Eutectic Resolves Lysolipid Paradox in Thermoresponsive Liposomes.

A better molecular understanding of the temperature-triggered drug release from lysolipid-based thermosensitive liposomes (LTSLs) is needed to overcome the recent setbacks in developing this important drug delivery system. Enhanced drug release was previously rationalized in terms of detergent-like effects of the lysolipid monostearyl lysophosphatidylcholine (MSPC), stabilizing local membrane defects upon LTSL lipid melting. This is highly surprising and here referred to as the 'lysolipid paradox,' because detergents usually induce the opposite effect─they cause leakage upon freezing, not melting. Here, we aim at better answers to (i) why lysolipid does not compromise drug retention upon storage of LTSLs in the gel phase, (ii) how lysolipids can enhance drug release from LTSLs upon lipid melting, and (iii) why LTSLs typically anneal after some time so that not all drug gets released. To this end, we studied the phase transitions of mixtures of dipalmitoylphosphatidylcholine (DPPC) and MSPC by a combination of differential scanning and pressure perturbation calorimetry and identified the phase structures with small- and wide-angle X-ray scattering (SAXS and WAXS). The key result is that LTSLs, which contain the standard amount of 10 mol % MSPC, are at a eutectic point when they release their cargo upon melting at about 41 °C. The eutectic present below 41 °C consists of a MSPC-depleted gel phase as well as small domains of a hydrocarbon chain interdigitated gel phase containing some 30 mol % MSPC. In these interdigitated domains, the lysolipid is stored safely without compromising membrane integrity. At the eutectic temperature, both the MSPC-depleted bilayer and interdigitated MSPC-rich domains melt at once to fluid bilayers, respectively. Intact, fluid membranes tolerate much less MSPC than interdigitated domains─where the latter have melted, the high local MSPC content causes transient pores. These pores allow for fast drug release. However, these pores disappear, and the membrane seals again as the MSPC distributes more evenly over the membrane so that its local concentration decreases below the pore-stabilizing threshold. We provide a pseudobinary phase diagram of the DPPC-MSPC system and structural and volumetric data for the interdigitated phase.

On the validity of fluorimetric intracellular calcium detection: Impact of lipid components.

We investigated the effects of different lipids on the activity of the angiotensin II type 1 receptor (AT1R). As calcium plays a key role in the signaling of the AT1R, we used the calcium-sensitive fluorescence indicators fura-2 to detect intracellular calcium release upon stimulation with the agonist angiotensin II. At first sight, cells preincubated with Very low-density lipoprotein (VLDL) showed a reduced calcium release triggered by angiontensin II compared to untreated control. However, on closer examination, this result seemed to be an artifact. Incubation with VLDL reduced also the amount of intracellular fura-2, as measured by fluorescence in the isosbestic point. Additionally, the maximal obtainable ratio, obtained after complete saturation with calcium ions, was reduced in cells preincubated with VLDL. These findings rendered our initial results questionable. We report the results of our work and our suggestions regarding the experimental setup to contribute to the understanding of the interpretation of fura-2 measurements and to avoid erroneous conclusions.

Lipoprotein turnover and possible remnant accumulation in preeclampsia: insights from the Freiburg Preeclampsia H.E.L.P.-apheresis study.

BACKGROUND: Preeclampsia is a life-threatening disease in pregnancy, and its complex pathomechanisms are poorly understood. In preeclampsia, lipid metabolism is substantially altered. In late onset preeclampsia, remnant removal disease like lipoprotein profiles have been observed. Lipid apheresis is currently being explored as a possible therapeutic approach to prolong preeclamptic pregnancies. Here, apheresis-induced changes in serum lipid parameters are analyzed in detail and their implications for preeclamptic lipid metabolism are discussed. METHODS: In the Freiburg H.E.L.P.-Apheresis Study, 6 early onset preeclamptic patients underwent repeated apheresis treatments. Serum lipids pre- and post-apheresis and during lipid rebound were analyzed in depth via ultracentrifugation to yield lipoprotein subclasses. RESULTS: The net elimination of Apolipoprotein B and plasma lipids was lower than theoretically expected. Lipids returned to previous pre-apheresis levels before the next apheresis even though apheresis was repeated within 2.9 ± 1.2 days. Apparent fractional catabolic rates and synthetic rates were substantially elevated, with fractional catabolic rates for Apolipoprotein B / LDL-cholesterol being 0.7 ± 0.3 / 0.4 ± 0.2 [day- 1] and synthetic rates being 26 ± 8 / 17 ± 8 [mg*kg- 1*day- 1]. The distribution of LDL-subclasses after apheresis shifted to larger buoyant LDL, while intermediate-density lipoprotein-levels remained unaffected, supporting the notion of an underlying remnant removal disorder in preeclampsia. CONCLUSION: Lipid metabolism seems to be highly accelerated in preeclampsia, likely outbalancing remnant removal mechanisms. Since cholesterol-rich lipoprotein remnants are able to accumulate in the vessel wall, remnant lipoproteins may contribute to the severe endothelial dysfunction observed in preeclampsia. TRIAL REGISTRATION: ClinicalTrails.gov, NCT01967355 .

