Suppression of metastatic organ colonization and antiangiogenic activity of the orally bioavailable lipid raft-targeted alkylphospholipid edelfosine.

Metastasis is the leading cause of cancer mortality. Metastatic cancer is notoriously difficult to treat, and it accounts for the majority of cancer-related deaths. The ether lipid edelfosine is the prototype of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs, and its antitumor activity involves lipid raft reorganization. In this study, we examined the effect of edelfosine on metastatic colonization and angiogenesis. Using non-invasive bioluminescence imaging and histological examination, we found that oral administration of edelfosine in nude mice significantly inhibited the lung and brain colonization of luciferase-expressing 435-Lung-eGFP-CMV/Luc metastatic cells, resulting in prolonged survival. In metastatic 435-Lung and MDA-MB-231 breast cancer cells, we found that edelfosine also inhibited cell adhesion to collagen-I and laminin-I substrates, cell migration in chemotaxis and wound-healing assays, as well as cancer cell invasion. In 435-Lung and other MDA-MB-435-derived sublines with different organotropism, edelfosine induced G2/M cell cycle accumulation and apoptosis in a concentration- and time-dependent manner. Edelfosine also inhibited in vitro angiogenesis in human and mouse endothelial cell tube formation assays. The antimetastatic properties were specific to cancer cells, as edelfosine had no effects on viability in non-cancerous cells. Edelfosine accumulated in membrane rafts and endoplasmic reticulum of cancer cells, and membrane raft-located CD44 was downregulated upon drug treatment. Taken together, this study highlights the potential of edelfosine as an attractive drug to prevent metastatic growth and organ colonization in cancer therapy. The raft-targeted drug edelfosine displays a potent activity against metastatic organ colonization and angiogenesis, two major hallmarks of tumor malignancy.

Antitumor activity of alkylphospholipid edelfosine in prostate cancer models and endoplasmic reticulum targeting.

Prostate cancer is the second most frequent cancer and the fifth leading cause of cancer death among men worldwide. While the five-year survival in local and regional prostate cancer is higher than 99%, it falls to about 28% in advanced metastatic prostate cancer. The ether lipid edelfosine is considered the prototype of a family of promising antitumor drugs collectively named as alkylphospholipid analogs. Here, we found that edelfosine was the most potent alkylphospholipid analog in inducing apoptosis in three different human prostate cancer cell lines (LNCaP, PC3, and DU145) with distinct androgen dependency, and differing in tumor suppressor phosphatase and tensin homolog (PTEN) and p53 status. Edelfosine accumulated in the endoplasmic reticulum of prostate cancer cells, leading to endoplasmic reticulum stress and cell death in the three prostate cancer cells. Inhibition of autophagy potentiated the pro-apoptotic activity of edelfosine in LNCaP and PC3 cells, where autophagy was induced as a survival response. Edelfosine induced a slight and transient inhibition of AKT in PTEN-negative LNCaP and PC3 cells, but not in PTEN-positive DU145 cells. Daily oral administration of edelfosine in murine prostate restricted AKT kinase transgenic mice, expressing active AKT in a prostate-specific manner, and in a DU145 xenograft mouse model resulted in significant tumor regression and apoptosis in tumor cells. Taken together, these results show a significant in vitro and in vivo antitumor activity of edelfosine against prostate cancer, and highlight the endoplasmic reticulum as a novel and promising therapeutic target in prostate cancer.

Induction of Apoptosis in Human Pancreatic Cancer Stem Cells by the Endoplasmic Reticulum-Targeted Alkylphospholipid Analog Edelfosine and Potentiation by Autophagy Inhibition.

