Thymidine Levels Research Articles

Serum metabolic profiles in quails: Effects of age, breed, and sex

Over the past decade, metabolomic research in livestock and poultry has gained considerable momentum; however, quail metabolomics still lags behind that of livestock species such as chickens, pigs, and cattle. Quails are important models due to their low-cost protein sources—both eggs and meat—and practical benefits such as minimal space requirements, high egg production, disease resistance, and rapid reproduction. Therefore, it is necessary to systematically understand the effects of various factors on quail metabolism to provide a theoretical basis for accurate feeding and breeding practices. In this study, liquid chromatography with tandem mass spectroscopy (LC-MS/MS)-based metabolomics was used to examine the effects of age, breed, and sex on the serum metabolic profile of quails. A total of 550 metabolites were identified. Relative to breed and sex, we found that age played a crucial role in influencing quail serum metabolites. At 20 days of age (D20), quails had high levels of serum thymidine and alpha-D-glucose, while at 70 days of age (D70), the lipids, including 3-isothujone, 15-deoxy-d-12,14-PGJ2, and 2-aminobut-2-enoate dominated the serum. Additionally, xanthine, hypoxanthine, diaminopimelic acid, and 2-deoxy-scyllo-inosose appeared to be specific metabolites of Japanese quail (JAPQ). Serum levels of N-acetylglutamic acid, hydroxypyruvic acid, carnosine, alloepipregnanolone, lumichrome, 6-hydroxynicotinate, and myristic acid were higher in D70 Hengyan white feather quails (HYWQ) than those in D70 JAPQ. Notably, this study also identified 2-hydroxy-2-ethylsuccinic acid and riboflavin as potential specific metabolites in female quails. Furthermore, integration analysis showed that amino acid biosynthesis and metabolism, as well as ABC transporters, were the key pathways distinguishing D20 from D70. Purine metabolism, pyrimidine metabolism, ABC transporters, and TCA cycle were the key pathways distinguishing HYWQ from JAPQ. Differences in energy metabolism and amino acid biosynthesis and metabolism were observed between males and females. These findings enhance our understanding of the dynamic changes in quail serum metabolites influenced by various factors and address the knowledge gap regarding serum metabolic changes at different stages in quails.

Depletion of plasma thymidine results in growth retardation and mitochondrial myopathy in mice overexpressing human thymidine phosphorylase

Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human thymidine phosphorylase to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by 4weeks of age with a decrease in plasma thymidine level compared with the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when thymidine phosphorylase inhibitor administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice.

A new mutation in the TYMP-gene: clinical and morphological characteristics of a patient with MNGIE syndrome

Mitochondrial neurogastrointestinal encephalomyopathy is an extremely rare (1–9:1 000 000, Orphanet, 2021) multisystem genetic disease caused by mutations in the TYMP gene encoding the enzyme thymidine phosphorylase.The article presents the data of a thirteen‑year survey on 40‑year‑old patient D. with clinical manifestations of mitochondrial neurogastrointestinal encephalomyopathy syndrome associated with the previously undescribed missense mutation c.1301G>T (p.Gly434Val) of the TYMP gene. Detailed clinical picture (gastrointestinal dysfunction, cachexia, blepharoptosis, ophthalmoparesis, peripheral polyneuropathy and leukoaraiosis), electroneuromyography data (demyelination with secondary axonopathy), high blood serum level of dihydrothymine together with normal levels of thymidine and deoxyuridine made it possible to verify the diagnosis. Histopathological examination revealed atrophy of the longitudinal (outer) muscle layer of the small and large intestines and a significant decrease in the number of CD117+ cells (telocytes), signs of damage to the striated skeletal muscles of a mixed nature with a predominance of the myogenic pattern, as well the destruction of the myelin sheaths of peripheral nerves. Histochemical examination did not reveal “ragged red fibers” characteristic of mitochondrial pathology. Transmission electron microscopy demonstrated the presence of megalomitochondria in the myocardium.

Regulator Of G Protein Signaling 14 Disruption Affects The Gut Microbiota And Metabolome In Mice

The gut microbiota is linked to brown adipose tissue (BAT), but the mechanisms and microbes facilitating BAT production or function are unknown. A novel mouse model containing a gene knock-out of regulator of G protein signaling 14 (RGS14 KO) has increased BAT. Our early studies found distinct microbes in RGS14 KO mice and BAT-specific metabolites. PURPOSE: To identify key gut microbial species and uncover BAT-specific metabolites in RGS14 KO mice. Further, we aim to identify and associate metabolites produced in specific tissue samples with these key gut microbes. METHODS: Twenty-two mice (N = 13 RGS14 KO, N = 9 Wild type (WT)) were used to identify predominant microbes and metabolites. Gut microbiota profiles were obtained by sequencing bacterial ribosomal operons. Metabolomics used ultra-high performance liquid chromatography to evaluate polar and nonpolar untargeted metabolites in fecal, cecal, brain, and BAT samples. Microbiome analysis used Kulczynski distance to compare WT to RGS14 KO reads in two-dimensional non-metric multidimensional scaling (NMDS) plots. Two-tailed t-tests were used to compare WT and RGS14 KO metabolite means (p < 0.05). MetaboAnalyst 5.0 was used to identify significant metabolite pathways in tissue samples and generate pathway plots. RESULTS: Approximately 500 k rRNA reads post QA/QC ((84% identify; >1000 bp alignment) were obtained from all samples by MegaBlast. NMDS plots showed significant bacterial community differences (Genus: p = 0.035; species: p = 0.028; strain: p = 0.037) between WT and RGS14 KO mice. Specifically, RGS14 KO mice housed two unique strains of Akkermansia muciniphilia (A. muciniphilia BIOML-A22 and A. muciniphilia AN78) while WT animals contained A. muciniphilia EB-AMDK-1. Untargeted metabolomics identified 82 significantly different (p < 0.05) unique metabolites were between RGS14 KO and WT mice in one of the four samples. Specifically, RGS14 KO animals had significantly higher levels of G6P, glycyl-l-proline, glyercophosphocholine, isoleucine, pipecolic acid, thymidine, UDP-D-glucose, malate, leucic acid, NADP+, and guanosine in BAT compared to WT animals. CONCLUSION: The gut microbiome differs between RGS14 KO and WT mice from genus to strain level. Unique metabolites found in RGS14 KO BAT may be linked to BAT function. Supported by ONR Grant 826640

Mitochondrial neurogastrointestinal encephalomyopathy: Clinical and biochemical impact of allogeneic stem cell transplantation in a Greek patient with one novel TYMP mutation

We describe the case of a Greek female patient with the Classic form of the ultra- rare and fatal autosomal recessive disorder Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and the impact of allogeneic hematopoietic stem cell transplantation on the biochemical and clinical aspects of the disease.The patient presented at the age of 15 years with severe gastrointestinal symptoms, cachexia, peripheral neuropathy and diffuse leukoencephalopathy. The diagnosis of MNGIE disease was established by the increased levels of thymidine and deoxyuridine in plasma and the complete deficiency of thymidine phosphorylase activity. The novel c.[978dup] (p.Ala327Argfs*?) variant and the previously described variant c.[417 + 1G > A] were identified in TYMP. The donor for the allogeneic hematopoietic stem cell transplantation was her fully compatible sister, a carrier of the disease. The patient had a completely uneventful post- transplant period and satisfactory PB chimerism levels. A marked and rapid decrease in thymidine and deoxyuridine plasma levels and an increase of the thymidine phosphorylase activity to the levels measured in her donor sister was observed and is still present sixteen months post-transplant. Disease symptoms stabilized and some improvement was also observed both in her neurological and gastrointestinal symptoms. Follow up studies will be essential for determining the long term impact of allogeneic hematopoietic stem cell transplantation in our patient.