Dietary supplementation with n-3-fatty acids in patients with pancreatic cancer and cachexia: marine phospholipids versus fish oil - a randomized controlled double-blind trial.

BACKGROUND: Like many other cancer patients, most pancreatic carcinoma patients suffer from severe weight loss. As shown in numerous studies with fish oil (FO) supplementation, a minimum daily intake of 1.5 g n-3-fatty acids (n-3-FA) contributes to weight stabilization and improvement of quality of life (QoL) of cancer patients. Given n-3-FA not as triglycerides (FO), but mainly bound to marine phospholipids (MPL), weight stabilization and improvement of QoL has already been seen at much lower doses of n-3-FA (0,3 g), and MPL were much better tolerated. The objective of this double-blind randomized controlled trial was to compare low dose MPL and FO formulations, which had the same n-3-FA amount and composition, on weight and appetite stabilization, global health enhancement (QoL), and plasma FA-profiles in patients suffering from pancreatic cancer. METHODS: Sixty pancreatic cancer patients were included into the study and randomized to take either FO- or MPL supplementation. Patients were treated with 0.3 g of n-3-fatty acids per day over six weeks. Since the n-3-FA content of FO is usually higher than that of MPL, FO was diluted with 40% of medium chain triglycerides (MCT) to achieve the same capsule size in both intervention groups and therefore assure blinding. Routine blood parameters, lipid profiles, body weight, and appetite were measured before and after intervention. Patient compliance was assessed through a patient diary. Quality of life and nutritional habits were assessed with validated questionnaires (EORTC-QLQ-C30, PAN26). Thirty one patients finalized the study protocol and were analyzed (per-protocol-analysis). RESULTS: Intervention with low dose n-3-FAs, either as FO or MPL supplementation, resulted in similar and promising weight and appetite stabilization in pancreatic cancer patients. MPL capsules were slightly better tolerated and showed fewer side effects, when compared to FO supplementation. CONCLUSION: The similar effects between both interventions were unexpected but reliable, since the MPL and FO formulations caused identical increases of n-3-FAs in plasma lipids of included patients after supplementation. The effects of FO with very low n-3-FA content might be explained by the addition of MCT. The results of this study suggest the need for further investigations of marine phospholipids for the improvement of QoL of cancer patients, optionally in combination with MCT.

Saturated and mono-unsaturated lysophosphatidylcholine metabolism in tumour cells: a potential therapeutic target for preventing metastases.

BACKGROUND: Metastasis is the leading cause of mortality in malignant diseases. Patients with metastasis often show reduced Lysophosphatidylcholine (LysoPC) plasma levels and treatment of metastatic tumour cells with saturated LysoPC species reduced their metastatic potential in vivo in mouse experiments. To provide a first insight into the interplay of tumour cells and LysoPC, the interactions of ten solid epithelial tumour cell lines and six leukaemic cell lines with saturated and mono-unsaturated LysoPC species were explored. METHODS: LysoPC metabolism by the different tumour cells was investigated by a combination of cell culture assays, GC and MS techniques. Functional consequences of changed membrane properties were followed microscopically by detecting lateral lipid diffusion or cellular migration. Experimental metastasis studies in mice were performed after pretreatment of B16.F10 melanoma cells with LysoPC and FFA, respectively. RESULTS: In contrast to the leukaemic cells, all solid tumour cells show a very fast extracellular degradation of the LysoPC species to free fatty acids (FFA) and glycerophosphocholine. We provide evidence that the formerly LysoPC bound FFA were rapidly incorporated into the cellular phospholipids, thereby changing the FA-compositions accordingly. A massive increase of the neutral lipid amount was observed, inducing the formation of lipid droplets. Saturated LysoPC and to a lesser extent also mono-unsaturated LysoPC increased the cell membrane rigidity, which is assumed to alter cellular functions involved in metastasis. According to that, saturated and mono-unsaturated LysoPC as well as the respective FFA reduced the metastatic potential of B16.F10 cells in mice. Application of high doses of liposomes mainly consisting of saturated PC was shown to be a suitable way to strongly increase the plasma level of saturated LysoPC in mice. CONCLUSION: These data show that solid tumours display a high activity to hydrolyse LysoPC followed by a very rapid uptake of the resulting FFA; a mechanistic model is provided. In contrast to the physiological mix of LysoPC species, saturated and mono-unsaturated LysoPC alone apparently attenuate the metastatic activity of tumours and the artificial increase of saturated and mono-unsaturated LysoPC in plasma appears as novel therapeutic approach to interfere with metastasis.

