborate-derived tyrosine (denoted as 18F-FBY) has been investigated. It is designed as a first-in-human study in healthy volunteers and to assay LAT-1 expression level in glioma patients. METHODS: Six healthy volunteers (3 M, 3 F) underwent whole-body PET acquisitions at multiple time points after bolus injection of 18F-FBY. Regions of interest (ROIs) were mapped manually on major organs, and then the time-activity curves (TACs) were obtained. Dosimetry was calculated with the OLINDA/EXM software. Thirteen patients who were suspected of glioma were scanned with PET/CT at 30 min after 18F-FBY injection. Within 7 days after PET/CT, the tumor was removed surgically, and LAT-1 immunohistochemical staining for LAT-1 was performed on tumor samples and correlated with 18F-FBY PET imaging. RESULTS: 18F-FBY was well tolerated by all healthy volunteers, and no adverse symptoms were observed or reported. 18F-FBY is rapidly cleared from the blood circulation and excreted mainly through the kidneys and urinary tract. The effective dose (ED) was 0.0039 ± 0.0006 mSv/MBq. In 14 surgical confirmed gliomas (one of the patiens had two gliomas), 18F-FBY uptake increased consistently with tumor grade, with maximum standard uptake values (SUVmax) of 0.28 ± 0.14 and 2.84 ± 0.46 and tumor-to-normal contralateral activity (T/N) ratio of 2.30 ± 1.26 and 24.56 ± 6.32 in low- and high-grade tumors, respectively. In addition to the significant difference in the uptakes between low- and high-grade gliomas (P < 0.001), the immunohistochemical staining confirmed the positive correlations between the SUVmax, LAT-1 expression (r2 = 0.80, P < 0.001), and Ki-67 labeling index (r2 = 0.79, P < 0.001). CONCLUSION: 18F-FBY is a PET tracer with favorable dosimetry profile and pharmacokinetics. It has the potential to assay LAT-1 expression in glioma patients and may provide imaging guidance for further boron neutron capture therapy of gliomas. TRIAL REGISTRATION: clinicaltrials.gov (NCT03980431).

Potent-By-Design: Amino Acids Mimicking Porous Nanotherapeutics with Intrinsic Anticancer Targeting Properties.

Exogenous sources of amino acids are essential nutrients to fuel cancer growth. Here, the increased demand for amino acid displayed by cancer cells is unconventionally exploited as a design principle to replete cancer cells with apoptosis inducing nanoscopic porous amino acid mimics (Nano-PAAM). A small library consisting of nine essential amino acids nanoconjugates (30 nm) are synthesized, and the in vitro anticancer activity is evaluated. Among the Nano-PAAMs, l-phenylalanine functionalized Nano-PAAM (Nano-pPAAM) has emerged as a novel nanotherapeutics with excellent intrinsic anticancer and cancer-selective properties. The therapeutic efficacy of Nano-pPAAM against a panel of human breast, gastric, and skin cancer cells could be ascribed to the specific targeting of the overexpressed human large neutral amino acid transporter SLC7A5 (LAT-1) in cancer cells, and its intracellular reactive oxygen species (ROS) inducing properties of the nanoporous core. At the mechanistic level, it is revealed that Nano-pPAAM could activate both the extrinsic and intrinsic apoptosis pathways to exert a potent "double-whammy" anticancer effect. The potential clinical utility of Nano-pPAAM is further investigated using an MDA-MB-231 xenograft in NOD scid gamma mice, where an overall suppression of tumor growth by 60% is achieved without the aid of any drugs or application of external stimuli.

Optimising amino acid absorption: essential to improve nitrogen balance and metabolic control in phenylketonuria.

