Distinctive metabolic remodeling in TYMP deficiency beyond mitochondrial dysfunction.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). As a cytosolic metabolic enzyme, TP defects affect biological processes that are thought to not be limited to the abnormal replication of mitochondrial DNA. This study aimed to elucidate the characteristic metabolic alterations and associated homeostatic regulation caused by TYMP deficiency. The pathogenicity of novel TYMP variants was evaluated in terms of clinical features, genetic analysis, and structural instability. We analyzed plasma samples from three patients with MNGIE; three patients with m.3243A > G mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); and four healthy controls (HC) using both targeted and untargeted metabolomics techniques. Transcriptomics analysis and bioenergetic studies were performed on skin fibroblasts from participants in these three groups. A TYMP overexpression experiment was conducted to rescue the observed changes. Compared with controls, specific alterations in nucleosides, bile acids, and steroid metabolites were identified in the plasma of MNGIE patients. Comparable mitochondrial dysfunction was present in fibroblasts from patients with TYMP deficiency and in those from patients with the m.3243A > G mutation. Distinctively decreased sterol regulatory element binding protein (SREBP) regulated cholesterol metabolism and fatty acid (FA) biosynthesis as well as reduced FA degradation were revealed in fibroblasts with TYMP deficiency. The restoration of thymidine phosphorylase activity rescued the observed changes in MNGIE fibroblasts. Our findings indicated that more widespread metabolic disturbance may be caused by TYMP deficiency in addition to mitochondrial dysfunction, which expands our knowledge of the biochemical outcome of TYMP deficiency. KEY MESSAGES: Distinct metabolic profiles in patients with TYMP deficiency compared to those with m.3243A > G mutation. TYMP deficiency leads to a global disruption of nucleoside metabolism. Cholesterol and fatty acid metabolism are inhibited in individuals with MNGIE. TYMP is functionally related to SREBP-regulated pathways. Potential metabolite biomarkers that could be valuable clinical tools to improve the diagnosis of MNGIE.

A Novel Mutation in an MNGIE Patient Presenting with More Prominent Neurological Symptoms than GI Symptoms.

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is an autosomal recessive disease associated with the mutation of the TYMP gene. MNGIE causes gastrointestinal and neurological symptoms, and the gastrointestinal symptoms are usually notable, which may lead to misdiagnosis. However, we herein report a 29-year-old female who presented with prominent neurological symptoms, while her gastrointestinal symptoms were mild. Brain MRI revealed prominent diffused leukoencephalopathy and peripheral neuropathy was confirmed by the nerve conduction velocity test. Biochemical tests showed elevated plasma thymidine, deoxyuridine, and lactate levels. Molecular genetic testing demonstrated a novel homozygous TYMP c. 447 dupG mutation and the patient's mother was heterozygous for the mutation but had no clinical features. MNGIE was diagnosed based on the results. Unlike other patients who had notable gastrointestinal symptoms, this patient presented with more prominent neurological symptoms than gastrointestinal symptoms, which might have been caused by the novel mutation in the TYMP gene.

A very early onset MNGIE-like syndrome with POLG1 mutation and accompanying leukoencephalopathy.

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a well-known mitochondrial depletion syndrome. Since Van Goethem et al. described MNGIE syndrome with pathogenic POLG1 mutations in 2003, POLG1 gene became a target for MNGIE patients. Cases with POLG1 mutations strikingly differ from classic MNGIE patients due to a lack of leukoencephalopathy. Here we present a female patient with very early onset disease and leukoencephalopathy compatible with classic MNGIE disease who turned out to have homozygous POLG1 mutation compatible with MNGIE-like syndrome, mitochondrial depletion syndrome type 4b.

Thymidine phosphorylase facilitates retinoic acid inducible gene-I induced endothelial dysfunction.

