During the course of purifying nerve growth factor from the submaxillary gland of the mouse, Cohen (1960) and Levi-Montalcini and Cohen (1960) noticed that daily injections of certain gland extract fractions into newborn mice produced developmental changes that could not be ascribed to nerve growth factor. These changes included precocious opening of the eyelids (7 days compared to the usual 14 days) and a similar early eruption of the incisors. Using these gross anatomical changes as an assay, Cohen (1962) proceeded to isolate the active factor — a polypeptide which he termed epidermal growth factor (EGF).

Epidermal growth factor

Most cancer cells exhibit increased glycolysis and use this metabolic pathway for generation of ATP as a main source of their energy supply. This phenomenon is known as the Warburg effect and is considered as one of the most fundamental metabolic alterations during malignant transformation. In recent years, there are significant progresses in our understanding of the underlying mechanisms and the potential therapeutic implications. Biochemical and molecular studies suggest several possible mechanisms by which this metabolic alteration may evolve during cancer development. These mechanisms include mitochondrial defects and malfunction, adaptation to hypoxic tumor microenvironment, oncogenic signaling, and abnormal expression of metabolic enzymes. Importantly, the increased dependence of cancer cells on glycolytic pathway for ATP generation provides a biochemical basis for the design of therapeutic strategies to preferentially kill cancer cells by pharmacological inhibition of glycolysis. Several small molecules have emerged that exhibit promising anticancer activity in vitro and in vivo, as single agent or in combination with other therapeutic modalities. The glycolytic inhibitors are particularly effective against cancer cells with mitochondrial defects or under hypoxic conditions, which are frequently associated with cellular resistance to conventional anticancer drugs and radiation therapy. Because increased aerobic glycolysis is commonly seen in a wide spectrum of human cancers and hypoxia is present in most tumor microenvironment, development of novel glycolytic inhibitors as a new class of anticancer agents is likely to have broad therapeutic applications.

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Glycolysis inhibition for anticancer treatment

Free radicals and diabetes

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

/pdf/recent-advances-in-2d-and-3d-in-vitro-systems-using-primary-46n7hrqknx.pdf

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Protein phosphorylation is now recognized to be the major general mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli However, only recently has the idea that different cellular functions are controlled by common protein kinases and protein phosphatases started to gain widespread acceptance Thus there is an integrated network of regulatory pathways, mediated by phosphorylation-dephosphorylation, that allows diverse cellular events to be coordinated by neural and hormonal stimuli The evidence that supports this concept is reviewed, with emphasis on the role of protein phosphorylation in enzyme regulation

The role of protein phosphorylation in neural and hormonal control of cellular activity

Adult rat hepatocytes have been previously isolated and maintained in monolayer culture, but attempts to stimulate DNA synthesis have been unsuccessful. Hormonal conditions are now described which induce DNA synthesis in cultured hepatocytes from partially hepatectomized rats. DNA synthesis was determined autoradiographically by the incorporation of [3H]thymidine into nuclei of morphologically distinct hepatocytes. Insulin (4-4000 nM) or epidermal growth factor (10 ng/ml) alone caused significant increases in the labeling index. The two hormones together acted synergistically to produce labeling indices of 35-50% on the third day of culture, compared with 2-7% in control cultures. The addition of glucagon (400 nM) further increased the labeling indes. Dexamethasone (80 ng/ml) inhibited DNA synthesis but, under certain conditions, enhanced cell attachment. Growth hormone and triiodothyronine had no significant effect on DNA synthesis. The mixture of epidermal growth factor, insulin, and glucagon also stimulated incorporation of [3H]thymidine into phenol-extracted DNA. Although DNA synthesis was stimulated, cell division occurred infrequently. These data suggest a prominent role for epidermal growth factor in promoting hepatic DNA synthesis by acting in concert with insulin and glucagon.

https://www.pnas.org/content/pnas/73/10/3589.full.pdf

Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes.

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats.

Publisher Summary Fructose 2,6-bisphosphate (fructose 2,6-P 2 ) is a unique sugar diphosphate that is an important regulator of hepatic carbohydrate metabolism. This chapter describes the discovery and identification of the compound and its chemical synthesis; discusses the regulation of its concentration in liver and of the enzymes responsible for its metabolism; summarizes what is known of the mechanism of the effect of fructose 2,6-P 2 in the regulation of 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase activity; and describes the role of this effector in the regulation of hepatic carbohydrate metabolism. Fructose 2,6-P 2 has been found in all mammalian tissues that have been studied. The highest concentrations have been found in liver, brain, and heart muscle. Lower concentrations have been found in skeletal muscle, kidney, and epididymal fat. It has also been detected in rat pancreatic islets, and its concentration increases upon exposure of the islets to glucose. The compound has been isolated also from Saccharomyces cerevisiae grown on glucose, from mung beans, and from spinach leaves.

Simon J. Pilkis

Papers

Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes.

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats.

The Role of Fructose 2,6-Bisphosphate in the Regulation of Carbohydrate Metabolism

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase.