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.

Raft platforms highly enriched in cholesterol: major scaffolds for IL-6 signalling assembly with implications in inflammation and cancer.

Interleukin-6 (IL-6) is a pleiotropic cytokine with complex and major roles in inflammation, which could be linked to the different ways IL-6 signals at the plasma membrane. In this issue of FEBS Journal, Woo and co-workers present evidence for the involvement of Eps15 homology domain-containing protein 1 (EHD1)-mediated lipid raft platforms, highly enriched in cholesterol, in the IL-6 signalling pathway. Because of the strong connection between IL-6, inflammation and cancer, one implication of this report is that agents or approaches targeting cholesterol-rich raft platforms may assist the development of novel strategies to treat inflammatory and malignant diseases. Comment on: https://doi.org/10.1111/febs.16458.

Identification of new FK866 analogues with potent anticancer activity against pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases for which chemotherapy has not been very successful yet. FK866 ((E)-N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) is a well-known NAMPT (nicotinamide phosphoribosyltransferase) inhibitor with anti-cancer activities, but it failed in phase II clinical trials. We found that FK866 shows anti-proliferative activity in three PDAC cell lines, as well as in Jurkat T-cell leukemia cells. More than 50 FK866 analogues were synthesized that introduce substituents on the phenyl ring of the piperidine benzamide group of FK866 and exchange its buta-1,4-diyl tether for 1-oxyprop-3-yl, (E)-but-2-en-1,4-diyl and 2- and 3-carbon tethers. The pyridin-3-yl moiety of FK866 was exchanged for chlorinated and fluorinated analogues and for pyrazin-2-yl and pyridazin-4-yl groups. Several compounds showed low nanomolar or sub-nanomolar cell growth inhibitory activity. Our best cell anti-proliferative compounds were the 2,4,6-trimethoxybenzamide analogue of FK866 ((E)-N-(4-(1-(2,4,6-trimethoxybenzoyl)piperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) (9), the 2,6-dimethoxybenzamide (8) and 2-methoxybenzamide (4), which exhibited an IC50 of 0.16 nM, 0.004 nM and 0.08 nM toward PDAC cells, respectively.

Clusters of apoptotic signaling molecule-enriched rafts, CASMERs: membrane platforms for protein assembly in Fas/CD95 signaling and targets in cancer therapy.

Mammalian cells show the ability to commit suicide through the activation of death receptors at the cell surface. Death receptors, among which Fas/CD95 is one of their most representative members, lack enzymatic activity, and depend on protein-protein interactions to signal apoptosis. Fas/CD95 death receptor-mediated apoptosis requires the formation of the so-called death-inducing signaling complex (DISC), bringing together Fas/CD95, Fas-associated death domain-containing protein and procaspase-8. In the last two decades, cholesterol-rich lipid raft platforms have emerged as scaffolds where Fas/CD95 can be recruited and clustered. The co-clustering of Fas/CD95 and rafts facilitates DISC formation, bringing procaspase-8 molecules to be bunched together in a limited membrane region, and leading to their autoproteolytic activation by oligomerization. Lipid raft platforms serve as a specific region for the clustering of Fas/CD95 and DISC, as well as for the recruitment of additional downstream signaling molecules, thus forming the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER. These raft/CASMER structures float in the membrane like icebergs, in which the larger portion lies inside the cell and communicates with other subcellular structures to facilitate apoptotic signal transmission. This allows an efficient spatiotemporal compartmentalization of apoptosis signaling machinery during the triggering of cell death. This concept of proapoptotic raft platforms as a basic chemical-biological structure in the regulation of cell death has wide-ranging implications in human biology and disease, as well as in cancer therapy. Here, we discuss how these raft-centered proapoptotic hubs operate as a major linchpin for apoptosis signaling and as a promising target in cancer therapy.

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.

New indolesulfonamide derivatives targeting the colchicine site of tubulin: synthesis, anti-tumour activity, structure-activity relationships, and molecular modelling.