It has been nearly 70 years since the discovery that strict adherence to a diet low in phenylalanine prevents severe neurological sequelae in patients with phenylalanine hydroxylase deficiency (phenylketonuria; PKU). Today, dietary treatment with restricted phenylalanine intake supplemented with non-phenylalanine amino acids to support growth and maintain a healthy body composition remains the mainstay of therapy. However, a better understanding is needed of the factors that influence N balance in the context of amino acid supplementation. The aim of the present paper is to summarise considerations for improving N balance in patients with PKU, with a focus on gaining greater understanding of amino acid absorption, disposition and utilisation. In addition, the impact of phenylalanine-free amino acids on 24 h blood phenylalanine/tyrosine circadian rhythm is evaluated. We compare the effects of administering intact protein v. free amino acid on protein metabolism and discuss the possibility of improving outcomes by administering amino acid mixtures so that their absorption profile mimics that of intact protein. Protein substitutes with the ability to delay absorption of phenylalanine and tyrosine, mimicking physiological absorption kinetics, are expected to improve the rate of assimilation into protein and minimise fluctuations in quantitative plasma amino acid levels. They may also help maintain normal glycaemia and satiety sensation. This is likely to play an important role in improving the management of patients with PKU.

l-Citrulline restores nitric oxide level and cellular uptake at the brain capillary endothelial cell line (TR-BBB cells) with glutamate cytotoxicity.

OBJECTIVE: Glutamate excitotoxicity provokes neuronal cell damage and death, leading to collapse of the blood-brain barrier (BBB). Recently, it has been reported that l-citrulline, a neutral amino acid and a major precursor of l-arginine in the nitric oxide (NO) cycle, can prevent both neuronal cell death and cerebrovascular cell loss in brain ischemia. Therefore, the objective of this study was to investigate the effect of l-citrulline on glutamate cytotoxicity in the BBB using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells) as an in vitro model of the BBB. METHODS: Cell viability was determined using MTT assay. Cellular uptake of [14C] l-citrulline and expression levels of rat large neutral amino acid transporter 1 (rLAT1), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) at mRNA level were performed using quantitative real-time polymerase chain reaction (PCR) analysis. NO production from TR-BBB cells was measured using Griess reagents. All experiments were performed after treatment of TR-BBB cells with glutamate alone or co-treatment with l-citrulline, l-arginine, and/or taurine for 24 h. RESULTS: l-Citrulline treatment increased cell viability, [14C] l-citrulline uptake, and the mRNA levels of LAT1 and eNOS in TR-BBB cells treated with glutamate. However, iNOS mRNA expression was inhibited by l-citrulline. NO production and transcript level of iNOS were markedly increased by glutamate treatment alone. However, co-treatment with l-citrulline, taurine, or both l-citrulline and taurine decreased NO levels and mRNA levels of iNOS in TR-BBB cells treated with glutamate. In co-treatment of TR-BBB cells with l-arginine, a NO donor, and glutamate, NO levels were increased and expression levels of iNOS mRNA were similar compared to those in cells treated with glutamate alone. CONCLUSION: l-Citrulline can restore NO level and its cellular uptake in TR-BBB cells with glutamate cytotoxicity. Supplying l-citrulline at the BBB may provide neuroprotective effect to improve cerebrovascular dysfunction such as a brain ischemia.

Up-Regulation of LAT1 during Antiandrogen Therapy Contributes to Progression in Prostate Cancer Cells.

PURPOSE: Cancer cells require massive amounts of amino acids for survival. LAT1 (L-type amino acid transporter 1) transports essential amino acids, including leucine, which trigger the downstream mTOR (mammalian target of rapamycin) pathway. We examined the association between androgen receptor and LAT1, and the association between LAT1 expression and the acquisition of castration resistance. MATERIALS AND METHODS: Western blot and real-time polymerase chain reaction were performed to study protein and mRNA expression. siRNA was used to knock down target genes. A total of 92 prostate biopsy specimens of patients who underwent androgen deprivation therapy were used for immunohistochemical analyses. Cox hazard proportional models and the Kaplan-Meier method were used for statistical analyses. RESULTS: LAT1 was highly expressed in hormone resistant prostate cancer cell lines. Knockdown of LAT1 in LNCaP and C4-2 cells significantly suppressed cell proliferation, migration and invasion. Androgen receptor siRNA or androgen receptor blocking through bicalutamide (10 µM) or MDV3100 (10 µM) significantly increased LAT1 expression (p <0.01). Treatment with dihydrotestosterone (0.1 to 10 nM) reduced LAT1 expression in a dose dependent manner (p <0.01). Bicalutamide/MDV3100 plus siLAT1 synergistically suppressed prostate cancer cell proliferation compared to single inhibition by androgen receptor or LAT1 (p <0.01). High LAT1 expression correlated with significantly shorter prostate specific antigen recurrence-free survival in patients receiving androgen deprivation therapy (p <0.0001). LAT1 expression was an independent predictor of castration resistance on multivariate analysis (HR 3.56, p = 0.0133). CONCLUSIONS: The current data may indicate a novel mechanism to acquire castration resistance through activation of the amino acid transporter LAT1.

