ucn-1028-c and safingol

A large body of evidence suggests that the abnormal phenotype of neoplastic astrocytes, including their excessive proliferation rate and high propensity to invade surrounding tissues, results from mutations in critical genes involved in key cellular events. These genetic alterations can affect cell-surface-associated receptors, elements of signaling pathways, or components of the cell cycle clock, conferring a gain or a loss of relevant metabolic functions of the cells. The understanding of such phenomena may allow the development of more efficacious forms of cancer treatment. Examples are therapies specifically directed against overexpressed epidermal growth factor receptor, hyperactive Ras, excessively stimulated Raf-1, overproduced ornithine decarboxylase, or aberrantly activated cyclin-dependent kinases. The applicability of some of these approaches is now being assessed in patients suffering from primary malignant central nervous system tumors that are not amenable to current therapeutic modalities. Another potentially useful therapeutic strategy against such tumors involves the inhibition of hyperactive or overexpressed protein kinase C (PKC). This strategy is justified by the decrease in cell proliferation and invasion following inhibition of the activity of this enzyme observed in preclinical glioma models. Thus, interference with PKC activity may represent a novel form of experimental cancer treatment that may simultaneously restrain the hyperproliferative state and the invasive capacity of high-grade malignant gliomas without inducing the expected toxicity of classical cytotoxic agents. Of note, the experimental use of PKC-inhibiting agents in patients with refractory high-grade malignant gliomas has indeed led to some clinical responses. The present paper reviews the current status of the biochemistry and molecular biology of PKC, as well as the possibilities for developing novel anti-PKC-based therapies for central nervous system malignancies.

Topics: Adolescent; Adult; Antineoplastic Agents; Apoptosis; Bryostatins; Cell Division; Child; Child, Preschool; Enzyme Activation; Enzyme Inhibitors; Glioma; Humans; Infant; Lactones; Macrolides; Mutation; Naphthalenes; Neoplasm Invasiveness; Neoplasm Proteins; Phenotype; Protein Kinase C; Sphingosine; Staurosporine

Neoplastic cell survival is governed by a balance between pro-apoptotic and anti-apoptotic signals. Noteworthy among several anti-apoptotic signaling elements is the protein kinase C (PKC) isoenzyme family, which mediates a central cytoprotective effect in the regulation of cell survival. Activation of PKC, and subsequent recruitment of numerous downstream elements such as the mitogen-activated protein kinase (MAPK) cascade, opposes initiation of the apoptotic cell death program by diverse cytotoxic stimuli. The understanding that the lethal actions of numerous antineoplastic agents are, in many instances, antagonized by cytoprotective signaling systems has been an important stimulus for the development of novel antineoplastic strategies. In this regard, inhibition of PKC, which has been shown to initiate apoptosis in a variety of malignant cell types, has recently been the focus of intense interest. Furthermore, there is accumulating evidence that selective targeting of PKC may prove useful in improving the therapeutic efficacy of established antineoplastic agents. Such chemosensitizing strategies can involve either (a) direct inhibition of PKC (e.g., following acute treatment with relatively specific inhibitors such as the synthetic sphingoid base analog safingol, or the novel staurosporine derivatives UCN-01 and CGP-41251) or (b) down-regulation (e.g., following chronic treatment with the non-tumor-promoting PKC activator bryostatin 1). In preclinical model systems, suppression of the cytoprotective function(s) of PKC potentiates the activity of cytotoxic agents (e.g., cytarabine) as well as ionizing radiation, and efforts to translate these findings into the clinical arena in humans are currently underway. Although the PKC-driven cytoprotective signaling systems affected by these treatments have not been definitively characterized, interference with PKC activity has been associated with loss of the mitogen-activated protein kinase (MAPK) response. Accordingly, recent pre-clinical studies have demonstrated that pharmacological disruption of the primary MEK-ERK module can mimic the chemopotentiating and radiopotentiating actions of PKC inhibition and/or down-regulation.

