Sepiapterin Research Articles

Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism.

HER2+ breast tumors have abundant immune-suppressive cells, including M2-type tumor-associated macrophages (TAMs). Although TAMs consist of the immune-stimulatory M1 type and immune-suppressive M2 type, the M1/M2-TAM ratio is reduced in immune-suppressive tumors, contributing to their immunotherapy refractoriness. M1- versus M2-TAM formation depends on differential arginine metabolism, where M1-TAMs convert arginine to nitric oxide (NO) and M2-TAMs convert arginine to polyamines (PAs). We hypothesize that such distinct arginine metabolism in M1- versus M2-TAMs is attributed to different availability of BH4 (NO synthase cofactor) and that its replenishment would reprogram M2-TAMs to M1-TAMs. Recently, we reported that sepiapterin (SEP), the endogenous BH4 precursor, elevates the expression of M1-TAM markers within HER2+ tumors. Here, we show that SEP restores BH4 levels in M2-like macrophages, which then redirects arginine metabolism to NO synthesis and converts M2 type to M1 type. The reprogrammed macrophages exhibit full-fledged capabilities of antigen presentation and induction of effector T cells to trigger immunogenic cell death of HER2+ cancer cells. This study substantiates the utility of SEP in the metabolic shift of the HER2+ breast tumor microenvironment as a novel immunotherapeutic strategy.

NOS cofactor tetrahydrobiopterin contributes to anesthetic preconditioning induced myocardial protection in the isolated exvivo rat heart.

Anesthetic preconditioning (APC) may decrease the myocardium injury nearly 50% following ischemia/reperfusion (I/R) by enhancing recovery of cardiac function, reducing myocardial enzyme release and lowering infarct size when utilized as pretreatment or posttreatment agents. I/R increases nitric oxide (NO) production through endothelial NO synthase (NOS3) and heat shock protein 90 (HSP90). The present study aimed to observe the role of BH4 availability and the association of HSP90 with NOS3 in APC‑mediated cardioprotection against I/R injury. Isolated rat hearts were subjected to no‑flow ischemia for 30min and reperfusion for 120min. Sevoflurane (3.5%) was administered for 15min followed by a 15min washout prior to ischemia. 2,4-Diamino-6-hydroxypyrimidine(DAHP) or sepiapterin(SP) was administered for 40min until the onset of ischemia. The results revealed that compared with pre‑ischemic basal levels, BH4 levels decreased and BH2 levels increased following I/R. BH4 levels were significantly increased and BH2 levels were significantly decreased in the APC+I/R hearts compared with the I/R group hearts. The BH4:BH2 ratio in the APC‑treated hearts was also increased compared with that in the I/R group hearts. SP increased the recovery of contractile function and the production of NO, and decreased the production of superoxide anion (O2·‑) in I/R heart, but did not elicit these effects in APC‑treated hearts. DAHP treatment inhibited the APC‑mediated recovery of contractile function, increased O2·‑ levels and decreased NO production, but had no effect in I/R hearts. The cardioprotection of APC was demonstrated to be modulated by the BH4 precursor SP, which increased BH4 levels, or DAHP, which inhibited GTP cyclohydrolase I. Both APC and SP treatments increased the combination of HSP90 and NOS3, which improved the NOS3 activity and function. The results suggested that BH4, which servesas a cofactor for NOS, mediated the resistance of APC to I/R injury by promoting the binding of HSP90 and NOS3.

Enhanced Nanoencapsulation of Sepiapterin within PEG-PCL Nanoparticles by Complexation with Triacetyl-Beta Cyclodextrin.

