Involvement of cytoplasmic pH in the production of ethylene,a potent inducer of sexual development inDictyostelium mucoroides

Summary Dictyostelium mucoroides-7 (Dm 7) and a mutant MF 1 derived from it exhibit two developmental pathways: sorocarp formation occurs during the asexual process, and macrocyst formation during the sexual cycle. The two developmental pathways are mainly regulated by two chemical substances: 3,5-cyclic adenosine monophosphate (cAMP) and ethylene. Recently, we have demonstrated that cytoplasmic pH (pH_i) has a critical role for the choice of developmental pathways, higher pH_i being favourable to macrocyst formation. Thereupon, attention was riveted to the relation of pH_i to biosynthesis of cAMP and ethylene. Effect of pH_i on the production and release of ethylene, a potent inducer of macrocyst formation, was examined, using the two facing culture method. The result showed that lowered pH_i inhibits ethylene production, thus resulting in a failure of cells to form macrocysts. The accumulation of cAMP, an inhibitor of macrocyst formation, was found to vary depending on extracellular pH (pH_o), but diethylstilbestrol (DES) that is a proton pump inhibitor and also an inhibitor of macrocyst formation had no significant effect on the accumulation. Taken together these results indicate that higher pH_i may induce macrocyst formation through enhancement of ethylene production rather than inhibition of cAMP synthesis.Abbreviations cAMP 3,5-cyclic adenosine monophosphate - pH_i cytoplasmic pH - pH_o extracellular pH - ACC 1-1-aminocyclopropane-1-carboxylic acid     

An endogenous inducer of sexual development in Aspergillus nidulans

During development of the homothallic ascomycete Aspergillus nidulans, asexual sporulation is followed by sexual sporulation. We report here the detection of a solvent-extractable activity which inhibits asexual sporulation and stimulates premature sexual sporulation. This activity, called precocious sexual inducer (psi), is overproduced by certain mutants that are blocked in both modes of sporulation. Using partially purified preparations of psi, biological response could be elicited with as little as 50 ng of material. We suggest that psi is a hormone-precursor which is converted to a hormone by normal sporulating strains that respond to psi, but not by the asporogenous mutants that overproduce psi. The stability of psi activity gives promise that the compound can be purified and identified.     

Bufalin as a potent inducer of differentiation of human myeloid leukemia cells.

Bufalin was found to be a potent inducer of differentiation in human erythroleukemia K562 cells by examination of various differentiation markers (as assessed by the morphology, histochemistry, and the abilities to phagocytose latex particles, to reduce nitro-blue tetrazolium and to develop Fc receptors). Bufalin, at a concentration as low as 10 nM, also produced a strong differentiation-inducing activity in three other human leukemia-derived cell lines (human promyelocytic HL60, monoblastic U937 and myeloblastic ML1). Treatment of K562 cells with other cardiotonic steroids, such as cinobufagin, ouabain and digitoxigenin, at the concentration of 10 nM for four days resulted in weak or no effect on the cells. These findings suggest that bufalin might have potentiality as a new agent in the differentiation therapy for human myelogenous leukemia.     

Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay

Gambogic acid (2), a natural product isolated from the resin of Garcinia hurburyi tree, was discovered to be a potent apoptosis inducer using our cell- and caspase-based high-throughput screening assays. Gambogic acid was found to have an EC(50) of 0.78 microM in the caspase activation assay in T47D breast cancer cells. The apoptosis-inducing activity of gambogic acid was further characterized by a nuclear fragmentation assay and flow cytometry analysis in human breast tumor cells T47D. Gambogic acid was found to induce apoptosis independent of cell cycle, which is different from paclitaxel that arrests cells in the G2/M phase. To understand the structure-activity relationship (SAR) of gambogic acid, derivatives of 2 with modifications to different function groups were prepared. SAR studies of gambogic acid, as measured by the caspase activation assay, showed that the 9,10 carbon-carbon double bond of the alpha,beta-unsaturated ketone is important for biological activity, while the 6-hydroxy and 30-carboxy group can tolerate a variety of modifications. The importance of the 9,10 carbon-carbon double bond was confirmed by the traditional growth inhibition assay. The high potency of 2 as an inducer of apoptosis, its novel mechanism of action, easy isolation and abundant supply, as well as the fact that it is amenable to chemical modification, makes gambogic acid an attractive molecule for the development of anticancer agents.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Molecular cloning and chromosomal mapping of mouse intronless myc gene acting as a potent apoptosis inducer