Inhibition of Large Neutral Amino Acid Transporters Suppresses Kynurenic Acid Production Via Inhibition of Kynurenine Uptake in Rodent Brain.

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor and N-methyl-D-aspartic acid receptor at endogenous brain concentrations. Recent studies have suggested that increases of brain KYNA levels are involved in psychiatric disorders such as schizophrenia and depression, and regulation of KYNA production has become a new target for treatment of these diseases. Kynurenine (KYN), the immediate precursor of KYNA, is transported into astrocytes via large neutral amino acid transporters (LATs). In the present study, the effect of LATs regulation on KYN uptake and KYNA production was investigated in vitro and in vivo using an LATs inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). In the in vitro study, cortical slices of rat brain were incubated with a physiological concentration of KYN and 3 µmol/L-3 mmol/L BCH. BCH inhibited KYNA production and KYN uptake in a dose-dependent manner, and their IC50 values were 90.7 and 97.4 µmol/L, respectively. In the in vivo study, mice were administered KYN (50 mg/kg BW) orally and BCH (200 mg/kg BW) intravenously. Administration of KYN increased brain KYN and KYNA levels compared with the mice treated with vehicle, whereas additional administration of BCH suppressed KYN-induced elevations in KYN and KYNA levels to 50 and 70 % in the brain. These results suggest that inhibition of LATs prevented the increase of KYNA production via blockade of KYN uptake in the brain in vitro and in vivo. LATs can be a target to modulate brain function by regulation of KYNA production in the brain.

Effect of Various Pathological Conditions on Nitric Oxide Level and L-Citrulline Uptake in Motor Neuron-Like (NSC-34) Cell Lines.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a non-essential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [14C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [14C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [14C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [14C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

Fluctuations in phenylalanine concentrations in phenylketonuria: a review of possible relationships with outcomes.

Fluctuations in blood phenylalanine concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). This review evaluates the studies on phenylalanine fluctuations, factors affecting fluctuations, and if stabilizing phenylalanine concentrations affects outcomes, particularly neurocognitive outcome. Electronic literature searches of Embase and PubMed were performed for English-language publications, and the bibliographies of identified publications were also searched. In patients with PKU, phenylalanine concentrations are highest in the morning. Factors that can affect phenylalanine fluctuations include age, diet, timing and dosing of protein substitute and energy intake, dietary adherence, phenylalanine hydroxylase genotype, changes in dietary phenylalanine intake and protein metabolism, illness, and growth rate. Even distribution of phenylalanine-free protein substitute intake throughout 24h may reduce blood phenylalanine fluctuations. Patients responsive to and treated with 6R-tetrahydrobiopterin seem to have less fluctuation in their blood phenylalanine concentrations than controls. An increase in blood phenylalanine concentration may result in increased brain and cerebrospinal fluid phenylalanine concentrations within hours. Although some evidence suggests that stabilization of blood phenylalanine concentrations may have benefits in patients with PKU, more studies are needed to distinguish the effects of blood phenylalanine fluctuations from those of poor metabolic control.

Characterization of 2-(methylamino)alkanoic acid capacity to restrict blood-brain phenylalanine transport in Pah enu2 mice: preliminary findings.