In this work, we aimed to improve the encapsulation efficiency of sepiapterin (SP), the natural precursor of the essential cofactor tetrahydrobiopterin (BH4) that displays mild water-solubility and a short biological half-life, within methoxy-poly(ethylene-glycol)-poly(epsilon-caprolactone)(mPEG-PCL) nanoparticles (NPs) by means of its complexation and hydrophobization with 2,3,6-triacetyl-β-cyclodextrin (TAβCD). For this, SP/TAβCD complexes were produced by spray-drying of SP/TAβCD binary solutions in ethanol using the Nano Spray Dryer B-90 HP. Dry powders were characterized by differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), and transmission and scanning electron microscopy (TEM and SEM, respectively) and compared to the pristine components and their physical mixtures (PMs). Next, SP was encapsulated within mPEG-PCL NPs by nano-precipitation of an SP/TAβCD complex/mPEG-PCL solution. In addition to the nano-encapsulation of a preformed complex within the polymeric NPs, we assessed an alternative encapsulation approach called drying with copolymer (DWC) in which pristine SP, TAβCD, and mPEG-PCL were co-dissolved in a mixture of acetone and methanol at the desired weight ratio, dried under vacuum, re-dissolved, and nano-precipitated in water. The dissolution-drying step was aimed to promote the formation of molecular hydrophobic interactions between SP, TAβCD, and the PCL blocks in the copolymer. SP-loaded mPEG-PCL NPs were characterized by dynamic light scattering (DLS) and SEM. NPs with a size of 74–75 nm and standard deviation (S.D., a measure of the peak width) of 21–22 nm were obtained when an SP:TAβCD (1:1 molar ratio) spray-dried complex was used for the nano-encapsulation and SEM analysis revealed the absence of free SP crystals. The encapsulation efficiency (%EE) and drug loading (%DL) were 85% and 2.6%, respectively, as opposed to the much lower values (14% and 0.6%, respectively) achieved with pristine SP. Moreover, the NPs sustained the SP release with relatively low burst effect of 20%. Overall, our results confirmed that spray-drying of SP/TAβCD solutions at the appropriate molar ratio leads to the hydrophobization of the relatively hydrophilic SP molecule, enabling its encapsulation within mPEG-PCL NPs and paves the way for the use of this strategy in the development of novel drug delivery systems of this vital biological precursor.

Sepiapterin alleviates impaired gastric nNOS function in spontaneous diabetic female rodents through NRF2 mRNA turnover and miRNA biogenesis pathway.

An impaired nitrergic system and altered redox signaling contribute to gastric dysmotility in diabetics. Our earlier studies show that NF-E2-related factor 2 (NRF2) and phase II antioxidant enzymes play a vital role in gastric neuronal nitric oxide synthase (nNOS) function. This study aims to investigate whether supplementation of sepiapterin (SEP), a precursor for tetrahydrobiopterin (BH4) (a cofactor of NOS) via the salvage pathway, restores altered nitrergic systems and redox balance in spontaneous diabetic (DB) female rats. Twelve-week spontaneous DB and age-matched, non-DB rats, with and without dietary SEP (daily 20 mg/kg body wt for 10 days) treatment, were used in this study. Gastric antrum muscular tissues were excised to investigate the effects of SEP in nitrergic relaxation and the nNOS-nitric oxide (NO)-NRF2 pathway(s). Dietary SEP supplementation significantly ( P < 0.05) reverted diabetes-induced changes in nNOS dimerization and function; nitric oxide (NO) downstream signaling molecules; HSP-90, a key regulator of nNOSα activity and dimerization; miRNA-28 that targets NRF2 messenger RNA (mRNA), and levels of microRNA (miRNA) biogenesis pathway components, such as DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and TRBP (HIV1-1 transactivating response RNA-binding protein). These findings emphasize the importance of the BH4 pathway in regulating gastric motility functions in DB animals by modulating nNOSα dimerization in association with changes in enteric NRF2 and NO downstream signaling. Our results also identify a new pathway, wherein SEP regulates NRF2 mRNA turnover by suppressing elevated miRNA-28, which could be related to alterations in miRNA biogenesis pathway components. NEW & NOTEWORTHY This study is the first to show a causal link between NF-E2-related factor 2 (NRF2) and neuronal nitric oxide synthase (nNOS) in gastric motility function. Our data demonstrate that critical regulators of the miRNA biosynthetic pathway are upregulated in the diabetic (DB) setting; these regulators were rescued by sepiapterin (SEP) treatment. Finally, we show that low dihydrofolate reductase expression may lead to impaired nNOS dimerization/function-reduced nitric oxide downstream signaling and elevate oxidative stress by suppressing the NRF2/phase II pathway through miRNA; SEP treatment restored all of the above in DB gastric muscular tissue. We suggest that tetrahydrobiopterin supplementation may be a useful therapy for patients with diabetes, as well as women with idiopathic gastroparesis.

Sepiapterin Enhances Tumor Radio- and Chemosensitivities by Promoting Vascular Normalization.

