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 µ 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 µM, 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.
Highlights
The cellular slime mold Dictyostelium discoideum is a soil microorganism that forms a fruiting body consisting of spores and a multicellular stalk at the end of its life cycle
We show that Differentiation-inducing factor-3 (DIF-3), Bu-DIF-3, and BODIPY-DIF-3 have potential as potent mitochondrial uncouplers to induce morphological change and dysfunction of mitochondria
BODIPY-DIF-3 at 20 mM was rapidly incorporated into the cells within several minutes of exposure and a fluorescence signal was detected within 30 min (Figure 2A)
Summary
The cellular slime mold Dictyostelium discoideum is a soil microorganism that forms a fruiting body consisting of spores and a multicellular stalk at the end of its life cycle. The precise mechanisms underlying the actions of DIF-1 remain to be elucidated, and there have been no receptor(s) identified for DIF-1. In addition to their physiological activity in D. discoideum, DIF-1 and DIF-3 possess anti-tumor activity by suppressing cell growth and, in some cases, by inducing or promoting the differentiation of de-differentiated tumor cells in vitro [5,6,7,8,9,10,11]. We showed that several chemically synthesized derivatives of DIF-3 with a modification at the methoxy group, such as butoxy-DIF-3 (Bu-DIF-3) (Figure 1A), have a potent antiproliferative effect in K562 cells [13] These findings suggest that derivatives of DIF-3 are potential candidates for development as novel anti-cancer drugs. The mechanisms underlying the actions of DIF-like molecules have been investigated by using several approaches [6,7,8,9,10,11], [14] but remain to be elucidated
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