Right Ventricle Remodeling Metabolic Signature in Experimental Pulmonary Hypertension Models of Chronic Hypoxia and Monocrotaline Exposure.

Introduction: Over time and despite optimal medical management of patients with pulmonary hypertension (PH), the right ventricle (RV) function deteriorates from an adaptive to maladaptive phenotype, leading to RV failure (RVF). Although RV function is well recognized as a prognostic factor of PH, no predictive factor of RVF episodes has been elucidated so far. We hypothesized that determining RV metabolic alterations could help to understand the mechanism link to the deterioration of RV function as well as help to identify new biomarkers of RV failure. Methods: In the current study, we aimed to characterize the metabolic reprogramming associated with the RV remodeling phenotype during experimental PH induced by chronic-hypoxia-(CH) exposure or monocrotaline-(MCT) exposure in rats. Three weeks after PH initiation, we hemodynamically characterized PH (echocardiography and RV catheterization), and then we used an untargeted metabolomics approach based on liquid chromatography coupled to high-resolution mass spectrometry to analyze RV and LV tissues in addition to plasma samples from MCT-PH and CH-PH rat models. Results: CH exposure induced adaptive RV phenotype as opposed to MCT exposure which induced maladaptive RV phenotype. We found that predominant alterations of arginine, pyrimidine, purine, and tryptophan metabolic pathways were detected on the heart (LV+RV) and plasma samples regardless of the PH model. Acetylspermidine, putrescine, guanidinoacetate RV biopsy levels, and cytosine, deoxycytidine, deoxyuridine, and plasmatic thymidine levels were correlated to RV function in the CH-PH model. It was less likely correlated in the MCT model. These pathways are well described to regulate cell proliferation, cell hypertrophy, and cardioprotection. These findings open novel research perspectives to find biomarkers for early detection of RV failure in PH.

High-Dose Thymidine Infusions in Patients With Leukemia and Lymphoma

We have recently explored the cytokinetic effects of thymidine given by continuous infusion in an animal model and have demonstrated an arrest of cell cycle traverse in rapidly proliferating tissues. Similar biologic effects have been observed in clinical trials upon infusing thymidine at a dose rate of 75 g/sq m/day. Fifteen courses of at least 5 days in duration have been administered to 11 patients with either leukemia or lymphoma. Steady-state serum thymidine levels were achieved in the range of 1-2 mM and the serum half-life of thymidine was approximately 90 min upon completion of the infusion. The associated toxicity included myelosuppression, headache, anorexia, nausea, vomiting, and diarrhea. The antitumor effect was dependent on the disease being treated and the percentage of blasts in the peripheral blood. Three patients with AML and three patients with T-cell leukemia responded to the thymidine infusions with an abrupt decrease in peripheral blast count. In contrast, no response was observed in two patients with poorly differentiated lymphocytic lymphoma of B-cell origin in a leukemic phase. One patient with T-cell leukemia had a marked clinical response with resolution of hepatosplenomegaly and another patient with the same diagnosis had partial clearing of the bone marrow. The cytokinetic effect on the bone marrow or peripheral blasts was monitored by microfluorimetry and labeling with precursors to monitor DNA synthesis. These data indicate an arrest of DNA synthesis during the period of thymidine infusion. The clinical effects observed with the high-dose thymidine infusions are correlated with measurements of thymidine kinase and phosphorylase levels detected in the blasts obtained from the leukemic patients. The relative ratios of thymidine kinase to phosphorylase are predictive of cellular sensitivity to the cytokinetic effects of thymidine.