Searching for improved indolesulfonamides with higher polarities, 45 new analogues with modifications on the sulfonamide nitrogen, the methoxyaniline, and/or the indole 3-position were synthesised. They show submicromolar to nanomolar antiproliferative IC50 values against four human tumour cell lines and they are not P-glycoprotein substrates as their potencies against HeLa cells did not improve upon cotreatment with multidrug resistance (MDR) inhibitors. The compounds inhibit tubulin polymerisation in vitro and in cells, thus causing a mitotic arrest followed by apoptosis as shown by cell cycle distribution studies. Molecular modelling studies indicate binding at the colchicine site. Methylated sulfonamides were more potent than those with large and polar substitutions. Amide, formyl, or nitrile groups at the indole 3-position provided drug-like properties for reduced toxicity, with Polar Surface Areas (PSA) above a desirable 75 Å2. Nitriles 15 and 16 are potent polar analogues and represent an interesting class of new antimitotics.

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.

Neutrophils drive endoplasmic reticulum stress-mediated apoptosis in cancer cells through arginase-1 release.

Human neutrophils constitutively express high amounts of arginase-1, which depletes arginine from the surrounding medium and downregulates T-cell activation. Here, we have found that neutrophil arginase-1, released from activated human neutrophils or dead cells, induced apoptosis in cancer cells through an endoplasmic reticulum (ER) stress pathway. Silencing of PERK in cancer cells prevented the induction of ER stress and apoptosis. Arginase inhibitor Nω-hydroxy-nor-arginine inhibited apoptosis and ER stress response induced by conditioned medium from activated neutrophils. A number of tumor cell lines, derived from different tissues, were sensitive to neutrophil arginase-1, with pancreatic, breast, ovarian and lung cancer cells showing the highest sensitivity. Neutrophil-released arginase-1 and arginine deprivation potentiated the antitumor action against pancreatic cancer cells of the ER-targeted antitumor alkylphospholipid analog edelfosine. Our study demonstrates the involvement of neutrophil arginase-1 in cancer cell killing and highlights the importance and complex role of neutrophils in tumor surveillance and biology.

First and Second Waves of Coronavirus Disease 2019 in Madrid, Spain: Clinical Characteristics and Hematological Risk Factors Associated With Critical/Fatal Illness.

OBJECTIVES: This study aims to determine similarities and differences in clinical characteristics between the patients from two waves of severe acute respiratory syndrome coronavirus-2 infection at the time of hospital admission, as well as to identify risk biomarkers of coronavirus disease 2019 severity. DESIGN: Retrospective observational study. SETTING: A single tertiary-care center in Madrid. PATIENTS: Coronavirus disease 2019 adult patients admitted to hospital from March 4, 2020, to March 25, 2020 (first infection wave), and during July 18, 2020, and August 20, 2020 (second infection wave). INTERVENTIONS: Treatment with a hospital-approved drug cocktail during hospitalization. MEASUREMENTS AND MAIN RESULTS: Demographic, clinical, and laboratory data were compared between the patients with moderate and critical/fatal illness across both infection waves. The median age of patients with critical/fatal coronavirus disease 2019 was 67.5 years (interquartile range, 56.75-78.25 yr; 64.5% male) in the first wave and 59.0 years (interquartile range, 48.25-80.50 yr; 70.8% male) in the second wave. Hypertension and dyslipidemia were major comorbidities in both waves. Body mass index over 25 and presence of bilateral pneumonia were common findings. Univariate logistic regression analyses revealed an association of a number of blood parameters with the subsequent illness progression and severity in both waves. However, some remarkable differences were detected between both waves that prevented an accurate extrapolation of prediction models from the first wave into the second wave. Interleukin-6 and d-dimer concentrations at the time of hospital admission were remarkably higher in patients who developed a critical/fatal condition only during the first wave (p < 0.001), although both parameters significantly increased with disease worsening in follow-up studies from both waves. Multivariate analyses from wave 1 rendered a predictive signature for critical/fatal illness upon hospital admission that comprised six blood biomarkers: neutrophil-to-lymphocyte ratio (≥ 5; odds ratio, 2.684 [95% CI, 1.143-6.308]), C-reactive protein (≥ 15.2 mg/dL; odds ratio, 2.412 [95% CI, 1.006-5.786]), lactate dehydrogenase (≥ 411.96 U/L; odds ratio, 2.875 [95% CI, 1.229-6.726]), interleukin-6 (≥ 78.8 pg/mL; odds ratio, 5.737 [95% CI, 2.432-13.535]), urea (≥ 40 mg/dL; odds ratio, 1.701 [95% CI, 0.737-3.928]), and d-dimer (≥ 713 ng/mL; odds ratio, 1.903 [95% CI, 0.832-4.356]). The predictive accuracy of the signature was 84% and the area under the receiver operating characteristic curve was 0.886. When the signature was validated with data from wave 2, the accuracy was 81% and the area under the receiver operating characteristic curve value was 0.874, albeit most biomarkers lost their independent significance. Follow-up studies reassured the importance of monitoring the biomarkers included in the signature, since dramatic increases in the levels of such biomarkers occurred in critical/fatal patients over disease progression. CONCLUSIONS: Most parameters analyzed behaved similarly in the two waves of coronavirus disease 2019. However, univariate logistic regression conducted in both waves revealed differences in some parameters associated with poor prognosis in wave 1 that were not found in wave 2, which may reflect a different disease stage of patients on arrival to hospital. The six-biomarker predictive signature reported here constitutes a helpful tool to classify patient's prognosis on arrival to hospital.

Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence Microscopy: The Use of Combined Techniques to Assess Fas/CD95 Location in Rafts During Apoptosis Triggering.

Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.

Methylsulfanylpyridine based diheteroaryl isocombretastatin analogs as potent anti-proliferative agents.

Isocombretastatins are the not isomerizable 1,1-diarylethene isomers of combretastatins. Both families of antimitotics are poorly soluble and new analogs with improved water solubility are needed. The ubiquitous 3,4,5-trimethoxyphenyl ring and most of its replacements contribute to the solubility problem. 39 new compounds belonging to two series of isocombretastatin analogs with 2-chloro-6-methylsulfanyl-4-pyridinyl or 2,6-bis(methylsulfanyl)-4-pyridinyl moieties replacing the 3,4,5-trimethoxyphenyl have been synthesized and their antimitotic activity and aqueous solubility have been studied. We show here that 2-chloro-6-methylsulfanylpyridines are more successful replacements than 2,6-bis(methylsulfanyl)pyridines, giving highly potent tubulin inhibitors and cytotoxic compounds with improved water solubilities. The optimal combination is with indole rings carrying polar substitutions at the three position. The resulting diheteroaryl isocombretastatin analogs showed potent cytotoxic activity against human cancer cell lines caused by tubulin inhibition, as shown by in vitro tubulin polymerization inhibitory assays, cell cycle analysis, and confocal microscopy studies. Cell cycle analysis also showed apoptotic responses following G2/M arrest after treatment. Conformational analysis and docking studies were applied to propose binding modes of the compounds at the colchicine site of tubulin and were in good agreement with the observed SAR. 2-Chloro-6-methylsulfanylpyridines represent a new and successful trimethoxyphenyl ring substitution for the development of improved colchicine site ligands.

Upregulated expression and function of the α4ß1 integrin in multiple myeloma cells resistant to bortezomib.

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival, and resistance to different anti-MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4ß1 integrin is a main adhesion receptor mediating MM cell-stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion-mediated drug resistance (CAM-DR) and MM cell apoptosis. Whether decreased α4ß1 expression and activity are maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4ß1 function could boost MM cell survival and disease progression. Using BTZ-resistant MM cells, we found that they not only rescue their α4ß1 expression, but its levels were higher than in parental cells. Increased α4ß1 expression in resistant cells correlated with enhanced α4ß1-mediated cell lodging in the BM, and with disease progression. BTZ-resistant MM cells displayed enhanced NF-κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4ß1-dependent MM cell adhesion. These data emphasize the upregulation of α4ß1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM-DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin ß1) expression in MM and poor progression-free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high-risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Potent colchicine-site ligands with improved intrinsic solubility by replacement of the 3,4,5-trimethoxyphenyl ring with a 2-methylsulfanyl-6-methoxypyridine ring.