Click modification of multifunctional liposomes bearing hyperbranched polyether chains.

Aiming at controlled modification of liposomal surface structures, we describe a postpreparational approach for surface derivatization of a new type of multifunctional, sterically stabilized liposomes. Application of dual centrifugation (DC) resulted in high encapsulation efficiencies above 50% at very small batch sizes with a total volume of 150 µL, which were conductive to fast and efficient optimization of variegated surface modification reactions. Cholesterol-polymer amphiphiles, including complex hyperbranched polyether structures bearing 1-4 terminal alkynes, were used in DC formulations to provide steric stabilization. The alkyne moieties were explored as anchors for the conjugation of small molecules to the liposomal surface via click chemistry, binding 350-450 fluorophores per liposome as examples for surface active molecules. Using Förster resonance energy transfer (FRET) spectroscopy, the conjugation reaction as well as the uptake of FRET-labeled liposomes by RBE4 cells was monitored, and the distribution of the fluorescent lipids among cellular structures and membranes could be studied. Thus, the combination of clickable hyperbranched amphiphiles and dual centrifugation provides access to well-defined liposomal formulations with a variety of surface moieties.

Dual centrifugation as a novel and efficient method for the preparation of lipodisks.

Polyethylene glycol (PEG)-stabilized lipodisks have emerged as innovatiive, promising nanocarriers for several classes of drugs. Prior research underscores the important role of lipid composition and preparation method in determining the lipodisk size, uniformity, and drug loading capacity. In this study, we investigate dual centrifugation (DC) as a novel technique for the production of PEG-stabilized lipodisks. Moreover, we explore the potential use of DC for the encapsulation of two model drugs, curcumin and doxorubicin, within the disks. Our results show that by a considerate choice of experimental conditions, DC can be used as a fast and straightforward means to produce small and homogenous lipodisks with a hydrodynamic diameter of 20-30 nm. Noteworthy, the technique works well for the production of both cholesterol-free and cholesterol-containing disks and does not require pre-mixing of the lipids in organic solvent. Furthermore, our investigations confirm the efficacy of DC in formulating curcumin and doxorubicin within these lipodisks. For doxorubicin, careful control and optimization of the experimental conditions resulted in formulations displaying an encouraging encapsulation efficiency of 84 % and a favourable drug-to-lipid ratio of 0.13 in the disks.

Preparation of Nanosized Pharmaceutical Formulations by Dual Centrifugation.

Dual centrifugation (DC) is an innovative in-vial homogenization and in-vial nanomilling technique that has been in use for the preparation of liposomes for more than one decade. Since then, DC has continuously been developed for preparing various liposomes and other lipid nanoparticles including emulsions and solid lipid nanoparticles (SLNs) as well as polymersomes and nanocrystals. Improvements in equipment technology have been achieved over the past decade, so that DC is now on its way to becoming the quasi-standard for the simple, fast, and aseptic production of lipid nanoparticles and nanocrystals in small and medium batch sizes, including the possibility of simple and fast formulation screening or bedside preparations of therapeutic nanoparticles. More than 68 publications in which DC was used to produce nanoparticles have appeared since then, justifying an initial review of the use of DC for pharmaceutical nanotechnology.

Tailoring the Lamellarity of Liposomes Prepared by Dual Centrifugation.

Dual centrifugation (DC) is a new and versatile technique for the preparation of liposomes by in-vial homogenization of lipid-water mixtures. Size, size distribution, and entrapping efficiencies are strongly dependent on the lipid concentration during DC-homogenization. In this study, we investigated the detailed structure of DC-made liposomes. To do so, an assay to determine the ratio of inner to total membrane surfaces of liposomes (inaccessible surface) was developed based on either time-resolved or steady-state fluorescence spectroscopy. In addition, cryogenic electron microscopy (cryo-EM) was used to confirm the lamellarity results and learn more about liposome morphology. One striking result leads to the possibility of producing a novel type of liposome-small multilamellar vesicles (SMVs) with low PDI, sizes of the order of 100 nm, and almost completely filled with bilayers. A second particularly important finding is that VPGs can be prepared to contain open bilayer structures that will close spontaneously when, after storage, more aqueous phase is added and liposomes are formed. Through this process, a drug can effectively be entrapped immediately before application. In addition, dual centrifugation at lower lipid concentrations is found to produce predominantly unilamellar vesicles.