Successful liver transplantation in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal disorder characterized by progressive gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, skeletal myopathy, ophthalmoparesis, and ptosis. MNGIE stems from deficient thymidine phosphorylase activity (TP) leading to toxic elevations of plasma thymidine. Hematopoietic stem cell transplant (HSCT) restores TP activity and halts disease progression but has high transplant-related morbidity and mortality. Liver transplant (LT) was reported to restore TP activity in two adult MNGIE patients. We report successful LT in four additional MNGIE patients, including a pediatric patient.Our patients were diagnosed between ages 14 months and 36 years with elevated thymidine levels and biallelic pathogenic variants in TYMP. Two patients presented with progressive gastrointestinal dysmotility, and three demonstrated progressive peripheral neuropathy with two suffering limitations in ambulation. Two patients, including the child, had liver dysfunction and cirrhosis.Following LT, thymidine levels nearly normalized in all four patients and remained low for the duration of follow-up. Disease symptoms stabilized in all patients, with some manifesting improvements, including intestinal function. No patient died, and LT appeared to have a more favorable safety profile than HSCT, especially when liver disease is present. Follow-up studies will need to document the long-term impact of this new approach on disease outcome.Take Home Message: Liver transplantation is effective in stabilizing symptoms and nearly normalizing thymidine levels in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and may have an improved safety profile over hematopoietic stem cell transplant.

Metabolomic profiling to evaluate the efficacy of proxalutamide, a novel androgen receptor antagonist, in prostate cancer cells

Proxalutamide is a newly developed androgen receptor (AR) antagonist for the treatment of castration-resistant prostate cancer (PCa) that has entered phase III clinical trials. In the present study, we intended to elucidate the antitumor efficacy of proxalutamide through the metabolomic profiling of PCa cells. Two AR-positive PCa cell lines and two AR-negative PCa cell lines were investigated. Cell viability assays based on ATP quantitation were conducted. LC-Q/TOF-MS was used to analyze intracellular metabolites before or after the administration of proxalutamide and two other clinical AR antagonists (bicalutamide and enzalutamide). The results of this study showed that the inhibitory effect of proxalutamide on PCa cell proliferation was better than that of bicalutamide and enzalutamide, and proxalutamide preferentially affected AR-positive PCa cells over AR-negative cells. The metabolic composition of PCa cells changed significantly after proxalutamide administration, and these changes in response to proxalutamide were significantly different from those in the presence of the two other AR antagonists. In AR-positive cells, proxalutamide significantly decreased the intracellular levels of glutamine, glutamate, glutathione, cysteine, glycine, aspartate, uridine, cytidine and thymidine. However, the effects of the two other antagonists on these discriminant metabolites were ambiguous, and no changes in these metabolites were found in AR-negative cells. Our findings indicate that proxalutamide has inhibitory effects on glutamine metabolism, redox homeostasis and de novo pyrimidine synthesis in AR-positive PCa cells that enhance the cellular sensitivity to proxalutamide.

Mitochondrial neurogastrointestinal encephalopathy: a clinicopathological mimic of Crohn\u2019s disease

BackgroundMitochondrial neurogastrointestinal encephalopathy (MNGIE), due to mutations in TYMP, often presents with gastrointestinal symptoms. Two sisters, initially managed for Crohn’s disease based upon clinical, imaging and pathological findings, were later found to have MNGIE. The cases provide novel clinicopathological insight, for two further reasons: both sisters remain ambulant and in employment in their late 20s and 30s; diagnosis in one sister was made after a suspected azathioprine-precipitated acute illness.Case presentationA 25-year-old female presented with diarrhoea, vomiting, abdominal pain, and bloating. Faecal calprotectin, colonic biopsies and magnetic resonance enterography were consistent with a diagnosis of Crohn’s disease. Azathioprine initiation preceded admission with a sore throat, headache, myalgia, and pyrexia. Withdrawal led to rapid clinical improvement. MRI brain revealed persistent, extensive white matter changes. Elevated plasma and urine thymidine and deoxyuridine, and genetic testing for TYMP variants, confirmed MNGIE. Testing of the patient’s sister, also diagnosed with Crohn’s disease, revealed identical variants. In this context, retrospective review of colonic biopsies identified histological findings suggestive of MNGIE.ConclusionsAzathioprine interference in nucleic acid metabolism may interact with the mitochondrial DNA depletion of MNGIE. Nucleotide supplementation, proposed for treatment by manipulating mitochondrial nucleoside pools, may require caution. The late onset and mild phenotype observed confirms presentation can occur later in life, and may reflect residual thymidine phosphorylase activity. Clinicians should consider measuring plasma thymidine levels in suspected Crohn’s disease to rule out MNGIE, particularly if white matter abnormalities are identified on neuroimaging.

