A Novel Chinese Medicine Formula Inhibits Non-small Cell Lung Cancer by Triggering Oxidative Stress Dependent on Pentose Phosphate Pathway / 中国肺癌杂志

BACKGROUND@#Non-small cell lung cancer (NSCLC) is one of the most lethal malignancies worldwide. A novel Chinese medicine formula-01 (NCHF-01) has shown significant clinical efficacy in the treatment of NSCLC, but the mechanism of this formula in the treatment of NSCLC is not fully understood. The aim of this study is to investigate the molecular mechanism of NCHF-01 in inhibiting NSCLC.@*METHODS@#Lewis lung cells (LLC) tumor bearing mice were established to detect the tumor inhibitory effect of NCHF-01. The morphological changes of tissues and organs in LLC tumor-bearing mice were detected by hematoxylin-eosin (HE) staining. NSCLC cells were treated by NCHF-01. The effects of cell viability and proliferation were detected by MTT and crystal violet staining experiment. Flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Network pharmacology was used to predict the mechanism of its inhibitory effect of NSCLC. Western blot and immunohistochemistry (IHC) were used to detect the expression of related proteins.@*RESULTS@#NCHF-01 can inhibit tumor growth in LLC tumor-bearing mice, and has no obvious side effects on other tissues and organs. NCHF-01 could inhibit cell viability and proliferation, induce G2/M phase arrest and apoptosis, and promote the increase of ROS level. Network pharmacological analysis showed that NCHF-01 exerts anti-NSCLC effects through various biological processes such as oxidative stress and central carbon metabolism. NCHF-01 can reduce the protein expression and enzyme activity of the key enzymes 6-phosphate glucose dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in the pentose phosphate pathway (PPP).@*CONCLUSIONS@#NCHF-01 can inhibit NSCLC through oxidative stress dependent on the PPP.

PRMT6 promotes tumorigenicity and cisplatin response of lung cancer through triggering 6PGD/ENO1 mediated cell metabolism

Metabolic reprogramming is a hallmark of cancer, including lung cancer. However, the exact underlying mechanism and therapeutic potential are largely unknown. Here we report that protein arginine methyltransferase 6 (PRMT6) is highly expressed in lung cancer and is required for cell metabolism, tumorigenicity, and cisplatin response of lung cancer. PRMT6 regulated the oxidative pentose phosphate pathway (PPP) flux and glycolysis pathway in human lung cancer by increasing the activity of 6-phospho-gluconate dehydrogenase (6PGD) and α-enolase (ENO1). Furthermore, PRMT6 methylated R324 of 6PGD to enhancing its activity; while methylation at R9 and R372 of ENO1 promotes formation of active ENO1 dimers and 2-phosphoglycerate (2-PG) binding to ENO1, respectively. Lastly, targeting PRMT6 blocked the oxidative PPP flux, glycolysis pathway, and tumor growth, as well as enhanced the anti-tumor effects of cisplatin in lung cancer. Together, this study demonstrates that PRMT6 acts as a post-translational modification (PTM) regulator of glucose metabolism, which leads to the pathogenesis of lung cancer. It was proven that the PRMT6-6PGD/ENO1 regulatory axis is an important determinant of carcinogenesis and may become a promising cancer therapeutic strategy.

Research Progress on Correlation of Glucose Metabolism Mechanism with Invasion and Metastasis of Nasopharyngeal Carcinoma Cells / 肿瘤防治研究

Metastasis is the main cause of cancer-related death. Growing evidence has shown that changes in glucose metabolism in nasopharyngeal carcinoma cells affect the invasion and metastasis of nasopharyngeal carcinoma through many pathways. This review summarizes the molecular mechanism underlying abnormal glucose metabolism in nasopharyngeal carcinoma cells and analyzes its relationship with the invasion and metastasis of nasopharyngeal carcinoma, including aerobic glycolysis, aerobic oxidation, and pentose phosphate pathway. The aim is to provide novel approaches using the relationships among glucose metabolism, invasion, and metastasis in the targeted therapy of nasopharyngeal carcinoma.

