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Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.
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Citoesqueleto de Actina/metabolismo , Forminas/metabolismo , Gonorreia/microbiologia , Macrófagos/microbiologia , Neisseria gonorrhoeae/patogenicidade , Gonorreia/metabolismo , Humanos , Macrófagos/metabolismo , PolimerizaçãoRESUMO
BACKGROUND: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. METHODS: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. RESULTS: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbß3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbß3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.
Assuntos
Anexina A1/genética , Plaquetas/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Inflamação/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transdução de SinaisRESUMO
Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.
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Interações Hospedeiro-Patógeno/fisiologia , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Modelos Animais de Doenças , Imunofluorescência , Homeostase/fisiologia , Imageamento Tridimensional , Immunoblotting , Doença dos Legionários/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia/métodos , Reação em Cadeia da Polimerase em Tempo Real , Vacúolos/metabolismoRESUMO
Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions.
Assuntos
Extensões da Superfície Celular/metabolismo , Listeriose/transmissão , Fosfatidilinositol 3-Quinases/metabolismo , Shigella flexneri/patogenicidade , Vacúolos/microbiologia , Sistemas de Secreção Bacterianos/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Extensões da Superfície Celular/microbiologia , Disenteria Bacilar/patologia , Disenteria Bacilar/transmissão , Células HT29 , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/microbiologia , Listeria monocytogenes/patogenicidade , Listeriose/patologia , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Vacúolos/metabolismoRESUMO
Shigella flexneri is an intracellular pathogen that disseminates in the intestinal epithelium by displaying actin-based motility. We found that although S. flexneri displayed comparable actin-based motilities in the cytosols of HeLa229 and HT-29 epithelial cell lines, the overall dissemination process was much more efficient in HT-29 cells. Time-lapse microscopy demonstrated that as motile bacteria reached the cell cortex in HT-29 cells, they formed membrane protrusions that resolved into vacuoles, from which the bacteria escaped and gained access to the cytosol of adjacent cells. In HeLa229 cells, S. flexneri also formed membrane protrusions that extended into adjacent cells, but the protrusions rarely resolved into vacuoles. Instead, the formed protrusions collapsed and retracted, bringing the bacteria back to the cytosol of the primary infected cells. Silencing the serine/threonine kinase STK11 (also known as LKB1) in HT-29 cells decreased the efficiency of protrusion resolution into vacuoles. Conversely, expressing STK11 in HeLa229 cells, which lack the STK11 locus, dramatically increased the efficiency of protrusion resolution into vacuoles. S. flexneri dissemination in HT-29 cells led to the local phosphorylation of tyrosine residues in protrusions, a signaling event that was not observed in HeLa229 cells but was restored in STK11-expressing HeLa229 cells. Treatment of HT-29 cells with the tyrosine kinase inhibitor imatinib abrogated tyrosine kinase signaling in protrusions, which correlated with a severe decrease in the efficiency of protrusion resolution into vacuoles. We suggest that the formation of STK11-dependent lateral cell-cell contacts competent for tyrosine kinase signaling promotes S. flexneri dissemination in epithelial cells.
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Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Shigella flexneri/fisiologia , Quinases Proteína-Quinases Ativadas por AMP , Actinas , Inativação Gênica , Células HT29 , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Transdução de Sinais , VacúolosRESUMO
The interaction between the tumor microenvironment (TME) and the cancer cells is a complex and mutually beneficial system that leads to rapid cancer cells proliferation, metastasis, and resistance to therapy. It is now recognized that cancer cells are not isolated, and tumor progression is governed among others, by many components of the TME. The reciprocal cross-talk between cancer cells and their microenvironment can be indirect through the secretion of extracellular matrix (ECM) proteins and paracrine signaling through exosomes, cytokines, and growth factors, or direct by cell-to-cell contact mediated by cell surface receptors and adhesion molecules. Among TME components, cancer-associated fibroblasts (CAFs) are of unique interest. As one of the most abundant components of the TME, CAFs play key roles in the reorganization of the extracellular matrix, facilitating metastasis and chemotherapy evasion. Both direct and indirect roles have been described for CAFs in modulating tumor progression. In this review, we focus on recent advances in understanding the role of direct contact between cancer cells and cancer-associated fibroblasts (CAFs) in driving tumor development and metastasis. We also summarize recent findings on the role of direct contact between cancer cells and CAFs in chemotherapy resistance.