Metabolic profiling study on potential toxicity in male mice treated with Dechlorane 602 using UHPLC-ESI-IT-TOF-MS

Dechlorane 602 (Dec 602), a chlorinated flame retardant, has been widely detected in different environmental matrices and biota. However, toxicity data for Dec 602 seldom have been reported. A metabolomics study based on ultra-high performance liquid chromatography coupled with ion trap time-of-flight mass spectrometry was employed to study the urine and sera metabolic profiles of mice administered with Dec 602 (0, 0.001, 0.1, and 10 mg/kg body weight per day) for 7 days. A significant difference in metabolic profiling was observed between the Dec 602 treated group and the control group by multivariate analysis, which directly reflected the metabolic perturbations caused by Dec 602. The metabolomics analyses of urine from Dec 602-exposed animals exhibited an increase in the levels of thymidine and tryptophan as well as a decrease in the levels of tyrosine, 12,13-dihydroxy-9Z-octadecenoic acid, 2-hydroxyhexadecanoic acid and cuminaldehyde. The metabolomics analyses of sera showed a decrease in the levels of kynurenic acid, daidzein, adenosine, xanthurenic acid and hypoxanthine from Dec 602-exposed animals. These findings indicated Dec 602 induced disturbance in phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, tyrosine metabolism, pyrimidine metabolism, purine metabolism, ubiquinone and other terpenoid-quinone biosynthesis; phenylalanine metabolism and aminoacyl-tRNA biosynthesis. Significant alterations of immune and neurotransmitter-related metabolites (tyrosine, tryptophan, kynurenic acid, and xanthurenic acid) suggest that the toxic effects of Dec 602 may contribute to its interactions with the immune and neuronal systems. This study demonstrated that the UHPLC-ESI-IT-TOF-MS-based metabolomic approach can obtain more specific insights into the potential toxic effects of Dec 602 at molecular level.

Drawbacks of the use of cotrimoxazole in foreign-body infections

IntroductionThe anti-staphylococcal efficacy of cotrimoxazole in the setting of difficult-to-treat infections seems to be compromised by large amounts of pus and devitalized tissue, and, therefore, high levels of thymidine. Our objective was to evaluate the activity of cotrimoxazole against a staphylococcal foreign-body infection experimental model, which also yields significant quantities of thymidine. Material and methodsWe used a rat tissue-cage model of infection (with high inherent thymidine levels) caused by a strain of methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 29213). MIC values were determined (microdilution method) and compared in the presence or absence of tissue-cage fluid samples. ResultsThe inefficacy of cotrimoxazole was found to be similar to that of the control group. The MIC of cotrimoxazole was 4•8 fold higher in the presence of rat tissue-cage fluid. ConclusionsThe inefficacy of cotrimoxazole in our foreign-body infection model by MSSA, and the probable negative impact of the presence of thymidine on its efficacy, challenge the use of this drug in acute phases of foreign-body infections. It should be reserved as an alternative treatment when the infection is more controlled.

3'-Deoxy-3'-[18F]Fluorothymidine Uptake Is Related to Thymidine Phosphorylase Expression in Various Experimental Tumor Models.

We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) uptake in four untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models. Lysates from n=15 different tumor models originating from n=6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to [18F]FLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations. Expression of TP correlated positively with [18F]FLT SUVmax (ρ=0.549, P<0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (ρ=- 0.939, P<0.001). In a broad range of tumors, [18F]FLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding [18F]FLT uptake.

