Cannabinoid-Induced Immunogenic Cell Death of Colorectal Cancer Cells Through De Novo Synthesis of Ceramide Is Partially Mediated by CB2 Receptor.

Our recent studies have identified a link between sphingolipid metabolites and the induction of a specialized form of regulated cell death termed immunogenic cell death (ICD). We have recently demonstrated that the synthetic cannabinoid (±) 5-epi CP 55,940 (5-epi) stimulates the accumulation of ceramide (Cer), and that inhibition of sphingosine kinase 1 (SphK1) enhances Cer accumulation and ICD-induction in human colorectal cancer (CRC) cell lines. We employed flow-cytometric, western blot analyses, pharmacological inhibitors of the sphingolipid metabolic pathway and small molecule agonists and antagonists of the CB receptors to further analyze the mechanism by which 5-epi induces Cer accumulation.

Fanning the Flames of Endoplasmic Reticulum (ER) Stress: Can Sphingolipid Metabolism Be Targeted to Enhance ER Stress-Associated Immunogenic Cell Death in Cancer?

The three arms of the unfolded protein response (UPR) surveil the luminal environment of the endoplasmic reticulum (ER) and transmit information through the lipid bilayer to the cytoplasm to alert the cell of stress conditions within the ER lumen. That same lipid bilayer is the site of de novo synthesis of phospholipids and sphingolipids. Thus, it is no surprise that lipids are modulated by and are modulators of ER stress.

Characterization of Anticancer Effects of the Analogs of DJ4, a Novel Selective Inhibitor of ROCK and MRCK Kinases.

The Rho associated coiled-coil containing protein kinase (ROCK1 and ROCK2) and myotonic dystrophy-related Cdc-42 binding kinases (MRCKα and MRCKβ) are critical regulators of cell proliferation and cell plasticity, a process intimately involved in cancer cell migration and invasion. Previously, we reported the discovery of a novel small molecule (DJ4) selective multi-kinase inhibitor of ROCK1/2 and MRCKα/β. Herein, we further characterized the anti-proliferative and apoptotic effects of DJ4 in non-small cell lung cancer and triple-negative breast cancer cells.

Regulatory Role of Sphingosine-1-Phosphate and C16:0 Ceramide, in Immunogenic Cell Death of Colon Cancer Cells Induced by Bak/Bax-Activation.

We recently identified the sphingosine kinases (SphK1/2) as key intracellular regulators of immunogenic cell death (ICD) in colorectal cancer (CRC) cells. To better understand the mechanism by which SphK inhibition enhances ICD, we focused on the intracellular signaling pathways leading to cell surface exposure of calreticulin (ectoCRT). Herein, we demonstrate that ABT-263 and AZD-5991, inhibitors of Bcl-2/Bcl-X and Mcl-1, respectively, induce the production of ectoCRT, indicative of ICD.

Inhibition of Sphingosine Kinase Activity Enhances Immunogenic Cell Surface Exposure of Calreticulin Induced by the Synthetic Cannabinoid 5-epi-CP-55,940.

Endogenous and synthetic cannabinoids have been shown to induce cancer cell death through the accumulation of the sphingolipid, ceramide (Cer). Recently, we have demonstrated that Cer accumulation enhances the induction of immunogenic cell death (ICD). The primary objective of this study was to demonstrate that (±) 5-epi CP 55,940 (5-epi), a by-product of the chemical synthesis of the synthetic cannabinoid CP 55,940, induces ICD in colorectal cancer (CRC) cells, and that modulation of the sphingolipid metabolic pathway through inhibition of the sphingosine kinases (SphKs) enhances these effects.

Sphingosine Kinase Inhibition Enhances Dimerization of Calreticulin at the Cell Surface in Mitoxantrone-Induced Immunogenic Cell Death.

