Alkyl Phospholipids Research Articles

Investigation of miltefosine-model membranes interactions at the molecular level for two different PS levels modeling cancer cells

Miltefosine (MLT) is a broad-spectrum drug included in the alkylphospholipids (APL) used against leishmania and various types of cancer. The most crucial feature of APLs is that they are thought to only kill cancerous cells without harming normal cells. However, the molecular mechanism of action of APLs is not completely understood. The increase in the phosphatidylserine (PS) ratio is a marker showing the stage of cancer and even metastasis. The goal of this research was to investigate the molecular effects of miltefosine at the molecular level in different PS ratios. The effects of MLT on membrane phase transition, membrane orders, and dynamics were studied using DPPC/DPPS (3:1) and DPPC/DPPS (1:1) multilayer (MLV) vesicles mimicking DPPS ratio variation, Differential Scanning Calorimetry (DSC), and Fourier Transform Infrared spectroscopy (FTIR). Our findings indicate that miltefosine is evidence at the molecular level that it is directed towards the tumor cell and that the drug’s effect increases with the increase of anionic lipids in the membrane depending on the stage of cancer.

Abstract A025: Cytostatic effects and expression modulations in cell cycle genes by a 3rd generation alkyl-phospholipid (erufosine) in liver and lung cancer cells

Abstract Introduction: Alkyl-phospholipids (ALPs) are a family of compounds that have affinity to cell membranes and have demonstrated antineoplastic potential. Erufosine is a 3rd generation ALP that can be injected intravenously for its long (22-carbon) chain with a cis-13,14 double bond. Erufosine, like other ALPs, causes changes in the lipid rafts of the cell membrane and exerts its anticancer effects through cytotoxic and cytostatic mechanisms. In this study, intentions were to investigate erufosine mediated cytostatic effects in liver and lung cancer cells at functional and molecular levels. Materials and Methods: Liver (HepG2) and lung (H1299) cancer cell lines were exposed to various concentrations of erufosine (1.56-50µM) and resulting anti-proliferative effects were monitored via MTT dye reduction assay. Cytostatic effects of erufosine on the two cancer cell lines were investigated via propidium iodide-based DNA staining and FACS analysis. A real time PCR based panel was used to decipher the impact of erufosine exposure on 84 cell cycle related genes in the two cancer cell lines. Fold changes were identified via the Livak method while untreated cells were used as controls. Results: Erufosine induced the anti-proliferative effects in both cancer cell lines, however, lung cancer cells were more sensitive (IC50: 6.25µM) as compared to liver cancer cells (IC50: 33µM). The compound induced a comparable cell cycle arrest in S phase in the cancer cell lines as reflected by more accumulation of the cells in this particular phase. Exposure with erufosine (IC50/48 hours) de-regulated the cell cycle related genes in both cancer cell lines. Again, the lung cancer cells were more responsive in comparison to liver cancer cells as reflected by a substantial de-regulation (≥2fold) of 35/84 (42%) and 13/84 (15%) genes, respectively. Conclusion: The findings of this study confirmed erufosine mediated anti-proliferative and cytostatic effects in the liver and lung cancer cells. Furthermore, the compound induced substantial alterations in cell cycle related genes in the two cancer cell lines. Present findings foster further research to investigate the antineoplastic effects induced by erufosine in liver and lung cancer cells to support its clinical utilization. Citation Format: Asim Pervaiz, Marriyam Nasim, Nazakat Shafiq, Martin R. Berger. Cytostatic effects and expression modulations in cell cycle genes by a 3rd generation alkyl-phospholipid (erufosine) in liver and lung cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A025.

Mitochondrial Functionality Is Regulated by Alkylphospholipids in Human Colon Cancer Cells.

