Authors: Jiyeon Kim, Hyun Min Lee, Feng Cai...

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumour suppressor STK11 (also known as LKB1) encoding the kinase LKB1 result in aggressive tumours prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analysed tumour metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KRAS/LKB1 co-mutant tumours. KRAS/LKB1 co-mutant cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2). GFPT2 inhibition selectively reduced KRAS/LKB1 co-mutant tumour cell growth in culture, xenografts and genetically modified mice. Our results define a new metabolic vulnerability in KRAS/LKB1 co-mutant tumours and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Antineoplastic Agents; Azaserine; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Hexosamines; Humans; Lung Neoplasms; Metabolic Networks and Pathways; Metabolomics; Mice; Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Survival Analysis; Tumor Stem Cell Assay

PubMed: 33257855
DOI: 10.1038/s42255-020-00316-0

Authors: Zhiwen Xiao, Shengming Deng, He Liu...

Ferroptosis is a type of programmed cell death closely related to amino acid metabolism. Pancreatic cancer cells have a strong dependence on glutamine, which serves as a carbon and nitrogen substrate to sustain rapid growth. Glutamine also aids in self-protection mechanisms. However, the effect of glutamine on ferroptosis in pancreatic cancer remains largely unknown. Here, we aim to explore the association between ferroptosis and glutamine deprivation in pancreatic cancer. The growth of pancreatic cancer cells in culture media with or without glutamine is evaluated using Cell Counting Kit-8. Reactive oxygen species (ROS) are measured by 2',7'-dichlorodihydrofluorescein diacetate staining. Ferroptosis is assessed by BODIPY-C11 dye using confocal microscopy and flow cytometry. Amino acid concentrations are measured using ultrahigh-performance liquid chromatography-tandem mass spectrometry. Isotope-labelled metabolic flux analysis is performed to track the metabolic flow of glutamine. Additionally, RNA sequencing is performed to analyse the genetic alterations. Glutamine deprivation inhibits pancreatic cancer growth and induces ferroptosis both and . Additionally, glutamine decreases ROS formation via glutathione production in pancreatic cancer cells. Interestingly, glutamine inhibitors (diazooxonorleucine and azaserine) promotes ROS formation and ferroptosis in pancreatic cancer cells. Furthermore, ferrostatin, a ferroptosis inhibitor, rescues ferroptosis in pancreatic cancer cells. Glutamine deprivation leads to changes in molecular pathways, including cytokine-cytokine receptor interaction pathways ( , , , , , and ). Thus, exogenous glutamine is required for the detoxification of ROS in pancreatic cancer cells, thereby preventing ferroptosis.

Topics: Humans; Ferroptosis; Glutamine; Reactive Oxygen Species; Apoptosis; Pancreatic Neoplasms

PubMed: 36942991
DOI: 10.3724/abbs.2023029

Authors: Susanne M Geisen, Claudia M N Aloisi, Sabrina M Huber...

The -alkylguanosine adduct -carboxymethyldeoxyguanosine (-CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of -CMdG as well as the corresponding methyl adduct -methyldeoxyguanosine (-MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize -CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of -CMdG and -MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an -serine-CMdG (-Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that -Ser-CMdG forms as an intermediate that spontaneously decomposes to form -CMdG. Finally, we quantified levels of -CMdG and -MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.

Topics: Alkylation; Animals; Azaserine; Cells, Cultured; Deoxyguanosine; Humans; Hydrogen-Ion Concentration; Molecular Structure; Swine

PubMed: 34061515
DOI: 10.1021/acs.chemrestox.0c00471

Authors: Serhat Doğan, Murat Çakir, Adil Kartal...

