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    • In soft agar colony formation

    2021-09-17

    In soft agar colony formation assay, transformed pyridoxal phosphate indicate anchorage-independent growth and colony formation without attachment on the bottom of cell culture plate. This assay can investigate transformation and tumorigenic activities [24]. Our recent study showed that the long-term 5-FU treatment induced the large sized colonies in DLD1 cells [12]. In this study, the large sized colonies were formed in DLD-5FU-G and hmDLD1 cells. The colony formation activity of DLD-5FU-G cells was enhanced by FFA4 knockdown. In addition, FFA1 knockdown reduced the colony formation activity in hmDLD1 cells. These findings suggest that FFA1 positively and FFA4 negatively regulate the colony formation activity in DLD1 cells. In conclusion, this study showed that FFA1- and FFA4-mediated signaling are involved in the regulation of cellular functions during tumor progression in DLD1 cells. In addition to FFA1 and FFA4, several GPCRs have been identified as FFARs, such as FFA2 and FFA3 which are activated by short chain FFAs [2], [3], [25]. To better understand the roles of FFARs in the pathogenesis of cancer cells, we are currently investigating the roles of other FFARs in cellular functions of colon cancer cells.
    Acknowledgements This work was supported by JSPS KAKENHI Grant Number 24590493 and by Grants from the Faculty of Science and Engineering, Kindai University.
    Introduction The development of hyperlipidemia and chronic exposure to elevated levels of fatty acids (FAs) is a contributor to the pathogenesis of the metabolic syndrome and type 2 diabetes (T2D), promoting cellular dysfunction and/or cell death of the insulin producing beta cells. Lipotoxicity in pancreatic beta cells result from accumulation of long chain saturated FAs (LCSFAs), and remarkably, cosupplementation with monounsaturated FAs (MUFAs) counteract saturated FA (SFA) induced lipoapoptosis [1]. In fact, numerous studies in various cell types demonstrate that the extent and nature of the effects induced by FAs are dependent on the molecular species of FA and the length of exposure (refs). Reduced plasma levels of the insulin secretagogue Glucagon like peptide-1 (GLP-1) are detected in T2D, insulin resistance, increased BMI and obesity independent of diabetes [2,3]. A hallmark of these conditions is hyperlipidemia. FAs, and especially monounsaturated FAs (MUFAs) stimulate GLP-1 release in vivo [4] and in vitro [5,6]. However, a reduced number of intestinal GLP-1-producing cells are detected following hyperlipidemia and insulin resistance induced by a diet rich in saturated fat in vivo [7,8], and long term exposure to elevated levels of SFA (16:0) induce apoptosis of GLP-1-producing cells in vitro [[9], [10], [11], [12]]. In contrast, MUFA (18:1) does not induce apoptosis of GLP-1 secreting cells, but - in similarity to effects on the pancreatic beta cells - protect against the SFA induced lipotoxicity [6]. These findings support the hypothesis that lipotoxic reductions in L-cell number may reduce plasma levels of GLP-1 following long-term hyperlipidemia. Hyperlipidemia may thus reduce insulin secretion and beta cell viability by dual mechanisms, i.e. by direct actions on the beta cells and by reducing the potentiating/protective effects of GLP-1 on insulin secretion/beta cell viability [13]. FAs are taken up into cells mainly by membrane-associated FA-binding proteins and stored as triglycerides (TG) in times of abundant nutrient supply. However non-adipose cells have a limited capacity for TG storage and an abundant supply of FAs may thus instead increase FA oxidation (FAO), or generate lipid derivatives such as diacylglycerol (DAG) and ceramide, the latter implicated in propagation of lipotoxic effects in insulin secreting and GLP-1 secreting cells [6,14]. De novo synthesis of ceramide occurs in the endoplasmic reticulum (ER) - and to a lesser extent in the mitochondrial membrane [15]. However, the mitochondria-associated membranes facilitate transfer of ceramide from the ER to mitochondria [16], which play a central role in the regulation of ceramide-induced apoptosis [15]. Whereas ceramide induced ER stress has been implicated in the lipotoxic effects of SFAs [17], ceramides also inhibit the mitochondrial electron transport chain, thereby increasing generation of reactive oxygen species (ROS) [18,19]. However, a ROS increase – perhaps stimulated by increased fatty acid oxidation (FAO) in the presence of elevated substrate concentrations [20] - may also stimulate ceramide-releasing enzymes and whether ceramide is an upstream or downstream event of ROS in ceramide-induced apoptosis depends on the cell type and stimuli [15,21].