BACKGROUND: Our laboratory seeks a pharmacotherapeutic intervention for PKU that utilizes non-physiological amino acids (NPAAs) to block the accumulation of phenylalanine (Phe) in the brain. In previous studies (Vogel et al. 2013), methylation of the amino group of 2-aminoisobutyrate (AIB) provided an enhanced degree of selectivity for Phe restriction into the brain of Pah(enu2) mice in comparison to unmethylated AIB, leading to the hypothesis that 2-(methylamino)alkanoic acid analogs of AIB might represent targeted inhibitors of Phe accretion into the brain. METHODS: Pah(enu2) and control mice were intraperitoneally administered (500-750 mg/kg body weight, once daily; standard 19% protein diet) AIB, methyl AIB (MAIB), isovaline, and two MAIB analogs, 2-methyl-2-(methylamino)butanoic (MeVal) and 3-methyl-2-(methylamino)pentanoic (MePent) acids for one week, followed by brain and blood isolation for amino acid analyses using UPLC. RESULTS: In the brain, AIB significantly reduced Phe accretion in Pah(enu2) mice, while MeVal significantly improved glutamine and aspartic acids. Four of five test compounds improved brain threonine and arginine levels. AIB, MAIB and IsoVal significantly reduced blood Phe, with no effect of any drug intervention on other sera amino acids. CONCLUSIONS: Further evaluation of AIB and the 2-(methylamino)alkanoic acids as inhibitors of brain Phe accumulation in Pah(enu2) mice is warranted, with more detailed evaluations of route of administration, combinatorial intervention, and detailed toxicity studies.

SLC7A5 is linked to increased expression of genes related to proliferation and hypoxia in estrogen­receptor­positive breast cancer.

The amino acid transporter named solute carrier family 7 member 5 (SLC7A5) is suggested to play a part in altered cell metabolism and proliferative signaling and has been reported to be overexpressed in various types of cancer, including breast cancer. Estrogen­receptor­positive (ER+) breast cancers constitute the most common type of breast malignancies and are often treated with anti­estrogenic therapies. In this group of patients, endocrine resistance is a challenging problem that could lead to recurrent disease. To overcome this, additional prognostic biomarkers are needed. The present study aimed therefore to determine whether SLC7A5 may be considered as a possible prognostic marker in ER+ breast cancer and to investigate its relation with certain cancer­related genes. We used a local breast cancer cohort (n=154) and immunohistochemistry to analyze the expression of SLC7A5 in association with clinicopathological characteristics and patient outcome. In addition, gene expression analysis was performed on 80 of these tumors. Furthermore, the METABRIC dataset was used for correlation analyses between expression of SLC7A5 and several genes related to breast cancer biology. The results demonstrated that overexpression of SLC7A5 was significantly associated with histopathological grade in patients with breast cancer, and that SLC7A5 mRNA expression was positively correlated with the expression of marker of proliferation Ki­67 and hypoxia inducible factor 1 subunit alpha. Overexpression of SLC7A5 may therefore play a role in the biology of endocrinologically­driven disease. However, when further assessing SLC7A5 using the METABRIC dataset, SLC7A5 mRNA expression level was more significantly increased in ER­ subgroups compared with ER+ disease. All breast cancer subtypes included, SLC7A5 mRNA expression was correlated with a higher number of cancer­related genes than in estrogen receptor positive tumors alone. The present study suggested that SLC7A5 expression may be of importance for breast cancer cell proliferation and survival. In order to further establish the biological and clinical role of SLC7A5 in breast cancer, further investigation using different breast cancer subgroups is required.

Starting the conversation on gene therapy for phenylketonuria: Current perspectives of patients, caregivers, and advocates.

