Induction of ER and mitochondrial stress by the alkylphosphocholine erufosine in oral squamous cell carcinoma cells.

Endoplasmic reticulum (ER) plays an essential role in cell function and survival. Accumulation of unfolded or misfolded proteins in the lumen of the ER activates the unfolded protein response (UPR), resulting in ER stress and subsequent apoptosis. The alkylphosphocholine erufosine is a known Akt-mTOR inhibitor in oral squamous cell carcinoma (OSCC). In the present study, we evaluate erufosine's role to induce ER and mitochondrial stress leading to autophagy, apoptosis, and ROS induction. The cellular toxicity of erufosine was determined in two OSCC cell lines and gene expression and enrichment analyses were performed. A positive enrichment of ER stress upon erufosine exposure was observed, which was verified at protein levels for the ER stress sensors and their downstream mediators. Knockdown and pharmacological inhibition of the ER stress sensors PERK and XBP1 revealed their involvement into erufosine's cellular effects, including proliferation, apoptosis, and autophagy induction. Autophagy was confirmed by increased acidic vacuoles and LC3-B levels. Upon erufosine exposure, calcium influx into the cytoplasm of the two OSCC cell lines was seen. Apoptosis was confirmed by nuclear staining, Annexin-V, and immunoblotting of caspases. The induction of mitochondrial stress upon erufosine exposure was predicted by gene set enrichment analysis (GSEA) and shown by erufosine's effect on mitochondrial membrane potential, ATP, and ROS production in OSCC cells. These data show that ER and mitochondrial targeting by erufosine represents a new facet of its mechanism of action as well as a promising new framework in the treatment of head and neck cancers.

Gemcitabine treatment of rat soft tissue sarcoma with phosphatidyldiglycerol-based thermosensitive liposomes.

PURPOSE: The pyrimidine analogue gemcitabine (dFdC) is frequently used in the treatment of patients with solid tumors. However, after i.v. application dFdC is rapidly inactivated by metabolization. Here, the potential of thermosensitive liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2-TSL) were investigated as carrier and targeting system for delivery of dFdC in combination with local hyperthermia (HT). METHODS: DPPG2-TSL were prepared by the lipid film hydration and extrusion method and characterized by dynamic light scattering, thin layer chromatography, phosphate assay and HPLC. In vivo experiments were performed in Brown Norway rats with a syngeneic soft tissue sarcoma. Local HT treatment was performed by light exposure. RESULTS: DPPG2-TSL were stable at 37°C in serum and showed a temperature dependent dFdC release >40°C. Plasma half-life of dFdC was strongly increased from 0.07 h (non-liposomal) to 0.53 h (liposomal, vesicle size 105 nm) or 2.59 h (liposomal, 129 nm). Therapy of BN175 tumors with dFdC encapsulated in DPPG2-TSL + HT showed significant improvement in tumor growth delay compared to non-liposomal dFdC without HT (p < 0.05), non-liposomal dFdC with HT (p < 0.01), and liposomal dFdC without HT (p < 0.05), respectively. CONCLUSIONS: Gemcitabine encapsulated in DPPG2-TSL in combination with local HT is a promising tool for the treatment of solid tumors. Therefore, these encouraging results ask for further investigation and evaluation.

Effects of ionizing radiation in combination with Erufosine on T98G glioblastoma xenograft tumours: a study in NMRI nu/nu mice.