Pancreatic cancer is one of the most lethal malignancies with a poor and gloomy prognosis and the highest mortality-to-incidence ratio. Pancreatic cancer remains an incurable malignancy, and current therapies are ineffective. We isolated cancer stem cells (CSCs) from the human PANC-1 pancreatic cancer cell line as CD44+CD24+EpCAM+ cells. These CSCs form pancreatic cancer spheres or spheroids and develop tumors in SCID mice after subcutaneous injection of as few as 100 cells per mouse. Here, we found that the alkylphospholipid analog edelfosine inhibited CSC pancreatic cancer spheroid formation and induced cell death, as assessed by an increase in the percentage of cells in the sub-G0/G1 region by means of flow cytometry, indicative of DNA breakdown and apoptosis. This correlated with an increase in caspase-3 activity and PARP breakdown, as a major substrate of caspase-3, following PANC-1 CSC treatment with edelfosine. The antitumor ether lipid edelfosine colocalized with the endoplasmic reticulum in both PANC-1 cells as well as PANC-1 CSCs by using a fluorescent edelfosine analog, and induced an endoplasmic reticulum stress response in both PANC-1 cells and PANC-1 CSCs, with a potent CHOP/GADD153 upregulation. Edelfosine elicited a strong autophagy response in both PANC-1 cells and PANC-1 CSCs, and preincubation of CSCs with autophagy inhibitors, chloroquine or bafilomycin A1, enhanced edelfosine-induced apoptosis. Primary cultures from pancreatic cancer patients were sensitive to edelfosine, as well as their respective isolated CSCs. Nontumorigenic pancreatic human cell line HPNE and normal human fibroblasts were largely spared. These data suggest that pancreatic CSCs isolated from established cell lines and pancreatic cancer patients are sensitive to edelfosine through its accumulation in the endoplasmic reticulum and induction of endoplasmic reticulum stress.

Direct Endoplasmic Reticulum Targeting by the Selective Alkylphospholipid Analog and Antitumor Ether Lipid Edelfosine as a Therapeutic Approach in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), the most common malignancy of the pancreas, shows a dismal and grim overall prognosis and survival rate, which have remained virtually unchanged for over half a century. PDAC is the most lethal of all cancers, with the highest mortality-to-incidence ratio. PDAC responds poorly to current therapies and remains an incurable malignancy. Therefore, novel therapeutic targets and drugs are urgently needed for pancreatic cancer treatment. Selective induction of apoptosis in cancer cells is an appealing approach in cancer therapy. Apoptotic cell death is highly regulated by different signaling routes that involve a variety of subcellular organelles. Endoplasmic reticulum (ER) stress acts as a double-edged sword at the interface of cell survival and death. Pancreatic cells exhibit high hormone and enzyme secretory functions, and thereby show a highly developed ER. Thus, pancreatic cancer cells display a prominent ER. Solid tumors have to cope with adverse situations in which hypoxia, lack of certain nutrients, and the action of certain antitumor agents lead to a complex interplay and crosstalk between ER stress and autophagy-the latter acting as an adaptive survival response. ER stress also mediates cell death induced by a number of anticancer drugs and experimental conditions, highlighting the pivotal role of ER stress in modulating cell fate. The alkylphospholipid analog prototype edelfosine is selectively taken up by tumor cells, accumulates in the ER of a number of human solid tumor cells-including pancreatic cancer cells-and promotes apoptosis through a persistent ER-stress-mediated mechanism both in vitro and in vivo. Here, we discuss and propose that direct ER targeting may be a promising approach in the therapy of pancreatic cancer, opening up a new avenue for the treatment of this currently incurable and deadly cancer. Furthermore, because autophagy acts as a cytoprotective response to ER stress, potentiation of the triggering of a persistent ER response by combination therapy, together with the use of autophagy blockers, could improve the current gloomy expectations for finding a cure for this type of cancer.

Mitochondrial Targeting Involving Cholesterol-Rich Lipid Rafts in the Mechanism of Action of the Antitumor Ether Lipid and Alkylphospholipid Analog Edelfosine.

