Orotate Phosphoribosyltransferase Research Articles

Orotate phosphoribosyltransferase localizes to the Golgi complex and its expression levels affect the sensitivity to anti-cancer drug 5-fluorouracil.

Orotate phosphoribosyltransferase (OPRT) is engaged in de novo pyrimidine synthesis. It catalyzes oronitine to uridine monophosphate (UMP), which is used for RNA synthesis. De novo pyrimidine synthesis has long been known to play an important role in providing DNA/RNA precursors for rapid proliferative activity of cancer cells. Furthermore, chemotherapeutic drug 5-fluorouracil (5-FU) is taken up into cancer cells and is converted to 5-fluoro-UMP (FUMP) by OPRT or to 5-fluoro-dUMP (FdUMP) through intermediary molecules by thymidine phosphorylase. These 5-FU metabolites are misincorporated into DNA/RNA, thereby producing dysfunction of these information processing. However, it remains unclear how the subcellular localization of OPRT and how its variable expression levels affect the response to 5-FU at the cellular level. In this study, immunocytochemical analysis reveals that OPRT localizes to the Golgi complex. Results also show that not only overexpression but also downregulation of OPRT render cells susceptible to 5-FU exposure, but it has no effect on DNA damaging agent doxorubicin. This study provides clues to elucidate the cellular response to 5-FU chemotherapy in relation to the OPRT expression level.

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: A prospective multi-institutional study in Japan

ObjectivesThis investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. Materials and methods236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2cm or greater), IB, and II tumors were given a dose of 250mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Results and conclusionAmong 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS.The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery.

Successful curative resection of gallbladder cancer following S-1 chemotherapy: A case report and review of the literature.

The symptoms of gallbladder cancer (GBC) are vague and non-specific. Therefore, GBC is often detected at an advanced or metastatic stage. The most effective treatment for GBC is surgical resection, however the majority of GBC cases are unresectable at the time of diagnosis. Therefore, numerous GBC patients undergo chemotherapy. This study reports the case of a 60-year-old female with GBC who underwent successful surgical curative resection following a single dose of the chemotherapeutic agent, S-1, twice daily for 4 weeks followed by a 14-day rest period for 36 months. S-1 is a novel orally administered drug composed of a combination of the 5-fluorouracil (5-FU) prodrug, tegafur, 5-chloro-2,4-dihydroxypyridine (CDHP) and oteracil potassium in a 1:0.4:1 molar concentration ratio. The focus of the present study was the candidate factors that affect the therapeutic efficacy of S-1-based chemotherapy. In particular, the gene expression involved in the S-1 metabolic pathway was investigated by assessing the intratumoral dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS) and orotate phosphoribosyltransferase gene expression. The surgical specimen exhibited high intratumoral DPD gene expression levels compared with those observed in previously reported non S-1 responsive cases of biliary tract cancer. Due to the results obtained in the current study, we hypothesize that CDHP enhanced the antitumor efficacy of 5-FU by inhibiting the excess DPD protein produced by the tumor.

Expression level of dihydropyrimidine dehydrogenase is associated with clinical outcome in patients with T1G3 bladder cancer treated with Bacillus Calmette-Guerin

BackgroundRecently, it has been shown that 5-fluorouracil (5-FU) with a strong dihydropyrimidine dehydrogenase (DPD) inhibitor elicits a significant response in bladder cancer with a high level of DPD. However, only a few studies investigated the association between the level of the enzyme that regulates the metabolism of 5-FU and prognosis in bladder cancer. Furthermore, to our knowledge, there has also been no such report in T1G3 bladder tumors treated with BCG. Therefore, we evaluated enzymes that regulate the metabolism of 5-FU in T1G3 tumors treated with BCG immunotherapy using the Danenberg tumor profile (DTP) method, a highly accurate measurement of RNA from paraffin-embedded specimens.MethodsThis study included 28 patients with T1G3 bladder cancer, each of whom underwent complete transurethral tumor resection and BCG intravesical instillation at our institution. The median follow-up period was 39 months (range, 3 to 159 months). The DTP method was used to analyze the mRNA expression of 3 enzymes related to 5-FU: DPD, orotate phosphoribosyltransferase (OPRT), and thymidylate synthase (TS).ResultsAmong the 28 patients, 13 developed recurrences (46.4%) and 5 experienced disease progression (17.9%). An elevated DPD mRNA level was significantly associated with recurrence (p = 0.048) and progression (p = 0.045). However, TS and OPRT mRNA levels were not significantly associated with any other clinical features or outcomes. Furthermore, the high DPD group had a significantly lower recurrence-free survival rate than the low DPD group (p = 0.047). Among patients with low DPD, the 2- and 5-year recurrence-free survival rates were 88.9% and 74.1%, respectively; while among patients with high DPD, the corresponding rates were 61.3% and 36.8%, respectively. TS and OPRT were not significantly associated with recurrence-free survival rates.ConclusionDPD is significantly associated with recurrence and progression among T1G3 bladder cancer patients treated with BCG.

Chemotherapy for Dihydropyrimidine Dehydrogenase-Deficient Patient

ABSTRACT Aim: 5-FU is metabolized by thymidine phosphorylase and orotate phosphoribosyl transferase and is principally degraded in the blood or liver by dihydropyrimidine dehydrogenase (DPD). DPD, the catalyzing enzyme in the first and rate-limiting step of the degradation process, degrades approximately 85% of the administered 5-FU. DPD activity in peripheral blood mononuclear cells has been reported to correlate inversely with 5-FU clearance. In reports of DPD deficiency, the prognosis is poor, and approximately 60% patients die. With a reported high mortality rate, chemotherapy is generally contraindicated for patients with DPD deficiency, and there have been no reports of continued chemotherapy in patients with a DPD activity level of Methods: Chemotherapy for a 73-year old man with jejunal cancer was initiated. Capecitabine was administered in incrementally increasing doses, beginning with a single pill (dose-escalation method), while monitoring plasma 5-FU concentration, leukocyte, neutrophil, and platelet counts. Results: DPD protein measurement in the peripheral blood mononuclear cells yielded a level of 2.35 U/mg using the ELISA method (the same method yielded DPD measurements ranging from 33.6 to 183.6 U/mg in peripheral blood mononuclear cells from 10 healthy individuals). Ultimately, after increasing the Capecitabine dose to 1800 mg, oxaliplatin and bevacizumab were added and XELOX+bevacizumab treatment was initiated. Subsequent DPD protein measurement showed that the level had increased to approximately 10-fold the level before chemotherapy. Furthermore, the peritoneal metastases disappeared following chemotherapy. Conclusions: Because of serious adverse events, the chemotherapy-associated mortality rate among DPD-deficient patients is high, and there have been no reports of continuous chemotherapy in patients with DPD activity levels of Disclosure: All authors have declared no conflicts of interest.

Evaluation of 5-fluorouracil metabolic enzymes as predictors of response to adjuvant chemotherapy outcomes in patients with stage II/III colorectal cancer: a decision-curve analysis.

The effectiveness of 5-fluorouracil (5-FU)-based adjuvant chemotherapy is reported in patients with colorectal cancer (CRC), but the usefulness of 5-FU metabolic enzymes as predictive biomarkers of the efficacy of this chemotherapy remains unclear. This study aims to verify whether 5-FU metabolic enzymes are predictive biomarkers in the clinical setting of adjuvant chemotherapy for stage II/III CRC. In total, 179 patients with stage II/III CRC who were treated at our institute between 2000 and 2010 were enrolled. Messenger RNA (mRNA) expression of major 5-FU metabolic enzymes, namely thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase (TP), orotate phosphoribosyl transferase, and β-actin (control) was evaluated using the Danenberg Tumor Profile method. mRNA expression and other clinicopathological data were investigated with regard to CRC relapse. A total of 78 patients underwent surgery alone, while 101 underwent adjuvant chemotherapy (5-FU plus leucovorin [LV] or tegafur plus uracil /LV) following surgery. Relapse-free survival was longer and risk of recurrence was lower in association with high TP mRNA expression than in association with low TP mRNA expression in the adjuvant chemotherapy group (hazard ratio 0.66; 95 % confidence interval 0.47-0.92; p = 0.016), but not in the surgery alone group. mRNA expression of no other enzymes was associated with relapse in both groups. In decision-curve analyses, the predictive efficiency of TP mRNA expression plus clinicopathological factors was slightly better than that of clinicopathological factors only. TP mRNA expression in tumors predicted the effects of adjuvant chemotherapy for stage II/III CRC, although the beneficial effects were marginal.

Impact of 5-fluorouracil metabolizing enzymes on chemotherapy in patients with resectable colorectal cancer.

Although 5-fluorouracil (5-FU) is an important drug for colorectal cancer (CRC) treatment, no useful biomarker is currently available to predict treatment response. Since 5-FU is converted into active or inactive forms by orotate phosphoribosyltransferase (OPRT) or dihydropyrimidine dehydrogenase (DPD), a correlation between these enzymes and response to 5-FU has been suggested. However, such a correlation has not been investigated prospectively. Therefore, in the present study, we aimed to prospectively evaluate whether OPRT and DPD were predictive factors of the response to 5-FU treatment in patients with resectable CRC. The present investigation was designed as a multicenter prospective cohort study. OPRT and DPD activities were assessed in biopsy samples, obtained surgically from patients with resectable CRC. The OPRT/DPD ratio was calculated and the cut-off values for this ratio were determined for 5-year disease-free survival (DFS) and overall survival (OS). Patients were treated with 5-FU/leucovorin (LV) regimens and oral 5-FU. The endpoint of this study was the correlation between the OPRT/DPD ratio and 5-year DFS and OS. The cut-off value for the OPRT/DPD ratio was determined by using the maximum χ2 statistic method against 5-year DFS and OS. Sixty-eight patients were enrolled from July 2003 to May 2005. The median follow-up period was 1925 days. The OPRT/DPD ratio cut-off values for 5-year DFS and OS were 0.015 and 0.013, respectively. During the 5-year DFS and OS periods, patients with higher cut-off values had a better prognosis than those with lower ratios (P=0.03 and 0.02, respectively). In conclusion, our results suggest that the OPRT/DPD ratio could be a predictive factor for response to 5-FU/LV adjuvant chemotherapy.

Prognostic significance of thymidylate synthase in postoperative non-small cell lung cancer patients.

The aim of the present study was to investigate the clinicopathologic/prognostic significance of thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) proteins in postoperative non-small cell lung cancer (NSCLC) patients. Microarray slides from a set of 178 NSCLC patients were used for the detection of TS, OPRT, and TP expression by immunohistochemistry. The correlation between clinicopathologic factors and protein expression of three proteins was analyzed. Ninety seven carcinomas (57.4%) were TS-positive, 90 carcinomas (53.9%) were OPRT-positive, and 102 carcinomas (69.4%) were TP-positive. Compared with the TS-positive patients, the overall survival (OS) was significantly lower in the TS-negative patients (hazard ratio [HR] =1.766, 95% confidence interval [CI] =1.212–2.573, P=0.003). Significant differences between TS-positive and TS-negative patients was also observed in the following stratified analyses: 1) adenocarcinoma subgroup (HR =2.079, 95% CI =1.235–3.500, P=0.006); 2) less than 60-year-old subgroup (HR =1.890, 95% CI =1.061–3.366, P=0.031); 3) stage II/III subgroup (HR =1.594, 95% CI =1.036–2.453, P=0.034); and 4) surgery plus adjuvant therapy subgroup (HR =1.976, 95% CI =1.226–3.185, P=0.005). However, the OS was not significantly correlated with OPRT or TP protein expression. This study demonstrates that the TS level in tumor tissues may be a useful marker to predict the postoperative OS in NSCLC patients.

Pyrimidine nucleotide synthesis in Pseudomonas nitroreducens and the regulatory role of pyrimidines

Control of pyrimidine biosynthesis in the commercially important, hydrocarbon-utilizing bacterium Pseudomonas nitroreducens ATCC 33634 was investigated. When glucose-grown wild-type cells were supplemented with uracil or orotic acid, the pyrimidine biosynthetic activities were depressed. Pyrimidine limitation of glucose-grown cells of an orotate phosphoribosyltransferase mutant caused aspartate transcarbamoylase and dihydroorotase activities to increase by about 4-fold while the other enzyme activities about doubled. In succinate-grown phosphoribosyltransferase mutant cells subjected to pyrimidine limitation, transcarbamoylase and dehydrogenase activities rose by about 5-fold while dihydroorotase activity more than tripled. In an OMP decarboxylase mutant, pyrimidine limitation of glucose-grown cells increased transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities by 4-, 10-, 6- and 3.8-fold, respectively. Pyrimidine limitation of the succinate-grown decarboxylase mutant cells increased aspartate transcarbamoylase or dihydroorotase by more than 4-fold and the other activities by about 2-fold. Pyrimidine biosynthetic enzyme synthesis appeared to be regulated by pyrimidines with the regulation being influenced by the carbon source present. Aspartate transcarbamoylase activity in Ps. nitroreducens was regulated at the level of enzyme activity since the enzyme was strongly inhibited by UDP, pyrophosphate, ATP and ADP. Overall, the regulation of pyrimidine biosynthesis in Ps. nitroreducens can be used to differentiate it from other taxonomically related species of Pseudomonas.

Effects of smoking and alcohol consumption on 5-fluorouracil-related metabolic enzymes in oral squamous cell carcinoma.

Lifestyle, particularly smoking and alcohol consumption, may induce and/or inhibit drug metabolism. In order to reveal the effects of smoking and alcohol consumption on the 5-fluorouracil (5-FU)-related metabolic enzymes, namely thymidylate synthase, dihydropyrimidine dehydrogenase (DPD; a sole catabolic enzyme of 5-FU), orotate phosphoribosyl transferase (OPRT) and thymidine phosphorylase, in oral squamous cell carcinomas, the mRNA expression of these enzymes was investigated in 29 surgical specimens and compared by the Brinkman index and drinking years. The surgical specimens were divided into normal and tumor regions and were independently analyzed using quantitative reverse transcription-polymerase chain reaction. There was a significantly positive correlation between DPD mRNA expression in these tissues and Brinkman index/drinking years, with OPRT mRNA expression being significantly correlated to the Brinkman index in tumor tissues. These results revealed that lifestyle habits, including smoking and alcohol consumption, may vary the activity of the 5-FU-related metabolic enzymes. DPD is the initial and rate-limiting enzyme in the catabolic pathway of 5-FU. Therefore, smoking and alcohol consumption may reduce the anticancer activity of 5-FU, possibly through the induction of DPD activity.

A COX-2 inhibitor enhances the antitumor effects of chemotherapy and radiotherapy for esophageal squamous cell carcinoma

Cyclooxygenase-2 (COX-2) is a key enzyme of prostaglandin (PG) synthesis that has been demonstrated to be overexpressed in several types of cancers. The function of COX-2 in tumor progression has been recently elucidated. In tumors in which COX-2 is overexpressed, the antitumor effects are suppressed. We examined the effects of celecoxib, a COX-2 inhibitor, in enhancing the antitumor effects of chemotherapy and radiotherapy for esophageal squamous cell carcinoma (ESCC) by reducing the COX-2 activity. We used the human esophageal squamous cell lines TE2 and T.Tn treated with celecoxib and 5-FU/radiation, after which cell viability assays were performed. Changes in the expressions of dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT) mRNA and PGE2 were also measured. In addition, apoptotic changes, and the invasion and migration activity in both the celecoxib and 5-FU treated cells were evaluated. The experiments showed that T.Tn and TE2 proliferation was strongly inhibited by the combination of 5-FU/radiation and the COX-2 inhibitor. Inhibiting the COX-2 activity induced a reduction in PGE2 levels in TE2/T.Tn cells. Following treatment with the COX-2 inhibitor and 5-FU, the OPRT expression was upregulated and the DPD expression was downregulated in the resistant cells. In addition, the combination treatment with the COX-2 inhibitor and 5-FU markedly inhibited both the cell invasion and migration activity. Therefore, COX-2 inhibitors can be useful enhancers of antitumor drugs and radiotherapy for ESCC.

RAD-6: pyrimidine synthesis and radiation sensitivity in Caenorhabditis elegans

The Caenorhabditis elegans rad-6 (radiation-sensitive-6) mutant was isolated over 25 years ago in a genetic screen that identified mutants with enhanced sensitivity to DNA damaging agents. In the present paper we describe the molecular identification of the rad-6 gene and reveal that it encodes the bifunctional UMP synthase protein, which carries catalytic activities for OPRTase (orotate phosphoribosyltransferase) and ODCase (orotate monophosphate decarboxylase), key enzymes in the de novo pathway of pyrimidine synthesis. Mutations in genes encoding de novo pathway enzymes cause varying degrees of lethality and pleiotropic phenotypes in many organisms, including humans. We have examined how the absence of rad-6 activity leads to both UV-C hypersensitivity and a decline in both metabolic rate and lifespan. We discuss how rad-6 mutants adapt to the loss of the de novo pathway through a dependency on pyrimidine salvage. We establish further that rad-6(mn160) mutants lack ODCase activity because they are resistant to the cytotoxic effects of 5-FOA (5-fluoroorotic acid). Our results have also led to the identification of a metabolic sensor affecting survival and metabolism, which is dependent on the maternal rad-6 genotype.

A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

ObjectiveTo investigate the multienzyme complex formation of human malaria parasite Plasmodium falciparum (P. falciparum) orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzyme of the de novo pyrimidine biosynthetic pathway. Previously, we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric (OPRT)2(OMPDC)2 complex containing two subunits each of OPRT and OMPDC, and that the complex have catalytic kinetic advantages over the monofunctional enzyme. MethodsBoth enzymes were cloned and expressed as recombinant proteins. The protein-protein interaction in the enzyme complex was identified using bifunctional chemical cross-linker, liquid chromatography-mass spectrometric analysis and homology modeling. ResultsThe unique insertions of low complexity region at the α 2 and α 5 helices of the parasite OMPDC, characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms, was located on the OPRT-OMPDC interface. The structural models for the protein-protein interaction of the heterotetrameric (OPRT)2(OMPDC)2 multienzyme complex were proposed. ConclusionsBased on the proteomic data and structural modeling, it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction. The structural complex of the parasite enzymes, thus, represents an efficient functional kinetic advantage, which in line with co-localization principles of evolutional origin, and allosteric control in protein-protein-interactions.

Isolation of Mutants of the Nitrogen-Fixing Actinomycete <i>Frankia</i>

Frankia is a nitrogen (N)-fixing multicellular actinomycete which establishes root-nodule symbiosis with actinorhizal plants. Several aspects of Frankia N fixation and symbiosis are distinct, but genes involved in the specific features are largely unknown because of the lack of an efficient mutant screening method. In this study, we isolated mutants of Frankia sp. strain CcI3 using hyphae fragments mutagenized by chemical mutagens. Firstly, we isolated uracil auxotrophs as gain-of-function mutants resistant to 5-fluoroorotic acid (5-FOA). We obtained seven 5-FOA resistant mutants, all of which required uracil for growth. Five strains carried a frame shift mutation in orotidine-5′-phosphate decarboxylase gene and two carried an amino acid substitution in the orotate phosphoribosyltransferase gene. Secondly, we isolated mutants showing loss-of-function phenotypes. Mutagenized hyphae were fragmented by ultrasound and allowed to multiply at their tips. Hyphae were fragmented again and short fragments were enriched by filtration through 5 μm pores filters. Next-generation and Sanger sequencing revealed that colonies formed from the short hyphae fragments consisted of cells with an identical genotype. From the mutagenized colony population, we isolated three pigmentation mutants and a mutant with reduced N-fixation activity. These results indicate that our procedure is useful for the isolation of loss-of-function mutants using hyphae of Frankia.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

Cinnamaldehyde/chemotherapeutic agents interaction and drug-metabolizing genes in colorectal cancer

Cinnamaldehyde is an active monomer isolated from the stem bark of Cinnamomum cassia, a traditional oriental medicinal herb, which is known to possess marked antitumor effects in vitro and in vivo. The aim of the present study was to examine the potential advantages of using cinnamaldehyde in combination with chemotherapeutic agents commonly used in colorectal carcinoma (CRC) therapy, as well as to investigate the effect of cinnamaldehyde on chemotherapeutic-associated gene expression. The synergistic interaction of cinnamaldehyde and chemotherapeutic agents on human CRC HT-29 and LoVo cells was evaluated using the combination index (CI) method. The double staining with Annexin V conjugated to fluorescein-isothiocyanate and phosphatidylserine was employed for apoptosis detection. The expression of drug-metabolizing genes, including excision repair cross‑complementing 1 (ERCC1), orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), breast cancer susceptibility gene 1 (BRCA1) and topoisomerase 1 (TOPO1), all in HT-29 and LoVo cells, with or without the addition of cinnamaldehyde, was examined by quantitative polymerase chain reaction (PCR). Cinnamaldehyde had a synergistic effect on the chemotherapeutic agents cytotoxicity in HT-29 and LoVo cells. In addition, cinnamaldehyde suppressed BRCA1, TOPO1, ERCC1 and TS mRNA expression, except for OPRT expression, which was markedly upregulated. Our findings indicate that cinnamaldehyde appears to be a promising candidate as an adjuvant in combination therapy with 5-fluorouracil (5-FU) and oxaliplatin (OXA), two chemotherapeutic agents used in CRC treatment. The possible mechanisms of its action may involve the regulation of drug‑metabolizing genes.