Hydatid cyst is a zoonotic infestation caused by Echinococcus granulosus, and it is known that some parasites found in humans cause cancer in humans or some may have a protective effect against cancer. Cancer is one of the most serious health problems of today and it has been shown in some studies that parasites such as Echinococcus granulosus can have an inhibitory effect. The aim of this study was determined as whether Echinococcus granulosus has an inhibitory effect on exocrine pancreatic cancer with the help of the azaserine-rat model used in different cancer studies.  Material and Methods: During experimental process a total of 45 male Wistar rats used, 14-day-old male Wistar rats were divided into groups according to the experimental protocol, administered azaserine injection protocol or kept as a control group without azaserine injection. Animals are grouped as Group 1, Control Group (group not treated with Azaserine and not injected with protoscolex.) (E-A-) (n=7); Group 2, Group injected with (IP) Azaserine only (30mg/kg) (E-A+)  (n=8);Group 3, Group injected (IP) with protoscolex suspension of 1 cc only (E+A-) (n=15);Group 4, Group injected both Azaserine (IP) and protoscolex suspension (IP) (E+A+) (n=15). Atypical Acinar Cell Foci (AACF) load in the exocrine pancreas of each rat was measured quantitatively with the help of a video image analyzer and the AACF load was calculated with the help of a mathematical model. Results: Findings showed that the Atypical Acinar Cell Foci (AACF) burden was statistically significantly lower in the Azaserine+ protoscolex (Azaserine-injected-protoscolex-implanted) rat group compared to the other groups, suggesting that Echinococcosis in the azaserine-rat model could inhibit the development of precursor foci of neoplastic changes in the exocrine pancreas. Conclusion: The most significant aspect of our study is that it contributes new insights into the controversy that Echinococcosis suppresses pancreatic cancer.

Topics: Humans; Rats; Male; Animals; Rats, Wistar; Echinococcus granulosus; Azaserine; Pancreatic Neoplasms; Echinococcosis; Pancreas

PubMed: 37116153
DOI: 10.31557/APJCP.2023.24.4.1307

Authors: Devon Van Cura, Tai L Ng, Jing Huang...

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.

Topics: Azaserine; Biosynthetic Pathways; Methane; Oxidation-Reduction; Multigene Family

PubMed: 37151182
DOI: 10.1002/anie.202304646

Authors: Jing Huang, Andrew Quest, Pablo Cruz-Morales...

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Topics: Biological Products; Multigene Family; Biocatalysis; Streptomyces; Azaserine; Cyclopropanes; Styrene; Cytochrome P-450 Enzyme System; Coenzymes

PubMed: 37138074
DOI: 10.1038/s41586-023-06027-2

Review

Authors: Fuller W Bazer, Jinyoung Kim, Hakhyun Ka...

Interferon tau (IFNT) is the pregnancy recognition signal from ruminant conceptuses. IFNT also acts with P4 to induce expression of genes for transport of nutrients, such as glucose (Gluc) and arginine (Arg) into the uterine lumen to activate mechanistic mammalian target of rapamycin (MTOR) cell signaling that stimulates proliferation, migration, gene transcription and mRNA translation by conceptus trophectoderm (Tr). In ewes, Arg and Gluc increase significantly in the uterine lumen between Days 10 and 15 of pregnancy due to increased expression of transporters for Gluc (SLC2A1 and SLC5A1) and Arg (SLC7A2B) by uterine epithelia. Arg and Gluc stimulate proliferation, migration and mRNA translation by Tr. Arg increases expression of GTP cyclohydrolase 1 (GCH1) and IFNT mRNAs while Arg and Gluc increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1 mRNAs and proteins by Tr cells. GCH1 is required for synthesis of tetrahydrobiopterin, an essential cofactor for all NOS isoforms. Arg is metabolized to nitric oxide and polyamines that increase proliferation and migration of Tr cells. In pigs, Gluc, Arg, leucine (Leu) and glutamine (Gln) increase in the uterine lumen between Days 12 and 15 of pregnancy due to enhanced expression of transporters for Gluc and amino acids. Transporters for Gluc in porcine uterine LE (SLC2A1) and conceptus trophectoderm (SLC2A2) are abundant. Transporters for glutamate and neutral (SLC1A1, SLC1A4) and cationic (SLC7A1, SLC7A2, SLC7A7, SLC7A9) amino acids are expressed in uterine LE and SLC7A3 mRNA is expressed in conceptus Tr. Arg and Leu increase MTOR cell signaling and proliferation of pig Tr, as do Gluc and fructose. Azaserine, an inhibitor of hexosamine biosynthesis, inhibits effects of Gluc and fructose. Thus, select nutrients in the uterine lumen affect gene transcription and mRNA translation to affect conceptus development.