Health effects of dietary phospholipids.

Beneficial effects of dietary phospholipids (PLs) have been mentioned since the early 1900's in relation to different illnesses and symptoms, e.g. coronary heart disease, inflammation or cancer. This article gives a summary of the most common therapeutic uses of dietary PLs to provide an overview of their approved and proposed benefits; and to identify further investigational needs.From the majority of the studies it became evident that dietary PLs have a positive impact in several diseases, apparently without severe side effects. Furthermore, they were shown to reduce side effects of some drugs. Both effects can partially be explained by the fact that PL are highly effective in delivering their fatty acid (FA) residues for incorporation into the membranes of cells involved in different diseases, e.g. immune or cancer cells. The altered membrane composition is assumed to have effects on the activity of membrane proteins (e.g. receptors) by affecting the microstructure of membranes and, therefore, the characteristics of the cellular membrane, e.g. of lipid rafts, or by influencing the biosynthesis of FA derived lipid second messengers. However, since the FAs originally bound to the applied PLs are increased in the cellular membrane after their consumption or supplementation, the FA composition of the PL and thus the type of PL is crucial for its effect. Here, we have reviewed the effects of PL from soy, egg yolk, milk and marine sources. Most studies have been performed in vitro or in animals and only limited evidence is available for the benefit of PL supplementation in humans. More research is needed to understand the impact of PL supplementation and confirm its health benefits.

Screening for Optimal Liposome Preparation Conditions by Using Dual Centrifugation and Time-Resolved Fluorescence Measurements.

Dual centrifugation (DC) is a novel in-vial homogenization technique for the preparation of liposomes in small batch sizes under gentle and sterile conditions which allows encapsulation efficiencies (EE) for water soluble compounds of >50%. Since liposome size, size distribution (PDI), and EE depend on the lipid concentration used in the DC process, a screening method to find optimal lipid concentrations for a defined lipid composition was developed. Four lipid mixtures consisting of cholesterol, hydrogenated or non-hydrogenated egg PC, and/or PEG-DSPE were screened and suitable concentration ranges could be identified for optimal DC homogenization. In addition to the very fast and parallel liposome preparation of up to 40 samples, the screening process was further accelerated by the finding that DC generates homogeneously mixed liposomes from a macroscopic lipid mixture without the need to initially prepare a molecularly mixed lipid film from an organic solution of all components. This much simpler procedure even works for cholesterol containing lipid blends, which could be explained by a nano-milling of the cholesterol crystals during DC homogenization. Furthermore, EE determination was performed by time-resolved fluorescence measurements of calcein-loaded liposomes without removing the non-entrapped calcein. The new strategy allows the rapid characterization of a certain lipid composition for the preparation of liposomes within a working day.

Marine phospholipids--a promising new dietary approach to tumor-associated weight loss.

GOALS OF WORK: Advanced tumor disease very often evokes excessive loss of body weight. Among others, fish oil or marine fatty acid ethyl esters were investigated for treatment of cancer cachexia with controversial results. In this study, a new formulation of marine fatty acids was investigated, the marine phospholipids, with more than 50% of phospholipid-bound fatty acids being eicosapentaenoic and docosahexaenoic acid. MATERIALS AND METHODS: Thirty-one tumor patients with various tumor entities suffering from weight loss were asked to take marine phospholipids (1.5 g/day) as softgel capsules for a period of 6 weeks. Compliance, body weight, appetite, and quality of life as well as the fatty acid profile in plasma and blood cells were monitored; 17 patients could be analyzed. MAIN RESULTS: Marine phospholipids were very well accepted; low-dose supplementation resulted in a significant increase of eicosapentaenoic and docosahexaenoic acid in plasma phospholipids; therefore, significantly reducing the n-6 to n-3 fatty acid ratio. A stabilization of body weight was achieved (median weight change of +0.6% after 6 weeks), while appetite and quality of life improved. CONCLUSIONS: These promising first results encourage further investigation of marine phospholipids in cancer care.

