Differentiation-inducing Factor Research Articles

A longer-chain acylated derivative of Dictyostelium differentiation-inducing factor-1 enhances the antimalarial activity against Plasmodium parasites

The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50–100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20–30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment.

Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2-10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2-20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures invitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages invitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

Differentiation-inducing factor-1 reduces lipopolysaccharide-induced vascular cell adhesion molecule-1 by suppressing mTORC1-S6K signaling in vascular endothelial cells

AimsDifferentiation-inducing factor-1 (DIF-1), a compound in Dictyostelium discoideum, exhibits anti-cancer effects by inhibiting cell proliferation and motility of various mammalian cancer cells in vitro and in vivo. In addition, DIF-1 suppresses lung colony formation in a mouse model, thus impeding cancer metastasis. However, the precise mechanism underlying its anti-metastatic effect remains unclear. In the present study, we aim to elucidate this mechanism by investigating the adhesion of circulating tumor cells to blood vessels using in vitro and in vivo systems. Main methodsMelanoma cells (1.0 × 105 cells) were injected into the tail vein of 8-week-old male C57BL/6 mice after administration of DIF-1 (300 mg/kg per day) and/or lipopolysaccharide (LPS: 2.5 mg/kg per day). To investigate cell adhesion and molecular mechanisms, cell adhesion assay, western blotting, immunofluorescence staining, and flow cytometry were performed. Key findingsIntragastric administration of DIF-1 suppressed lung colony formation. DIF-1 also substantially inhibited the adhesion of cancer cells to human umbilical vein endothelial cells. Notably, DIF-1 did not affect the expression level of adhesion-related proteins in cancer cells, but it did decrease the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells by suppressing its mRNA-to-protein translation through inhibition of mTORC1-p70 S6 kinase signaling. SignificanceDIF-1 reduced tumor cell adhesion to blood vessels by inhibiting mTORC1-S6K signaling and decreasing the expression of adhesion molecule VCAM-1 on vascular endothelial cells. These findings highlight the potential of DIF-1 as a promising compound for the development of anti-cancer drugs with anti-metastatic properties.

Direct Differentiation of Bone Marrow Mononucleated Cells Into Insulin-Producing Cells Using 4 Specific Soluble Factors.

Bone marrow-derived stem cells are self-renewing and multipotent adult stem cells that differentiate into several types of cells. Here, we investigated a unique combination of 4 differentiation-inducing factors (DIFs), including putrescine (Put), glucosamine (GlcN), nicotinamide, and BP-1-102, to develop a differentiation method for inducing mature insulin-producing cells (IPCs) and apply this method to bone marrow mononucleated cells (BMNCs) isolated from mice. BMNCs, primed with the 4 soluble DIFs, were differentiated into functional IPCs. BMNCs cultured under the defined conditions synergistically expressed multiple genes, including those for PDX1, NKX6.1, MAFA, NEUROG3, GLUT2, and insulin, related to pancreatic beta cell development and function. They produced insulin/C-peptide and PDX1, as assessed using immunofluorescence and flow cytometry. The induced cells secreted insulin in a glucose-responsive manner, similar to normal pancreatic beta cells. Grafting BMNC-derived IPCs under kidney capsules of mice with streptozotocin (STZ)-induced diabetes alleviated hyperglycemia by lowering blood glucose levels, enhancing glucose tolerance, and improving glucose-stimulated insulin secretion. Insulin- and PDX1-expressing cells were observed in the IPC-bearing graft sections of nephrectomized mice. Therefore, this study provides a simple protocol for BMNC differentiation, which can be a novel approach for cell-based therapy in diabetes mellitus.

Derivatives of Differentiation-Inducing Factor 1 Differentially Control Chemotaxis and Stalk Cell Differentiation in Dictyostelium discoideum.

Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation-inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum.

DIF-1 exhibits anticancer activity in breast cancer via inhibition of CXCLs/CXCR2 axis-mediated communication between cancer-associated fibroblasts and cancer cells.