The ether lipid edelfosine induces apoptosis selectively in tumor cells and is the prototypic molecule of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs. Cumulative evidence shows that edelfosine interacts with cholesterol-rich lipid rafts, endoplasmic reticulum (ER) and mitochondria. Edelfosine induces apoptosis in a number of hematological cancer cells by recruiting death receptors and downstream apoptotic signaling into lipid rafts, whereas it promotes apoptosis in solid tumor cells through an ER stress response. Edelfosine-induced apoptosis, mediated by lipid rafts and/or ER, requires the involvement of a mitochondrial-dependent step to eventually elicit cell death, leading to the loss of mitochondrial membrane potential, cytochrome c release and the triggering of cell death. The overexpression of Bcl-2 or Bcl-xL blocks edelfosine-induced apoptosis. Edelfosine induces the redistribution of lipid rafts from the plasma membrane to the mitochondria. The pro-apoptotic action of edelfosine on cancer cells is associated with the recruitment of F1FO-ATP synthase into cholesterol-rich lipid rafts. Specific inhibition of the FO sector of the F1FO-ATP synthase, which contains the membrane-embedded c-subunit ring that constitutes the mitochondrial permeability transcription pore, hinders edelfosine-induced cell death. Taking together, the evidence shown here suggests that the ether lipid edelfosine could modulate cell death in cancer cells by direct interaction with mitochondria, and the reorganization of raft-located mitochondrial proteins that critically modulate cell death or survival. Here, we summarize and discuss the involvement of mitochondria in the antitumor action of the ether lipid edelfosine, pointing out the mitochondrial targeting of this drug as a major therapeutic approach, which can be extrapolated to other alkylphospholipid analogs. We also discuss the involvement of cholesterol transport and cholesterol-rich lipid rafts in the interactions between the organelles as well as in the role of mitochondria in the regulation of apoptosis in cancer cells and cancer therapy.

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 M E12 and P E12 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.

Synthesis and Evaluation of Antitumor Alkylphospholipid Prodrugs.

PURPOSE: Hemolysis is a serious side effect of antitumor alkylphospholipids (APLs) that limits dose levels and is a constraint in their use in therapeutic regimen. Nine prodrugs of promising APLs (miltefosine, perifosine, and erufosine) were synthesized so as to decrease their membrane activity and improve their toxicity profile while preserving their antineoplastic potency. METHODS: The synthesis of the pro-APLs was straightforwardly achieved in one step starting from the parent APLs. The critical aggregation concentration of the prodrugs, their hydrolytic stability under various pH conditions, their blood compatibility and cytotoxicity in three different cell lines were determined and compared to those of the parent antitumor lipids. RESULTS: The APL prodrugs display antitumor activity which is similar to that of the parent alkylphospholipids but without associated hemolytic toxicity. CONCLUSION: The pro-APL compounds may be considered as intravenously injectable derivatives of APLs. They could thus address one of the major issues met in cancer therapies involving antitumor lipids and restricting their utilization to oral and topical administration because of limited maximum tolerated dose.

Alkylphospholipids: An update on molecular mechanisms and clinical relevance.

Alkylphospholipids (APLs) represent a new class of drugs which do not interact directly with DNA but act on the cell membrane where they accumulate and interfere with lipid metabolism and signalling pathways. This review summarizes the mode of action at the molecular level of these compounds. In this sense, a diversity of mechanisms has been suggested to explain the actions of clinically-relevant APLs, in particular, in cancer treatment. One consistently reported finding is that APLs reduce the biosynthesis of phosphatidylcholine (PC) by inhibiting the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CT). APLs also alter intracellular cholesterol traffic and metabolism in human tumour-cell lines, leading to an accumulation of cholesterol inside the cell. An increase in cholesterol biosynthesis associated with a decrease in the synthesis of choline-containing phospholipids and cholesterol esterification leads to a change in the free-cholesterol:PC ratio in cells exposed to APLs. Akt phosphorylation status after APL exposure shows that this critical regulator for cell survival is modulated by changes in cholesterol levels induced in the plasma membrane by these lipid analogues. Furthermore, APLs produce cell ultrastructural alterations with an abundant autophagic vesicles and autolysosomes in treated cells, indicating an interference of autophagy process after APL exposure. Thus, antitumoural APLs interfere with the proliferation of tumour cells via a complex mechanism involving phospholipid and cholesterol metabolism, interfere with lipid-dependent survival-signalling pathways and autophagy. Although APLs also exert antiparasitic, antibacterial, and antifungal effects, in this review we provide a summary of the antileishmanial activity of these lipid analogues. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

A phase I/Ib trial targeting the Pi3k/Akt pathway using perifosine: Long-term progression-free survival of patients with resistant neuroblastoma.