Activation of nucleic acid sensors in endothelial cells (ECs) has been shown to drive inflammation across pathologies including cancer, atherosclerosis and obesity. We previously showed that enhancing cytosolic DNA sensing by inhibiting three prime exonuclease 1 (TREX1) in ECs led to EC dysfunction and impaired angiogenesis. Here we show that activation of a cytosolic RNA sensor, Retinoic acid Induced Gene 1 (RIG-I) diminishes EC survival, angiogenesis and triggers tissue specific gene expression programs. We discovered a RIG-I dependent 7 gene signature that affects angiogenesis, inflammation and coagulation. Among these, we identified the thymidine phosphorylase TYMP as a key mediator of RIG-I induced EC dysfunction via its regulation of a subset of interferon stimulated genes. Our RIG-I induced gene signature was also conserved in the context of human diseases - in lung cancer vasculature and herpesvirus infection of lung endothelial cells. Pharmacological or genetic inhibition of TYMP rescues RIG-I induced EC death, migration arrest and restores sprouting angiogenesis. Interestingly, using RNAseq we identified a gene expression program that was RIG-I induced but TYMP dependent. Analysis of this dataset indicated that IRF1 and IRF8 dependent transcription is diminished in RIG-I activated cells when TYMP is inhibited. Functional RNAi screen of our TYMP dependent EC genes, we found that a group of 5 genes - Flot1, Ccl5, Vars2, Samd9l and Ube2l6 are critical for endothelial cell death mediated by RIG-I activation. Our observations identify mechanisms by which RIG-I drives EC dysfunction and define pathways that can be pharmacologically targeted to ameliorate RIG-I induced vascular inflammation.

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 4 weeks 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.

Capecitabine induces hand-foot syndrome through elevated thymidine phosphorylase-mediated locoregional toxicity and GSDME-driven pyroptosis that can be relieved by tipiracil.

BACKGROUND: Hand-foot syndrome (HFS) is a serious dose-limiting cutaneous toxicity of capecitabine-containing chemotherapy, leading to a deteriorated quality of life and negative impacts on chemotherapy treatment. The symptoms of HFS have been widely reported, but the precise molecular and cellular mechanisms remain unknown. The metabolic enzyme of capecitabine, thymidine phosphorylase (TP) may be related to HFS. Here, we investigated whether TP contributes to the HFS and the molecular basis of cellular toxicity of capecitabine. METHODS: TP-/- mice were generated to assess the relevance of TP and HFS. Cellular toxicity and signalling mechanisms were assessed by in vitro and in vivo experiments. RESULTS: TP-/- significantly reduced capecitabine-induced HFS, indicating that the activity of TP plays a critical role in the development of HFS. Further investigations into the cellular mechanisms revealed that the cytotoxicity of the active metabolite of capecitabine, 5-DFUR, was attributed to the cleavage of GSDME-mediated pyroptosis. Finally, we demonstrated that capecitabine-induced HFS could be reversed by local application of the TP inhibitor tipiracil. CONCLUSION: Our findings reveal that the presence of elevated TP expression in the palm and sole aggravates local cell cytotoxicity, further explaining the molecular basis underlying 5-DFUR-induced cellular toxicity and providing a promising approach to the therapeutic management of HFS.

Mitochondrial neurogastrointestinal encephalomyopathy in a Pakistani female: a case report.

BACKGROUND: Mitochondrial neurogastrointestinal encephalopathy is a rare multisystem autosomal recessive disease caused by mutations in the TYMP gene, that encodes for thymidine phosphorylase. Mitochondrial neurogastrointestinal encephalopathy is a progressive degenerative disease characterized by a distinctive tetrad of gastrointestinal dysmotility, peripheral neuropathy, ophthalmoplegia with ptosis, and asymptomatic leukoencephalopathy. It provides a diagnostic dilemma to physicians in regions like Pakistan because of a lack of genetic study availability and associated financial constraints of the population. However, with careful examination and a few basic investigations, mitochondrial neurogastrointestinal encephalopathy can be diagnosed by ruling out most of the close differentials. CASE PRESENTATION: We report the case of a 23-year-old Asian female whose chief complaints were epigastric pain, bilious emesis, weight loss for 3 months, and bilateral lower limb weakness for 20 days. All clinical signs and symptoms along with relevant investigations including nerve conduction studies, electromyography, and magnetic resonance imaging of the brain were highly suggestive of mitochondrial neurogastrointestinal encephalopathy syndrome. Because of financial constraints, genetic studies could not be performed. The patient was managed with a multidisciplinary approach involving gastroenterology, physiotherapy, and nutrition departments. Currently, therapeutic options for the disease include allogeneic hematopoietic stem cell transplant and carrier erythrocyte entrapped thymidine phosphorylase; however, these could not be provided to the patient owing to certain limitations. CONCLUSIONS: As misdiagnosis and delayed diagnosis are quite common in this disease, the prime objective of this case report is to increase the basic understanding of this disease, especially its signs and symptoms, and address the limitations regarding the diagnostic investigations and management of patients with mitochondrial neurogastrointestinal encephalopathy.

Heat shock protein 90 inhibitor 17-AAG down-regulates thymidine phosphorylase expression and potentiates the cytotoxic effect of tamoxifen and erlotinib in human lung squamous carcinoma cells.