Agents that induce immunogenic cell death (ICD) alter the cellular localization of calreticulin (CRT), causing it to become cell surface-exposed within the plasma membrane lipid raft microdomain [cell surface-exposed CRT (ectoCRT)] where it serves as a damage associated-molecular pattern that elicits an antitumor immune response. We have identified the sphingolipid metabolic pathway as an integral component of the process of ectoCRT exposure. Inhibition of the sphingosine kinases (SphKs) enhances mitoxantrone-induced production of hallmarks of ICD, including ectoCRT production, with an absolute mean difference of 40 MFI (95% CI: 19-62; = 0.

Analysis of selective target engagement by small-molecule sphingosine kinase inhibitors using the Cellular Thermal Shift Assay (CETSA).

The recently renewed interest in scientific rigor and reproducibility is of critical importance for both scientists developing new targeted small-molecule inhibitors and those employing these molecule in cellular studies, alike. While off-target effects are commonly considered as limitations for any given small-molecule inhibitor, the ability of a given compound to distinguish between enzyme isoforms is often neglected when employing compounds in cellular studies. To call attention to this issue, we have compared the results of an assay for "direct target engagement", the Cellular Thermal Shift Assay (CETSA), to the published isoform selectivity of 12 commercially available sphingosine kinase 1 and 2 (SphK 1 and SphK2) inhibitors.

Development of SKI-349, a dual-targeted inhibitor of sphingosine kinase and microtubule polymerization.

Our sphingosine kinase inhibitor (SKI) optimization studies originated with the optimization of the SKI-I chemotype by replacement of the substituted benzyl rings with substituted phenyl rings giving rise to the discovery of SKI-178. We have recently reported that SKI-178 is a dual-targeted inhibitor of both sphingosine kinase isoforms (SphK1/2) and a microtubule disrupting agent (MDA). In mechanism-of-action studies, we have shown that these two separate actions synergize to induce cancer cell death in acute myeloid leukemia (AML) cell and animal models.

Transdifferentiation of Human Circulating Monocytes Into Neuronal-Like Cells in 20 Days and Without Reprograming.

Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming.

SKI-178: A Multitargeted Inhibitor of Sphingosine Kinase and Microtubule Dynamics Demonstrating Therapeutic Efficacy in Acute Myeloid Leukemia Models.

Aim: To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178.

Methods: Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, and assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML).

Results: Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2.

Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking.

Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids.

The regulatory roles of ROCK and MRCK kinases in the plasticity of cancer cell migration.

Metastatic cancer cells show great plasticity in their migratory mechanisms. In this review we briefly describe the signal transduction pathways associated with the ROCK and MRCK kinases and their roles in cancer cell migration and in its plasticity. With respect to therapeutic strategies targeting metastatic cancers, selectively blocking a single target, such as ROCK or MRCK, can induce alternate modes of cancer cell migration (i.

The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines.

We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis.

A novel selective multikinase inhibitor of ROCK and MRCK effectively blocks cancer cell migration and invasion.

Two structurally related protein kinase families, the Rho kinases (ROCK) and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) are required for migration and invasion of cancer cells. We hypothesized that simultaneous targeting of these two kinase families might represent a novel therapeutic strategy to block the migration and invasion of metastatic cancers. To this end, we developed DJ4 as a novel small molecule inhibitor of these kinases.

Gβ2 and Gβ4 participate in the opioid and adrenergic receptor-mediated Ca2+ channel modulation in rat sympathetic neurons.

Cardiac function is regulated in part by the sympathetic branch of the autonomic nervous system via the stellate ganglion (SG) neurons. Neurotransmitters, such as noradrenaline (NA), and neuropeptides, including nociceptin (Noc), influence the excit ability of SG neurons by modulating Ca(2+) channel function following activation of the adrenergic and nociceptin/orphanin FQ peptide (NOP) opioid receptors, respectively. The regulation of Ca(2+) channels is mediated by Gβγ, but the specific Gβ subunit that modulates the channels is not known.

Sphingosine kinase: a key to solving the 'French Paradox'?

A host of beneficial effects have been attributed to the red wine polyphenol, resveratrol. Foremost, among these are its anti-cancer properties. Yet, the mechanism by which resveratrol achieves these effects are unknown.

Targeting sphingosine kinase-1 to inhibit melanoma.

Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient's tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas.