Phenylketonuria (PKU) is a rare genetic condition caused by inborn error(s) in the gene for the enzyme phenylalanine hydroxylase. Resulting loss of phenylalanine (Phe) metabolism requires strict dietary therapy and/or medication to prevent toxic accumulation of Phe. Novel investigational therapies, including gene therapies that aim to address underlying causes of PKU, are now entering clinical trials. However, perceptions of this technology in the PKU community have not been assessed. We conducted a qualitative survey recruiting adult patients, caregivers, and patient advocates from the US and 3 EU countries to assess the impact of living with PKU and the perceptions of gene therapy. Telephone interviews were conducted for up to 60 min following a standardized discussion guide. Interviewers classified each participant by their level of knowledge regarding gene therapy as either: low (little or no prior awareness); moderate (awareness of gene therapy as a concept in PKU); or high (working knowledge of gene therapy, e.g., vectors). In total, 33 participants were recruited (patients, n = 24; caregivers, n = 5; advocates, n = 4). The patient sample was well balanced among age groups, sex, and US/EU geographies. The participants' experiences and burden of living with PKU were largely negative, characterized by frustrations with current management consistent with prior reports. Most participants (n = 18/33) were identified as displaying moderate gene-therapy knowledge, 10/33 as displaying high knowledge, and 5/33 as displaying low knowledge. Both positive and negative perceptions were observed; positive perceptions were often linked to "hope" that gene therapy may represent a cure, whereas negative perceptions were linked to the "uncertainty" of outcomes. High knowledge of gene therapy appeared to trend with negative perceptions; 7/10 participants from this group reported high levels of concern over gene therapy. In contrast, participants who displayed low knowledge reported low (n = 3/5) or moderate (n = 2/5) concern, with predominantly positive perceptions. These data highlight the need for education around the theoretical risk:benefit profile of gene therapy. Despite current unknowns around gene therapy, our study demonstrates the important role of healthcare providers as educators who can use available data to provide balanced information to patients and caregivers.

L-Citrulline Level and Transporter Activity Are Altered in Experimental Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease caused by the death of the neurons regulating the voluntary muscles which leads to the progressive paralysis. We investigated the difference of transport function of L-citrulline in ALS disease model (NSC-34/hSOD1G93A, MT) and a control model (NSC-34/hSOD1wt, WT). The [14C]L-citrulline uptake was significantly reduced in MT cells as compared with that of control. The Michaelis-Menten constant (Km) for MT cells was 0.67 ± 0.05 mM, whereas it was 1.48 ± 0.21 mM for control. On the other hand, the Vmax values for MT and control were 10.9 ± 0.8 nmol/mg protein/min and 18.3 ± 2.9 nmol/mg protein/min, respectively. The Km and Vmax values showed the high affinity and low capacity for MT as compared with control. Moreover, the uptake of [14C]L-citrulline was significantly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and harmaline which is the inhibitor of the large neutral amino acid transporter1 (LAT1) in NSC-34 cell lines. Furthermore, [14C]L-citrulline uptakes took place in Na+-independent manner. It was also inhibited by the neutral amino acids such as citrulline and phenylalanine. Likewise, L-dopa, gabapentin, and riluzole significantly inhibited the [14C]L-citrulline uptake. It shows the competitive inhibition for L-dopa in ALS cell lines. On the other hand, [14C]L-citrulline uptake in the presence of riluzole showed competitive inhibition in WT cells, whereas it was uncompetitive for MT cells. The small interfering RNA experiments showed that LAT1 is involved in the [14C]L-citrulline uptake in NSC-34 cell lines. On the other hand, in the examination of the alteration in the expression level of LAT1, it was significantly lower in MT cells as compared with that of control. Similarly, in the spinal cord of ALS, transgenic mice revealed a slight but significant decrease in LAT1 immunoreactivity in motor neurons of ALS mice compared with control. However, the LAT1 immunoreactivity in non-motor neurons and in astrocytes was relatively increased in the spinal cord gray matter of ALS mice. The experimental evidences of our results suggest that the change of transport activity of [14C]L-citrulline may be partially responsible for the pathological alteration in ALS.

Possibility of Amino Acid Treatment to Prevent the Psychiatric Disorders via Modulation of the Production of Tryptophan Metabolite Kynurenic Acid.