Alkylphospholipids (APLs) have been studied as anticancer drugs that interfere with biological membranes without targeting DNA. Although their mechanism of action is not fully elucidated yet, it is known that they disrupt the intracellular trafficking of cholesterol and its metabolism. Here, we analyzed whether APLs could also interfere with mitochondrial function. For this purpose, we used HT29 colorectal cancer cells, derived from a primary tumor, and SW620 colorectal cancer cells, derived from a metastasis site. After treatment with the APLs miltefosine and perifosine, we analyzed various mitochondrial parameters, including mitochondrial mass, cardiolipin content, mitochondrial membrane potential, H2O2 production, the levels of oxidative phosphorylation (OXPHOS) complexes, metabolic enzymes activity, the oxygen consumption rate, and the levels of apoptosis and autophagy markers. APLs, especially perifosine, increased mitochondrial mass while OXPHOS complexes levels were decreased without affecting the total oxygen consumption rate. Additionally, we observed an increase in pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH) levels and a decrease in lactate dehydrogenase (LDH) activity, suggesting a metabolic rewiring induced by perifosine. These alterations led to higher mitochondrial membrane potential, which was potentiated by decreased uncoupling protein 2 (UCP2) levels and increased reactive oxygen species (ROS) production. Consequently, perifosine induced an imbalance in mitochondrial function, resulting in higher ROS production that ultimately impacted cellular viability.

Effect of Polar Head Group Modifications on the Tumor Retention of Phospholipid Ether Analogs: Role of the Quaternary Nitrogen.

We have previously described the remarkable capacity of radioiodinated alkyl phospholipids to be sequestered and retained by a variety of tumors in vivo. We have already established the influence of certain structural parameters of iodinated alkyl phospholipids on tumor avidity, such as stereochemistry at the sn-2 carbon of alkylglycerol phosphocholines, meta-or para-position of iodine in the aromatic ring of phenylalkyl phosphocholines, and the length of the alkyl chain in alkyl phospholipids. In order to determine the additional structural requirements for tumor uptake and retention, three new radioiodinated alkylphospholipid analogs, 2-4, were synthesized as potential tumor imaging agents. Polar head groups were modified to determine structure-tumor avidity relationships. The trimethylammonio group in 1 was substituted with a hydrogen atom in 2, an ammonio group in 3 and a tertiary butyl group in 4. All analogs were separately labeled with iodine-125 or iodine-124 and administered to Walker 256 tumor-bearing rats or human PC-3 tumor-bearing SCID mice, respectively. Tumor uptake was assessed by gamma-camera scintigraphy (for [I-125]-labeled compounds) and high-resolution micro-PET scanning (for [I-124]-labeled compounds). It was found that structural modifications in the polar head group of alkyl phospholipids strongly influenced the tumor uptake and tissue distribution of these compounds in tumor-bearing animals. Phosphoethanolamine analog 3 (NM401) displayed a very slight accumulation in tumor as compared with phosphocholine analog 1 (NM346). Analogs 2 (NM400) and 4 (NM402) lacking the positively charged nitrogen atom failed to display any tumor uptake and localized primarily in the liver. This study provided important insights regarding structural requirements for tumor uptake and retention. Replacement of the quaternary nitrogen in the alkyl phospholipid head group with non-polar substituents resulted in loss of tumor avidity.

Analytical Method Development and Validation of Trimethylazanium containing Anti-leishmanial Phospholipid Drug Miltefosine by Liquid Chromatography Quadruple Tandem Mass Spectrometry (LC-ESI-MS/MS) API-2000

The phosphocholine and phospholipid that is hexadecyl monoester of phosphocholine (alkyl phospholipid) is a miltefosine drug used to treat all types of leishmaniasis. It is a broad-spectrum antimicrobial, anti-leishmanial, anti-cancer phospholipid drug. It is used for the treatment of breast cancer and cutaneous metastasis. This present study of miltefosine was given to develop. It validated the analytical method by LC-MS/MS (API-2000). It will be applied to the estimation of miltefosine from its solubility sample, force degradation sample, dissolution sample, and impurity analysis. The calibration concentrations of miltefosine were 125, 250, 500, 1000, 2000, and 4000 ng/ml which accuracy was 90.903 – 109.077% and reported stability was 90.71- 97.99%, 92.04 – 98.02%, 90.00 – 95.52%, 89.72 – 98.54% for freeze-thaw, short term, benchtop, autosampler stability respectively. The developed method for determining and quantifying miltefosine in the sample was also validated as per the US-FDA and EMA guidelines. The validation parameters found within the specified regulatory limit, hence acceptable. The present method also has a short run time (6.00 min). The method is simple, specific, highly selective, sensitive, and reproducible.

