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.

Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

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.

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.

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.

Derivatives of Dictyostelium differentiation-inducing factors suppress the growth of Plasmodium parasites in vitro and in vivo.

Malaria, which is caused by protozoa of the genus Plasmodium, remains a major endemic public health problem worldwide. Since artemisinin combination therapies are used as a first-line treatment in all endemic regions, the emergence of parasites resistant to these regimens has become a serious problem. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone originally found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivatives exhibit a range of biological activities. In the present study, we investigated the effects of 41 DIF derivatives on the growth of Plasmodium falciparum in vitro using four laboratory strains and 12 field isolates. Micromolar concentrations of several DIF derivatives strongly suppressed the growth of the four laboratory strains, including strains that exhibited resistance to chloroquine and artemisinin, as well as strains that were susceptible to these drugs. In addition, DIF-1(+2), the most potent derivative, strongly suppressed the growth of 12 field isolates. We also examined the effects of DIF-1(+2) on the activity of the rodent malarial parasite Plasmodium berghei in mice. Intraperitoneal administration of DIF-1(+2) over 4 days (50 or 70 mg/kg/day) significantly suppressed the growth of the parasite in the blood with no apparent adverse effects, and a dose of 70 mg/kg/day significantly prolonged animal survival. These results suggest that DIF derivatives, such as DIF-1(+2), could serve as new lead compounds for the development of antimalarial agents.

Dictyostelium Differentiation-Inducing Factor-1 Promotes Glucose Uptake, at Least in Part, via an AMPK-Dependent Pathway in Mouse 3T3-L1 Cells.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (a polyketide) found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivative, DIF-1(3M) promote glucose consumption in vitro in mammalian cells and in vivo in diabetic rats; they are expected to be the leading antiobesity and antidiabetes compounds. In this study, we investigated the mechanisms underlying the actions of DIF-1 and DIF-1(3M). In isolated mouse liver mitochondria, these compounds at 2-20 µM promoted oxygen consumption in a dose-dependent manner, suggesting that they act as mitochondrial uncouplers, whereas CP-DIF-1 (another derivative of DIF-1) at 10-20 µM had no effect. In confluent mouse 3T3-L1 fibroblasts, DIF-1 and DIF-1(3M) but not CP-DIF-1 induced phosphorylation (and therefore activation) of AMP kinase (AMPK) and promoted glucose consumption and metabolism. The DIF-induced glucose consumption was reduced by compound C (an AMPK inhibitor) or AMPK knock down. These data suggest that DIF-1 and DIF-1(3M) promote glucose uptake, at least in part, via an AMPK-dependent pathway in 3T3-L1 cells, whereas cellular metabolome analysis revealed that DIF-1 and DIF-1(3M) may act differently at least in part.

Two New Terpenes Isolated from Dictyostelium Cellular Slime Molds.

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.

Melibiosamine, a novel oligosaccharide, suppresses mitogen-induced IL-2 production via inactivation of NFAT and NFκB in Jurkat cells.

d-Glucosamine (GlcNH2) and several of its derivatives are known to possess immunosuppressive activities in various immune cell lines. The novel GlcNH2-containing oligosaccharide Galα1-6GlcNH2 (designated melibiosamine; MelNH2) is expected to be immunosuppressive also. In Jurkat cells (immortalized human T lymphocytes), interleukin 2 (IL-2) production (an index of the T-cell immune response) can be induced by stimulation with a mitogen, such as concanavalin A. Here, we compared the effects of GlcNH2 and MelNH2 on concanavalin A-induced IL-2 production (CIIP) in Jurkat cells and found that GlcNH2 and MelNH2 at millimolar levels both significantly suppressed CIIP without affecting cell viability. When we examined the effects of GlcNH2 and MelNH2 on the activation of the three transcription factors required for CIIP-NFAT (nuclear factor of activated T-cells), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), and AP-1 (activator protein 1)-we found that GlcNH2 and MelNH2 both suppressed CIIP by inhibiting the activation of NFAT and NFκB, but, unlike GlcNH2, MelNH2 also promoted the activation of AP-1. These results suggest that MelNH2 may be a potentially useful lead compound for development as an immunosuppressive or anti-inflammatory drug.

