Cholestane-3ß,5α,6ß-triol Induces Multiple Cell Death in A549 Cells via ER Stress and Autophagy Activation.

Cholestane-3ß,5α,6ß-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases.

Studying the trafficking of labeled trodusquemine and its application as nerve marker for light-sheet and expansion microscopy.

Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

The aminosterol Claramine inhibits ß-secretase 1-mediated insulin receptor cleavage.

The cleavage of the insulin receptor by ß-secretase 1 (BACE1) in the liver increases during diabetes, which contributes to reduce insulin receptor levels and impair insulin signaling. However, the precise signaling events that lead to this increased cleavage are unclear. We showed that BACE1 cleaves the insulin receptor in the early secretory pathway. Indeed, coimmunoprecipitation experiments reveal the interaction of the proforms of the two proteins. Moreover, fragments of insulin receptor are detected in the early secretory pathway and a mutated form of BACE1 that retains its prodomain cleaves an early secretory pathway-resident form of the insulin receptor. We showed that BACE1 proform levels are regulated by proteasome and/or lysosome-dependent degradation systems whose efficiencies are dependent on the O-GlcNacylation process. Our results showed that enhanced O-GlcNacylation reduces the efficiency of intracellular protein degradation systems, leading to the accumulation of the proform of BACE1 in the early secretory pathway where it cleaves the precursor of the insulin receptor. All these dysregulations are found in the livers of diabetic mice. In addition, we performed a screen of molecules according to their ability to increase levels of the insulin receptor at the surface of BACE1-overexpressing cells. This approach identified the aminosterol Claramine, which accelerated intracellular trafficking of the proform of BACE1 and increased autophagy. Both of these effects likely contribute to the reduced amount of the proform of BACE1 in the early secretory pathway, thereby reducing insulin receptor cleavage. These newly described properties of Claramine are consistent with its insulin sensitizing effect.

Neuronal Protein Tyrosine Phosphatase 1B Hastens Amyloid ß-Associated Alzheimer's Disease in Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, resulting in the progressive decline of cognitive function in patients. Familial forms of AD are tied to mutations in the amyloid precursor protein, but the cellular mechanisms that cause AD remain unclear. Inflammation and amyloidosis from amyloid ß (Aß) aggregates are implicated in neuron loss and cognitive decline. Inflammation activates the protein-tyrosine phosphatase 1B (PTP1B), and this could suppress many signaling pathways that activate glycogen synthase kinase 3ß (GSK3ß) implicated in neurodegeneration. However, the significance of PTP1B in AD pathology remains unclear. Here, we show that pharmacological inhibition of PTP1B with trodusquemine or selective ablation of PTP1B in neurons prevents hippocampal neuron loss and spatial memory deficits in a transgenic AD mouse model with Aß pathology (hAPP-J20 mice of both sexes). Intriguingly, while systemic inhibition of PTP1B reduced inflammation in the hippocampus, neuronal PTP1B ablation did not. These results dissociate inflammation from neuronal loss and cognitive decline and demonstrate that neuronal PTP1B hastens neurodegeneration and cognitive decline in this model of AD. The protective effect of PTP1B inhibition or ablation coincides with the restoration of GSK3ß inhibition. Neuronal ablation of PTP1B did not affect cerebral amyloid levels or plaque numbers, but reduced Aß plaque size in the hippocampus. In summary, our preclinical study suggests that targeting PTP1B may be a new strategy to intervene in the progression of AD.SIGNIFICANCE STATEMENT Familial forms of Alzheimer's disease (AD) are tied to mutations in the amyloid precursor protein, but the cellular mechanisms that cause AD remain unclear. Here, we used a mouse model expressing human amyloid precursor protein bearing two familial mutations and asked whether activation of a phosphatase PTP1B participates in the disease process. Systemic inhibition of this phosphatase using a selective inhibitor prevented cognitive decline, neuron loss in the hippocampus, and attenuated inflammation. Importantly, neuron-targeted ablation of PTP1B also prevented cognitive decline and neuron loss but did not reduce inflammation. Therefore, neuronal loss rather than inflammation was critical for AD progression in this mouse model, and that disease progression could be ameliorated by inhibition of PTP1B.

Inhibition of lipid phosphatase SHIP1 expands myeloid-derived suppressor cells and attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

Ketamine's schizophrenia-like effects are prevented by targeting PTP1B.

Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.

Tyrosine phosphatase PTP1B impairs presynaptic NMDA receptor-mediated plasticity in a mouse model of Alzheimer's disease.