The tumor microenvironment (TME), largely composed of tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), plays a key role in cancer progression. A small molecule, differentiation-inducing factor-1 (DIF-1) secreted by Dictyostelium discoideum, is known to exhibit anticancer activity; however, its effect on the TME remains unknown. In this study, we investigated the effect of DIF-1 on the TME using mouse triple-negative breast cancer 4T1-GFP cells, mouse macrophage RAW 264.7 cells, and mouse primary dermal fibroblasts (DFBs). Polarization of 4T1 cell-conditioned medium-induced macrophage into TAMs was not affected by DIF-1. In contrast, DIF-1 decreased 4T1 cell co-culturing-induced C-X-C motif chemokine ligand 1 (CXCL1), CXCL5, and CXCL7 expression in DFBs and suppressed DFB differentiation into CAF-like cells. Additionally, DIF-1 inhibited C-X-C motif chemokine receptor 2 (CXCR2) expression in 4T1 cells. Immunohistochemical analyses of tumor tissue samples excised from breast cancer-bearing mice showed that DIF-1 did not affect the number of CD206-positive TAMs; however, it decreased the number of α-smooth muscle actin-positive CAFs and CXCR2 expression. These results indicated that the anticancer effect of DIF-1 was partially attributed to the inhibition of CXCLs/CXCR2 axis-mediated communication between breast cancer cells and CAFs.

Differentiation-inducing factor 1 activates cofilin through pyridoxal phosphatase and AMP-activated protein kinase, resulting in mitochondrial fission

Differentiation-inducing factor 1 (DIF-1) is a morphogen produced by Dictyostelium discoideum that inhibits the proliferation and migration of both D. discoideum and most mammalian cells. Herein, we assessed the effect of DIF-1 on mitochondria, because DIF-3, which is similar to DIF-1, reportedly localizes in the mitochondria when added exogenously, however the significance of this localization remains unclear. Cofilin is an actin depolymerization factor that is activated by dephosphorylation at Ser-3. By regulating the actin cytoskeleton, cofilin induces mitochondrial fission, the first step in mitophagy. Here, we report that DIF-1 activates cofilin and induces mitochondrial fission and mitophagy mainly using human umbilical vein endothelial cells (HUVECs). AMP-activated kinase (AMPK), a downstream molecule of DIF-1 signaling, is required for cofilin activation. Pyridoxal phosphatase (PDXP)—known to directly dephosphorylate cofilin—is also required for the effect of DIF-1 on cofilin, indicating that DIF-1 activates cofilin through AMPK and PDXP. Cofilin knockdown inhibits mitochondrial fission and decreases mitofusin 2 (Mfn2) protein levels, a hallmark of mitophagy. Taken together, these results indicate that cofilin is required for DIF-1- induced mitochondrial fission and mitophagy.

<i>Dictyostelium</i> Differentiation-inducing Factor Derivatives Reduce the Glycosylation of PD-L1 in MDA-MB-231 Human Breast Cancer Cells

Objectives Triple-negative breast cancer (TNBC) is a metastatic and intractable cancer with limited treatment options. Refractory cancer cells often express the immune checkpoint molecules programmed death-ligand 1 (PD-L1) and PD-L2, which inhibit the anticancer effects of T cells. Differentiation-inducing factors, originally found in Dictyostelium discoideum, and their derivatives possess strong antiproliferative activity, at least in part by reducing cyclin D1 expression in various cancer cells, but their effects on PD-L1/PD-L2 have not been examined. In this study, we investigate the effects of six DIF compounds (DIFs) on the expression of PD-L1/PD-L2 and cyclin D1/D3 in MDA-MB-231 cells, a model TNBC cell line.Methods MDA-MB-231 cells were incubated for 5 or 15 h with or without DIFs, and the mRNA expression of cyclin D1, PD-L1, and PD-L2 were assessed by quantitative polymerase chain reaction (qPCR). Whereas, MDA-MD-231 cells were incubated for 12 or 24 h with or without DIFs, and the protein expression of cyclins D1 and D3, PD-L1, and PD-L2 were assessed by Western blotting.Results As expected, some DIFs strongly reduced cyclin D1/D3 protein expression in MDA-MB-231 cells. Contrary to our expectation, DIFs had little effect on PD-L1 mRNA expression or increased it transiently. However, some DIFs partially reduced glycosylated PD-L1 and increased non-glycosylated PD-L1 in MDA-MB-231 cells. The level of PD-L2 was very low in these cells.Conclusion Since PD-L1 glycosylation plays an important role in preventing T cells from attacking cancer cells, such DIFs may promote T cell attack on cancer cells in vivo.

SPDR-2 DEVELOPMENT OF A NOVEL THERAPEUTIC STRATEGY AGAINST MEDULLOBLASTOMA TARGETING MYC

Abstract Medulloblastoma (MB) is the most common pediatric malignant brain tumor, with aggressive behavior and central nervous system dissemination. Standard of care for medulloblastoma include surgery, chemotherapy with platinum agent, and craniospinal radiation. If patients survived with these intensive treatments, frequently faced treatment-related adverse events. Therefore, development of novel therapeutic strategy is an important issue. Driver mutation underlying MB pathogenesis have been discovered, and molecular profiling led to the identification of subgroups; Wingless (WNT), Sonic Hedgehog(SHH), Group3, and Group4. Group 3, especially the group with MYC/MYCN amplification/overexpression confer a poor prognosis. Myc is attracting as a therapeutic target, however, to date, no drugs have been developed that target myc. We had found that N-myc downstream regulated gene-1 (NDRG1) protein expression is a good prognostic factor in glioblastoma, a representative of adult malignant brain tumors, and that Differentiation inducing factor-1 (DIF-1) increased NDRG1 expression. We reported that DIF-1 crossed the Blood-Brain Barrier and could be an effective treatment for GBM in vivo. In this research process, we found that DIF-1 markedly reduced Myc expression in patient-derived glioblastoma stem cells with MYC/MYCN amplification. Based on this result, we found that DIF-1 decreased Myc expression and inhibited cell survival/proliferation even in medulloblastoma cell lines with MYC/MYCN amplification. In this presentation, we will present the results obtained in the research of a novel Myc-targeted therapy using DIF-1 for MB with MYC/MYCN amplification.

CSIG-04. BMP4 INDUCTION OF A SENESCENCE-LIKE PHENOTYPE IN HUMAN GLIOBLASTOMA CELLS IS PARTIALLY DEPENDENT ON P21

Abstract Bone morphogenetic protein 4 (BMP4) was initially suggested as a potential differentiation-inducing factor to be used in the therapy of glioblastoma. We and others have however demonstrated that the response is reversible, variable among patient samples, and heterogeneous within the same cell line. To deepen our knowledge on how BMP4 affects different types of glioblastoma cells, we treated phenotypically different clones from the same patient tumor—a multitherapy-sensitive/proneural-like (SENS/PN) clone and a multitherapy-resistant/mesenchymal-like (RES/MES) clone—with BMP4. In the SENS/PN clone, BMP4 turned on a mesenchymal-related gene program, whereas this response was less prominent in the RES/MES clone. Untreated SENS/PN cells were smaller than RES/MES cells, but both clones responded to BMP4 by cell size enlargement. Increase in cell size has been suggested to precede senescence; young cells are smaller than old cells that eventually enter replicative senescence. BMP4 induced a senescence-like phenotype in a subpopulation of cells, demonstrated by induction of senescence-associated (SA)-β-gal, p21 up-regulation, lamin B1 down-regulation, as well as increased lysosomal mass and granularity. This was more pronounced in the RES/MES clone than in the SENS/PN clone, and it was dependent on canonical SMAD signaling. Senolytic treatment ablated the SA-β-gal positive cells and reduced the p21 level at the population level. Targeted deletion of p21 abolished BMP4-induced SA-β-gal and increase in cell growth, while lamin B1 down-regulation remained, demonstrating that p21 signaling is crucial for one part of the senescence induction by BMP4. We are currently further investigating a connection between cell size, mesenchymality and senescence. We hypothesize that large mesenchymal-like cells are closer to senescence than smaller proneural cells within the cell culture. A combination of senescence-induction and senolytic treatment may open treatment opportunities to target therapy-resistant glioblastoma cells.

