ABSTRACT
To identify recurrent inflammation in hemophilia, we assessed the acute-phase response in the blood of patients with hemophilia A and B. Compared to age- and weight-matched controls, blood levels of interleukin-6 (IL-6), C-reactive protein (CRP), and LPS-binding protein (LBP) were significantly elevated in the entire cohort of hemophilia patients but exhibited a particularly pronounced increase in obese hemophilia patients with a body mass index (BMI) ≥30. Subgroup analysis of the remaining nonobese hemophilia patients (BMI: 18-29.9) revealed a significant spike of IL-6, CRP, and LBP in connection with a de-novo increase of soluble IL-6 receptor α (sIL-6Rα) in patients with bleeding events within the last month. Hemophilia patients who did not experience recent bleeding had IL-6, CRP, and sIL-6Rα blood levels similar to healthy controls. We did not find increased IL-6 or acute-phase reactants in hemophilia patients with arthropathy or infectious disease. The role of IL-6 as a marker of bleeding in hemophilia was confirmed in hemophilia patients with acute bleeding events as well as in transgenic hemophilia mice after needle puncture of the knee, which exhibited an extensive hematoma and a 150-fold increase of IL-6 blood levels within 7 days of the injury compared to needle-punctured control mice. Notably, IL-6 blood levels shrunk to a fourfold elevation in hemophilia mice over controls after 28 days, when the hematoma was replaced by arthrofibrosis. These findings indicate that acute-phase reactants in combination with sIL-6Rα could be sensitive biomarkers for the detection of acute and recent bleeding events in hemophilia.
Subject(s)
Hemophilia A , Mice , Animals , Hemophilia A/diagnosis , Interleukin-6/metabolism , Acute-Phase Reaction , C-Reactive Protein/metabolism , Mice, Transgenic , HematomaABSTRACT
Macrophages make important contributions to inflammation and wound healing. We show here that macrophage polarization is deregulated in haemophilia in response to macrophage colony-stimulating factor (M-CSF) and partially in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). As a result, haemophilia macrophages exhibit a specific impairment of M-CSF-mediated functions involved in wound healing such as clot invasion and phagocytosis. Haemophilia monocytes express reduced amounts of the receptors for M-CSF and GM-CSF, which correlates with a failure to express tumour necrosis factor α (TNFα) and CD163 in M-CSF-treated haemophilia macrophages and reduced expression of TNFα and CD206 after treatment with GM-CSF. Protein expression in response to M-CSF was regained with respect to CD163 and CD206 after embedding haemophilia monocytes in clotted plasma suggesting that a functioning coagulation system has positive effects on macrophage M2 polarization. Mimicking the functional deficits of haemophilia macrophages in normal macrophages was possible by adding leptin, which we found to be elevated in the blood of haemophilia patients, to a monocyte cell line. The increase of leptin occurred in conjunction with C-reactive protein in a body mass index-controlled cohort suggesting that haemophilia patients harbour chronic low-grade inflammation. Together, our data indicate that impaired clotting in haemophilia patients leads to increased inflammation and a deregulation in macrophage differentiation, which may explain the commonly observed deficits in wound healing and tissue regeneration.
Subject(s)
Blood Coagulation , Hemophilia A/blood , Macrophages/cytology , Adult , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Blood Circulation , Cell Differentiation , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Erythrocytes/cytology , Fibrin/metabolism , Flow Cytometry , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Hemophilia A/drug therapy , Humans , Lectins, C-Type/metabolism , Leukocytes, Mononuclear/cytology , Macrophage Colony-Stimulating Factor/therapeutic use , Male , Mannose Receptor , Mannose-Binding Lectins/metabolism , Microscopy, Fluorescence , Monocytes/cytology , Phagocytosis , Receptors, Cell Surface/metabolism , Tumor Necrosis Factor-alpha/metabolism , Young AdultABSTRACT
Fibrin plays an important role in lung metastasis. Here we show that fibrin promotes colony formation in primary kidney tumor cells from patients with kidney metastasis. In addition, we found that inhibition of fibrin formation with the thrombin inhibitor hirudin in nude mice in vivo significantly reduced the metastatic outgrowth of kidney tumor cells. Colony formation was significantly more efficient in tumor cells embedded in fibrin compared to matrigel and this effect correlates with the capacity of tumor cells to assemble a fibronectin matrix and generate stress fibers. Interestingly, stress fiber formation in fibrin was a specific function of metastatic kidney tumor cells while non-metastatic cells remained round. Inhibition of stress fiber formation with the Rho kinase inhibitor Y-27632, in turn, reduced fibronectin matrix assembly and colony formation in fibrin suggesting that spreading is a critical mechanism for the outgrowth of metastatic kidney tumor cells. Overall, our results indicate that adhesive interactions with fibrin play an important role for the progression of renal cell carcinoma and that inhibiting these interactions could be a promising strategy for treatment and prevention of kidney cancer metastasis.