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, in cancer cells, are related to a poor prognosis in a variety of cancers. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is involved in the stabilization and maturation of many oncogenic proteins. The aim of this study is to elucidate whether Hsp90 inhibitor 17-AAG could enhance tamoxifen- and erlotinib-induced cytotoxicity in nonsmall cell lung cancer (NSCLC) cells via modulating TP expression in two squamous NSCLC cell lines, H520 and H1703. We found that 17-AAG reduced TP expression via inactivating the MKK1/2-ERK1/2-mitogen-activated protein kinase (MAPK) pathway. TP knockdown with siRNA or ERK1/2 MAPK inactivation with the pharmacological inhibitor U0126 could enhance the cytotoxic and growth inhibitory effects of 17-AAG. In contrast, MKK1-CA or MKK2-CA (a constitutively active form of MKK1/2) vector-enforced expression could reduce the cytotoxic and cell growth inhibitory effects of 17-AAG. Furthermore, 17-AAG enhanced the cytotoxic and cell growth inhibitory effects of tamoxifen and erlotinib in NSCLC cells, which were associated with TP expression downregulation and MKK1/2-ERK1/2 signal inactivation. Taken together, Hsp90 inhibition downregulates TP, enhancing the tamoxifen- and erlotinib-induced cytotoxicity in H520 and H1703 cells.

A novel thymidine phosphorylase mutation in a family with Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Molecular docking, dynamic simulation and computational investigations.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) is a rare inherited metabolic disorder mostly caused by mutations in TYMP gene encoding thymidine phosphorylase (TP) protein that affects the mitochondrial nucleotide metabolism. TP, functionally active as a homodimer, is involved in the salvage pathway of pyrimidine nucleosides. MNGIE-like syndrome having an overlapping phenotype of MNGIE was also described and has been associated with mutations in POLG and RRM2B genes. In the present study, we report the molecular investigation of a consanguineous family including two patients with clinical features suggestive of MNGIE syndrome. Bioinformatics analyses were carried out in addition to mtDNA deletion screening and copy number quantification in the blood of the two patients. Whole exome sequencing and Sanger sequencing analyses revealed the segregation in the affected family a novel mutation c.1205T>A (p.L402Q) within the exon 9 of the TYMP gene. In addition, mtDNA analysis revealed the absence of mtDNA deletions and a decrease of the copy number in the blood of the two patients of the studied family. The p.Leu402Gln mutation was located in a conserved amino acid within the α/ß domain of the TP protein and several software supported its pathogenicity. In addition, and based on docking and molecular dynamic simulation analyses, results revealed that L402Q caused a conformational change in TP mutated structure and could therefore alter its flexibility and stability. These changes prevent also the formation of stable homodimer leading to non-functional protein with partial or complete loss of its catalytic activity.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer.

BACKGROUND: Tumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance. METHODS: Stage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma. RESULTS: TYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09-2.56; p < 0.05). CONCLUSIONS: TYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Loss of thymidine phosphorylase activity disrupts adipocyte differentiation and induces insulin-resistant lipoatrophic diabetes.

BACKGROUND: Thymidine phosphorylase (TP), encoded by the TYMP gene, is a cytosolic enzyme essential for the nucleotide salvage pathway. TP catalyzes the phosphorylation of the deoxyribonucleosides, thymidine and 2'-deoxyuridine, to thymine and uracil. Biallelic TYMP variants are responsible for Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE), an autosomal recessive disorder characterized in most patients by gastrointestinal and neurological symptoms, ultimately leading to death. Studies on the impact of TYMP variants in cellular systems with relevance to the organs affected in MNGIE are still scarce and the role of TP in adipose tissue remains unexplored. METHODS: Deep phenotyping was performed in three patients from two families carrying homozygous TYMP variants and presenting with lipoatrophic diabetes. The impact of the loss of TP expression was evaluated using a CRISPR-Cas9-mediated TP knockout (KO) strategy in human adipose stem cells (ASC), which can be differentiated into adipocytes in vitro. Protein expression profiles and cellular characteristics were investigated in this KO model. RESULTS: All patients had TYMP loss-of-function variants and first presented with generalized loss of adipose tissue and insulin-resistant diabetes. CRISPR-Cas9-mediated TP KO in ASC abolished adipocyte differentiation and decreased insulin response, consistent with the patients' phenotype. This KO also induced major oxidative stress, altered mitochondrial functions, and promoted cellular senescence. This translational study identifies a new role of TP by demonstrating its key regulatory functions in adipose tissue. CONCLUSIONS: The implication of TP variants in atypical forms of monogenic diabetes shows that genetic diagnosis of lipodystrophic syndromes should include TYMP analysis. The fact that TP is crucial for adipocyte differentiation and function through the control of mitochondrial homeostasis highlights the importance of mitochondria in adipose tissue biology.

Multifunctional role of thymidine phosphorylase in cancer.

Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine, maintaining nucleoside homeostasis for DNA repair and replication. In many cancers TP is expressed at high levels and promotes thymidine catabolism, ultimately generating 2-deoxyribose (2dDR) that can support multiple procancer processes, including glycation of proteins, alternative metabolism, extracellular matrix remodeling, and angiogenesis. Therefore, inhibition of TP is an attractive anticancer strategy; however, an alternative approach that exploits the catalytic activity of TP to activate 5-fluorouracil (5-FU) prodrugs has been clinically successful. Here, we review the structure, function, and regulation of TP, its multiple supporting roles in cancer growth and survival. We summarize TP inhibitor and prodrug development and propose TP-targeting strategies that could potentiate the action of current therapies.

The Janus kinase inhibitor (baricitinib) suppresses the rheumatoid arthritis active marker gliostatin/thymidine phosphorylase in human fibroblast-like synoviocytes.

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities in the pathogenesis of rheumatoid arthritis (RA). The novel oral Janus kinase (JAK) inhibitor baricitinib has demonstrated high efficacy in RA. However, the effect of baricitinib on fibroblast-like synoviocytes (FLSs), a key component of invasive synovitis, has not been still elucidated. This study investigated whether GLS/TP production could be regulated by JAK/signal transducers and activators of transcription (STAT) signaling in FLSs derived from patients with RA. FLSs were cultured and stimulated by interferon (IFN)γ in the presence of baricitinib. Expression levels of GLS/TP were determined using reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry. Phosphorylation of STAT proteins was investigated by Western blot. In cultured FLSs, GLS/TP mRNA and protein levels were significantly induced by treatment with IFNγ and these inductions were suppressed by baricitinib treatment. Baricitinib inhibited IFNγ-induced STAT1 phosphorylation, while JAK/STAT activation played a pivotal role in IFNγ-mediated GLS/TP upregulation in RA. These results suggested that baricitinib suppressed IFNγ-induced GLS/TP expression by inhibiting JAK/STAT signaling, resulting in the attenuation of neovascularization, synovial inflammation, and cartilage destruction.

Effect of Thymidine Phosphorylase Gene Demethylation on Sensitivity to 5-Fluorouracil in Colorectal Cancer Cells.

BACKGROUND/AIM: Chemotherapy is used for recurrent and metastatic colorectal cancer, but the response rate of 5-fluorouracil (5-FU), the standard treatment for colorectal cancer, is low. We hypothesized that thymidine phosphorylase (TYMP) expression, a rate-limiting activating enzyme of 5-FU, is regulated by methylation of the gene promoter region, and demethylation of TYMP would increase sensitivity to 5-FU. MATERIALS AND METHODS: HCT116 colon cancer cells were treated with 5-aza-2'-deoxycytidine, a demethylating agent, and changes in TYMP transcription and sensitivity to 5-FU were evaluated. RESULTS: TYMP expression increased over 54-fold in HCT116 transfected with TYMP. The cytotoxicity of 5-FU increased up to 5.5-fold. In comparison, in HCT116 treated with 5-aza-2'-deoxycytidine, TYMP expression increased 5.8-fold. However, the cytotoxicity of 5-FU remained unchanged. CONCLUSION: Demethylating agent alone did not promote the cytotoxicity of 5-FU against colorectal cancer. To further increase the sensitivity to 5-FU, combination with adjuvant therapy focusing on metabolic pathways other than the TYMP pathway appear necessary.

Thymidine phosphorylase induction by ionizing radiation antagonizes 5-fluorouracil resistance in human ductal pancreatic adenocarcinoma.