BACKGROUND: Erufosine is a promising anticancer drug that increases the efficacy of radiotherapy in glioblastoma cell lines in vitro. Moreover, treatment of nude mice with repeated intraperitoneal or subcutaneous injections of Erufosine is well tolerated and yields drug concentrations in the brain tissue that are higher than the concentrations required for cytotoxic drug effects on glioblastoma cell lines in vitro. METHODS: In the present study we aimed to evaluate the effects of a combined treatment with radiotherapy and Erufosine on growth and local control of T98G subcutaneous glioblastoma xenograft-tumours in NMRI nu/nu mice. RESULTS: We show that repeated intraperitoneal injections of Erufosine resulted in a significant drug accumulation in T98G xenograft tumours on NMRI nu/nu mice. Moreover, short-term treatment with 5 intraperitoneal Erufosine injections caused a transient decrease in the growth of T98G tumours without radiotherapy. Furthermore, an increased radiation-induced growth delay of T98G xenograft tumours was observed when fractionated irradiation was combined with short-term Erufosine-treatment. However, no beneficial drug effects on fractionated radiotherapy in terms of local tumour control were observed. CONCLUSIONS: We conclude that short-term treatment with Erufosine is not sufficient to significantly improve local control in combination with radiotherapy in T98G glioblastoma xenograft tumours. Further studies are needed to evaluate efficacy of extended drug treatment schedules.

Proteins and cholesterol lipid vesicles are mediators of drug release from thermosensitive liposomes.

Thermosensitive liposomes (TSL) are a promising tool for triggered drug delivery in combination with local hyperthermia. Objective of this study was to investigate the influence of serum on TSL in more detail and to identify serum components which are responsible for increasing drug release. Four different formulations were investigated: DPPC/DSPC/1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG(2)) 50/20/30 (mol/mol) (DPPG(2)-TSL); DPPC/DSPC/DPPG(2)/DSPE-PEG2000 50/15/30/5 (mol/mol) (DPPG(2)/PEG-TSL), DPPC/P-Lyso-PC/DSPE-PEG2000 90/10/4 (mol/mol) (PEG/Lyso-TSL), and DPPC/DSPC/DSPE-PEG2000 80/15/5 (mol/mol) (PEG-TSL). DPPG(2)-TSL was the only formulation which was unaffected by osmotic stress. All formulations tested were influenced by serum components but the susceptibility was depended on the lipid composition of the vesicle. Presence of albumin (HSA) or cholesterol-containing lipid vesicles (DPPC/Chol-LLV) increased the membrane permeability for all tested formulations at temperatures around and above T(m) in a concentration based manner. PEGylation was not able to prevent the observed effect. PEG-TSL and PEG/Lyso-TSL were more susceptible to DPPC/Chol-LLV than DPPG(2)-containing TSL. In contrast, immunoglobulin type G (IgG) affected only anionic formulations. The membrane of DPPG(2)-TSL and DPPG(2)/PEG-TSL was more susceptible toward IgG as compared to HSA. DPPG(2)-TSL and PEG/Lyso-TSL were differentially influenced by fetal calf serum (FCS). As DPPG(2)-TSL was stabilized by pre-incubation with FCS at 37°C, this was the opposite for PEG/Lyso-TSL which were destabilized under these conditions. Individual serum components were unable to mimic the complex situation in full serum. Hence, the use of plasma or serum is still inevitable to investigate stability and release properties of novel TSL formulations until all serum components have been identified that alter TSL integrity.

Erufosine suppresses breast cancer in vitro and in vivo for its activity on PI3K, c-Raf and Akt proteins.

PURPOSE: This study investigated the antineoplastic effect of the membrane active alkylphosphocholine erufosine in breast carcinoma models in vitro and in vivo and determined its influence on the PI3K/Akt and Ras/Raf/MAPK signaling pathways. METHODS: The antiproliferative effect of erufosine in vitro was determined by the MTT dye reduction assay, and the antineoplastic efficacy on tumor growth was investigated by relating the mean total tumor volumes of treated and control rats. Immunoblot analysis was used for detecting changes in the expression level of the signal molecules p-PI3K (p-p85), p-Akt at Thr 308 and p-cRaf. RESULTS: Based on their IC(50) (40 µM, respectively), the breast carcinoma cell lines MCF-7 and MDA-MB 231, which are estrogen receptor positive and negative, respectively, were equally sensitive to erufosine. In addition, erufosine caused dose-dependent decreases in the phosphorylation of PI3K (p85), Akt (PKB) at Thr 308 and cRaf in both cell lines. Moreover, administration of erufosine to rats bearing autochthonous methylnitrosourea-induced rat mammary carcinomas caused a significant dose-related tumor remission by more than 85 % (p < 0.05), which was well tolerated, as evidenced by a body weight loss of maximally 7 % and reduced tumor-related mortality (2 of 35 instead of 6 of 18 controls, p < 0.002). CONCLUSIONS: The results clearly indicate that erufosine possesses high antineoplastic activity not only in human breast cancer cell lines in vitro but also in rat mammary carcinoma in vivo. In addition, it can be derived that the mechanism of action of erufosine involves influence on both, PI3K/Akt and Ras/Raf/MAPK signaling pathways.

The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation.

BACKGROUND AND PURPOSE: The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined. MATERIALS AND METHODS: Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis. RESULTS: The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy. CONCLUSIONS: Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines - one which is sensitive to the Akt-inhibitor ErPC3 and one which is more sensitive to the PI3K-inhibitor LY294002. Our findings underline the importance for the definition of predictive biomarkers that allow the selection patients that may benefit from the treatment with a specific signal transduction modifier.

Size of thermosensitive liposomes influences content release.

Thermosensitive liposomes (TSL) in combination with regional hyperthermia represent a powerful tool for tumor specific drug delivery. The objective of this study was to investigate the influence of vesicle size on the biophysical properties of TSL. TSL were composed of DPPC/DSPC/1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPG(2)) 50:20:30 (mol/mol) (DPPG(2)-TSL) and DPPC/P-Lyso-PC/DSPE-PEG2000 90:10:4 (mol/mol) (PEG/Lyso-TSL) with encapsulated fluorescent dye carboxyfluorescein, anticancer drug doxorubicin or magnetic resonance contrast agent gadodiamide. Extrusion was performed with polycarbonate filters of distinct pore size to obtain TSL with different diameters (50 to 200nm). Phase transition temperature (T(m)) of the bilayer forming phospholipids was not influenced by vesicle size in the tested range. However, vesicle size had a major impact on in vitro content release properties of TSL in the investigated temperature range between 30 and 45°C. Generally, vesicle size was inversely related to content release properties with increased content release rates for decreased vesicle sizes. Size dependency of content release properties varied between all tested formulations and DPPG(2)-TSL were generally less affected by size changes in the range of 100 to 150nm as compared to PEG/Lyso-TSL. Independent from gadodiamide release, vesicle size influenced the signal intensity of DPPG(2)-TSL also at temperatures below T(m) due to improved water exchange for smaller vesicles. Liposomes around 100nm in size are routinely used in vivo, hence a quality control for TSL preparations is required prior to use. Even small changes in size or a wider size distribution might affect stability and release properties and thus yield in decreased efficacy or unwanted side effects of drug loaded TSL during in vivo applications.

Pharmacokinetics and biodistribution of Erufosine in nude mice--implications for combination with radiotherapy.

BACKGROUND: Alkylphosphocholines represent promising antineoplastic drugs that induce cell death in tumor cells by primary interaction with the cell membrane. Recently we could show that a combination of radiotherapy with Erufosine, a paradigmatic intravenously applicable alkylphosphocholine, in vitro leads to a clear increase of irradiation-induced cell death. In view of a possible combination of Erufosine and radiotherapy in vivo we determined the pharmacokinetics and bioavailability as well as the tolerability of Erufosine in nude mice. METHODS: NMRI (nu/nu) nude mice were treated by intraperitoneal or subcutaneous injections of 5 to 40 mg/kg body weight Erufosine every 48 h for one to three weeks. Erufosine-concentrations were measured in brain, lungs, liver, small intestine, colon, spleen, kidney, stomach, adipoid tissue, and muscle by tandem-mass spectroscopy. Weight course, blood cell count and clinical chemistry were analyzed to evaluate general toxicity. RESULTS: Intraperitoneal injections were generally well tolerated in all dose groups but led to a transient loss of the bodyweight (<10%) in a dose dependent manner. Subcutaneous injections of high-dose Erufosine caused local reactions at the injection site. Therefore, this regimen at 40 mg/kg body weight Erufosine was stopped after 14 days. No gross changes were observed in organ weight, clinical chemistry and white blood cell count in treated compared to untreated controls except for a moderate increase in lactate dehydrogenase and aspartate-aminotransferase after intensive treatment. Repeated Erufosine injections resulted in drug-accumulation in different organs with maximum concentrations of about 1000 nmol/g in spleen, kidney and lungs. CONCLUSION: Erufosine was well tolerated and organ-concentrations surpassed the cytotoxic drug concentrations in vitro. Our investigations establish the basis for a future efficacy testing of Erufosine in xenograft tumor models in nude mice alone and in combination with chemo- or radiotherapy.

