Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms.

Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6-12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34(+) leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34(+) CD38(Low/Neg) CD123(+)) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.

Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: consequences for erucylphosphocholine-induced apoptosis.

Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8-33% (Apaf-1) and to 30-50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17-33% (Apaf-1) and to 38-50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.

Bcl-2 mediated inhibition of erucylphosphocholine-induced apoptosis depends on its subcellular localisation.

The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2. Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood. To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/DeltaTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria. The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.

Miltefosine (Impavido): the first oral treatment against leishmaniasis.

Miltefosine is a novel antileishmanial drug that has significant selectivity in both in vitro and in vivo models. Clinical efficacy was demonstrated for the treatment of visceral leishmaniasis with the advantage of oral administration over the currently recommended antileishmanial drugs that require parenteral administration. Miltefosine produces high cure rates also in patients resistant to the standard antimonial therapy.

Induction of differentiation and tetraploidy by long-term treatment of C6 rat glioma cells with erucylphosphocholine.

Induction of differentiation represents a promising concept for chemotherapy of malignant gliomas, which are often refractory even to the combined treatment with surgery, irradiation and chemotherapy. Since anti-neoplastic alkylphosphocholines can induce differentiation of leukemic cell lines, the effects of the intravenously applicable alkylphosphocholine-derivative erucylphosphocholine (ErPC) on proliferation, morphology and differentiation of the rat glioma cell line C6 was examined in vitro. Short-term exposure to ErPC induced accumulation of the cells in the G2/M-phase of the cell cycle and apoptotic cell death. In contrast, continuous exposure of C6 rat glioma cells to sublethal ErPC doses (30 and 50 microM) caused both the formation of a slower growing tetraploid cell population and astrocytic differentiation. No resistance to in vivo obtainable ErPC concentrations was observed during this treatment. We conclude that ErPC-induced differentiation might be beneficial for a long-term adjuvant chemotherapy of low grade glioma.

Separation and quantitation of alkylphosphocholines and analogues of different liposome formulations by HPTLC.

High-performance thin-layer chromatographic (HPTLC) analysis of non UV-active phospholipids in biological matrixes is a common method for separation, detection, and quantitation. Liposomes containing new alkylphosphocholines and analogues with enhanced cytostatic activity had been prepared. The liposomal formulations were designed to enable the intravenous application of the alkylphosphocholines and analogues and to reduce dose-limiting toxicities observed after oral administration. For quality control the liposomes were analyzed by HPTLC for content of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), cholesterol, alkylphosphocholines, and analogues and their related compounds (main degradation products). Due to the differences in lipophily of the compounds, different mobile phases were necessary to achieve separation. Automated Multiple Development was used to reduce the number of plates and to improve the selectivity and the capacity of the chromatographic system to separate the described alkylphosphocholines and analogues from DPPG and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in one chromatographic system.

Effects of miltefosine and other alkylphosphocholines on human intestinal parasite Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess. It is therefore responsible for significant morbidity and mortality in a number of countries. Infections with E. histolytica are treated with nitroimidazoles, primarily with metronidazole. At this time, there is a lack of useful alternative classes of substances for the treatment of invasive amoebiasis. Alkylphosphocholines (alkyl-PCs) such as hexadecyl-PC (miltefosine) were originally developed as antitumor agents, but recently they have been successfully used for the treatment of visceral leishmaniasis in humans. We examined hexadecyl-PC and several other alkyl-PCs with longer alkyl chains, with and without double bond(s), for their activity against two strains of E. histolytica. The compounds with the highest activity were oleyl-PC, octadecyl-PC, and nonadecenyl-PC, with 50% effective concentrations for 48 h of treatment between 15 and 21 microM for strain SFL-3 and between 73 and 98 microM for strain HM-1:IMSS. We also tested liposomal formulations of these alkyl-PCs and miltefosine. The alkyl-PC liposomes showed slightly lower activity, but are expected to be well tolerated. Liposomal formulations of oleyl-PC or closely related alkyl-PCs could be promising candidates for testing as broad-spectrum antiprotozoal and antitumor agents in humans.

Erucylphosphocholine-induced apoptosis in chemoresistant glioblastoma cell lines: involvement of caspase activation and mitochondrial alterations.

Intrinsic chemoresistance constitutes a major problem in the therapy of malignant gliomas. In vitro experiments with four astrocytoma/glioblastoma (AC/GBM) cell lines revealed that the chemoresistance of two cell lines, A172 and T98G, to cisplatin and etoposide was due to resistance to drug-induced apoptosis. In contrast, all the AC/GBM cell lines tested were sensitive to treatment with the lipophilic ether lipid erucylphosphocholine, ErPC. ErPC-induced apoptosis was independent of wild-type p53-signaling and triggering of the CD95/CD95 ligand (CD95L) system. Inhibition of protein and RNA synthesis by cycloheximide and actinomycin D did not abrogate ErPC-induced apoptosis. However, expression of members of the bcl-2 protein family was modulated during ErPC treatment. Activation of caspase 3 and mitochondrial alterations were central to ErPC-induced apoptosis. We conclude that ErPC-induced activation of the mitochondrial pathway enables cell death in the chemoresistant AC/GBM cells.

Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography.

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.

Transient and controllable opening of the blood-brain barrier to cytostatic and antibiotic agents by alkylglycerols in rats.

The blood-brain barrier hinders progress in the chemotherapy of brain tumors due to insufficient penetration of anticancer drugs into the brain tissue. Short-chain alkylglycerols affect the physicochemical properties of biological membranes. The enhancement of the blood-brain barrier permeability by intra-arterial administration of alkylglycerols was investigated in tumor-free and C6 astroglioma bearing rats. The antineoplastic agents cisplatin and methotrexate and the antibiotics vancomycin and gentamicin were selectively injected into the right internal carotid artery in the absence and presence of various alkylmono-, alkyldi-, and alkyltriglycerols. In normal rats the intra-arterial administration of the drugs without alkylglycerols resulted in low drug concentrations in brain tissue. In the presence of alkylglycerols (0.01-0.3 M) a reversible (within minutes) and concentration-dependent enrichment of the coinjected agents was found, preferentially in the ipsilateral hemisphere. The extent of drug accumulation in the brain was modified by changes in the chemical structure of the alkylglycerols. The effect increased with the chain length of the alkyl group, decreased with the number of glycerols, and varied from 2- to more than 230-fold compared to controls. In rats with C6 tumors 1-O-pentylglycerol increased the delivery of methotrexate 18-fold in the tumor, 28-fold in the surrounding brain, 18-fold in the contralateral brain, and 19-fold in the cerebellum compared to controls with methotrexate in the absence of pentylglycerol. In conclusion, the intra-arterial administration of alkylglycerols represents a novel and well controllable method for enhanced drug delivery to the brain and to brain tumors.

BCR-ABL influences the antileukaemic efficacy of alkylphosphocholines.

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr-abl rearrangement (K-562, LAMA-84, CML-T1 and BV-173) and two other leukaemic cell lines (HL-60 and SKW-3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR-ABL-positive cell lines than the two reference APC. It induced programmed cell death in HL-60 and SKW-3 cells after exposure for 24 h, and in bcr-abl expressing cells after a prolonged incubation period (48 h). LAMA-84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast-like phenotype. Experiments with a cell-free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT-PCR we confirmed that K-562 and LAMA-84 cells carry the b3a2 fusion type, and CML-T1 and BV-173 the b2a2 variant. BV-173 cells had the lowest level of bcr-abl mRNA which correlated with their increased sensitivity. Transfection of K-562 cells with antisense oligonucleotides directed against bcr-abl caused a specific suppression of K-562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR-ABL-positive blasts as compared with miltefosine and perifosine. The expression of BCR-ABL cannot prevent apoptosis but delays erucylphosphocholine-induced programmed cell death. Transfection with bcr-abl directed antisense oligonucleotides reduces the clonogenicity of K-562 cells.

Erucylphosphocholine: pharmacokinetics, biodistribution and CNS-accumulation in the rat after intravenous administration.

The clinical use of alkylphosphocholines (APC) in cancer patients is restricted because of the high gastrointestinal toxicity and the need for oral administration. Therefore we evaluated the clinical pharmacology of erucylphosphocholine (ErPC), the first derivative of the APC family suitable for intravenous administration with strong antineoplastic activity, in vitro and in vivo in rats. The pharmacokinetic parameters after a single intravenous dose of 40 mg/kg were calculated using a two-compartment model: C(max) = 1.6 +/- 0.3 micromol/ml, T(1/2alpha) = 0.18 +/- 0.09 h, T(1/2beta) = 3.3 +/- 0.88 h, clearance = 9.7 +/- 1.2 ml/h, AUC = 2.5 +/- 0.3 micromol/ml per h and Vss = 40.4 +/- 7.9 ml. Biodistribution studies were performed after repeated ErPC administration at different doses. Intravenous injections of 20 mg/kg given at intervals of 48 h for up to 4 weeks were well tolerated. Neither clinical evaluation nor laboratory parameters (haematology and clinical chemistry) revealed toxic side effects. In contrast, higher doses of ErPC (40 mg/kg per 48 h) led to weight loss. After 2 and 4 weeks of therapy with 20 mg/kg per 48 h a high ErPC accumulation was found in the adrenal glands, small intestine and brain. The brain to serum concentration ratios averaged 2.1 after 2 weeks and 4.5 after 4 weeks. Significant leucocytosis and thrombocytosis were observed after 4 weeks of ErPC treatment. The findings suggest that ErPC is a suitable candidate for clinical trials. In particular, owing to the high accumulation in brain tissue, ErPC is a potential agent for chemotherapy against malignant brain tumours.

Erucylphosphocholine, a novel antineoplastic ether lipid, blocks growth and induces apoptosis in brain tumor cell lines in vitro.