Drawbacks of the use of cotrimoxazole in foreign-body infections

IntroductionThe anti-staphylococcal efficacy of cotrimoxazole in the setting of difficult-to-treat infections seems to be compromised by large amounts of pus and devitalized tissue, and, therefore, high levels of thymidine. Our objective was to evaluate the activity of cotrimoxazole against a staphylococcal foreign-body infection experimental model, which also yields significant quantities of thymidine. Material and methodsWe used a rat tissue-cage model of infection (with high inherent thymidine levels) caused by a strain of methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 29213). MIC values were determined (microdilution method) and compared in the presence or absence of tissue-cage fluid samples. ResultsThe inefficacy of cotrimoxazole was found to be similar to that of the control group. The MIC of cotrimoxazole was 4–8 fold higher in the presence of rat tissue-cage fluid. ConclusionsThe inefficacy of cotrimoxazole in our foreign-body infection model by MSSA, and the probable negative impact of the presence of thymidine on its efficacy, challenge the use of this drug in acute phases of foreign-body infections. It should be reserved as an alternative treatment when the infection is more controlled.

Abstract B16: Activation of NRF2 and adaptive resistance to chemotherapy

Abstract Nuclear factor-erythroid-2-related factor 2 (NRF2), a member of the cap ‘n’ collar family of bZIP transcription factors, confers protection against oxidative and electrophilic stress. NRF2 is of great interest in cancer research, due to its role in response to chemotherapy, including the class of drugs targeting thymidylate synthase (TYMS). It has long been known that inhibition of TYMS leads to depletion of thymidine levels and the onset of programmed cell death, deriving from the enzyme's function as the sole de novo source of thymidine for DNA replication and repair. Exposing cells to TYMS inhibitors such as fluoropyrimidine antimetabolites (5-fluorouracil, or FUra; 5'-fluoro-2'-deoxyuridine, or FdUrd), as well as anti-folate analogs (raltitrexed, or RTX), induce intracellular concentrations of reactive oxygen species, which are a primary cause of drug-mediated toxicity. This prompted our focus on assessing the impact of NRF2 on cellular response to TYMS inhibitors. Using human colon tumor-derived cell line HCT116, we have shown by gene expression profiling that drug exposure induces expression of a number of genes that are modulated by NRF2. Quantitative PCR assays of several colon tumor cell lines verified that FUra, FdUrd, and RTX induce transcription of several genes known to be NRF2-targets, including AKR1B10, ALDH3A1, HSPB8, HMOX1, and SERPINE1, among others. Such induction mirrors that in response to the known NRF2 activator tert-butylhydroquinone (tBHQ). NRF2 protein concentration and nuclear localization are increased by FdUrd treatment, though not to the same extent as with tBHQ. Reporter gene constructs were used to show that both FdUrd and tBHQ induce transcription mediated by the NRF2-binding antioxidant response element (ARE). Furthermore, chromatin-immunoprecipitation experiments revealed that TYMS inhibitors promote occupancy of the ARE regions of several genes by NRF2; again, tBHQ had a much greater effect. Further analysis showed an association between gene induction by TYMS inhibitors and patterns of histone acetylation within the NRF2-binding ARE regions. Finally, we observed that increases in the apoptotic index following exposure to TYMS inhibitors were greater in cells in which the transcription factor was subjected to siRNA-mediated “knockdown” or CRISPR/Cas9-mediated “knockout”, indicating that reduced NRF2 expression sensitizes cells to TYMS inhibitors. Overall, we conclude that TYMS inhibitors activate NRF2 and its downstream target genes, thereby constraining drug response. Reducing such activation of NRF2 or its consequences may be an effective strategy to sensitizing tumor cells to chemotherapy. Citation Format: Sarah A. Clinton, Karen W. Barbour, Franklin G. Berger. Activation of NRF2 and adaptive resistance to chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B16.