The effects of miltefosine on the structure and dynamics of DPPC and DPPS liposomes mimicking normal and cancer cell membranes: FTIR and DSC studies

Miltefosine (MLT,hexadecylphosphocholine (HePC)) is an alkylphospholipid (APL) clinically used against leishmaniasis and the topical treatment of cancer. Although the MLT mechanism of action has not yet been clarified, it is thought to be related to its incorporation into the plasma membrane, affecting lipid homeostasis. An essential feature of APLs is that they possess a lethal effect that acts only on malignant cells without affecting normal cells; this property indicates the potential selective antitumor properties of these types of drugs. However, the exact mechanism of APLs action at the molecular level is not yet known in detail. This study aimed to elucidate the molecular mechanisms of the interaction of the antitumor alkylphospholipid drug miltefosine with molecular models of normal and cancer cell membranes. The interaction of MLT with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) mimicking normal cell membrane and dipalmitoyl phosphatidylserine (DPPS) multilamellar liposomes (MLVs) mimicking cancer cell membrane was studied in the presence of (1 mol%,3 mol%, 6 mol%, 9 mol%, 15 mol%, 24 mol%, 30 mol% and 35 mol%) and in the absence of MLT by using two different non-invasive techniques; Fourier Transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). The comparative results show that miltefosine affects DPPS/MLT systems modeled cancer cells more than the DPPC/MLT system modeled healthy cells. Our results may be proof at the molecular level that miltefosine has selective antitumor properties.

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via a nongenomic PPARγ-dependent pathway

Oxidized phospholipids have been shown to exhibit pleiotropic effects in numerous biological contexts. For example, 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxidized phospholipid formed from alkyl phosphatidylcholines, is a peroxisome proliferator–activated receptor gamma (PPARγ) nuclear receptor agonist. Although it has been reported that PPARγ agonists including thiazolidinediones can induce plasma volume expansion by enhancing renal sodium and water retention, the role of azPC in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues and also investigated the effect of azPC on renal sodium handling in vivo. We showed using a microperfusion technique that azPC rapidly stimulated Na+/HCO3− cotransporter 1 (NBCe1) and luminal Na+/H+ exchanger (NHE) activities in a dose-dependent manner at submicromolar concentrations in isolated PTs from rats and humans. The rapid effects (within a few minutes) suggest that azPC activates NBCe1 and NHE via nongenomic signaling. The stimulatory effects were completely blocked by specific PPARγ antagonist GW9662, ERK kinase inhibitor PD98059, and CD36 inhibitor sulfosuccinimidyl oleate. Treatment with an siRNA against PPAR gamma completely blocked the stimulation of both NBCe1 and NHE by azPC. Moreover, azPC induced ERK phosphorylation in rat and human kidney cortex tissues, which were completely suppressed by GW9662 and PD98059 treatments. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via a CD36/PPARγ/mitogen-activated protein/ERK kinase/ERK pathway. We conclude that the stimulatory effects of azPC on PT transport may be partially involved in volume expansion.

Lipid raft-disrupting miltefosine preferentially induces the death of colorectal cancer stem-like cells.

BackgroundLipid rafts (LRs), cholesterol‐enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem‐like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear.MethodsThis study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR‐disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses.ResultsMiltefosine suppresses CSC populations and their self‐renewal activities in CRC cells, a CSC‐targeting effect leading to irreversible disruption of tumour‐initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44high CSCs, we found that CSCs exhibited stronger therapy resistance than non‐CSC counterparts by preventing cell death through CHEK1‐mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death.ConclusionOur findings underscore the therapeutic potential of LR‐targeting APLs for CRC treatment that overcomes the therapy‐resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.