Cloning, expression and characterization of L-arabinose isomerisefrom thermophilic Anoxybacillus kestanbolensis AC26Sari strain:Bioconversation of L-arabinose to L-ribulose

L-Arabinose isomerase (L-AI) is a pivotal enzyme in the microbial pentose phosphate pathway. It is considered as asignificant biological catalyst in rare sugar production. This enzyme can isomerize L-arabinose into L-ribulose and alsoD-galactose into D-tagatose. Here, we cloned the araA gene encoding L-arabinose isomerase from Anoxybacilluskestanbolensis AC26Sari strain, sequenced and over-expressed in E. coli BL21 (DE3): pLysS. This gene is involved inL-arabinose operon in A. kestanbolensis AC26Sari. DNA sequence analysis revealed an open reading frame of 1,506 bp,capable of encoding a polypeptide of 502 amino acid residues with calculated molecular weight of 55.6776 kDa. Therecombinant was purified by heat treatment and Ni-HisTaq chromatography. The purified enzyme showed maximal activityat pH 8.5 and 65ºC and required divalent cations such as Co2+ and Mn2+ for its activity and thermostability. The apparent Kmvalue of the enzyme for L-arabinose was 6.5 mM (Vmax, 140.1002 U/mg) as determined in the precence of both 1 mM Co2+and Mn2+.

Neonatal transaldolase deficiency: a case report / 中华围产医学杂志

We report a neonate with transaldolase deficiency caused by compound heterozygous variation of the TALDO1 gene. A pregnant woman, who had an adverse pregnancy history, was found with multiple fetal abnormalities on prenatal ultrasound and the following whole exon sequencing indicated a likely pathogenic heterozygous variation of c.462-2A>G and c.574C>T(p.R192C) in TALDO1 gene in the fetus. A body was born at 38 +1 weeks and presented dysmorphic features (cutis laxa/wrinkled skin and low-set ears, etc.), splenomegaly, anemia, abnormal liver function and coagulation. In combination with the prenatal testing results, transaldolase deficiency was diagnosed. The patients still had cutis laxa/wrinkled skin on the back of both hands and neck at one year and three months old. Therefore, for babies with hepatosplenomegaly, anemia, thrombocytopenia, coagulation dysfunction, at the same time with dysmorphic features such as cutis laxa, low-set ears, attention should be paid to the investigation of transaldolase deficiency.

Dihydrolipoamide Acetyltransferase Promotes Nucleic Acid Synthesis by Controlling Phosphogluconate Dehydrogenase Acetylation / 中国生物化学与分子生物学报

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells‚ decarboxylation of glycolytic intermediate pyruvate to acetyl coenzyme A (acetyl-CoA) in mitochondria. Dihydrolipoyl acetyltransferase (DLAT) is a subunit of the pyruvate dehydrogenase complex. Here‚ we reported that DLAT was commonly increased in lung cancer and its expression was associated with worse clinical outcomes. We found that suppression of DLAT in lung cancer cells resulted in reduced nucleic acid biosynthesis and attenuated cancer cell proliferation through controlling acetylation level of 6-phosphogluconate dehydrogenase (6PGD) ‚ the third enzyme in the oxidative pentose phosphate pathway (PPP) ‚ in which ribulose-5-phosphate (Ru-5-P) is produced for nucleic acid biosynthesis. Together‚ our study contributes to recent interest and discussion cross talk in cancer metabolism‚ which contributes to tumor growth. Future mechanistic studies should lead to the elucidation of the mode of action of DLAT in human lung cancer and establish DLAT as a viable drug target.

Betaine supplementation improved L-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

BACKGROUND: The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an L-threonine producer, was used to examine the production of L-threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. RESULTS: Betaine supplementation in the medium of E. coli THRD significantly improved L-threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for L-threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while L-threonine fermentation of the new strain was promoted by betaine addition. Conclusions: We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of L-threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains.