Development of novel naphthalimide derivatives and their evaluation as potential melanoma therapeutics.

Synthesis and anti-melanoma activity of various naphthalimide analogs, rationally modified by introducing isothiocyanate (ITC) and thiourea (TU) functionalities, found in well-known anti-cancer agents, is described. The structure-activity relationship comparison of the novel agents in inhibiting cancer cell growth was evaluated in various melanoma cell lines. Both ITC and TU analogs effectively inhibited cell viability and induced apoptosis in various human melanoma cells.

Transcriptional regulation of the human neutral ceramidase gene.

Ceramidases play a critical role in generating sphingosine-1-phosphate by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. In order to elucidate its transcriptional regulation, we identify here a putative promoter region in the 5'-UTR of the human neutral CDase (nCDase) gene. Using human genomic DNA, we cloned a 3000 bp region upstream of the translational start site of the nCDase gene.

AP-1 binding transcriptionally regulates human neutral ceramidase.

Many forms of cellular stress cause an elevation of endogenous ceramide levels leading to growth arrest or apoptosis. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine-1-phosphate. Growth factor induction of neutral CDase (nCDase) has been shown to have a cytoprotective effect against cytokine-induced increases in ceramide levels.

Development of a sphingosine kinase 1 specific small-molecule inhibitor.

The sphingolipid metabolic pathway represents a potential source of new therapeutic targets for numerous hyperproliferative/inflammatory diseases. Targets such as the sphingosine kinases (SphKs) have been extensively studied and numerous strategies have been employed to develop inhibitors against these enzymes. Herein, we report on the optimization of our novel small-molecule inhibitor SKI-I (N'-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide) and the identification of a SphK1-specific analog, SKI-178, that is active in vitro and in vivo.

Enhancement of sphingosine kinase 1 catalytic activity by deletion of 21 amino acids from the COOH-terminus.

Sphingosine kinase 1 (SphK1) responds to a variety of growth factor signals by increasing catalytic activity as it translocates to the plasma membrane (PM). Several studies have identified amino acids residues involved in translocation yet how SphK1 increases its catalytic activity remains to be elucidated. Herein, we report that deletion of 21 amino acids from the COOH-terminus of SphK1 (1-363) results in increased catalytic activity relative to wild-type SphK1 (1-384) which is independent of the phosphorylation state of Serine 225 and PMA stimulation.

Sphingosine kinase 1 localized to the plasma membrane lipid raft microdomain overcomes serum deprivation induced growth inhibition.

Several studies have demonstrated that sphingosine kinase 1 (SphK1) translocates to the plasma membrane (PM) upon its activation and further suggested the plasma membrane lipid raft microdomain (PMLRM) as a target for SphK1 relocalization. To date, however, direct evidence of SphK1 localization to the PMLRM has been lacking. In this report, using multiple biochemical and subcellular fractionation techniques we demonstrate that endogenous SphK1 protein and its substrate, D-erythro-sphingosine, are present within the PMLRM.

Encapsulation of organic molecules in calcium phosphate nanocomposite particles for intracellular imaging and drug delivery.

Encapsulation of imaging agents and drugs in calcium phosphate nanoparticles (CPNPs) has potential as a nontoxic, bioresorbable vehicle for drug delivery to cells and tumors. The objectives of this study were to develop a calcium phosphate nanoparticle encapsulation system for organic dyes and therapeutic drugs so that advanced fluoresence methods could be used to assess the efficiency of drug delivery and possible mechanisms of nanoparticle bioabsorption. Highly concentrated CPNPs encapsulating a variety of organic fluorophores were successfully synthesized.

Network model of survival signaling in large granular lymphocyte leukemia.

T cell large granular lymphocyte (T-LGL) leukemia features a clonal expansion of antigen-primed, competent, cytotoxic T lymphocytes (CTL). To systematically understand signaling components that determine the survival of CTL in T-LGL leukemia, we constructed a T-LGL survival signaling network by integrating the signaling pathways involved in normal CTL activation and the known deregulations of survival signaling in leukemic T-LGL. This network was subsequently translated into a predictive, discrete, dynamic model.