Liposomal gemcitabine (GemLip)-efficient drug against hormone-refractory Du145 and PC-3 prostate cancer xenografts.

BACKGROUND: Gemcitabine (Gemc) is an efficient chemotherapeutic drug in various cancer types (e.g., pancreas) but has only limited effects on hormone-refractory prostate cancer (HRPCa). Since HRPCa cells are highly sensitive to even low doses of Gemc in vitro, the lack of clinical effects might be due to rapid degradation of Gemc by deaminases combined with impaired accumulation in tumor tissue and PCa cells. Liposomal formulation (GemLip) is expected to protect the entrapped cytotoxic substance from enzymatic degradation and furthermore augment its accumulation within tumor tissues due to an enhanced permeability of the tumor vessels. METHODS: Anti-tumoral and anti-metastatic activity of GemLip and Gemc were investigated in two luciferase-expressing, human hormone-refractory PC-3 and Du145 HRPCa xenograft models in immunodeficient mice. Tumor growth was monitored by in vivo luminescence imaging (orthotopic) or callipering (subcutaneous). Anti-metastatic effects of treatment were determined by in vitro luciferase assay of the tissues. RESULTS: Tumor growth of subcutaneous Du145 xenografts was significantly inhibited only by GemLip (8 mg/kg: P = 0.014 and 6 mg/kg: P = 0.011) but not by conventional Gemc (360 mg/kg). In contrast, growth of orthotopic PC-3 xenografts was significantly inhibited by both, GemLip (P = 0.041) and Gemc (P = 0.002). The drugs furthermore strongly reduced spleen and liver metastases in this model. CONCLUSIONS: As shown by the very low efficient concentration of GemLip, liposomal entrapment of Gemc greatly enhances its activity. GemLip has, even at very low doses, a significant anti-tumoral and anti-metastatic therapeutic effect in HRPCa xenografts in vivo and was beneficial even when the conventional Gemc failed.

Surveillance of siRNA integrity by FRET imaging.

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.

Rapid development of API nano-formulations from screening to production combining dual centrifugation and wet agitator bead milling.

Various wet ball nanomilling-screening tools for poorly soluble APIs are available which differ in their milling principle, batch size and number of samples. Here, the transferability of results from screening (small to medium-scale) to pharmaceutical production (largescale) was investigated. Wet ball milling in a dual centrifuge (DC) (10-100 mg API, 40 samples in parallel) was used to identify stable nanoformulations. In addition different sized agitator bead mills were used for scale-up to industrial scales. DC-and small-scale agitator milling (AM) resulted in small and virtually identical API-particles. Additionally, similar API-particles were obtained using two different sized agitator bead mills (batch size 1.5 and 30 kg) and applying comparable specific grinding energies (SGE). The SGE used in the trials represents the grinding limit for this API-suspension. Using lower SGEs, AM results in larger API-particles. All used milling tools had no influence on the APIs crystal structure and wear of grinding media (Zr/Y) is low. The study confirmed the importance to choose the right formulation and process parameters, which positively affect grinding efficacy, particle size distribution and wear contamination. The excellent comparability of results obtained from DC-milling and AM significantly reduces the duration for successful and predictable formulation development.

Plasma lyso-phosphatidylcholine concentration is decreased in cancer patients with weight loss and activated inflammatory status.

BACKGROUND: It has been observed that ras-transformed cell lines in culture have a higher phosphatidylcholine (PC) biosynthesis rate as well as higher PC-degradation rate (increased PC-turnover) than normal cells. In correspondence to these findings, the concentrations of the PC-degradation product lyso-phosphatidylcholine (LPC) in cancer patients were found to be decreased. Our objective was the systematic investigation of the relationship between LPC and inflammatory and nutritional parameters in cancer patients. Therefore, plasma LPC concentrations were assessed in 59 cancer patients and related to nutritional and inflammatory parameters. To determine LPC in blood plasma we developed and validated a HPTLC method. RESULTS: Average plasma LPC concentration was 207 +/- 59 microM which corresponds to the lower limit of the reported range in healthy subjects. No correlation between LPC and age, performance status, body mass index (BMI) or fat mass could be seen. However, LPC correlated inversely with plasma C-reactive protein (CRP) and whole blood hydrogen peroxides (HPO). Further, a negative correlation could be observed between LPC and whole body extra cellular fluid volume (ECF) as well as with relative change in body weight since cancer diagnosis. CONCLUSION: In conclusion, LPC concentrations were decreased in cancer patients. LPC plasma concentrations correlated with weight loss and inflammatory parameters and, therefore, might be a general indicator of severity of malignant disease.