Halogen-Substituted Derivatives of Dictyostelium Differentiation-Inducing Factor-1 Suppress Serum-Induced Cell Migration of Human Breast Cancer MDA-MB-231 Cells in Vitro.

Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 M) with negligible effects on cell proliferation (IC50, >20 M). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 M, respectively, without affecting cell proliferation (IC50, >20 M). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.

Antimicrobial Activities of Dictyostelium Differentiation-Inducing Factors and Their Derivatives.

At the end of its life cycle, the cellular slime mold Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli (MIC, >100 µM). Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.

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

The cellular slime mold Dictyostelium discoideum is an excellent model organism for the study of cell and developmental biology because of its simple life cycle and ease of use. Recent findings suggest that Dictyostelium and possibly other genera of cellular slime molds, are potential sources of novel lead compounds for pharmacological and medical research. In this review, we present supporting evidence that cellular slime molds are an untapped source of lead compounds by examining the discovery and functions of polyketide differentiation-inducing factor-1, a compound that was originally isolated as an inducer of stalk-cell differentiation in D. discoideum and, together with its derivatives, is now a promising lead compound for drug discovery in several areas. We also review other novel compounds, including secondary metabolites, that have been isolated from cellular slime molds.

Biological Activities of Novel Derivatives of Differentiation-Inducing Factor 3 from Dictyostelium discoideum.

Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for the development of new medicines; DIF-3 and its derivatives possess several beneficial biological activities, including anti-tumor, anti-Trypanosoma cruzi, and immunoregulatory effects. To assess the relationship between the biological activities of DIF-3 and its chemical structure, particularly in regard to its alkoxy group and the length of the alkyl chains at the acyl group, we synthesized two derivatives of DIF-3, 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)-hexan-1-one (Hex-DIF-3), and investigated their biological activities in vitro. At micro-molar levels, DIF-3(+3) and Hex-DIF-3 exhibited strong anti-proliferative effects in tumor cell cultures, but their anti-T. cruzi activities at 1 µM in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5 µM significantly suppressed mitogen-induced interleukin-2 production in vitro in Jurkat T cells. These results suggest that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immunosuppressive agents.

Isolation, Synthesis, and Biological Activity of Chlorinated Alkylresorcinols from Dictyostelium Cellular Slime Molds.

Eight chlorinated alkylresorcinols, monochasiol A-H (1-8), were isolated from the fruiting bodies of Dictyostelium monochasioides. Compounds 1-8 were synthesized to confirm their structures and to obtain sufficient material for performing biological tests. Monochasiol A (1) selectively inhibited the concanavalin A-induced interleukin-2 production in Jurkat cells, a human T lymphocyte cell line. Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides, MPBD and DIF-1, produced by Dictyostelium discoideum.

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D. discoideum.

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25-50 nM, and dinitrophenol (DNP), at 2.5-5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1-2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity.

Identification of brefelamide as a novel inhibitor of osteopontin that suppresses invasion of A549 lung cancer cells.

The contribution of aberrant osteopontin (OPN) expression to tumor progression and metastasis has been documented in a wide spectrum of malignancies, and targeted inhibition of OPN has therefore emerged as an attractive strategy for cancer therapy. Transcription of OPN is regulated by various transcription factors, and our recently published study demonstrated that downregulation of OPN is an important event in the TGF­ß cytostatic program. We report here that brefelamide exerts an inhibitory effect on OPN expression and function in A549 human lung carcinoma cells. The promoter, RNA, and protein levels of OPN were decreased in brefelamide­treated A549 cells, which was accompanied by reduced invasive ability in vitro. OPN inhibition by brefelamide was largely abrogated by disruption of a putative TGF­ß inhibitory element in the OPN promoter. Treatment with brefelamide induced Smad4 expression, and knockdown of Smad4 by RNA interference partially diminished the inhibitory effect of brefelamide on OPN. These results indicate that brefelamide inhibited OPN­mediated cell invasion through restoration of the OPN repression by TGF­ß/Smad signaling. Together with the reported antiproliferative property, our findings suggest that brefelamide might serve as a potential candidate for the development of a new antitumor and antimetastatic agent.

Oral administration of Dictyostelium differentiation-inducing factor 1 lowers blood glucose levels in streptozotocin-induced diabetic rats.