Our previous findings suggest that the activation of the rat intronless myc gene provides a selective advantage in tumor suppression through apoptosis induction. In the present study, to examine whether intronless myc gene acting as an apoptosis inducer is evolutionarily conserved in mammalian cells, we isolated the mouse intronless myc gene and characterized it. A sequence analysis demonstrated that mouse intronless myc gene, ms-myc, has a linearly opened translatable frame consisting of 1293 bp with 90% homology with that of rat s-myc. The chromosomal locus of ms-myc was identified on chromosome 19B by a fluorescent in situ hybridization (FISH) analysis. Gene transfection experiments showed that the transient overexpression of ms-Myc with transactivation activity effectively induces cell death in a wild-type p53-independent manner. In addition, cells stably expressing transfected ms-myc became more susceptible to apoptosis induced by genotoxic stress such as UV-irradiation and hydrogen peroxide compared with untransfected control cells. These observations suggest that the rodents commonly contain an s-myc-type of intronless myc gene with apoptosis-inducing activity.     

Lasonolide A,a potent and reversible inducer of chromosome condensation

Lasonolide A (LSA) is a natural product with high and selective cytotoxicity against mesenchymal cancer cells, including leukemia, melanomas and glioblastomas. Here, we reveal that LSA induces rapid and reversible premature chromosome condensation (PCC) associated with cell detachment, plasma membrane smoothening and actin reorganization. PCC is induced at all phases of the cell cycle in proliferative cells as well as in circulating human lymphocytes in G₀. It is independent of Cdk1 signaling, associated with cyclin B downregulation and induced in cells at LSA concentrations that are three orders of magnitude lower than those required to block phosphatases 1 and 2A in vitro. At the epigenetic level, LSA-induced PCC is coupled with histone H3 and H1 hyperphosphorylation and deacetylation. Treatment with SAHA reduced LSA-induced PCC, implicating histone deacetylation as one of the PCC effector mechanisms. In addition, PCC is coupled with topoisomerase II (Top2) and Aurora A hyperphosphorylation and activation. Inhibition of Top2 or Aurora A partially blocked LSA-induced PCC. Our findings demonstrate the profound epigenetic alterations induced by LSA and the potential of LSA as a new cytogenetic tool. Based on the unique cellular effects of LSA, further studies are warranted to uncover the cellular target of lasonolide A (“TOL”).     

Lasonolide A,a potent and reversible inducer of chromosome condensation

Lasonolide A (LSA) is a natural product with high and selective cytotoxicity against mesenchymal cancer cells, including leukemia, melanomas and glioblastomas. Here, we reveal that LSA induces rapid and reversible premature chromosome condensation (PCC) associated with cell detachment, plasma membrane smoothening and actin reorganization. PCC is induced at all phases of the cell cycle in proliferative cells as well as in circulating human lymphocytes in G₀. It is independent of Cdk1 signaling, associated with cyclin B downregulation and induced in cells at LSA concentrations that are three orders of magnitude lower than those required to block phosphatases 1 and 2A in vitro. At the epigenetic level, LSA-induced PCC is coupled with histone H3 and H1 hyperphosphorylation and deacetylation. Treatment with SAHA reduced LSA-induced PCC, implicating histone deacetylation as one of the PCC effector mechanisms. In addition, PCC is coupled with topoisomerase II (Top2) and Aurora A hyperphosphorylation and activation. Inhibition of Top2 or Aurora A partially blocked LSA-induced PCC. Our findings demonstrate the profound epigenetic alterations induced by LSA and the potential of LSA as a new cytogenetic tool. Based on the unique cellular effects of LSA, further studies are warranted to uncover the cellular target of lasonolide A (“TOL”).     