Previously, we demonstrated that nitric oxide (NO) synthase (NOS) is uncoupled in a wide range of solid tumors and that restoring NOS coupling with the tetrahydrobiopterin precursor sepiapterin (SP) inhibits tumor progression. Endothelial dysfunction characterizes the poorly functional vasculature of solid tumors, and since NO is critical for regulation of endothelial function we asked whether SP, by recoupling NOS, improves tumor vasculature structure and function-enhancing chemotherapeutic delivery and response to radiotherapy. MMTV-neu mice with spontaneous breast tumors were treated with SP by oral gavage and evaluated by multispectral optoacoustic tomographic analysis of tumor HbO2 and by tissue staining for markers of hypoxia, blood perfusion, and markers of endothelial and smooth muscle proteins. Recoupling tumor NOS activity results in vascular normalization observed as reduced tumor hypoxia, improved tumor percentage of HbO2 and perfusion, as well as increased pericyte coverage of tumor blood vessels. The normalized vasculature and improved tumor oxygenation led to a greater than 2-fold increase in radiation-induced apoptosis compared with radiation or SP alone. High-performance liquid chromatography analysis of tumor doxorubicin levels showed a greater than 50% increase in doxorubicin uptake and a synergistic effect on tumor cell apoptosis. This study highlights for the first time the importance of NOS uncoupling and endothelial dysfunction in the development of tumor vasculature and presents a new approach for improving the tumoricidal efficacies of chemotherapy and radiotherapy.

Abstract 5196: Recoupling nitric oxide synthetase to normalize tumor vasculature and enhance tumor radiosensitivity and anti-tumor drug uptake

Abstract The aberrant nature of tumor vasculature results in uneven, heterogeneous blood flow and leaky, hemorrhagic blood vessels. Due to the unusual nature of tumor vessels, areas of hypoxia develop that contribute to radioresistance and inefficiency of therapeutic drug delivery. Our current studies examine the role of nitric oxide synthase (NOS) in tumor vasculature. NOS has been demonstrated to be “uncoupled” in tumors and isolated tumor cells due to reduced levels of tetrahydrobiopterin (BH4), a necessary cofactor, resulting in superoxide and peroxynitrite formation in lieu of NO (Rabender et al 2015 Molecular Cancer Research). NO signaling is critical for vascular function and thus uncoupling of eNOS in tumor endothelial cells may partly explain the poor vasculature found within solid tumors. Having previously demonstrated that NOS can be “recoupled” and NO production restored through treatment of tumor cells with Sepiapterin (SP), a BH4 precursor, we examined whether SP could normalize tumor vasculature, promoting radiosensitivity and improving drug uptake. Multispectral optoacoustic tomography analysis of both flank tumor xenografts and a spontaneous breast tumor model (MMTV) demonstrate that SP given by oral gavage once a day for 7 days significantly enhances tumor BH4 levels and the percent of oxyhemoglobin in the tumor. Immunohistochemical analysis of tumors from mice treated with SP showed a significant reduction in CD31 staining and a significant increase in smooth muscle actin (SMA), both hallmarks of vascular normalization. A significant decrease in pimonidazole staining and concomitant increase in Hoescht staining in MMTV mice treated with SP confirms the reduction in tumor hypoxia as well as increased tumor perfusion. Increased tumor perfusion was also demonstrated through enhanced doxorubicin uptake in tumors of mice treated with SP. Lastly, the enhanced tumor oxygenation correlated with more than a 2-fold increase in radiation induced cell killing measured by ex vivo clonogenic assay. These preliminary data demonstrate the need for continued study of NOS uncoupling in solid tumors, especially when we take into consideration that SP has been demonstrated to be cytotoxic to both breast and colon tumors. On-going studies are examining the consequences of enhanced drug uptake on both normal and tumor tissue as well as the mechanism behind the vascular normalization. Note: This abstract was not presented at the meeting. Citation Format: Christopher S. Rabender, Sundaresan Gobalakrishnan, Li Wang, Jamal Zweit, Ross B. Mikkelsen. Recoupling nitric oxide synthetase to normalize tumor vasculature and enhance tumor radiosensitivity and anti-tumor drug uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2017-5196

Abstract 3383: Tumor vasculature normalization by recoupling nitric oxide synthase with a tetrahydrobiopterin precursor