ABSTRACT
UNLABELLED: Chloride intracellular channel 1 (CLIC1) has been shown to be upregulated in various malignancies but its exact function remains unclear. Here, it is revealed that CLIC1 is critical for the stability of invadopodia in endothelial and tumor cells embedded in a 3-dimensional (3D) matrix of fibrin. Invadopodia stability was associated with the capacity of CLIC1 to induce stress fiber and fibronectin matrix formation following its ß3 integrin (ITGB3)-mediated recruitment into invadopodia. This pathway, in turn, was relevant for fibrin colonization as well as slug (SNAI2) expression and correlated with a significant role of CLIC1 in metastasis in vivo. Mechanistically, a reduction of myosin light chain kinase (MYLK) in CLIC1-depleted as well as ß3 integrin-depleted cells suggests an important role of CLIC1 for integrin-mediated actomyosin dynamics in cells embedded in fibrin. Overall, these results indicate that CLIC1 is an important contributor to tumor invasion, metastasis, and angiogenesis. IMPLICATIONS: This study uncovers an important new function of CLIC1 in the regulation of cell-extracellular matrix interactions and ability of tumor cells to metastasize to distant organs.
Subject(s)
Chloride Channels/metabolism , Extracellular Matrix/pathology , Neoplasm Metastasis/pathology , Neovascularization, Pathologic/pathology , Adult , Aged , Cell Line, Tumor , Chloride Channels/genetics , Female , Human Umbilical Vein Endothelial Cells , Humans , Integrin beta3/metabolism , Male , Middle Aged , Neoplasm Transplantation , Neovascularization, Pathologic/metabolism , Signal Transduction , Snail Family Transcription Factors , Transcription Factors/metabolismABSTRACT
Blood clotting specifically supports the metastatic dissemination of malignant cells to the lung. We have recently demonstrated that 2 tumor types that are prone to form lung metastases, renal cell carcinoma and soft tissue sarcoma, share specific adhesive mechanisms that support the invasion and colonization of blood clots in the pulmonary vasculature.
ABSTRACT
The blood clotting cascade is selectively involved in lung metastasis, but the reason for this selectivity is unclear. Here, we show that tumor cells that metastasize predominantly to the lung, such as renal cell carcinoma (RCC) and soft tissue sarcoma (STS), have an inherent capacity to generate extensive invadopodia when embedded in a blood clot. Compared with other metastatic cancer cells tested, RCC and STS cells exhibited increased levels of expression of fibronectin and an activated form of the integrin αvß3, which coordinately supported the generation of an elaborate fibronectin matrix and actin stress fibers in fibrin-embedded tumor cells. Together, fibronectin and αvß3 induced upregulation of the transcription factor Slug, which mediates epithelial-mesenchymal transition as well as fibrin invasion and lung metastasis. This mechanism is clinically significant, because primary cancer cells from patients with metastatic RCC strongly invaded fibrin and this correlated with fibronectin matrix formation and Slug expression. In contrast, tumor cells from patients with localized RCC were largely noninvasive. Together, our findings establish that activated integrin αvß3 and fibronectin promote lung metastasis by upregulating Slug, defining a mechanism through which cancer cells can colonize blood clots in the lung vasculature.