Chemoresistance in pancreatic ductal adenocarcinoma (PDAC) frequently contributes to failure of systemic therapy. While the radiosensitizing properties of 5-fluorouracil (FU) are well known, it is unknown whether ionizing radiation (IR) sensitizes towards FU cytotoxicity. Here, we hypothesize that upregulation of thymidine phosphorylase (TP) by IR reverses FU chemoresistance in PDAC cells. The FU resistant variant of the human PDAC cell line AsPC-1 (FU-R) was used to determine the sensitizing effects of IR. Proliferation rates of FU sensitive parental (FU-S) and FU-R cells were determined by WST-1 assays after low (0.05 Gy) and intermediate dose (2.0 Gy) IR followed by FU treatment. TP protein expression in PDAC cells before and after IR was assessed by Western blot. To analyze the specificity of the FU sensitizing effect, TP was ablated by siRNA. FU-R cells showed a 2.7-fold increase of the half maximal inhibitory concentration, compared to FU-S parental cells. Further, FU-R cells showed a concomitant IR resistance towards both doses applied. When challenging both cell lines with FU after IR, FU-R cells had lower proliferation rates than FU-S cells, suggesting a reversal of chemoresistance by IR. This FU sensitizing effect was abolished when TP was blocked by anti-TP siRNA before IR. An increase of TP protein expression was seen after both IR doses. Our results suggest a TP dependent reversal of FU-chemoresistance in PDAC cells that is triggered by IR. Thus, induction of TP expression by low dose IR may be a therapeutic approach to potentially overcome FU chemoresistance in PDAC.

Vesicular IFN-γ as a cooperative attacker to enhance anti-cancer effect of 5-fluorouracil via thymidine phosphorylase upregulation and tumor microenvironment normalization.

On the basis of immuno-modulating effect and upregulating the activity of thymidine phosphorylase (TP), interferon-γ (IFN-γ) as a cooperative attacker was explored to enhance the anticancer activity of 5-fluorouracil (5-FU). We designed and prepared a self-assembled nano-vesicular system IFN-γ-EDP formulated by amphiphilic poly((polyethylene glycol)(dodecylphosphoethanolamine)phosphazene) (EDP) to entrap IFN-γ in the hydrophilic cavity. The IFN-γ-EDP vesicles allowed IFN-γ to accumulate at the tumor site and be taken up by tumor cells, resulting in significantly upregulated expression level of TP, distinct inhibition of cell growth, more cellular apoptosis and more serious cell cycle arrest when administrated combined with 5-FU. Moreover, IFN-γ-EDP could normalize the tumor microenvironment by enhancing the CD4+ and CD8+ T cell populations, promoting the IL-12 secretion and suppressing the IL-10 secretion in tumor. As a consequence, the combination therapy of IFN-γ-EDP with 5-FU achieved remarkably enhanced tumor inhibition rate of 56.9% against CT26 colorectal cancer.

Synthetic and Naturally Occurring Heterocyclic Anticancer Compounds with Multiple Biological Targets.

Cancer is a complex group of diseases initiated by abnormal cell division with the potential of spreading to other parts of the body. The advancement in the discoveries of omics and bio- and cheminformatics has led to the identification of drugs inhibiting putative targets including vascular endothelial growth factor (VEGF) family receptors, fibroblast growth factors (FGF), platelet derived growth factors (PDGF), epidermal growth factor (EGF), thymidine phosphorylase (TP), and neuropeptide Y4 (NY4), amongst others. Drug resistance, systemic toxicity, and drug ineffectiveness for various cancer chemo-treatments are widespread. Due to this, efficient therapeutic agents targeting two or more of the putative targets in different cancer cells are proposed as cutting edge treatments. Heterocyclic compounds, both synthetic and natural products, have, however, contributed immensely to chemotherapeutics for treatments of various diseases, but little is known about such compounds and their multimodal anticancer properties. A compendium of heterocyclic synthetic and natural product multitarget anticancer compounds, their IC50, and biological targets of inhibition are therefore presented in this review.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

Novel Mutations of the TYMP Gene in Mitochondrial Neurogastrointestinal Encephalomyopathy: Case Series and Literature Review.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a multi-system disorder caused by several homozygous or compound heterozygous mutations, mostly in the nuclear gene of TYMP. Our current knowledge on the underlying pathology of the disease is derived through the study of about 200 cases of different ethnicities. Clinical presentations include severe cachexia, weakness, ptosis, diplopia, abdominal cramps or digestive tract disorders, hearing impairment, and paresthesia.Herein, we aim to present five novel mutations of the nuclear gene of TYMP in six Iranian patients diagnosed with MNGIE. In our population, age at the time of diagnosis was 18 to 49 years, while the onset of the symptoms varied from 13 to 20 years. We detected two pathogenic non-frameshift nonsense premature stop codon mutations (c.1013C > A, and c.130C > T), one variant of uncertain significance (VUS) non-frameshift missense mutation (c.345G > T), one likely pathogenic frameshift insertion (c.801_802insCGCG), and one likely benign homozygous non-frameshift deletion (c.1176_1187del) from two siblings. Our findings also confirm the autosomal recessive inheritance pattern of MNGIE in the Iranian population. The lack of knowledge in the area of nuclear gene-modifier genes shadows the genotype-phenotype relationships of MNGIE.

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.