Apoptotic DNA fragmentation is not related to the phosphorylation state of histone H1.

Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.

Increased cytotoxicity of ionizing radiation in combination with membrane-targeted apoptosis modulators involves downregulation of protein kinase B/Akt-mediated survival-signaling.

BACKGROUND AND PURPOSE: The membrane-targeted apoptosis modulators erucylphosphocholine (ErPC) and erucylphosphohomocholine (ErPC3) induce apoptosis in highly apoptosis resistant malignant glioma cell lines and enhance radiation-induced cell death and eradication of clonogenic tumor cells in vitro. Aim of the present study was to elucidate molecular mechanisms of combined action. MATERIALS AND METHODS: Induction of apoptosis was evaluated by determination of nuclear morphology (fluorescence microscopy), alteration of mitochondrial function and caspase-activation (flow cytometry, Western blot). Activity of protein kinase B (PKB/Akt) and key downstream effectors involved in apoptosis regulation was verified by Western blot analysis using activation-specific antibodies. RESULTS: Increased cytotoxicity of the combination was linked to a more efficient activation of the intrinsic apoptosis pathway with increased damage of the mitochondria and caspase-activation. Moreover, activity of the survival kinase PKB/Akt was downregulated upon treatment with ErPC/ErPC3 alone or in combination with ionizing radiation. Inhibition of PKB/Akt was associated with decreased phosphorylation and thus activation of the pro-apoptotic Bcl-2 protein Bad as well as dephosphorylation of the transcription factor FOXO3A (FKHRL1) that may be responsible for the observed increased expression of the pro-apoptotic Bcl-2 protein Bim. CONCLUSIONS: Our data suggest a role for inhibition of PKB/Akt-mediated anti-apoptotic signaling in increased efficacy of the combination.

The membrane targeted apoptosis modulators erucylphosphocholine and erucylphosphohomocholine increase the radiation response of human glioblastoma cell lines in vitro.

BACKGROUND: Alkylphosphocholines constitute a novel class of antineoplastic synthetic phospholipid derivatives that induce apoptosis of human tumor cell lines by targeting cellular membranes. We could recently show that the first intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC) is a potent inducer of apoptosis in highly resistant human astrocytoma/glioblastoma cell lines in vitro. ErPC was shown to cross the blood brain barrier upon repeated intravenous injections in rats and thus constitutes a promising candidate for glioblastoma therapy. Aim of the present study was to analyze putative beneficial effects of ErPC and its clinically more advanced derivative erucylphosphohomocholine (erucyl-N, N, N-trimethylpropanolaminphosphate, ErPC3, Erufosinetrade mark on radiation-induced apoptosis and eradication of clonogenic tumor cells in human astrocytoma/glioblastoma cell lines in vitro. RESULTS: While all cell lines showed high intrinsic resistance against radiation-induced apoptosis as determined by fluorescence microscopy, treatment with ErPC and ErPC3 strongly increased sensitivity of the cells to radiation-induced cell death (apoptosis and necrosis). T98G cells were most responsive to the combined treatment revealing highly synergistic effects while A172 showed mostly additive to synergistic effects, and U87MG cells sub-additive, additive or synergistic effects, depending on the respective radiation-dose, drug-concentration and treatment time. Combined treatment enhanced therapy-induced damage of the mitochondria and caspase-activation. Importantly, combined treatment also increased radiation-induced eradication of clonogenic T98G cells as determined by standard colony formation assays. CONCLUSION: Our observations make the combined treatment with ionizing radiation and the membrane targeted apoptosis modulators ErPC and ErPC3 a promising approach for the treatment of patients suffering from malignant glioma. The use of this innovative treatment concept in an in vivo xenograft setting is under current investigation.