A potential benefit of alkylphosphocholines in brain tumor therapy was evaluated. The in vitro effects of the intravenously applicable erucylphosphocholine (ErPC) on proliferation, viability, morphology and cell cycle distribution of a rat glioma, four human astrocytoma/glioblastoma and a human medulloblastoma cell line were analyzed daily after continuous drug-exposure for up to six days. ErPC exerted strong cytostatic and direct cytotoxic effects on all cell lines tested at drug concentrations that are achieved in the rat brain after repeated intravenous injections of nontoxic drug doses. Concentrations of 70 microM (T98G, A172, 85HG66, 86HG39) and 110 microM (C6, D283 Med) led to complete cell death within 48-96 h. Particular characteristics of ErPC action are i) the accumulation of cells with a 4n DNA content corresponding to the G2/M-phase of the cell cycle, ii) the formation of two- and multinucleated cells and iii) the induction of apoptosis.

Antitumor effects of erucylphosphocholine on brain tumor cells in vitro and in vivo.

The antineoplastic activity of erucylphosphocholine (ErPC) on C6 rat glioma cells and the human glioblastoma cell lines A172 and T98G was studied in vitro. ErPC exerted concentration- and time-dependent cytostatic and cytotoxic actions. The effects of ErPC on C6 cells were stronger than hexadecylphosphocholine and similar to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. The human cell lines were more sensitive to ErPC (LC50 = 36 microM and 29 microM) than C6 cells (LC50 = 70 microM) (48 hours, WST-1-test). Morphological characteristics of apoptotic cell death were observed. Rats bearing intracerebral and subcutaneous gliomas were treated with intravenous ErPC for 30 days to determine the antitumor effects of ErPC in vivo. ErPC accumulation in subcutaneous tumors was higher than in brain tissue. Peripheral tumors revealed a better response than CNS-tumors. There was a strong negative relationship between the concentration of ErPC and the weight of peripheral tumors. ErPC is considered to be promising for chemotherapy of malignant brain tumors.

Alkylphosphocholines induce apoptosis in HL-60 and U-937 leukemic cells.

Alkylphosphocholines (APC) represent a new group of ether-lipid-related compounds with remarkable activity against transformed cells in vitro and good tolerability in vivo. Their mechanism of action remains unknown. The aim of the present study was to investigate the effects of a series of APC on three human leukemic cell lines: K-562, HL-60, and U-937. The tetrazolium dye-reduction (MTT) assay and cell counting were used to determine the cytotoxicity of the APC used. DNA gel electrophoresis and enzyme-linked immunosorbent assay (ELISA) detection of oligonucleosomes were performed to identify and quantify DNA fragmentation. Electron and phase-contrast microscopy were used to detect morphologic changes specific for programmed cell death. HL-60 and U-937 cells were found to be sensitive, but K-562 cells were relatively resistant to APC exposure. APC with long alkyl chains exerted stronger cytotoxicity than did those with short alkyl chains. DNA fragmentation was found after treatment with APC in HL-60 and U-937 cells but not in K-562 cells. In HL-60 cells the increase in mono- and oligonucleosome formation as measured by ELISA was correlated with the length of the alkyl chains at 14 h of exposure to APC but plateaued at 20 h. The morphologic alterations in HL-60 and U-937 cell lines, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies, confirmed the induction of apoptosis after APC exposure. It is concluded that programmed cell death plays an important role in the cytotoxicity of APC against certain human leukemic cell lines. The antineoplastic profiles of APC with long alkyl chains render them attractive for further therapeutic application.

On the mechanism of lysophospholipase activity of secretory phospholipase A2 (EC 3.1.1.4): deacylation of monoacylphosphoglycerides by intrinsic sn-1 specificity and pH-dependent acyl migration in combination with sn-2 specificity.

We show for the first time that secreted low-molecular weight phospholipase A2 (EC 3.1.1.4) catalyzes the deacylation of monoacylphosphoglycerides directly from the sn-1 position, although at a very low rate: purified phospholipase A2 enzymes from bee venom, crotalus atrox venom, and porcine pancreas hydrolyze the sn-1 ester bond in 1-palmitoyl-2-O-methyl-sn-glycero-3-phosphorylcholine. Hydrolytic rates with the corresponding isomer, 1-O-methyl-2-palmitoyl-sn-glycero-3-phosphorylcholine, are about 3-4 orders of magnitude higher. The similarities in Ca2+ requirement and inactivation profiles suggest that deacylation, albeit with different rates, from both sn-1 and sn-2 positions is catalyzed by the same catalytic site of phospholipase A2. Furthermore, evidence is provided that phospholipase A2-catalyzed 1-acyl lysophospholipid deacylation is mediated by sn-1-directed action, but above pH 7 acyl migration with subsequent enzyme-catalyzed hydrolytic cleavage from the sn-2 position contributes to the overall deacylation of monoacylphosphoglycerides, acyl migration becoming eventually the rate-limiting factor.