Subject(s)
Fibronectins/metabolism , Integrin alphaVbeta3/metabolism , Neoplasms/blood , Neoplasms/pathology , Transcription Factors/metabolism , Animals , Cell Adhesion , Cell Line, Tumor , Female , Fibronectins/genetics , Gene Silencing , Humans , Integrin alphaVbeta3/genetics , Male , Mice, Nude , Microscopy, Confocal , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasms/metabolism , Snail Family Transcription Factors , Thrombosis/pathology , Transcription Factors/genetics , Up-RegulationABSTRACT
Our previous research has shown that plasma fibronectin promotes lung metastasis by facilitating tumor cell invasion in clotted plasma. To evaluate the role of clotted plasma for tumor cell survival, we treated B16F1 cells embedded in a 3-dimensional matrix of fibrin with tumor necrosis factor α (TNFα), a cytokine with anti-tumor activity. Under these conditions, TNFα caused significant cytotoxicity, which was prevented when we added plasma fibronectin to the fibrin clot. Fibronectin-mediated TNFα resistance was dependent on PI3-kinase, which also mediated the pro-adhesive and pro-invasive effects of plasma fibronectin on tumor cells. To further investigate the role of plasma fibronectin in tumor cell signaling, we performed a gene array that showed specific upregulation of Tie2 in B16F1 cells embedded in fibrin-fibronectin compared to fibrin. Importantly, inhibition of Tie2 resulted in decreased tumor cell invasion, reduced colony formation and increased tumor cell death in response to TNFα. Together, our findings indicate that plasma fibronectin induces tumor cell invasion and protects tumor cells from the cytotoxic effects of inflammatory mediators through up-regulation of Tie2.
ABSTRACT
High-grade non-muscle-invasive bladder cancer is commonly treated with Bacillus Calmette-Guérin, an immunotherapeutic that depends on fibronectin and tumor cell integrin α5ß1 for internalization into bladder cancer cells. We previously showed that the anti-angiogenic peptide CLT1 forms cytotoxic complexes with fibronectin that are cooperatively internalized into proliferating endothelium through ligation of integrins and chloride intracellular channel 1. While CLT1 has no effect on mature, differentiated cells, we show here that CLT1 is highly cytotoxic for a panel of bladder tumor cell lines as well as a variety of cell lines derived from kidney, lung, breast, and prostate cancer. Paralleling our previous results, we found CLT1-induced tumor cell death to be increased in the presence of fibronectin, which mediated CLT1 internalization and subsequent autophagic cell death in a mechanism that depends on tumor cell integrin α5ß1 and chloride intracellular channel 3 (CLIC3). This mechanistic link was further supported by our results showing upregulation of α5ß1 and CLIC3 in CLT1-responsive tumor cell lines and colocalization with CLT1 in tumor tissues. Incubating tumor tissue from patients with bladder cancer with fluorescein-conjugated CLT1 resulted in a strong and specific fluorescence whereas normal bladder tissue remained negative. On the basis of its affinity for bladder tumor tissue and strong antitumor effects, we propose that CLT1 could be useful for targeting bladder cancer.
Subject(s)
Integrin alpha5beta1/metabolism , Peptides, Cyclic/pharmacology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolism , Autophagy/drug effects , Cell Line, Tumor , Fibronectins/pharmacology , Gene Silencing , Humans , Integrin alpha5beta1/genetics , Molecular Targeted Therapy , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Oligopeptides/pharmacology , Peptides, Cyclic/pharmacokinetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Signal Transduction , Urinary Bladder Neoplasms/blood supply , Urinary Bladder Neoplasms/geneticsABSTRACT
Angiogenesis is important for tumor growth and metastasis. CLT1 (CGLIIQKNEC), a peptide that binds to tumor interstitial spaces in the presence of fibrin-fibronectin, has structural similarity to the anti-angiogenic ß-sheet peptides anastellin and anginex. This similarity is reflected in the ability of CLT1 to form co-aggregates with fibronectin that induce an unfolded protein response and cause autophagic cell death in proliferating endothelial cells. CLT1 cytotoxicity is mediated at least in parts by a novel CLT1 binding protein, Chloride Intracellular Channel 1 (CLIC1), which promotes internalization of CLT1-fibronectin co-aggregates in a mechanism that depends on the LIIQK amino acid sequence of CLT1. LIIQK encompasses amino acid residues relevant for CLT1 binding to CLIC1 and in addition, facilitates the formation of CLT1-fibronectin co-aggregates, which in turn promote translocation of CLIC1 to the endothelial cell surface through ligation of integrin αvß3. Paralleling the in vitro results, we found that CLT1 co-localizes with CLIC1 and fibronectin in angiogenic blood vessels in vivo, and that CLT1 treatment inhibited angiogenesis and tumor growth. Our findings show that CLT1 is a new anti-angiogenic compound, and its mechanism of action is to form co-aggregates with fibronectin, which bind to angiogenic endothelial cells through integrins, become internalized through CLIC1 and elicit a cytotoxic unfolded protein response. The simple structure and high potency of CLT1 make it a potentially useful compound for anti-angiogenic treatments.