Advances of relationship between protein O-GlcNAcylation and glucose metabolism in tumors / 中国药科大学学报

@#O-GlcNAcylation is the addition of a single N-acetylglucosamine(GlcNAc)moiety to the hydroxyl groups of serine or threonine residues of nuclear and cytoplasmic proteins. The transcription factors, kinases of the metabolic pathways and some cytoplasmic enzymes can be O-GlcNAcylated to affect cell transcription, signal transduction, cell metabolism and other biological functions. Abnormal glucose metabolism of tumors has been a hotspot in the research field of tumor pathogenesis and therapeutic targets recently. O-GlcNAclation regulates the glucose metabolism of tumor by affecting the activity of kinases in the metabolic pathway, which is closely associated with the abnormal glucose metabolism of tumor. The abnormal O-GlcNAcylation is one of the potential reasons of cancer. In this review, in order to provide a theoretical reference for developing anti-tumor targets and drugs targeting O-GlcNAc modification, we briefly summarized how O-GlcNAcylation regulated glucose metabolism on glucose metabolism, glucose uptake, glycolysis, pentose phosphate pathway and tricarboxylic acid cycle in cancer cell.

The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeutic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphorylation required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progression as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress.

Effect of knockdown or overexpression of G6PD on the proliferation and migration of hepatoma cells / 实用医学杂志

Objective To investigate the effect of knockdown or overexpression of G6PD on proliferation, growth and migration of human hepatocellular carcinoma cell PLC/PRF/5. Methods Lentivirus-mediated knock-down or overexpression of G6PD was achieved in human hepatocellular carcinoma cell line PLC/PRF/5. RT-PCR and Western blotting assay were used to detect the overexpression or knockdown of G6PD.Cell proliferation and mi-gration curves were recorded by real-time cell analysis system(RTCA),the cell proportion in the DNA replication phase can be directly displayed with EDU experiment,cell growth ability was detected by colony forming assay. Results The doubling time of cells in G6PD knockdown group was longer than that of the control group,and the cell growth rate decreased significantly,the proportion of cells in proliferative phase(43.2%)was lower than that in the control group,but the rates colony formation and migration were significantly decreased(P<0.05,respective-ly),and the migration curves separated apparently.While no significant differences in proliferation,growth and mi-gration of PLC/PRF/5 cells were found between the over-expressed strain and the control group. Conclusion The reduction of G6PD expression in HCC cells inhibits the proliferation and growth of HCC,which may lay a foun-dation for the further study of the pathogenesis and treatment of HCC.

Function of the pentose phosphate pathway and its key enzyme, transketolase, in the regulation of the meiotic cell cycle in oocytes / 대한생식의학회지

OBJECTIVE: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). METHODS: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). RESULTS: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. CONCLUSION: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da via em resposta à relação NADP/NADPH, é codificada por um número de genes por genoma haplóide e é induzida até 46-vezes por peróxido de hidrogênio em trypomastigotas metacíclicos. Os genes que codificam 6-fosfogluconolactonase, 6-fosfogluconato desidrogenase, transaldolase e transcetolase estão presentes no clone CL Brener como cópia única por genoma haplóide. 6-fosfogluconato desidrogenase é muito instável, mas foi estabilizada introduzindo duas pontes salinas por mutagênese sítio-dirigida. A Ribose-5-fosfato isomerase pertence ao Tipo B; genes que codificam enzimas Tipo A, presentes em mamíferos estão ausentes. A Ribulose-5-fosfato epimerase é codificada por dois genes. As enzimas da via têm um componente citosólico principal, embora várias delas tenham uma localização glicosomal secundária e também, localizações em menor número em outras organelas.

In Silico Identification of 6-Phosphogluconolactonase Genes that are Frequently Missing from Completely Sequenced Bacterial Genomes

6-Phosphogluconolactonase (6PGL) is one of the key enzymes in the ubiquitous pathways of central carbon metabolism, but bacterial 6PGL had been long known as a missing enzyme even after complete bacterial genome sequence information became available. Although recent experimental characterization suggests that there are two types of 6PGLs (DevB and YbhE), their phylogenetic distribution is severely biased. Here we present that proteins in COG group previously described as 3-carboxymuconate cyclase (COG2706) are actually the YbhE-type 6PGLs, which are widely distributed in Proteobacteria and Firmicutes. This case exemplifies how erroneous functional description of a member in the reference database commonly used in transitive genome annotation cause systematic problem in the prediction of genes even with universal cellular functions.