Colchicine site antimitotic agents typically suffer from low aqueous solubilities and are formulated as phosphate prodrugs of phenolic groups. These hydroxyl groups are the aim of metabolic transformations leading to resistance. There is an urgent need for more intrinsically soluble analogues lacking these hydroxyl groups. The 3,4,5-trimethoxyphenyl ring of combretastatin A-4 is a liability in terms of solubility but it is considered essential for high cytotoxic and tubulin polymerization inhibitory (TPI) activity. We have synthesized 36 new analogues of combretastatin A-4 replacing the trimethoxyphenyl moiety with more polar pyridine based moieties, measured their aqueous solubility, and studied their anti-proliferative effects against 3 human cancer cell lines. We show here that pyridine rings can be successful replacements for the trimethoxyphenyl ring, resulting in potent and more soluble analogues. The more straightforward replacement, a 2,6-dimethoxypyridine ring led to inactive analogues, but a 2-methoxy-6-methylsulfanylpyridine moiety led to active analogues when combined with different B rings. This replacement led to potent cytotoxic activity against sensitive human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies. Cell cycle analysis also showed apoptotic responses following treatment. Docking studies suggested binding at the colchicine site of tubulin and provided a good agreement with the observed SAR. A 2-methoxy-6-methylsulfanylpyridine moiety is a good trimethoxyphenyl ring replacement for the development of new colchicine site ligands.

Lipid rafts as signaling hubs in cancer cell survival/death and invasion: implications in tumor progression and therapy: Thematic Review Series: Biology of Lipid Rafts.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

Genome-wide miRNA profiling and pivotal roles of miRs 125a-5p and 17-92 cluster in human neutrophil maturation and differentiation of acute myeloid leukemia cells.

MicroRNAs (miRNAs, miRs) are short non-coding post-transcriptional regulators of gene expression in normal physiology and disease. Acute myeloid leukemia is characterized by accumulation of malignantly transformed immature myeloid precursors, and differentiation therapy, used to overcome this differentiation blockage, has become a successful therapeutic option. The human HL-60 acute leukemia cell line serves as a cell culture model for granulocytic maturation, and dimethyl sulfoxide (DMSO) incubation leads to its differentiation towards neutrophil-like cells, as assessed by biochemical, functional and morphological parameters. DMSO-induced HL-60 cell differentiation constitutes an excellent model to examine molecular processes that turn a proliferating immortal leukemic cell line into mature non-proliferating and apoptosis-prone neutrophil-like end cells. By performing genome-wide miRNA profiling and functional assays, we have identified a signature of 86 differentially expressed canonical miRNAs (51 upregulated; 35 downregulated) during DMSO-induced granulocytic differentiation of HL-60 cells. Quantitative real-time PCR was used to validate miRNA expression. Among these differentially expressed canonical miRNAs, we found miR-125a-5p upregulation and miR-17-92 cluster downregulation acted as major regulators of granulocytic differentiation in HL-60 cells. Enforced expression of miR-125a-5p promoted granulocytic differentiation in HL-60 cells, whereas miR-17-92 ectopic expression inhibited DMSO-induced HL-60 granulocytic differentiation. Ectopic expression of miR-125a-5p also promoted granulocytic differentiation in human acute promyelocytic leukemia NB4 cells, as well as in naïve human primary CD34+-hematopoietic progenitor/stem cells. These findings provide novel molecular insights into the identification of miRNAs regulating granulocytic differentiation of human leukemia cells and normal CD34+-hematopoietic progenitor/stem cells, and may assist in the development of novel miRNA-targeted therapies for leukemia.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Neutrophil Degranulation, Plasticity, and Cancer Metastasis.

Neutrophils are the first responders to inflammation and infection. Recently, an elevated neutrophil-to-lymphocyte ratio has generally become a prognostic indicator of poor overall survival in cancer. Accordingly, heterogeneous ill-defined neutrophil-like populations have been increasingly recognized as important players in cancer development. In addition, neutrophil granule proteins released upon cell activation have been associated with tumor progression; this differential granule mobilization may allow neutrophils - and possibly associated cancer cells - to leave the bloodstream and enter inflamed/infected tissues. This review discusses and proposes how granule mobilization may facilitate neutrophil-mediated transport of cancer cells into different tissues as well as leading to different cellular phenotypes that underlie remarkable neutrophil plasticity. This concept might inform novel neutrophil-centered approaches to putative cancer therapies.