MAP kinase pathways involved in glioblastoma response to erucylphosphocholine.

Erucylphosphocholine (ErPC) is a promising antineoplastic drug for the treatment of malignant brain tumors. It exerts strong anticancer activity and induces apoptosis even in chemoresistant glioma cells. In the present study, A172 and U373MG glioma cells were treated with ErPC to explore the contribution of MAP kinase family members ERK, JNK and p38 kinase to ErPC-induced cell death. The exposure to ErPC led to activation of JNK and concurrent inhibition of ERK in both cell lines. Using specific MAP kinase inhibitors we confirmed that in U373MG cells ERK was blocked and JNK was activated upon ErPC treatment. Both effects were dependent on caspase activation. In A172 cells, ErPC treatment resulted in an activation of the JNK pathway, whereas the situation with respect to ERK signalling was more complex. We conclude that inhibition of the ERK pathway by ErPC may be related to antiproliferative effects, while activation of the JNK pathway may be responsible for its pro-apoptotic action.

Alkylglycerol opening of the blood-brain barrier to small and large fluorescence markers in normal and C6 glioma-bearing rats and isolated rat brain capillaries.

1. The blood-brain barrier (BBB) represents the major impediment to successful delivery of therapeutic agents to target tissue within the central nervous system. Intracarotid alkylglycerols have been shown to increase the transfer of chemotherapeutics across the BBB. 2. We investigated the spatial distribution of intracarotid fluorescein sodium and intravenous lissamine-rhodamine B200 (RB 200)-albumin in the brain of normal and C6 glioma-bearing rats after intracarotid co-administration of 1-O-pentylglycerol (200 mm). To elucidate the mechanisms involved in the alkylglycerol-mediated BBB opening, intraluminal accumulation of fluorescein isothiocyanate (FITC)-dextran 40,000 was studied in freshly isolated rat brain capillaries using confocal microscopy during incubation with different alkylglycerols. Furthermore, 1-O-pentylglycerol-induced increase in delivery of methotrexate (MTX) to the brain was evaluated in nude mice. 3. Microscopic evaluation showed a marked 1-O-pentylglycerol-induced extravasation of fluorescein and RB 200-albumin in the ipsilateral normal brain. In glioma-bearing rats, increased tissue fluorescence was found in both tumor tissue and brain surrounding tumor. Confocal microscopy revealed a time- and concentration-dependent accumulation of FITC-dextran 40,000 within the lumina of isolated rat brain capillaries during incubation with 1-O-pentylglycerol and 2-O-hexyldiglycerol, indicating enhanced paracellular transfer via tight junctions. Intracarotid co-administration of MTX and 1-O-pentylglycerol (200 mm) in nude mice resulted in a significant increase in MTX concentrations in the ipsilateral brain as compared to controls without 1-O-pentylglycerol (P<0.005). 4. In conclusion, 1-O-pentylglycerol increases delivery of small and large compounds to normal brain and brain tumors and this effect is mediated at least in part by enhanced permeability of tight junctions.

Intracarotid administration of short-chain alkylglycerols for increased delivery of methotrexate to the rat brain.