AIMS: Differentiation-inducing factor 1 (DIF-1), originally discovered in the cellular slime mold Dictyostelium discoideum, and its derivatives possess pharmacological activities, such as the promotion of glucose uptake in non-transformed mammalian cells in vitro. Accordingly, DIFs are considered promising lead candidates for novel anti-diabetic drugs. The aim of this study was to assess the anti-diabetic and toxic effects of DIF-1 in mouse 3T3-L1 fibroblast cells in vitro and in diabetic rats in vivo. Main methods We investigated the in vitro effects of DIF-1 and DIF-1(3M), a derivative of DIF-1, on glucose metabolism in 3T3-L1 cells by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). We also examined the effects of DIF-1 on blood glucose levels in streptozotocin (STZ)-induced rats. KEY FINDINGS: CE-TOF-MS revealed that 20µM DIF-1 and 20µM DIF-1(3M) promoted glucose uptake and metabolism in 3T3-L1 cells. Oral administration of DIF-1 (30mg/kg) significantly lowered basal blood glucose levels in STZ-treated rats and promoted a decrease in blood glucose levels after oral glucose loading (2.5g/kg) in the rats. In addition, daily oral administration of DIF-1 (30mg/kg/day) for 1wk significantly lowered the blood glucose levels in STZ-treated rats but did not affect their body weight and caused only minor alterations in the levels of other blood analytes. SIGNIFICANCE: These results indicate that DIF-1 may be a good lead compound for the development of anti-diabetic drugs.

Differentiation-inducing factor 2 modulates chemotaxis via the histidine kinase DhkC-dependent pathway in Dictyostelium discoideum.

Differentiation-inducing factor 1(DIF-1) and DIF-2 are signaling molecules that control chemotaxis in Dictyostelium discoideum. Whereas DIF-1 suppresses chemotaxis in shallow cAMP gradients, DIF-2 enhances chemotaxis under the same conditions via a phosphodiesterase, response regulator A (RegA), which is a part of the DhkC-RdeA-RegA two-component signaling system. In this study, to investigate the mechanism of the chemotaxis regulation by DIF-2, we examined the effects of DIF-2 (and DIF-1) on chemotaxis in rdeA(-) and dhkC(-) mutant strains. In the parental wild-type strains, chemotactic cell movement was suppressed with DIF-1 and enhanced with DIF-2 in shallow cAMP gradients. In contrast, in both rdeA(-) and dhkC(-) strains, chemotaxis was suppressed with DIF-1 but unaffected by DIF-2. The results suggest that DIF-2 modulates chemotaxis via the DhkC-RdeA-RegA signaling system.

Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum.

In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography-tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4- and gst4OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4- cells were smaller than those of wild-type Ax2 cells, and those of gst4OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum.

A Derivative of Differentiation-Inducing Factor-3 Inhibits PAK1 Activity and Breast Cancer Cell Proliferation.

Differentiation-inducing factors 1-3 (DIFs 1-3), chlorinated alkylphenones identified in the cellular slime mold Dictyostelium discoideum, are considered anti-tumor agents because they inhibit proliferation of a variety of mammalian tumor cells in vitro. Although the anti-proliferative effects of DIF-1 and DIF-3 are well-documented, the precise molecular mechanisms underlying the actions of DIFs have not been fully elucidated. In this study, we examined the effects of DIFs and their derivatives on PAK1, a key serine-threonine kinase, which is activated by multiple ligands and regulates cell proliferation. We examined the effect of DIF derivatives on PAK1 kinase activity in cells. We also examined the effect of DIF-3(+1) derivative on PAK1 kinase activity in vitro, cyclin D1 promoter activity and breast cancer cell proliferation. It was found that some derivatives strongly inhibited PAK1 kinase activity in human breast cancer MCF-7 cells stably over expressing PAK1. Among the derivatives, DIF-3(+1) was most potent, which directly inhibited kinase activity of recombinant purified PAK1 in an in vitro kinase assay. Furthermore, DIF-3(+1) strongly inhibited both cyclin D1 promoter activity and proliferation of MCF-7 and T47D breast cancer cells stably over expressing PAK1 in response to prolactin, estrogen, epidermal growth factor and heregulin. In the present study we propose PAK1 as DIF-3(+1) target mediating its anti-proliferative effect.