Phospholipid Analogues as Chemotherapeutic Agents Against Trypanosomatids

Neglected tropical diseases (NTDs) represent a serious problem in a number of countries around the world and especially in Africa and South America, affecting mostly the poor population which has limited access to the healthcare system. The drugs currently used for the treatment of NTDs are dated many decades ago and consequently, present in some cases very low efficacy, high toxicity and development of drug resistance. In the search for more efficient chemotherapeutic agents for NTDs, a large number of different compound classes have been synthesized and tested. Among them, ether phospholipids, with their prominent member miltefosine, are considered one of the most promising. This review summarizes the literature concerning the development of antiparasitic phospholipid derivatives, describing the efforts towards more efficient and less toxic analogues while providing an overview of the mechanism of action of this compound class against trypanosomatids. Phospholipid analogues are already known for their antiprotozoal activity. Several studies have been conducted in order to synthesize novel derivatives with the aim to improve current treatments such as miltefosine, with promising results. Photolabeling and fluorescent alkyl phospholipid analogues have contributed to the clarification of the mode of action of this drug family.

Dual Gene Delivery Reagents From Antiproliferative Alkylphospholipids for Combined Antitumor Therapy.

Alkylphospholipids (APLs) have elicited great interest as antitumor agents due to their unique mode of action on cell membranes. However, their clinical applications have been limited so far by high hemolytic activity. Recently, cationic prodrugs of erufosine, a most promising APL, have been shown to mediate efficient intracellular gene delivery, while preserving the antiproliferative properties of the parent APL. Here, cationic prodrugs of the two APLs that are currently used in the clinic, miltefosine, and perifosine, are investigated and compared to the erufosine prodrugs. Their synthesis, stability, gene delivery and self-assembly properties, and hemolytic activity are discussed in detail. Finally, the potential of the pro-miltefosine and pro-perifosine compounds ME12 and PE12 in combined antitumor therapy is demonstrated using pUNO1-hTRAIL, a plasmid DNA encoding TRAIL, a member of the TNF superfamily. With these pro-APL compounds, we provide a proof of concept for a new promising strategy for cancer therapy combining gene therapy and APL-based chemotherapy.

Alkylphospholipids are Signal Transduction Modulators with Potential for Anticancer Therapy.

Alkylphospholipids (APLs) are synthetically derived from cell membrane components, which they target and thus modify cellular signalling and cause diverse effects. This study reviews the mechanism of action of anticancer, antiprotozoal, antibacterial and antiviral activities of ALPs, as well as their clinical use. A literature search was used as the basis of this review. ALPs target lipid rafts and alter phospholipase D and C signalling cascades, which in turn will modulate the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK pathways. By feedback coupling, the SAPK/JNK signalling chain is also affected. These changes lead to a G2/M phase cell cycle arrest and subsequently induce programmed cell death. The available knowledge on inhibition of AKT phosphorylation, mTOR phosphorylation and Raf down-regulation renders ALPs as attractive candidates for modern medical treatment, which is based on individualized diagnosis and therapy. Corresponding to their unusual profile of activities, their side effects result from cholinomimetic activity mainly and focus on the gastrointestinal tract. These aspects together with their bone marrow sparing features render APCs well suited for modern combination therapy. Although the clinical success has been limited in cancer diseases so far, the use of miltefosine against leishmaniosis is leading the way to better understanding their optimized use. Recent synthetic programs generate congeners with the increased therapeutic ratio, liposomal formulations, as well as diapeutic (or theranostic) derivatives with optimized properties. It is anticipated that these innovative modifications will pave the way for the further successful development of ALPs.

The lipid raft protein NTAL participates in AKT signaling in mantle cell lymphoma

Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold (n = 8) and decreased tumor weight by 2.7-fold (n = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.

Supplemental feeding of phospholipid-enriched alkyl phospholipid from krill relieves spontaneous atopic dermatitis and strengthens skin intercellular lipid barriers in NC/Nga mice.