1 The intracarotid administration of alkylglycerols has been reported previously by us to be a novel strategy for increased delivery of various chemotherapeutic drugs to the normal brain and brain tumors in rats. 2 Effectiveness and structure-activity relations of the most promising pentyl- and hexylglycerol derivatives have been elucidated in vivo by analyzing the transfer of methotrexate (MTX) across the blood-brain barrier (BBB) in normal rats. The effects were compared with BBB disruption using hypertonic mannitol or intracarotid infusion of bradykinin. Furthermore, toxicity of the alkylglycerols has been studied in long-term experiments. 3 Apart from 1-O-pentyldiglycerol, all alkylglycerols induced a concentration-dependent increase in MTX delivery to the brain varying from 1.1 to more than 300-fold compared to intra-arterial MTX alone. Enhanced barrier permeability rapidly approached baseline values within 5 and 120 min at the latest. Chemical structure, concentration, time schedule of injections and combination of different alkylglycerols were identified as instruments suited to regulate the MTX accumulation within a wide range. Mannitol 1.4 M resulted in very high MTX levels in the brain as observed using the highest concentrations of alkylglycerols. Intracarotid infusion of bradykinin had only a minor effect on the BBB. Using 1-O-pentylglycerol or 2-O-hexyldiglycerol, both cell culture experiments and long-term in vivo analyses including clinical, laboratory and histopathological evaluations revealed no signs of toxicity. 4 In summary, intracarotid short-chain alkylglycerols constitute a very effective and low toxic strategy for transient opening of the BBB to overcome the limited access of cytotoxic drugs to the brain.

Intracellular mediators of erucylphosphocholine-induced apoptosis.

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFalpha-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.

Induction of apoptosis by erucylphospho-N,N,N-trimethylammonium is associated with changes in signal molecule expressionand location.

At concentrations effecting apoptosis, the alkylphosphocholine ErPC3 induced increased expression of the Rb protein in breast cancer (MCF-7) and leukemia (SKW-3, AR-230) cell lines as well as hypophosphorylation (K-562, CMLT-1, DOHH-2) and fragmentation of Rb (BV-173, SKW-3) in leukemia cell lines. ErPC3 exerts at least part of its antineoplastic activity by apoptosis, and this chain of events comprises early changes in the lipid raft fraction of the cellular membrane as well as modulation of different signal molecules, such as Abl, Bcr-Abl (fusion protein), and Rb.

[Treatment of Acanthamoeba keratitis: possibilities, problems, and new approaches]. / Die behandlung der Acanthamoeba-Keratitis: Möglichkeiten, Probleme und neue Wege.

Acanthamoeba keratitis is a corneal disease associated predominantly with contact lens wear. The occurrence of Acanthamoeba keratitis has been rising since 1990 in correlation to the growing number of contact lens wearers. To date approximately 2000 cases have been published around the world. Due to the complicated diagnostics, the elaborate treatment and the usually bad compliance of the patients, Acanthamoeba keratitis unfortunately very often takes a serious progression, which may lead to serious visual loss and perforating keratoplasty. Today, local treatment with a combination of polyhexamethylene biguanide (PHMB) and propamidine isethionate (Brolene) is considered the first line therapy for Acanthamoeba keratitis. Alternatively also a combination of propamidine and chlorhexidine or neomycine achieves good therapeutic results. However, the complicated mode of application consistently remains a problem. The intensive local treatment, i.e. hourly application of therapeutics during the first three days day and night makes hospitalization inevitable. Moreover, sufficient efficacy can not always be achieved, and also resistance against propamidine has already been observed. Recently propamidine has sometimes been replaced by hexamidine, which seems to have a greater cysticidal activity. A new path might be struck by the application of alkylphosphocholines. These are phosphocholines esterified to aliphatic alcohols. They exhibit in vitro and in vivo antineoplastic activity and have been shown to be cytotoxic against Leishmania donovani, Trypanosoma cruzi, and Entamoeba histolytica. A recent study has demonstrated that particularly hexadecylphosphocholine is highly effective also against various strains of Acanthamoeba.