Chemomodulatory effect Melastoma Malabathricum Linn against chemically induced renal carcinogenesis rats via attenuation of inflammation, oxidative stress, and early markers of tumor expansion.

Melastoma malabathricum Linn (MM) has high valued for its commercial significance. Indian market (northeast) has great demand for the plants, which extended, its use as a traditional home remedy due to its anti-inflammatory effects. In this study, we scrutinize the therapeutic and protective effect of MM against diethylnitrosamine (DEN) and ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis, renal hyperproliferation, and oxidative stress in rats. Liquid chromatography mass spectroscopy (LC-MS) was used for identification of phytoconstituents. Administration of DEN confirmed the initiation the renal carcinogenesis via enhancing the expansion of tumor incidence. Intraperitoneally, administration of Fe-NTA boost the antioxidant enzymes (phase I), viz., superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and phase II, viz., quinone reductase (QR) and glutathione-S-transferase (GST). It also increased the content of renal lipid peroxidation (LPO), hydrogen peroxidase (H2O2) with decrease content in glutathione content (GSH). It also increased the renal biochemical and non-biochemical parameter. It also confirmed the augment the level of thymidine [3H] incorporation into renal DNA, ornithine decarboxylase (ODC) activity and increased the generation of proinflammatory (TNF-α, IL-6 and IL-β) and inflammatory mediator (PGE2). We also analyzed the macroscopic and histologic of renal tissue. In addition, the effect of phytoconstituent of MM extract was evaluated in silico and free radical scavenging activity against the DPPH and ABTS free radicals. LC-MS confirmed the presence of quercetin >gallic acid in MM extract. Renal carcinogenesis rats treated with MM (100, 250, and 500mg/kg) confirmed the significantly (P<0.001) protective effect via reduction the antioxidant (phase I and phase II) enzymes, biochemical parameter and restore the proinflammatory and inflammatory mediator at dose dependent manner. MM altered the ODC and thymidine activity in renal DNA. The chemoprotective effect of MM was confirmed via decreased the renal tumor incidence, which was confirmed by the macroscopic and histopathological observation. Consequently, our result suggests that MM is a potent chemoprotective agent and suppresses DEN+ Fe-NTA-induced renal carcinogenesis, inflammatory reaction, and oxidative stress injury in Wister rats.

Abstract 493: NRF2 modulates sensitivity to thymidylate synthase inhibitors in colon cancer cells

Abstract Thymidylate synthase (TYMS) catalyzes the reductive methylation of dUMP, and is the sole de novo source of thymidine for DNA replication and repair. As such, it is an important target of chemotherapeutic drugs, particularly the fluoropyrimidines 5-fluorouracil (FUra) and 5-fluoro-2’-deoxyuridine (FdUrd), as well as the folate analog raltitrexed (RTX). In cells, these drugs are metabolized to derivatives that bind to and inhibit TYMS, leading to depletion of thymidine levels, dysregulation of redox metabolism, generation of oxidative stress, and, eventually, apoptotic cell death. Gene expression profiles of FUra-treated HCT116 cells (a human colon cancer cell line) showed that a number of genes involved in redox metabolism are up-regulated following drug exposure. Several of these were recognized as known targets of nuclear factor-erythroid-2-related factor 2 (NRF2), a member of the cap ‘n’ collar family of bZIP transcription factors that confers protection against oxidative and electrophilic stress. Quantitative PCR assays verified that FUra, FdUrd, and RTX do indeed induce several NRF2-regulated genes, including AKR1B10, ALDH3A1, NQO1, and SERPINE1, among others. Overproduction of NRF2 via DNA transfection increased expression of some, but not all, of these genes, a phenomenon that was reversed by Kelch-like ECH-associated protein-1 (KEAP1), a known negative regulator of NRF2. The impact of TYMS inhibitors on expression of NRF2 target genes was maintained in the presence of exogenous thymidine and the antioxidant N-acetylcysteine, even though these agents completely ameliorated drug-induced apoptosis. This suggests that the response of NRF2 target genes to TYMS-directed drugs occurs independently of TYMS inhibition and subsequent oxidative stress. Utilizing siRNA technology, we examined the impact of NRF2 knockdown on drug-induced apoptosis, and found that increases in the apoptotic index following exposure to TYMS inhibitors were greater in NRF2-knockdown cells as compared to control cells. This was also observed in TYMS-overproducing cells that are profoundly drug resistant, and indicates that reduced NRF2 expression sensitizes cells to these TYMS inhibitors. Overall, we conclude that the induction of NRF2 target genes is a constraining factor in the cytotoxic response to TYMS inhibitors, and that inhibition of the transcription factor may be an effective strategy to enhance sensitivity to these agents. Citation Format: Sarah A. Clinton, Karen W. Barbour, Ufuk Ozer, Franklin G. Berger. NRF2 modulates sensitivity to thymidylate synthase inhibitors in colon cancer cells. [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 493. doi:10.1158/1538-7445.AM2014-493