Abstract The abnormal, irregular nature of tumor vasculature has been well documented. The aberrant nature of the vasculature results in uneven, heterogeneous blood flow and leaky, hemorrhagic blood vessels. Due to the unusual nature of tumor vessels, areas of hypoxia tend to develop that contribute to radioresistance and inefficiency of therapeutic drug delivery. Our current studies examine the role of nitric oxide synthase (NOS) in tumor vasculature. NOS has been demonstrated to be “uncoupled” in tumor cells due to reduced levels of tetrahydrobiopterin (BH4), a necessary cofactor, resulting in peroxynitrite (ONOO-) formation in lieu of nitric oxide (NO). NO signaling is critical for vascular function and thus uncoupling of eNOS in the endothelial cells may partly explain the poor vasculature structure found within solid tumors. Having previously demonstrated that NOS can be “recoupled” and NO production restored through treatment of tumor cells with Sepiapterin (SP), a BH4 precursor, we examined whether SP could normalize tumor vasculature, promoting radiosensitivity and improving drug uptake. Multispectral optoacoustic tomography analysis of both flank tumor xenografts and a spontaneous breast tumor model (MMTV) demonstrate that SP given orally significantly enhances the percent of oxyhemoglobin in the tumor. Immunohistochemical analysis of tumors treated with SP showed a significant reduction in CD31 staining and a significant increase in smooth muscle actin (SMA), both hallmarks of vascular normalization. Ex vivo analysis of tumors revealed that the enhanced tumor oxygenation resulted in over a two-fold increase in radiation induced cell killing. These preliminary data demonstrate great potential for SP as an adjuvant in the treatment of cancer, especially when we take into consideration that SP has been demonstrated to be cytotoxic to both breast and colon tumors. Future studies will examine drug uptake in tumors as well as the mechanism behind the vascular normalization. Citation Format: Christopher Rabender, Ninu Bruno, Ross B. Mikkelsen. Tumor vasculature normalization by recoupling nitric oxide synthase with a tetrahydrobiopterin precursor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3383.

40 Sepiapterin, a potential therapeutic approach to restore the metabolic phenotype switch induced by sFlt-1 in preeclampsia

Introduction Preeclampsia (PE) is an often fatal cardiovascular complication related to pregnancy with no current effective way of treatment. Evidence of early increased circulating anti-angiogenic factors (AAFs) support the onset of a multi-systemic disorder and widespread maternal endothelial dysfunction in PE. However, the molecular mechanisms are still not well understood. Objectives To determine the potential early metabolic perturbations and mitochondrial bioenergetics effects of sFlt-1 in bovine aortic endothelial cells (ECs) and first trimester extravillous trophoblasts (HTR-8/SVneo). Also, to evaluate the potential use of sepiapterin (SE), a precursor of eNOS’s cofactor tetrahydrobiopterin (BH4), in abrogating the effects of sFlt-1 in cells. Methods Metabolic perturbations and mitochondrial bioenergetics were assessed in ECs and HTR-8/SVneo in an in-vitro model of preeclampsia using exogenous sFlt-1 and serum from preeclamptic women. Mitochondrial bioenergetics was assessed using an XFe24 Extracellular Flux Analyzer. Nitric oxide (NO) was determined by chemiluminescence. Mitochondrial function and metabolism in sFlt-1-treated cells was evaluated also in galactose media. Mitochondrial membrane potential and superoxide was evaluated by JC-1 and Mito-Sox, respectively, by flow cytometry. Results We found that treatment with sFlt-1 affected the mitochondrial maximal respiration and spare respiratory capacity in ECs in a dose dependent manner leading to a metabolic phenotype switch to glycolysis. In contrast, HTR-8/SVneo, displayed an unexpected strong glycolytic metabolism. sFlt-1 was found not to disturb the trophoblast mitochondrial metabolic and bioenergetics profile even at relatively high doses. In addition, we found that sFlt-1 treatment caused concentration dependent decreases in mitochondrial membrane potential and diminished NO levels in ECs. Moreover, treatment of ECs with sFlt-1 in galactose strongly impaired cell viability suggesting the role of sFlt-1 as a mitochondrial disruptor. SE, protected ECs and HTR8/SVneo cells from sFlt-1-induced superoxide formation and restored the NO levels and metabolic phenotype switch induced by sFlt-1 and maternal PE serum in ECs. Conclusions sFlt-1 disrupts mitochondria bioenergetics and metabolism in both, ECs and HTR-8/SVneo. This evidence could explain the potential detrimental effects of AAFs in the maternal endothelium and the hallmark of hypertension in PE. Hence, we demonstrate that SE, by enhancing BH4/NO bioavailability and diminish mitochondrial superoxide formation, restored the metabolic phenotype switch induced by sFlt-1 and PE serum. Based on this evidence, we postulate the use of SE as a potential therapeutic approach to prevent or treat PE.