Identification of lysophosphatidylthreonine with an aromatic fatty acid surrogate as a potent inducer of mast cell degranulation

Upon various stimulations, mast cells (MCs) release a wide variety of chemical mediators stored in their cytoplasmic granules, which then initiates subsequent allergic reactions. Lysophosphatidylserine (LysoPS), a kind of lysophospholipid, potentiates the histamine release from MCs triggered by antigen stimulation. We previously showed through structure-activity studies of LysoPS analogs that LysoPS with a methyl group at the carbon of the serine residue, i.e., lysophosphatidylthreonine (LysoPT), is extremely potent in stimulating the MC degranulation. In this study, as our continuing study to identify more potent LysoPS analogs, we developed LysoPS analogs with fatty acid surrogates. We found that the substitution of oleic acid to an aromatic fatty acid surrogate (C3-pH-p-O-C11) in 2-deoxy-1-LysoPS resulted in significant increase in the ability to induce MCs degranulation compared with 2-deoxy-1-LysoPS with oleic acid. Conversion of the serine residue into the threonine residue further increased the activity of MC degranulation both in vitro and in vivo. The resulting super agonist, 2-deoxy-LysoPT with C3-pH-p-O-C11, will be a useful tool to elucidate the mechanisms of stimulatory effect of LysoPS on MC degranulation.     

The secretable form of trimeric TRAIL, a potent inducer of apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of the TNF family. Soluble recombinant TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to specifically limit tumor growth without damaging normal cells and tissues in vivo. These results suggest a strong potential of TRAIL as an anticancer therapy. Here we report an artificial TRAIL gene that expresses and secretes trimeric TRAIL into the culture supernatant. This novel TRAIL gene is composed of three functional elements, including a secretion signal, a trimerization domain, and an apoptosis-inducing moiety of TRAIL gene sequence. The expression vectors delivering this TRAIL gene produced secretable forms of trimeric TRAIL proteins. These TRAIL proteins showed greater apoptotic activity than the known TRAIL protein that does not contain an additional trimerization domain. Our data suggest that the gene therapy using our artificial TRAIL gene may be used as an anticancer therapy.     

IL-33, a potent inducer of adaptive immunity to intestinal nematodes

IL-33 (IL-1F11) binds ST2 (IL-1R4), both of which are associated with optimal CD4(+) Th2 polarization. Exogenous IL-33 drives induction of Th2-associated cytokines and associated pathological changes within the gut mucosa. Th2 polarization is also a prerequisite to expulsion of the intestinal-dwelling nematode Trichuris muris. In this study, we demonstrate that IL-33 mRNA is expressed early during parasite infection and susceptible mice can be induced to expel the parasite by a regime of exogenous IL-33 administration. IL-33 prevents an inappropriate parasite-specific Th1-polarized response and induces IL-4, IL-9, and IL-13. This redirection requires the presence of T cells and must occur at the initiation of the response to the pathogen. Interestingly, exogenous IL-33 also induced thymic stromal lymphopoietin mRNA within the infected caecum, an epithelial cell-restricted cytokine essential for the generation of Th2-driven parasite immunity. IL-33 also acts independently of T cells, altering intestinal pathology in chronically infected SCID mice, leading to an increased crypt length and intestinal epithelial cell proliferation, but reducing goblet cell hyperplasia. Thus, the ability of IL-33 to induce Th2 responses has functional relevance in the context of intestinal helminth infection, particularly during the initiation of the response.     

The marine cytotoxin portimine is a potent and selective inducer of apoptosis

Portimine is a recently discovered member of a class of marine micro-algal toxins called cyclic imines. In dramatic contrast to related compounds in this toxin class, portimine has very low acute toxicity to mice but is highly cytotoxic to cultured cells. In this study we show that portimine kills human Jurkat T-lymphoma cells and mouse embryonic fibroblasts (MEFs), with LC₅₀ values of 6 and 2.5 nM respectively. Treated cells displayed rapid caspase activation and phosphatidylserine exposure, indicative of apoptotic cell death. Jurkat cells overexpressing the anti-apoptotic protein Bcl-2 or Bax/Bak knockout MEFs were completely protected from portimine. This protection was apparent even at high concentrations of portimine, with no evidence of necrotic cell death, indicating that portimine is a selective chemical inducer of apoptosis. Treatment of the Bcl-2-overexpressing cells with both portimine and the Bcl-2 inhibitor ABT-737 proved a powerful combination, causing >90?% death. We conclude that portimine is one of the most potent naturally derived inducers of apoptosis to be discovered, and it displays strong selectivity for the induction of apoptotic pathways.     

