Correction: Mitochondria Are the Target Organelle of Differentiation-Inducing Factor-3, an Anti-Tumor Agent Isolated from Dictyostelium Discoideum

Abstract

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5-20 m M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 mM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1-3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.

Dictyostelium: An Important Source of Structural and Functional Diversity in Drug Discovery

“…The mechanisms underlying the antitumor activities of DIFs 1 and 3 and their derivatives have been partially elucidated (Figure 4); for example, it has been reported that (1) they rapidly increase intracellular calcium concentration in several tumor cell lines [62,63,64,68,69]; (2) they directly inhibit the activities of calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) [76] and p21-activated kinase 1 (PAK1) [74]; (3) they function as mitochondrial uncouplers and disrupt mitochondrial functions, possibly resulting in the induction of mitophagy and autophagy [69,77]; (4) they affect the activities of several crucial enzymes such as phosphatidylinositol 3-kinase (PI3K) and Akt kinase (protein kinase B) [78], extracellular signal-regulated kinase (Erk) [65,79] and glycogen synthase kinase-3β (GSK-3β) [66,80] in several tumor cell lines; and (5) they suppress the expression of cyclins D/E and reduce the phosphorylation of retinoblastoma protein (pRB), resulting in cell-cycle arrest at the G 1 /G 0 phase [70,79,81].…”

“…However, since DIF-1 promotes glucose uptake even in the presence of the PI3K inhibitors wortmannin and LY294002 in 3T3-L1 fibroblasts and 3T3-L1 adipocytes [84], the glucose uptake-promoting effect of DIF-1 is likely PI3K−Akt-independent. Also, DIF-1 and DIF-1 (3M) disturb mitochondrial activity, possibly by acting as uncouplers and promote cellular glucose metabolism in vitro [77,85]. During analysis of the antitumor activity of DIF-3, Dubois et al [69] found that DIF-3 at 20 µM induces a loss of mitochondrial membrane potential, possibly by acting as an uncoupler and decreases cellular ATP levels in K562 leukemia cells.…”

“…Since DIFs possess various biological activities, there would be multiple target molecules of the DIFs; some candidate targets of DIFs that may be involved in the functions of DIFs have been reported (Table 1). Direct inhibition of PDE1 and PAK1 by DIFs may cause antitumor effects [74,76], whereas uncoupling of mitochondrial activity by DIFs may cause antitumor effects and/or promote glucose consumption in mammalian cells [72,77,85]. Matsuda et al [99] have reported that DIF-1 but not DIF-3, directly inhibits mitochondrial malate dehydrogenase (mMDH), which may affect glucose consumption.…”

“…After the addition of one of the DIFs to tumor cells, the DIF rapidly (within several minutes) disturbs mitochondrial function [69,77], increases reactive oxygen species (ROS) production [69] and intracellular calcium concentration ([Ca 2+ ] i ) [63,64,69,77] and inhibits the activities of p21-activated kinase 1 (PAK1) [74] and calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) (resulting in an increase in cAMP levels) [76]. Over time (from several minutes to hours) the DIF also affects the activities of extracellular signal-regulated kinase (Erk), signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3-kinase (PI3K)–Akt, glycogen synthase kinase-3β (GSK-3β) and the Wnt/β-catenin pathway, which suppresses the expression of cyclins D/E (and promotes the degradation of cyclin D1) and the subsequent reduction of phospho-pRB [65,66,74,77,78,79,80,81]. At appropriate concentrations the DIFs have been found to induce growth arrest in all the tumor cell lines tested to date in vitro and in vivo and at higher concentrations they have induced caspase-independent cell death [67,68,69].…”

“…Stimulation with a DIF induces glucose transporter 1 (GLUT1) translocation from intracellular vesicles to the plasma membrane (PM) via a PI3K–Akt-independent pathway, resulting in the promotion of glucose uptake by confluent mammalian cells [84]; the DIFs activate PI3K and Akt but this is not related to the DIF glucose uptake-promoting activity [84]. The DIFs also function as mitochondrial uncouplers, promoting oxygen consumption [77] and glucose metabolism (glycolysis and subsequent degradation in the tricarboxylic acid (TCA) cycle) [85] by mitochondria; this further increases GLUT1 translocation and promotes glucose uptake into the cells. Chemical structure-activity relationship analysis revealed that DIF-1 and DIF-1 (3M) are promising lead compounds for the development of anti-diabetes and anti-obesity drugs [71,84,85].…”