Chronic Co-Administration of Sepiapterin and L-Citrulline Ameliorates Diabetic Cardiomyopathy and Myocardial Ischemia/Reperfusion Injury in Obese Type 2 Diabetic Mice.

Diabetic heart disease is associated with tetrahydrobiopterin oxidation and high arginase activity, leading to endothelial nitric oxide synthase dysfunction. Sepiapterin (SEP) is a tetrahydrobiopterin precursor, and L-citrulline (L-Cit) is converted to endothelial nitric oxide synthase substrate, L-arginine. Whether SEP and L-Cit are effective at reducing diabetic heart disease is not known. The present study examined the effects of SEP and L-Cit on diabetic cardiomyopathy and ischemia/reperfusion injury in obese type 2 diabetic mice. Db/db and C57BLKS/J mice at 6 to 8 weeks of age received vehicle, SEP, or L-Cit orally alone or in combination for 8 weeks. Cardiac function was evaluated with echocardiography. Db/db mice displayed hyperglycemia, obesity, and normal blood pressure and cardiac function compared with C57BLKS/J mice at 6 to 8 weeks of age. After vehicle treatment for 8 weeks, db/db mice had reduced ejection fraction, mitral E/A ratio, endothelium-dependent relaxation of coronary arteries, tetrahydrobiopterin concentrations, ratio of endothelial nitric oxide synthase dimers/monomers, and nitric oxide levels compared with vehicle-treated C57BLKS/J mice. These detrimental effects of diabetes mellitus were abrogated by co-administration of SEP and L-Cit. Myocardial infarct size was increased, and coronary flow rate and ± dP/dt were decreased during reperfusion in vehicle-treated db/db mice subjected to ischemia/reperfusion injury compared with control mice. Co-administration of SEP and L-Cit decreased infarct size and improved coronary flow rate and cardiac function in both C57BLKS/J and db/db mice. Co-administration of SEP and L-Cit limits diabetic cardiomyopathy and ischemia/reperfusion injury in db/db mice through a tetrahydrobiopterin/endothelial nitric oxide synthase/nitric oxide pathway.

Abstract 4250: Nitric oxide synthase activity and its modulation in the treatment of colorectal cancer

Abstract The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn's Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase. Under non-inflammatory conditions NOS generates Nitric Oxide. However, in an inflammatory environment, such as the oxidative tumor microenvironment, NOS's cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2). NOS bound to BH2 is said to be uncoupled and produces superoxide O2- and peroxyinitrite (ONOO-). Previous work in our and other's labs have shown that increased production of ROS/RNS leads to the activation of pro-inflammatory/proliferative molecules such as NFκB, Stat3, β-Catenin and Akt. NOS can be re-coupled by supplementing cells and animals with BH4 or its precursor Sepiapterin (SP). Herein Wwe have shownshow that by recoupling NOS with SP in HCT116, Caco-2 and HT29 cells, decreased tumor cell proliferation, increased β-Catenin degradation and decreased Akt activity. We have also seen increased tumor cell death measured via by in vitro clonogenic assays, as well decreased metabolic uptake in AOM/DSS induced colorectal cancer in vivo measured via by F18Deoxy-Glucose PET imaging. We believe by recoupling NOS both in vivo and in vitro we are modulatingion Wnt signaling via Akt and GSK-3β. Further studies are needed to elucidate the mechanism of decreased Akt activity and the downstream effectors of Wnt signaling which are affected by NOS recoupling. Lastly, we wish to combine radiation therapy with Sepiapterin SP to determine if the combination of these therapies can increase tumor cell deathincrease tumor control. Citation Format: Asim Alam, Chris Rabender, Li Wang, Sundaresan Gobalakrishnan, Jamal Zweit. Nitric oxide synthase activity and its modulation in the treatment of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2014-4250