Subject(s)
Chloride Channels/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fibronectins/metabolism , Neovascularization, Physiologic/drug effects , Peptides, Cyclic/pharmacology , Amino Acid Sequence , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Death/drug effects , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Proliferation/drug effects , Endocytosis/drug effects , Fibronectins/chemistry , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Male , Mice , Mice, Nude , Molecular Sequence Data , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Peptides, Cyclic/chemistry , Peptides, Cyclic/therapeutic use , Protein Structure, QuaternaryABSTRACT
The attachment of circulating tumor cells to the blood vessels of distant organs is an important step in metastasis. We show here that experimental lung metastasis by two cell lines, B16F1 melanoma and 3LL lung carcinoma, is greatly reduced in transgenic mice that lack plasma fibronectin. This multifunctional adhesive glycoprotein becomes cross-linked to fibrin during clotting. Here, we report that eliminating plasma fibronectin from the blood circulation reverses the prometastatic effects of blood clotting and tumor cell integrin alphavbeta3. In vitro studies showed that fibrin-fibronectin complexes, but not purified fibrin, supported tumor cell attachment and invasion. These functions correlate with the ability of fibrin-fibronectin complexes to induce the activation of integrin alphavbeta3. Our findings reveal an important contribution of plasma fibronectin in lung metastasis. Furthermore, they suggest that the previously noted effects of blood clotting on lung metastasis might be mediated in part by a fibronectin-alphavbeta3 integrin axis, in which plasma fibronectin has to be incorporated into the blood clot.
Subject(s)
Carcinoma, Lewis Lung/blood , Carcinoma, Lewis Lung/secondary , Fibronectins/blood , Lung Neoplasms/blood , Lung Neoplasms/secondary , Melanoma, Experimental/blood , Melanoma, Experimental/secondary , Animals , Blood Coagulation/physiology , Carcinoma, Lewis Lung/pathology , Cell Adhesion/physiology , Fibrin/metabolism , Integrin alphaVbeta3/blood , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasm InvasivenessABSTRACT
PURPOSE: We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling. EXPERIMENTAL DESIGN: Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice. RESULTS: c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa. CONCLUSIONS: These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Head and Neck Neoplasms/metabolism , Hepatocyte Growth Factor/metabolism , Proto-Oncogene Proteins c-met/metabolism , Signal Transduction/physiology , Animals , Blotting, Western , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/physiopathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Crizotinib , Dose-Response Relationship, Drug , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/physiopathology , Hepatocyte Growth Factor/pharmacology , Humans , Immunohistochemistry , Indoles/pharmacology , Mice , Mice, Nude , Neoplasm Transplantation , Paracrine Communication/physiology , Piperazines/pharmacology , Piperidines/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyrazoles , Pyridines/pharmacology , Signal Transduction/drug effects , Stress, Mechanical , Sulfonamides/pharmacology , Transplantation, Heterologous , Tumor Burden/drug effectsABSTRACT
Fatty-acid synthase (FAS) is up-regulated in a broad range of cancers, including those of the breast, prostate, and ovaries. In tumor cells, the inhibition of FAS elicits cell cycle arrest and apoptosis, so it is considered a potential drug target for oncology. Results from this study show that inhibition of FAS, by either knockdown with small interfering RNA or inhibition with the small molecule drug orlistat, leads to activation of the receptor-mediated apoptotic cascade (caspase-8-mediated) and ultimately to cell death. However, knockdown of two enzymes upstream of FAS, acetyl-CoA carboxylase-alpha and ATP-citrate lyase, fails to activate caspase-8 or to elicit apoptosis in tumor cells, even though palmitate synthesis was suppressed. Using differential gene analysis, we traced the unique apoptotic effect of FAS inhibition to up-regulation of DDIT4 (DNA damage-inducible transcript 4), a stress-response gene that negatively regulates the mTOR pathway. These findings indicate that suppression of palmitate synthesis is not sufficient for eliciting tumor cell death and suggest that the unique effect of inhibition of FAS results from negative regulation of the mTOR pathway via DDIT4.
Subject(s)
Apoptosis , Caspase 8/metabolism , Fatty Acid Synthases/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Transcription Factors/metabolism , Up-Regulation , Apoptosis/drug effects , Cell Line, Tumor , Enzyme Activation , Eukaryotic Initiation Factor-4E/metabolism , Fatty Acid Synthases/genetics , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/genetics , Phosphorylation , RNA, Small Interfering/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , fas Receptor/antagonists & inhibitors , fas Receptor/metabolismABSTRACT
Fatty acid synthase (FAS) is necessary for growth and survival of tumor cells and is a promising drug target for oncology. Here, we report on the syntheses and activity of novel inhibitors of the thioesterase domain of FAS. Using the structure of orlistat as a starting point, which contains a beta-lactone as the central pharmacophore, 28 novel congeners were synthesized and examined. Structural features such as the length of the alpha- and beta-alkyl chains, their chemical composition, and amino ester substitutions were altered and the resulting compounds explored for inhibitory activity toward the thioesterase domain of FAS. Nineteen congeners show improved potency for FAS in biochemical assays relative to orlistat. Three of that subset, including the natural product valilactone, also display an increased potency in inducing tumor cell death and improved solubility compared to orlistat. These findings support the idea that an orlistat congener can be optimized for use in a preclinical drug design and for clinical drug development.
Subject(s)
Antineoplastic Agents/chemical synthesis , Fatty Acid Synthases/antagonists & inhibitors , Lactones/chemical synthesis , Cell Death/drug effects , Cell Line, Tumor , Fibroblasts , Humans , Lactones/pharmacology , Neoplasms/drug therapy , Neoplasms/pathology , Orlistat , Solubility , Structure-Activity RelationshipABSTRACT
BACKGROUND: The lipogenic enzyme fatty acid synthase (FAS) is up-regulated in a wide variety of cancers, and is considered a potential metabolic oncogene by virtue of its ability to enhance tumor cell survival. Inhibition of tumor FAS causes both cell cycle arrest and apoptosis, indicating FAS is a promising target for cancer treatment. RESULTS: Here, we used gene expression profiling to conduct a global study of the cellular processes affected by siRNA mediated knockdown of FAS in MDA-MB-435 mammary carcinoma cells. The study identified 169 up-regulated genes (> or = 1.5 fold) and 110 down-regulated genes (< or = 0.67 fold) in response to knockdown of FAS. These genes regulate several aspects of tumor function, including metabolism, cell survival/proliferation, DNA replication/transcription, and protein degradation. Quantitative pathway analysis using Gene Set Enrichment Analysis software further revealed that the most pronounced effect of FAS knockdown was down-regulation in pathways that regulate lipid metabolism, glycolysis, the TCA cycle and oxidative phosphorylation. These changes were coupled with up-regulation in genes involved in cell cycle arrest and death receptor mediated apoptotic pathways. CONCLUSION: Together these findings reveal a wide network of pathways that are influenced in response to FAS knockdown and provide new insight into the role of this enzyme in tumor cell survival and proliferation.
Subject(s)
Breast Neoplasms/genetics , Fatty Acid Synthases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Genome, Human , Metabolic Networks and Pathways/genetics , RNA, Small Interfering/pharmacology , Apoptosis/genetics , Cell Cycle/genetics , Cell Line, Tumor , Down-Regulation , Female , Gene Expression Profiling , Humans , Up-RegulationABSTRACT
Anginex is a synthetic beta-sheet peptide with anti-angiogenic and anti-tumor activity. When added to cultured endothelial cells at concentrations ranging from 2.5 microM to 25 microM, anginex induced cell death, which was reflected by a strong increase of subdiploid cells and fragments, loss of cellular ATP, and LDH release. Cytotoxicity remained the same whether cells were treated with anginex at 4 degrees C or at 37 degrees C. At low temperatures, fluorescein-conjugated anginex accumulated on the endothelial surface, but did not reach into the cytoplasm, indicating that the cell membrane is the primary target for the peptide. Within minutes of treatment, anginex caused endothelial cells to take up propidium iodide and undergo depolarization, both parameters characteristic for permeabilization of the cell membrane. This process was amplified when cells were activated with hydrogen peroxide. Red blood cell membranes were essentially unaffected by anginex. Anginex bound lipid bilayers with high affinity and with a clear preference for anionic over zwitterionic phospholipids. Structural studies by circular dichroism and solid-state nuclear magnetic resonance showed that anginex forms a beta-sheet and adopts a unique and highly ordered conformation upon binding to lipid membranes. This is consistent with lipid micellization or the formation of pore-forming beta-barrels. The data suggest that the cytotoxicity of anginex stems from its ability to target and disrupt the endothelial cell membrane, providing a possible explanation for the angiostatic activity of the peptide.
Subject(s)
Cell Membrane/drug effects , Endothelial Cells/drug effects , Proteins/metabolism , Proteins/pharmacology , Cell Death , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Humans , Lipid Bilayers/chemistry , Liposomes , Peptides , Protein Conformation , Proteins/chemistryABSTRACT
In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins.