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DNA Repair Feb 2022Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The...
Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O-carboxymethylguanine (O-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O-CMG mutagenicity. To better address how cells detect and remove O-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O-CMG. We found that cells deficient in either the direct repair protein O-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O-CMG DNA damage.
Topics: DNA; DNA Adducts; DNA Damage; DNA Repair; Mutagenesis; Mutagens; O(6)-Methylguanine-DNA Methyltransferase
PubMed: 35030424
DOI: 10.1016/j.dnarep.2021.103262 -
BioMed Research International 2020Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis...
Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and contributes to the O-GlcNAcylation process. However, the spectrum of HBP-dependent tumors and the mechanisms by which the HBP promotes tumor aggressiveness remain areas of active investigation. In this study, we analyzed the activity of the HBP and its prognostic value across 33 types of human cancers. Increased HBP activity was observed in pancreatic ductal adenocarcinoma (PDAC), and higher HBP activity predicted a poor prognosis in PDAC patients. Genetic silencing or pharmacological inhibition of the first and rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), inhibited PDAC cell proliferation, invasive capacity, and triggered cell apoptosis. Notably, these effects can be restored by addition of UDP-GlcNAc. Moreover, similar antitumor effects were noticed by pharmacological inhibition of GFAT1 with 6-diazo-5-oxo-l-norleucine (DON) or Azaserine. PDAC is maintained by oncogenic Wnt/-catenin transcriptional activity. Our data showed that GFAT1 can regulate -catenin expression via modulation of the O-GlcNAcylation process. TOP/FOP-Flash and real-time qPCR analysis showed that GFAT1 knockdown inhibited -catenin activity and the transcription of its downstream target genes and . Ectopic expression of a stabilized form of -catenin restored the suppressive roles of GFAT1 knockdown on PDAC cell proliferation and invasion. Collectively, our findings indicate that higher GFAT1/HBP/O-GlcNAcylation exhibits tumor-promoting roles by maintaining -catenin activity in PDAC.
Topics: Cell Line, Tumor; Cell Proliferation; Databases, Genetic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Glycosylation; Hexosamines; Humans; Pancreatic Neoplasms; beta Catenin
PubMed: 32149084
DOI: 10.1155/2020/1921609 -
Organic Letters Jun 2023Azaserine () is a natural product and nonproteinogenic amino acid containing a diazo group. Here we report the biosynthetic gene cluster for from . We then use isotopic...
Azaserine () is a natural product and nonproteinogenic amino acid containing a diazo group. Here we report the biosynthetic gene cluster for from . We then use isotopic feeding, gene deletion, and biochemical experiments to support a pathway whereby hydrazinoacetic acid () and a peptidyl carrier protein-loaded serine () are intermediates on route to the final natural product .
Topics: Azaserine; Serine; Multigene Family; Hydrazines; Biological Products
PubMed: 37235858
DOI: 10.1021/acs.orglett.3c01229 -
Chembiochem : a European Journal of... Apr 2022During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/β hydrolase AzpM...
During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/β hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS-AzpF and DON-AzpF, but not between AZS-AzpF and DON. Possible reaction intermediates, DON-DON-AzpF and AZS-AZS-AzpF, were also detected during AzpM-catalyzed dipeptide formation from DON-AzpF and AZS-AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON-AzpF and subsequent hydrolysis to produce DON-DON. Thus, AzpM is an unprecedented α/β hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein-tethered amino acid.
Topics: Carrier Proteins; Diazooxonorleucine; Dipeptides; Hydrolases
PubMed: 35132756
DOI: 10.1002/cbic.202100700 -
Pancreas Apr 2021High-fat diet has been considered a risk factor for the development of pancreatic cancer. It is also shown to significantly impact composition and dysbiosis of gut...
OBJECTIVES
High-fat diet has been considered a risk factor for the development of pancreatic cancer. It is also shown to significantly impact composition and dysbiosis of gut microbiota in both humans and animals. However, there is little information on the effect of high-fat diet on the development of pancreatic cancer or upon the gut microbiota of patients with pancreatic cancer in humans or animal models.
METHODS
In this study, the effect of high-fat diet on cancer pathology and the gut microbiota was investigated by a carcinogen-induced pancreatic cancer mouse model.
RESULTS
Compared with carcinogen alone, mice with high-fat diet and carcinogen showed more obvious pathological changes in pancreatic tissue; increased levels of proinflammatory cytokine tumor necrosis factor-α, interleukin-6, interleukin-10, and carbohydrate antigen 242; and increased expression of cancer-associated biomarkers mucin-4 and claudin-4 in pancreatic tissue. Moreover, there is a significant change in the gut microbiota between the carcinogen group and the carcinogen with high-fat diet group. We identified that Johnsonella ignava especially existed in the carcinogen with high-fat diet group, which may contribute to pancreatic cancer development.
CONCLUSIONS
Our results revealed that high-fat diet changed the composition of the gut microbiota and was involved in carcinogen-induced pancreatic cancer progression.
Topics: Animals; Azaserine; Bacteria; Carcinogens; Claudin-4; Cytokines; Diet, High-Fat; Disease Models, Animal; Drug Synergism; Feces; Female; Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Mice, Inbred C57BL; Mucin-4; Pancreatic Neoplasms; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Mice
PubMed: 33939670
DOI: 10.1097/MPA.0000000000001797 -
Discovery Medicine Jun 2024Atypical acinar cell foci (AACF) seen in pancreatic cancer are fatal and have been studied with some causative agents. However, for the first time, the effect of...
BACKGROUND
Atypical acinar cell foci (AACF) seen in pancreatic cancer are fatal and have been studied with some causative agents. However, for the first time, the effect of acetylsalicylic acid with nitric oxide (NO-ASA) on AACF was examined in this study. Although NO-ASA has very successful inhibitory effects against some types of cancer, it has not been investigated whether they can exert their inhibition effects on AACFs.
METHODS
For experimental purposes, 21 14-day-old male Wistar albino rats were used. Azaserine (30 mg/kg) was dissolved in 0.9% NaCl solution and injected intraperitoneally (i.p.) into 14 rats, except for the Control group (Cont) rats, for three weeks. Rats that were injected with azaserine once a week for three weeks and those that did not receive treatment were divided into experimental groups. 15 days after the end of the azaserine injection protocol, NO-ASA was applied to azaserine with NO-ASA (Az+NO-ASA) group rats three consecutive times with an interval of 15 days by gavage. At the end of the 5-month period, pancreatic tissue was dissected and weighed. Pancreas preparations prepared from histological sections were examined for AACF burden and analyzed via a video image analyzer. One-way analysis of variance (ANOVA) non-parametric statistical analyses were performed to test whether there was a difference between the averages of the experimental and Control groups.
RESULTS
AACF burden in both groups injected with azaserine was found to be statistically significant in all categories compared to that of the Control group ( < 0.05). The average Calculated Estimated average AACF volume (mm) values, the Calculated estimated average AACF diameter (μm), the Estimated average number of AACF per unit volume, AACF rate as a % of Calculated Organ Volume were higher in the AzCont group rats than in the Az+NO-ASA group, when compared, and there was an important level statistical difference between the groups ( < 0.05). It was determined that for all parameters AACFs load in Az+NO-ASA group rats were significantly reduced compared to that of AzCont group rats ( < 0.05).
CONCLUSIONS
We observed that, as a result of the NO-ASA application, the experimental AACF focus ratio created by azaserine injection was significantly inhibited. The inhibitory effect of AACFs in Az+NO-ASA group rats may have resulted from the significant and independent chemopreventive and/or chemotherapeutic activity of NO-ASA against exocrine pancreatic AACF foci.
Topics: Animals; Male; Aspirin; Rats, Wistar; Nitric Oxide; Rats; Pancreatic Neoplasms; Acinar Cells; Pancreas, Exocrine
PubMed: 38926102
DOI: 10.24976/Discov.Med.202436185.106 -
Molecules (Basel, Switzerland) Apr 2020is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian...
is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to survival. Recently, we described that can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.
Topics: Animals; Azaserine; CHO Cells; Cell Cycle; Cell Proliferation; Cricetulus; Energy Metabolism; Glutamic Acid; Glutamine; Isoxazoles; Life Cycle Stages; Molecular Structure; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 32252252
DOI: 10.3390/molecules25071628 -
Journal of Plant Physiology Aug 2019In higher plants ammonium (NH) assimilation occurs mainly through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. Nevertheless, when plants are exposed...
In higher plants ammonium (NH) assimilation occurs mainly through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. Nevertheless, when plants are exposed to stress conditions, such as excess of ammonium, the contribution of alternative routes of ammonium assimilation such as glutamate dehydrogenase (GDH) and asparagine synthetase (AS) activities might serve as detoxification mechanisms. In this work, the in vivo functions of these pathways were studied after supplying an excess of ammonium to tomato (Solanum lycopersicum L. cv. Agora Hybrid F1) roots previously adapted to grow under either nitrate or ammonium nutrition. The short-term incorporation of labelled ammonium (NH) into the main amino acids was determined by GC-MS in the presence or absence of methionine sulphoximine (MSX) and azaserine (AZA), inhibitors of GS and GOGAT activities, respectively. Tomato roots were able to respond rapidly to excess ammonium by enhancing ammonium assimilation regardless of the previous nutritional regime to which the plant was adapted to grow. The assimilation of NH could take place through pathways other than GS/GOGAT, since the inhibition of GS and GOGAT did not completely impede the incorporation of the labelled nitrogen into major amino acids. The in vivo formation of Asn by AS was shown to be exclusively Gln-dependent since the root was unable to incorporate NH directly into Asn. On the other hand, an in vivo aminating capacity was revealed for GDH, since newly labelled Glu synthesis occurred even when GS and/or GOGAT activities were inhibited. The aminating GDH activity in tomato roots responded to an excess ammonium supply independently of the previous nutritional regime to which the plant had been subjected.
Topics: Amination; Ammonium Compounds; Fertilizers; Glutamate Dehydrogenase; Solanum lycopersicum; Plant Roots
PubMed: 31229903
DOI: 10.1016/j.jplph.2019.03.009 -
American Journal of Physiology.... Mar 2020The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin...
The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform () and mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.
Topics: Adenosine Triphosphate; Animals; Azaserine; Carrier Proteins; Cattle; Female; Gene Knockdown Techniques; Glucose; Glycolysis; In Vitro Oocyte Maturation Techniques; Infertility, Female; Intracellular Space; Mitochondria; Oocytes; Oxidation-Reduction; RNA Interference; RNA, Small Interfering
PubMed: 31935112
DOI: 10.1152/ajpendo.00057.2019 -
Carcinogenesis Aug 2021The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may...
The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.
Topics: Cell Line; Colon; DNA Damage; DNA Repair; Guanine; Humans; Intestinal Mucosa; O(6)-Methylguanine-DNA Methyltransferase
PubMed: 34115837
DOI: 10.1093/carcin/bgab049