Mutations in the beta-amyloid protein (APP) cause familial Alzheimer's disease. In hAPP-J20 mice expressing mutant APP, pharmacological inhibition or genetic ablation of the tyrosine phosphatase PTP1B prevents CA3 hippocampus neuron loss and cognitive decline. However, how targeting PTP1B affects the cellular mechanisms underlying these cognitive deficits remains unknown. Changes in synaptic strength at the hippocampus can affect information processing for learning and memory. While prior studies have focused on post-synaptic mechanisms to account for synaptic deficits in Alzheimer's disease models, presynaptic mechanisms may also be affected. Here, using whole cell patch-clamp recording, coefficient of variation (CV) analysis suggested a profound presynaptic deficit in long-term potentiation (LTP) of CA3:CA1 synapses in hAPP-J20 mice. While the membrane-impermeable ionotropic NMDA receptor (NMDAR) blocker norketamine in the post-synaptic recording electrode had no effect on LTP, additional bath application of the ionotropic NMDAR blockers MK801 could replicate the deficit in LTP in wild type mice. In contrast to LTP, the paired-pulse ratio and short-term facilitation (STF) were aberrantly increased in hAPP-J20 mice. These synaptic deficits in hAPP-J20 mice were associated with reduced phosphorylation of NMDAR GluN2B and the synaptic vesicle recycling protein NSF (N-ethylmaleimide sensitive factor). Phosphorylation of both proteins, together with synaptic plasticity and cognitive function, were restored by PTP1B ablation or inhibition by the PTP1B-selective inhibitor Trodusquemine. Taken together, our results indicate that PTP1B impairs presynaptic NMDAR-mediated synaptic plasticity required for spatial learning in a mouse model of Alzheimer's disease. Since Trodusquemine has undergone phase 1/2 clinical trials to treat obesity, it could be repurposed to treat Alzheimer's disease.

Galtonosides A-E: Antiproliferative and Antiplasmodial Cholestane Glycosides from Galtonia regalis.

An extract of Galtonia regalis from the Natural Products Discovery Institute showed moderate antiplasmodial activity, with an IC50 value less than 1.25 µg/mL. The two known cholestane glycosides 1 and 2 and the five new cholestane glycosides galtonosides A-E (3-7) were isolated after bioassay-directed fractionation. The structures of the new compounds were determined by interpretation of their NMR and mass spectra. Among these compounds, galtonoside B (4) displayed the most potent antiplasmodial activity, with an IC50 value of 0.214 µM against the drug-resistant Dd2 strain of Plasmodium falciparum.

Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.

Structural Characterization of Cholestane Rhamnosides from Ornithogalum saundersiae Bulbs and Their Cytotoxic Activity against Cultured Tumor Cells.

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1-12) and seven known compounds (13-19). The structures of the new compounds (1-12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1-19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10-12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction.

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, has a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We define a new mechanism of allosteric inhibition that targets the C-terminal, noncatalytic segment of PTP1B. We present what is to our knowledge the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small-molecule inhibitor MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Polyhydroxylated sulfated steroids derived from 5α-cholestanes as antiviral agents against herpes simplex virus.

Twelve polyhydroxylated sulfated steroids synthesized from a 5α-cholestane skeleton with different substitutions in C-2, C-3 and C-6 were evaluated for cytotoxicity and antiviral activity against herpes simplex virus (HSV) by a virus plaque reduction assay. Four compounds elicited a selective inhibitory effect against HSV. The disodium salt of 2ß,3α-dihydroxy-6E-hydroximine-5α-cholestane-2,3-disulfate, named compound 7, was the most effective inhibitor of HSV-1, HSV-2 and pseudorabies virus (PrV) strains, including acyclovir-resistant variants, in human and monkey cell lines. Preliminary mechanistic studies demonstrated that compound 7 did not affect the initial steps of virus entry but inhibited a subsequent event in the infection process of HSV.

Functional properties of Claramine: a novel PTP1B inhibitor and insulin-mimetic compound.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-ß (IRß), Akt and GSK3ß. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

Protein-tyrosine phosphatase 1B antagonized signaling by insulin-like growth factor-1 receptor and kinase BRK/PTK6 in ovarian cancer cells.

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R ß-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R ß-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rß signaling in ovarian cancer cells.

The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules.

In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich strand of the cis-regulatory element in the BCL2 promoter element is highly dynamic in nature and can form either an i-motif or a flexible hairpin. Under physiological conditions these two secondary DNA structures are found in an equilibrium mixture, which can be shifted by the addition of small molecules that trap out either the i-motif (IMC-48) or the flexible hairpin (IMC-76). In cellular experiments we demonstrate that the addition of these molecules has opposite effects on BCL2 gene expression and furthermore that these effects are antagonistic. In this contribution we have identified a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription. The molecular basis for the recognition of the i-motif by hnRNP LL is determined, and we demonstrate that the protein unfolds the i-motif structure to form a stable single-stranded complex. In subsequent experiments we show that IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnRNP LL-i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter. For the first time we propose that the i-motif acts as a molecular switch that controls gene expression and that small molecules that target the dynamic equilibrium of the i-motif and the flexible hairpin can differentially modulate gene expression.

Synthesis and evaluation of some new aza-B-homocholestane derivatives as anticancer agents.