Sepiapterin Ameliorates Chemically Induced Murine Colitis and Azoxymethane-Induced Colon Cancer

The effects of modulating tetrahydrobiopterin (BH4) levels with a metabolic precursor, sepiapterin (SP), on dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)-induced colorectal cancer were studied. SP in the drinking water blocks DSS-induced colitis measured as decreased disease activity index (DAI), morphologic criteria, and recovery of Ca(2+)-induced contractility responses lost as a consequence of DSS treatment. SP reduces inflammatory responses measured as the decreased number of infiltrating inflammatory macrophages and neutrophils and decreased expression of proinflammatory cytokines interleukin 1β (IL-1β), IL-6, and IL-17A. High-performance liquid chromatography analyses of colonic BH4 and its oxidized derivative 7,8-dihydrobiopterin (BH2) are inconclusive although there was a trend for lower BH4:BH2 with DSS treatment that was reversed with SP. Reduction of colonic cGMP levels by DSS was reversed with SP by a mechanism sensitive to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). ODQ abrogates the protective effects of SP on colitis. This plus the finding that SP reduces DSS-enhanced protein Tyr nitration are consistent with DSS-induced uncoupling of NOS. The results agree with previous studies that demonstrated inactivation of sGC in DSS-treated animals as being important in recruitment of inflammatory cells and in altered cholinergic signaling and colon motility. SP also reduces the number of colon tumors in AOM/DSS-treated mice from 7 to 1 per unit colon length. Thus, pharmacologic modulation of BH4 with currently available drugs may provide a mechanism for alleviating some forms of colitis and potentially minimizing the potential for colorectal cancer in patients with colitis.

Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. Dihydrofolate reductase (DHFR) can catalyze 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin (BH4) in the salvage pathway of BH4 synthesis from sepiapterin (SP), a major pigment component contained in the integument of silkworm Bombyx mori mutant lemon (lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkworm B. mori (BmDhfr) and identification of enzymatic properties of BmDHFR. BmDhfr is located on scaffold Bm_199 with a predicted gene model BGIBMGA013340, which encodes a 185-aa polypeptide with a predicted molecular mass of about 21 kDa. Biochemical analyses showed that the recombinant BmDHFR protein exhibited high enzymatic activity and suitable parameters to substrate. Together with our previous studies on SP reductase of B. mori (BmSPR) and the lem mutant, it may be an effective way to industrially extract SP from the lem silkworms in large scale to produce BH4 in vitro by co-expressing BmSPR and BmDHFR and using the extracted SP as a substrate in the future.

Abstract 2057: Tumor suppressor effects of tetrahydrobiopterin are mediated through beta-catenin in breast cancer cells

Abstract Nitric Oxide (NO) can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2− instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO−), which is produced rapidly by the reaction of O2− with NO. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7 and MDA-231 cells in tissue culture and in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is around 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. To ensure that increasing the BH4:BH2 ratio leads to enhanced NOS coupling, the production of NO was measured by cGMP formation and O2− levels were assessed by HPLC. As hypothesized NO levels went up and O2− levels went down. To directly examine the effects of manipulating the BH4:BH2 ratio cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. To assess the effects of SP on xenografts, we used an ex-vivo clonogenic assay. This assay also showed that colony formation is significantly reduced upon treatment of the animals with SP. Previous studies in colon cancer cells have shown that when PKG, the major pathway activated by NO, is activated β-catenin levels are reduced. In our experiments MCF7 xenografts showed a significant decrease in β-catenin levels after treatment with SP. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2057. doi:1538-7445.AM2012-2057

Time course of development of erectile dysfunction and coronary artery endothelial dysfunction in response to a western diet: influence of endothelial nitric oxide synthase uncoupling