6-hydroximino-4-aza-A-homo-cholest-3-one and related analogue as a potent inducer of apoptosis in cancer cells

Here we report that 6-hydroximino-4-aza-A-homo-cholest-3-one and 6-hydroxyl-4-aza-A-homo-cholest-3-one, new steroidal lactams were synthesized recently, displayed antiproliferative activity against some cancer cells through inducing cancer cell apoptosis by activation of the intrinsic pathway. The apoptotic function of the compounds was demonstrated by release of cytochrome C, activation of caspase 3 and annexin V labeling. Furthermore, the compound was able to inhibit tumor growth in an athymic mouse model.     

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.     

Butyric acid,a potent inducer of erythroid differentiation in cultured erythroleukemic cells

Butyric acid is an unusually potent inducer of erythroid differentiation in cultured erythroleukemic cells. It is effective at one hundredth the concentration required of dimethylsulfoxide, a most effective inducing agent. Studies using a variety of analogues and metabolites suggest that the structural features of butyric acid are rather stringently required for induction. This effect is considered in view of the fact that butyric acid is a naturally occurring fatty acid, is effective in relatively low concentrations, and is widely used to form derivatives of cAMP.     

BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts

The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, expression of non-apoptotic levels of either ncBID or wtBID in Bid -/- MEFs induced a similar and significant enhancement in apoptosis in response to a variety of death signals, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.     

Triwaglerin: a potent platelet aggregation inducer purified from Trimeresurus wagleri snake venom

Trimeresurus wagleri venom is the most potent inducer of platelet aggregation among the seven Trimeresurus snake venoms tested. By means of CM-Sephadex C-50 column chromatography, T. wagleri venom was separated into 19 fractions. Fraction XVI possessed the strongest aggregating activity and was further purified by Sephadex G-75 and on heparin-agarose columns, and finally Triwaglerin, with a molecular weight of 68000, was obtained. Its aggregating and ATP-releasing activity was dose-dependent and 10-times more potent than the crude venom. Triwaglerin was devoid of any of the enzymatic activities possessed by the crude venom. Triwaglerin-induced aggregation was not affected by indomethacin, creatine phosphate/creatine phosphokinase (CP/CPK), platelet-activating factor (PAF) antagonists, verapamil or heparin, but was inhibited completely by mepacrine, imipramine and forskolin and markedly by tetracaine and sodium nitroprusside. Thromboxane B2 formation caused by Triwaglerin was suppressed by mepacrine, imipramine and indomethacin. R59022 and TMB-8 caused a synergistic inhibitory effect against Triwaglerin-induced aggregation. These data suggest that Triwaglerin activates platelets in a unique action which is independent of formation of thromboxane A2 and PAF, or release of ADP.     

Ethylene: indicator but not inducer of phytoalexin synthesis in soybean

Cell wall preparations (elicitors) from Phytophthora megasperma var. sojae increase C₂H₄ formation, phenylalanine ammonia lyase activity, and glyceollin accumulation in soybean cotyledons within about 1.5, 3, and 6 hours after treatment, respectively. The immediate precursor of C₂H₄, 1-aminocyclopropane-1-carboxylic acid, stimulates C₂H₄ formation like the elicitor within 1.5 hours after administration, whereas phenylalanine ammonia lyase activity and glyceollin concentration remain unchanged. Aminoethoxyvinylglycine, a specific inhibitor of C₂H₄ formation in higher plants, inhibits elicitor-induced C₂H₄ formation by about 95% but has no effects on phenylalanine ammonia lyase or glyceollin accumulation. It was concluded that C₂H₄ is a signal accompanying the specific recognition process which finally leads to the induction of phytoalexin formation, but it is not functioning as a link or messenger in the induction sequence of glyceollin accumulation.