Variability of Proliferation and Diffusion in Different Lung Cancer Models as Measured by 3'-Deoxy-3'-¹⁸F-Fluorothymidine PET and Diffusion-Weighted MR Imaging.

Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts. Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by (18)F-FDG and (18)F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2'-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography-mass spectrometry. Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. (18)F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in (18)F-FDG uptake were only marginal. No clear relationship between (18)F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence (18)F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death. A direct relationship of (18)F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings.

Thymidine phosphorylase influences [18F]fluorothymidine uptake in cancer cells and patients with non-small cell lung cancer

Thymidine phosphorylase (TP), a key enzyme in the pyrimidine nucleoside salvage pathway, catalyses the reversible phosphorylation of thymidine, thereby generating thymine and 2-deoxy-D-ribose-1-phosphate. By regulating the levels of endogenous thymidine, TP may influence [(18)F]fluorothymidine ([(18)F]FLT) uptake. We investigated the effect of TP activity on [(18)F]FLT uptake by tumours. Uptake of [(3)H]FLT and [(3)H]thymidine ([(3)H]Thd) and the activities of TP, thymidine kinase 1 (TK1), and equilibrative nucleoside transporter 1 (ENT1) were determined in exponentially growing A431, A549, HT29, HOP92, ACHN, and SKOV3 cells in the presence or absence of tipiracil hydrochloride, a TP inhibitor. Eighty-five non-small cell lung cancer tissues from a patient cohort that was previously studied with [(18)F]FLT positron emission tomography (PET) were retrieved and subjected to immunohistochemical analysis of TP expression. Factors that affected the maximum standardised uptake value (SUVmax) of [(18)F]FLT-PET were identified by multiple linear regression analysis. A431 cells had the highest TP activity; A549 and HT29 cells had moderate TP activity; and ACHN, SKOV3, and HOP92 cells had little detectable TP activity. Cell lines with high TP activity took up more [(3)H]FLT than [(3)H]Thd, whereas cells with little TP activity took up more [(3)H]Thd than [(3)H]FLT. In cells with high TP activity, TP inhibition decreased [(3)H]FLT uptake and increased [(3)H]Thd uptake. However, TP inhibition had no effect on ACHN, SKOV3, and HOP92 cells. TP inhibition did not change TK1 or ENT1 activity, but did increase the intracellular level of thymidine. The SUVmax of [(18)F]FLT was affected by three independent factors: Ki-67 expression (P < 0.001), immunohistochemical TP score (P < 0.001), and tumour size (P = 0.015). TP activity influences [(18)F]FLT uptake, and may explain preferential uptake of [(18)F]FLT over [(3)H]Thd. These results provide important insights into the biology of [(18)F]FLT as a proliferation marker.

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.