AKT plays a pivotal role in driving the malignant phenotype of many cancers, including high-risk neuroblastoma (HR-NB). AKT signaling, however, is active in normal tissues, raising concern about excessive toxicity from its suppression. The oral AKT inhibitor perifosine showed tolerable toxicity in adults and in our phase I trial in children with solid tumors (clinicaltrials.gov NCT00776867). We now report on the HR-NB experience. HR-NB patients received perifosine 50-75 mg m-2  day-1 after a loading dose of 100-200 mg m-2 on day 1, and continued on study until progressive disease. The 27 HR-NB patients included three treated for primary refractory disease and 24 with disease resistant to salvage therapy after 1-5 (median 2) relapses; only one had MYCN-amplified HR-NB. Pharmacokinetic studies showed µM concentrations consistent with cytotoxic levels in preclinical models. Nine patients (all MYCN-non-amplified) remained progression-free through 43+ to 74+ (median 54+) months from study entry, including the sole patient to show a complete response and eight patients who had persistence of abnormal 123 I-metaiodobenzylguanidine skeletal uptake but never developed progressive disease. Toxicity was negligible in all 27 patients, even with the prolonged treatment (11-62 months, median 38) in the nine long-term progression-free survivors. The clinical findings (i) confirm the safety of therapeutic serum levels of an AKT inhibitor in children; (ii) support perifosine for MYCN-non-amplified HR-NB as monotherapy after completion of standard treatment or combined with other agents (based on preclinical studies) to maximize antitumor effects; and (iii) highlight the welcome possibility that refractory or relapsed MYCN-non-amplified HR-NB is potentially curable.

The alkylphospholipid edelfosine shows activity against Strongyloides venezuelensis and induces apoptosis-like cell death.

Strongyloidiasis is widely distributed in the tropical and subtropical areas. Ivermectin is the drug of choice for the treatment. However, the concerns about relying treatment on a single drug make identification of new molecules a priority. Alkylphospholipid analogues, including edelfosine, are a group of synthetic compounds that have shown activity against some parasites. The objective was to assess the in vitro and in vivo activity of edelfosine, miltefosine, perifosine against Strongyloides venezuelensis. Moreover, apoptosis-like mechanism in larvae after treatment was studied. Edelfosine displayed the highest activity and the best selectivity index (LD50=49.6 ± 5.4µM, SI=1.1) compared to miltefosine or perifosine. Third stage larvae after culture with edelfosine were not able to develop an infection in mice. Treatment of mice with edelfosine showed reduction of 47% in parasitic females allocated in the gut. Moreover, DNA fragmentation was observed by TUNEL staining in larvae treated with edelfosine. These results suggest that edelfosine could be an effective drug against strongyloidiasis, probably through induction of apoptosis-like cell death.

Modulation of the voltage-gated potassium channel Kv2.1 by the anti-tumor alkylphospholipid perifosine.

BACKGROUND: The aim of the present study was to assess the effects of perifosine-a third generation alkylphospholipid analog with anti-tumor properties-on the activity of Kv2.1 channels. METHODS: The whole-cell patch clamp technique was applied to follow the modulatory effect of perifosine on Kv2.1 channels expressed in HEK293 cells. RESULTS: Obtained data provide evidence that perifosine application decreases the whole cell Kv2.1 currents in a concentration-independent manner. Perifosine induces a hyperpolarizing shift in the voltage dependence of Kv2.1 channels inactivation without altering the voltage dependence of channels activation. The kinetics of Kv2.1 closed-state inactivation was accelerated by perifosine, with no significant effects on the recovery rate from inactivation. CONCLUSIONS: Taken together, these results show that perifosine modified the Kv2.1 inactivation gating resulting in a decrease of the current amplitude. These data will help to elucidate the mechanism of action of this promising anti-cancer drug on ion channels and their possible implications.

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice.

BACKGROUND AND AIM: We previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. METHODS AND RESULTS: Six-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (-28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (-78%, P = 5.11E-06) in the aorta. CONCLUSION: Plasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation.

ERK1/2 acts as a switch between necrotic and apoptotic cell death in ether phospholipid edelfosine-treated glioblastoma cells.