Dual centrifugation - A new technique for nanomilling of poorly soluble drugs and formulation screening by an DoE-approach.

The development of nanosuspensions of poorly soluble APIs takes a lot of time and high amount of active material is needed. In this publication the use of dual centrifugation (DC) for an effective and rapid API-nanomilling is described for the first time. DC differs from normal centrifugation by an additional rotation of the samples during centrifugation, resulting in a very fast and powerful movement of the samples inside the vials, which - in combination with milling beads - result in effective milling. DC-nanomilling was compared to conventional wet ball milling and results in same or even smaller particle sizes. Also drug concentrations up to 40% can be processed. The process is fast (typical 90min) and the temperature can be controlled. DC-nanomilling appears to be very gentle, experiments showed no change of the crystal structure during milling. Since batch sizes are very small (100-1000mg) and since 40 sample vials can be processed in parallel, DC is ideal for the screening of suitable polymer/surfactant combinations. Fenofibrate was used to investigate DC-nanomilling for formulation screening by applying a DoE-approach. The presented data also show that the results of DC-nanomilling experiments are highly comparable to the results obtained by common agitator mills.

Cytotoxic efficacy and influence on cellular phospholipid metabolism of 2-hydroxy- and 2-O-acetyl-octadecylphosphocholines.

The cytotoxic efficacy and influence on phospholipid composition of the new alkylphosphocholines (APC) 2-hydroxy and 2-O-acetyl-octadecylphosphocholines both synthesised in R- and S-configuration (R/S-OH and R/S-O-acetyl) were examined in vitro using HL-60 and MDA-MB-468 cells. IC50- and LC50-values were measured by MTT- and cell count assay. All tested APC showed higher or similar cytotoxic efficacy compared to the well known APC hexadecylphosphocholine (HePC). However, while S-configured APC (IC50) revealed considerably higher cytotoxic activities, only R- (natural)-configured APC caused significant changes in the phospholipid composition of tumour cells. Further investigations revealed an increase in R-O-acyl and loss of PC up to 70% in the membrane of both cell lines. Similar to PC, R-O-acyl bears two long non-polar hydrocarbon chains and stabilises cell membranes structurally, thus, possibly explaining less cytotoxicity and lack of apoptosis induction by R-configured APC. Nevertheless, an enrichment of R-O-acyl up to 70% at the expense of PC in cell membrane is tremendous and may inhibit tumour development by influencing the intracellular lipid signalling. In conclusion, our findings reveal the antitumoral efficacy of all tested new APC and offer new perspectives in drug development targeting phospholipid metabolism.

The molecular mechanism by which saturated lysophosphatidylcholine attenuates the metastatic capacity of melanoma cells.

Lysophophatidylcholine (LysoPC) is an abundant constituent in human plasma. Patients with malignant cancer diseases have attenuated LysoPC plasma levels, and thus LysoPC has been examined as a metabolic biomarker for cancer prediction. Preclinical studies have shown that solid tumor cells drastically degrade LysoPCs by incorporating their free fatty acids into cell membrane phospholipids. In this way, LysoPC C18:0 reduced the metastatic spread of murine melanoma B16.F10 cells in mice. Although membrane rigidification may have a key role in the attenuation of metastasis, evidence for this has yet to be shown. Therefore, the present study aimed to determine how LysoPC reduces the metastatic capacity of B16.F10 cells. Following cellular preincubation with LysoPC C18:0 at increasing concentrations and lengths of time, cell migration was most significantly attenuated with 450 µm LysoPC C18:0 at 72 h. Biosensor measurements suggest that, despite their abundance in B16.F10 cells, LysoPC-sensitive G protein-coupled receptors do not appear to contribute to this effect. Instead, the attenuated migration appears to result from changes in cell membrane properties and their effect on underlying signaling pathways, most likely the formation of focal adhesion complexes. Treatment with 450 µm LysoPC C18:0 activates protein kinase C (PKC)δ to phosphorylate syndecan-4, accompanied by deactivation of PKCα. Subsequently, focal adhesion complex formation was attenuated, as confirmed by the reduced activity of focal adhesion kinase (FAK). Interestingly, 450 µm LysoPC C18:1 did not affect FAK activity, explaining its lower propensity to affect migration and metastasis. Therefore, membrane rigidification by LysoPC C18:0 appears to prevent the formation of focal adhesion complexes, thus affecting integrin activity as a key for metastatic melanoma spread.