Catalytic site interactions in yeast OMP synthase

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330–5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5′-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.

Transition State Analogues of Plasmodium falciparum and Human Orotate Phosphoribosyltransferases

The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nM. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5'-phosphate bound to OPRTs with Kd values near 40 nM. Analogues designed with a C5-pyrimidine carbon-carbon bond to ribocation mimics gave Kd values in the range of 80-500 nM. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5'-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.

18F-FDG uptake on PET is a predictive marker of thymidylate synthase expression in patients with thoracic neoplasms

The aim of this study was to investigate the relationship between the expression levels of thymidylate synthase (TS) and 2-[¹⁸F]-fluoro-2-deoxy-D-glucose (¹⁸F-FDG) uptake on positron emission tomography (PET) in various thoracic neoplasms. In total, 392 patients [non-small cell lung cancer (NSCLC) (n=140), malignant pleural mesothelioma (MPM) (n=21), pulmonary metastatic tumors (PMT) (n=148), thymic epithelial tumors (n=49) and pulmonary neuroendocrine (NE) tumor (n=34)] who underwent ¹⁸F-FDG PET before treatment were included in this study. Tumor sections were stained using immunohistochemistry for determination of TS, orotate phosphoribosyltransferase (OPRT), dihydropyrimidine dehydrogenase (DPD), vascular endothelial growth factor (VEGF), microvessel density (MVD), CD34 and p53. The expression of TS in thoracic neoplasms had a positivity of 58% (230/392), and the positive rates of TS expression in NSCLC, PMT, thymic epithelial tumor, NE tumor and MPM samples were 56, 57, 57, 85 and 47%, respectively. The positivity of TS expression was significantly higher in NE tumors compared to that in other thoracic tumors. A statistically significant correlation between TS expression and ¹⁸F-FDG uptake was observed in thoracic neoplasms, in particular primary lung adenocarcinomas, high-grade NE tumors, thymomas and MPMs. Moreover, TS expression was closely associated with angiogenesis, DPD, OPRT and p53. Our results indicated that SUV(max) by ¹⁸F-FDG uptake may be an alternative biomarker for predicting TS expression in patients with primary lung adenocarcinoma, high-grade NE tumor, thymoma and MPM.

Protein-bound polysaccharide-K augments the anticancer effect of fluoropyrimidine derivatives possibly by lowering dihydropyrimidine dehydrogenase expression in gastrointestinal cancers

Protein-bound polysaccharide-K (PSK) enhances the antitumor effect of anticancer drug when used clinically in combination with such drugs. PSK is known to act by immune-mediated mechanisms; however, the relationship between PSK and metabolic enzymes of anticancer drugs is unknown. We used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) clinically to evaluate the sensitivity of anticancer drugs. In the present study, we modified the CD-DST by adding peripheral blood mononuclear cells (PBMCs) (immuno-CD-DST) and examined the antitumor effect of PSK in combination with anticancer drugs. First, HCT116 human colon cancer cells were cultured with PSK and 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouridine (5'-DFUR) in the presence or absence of PBMCs, and the antiproliferative effects were compared. In the presence of PBMCs, PSK augmented the inhibitory effects of 5-FU and 5'-DFUR on HCT116 cell proliferation. Next, using human gastric cancer and colon cancer cell lines, the effects of PSK on mRNA expression of various metabolic enzymes of fluoropyrimidines: dihydropyrimidine dehydrogenase (DPD), thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase, were examined by real-time PCR. PSK significantly enhanced DPD mRNA expression in all of the cancer cell lines tested, but not those of the other enzymes. Addition of IFN-α and TRAIL, cytokines known to inhibit DPD expression, to the cultures reduced DPD mRNA expression in the cancer cells. When PBMC samples collected from healthy volunteers were cultured with PSK, IFN-α mRNA expression increased in 3 of the 5 PBMC samples, while TRAIL mRNA expression was unchanged. The present results propose the possibility that PSK induces PBMCs to express IFN-α which inhibits DPD expression, and consequently augments the antitumor effect of 5-FU or 5'-DFUR. Immuno-CD-DST is useful for evaluating drugs with immunological mechanisms of action.