Glioblastoma is characterized by constitutive apoptosis resistance and survival signaling expression, but paradoxically is a necrosis-prone neoplasm. Incubation of human U118 glioblastoma cells with the antitumor alkylphospholipid analog edelfosine induced a potent necrotic cell death, whereas apoptosis was scarce. Preincubation of U118 cells with the selective MEK1/2 inhibitor U0126, which inhibits MEK1/2-mediated activation of ERK1/2, led to a switch from necrosis to caspase-dependent apoptosis following edelfosine treatment. Combined treatment of U0126 and edelfosine totally inhibited ERK1/2 phosphorylation, and led to RIPK1 and RelA/NF-κB degradation, together with a strong activation of caspase-3 and -8. This apoptotic response was accompanied by the activation of the intrinsic apoptotic pathway with mitochondrial transmembrane potential loss, Bcl-xL degradation and caspase-9 activation. Inhibition of ERK phosphorylation also led to a dramatic increase in edelfosine-induced apoptosis when the alkylphospholipid analog was used at a low micromolar range, suggesting that ERK phosphorylation acts as a potent regulator of apoptotic cell death in edelfosine-treated U118 cells. These data show that inhibition of MEK1/2-ERK1/2 signaling pathway highly potentiates edelfosine-induced apoptosis in glioblastoma U118 cells and switches the type of edelfosine-induced cell death from necrosis to apoptosis.

Caenorhabditis elegans as a platform to study the mechanism of action of synthetic antitumor lipids.

Drugs capable of specifically recognizing and killing cancer cells while sparing healthy cells are of great interest in anti-cancer therapy. An example of such a drug is edelfosine, the prototype molecule of a family of synthetic lipids collectively known as antitumor lipids (ATLs). A better understanding of the selectivity and the mechanism of action of these compounds would lead to better anticancer treatments. Using Caenorhabditis elegans, we modeled key features of the ATL selectivity against cancer cells. Edelfosine induced a selective and direct killing action on C. elegans embryos, which was dependent on cholesterol, without affecting adult worms and larvae. Distinct ATLs ranked differently in their embryonic lethal effect with edelfosine > perifosine > erucylphosphocholine >> miltefosine. Following a biased screening of 57 C. elegans mutants we found that inactivation of components of the insulin/IGF-1 signaling pathway led to resistance against the ATL edelfosine in both C. elegans and human tumor cells. This paper shows that C. elegans can be used as a rapid platform to facilitate ATL research and to further understand the mechanism of action of edelfosine and other synthetic ATLs.

Novel alkylphospholipid-DTC hybrids as promising agents against endocrine related cancers acting via modulation of Akt-pathway.

A new series of 2-(alkoxy(hydroxy)phosphoryloxy)ethyl dialkylcarbodithioate derivatives was synthesized and evaluated against endocrine related cancers, acting via modulation of Akt-pathway. Eighteen compounds were active at 7.24-100 µM against MDA-MB-231 or MCF-7 cell lines of breast cancer. Three compounds (14, 18 and 22) were active against MCF-7 cells at IC50 significantly better than miltefosine and most of the compounds were less toxic towards non-cancer cell lines, HEK-293. On the other hand, twelve compounds exhibited cell growth inhibiting activity against prostate cancer cell lines, either PC-3 or DU-145 at 14.69-95.20 µM. While nine of these were active against both cell lines. The most promising compounds 14 and 18 were about two and five fold more active than miltefosine against DU-145 and MCF-7 cell lines respectively and significantly down regulated phospho-Akt. Possibly anti-cancer and pro-apoptotic activity was mostly due to blockade of Akt-pathway.

AKT as a therapeutic target in multiple myeloma.

INTRODUCTION: Multiple myeloma remains an incurable malignancy with poor survival. Novel therapeutic approaches capable of improving outcomes in patients with multiple myeloma are urgently required. AKT is a central node in the phosphatidylinositol-3-kinase/AKT/mammalian target of rapamycin signaling pathway with high expression in advanced and resistant multiple myeloma. AKT contributes to multiple oncogenic functions in multiple myeloma which may be exploited therapeutically. Promising preclinical data has lent support for pursuing further development of AKT inhibitors in multiple myeloma. Lead drugs are now entering the clinic. AREAS COVERED: The rationale for AKT inhibition in multiple myeloma, pharmacological subtypes of AKT inhibitors in development, available results of clinical studies of AKT inhibitors and suitable drug partners for further development in combination with AKT inhibition in multiple myeloma are discussed. EXPERT OPINION: AKT inhibitors are a welcome addition to the armamentarium against multiple myeloma and promising clinical activity is being reported from ongoing trials in combination with established and/or novel treatment approaches. AKT inhibitors may be set to improve patient outcomes when used in combination with synergistic drug partners.