Topics: Animals; Embryonic Development; Endometrium; Extraembryonic Membranes; Female; Fetal Development; Pregnancy; Pregnancy Maintenance; Sheep, Domestic; Sus scrofa; Uterus

PubMed: 22738901
DOI: 10.1262/jrd.2011-019

Review

Authors: Sara Verdura, Elisabet Cuyàs, Verónica Ruiz-Torres...

The flavonolignan silibinin, the major bioactive component of the silymarin extract of (milk thistle) seeds, is gaining traction as a novel anti-cancer therapeutic. Here, we review the historical developments that have laid the groundwork for the evaluation of silibinin as a chemopreventive and therapeutic agent in human lung cancer, including translational insights into its mechanism of action to control the aggressive behavior of lung carcinoma subtypes prone to metastasis. First, we summarize the evidence from chemically induced primary lung tumors supporting a role for silibinin in lung cancer prevention. Second, we reassess the preclinical and clinical evidence on the effectiveness of silibinin against drug resistance and brain metastasis traits of lung carcinomas. Third, we revisit the transcription factor STAT3 as a central tumor-cell intrinsic and microenvironmental target of silibinin in primary lung tumors and brain metastasis. Finally, by unraveling the selective vulnerability of silibinin-treated tumor cells to drugs using CRISPR-based chemosensitivity screenings (e.g., the hexosamine biosynthesis pathway inhibitor azaserine), we illustrate how the therapeutic use of silibinin against targetable weaknesses might be capitalized in specific lung cancer subtypes (e.g., co-mutant tumors). Forthcoming studies should take up the challenge of developing silibinin and/or next-generation silibinin derivatives as novel lung cancer-preventive and therapeutic biomolecules.

PubMed: 34208282
DOI: 10.3390/ph14060559

Authors: Rebekka Vibjerg Jensen, Jacob Johnsen, Steen Buus Kristiansen...

OBJECTIVES

Through the hexosamine biosynthetic pathway (HBP) proteins are modified by O-linked-β-N-acetylglucosamine (O-GlcNAc), which acts as a stress sensor. Augmentation of O-GlcNAc confers cardioprotection against ischemia- reperfusion injury, but its role in ischemic preconditioning (IPC) is unknown. Azaserine and alloxan are unspecific blockers of the HBP and have been used to block the cardioprotective effects of O-GlcNAc. We hypothesized that IPC reduces infarct size and increases O-GlcNAc levels in hearts subjected to ischemia-reperfusion injury, and that these effects could be blocked by azaserine and alloxan.

DESIGN

Isolated rat hearts subjected to 40 min global ischemia and 120 min reperfusion were randomized to control, IPC, IPC + azaserine or alloxan, or control + azaserine or alloxan. The effects on infarct size, hemodynamic recovery, myocardial O-GlcNAc levels, and HBP enzyme activities were determined.

RESULTS

IPC reduced infarct size, increased O-GlcNAc levels, O-GlcNAc-transferase levels, and O-GlcNAc-transferase activity. Azaserine and alloxan did not block the effect of IPC on O-GlcNAc levels and O-GlcNAc-transferase activity.

CONCLUSIONS

IPC increased O-GlcNAc levels though increased O-GlcNAc-transferase expression and activity. Azaserine and alloxan failed to block these effects presumably due to poor specificity and sensitivity of the blockers, and IPC-mediated cardioprotection may therefore still be dependent on O-GlcNAc.

Topics: Acetylglucosamine; Alloxan; Animals; Azaserine; Disease Models, Animal; Glycosylation; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; N-Acetylglucosaminyltransferases; Rats; Rats, Wistar; Recovery of Function; Time Factors; Up-Regulation

PubMed: 23301939
DOI: 10.3109/14017431.2012.756984

Authors: Bilal Moiz, Jonathan Garcia, Sarah Basehore...

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with C metabolic flux analysis (C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed C enrichment differences in UDP-GlcNAc. C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.

PubMed: 33917224
DOI: 10.3390/metabo11040226