Plasmalogen (Pls) is a glycerophospholipid derived from alkyl phospholipid (Alk) with antioxidant functions in vivo. The present study investigated the effects of ether phospholipids, such as Pls and Alk, on intercellular lipid barriers in the skin of NC/Nga mice, a model of atopic dermatitis (AD). NC/Nga mice fed Alk showed increased plasma levels of Alk and Pls. The AD-related changes in ceramide composition in the skin were abrogated by oral administration of Alk. Moreover, Alk suppressed skin inflammation in AD mice. These results indicate that Alk partially fortifies the stratum corneum lipid barrier and may be an effective treatment for AD. Abbreviations: Pls: plasmalogen; PlsCho: choline plasmalogen; PlsEtn: ethanolamine plasmalogen; Alk: alkyl phospholipid; TJ: tight junction; FA: fatty acid; AD: atopic dermatitis; SO: soybean oil; FO: fish oil; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TG: triglyceride; PL: phospholipid; RF: retention factor; AlkCho: choline-type alkyl phospholipid; AlkEtn: ethanolamine-type alkyl phospholipid; LC-MS/MS: liquid chromatography-tandem mass spectrometry; FAR1: fatty acyl-coenzyme (Co)A reductase 1.

Preclinical Pharmacokinetics and Dosimetry Studies of 124I/131I-CLR1404 for Treatment of Pediatric Solid Tumors in Murine Xenograft Models.

Cancer is the second leading cause of death for children between the ages of 5 and 14 y. For children diagnosed with metastatic or recurrent solid tumors, for which the utility of external-beam radiotherapy is limited, the prognosis is particularly poor. The availability of tumor-targeting radiopharmaceuticals for molecular radiotherapy (MRT) has demonstrated improved outcomes in these patient populations, but options are nonexistent or limited for most pediatric solid tumors. 18-(p-iodophenyl)octadecylphosphocholine (CLR1404) is a novel antitumor alkyl phospholipid ether analog that broadly targets cancer cells. In this study, we evaluated the in vivo pharmacokinetics of 124I-CLR1404 (CLR 124) and estimated theranostic dosimetry for 131I-CLR1404 (CLR 131) MRT in murine xenograft models of the pediatric solid tumors neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Methods: Tumor-bearing mice were imaged with small-animal PET/CT to evaluate the whole-body distribution of CLR 124 and, correcting for differences in radioactive decay, predict that of CLR 131. Image volumes representing CLR 131 provided input for Geant4 Monte Carlo simulations to calculate subject-specific tumor dosimetry for CLR 131 MRT. Pharmacokinetics for CLR 131 were extrapolated to adult and pediatric humans to estimate normal-tissue dosimetry. In neuroblastoma, a direct comparison of CLR 124 with 124I-metaiodobenzylguanidine (124I-MIBG) in an MIBG-avid model was performed. Results: In vivo pharmacokinetics of CLR 124 showed selective uptake and prolonged retention across all pediatric solid tumor models investigated. Subject-specific tumor dosimetry for CLR 131 MRT presents a correlative relationship with tumor-growth delay after CLR 131 MRT. Peak uptake of CLR 124 was, on average, 22% higher than that of 124I-MIBG in an MIBG-avid neuroblastoma model. Conclusion: CLR1404 is a suitable theranostic scaffold for dosimetry and therapy with potentially broad applicability in pediatric oncology. Given the ongoing clinical trials for CLR 131 in adults, these data support the development of pediatric clinical trials and provide detailed dosimetry that may lead to improved MRT treatment planning.

Enhanced Radiosensitivity in Solid Tumors using a Tumor-selective Alkyl Phospholipid Ether Analog.