Novel RNAseq-Informed Cell-type Markers and Their Regulation Alter Paradigms of Dictyostelium Developmental Control.

Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis.

Chromatin Reorganization during Myoblast Differentiation Involves the Caspase-Dependent Removal of SATB2.

The induction of lineage-specific gene programs are strongly influenced by alterations in local chromatin architecture. However, key players that impact this genome reorganization remain largely unknown. Here, we report that the removal of the special AT-rich binding protein 2 (SATB2), a nuclear protein known to bind matrix attachment regions, is a key event in initiating myogenic differentiation. The deletion of myoblast SATB2 in vitro initiates chromatin remodeling and accelerates differentiation, which is dependent on the caspase 7-mediated cleavage of SATB2. A genome-wide analysis indicates that SATB2 binding within chromatin loops and near anchor points influences both loop and sub-TAD domain formation. Consequently, the chromatin changes that occur with the removal of SATB2 lead to the derepression of differentiation-inducing factors while also limiting the expression of genes that inhibit this cell fate change. Taken together, this study demonstrates that the temporal control of the SATB2 protein is critical in shaping the chromatin environment and coordinating the myogenic differentiation program.

Differentiation inducing factor-1 suppressed the epithelial-mesenchymal transition via degradation of Yes-associated protein

Differentiation-inducing factors (DIFs), produced by Dictyostelium discoideum, show anti-tumor activity via several different signaling molecules, including glycogen synthase kinase-3 (GSK-3) and signal transducer and activator of transcription 3 (STAT3). However, the exact mechanism of action of DIFs is still poorly understood. Therefore, to better understand the action of DIF, we performed DNA microarray analysis and found that the activity of the Hippo signaling pathway may be modified. Therefore, we analyzed mRNA expressions of Hippo signaling pathway target genes. Although the mRNA expression of the target genes including CTGF and Cyr61were elevated by DIF-1, surprisingly the protein expression themselves were reduced. In an attempt to elucidate the mechanism, we found that DIF promoted proteolysis of Yes-associated protein (YAP), a transcriptional co-activator of the Hippo signaling pathway, in the human cervical cell line HeLa and the human colon cancer cell line HCT-116. YAP has also been reported to promote epithelial-to-mesenchymal transition (EMT). Indeed, cell migration, cell invasion and expressions of EMT-related proteins (fibronectin, vimentin and N-cadherin) were reduced by DIF-1. These results suggest that DIF-1 suppresses EMT via degradation of YAP in human cancer cell lines.

Differentiation-inducing factor 1 induces mitochondrial fission via Cofilin activation in endothelial cells

Differentiation-inducing factor 1 induces mitochondrial fission via Cofilin activation in endothelial cells

Differentiation-inducing factor-1 inhibited cancer cell adhesion to vascular endothelial cells via curtailing VCAM-1 protein synthesis

Differentiation-inducing factor-1 inhibited cancer cell adhesion to vascular endothelial cells via curtailing VCAM-1 protein synthesis

Differentiation-inducing factor-1 affects cancer-associated fibroblasts (CAF) in triple negative breast cancer through CXCLs/CXCR2 axis

Differentiation-inducing factor-1 affects cancer-associated fibroblasts (CAF) in triple negative breast cancer through CXCLs/CXCR2 axis

Differentiation-inducing factor-1は腫瘍細胞と血管内皮細胞の接着抑制を介して抗転移効果を発揮する

Differentiation-inducing factor-1は腫瘍細胞と血管内皮細胞の接着抑制を介して抗転移効果を発揮する

The AMPK-mTORC1 system as a drug target: Anticancer mechanisms of the Dictyostelium differentiation-inducing factor DIF

The AMPK-mTORC1 system as a drug target: Anticancer mechanisms of the Dictyostelium differentiation-inducing factor DIF