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan catabolism, acts as an antagonist for both the α7 nicotinic acetylcholine receptor and glycine coagonist sites of the N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Elevation of brain kynurenic acid levels reduces the release of neurotransmitters such as dopamine and glutamate, and kynurenic acid is considered to be involved in psychiatric disorders such as schizophrenia and depression. Thus, the control of kynurenine pathway, especially kynurenic acid production, in the brain is an important target for the improvement of brain function or the effective treatment of brain disorders. Astrocytes uptake kynurenine, the immediate precursor of kynurenic acid, via large neutral amino acid transporters, and metabolize kynurenine to kynurenic acid by kynurenine aminotransferases. The former transport both branched-chain and aromatic amino acids, and the latter have substrate specificity for amino acids and their metabolites. Recent studies have suggested the possibility that amino acids may suppress kynurenic acid production via the blockade of kynurenine transport or via kynurenic acid synthesis reactions. This approach may be useful in the treatment and prevention of neurological and psychiatric diseases associated with elevated kynurenic acid levels.

Amino Acid Transporters as Potential Therapeutic Targets in Thyroid Cancer.

Thyroid cancer cells have a high amino acid demand for proliferation, invasion, and metastasis. Amino acids are taken up by thyroid cancer cells, both thyroid follicular cell and thyroid parafollicular cells (commonly called "C-cells"), via amino acid transporters. Amino acid transporters up-regulate in many cancers, and their expression level associate with clinical aggressiveness and prognosis. This is the review to discuss the therapeutic potential of amino acid transporters and as molecular targets in thyroid cancer.

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.

Studying the effect of large neutral amino acid supplements on oxidative stress in phenylketonuric patients.

Background Oxidative stress may be one of the causes responsible for mental retardation in phenylketonuria (PKU) patients. Phenylalanine (Phe) reduces antioxidant defense and promotes oxidative stress by causing increase in reactive oxygen-nitrogen species. Our study aimed to investigate the effect of different treatments (amino acid mixture/large neutral amino acid [LNAA] supplements) on oxidative stress which are applied to late-diagnosed patients. To the best of our knowledge, this is the first study to investigate the effect of LNAA supplements on oxidative stress. Methods Twenty late-diagnosed classic PKU patients were included in this study. Patients were classified into two groups: patients under Phe-restricted diet and using Phe-free amino acid mixtures (Group I) (mean age: 13.8 ± 2.8), and patients taking LNAA supplements (Group II) (mean age: 14.8 ± 3.8). Healthy controls (mean age: 13.6 ± 4.8) with ages consistent with the ages of the patients in the experimental groups were included. Results Glutathione peroxidase is lower in patients of taking LNAA supplements than the control group (p = 0.022). Coenzyme Q10 is lower in patients of using Phe-free amino acid mixtures than the control group and it is significantly higher in Group II than Group I (p = 0.0001, p = 0.028, respectively). No significant differences were detected in total antioxidant status, total oxidant status, oxidative stress index, paraoxonase 1 and L-carnitine levels. Conclusions Different treatments affect oxidative stress parameters in PKU patients. In this study, although patients were followed up with classic PKU, patient-specific adjuvant antioxidant therapies should be implemented in response to oxidative stress.

Propagation of Plasma L-Phenylalanine Concentration Fluctuations to the Neurovascular Unit in Phenylketonuria: An in silico Study.

Phenylketonuria (PKU) is an inherited metabolic disease characterized by abnormally high concentrations of the essential amino acid L-phenylalanine (Phe) in blood plasma caused by reduced activity of phenylalanine hydroxylase (PAH). While numerous studies have shown association between high plasma Phe concentration and intellectual impairment, it is not clear whether increased Phe fluctuations also observed in PKU affect the brain as well. To investigate this, time-resolved in vivo data on Phe and competing large neutral amino acid (LNAA) concentrations in neurons are needed, but cannot be acquired readily with current methods. We have used in silico modeling as an alternative approach to characterize the interactive dynamics of Phe and competing LNAAs (CL) in the neurovascular unit (NVU). Our results suggest that plasma Phe fluctuations can propagate into the NVU cells and change there the concentration of LNAAs, with the highest magnitude of this effect observed at low frequency and high amplitude-to-mean ratio of the plasma Phe concentration fluctuations. Our model further elucidates the effect of therapeutic LNAA supplementation in PKU, showing how abnormal concentrations of Phe and CL in the NVU move thereby toward normal physiologic levels.

Functional Polarity of Microvascular Brain Endothelial Cells Supported by Neurovascular Unit Computational Model of Large Neutral Amino Acid Homeostasis.

The homeostatic regulation of large neutral amino acid (LNAA) concentration in the brain interstitial fluid (ISF) is essential for proper brain function. LNAA passage into the brain is primarily mediated by the complex and dynamic interactions between various solute carrier (SLC) transporters expressed in the neurovascular unit (NVU), among which SLC7A5/LAT1 is considered to be the major contributor in microvascular brain endothelial cells (MBEC). The LAT1-mediated trans-endothelial transport of LNAAs, however, could not be characterized precisely by available in vitro and in vivo standard methods so far. To circumvent these limitations, we have incorporated published in vivo data of rat brain into a robust computational model of NVU-LNAA homeostasis, allowing us to evaluate hypotheses concerning LAT1-mediated trans-endothelial transport of LNAAs across the blood brain barrier (BBB). We show that accounting for functional polarity of MBECs with either asymmetric LAT1 distribution between membranes and/or intrinsic LAT1 asymmetry with low intraendothelial binding affinity is required to reproduce the experimentally measured brain ISF response to intraperitoneal (IP) L-tyrosine and L-phenylalanine injection. On the basis of these findings, we have also investigated the effect of IP administrated L-tyrosine and L-phenylalanine on the dynamics of LNAAs in MBECs, astrocytes and neurons. Finally, the computational model was shown to explain the trans-stimulation of LNAA uptake across the BBB observed upon ISF perfusion with a competitive LAT1 inhibitor.

Multibranched Gold Nanoparticles with Intrinsic LAT-1 Targeting Capabilities for Selective Photothermal Therapy of Breast Cancer.

Because of the critical role of the large neutral amino acid transporter-1 (LAT-1) in promoting tumor growth and proliferation, it is fast emerging as a highly attractive biomarker for the imaging and treatment of human malignancies, including breast cancer. While multibranched gold nanoparticles (AuNPs) have emerged as a promising modality in the photothermal therapy (PTT) of cancers, some of the key challenges limiting their clinical translation lie in the need to develop reproducible and cost-effective synthetic methods as well as the selective accumulation of sufficient AuNPs at tumor sites. In this study, we report a simple and direct seed-mediated synthesis of monodispersed multibranched AuNPs using the catechol-containing LAT-1 ligands, L- and D-dopa, to confer active cancer targeting. This route obviates the need for additional conjugation with targeting moieties such as peptides or antibodies. Nanoflower-like AuNPs (AuNF) with diameters of approximately 46, 70, and 90 nm were obtained and were found to possess excellent colloidal stability and biocompatibility. A significantly higher intracellular accumulation of the L- and D-dopa functionalized AuNFs was observed in a panel of breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, and MDA-MB-453) when compared to the nontargeting control AuNFs synthesized with dopamine and 4-ethylcatechol. Importantly, no significant difference in uptake between the targeting and nontargeting AuNFs was observed in a non-tumorigenic MCF-10A breast epithelial cell line, hence demonstrating tumor selectivity. For PTT of breast cancer, Ag+ was introduced during synthesis to obtain L-dopa functionalized nanourchin-like AuNPs (AuNUs) with strong near-infrared (NIR) absorbance. The L-dopa functionalized AuNUs mediated selective photothermal ablation of the triple negative MDA-MB-231 breast cancer cell line and sensitized the cells to the anticancer drugs cisplatin and docetaxel. This work brings forward an effective strategy for the facile preparation of cancer targeting multibranched AuNPs with potential for the in vivo PTT of breast cancer.