Epidemiological evidence suggests that men experiencing cardiac events may have preexisting erectile dysfunction (ED). Male Sprague‐Dawley rats (n = 5/group) were fed a control or western diet for 4, 8, or 12 weeks. Erectile function was evaluated by calculating the area under the curve (AUC) of intracavernosal pressure/mean arterial pressure in response to electrical field stimulation of the major pelvic ganglion, and again following intracavernosal injection of 10 μM sepiapterin (SA). Coronary artery segments were mounted on a wire myograph, pre‐constricted with serotonin and endothelial function was evaluated by cumulative doses of acetylcholine (ACh) (0.001 – 10 μM), and repeated in one segment following incubation in 10 μM SA. Erectile function was attenuated at 8 and 12 weeks (C: 286 ± 52, 4w: 232 ± 35, 8w: 123 ± 49, 12w: 160 ± 59 (AUC); p = 0.004). SA had no effect on erectile function at C or 4w, but showed improvement at 8w (225 ± 99, p = 0.13) and 12w (211 ± 80, p = 0.24). The western diet increased the coronary ACh EC50 at 12w (C: 0.20 ± 0.18, 4w: 0.25 ± 0.15, 8w: 0.50 ± 0.32, 12w: 0.84 ± 0.55 μM; p = 0.046). SA had no effect on C, 4w or 8w, but improved the EC50 38% at 12w (1.37 ± 2.06 vs 0.85 ± 0.77 μM). ED was manifest prior to coronary dysfunction. There is a trend for improved erectile and coronary function in the dysfunctional state with SA, suggesting that eNOS uncoupling is a common contributor to dysfunction in these vascular beds.

Increasing dihydrobiopterin causes dysfunction of endothelial nitric oxide synthase in rats in vivo

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.

Abstract 4062: Nitric Oxide synthase dysregulation in solid tumors: The therapeutic potential of tetrahydrobiopterin

Abstract Nitric Oxide (NO), in terms of cancer therapeutics, can be viewed as a double edge sword. Both anti-NO and NO-based anticancer treatments appear effective in several preclinical models. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2- instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO-), which is produced rapidly by the reaction of O2- with NO, and is a highly reactive species that can modify proteins, lipids and DNA. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7, Fadu, and A431 cells in vitro and Fadu and A431 cells in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is less than 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. Increasing the BH4:BH2 ratio leads to enhanced NO production as evidenced by an increase in cGMP formation. To directly examine the effects of manipulating the BH4:BH2 ratio, MCF7 and MDA cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. Current experiments focus on SP's effects in vivo and elucidating the mechanism of cell death and the signaling pathways involved. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2011-4062

Impairment of nitrergic system and delayed gastric emptying in low density lipoprotein receptor deficient female mice

In the current study, we have investigated whether low density lipoprotein receptor knockout mice (LDLR-KO), moderate oxidative stress model and cholesteremia burden display gastroparesis and if so whether nitrergic system is involved in this setting. In addition, we have investigated if sepiapterin (SEP) supplementation attenuated impaired nitrergic system and delayed gastric emptying. Gastric emptying and nitrergic relaxation were measured in overnight fasting mice. nNOSα dimerization, anti-oxidant markers such as Nrf2, GCLM, GCLC, HO-1, catalase (CAT), and superoxide dismutase (SOD1) were measured using standard methods. Biopterin levels and intestinal transit time were measured using HPLC and dye migration assay, respectively. Wild type (WT) and LDLR-KO were supplemented with SEP. In LDLR null stomachs: (i) significant reduction in rate of gastric emptying, gastric pyloric and fundus nitrergic relaxation and nNOSα dimerization, (ii) elevated oxidized biopterins and reduced ratio of BH(4) /BH(2) + B, (iii) reduced Nrf2 and GCLC protein expression and no change in GCLM, HO-1, CAT, SOD1, and (iv) accelerated small intestinal motility were noticed. Supplementation of SEP restored delayed gastric emptying, impaired pyloric and fundus nitrergic relaxation with restoration of nNOS dimerization and nNOS expression. This novel data suggests that hyperlipidemia and/or suppression of selective antioxidants may be a potential cause of developing gastroparesis in diabetic patients.

Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters: A plausible gateway for the salvage pathway of Tetrahydrobiopterin biosynthesis

Tetrahydrobiopterin (BH 4) is synthesized de novo in particular cells, but in the case of a systemic or local BH 4 deficiency, BH 4 supplementation therapy is applied. BH 4-responsive PKU has also been effectively treated with BH 4 supplementation. However, the rapid clearance of the supplemented BH 4 has prevented the therapy from being widely accepted. Deposition of BH 4 after supplementation involves oxidation of BH 4 to dihydrobiopterin (BH 2) and subsequent conversion to BH 4 by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH 4 across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH 2 or BH 4 using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP > BH 2 > BH 4. hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH 4 via uptake that is likely mediated by ENT2 (SP > BH 2 > BH 4 ). When exogenous BH 2 was given to mice, it was efficiently converted to BH 4 and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH 4 deposition after BH 2 administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH 2 was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH 2, and BH 4 across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH 4 biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH 2 uptake for BH 4 accumulation and the release for modifying the intracellular BH 2/BH 4 ratio.