Antitumor alkyl phospholipid (APL) analogs comprise a group of structurally related molecules with remarkable tumor selectivity. Some of these compounds have shown radiosensitizing capabilities. CLR127 is a novel, clinical-grade antitumor APL ether analog, a subtype of synthetic APL broadly targeting cancer cells with limited uptake in normal tissues. The purpose of this study was to investigate the effect of CLR127 to modulate radiation response across several adult and pediatric cancer types in vitro as well as in murine xenograft models of human prostate adenocarcinoma, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma. In vitro, CLR127 demonstrated selective uptake in cancer cells compared to normal cells. In cancer cells, CLR127 treatment prior to radiation significantly decreased clonogenic survival in vitro, and led to increased radiation-induced double-stranded DNA (dsDNA) breakage compared with radiation alone, which was not observed in normal controls. In animal models, CLR127 effectively increased the antitumor response to fractionated radiotherapy and led to delayed tumor regrowth at potentially clinically achievable doses. In conclusion, our study highlights the ability of CLR127 to increase radiation response in several cancer types. Given almost universal uptake of CLR127 in malignant cells, future research should test whether the observed effects can be extended to other tumor types. Our data provide a strong rationale for clinical testing of CLR127 as a tumor-targeted radiosensitizing agent. Mol Cancer Ther; 17(11); 2320-8. ©2018 AACR.

Abstract 4307: Molecular basis of cell cycle arrest induced by erufosine in metastatic breast cancer cells

Abstract Introduction: Breast cancer, a major health care burden with limited curative options for advanced metastatic phase, demands a search for novel therapeutic compounds. Synthetic alkyl-phospholipids (ALPs) comprise a promising class of anti-cancer agents in this context, where 1st and 2nd generation compounds are already in clinical use to treat various malignancies. Erufosine is the latest (3rd) generation of ALPs and has shown promising anti-tumour activities against breast cancer with minimal toxicity towards GI tract, negligible haemolytic activity and a good pharmacological profile in laboratory settings. Previously, we highlighted the significant potential of erufosine to induce arrest in G2/M phase of cell cycle in breast cancer cells. Currently, we decipher the mechanistic basis of these observed effects at molecular levels. Materials & Methods: Metastatic breast cancer cells (MDA-MB-231) were exposed to low (IC25, 12.3µM), medium (IC50, 19.5µM) and high (IC75, 30.2µM) concentrations of erufosine for 48h. Distribution of the cells in different phases of the cell cycle was determined by propidium iodide based labelling of the DNA and flow cytometry analysis. Expressional modulations in 84 cell cycle relevant genes in response to erufosine exposure (IC75, 48h) were studied by using a ready-made panel (Qiagen, Cat#330231) and real-time PCR methodology. Alterations in expressional profile of significantly altered genes (≥2fold) were used to design a signalling pathway with the help of Ingenuity Pathway Analysis at core facility of DKFZ, Heidelberg, Germany. Results: Exposure with erufosine induced a significant arrest in the G2/M phase of cell cycle in MDA-MB-231 cells in a concentration dependant format. Significant alterations (≥2fold) were found in 39% (33/84) of the cell cycle relevant genes incorporated in the ready-made panel. Most of the altered genes (28/33) were down-regulated in response to erufosine exposure. Significantly altered genes belong to various groups like kinases (ATM, AURKA, B), phosphatases (CDC25A, C), tumour suppressors (BRCA1), anti-apoptotic genes ( BCL2, BIRC5), cyclins (CCNA2, CCNB1, CCNB2, CCND2, CCNF, CCNG2), their target kinases (CDKs), activators (CDC6, MK167), inhibitors (CDKN1A, 2A, CDKN3, CKS1B), regulators (CDC20, MKI67), facilitators of the cell cycle (MCM2, 3, 4) and transcription factors (CKS2, E2F1). Conclusion: Erufosine is a significant cytostatic agent and induces a major halt in the G2/M phase of cell cycle in metastatic breast cancer cells. The compound imposes noteworthy alterations in various cell cycle relevant genes to cause these cytostatic effects. Erufosine, provided with further studies in laboratory (in vitro and in vivo) and clinical settings, could be a valuable therapeutic agent for metastatic breast cancer. Citation Format: Asim Pervaiz, Muhammad Shoaib Akhtar, Saqib Mahmood, Osheen Sajjad, Saba Khaliq, Martin R. Berger. Molecular basis of cell cycle arrest induced by erufosine in metastatic breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4307.