Using analogues of some marine steroidal oximes as precursors, a series of aza-B-homocholestane derivatives possessing different substituted groups at the 3-position of the steroidal nucleus were synthesized. Their biological activity against cancer cell proliferation was determined with multiple cancer cell lines. Aza-B-homocholestane derivatives possessing 3-hydroxyl, 3-hydroximino and 3-thiosemicarbazone groups displayed remarkable cytotoxicity to cancer cells via apoptosis inducing mechanism. Compounds 5, 10, 12, 15 and 18 exhibited better potency to inhibit cancer cell proliferation. In addition, compound 15 was further evaluated with three dimensional (3D) multicellular spheroids assay to determine its potency against spheroid growth. The structure-activity relationship (SAR) generated in the studies is valuable for the design of novel chemotherapeutic agents.

New 23-spirocholestane derivatives from Ypsilandra thibetica.

Three new unusual 23-spirocholestane derivatives, ypsilanogenin (1), ypsilanogenin 3-O-ß-D-glucopyranoside (2), and 4'-acetylypsilanogenin 3-O-ß-D-glucopyranoside (3), were isolated from the whole plants of Ypsilandra thibetica. The structures of compounds 1-3 were deduced by spectroscopic and chemical methods, and the structure of 1 was further confirmed by a single-crystal diffraction analysis. All isolates were evaluated for their inhibitory activities against HIV-1.

Therapeutic potential of SH2 domain-containing inositol-5'-phosphatase 1 (SHIP1) and SHIP2 inhibition in cancer.

Many tumors present with increased activation of the phosphatidylinositol 3-kinase (PI3K)-PtdIns(3,4,5)P(3)-protein kinase B (PKB/Akt) signaling pathway. It has long been thought that the lipid phosphatases SH2 domain-containing inositol-5'-phosphatase 1 (SHIP1) and SHIP2 act as tumor suppressors by counteracting with the survival signal induced by this pathway through hydrolysis or PtdIns(3,4,5)P(3) to PtdIns(3,4)P(2). However, a growing body of evidence suggests that PtdInd(3,4)P(2) is capable of, and essential for, Akt activation, thus suggesting a potential role for SHIP1/2 enzymes as proto-oncogenes. We recently described a novel SHIP1-selective chemical inhibitor (3α-aminocholestane [3AC]) that is capable of killing malignant hematologic cells. In this study, we further investigate the biochemical consequences of 3AC treatment in multiple myeloma (MM) and demonstrate that SHIP1 inhibition arrests MM cell lines in either G0/G1 or G2/M stages of the cell cycle, leading to caspase activation and apoptosis. In addition, we show that in vivo growth of MM cells is blocked by treatment of mice with the SHIP1 inhibitor 3AC. Furthermore, we identify three novel pan-SHIP1/2 inhibitors that efficiently kill MM cells through G2/M arrest, caspase activation and apoptosis induction. Interestingly, in SHIP2-expressing breast cancer cells that lack SHIP1 expression, pan-SHIP1/2 inhibition also reduces viable cell numbers, which can be rescued by addition of exogenous PtdIns(3,4)P(2). In conclusion, this study shows that inhibition of SHIP1 and SHIP2 may have broad clinical application in the treatment of multiple tumor types.

Chemo-ecological studies on hexactinellid sponges from the Southern Ocean.

Hexactinellids (glass sponges) are an understudied class with syncytial organization and poor procariotic associations, thought to lack defensive secondary metabolites. Poriferans, though, are outstanding sources of bioactive compounds; nonetheless, a growing suspicion suggests that many of these chemicals could be symbiont-derived. In Polar latitudes, sponges are readily invaded by diatoms, which could provide natural products. Hexactinellids are typical of deep waters; but in Antarctica, they dominate the upper shelf providing shelter and food supply to many opportunistic mesograzers and macroinvertebrates, which exert strong ecological pressures on them. Aiming to examine the incidence of defensive activities of hexactinellids against consumption, feeding experiments were conducted using their lipophilic fractions. Antarctic hexactinellid and demosponge extracts were tested against the asteroid Odontaster validus and the amphipod Cheirimedon femoratus as putative sympatric, omnivorous consumers. Hexactinellids yielded greater unpalatable activities towards the amphipod, while no apparent allocation of lipophilic defenses was noted. After chemical analyses on the lipophilic fractions from these Antarctic glass sponges, quite similar profiles were revealed, and no peculiar secondary metabolites, comparable to those characterizing other poriferans, were found. Instead, the lipidic compounds 5α(H)-cholestan-3-one and two glycoceramides were isolated for their particular outspread presence in our samples. The isolated compounds were further assessed in asteroid feeding assays, and their occurrence was evaluated for chemotaxonomical purposes in all the Antarctic samples as well as in glass sponges from other latitudes by NMR and MS. Characteristic sphingolipids are proposed as chemical markers in Hexactinellida, with possible contributions to the classification of this unsettled class.