Combination with an antisense oligonucleotide synergistically improves the antileukemic efficacy of erucylphospho-N,N,N-trimethylpropylammonium in chronic myeloid leukemia cell lines.

The aim of this study was to enhance the antileukemic efficacy of the alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) in chronic myeloid leukemia (CML)-derived cell lines by a bcr-directed antisense oligonucleotide (ASO-bcr). The mechanism was substantiated by Western blotting of the BCR-ABL expression level of CML cells, and the efficacy was substantiated by inhibition of colony formation compared with normal hematopoietic cells. The clonogenicity of K-562 cells expressing high levels of p210(BCR-ABL) was inhibited significantly by the ASO-bcr (T/C%, 30; P < 0.05) but not by ErPC3 (T/C%, 70). Combined sequential exposure to ErPC3 and the ASO-bcr, however, inhibited synergistically colony growth (T/C%, 3; P < 0.01). The colony growth of BV-173 cells expressing lower levels of p210(BCR-ABL) than K562 cells was inhibited to a greater extent by the ASO-bcr (T/C%, 15; P < 0.01). AR-230 cells that express high levels of p230(BCR-ABL) showed an intermediate decrease in colony formation in response to the ASO-bcr (T/C%, 20; P < 0.05). BCR-ABL levels of BV-173, CML-T1, and LAMA-84 cells were reduced in response to the ASO-bcr, as evidenced by Western blot. However, K-562 and AR-230 cells showed reduced BCR-ABL expression only after repeated treatment. ErPC3 and the ASO-bcr did not reduce colony formation (CFU-GM) of normal mouse bone marrow cells from long-term bone marrow cell cultures; instead, ErPC3 stimulated colony formation (P < 0.05) and did not induce chromosomal aberrations in mouse bone marrow. In conclusion, the combination of ErPC3 with a suitable antisense oligonucleotide inhibited synergistically colony formation of CML cell lines without damaging normal cells and thus might have a bearing on the purging of autologous hematopoietic transplants in CML patients.

Increased delivery of erucylphosphocholine to C6 gliomas by chemical opening of the blood-brain barrier using intracarotid pentylglycerol in rats.

BACKGROUND: Erucylphosphocholine (ErPC) has been shown to exert strong antineoplastic effects against various brain tumor cell lines in vitro. Since ErPC only enters the brain after long-term treatment, ineffective drug delivery to the tumor is considered to be the reason for the moderate responses to chemotherapy with ErPC observed in animal brain tumor models. We investigated a recently described method for chemically opening the blood-brain barrier (BBB) using intraarterial administration of alkylglycerols to increase the transfer of ErPC into the brain. METHODS: ErPC (40 mg/kg) was given to C6 glioma-bearing rats either as a single intracarotid bolus injection in the presence or absence of 1- O-pentylglycerol (300 m M) or as an intracarotid infusion in conjunction with bradykinin. Brain tissue concentrations were analyzed and compared to values obtained after intravenous ErPC treatment over 14 and 30 days (cumulative ErPC doses of 210 and 350 mg/kg, respectively). RESULTS: Pentylglycerol-induced BBB opening resulted in a significant increase in ErPC delivery to the tumor (17-fold) and, to a lesser extent, to the surrounding ipsilateral brain (7-fold) compared to intraarterial ErPC administration without alkylglycerol ( P<0.05). Furthermore, the resulting ErPC concentrations in the brain tumor exceeded those obtained in tumor and tumor-free brain after long-term intravenous ErPC administration. In contrast to this, intracarotid bradykinin did not increase the transfer of ErPC to the tumor or tumor-free brain. CONCLUSIONS: The intracarotid administration of pentylglycerol represents a novel and nontoxic method of overcoming the limited access of ErPC to both brain tumors and brain tissue adjacent to tumors. The present results provide further evidence that chemical opening of the BBB by intraarterial alkylglycerols is a promising new concept for improving delivery of chemotherapeutic agents to brain tumors.

Erucylphosphocholine-induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation.

Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.