Neutrophils (polymorphonuclear leukocytes) are short lived granulocytes that playa primordial role in host innate defense against invading pathogens. Freshly isolated neutrophils spontaneously undergo apoptosis when cultured, which is associated with oxidative stress. We found that there is a dramatic shift in the metabolism of the 5-lipoxygenase product 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from its biologically inactive o-oxidation product in freshly isolated neutrophils to the potent granulocyte chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in neutrophils cultured for 24 h. o-oxidation of the chemoattractant leukotriene B4 (LTB4) was also reduced in aging neutrophils incubated with arachidonic acid, resulting in higher levels of LTB4. The reduced o-oxidation activity appeared to be due to a decrease in active LTB4 20-hydroxylase. In contrast, the increased 5-oxo-ETE formation was not associated with an increase in the amount of active 5-hydroxyeicosanoid dehydrogenase, which is required for its formation, but rather with a dramatic increase in its cofactor NADP +. NAD+ levels also increased, but NADPH levels remained unchanged after 24 h. There was also evidence for increased oxidative stress (high GSSG/GSH) in aging neutrophils. The changes in 5-HETE metabolism and pyridine nucleotides in cultured neutrophils could be inhibited by neutrophil survival factors and antioxidants. These results suggest that in severe inflammation, aging neutrophils that have evaded rapid uptake by macrophages may produce increased amounts of the chemoattractants 5-oxo-ETE and LTB4, resulting in delayed resolution of inflammation. Similarly, we found that the NADPH oxidase activator PMA caused a very rapid and dramatic increase in NADP + levels in both freshly isolated and cultured neutrophils, accompanied by a rapid increase in 5-oxo-ETE synthesis and reduced o-oxidation activity. Surprisingly, this was not accompanied by a corresponding decline in NADPH levels, which instead initially increased, but rather by a precipitous reduction in NAD+, which mirrored the increase in NADP+. These results suggest that the phosphorylation of NAD+ by NAD kinase may be very important for providing both NADP+ for 5-oxo-ETE synthesis and NADPH for the respiratory burst.

Ethanolamine was shown to replace vitamin B6 in the nutrition of excised tomato roots grown in sterile culture (Boll, 1954a, 1959a). This replacement cannot be explained as a precursor effect (Boll, 1954a; Nillernot, 1962). An alternative explanation of the replacement was made by Boll (1954a, 1959a). The explanation involved negative feedback control, by ethanolamine, of a postulated serine decarboxylase yielding ethanolamine, and requiring pyridoxal phosphate as coenzyme. In this way, ethanolsmine would exert a sparing action on the limited amount of vitamin B6 which, as shown by Boll (1954a), is synthesized by the roots. [...]

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Glioblastoma (GBM), similar to many other cancers, exhibits enhanced aerobic glycolysis with concomitant lactate production, a phenomenon known as the Warburg effect. We have demonstrated that preferential expression of Hexokinase 2 (HK2) is a critical mediator of metabolic reprograming in GBMs and its inhibition is a potential therapeutic strategy for sensitization of GBM tumors to radiation (RAD) and/or temozolomide (TMZ). Our results indicate that conditional HK2 inhibition disrupts energy homeostasis and sensitizes GBMs to radiochemotherapy under hypoxia. In GBM xenografts, conditional HK2 loss sensitizes GBM tumors to concomitant RAD/TMZ and results in a significant survival benefit in the mice. Moreover, loss of HK2 resulted in GBM remodeling with HK2 knockdowns showing increased necrosis, hypoxia, inflammatory infiltration and reduced vascularization. We anticipate that targeting a key metabolic enzyme involved in the Warburg effect might improve the efficacy of current therapeutic regimen and provide a unique paradigm for the management of GBMs.

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. To better understand the mechanisms of sensitivity and resistance to mitochondrial translation inhibition, we treated TEX leukemia cells with increasing concentrations of tigecycline over 4 months, and selected a population of cells resistant to tigecycline (RTEX+TIG). Compared to their wild type counterparts, the resistant RTEX+TIG cells had an altered metabolic profile with diminished oxidative phosphorylation and a greater reliance on glycolysis. Upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation to wild type levels. At the molecular level, these cells had increased levels of HIF1a. Strikingly, tigecycline-resistant cells had decreased expression of CD34 and CD117, clonogenic growth potential and engraftment capabilities in vivo. Thus, chronic inhibition of mitochondrial translation leads to the establishment of rho-zero-like metabolic phenotype, and is associated with differentiation of leukemia cells.

Human leukemia cell K562 in culture displayed an absolute requirement for folate, proliferating at optimal rate only when the total intracellular folate concentration was greater than 1.5 uM. This critical intracellular concentration was maintained by extracellular 5-formyltetrahydrofolic acid or 5-methyltetrahydrofolic acid at a 100-fold lower concentration than folic acid. Growth rate of folate-deficient cells was proportional to intracellular concentration of fully-reduced, non-methylated folate but not to intracellular 5-methyltetrahydrofolate concentration. Growth of folate-deficient cells was stimulated by inosine but not by thymidine, suggesting that the primary cause of growth restriction in these cells was purine deficiency. Serine stimulated, and glycine inhibited, growth of K562 cells, likely by modulating the supply of one-carbon units for folate metabolism. Folate-deficient cells were larger in size than folate-replete cells, but megaloblastic morhology was not observed. DNA content of folate-deficient cells was predominantly characteristic of cells in S or G2/M phases of the cell cycle.