Topics: Animals; Antineoplastic Agents; Bryostatins; Cell Survival; Cytoprotection; Enzyme Inhibitors; Humans; Lactones; Macrolides; Mitogen-Activated Protein Kinase Kinases; Naphthalenes; Protein Kinase C; Sphingosine; Staurosporine

Previous studies have shown that fumonisin B1 (FB1) inhibits ceramide synthase, resulting in accumulation of free sphinganine and sphingosine. Tumor necrosis factor-alpha (TNFalpha) plays an important role in FB1 toxicity and the expression of TNFalpha mRNA in liver and kidney is increased following FB1 exposure in mice. The objective of the current study was to investigate whether these two events (sphingoid bases accumulation and TNFalpha induction) are dependent on each other. An increase in expression of TNFalpha mRNA was detected in LLC-PK1 cells as early as 4 h after FB1 treatment but decreased to the control levels after 8 h. A positive linear correlation was observed between the expression of TNFalpha mRNA and FB1 concentration. Increases of intracellular sphingoid bases were also detected after 4 h of FB1 treatment and progressively increased until 24 h. Exposure of the cells to sphinganine or sphingosine did not significantly alter the expression of TNFalpha. Inhibition of sphingoid base biosynthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, efficiently blocked the accumulation of free sphingoid bases in response to FB1, but it did not prevent the induction of TNFalpha expression. Results indicate that FB1-induced increase in TNFalpha expression is independent of sphingoid base accumulation-induced by ceramide synthase inhibition in LLC-PK1 cells.

Topics: Acyltransferases; Animals; Carboxylic Acids; Cell Line; Ceramides; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Gene Expression; Kidney; Kinetics; Mitogen-Activated Protein Kinases; Naphthalenes; Oxidoreductases; Protein Kinase C; RNA, Messenger; Serine C-Palmitoyltransferase; Sphingosine; Swine; Tumor Necrosis Factor-alpha

In osteoblast-like MC3T3-E1 cells, we have recently reported that sphingosine 1-phosphate among sphingomyelin metabolites acts as a second messenger for tumor necrosis factor-alpha (TNF)-induced interleukin-6 (IL-6) synthesis. In the present study, we investigated the effect of extracellular sphingomyelinase on IL-6 synthesis in MC3T3-E1 cells. Sphingomyelinase stimulated IL-6 synthesis in a time-dependent manner for up to 24 h. This stimulative effect was dose dependent in the range between 1 and 300 mU/ml. Calphostin C, a highly and potent inhibitor of protein kinase C, enhanced sphingomyelinase-induced IL-6 synthesis. DL-Threo-dihydrosphingosine, an inhibitor of sphingosine kinase, significantly inhibited the IL-6 synthesis induced by sphingomyelinase. Sphingomyelinase markedly elicited sphingomyelin hydrolysis. In addition, the effect of a combination of sphingomyelinase and TNF on IL-6 synthesis was synergistic. These results strongly suggest that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblasts, resulting in IL-6 synthesis, and that protein kinase C acts as a negative controller of the IL-6 synthesis.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Interleukin-6; Mice; Naphthalenes; Osteoblasts; Protein Kinase C; Sphingomyelin Phosphodiesterase; Sphingomyelins; Sphingosine; Time Factors; Tumor Necrosis Factor-alpha

Nerve terminals ("synaptosomes") isolated from rat brain hippocampus were loaded with the fluorescent Ca2+ indicator fura-2 and were subjected to depolarization with an elevated K+ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca2+ channels in the presynaptic membrane. Three components of Ca2+ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca2+ channels with different inactivation kinetics (tau of approximately 60 ms and 1 s, respectively) and Na+/Ca2+ exchange working in the "reverse" mode. The activity of both classes of voltage-dependent Ca2+ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca2+ channel activity, but the effect of DHS could be slowed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca2+ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.

Topics: Animals; Calcium; Calcium Channels; Fura-2; Hippocampus; Kinetics; Naphthalenes; Phorbol Esters; Phosphorylation; Polycyclic Compounds; Potassium; Presynaptic Terminals; Protein Kinase C; Rats; Sodium; Spectrometry, Fluorescence; Sphingosine; Tetradecanoylphorbol Acetate