Sepiapterin reverses the changes in gastric nNOS dimerization and function in diabetic gastroparesis

We have demonstrated previously that in vivo supplementation of tetrahydrobiopterin (BH₄); a co-factor for neuronal nitric oxide synthase (nNOS) significantly restored delayed gastric emptying and attenuated nitrergic relaxation in diabetic rat. In this study, we have investigated whether supplementation of sepiapterin (SEP), a precursor for BH₄ biosynthesis via salvage pathway restores gastric emptying and nitrergic system in female diabetic rats. Diabetic rats (streptozotocin-induced) were supplemented with BH₄ or SEP (20 mg kg⁻¹ body weight). Gastric nitrergic relaxation in the presence or absence of high glucose and SEP were measured by electric field stimulation. Gastric muscular strips from healthy or diabetic female rats were incubated in the presence or absence of high glucose, SEP and/or methotrexate (MTX). Nitric oxide release was measured colorimetrically by NO assay kit. The expression of nNOSα and dimerization was detected by Western blot. In vitro studies on gastric muscular tissues showed that MTX, an inhibitor of BH₄ synthesis via salvage pathway, significantly decreased NO release. In vivo treatment with MTX reduced both gastric nitrergic relaxation and nNOSα dimerization. Supplementation of SEP significantly attenuated delayed gastric emptying in diabetic rats. In addition, SEP supplementation restored impaired nitrergic relaxation, gastric nNOSα protein expression, and dimerization in diabetic rats. The above data suggests that supplementation of SEP accelerated gastric emptying and attenuated reduced gastric nNOSα expression, and dimerization. Therefore, SEP supplementation is a potential therapeutic option for female patients of diabetic gastroparesis.

Abstract 1324: Modulation of IR induced NFKB activity by manipulation of NOS coupling

Abstract The transcriptional activity of NFκβ is in part regulated by nitric oxide (NO) and reactive nitrogen species (RNS). Previously we demonstrated that low doses of ionizing radiation (IR) activate constitutive, Ca2+ dependent NO Synthases (NOS) and the generation of RNS, resulting in protein Tyr nitration and/or Cys s-nitrosylation. Previous studies in our lab demonstrated that nitration of IκBα is critical for the IR-induced activation of NFκβ whereas others have shown that s-nitrosylation of the p65 subunit leads to the inhibition of NFκβ. Here we show that exposing MCF-7 cells to 5Gy IR results not only in Tyr nitration of IκBα but also Cys s-nitrosylation of NFκβ both with a maximum at 30min. post IR. One explanation for the discordant results is that NOS activity is uncoupled, resulting in the production of increased levels of superoxide anion as opposed to NO. Tetrahydrobiopterin (BH4) is a necessary cofactor that stabilizes the enzyme and allows the generation of NO through oxidation of L-arginine. However, if BH4 levels become limiting as found in chronic inflammatory or oxidative conditions, electron transfer is instead shuttled to molecular oxygen resulting in the production of superoxide anion, a scenario in which NOS is described as “uncoupled.” When NOS is uncoupled the resulting increase in superoxide can lead to enhanced levels of peroxynitrite formation, resulting in enhanced IκBα Tyr nitration and NFκβ activity. This evidence has led to the hypothesis that by altering cellular levels of BH4, the levels of Tyr nitration and Cys s-nitrosylation can be manipulated, leading to changes in NFκβ activity. To determine the effects of exogenous BH4 on IκBα nitration in MCF-7 cells, cells were treated with varying concentrations of cell permeable sepiapterin (SP), a precursor of BH4, and exposed to 5Gy IR. The results show that between 100nM-500nM IκBα nitration is all but completely eliminated, resulting in decreased NFκβ activity as assessed by luciferase assay. Initial experiments looking at the effects of SP on p65 s-nitrosylation have been variable. At a minimum, levels of s-nitrosylation remain the same as untreated cells; however, enhancement of s-nitrosylation is seen in some experiments. Studies are ongoing to clarify SP's effects on p65 s-nitrosylation as well as studies to determine SP's effects on tumor cell survival post IR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1324.