The sp2-iminosugar glycolipid 1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO2-ONJ) as selective anti-inflammatory agent by modulation of hemeoxygenase-1 in Bv.2 microglial cells and retinal explants

Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp2-iminosugar glycolipid (sp2-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO2-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO2-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO2-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO2-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO2-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO2-ONJ in microglia.

Adsorption characteristics of the alkyl phospholipid Inositol-C2-PAF at the solution/air interface

On the basis of surface tension and surface visco-elasticity measurements performed using profile analysis tensiometry with single pendent drops the surface properties of the alkyl phospholipid Inositol-C2-PAF were determined. Because of the very low bulk concentrations of Inositol-C2-PAF used in the studies, the obtained adsorption data had to be analysed including the depletion of the bulk concentration due to adsorption at the surface of the measured drops. The determined critical micellar concentration (CMC) of aqueous Inositol-C2-PAF solutions was found to be 1.1μM, similar to other anti-tumour and anti-inflammatory substances.

AKT1 Activation is Obligatory for Spontaneous BCC Tumor Growth in a Murine Model that Mimics Some Features of Basal Cell Nevus Syndrome.

Patients with basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, develop numerous basal cell carcinomas (BCC) due to germline mutations in the tumor suppressor PTCH1 and aberrant activation of Hedgehog (Hh) signaling. Therapies targeted at components of the Hh pathway, including the smoothened (SMO) inhibitor vismodegib, can ablate these tumors clinically, but tumors recur upon drug discontinuation. Using SKH1-Ptch1+/- as a model that closely mimics the spontaneous and accelerated growth pattern of BCCs in patients with BCNS, we show that AKT1, a serine/threonine protein kinase, is intrinsically activated in keratinocytes derived from the skin of newborn Ptch1+/- mice in the absence of carcinogenic stimuli. Introducing Akt1 haplodeficiency in Ptch1+/- mice (Akt1+/- Ptch1+/-) significantly abrogated BCC growth. Similarly, pharmacological inhibition of AKT with perifosine, an alkyl phospholipid AKT inhibitor, diminished the growth of spontaneous and UV-induced BCCs. Our data demonstrate an obligatory role for AKT1 in BCC growth, and targeting AKT may help reduce BCC tumor burden in BCNS patients. Cancer Prev Res; 9(10); 794-802. ©2016 AACR.

Miltefosine Suppresses Hepatic Steatosis by Activating AMPK Signal Pathway.

Background and PurposeIt has been accepted that AMPK (Adenosine monophosphate–activated protein kinase) activation exhibits many beneficial effects on glucolipid metabolism. Lysophosphatidylcholine (LPC) is an important lysophospholipid which can improve blood glucose levels in diabetic mice and attenuate inflammation by activating AMPK signal pathway in macrophages. Synthetic alkylphospholipids (ALPs), such as miltefosine, is used as an alternate of LPC for the clinical application. Here, we investigated whether miltefosine could have an impact on hepatic steatosis and related metabolic disorders.Experimental ApproachMice were fed with high fat diet (HFD) for 16 weeks to generate an obese model. Next, the obese mice were randomly divided into three groups: saline-treated and miltefosine-treated (2.5 or 5 mg/kg/d) groups. Miltefosine was intraperitoneally administrated into mice for additional 4 weeks plus HFD treatment.Key ResultsIt was shown that miltefosine treatment could substantially improve glucose metabolism, prevented hepatic lipid accumulation, and inhibited liver inflammation in HFD-fed mice by activating AMPK signal pathway. In vitro, miltefosine stimulated AMPKα phosphorylation both in time and dose dependent manner and decreased lipid accumulation in liver cells. When a specific AMPK inhibitor compound C was used to treat mice, the antagonistic effects of miltefosine on HFD-induced mouse hyperlipidaemia and liver steatosis were abolished. Treatment with miltefosine also dramatically inhibited the HFD-induced liver inflammation in mice.Conclusions and ImplicationsHere we demonstrated that miltefosine might be a new activator of AMPK signal pathway in vivo and in vitro and be useful for treatment of hepatic steatosis and related metabolic disorders.