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Neisseria gonorrhoeae (Ng) is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. Ng has developed numerous mechanisms to avoid and actively suppress innate and adaptive immune responses. Ng successfully colonizes and establishes topologically distinct colonies in human macrophages and avoids phagocytic killing. During colonization, Ng manipulates the actin cytoskeleton to invade and create an intracellular niche supportive of bacterial replication. The cellular reservoir(s) supporting bacterial replication and persistence in gonorrhea infections are poorly defined. The manner in which gonococci colonize macrophages points to this innate immune phagocyte as a strong candidate for a cellular niche during natural infection. Here we investigate whether nutrients availability and immunological polarization alter macrophage colonization by Ng. Differentiation of macrophages in pro-inflammatory (M1-like) and tolerogenic (M2-like) phenotypes prior to infection reveals that Ng can invade macrophages in all activation states, albeit with lower efficiency in M1-like macrophages. These results suggest that during natural infection, bacteria could invade and grow within macrophages regardless of the nutrients availability and the macrophage immune activation status.
Assuntos
Macrófagos , Neisseria gonorrhoeae , Nutrientes , Neisseria gonorrhoeae/imunologia , Macrófagos/microbiologia , Macrófagos/imunologia , Humanos , Gonorreia/microbiologia , Gonorreia/imunologia , Ativação de Macrófagos , Interações Hospedeiro-Patógeno/imunologiaRESUMO
Neisseria gonorrhoeae (Ng) is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. Ng has developed numerous mechanisms to avoid and actively suppress innate and adaptive immune responses. Ng successfully colonizes and establishes topologically distinct colonies in human macrophages and avoids phagocytic killing. During colonization, Ng manipulates the actin cytoskeleton to invade and create an intracellular niche supportive of bacterial replication. The cellular reservoir(s) supporting bacterial replication and persistence in gonorrhea infections are poorly defined. The manner in which gonococci colonize macrophages points to this innate immune phagocyte as a strong candidate for a cellular niche during natural infection. Here we investigate whether nutrients availability and immunological polarization alter macrophage colonization by Ng . Differentiation of macrophages in pro-inflammatory (M1-like) and tolerogenic (M2-like) phenotypes prior to infection reveals that Ng can invade macrophages in all activation states, albeit with lower efficiency in M1-like macrophages. These results suggest that during natural infection, bacteria could invade and grow within macrophages regardless of the nutrients availability and the macrophage immune activation status.
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BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a developmental program that consists of the loss of epithelial features concomitant with the acquisition of mesenchymal features. Activation of EMT in cancer facilitates the acquisition of aggressive traits and cancer invasion. EMT plasticity (EMP), the dynamic transition between multiple hybrid states in which cancer cells display both epithelial and mesenchymal markers, confers survival advantages for cancer cells in constantly changing environments during metastasis. METHODS: RNAseq analysis was performed to assess genome-wide transcriptional changes in cancer cells depleted for histone regulators FLASH, NPAT, and SLBP. Quantitative PCR and Western blot were used for the detection of mRNA and protein levels. Computational analysis was performed on distinct sets of genes to determine the epithelial and mesenchymal score in cancer cells and to correlate FLASH expression with EMT markers in the CCLE collection. RESULTS: We demonstrate that loss of FLASH in cancer cells gives rise to a hybrid E/M phenotype with high epithelial scores even in the presence of TGFß, as determined by computational methods using expression of predetermined sets of epithelial and mesenchymal genes. Multiple genes involved in cell-cell junction formation are similarly specifically upregulated in FLASH-depleted cells, suggesting that FLASH acts as a repressor of the epithelial phenotype. Further, FLASH expression in cancer lines is inversely correlated with the epithelial score. Nonetheless, subsets of mesenchymal markers were distinctly up-regulated in FLASH, NPAT, or SLBP-depleted cells. CONCLUSIONS: The ZEB1low/SNAILhigh/E-cadherinhigh phenotype described in FLASH-depleted cancer cells is driving a hybrid E/M phenotype in which epithelial and mesenchymal markers coexist.
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Shigella flexneri is a Gram-negative intracellular pathogen that infects the intestinal epithelium and utilizes actin-based motility to spread from cell to cell. S. flexneri actin-based motility has been characterized in various cell lines, but studies in intestinal cells are limited. Here we characterized S. flexneri actin-based motility in HT-29 intestinal cells. In agreement with studies conducted in various cell lines, we showed that S. flexneri relies on neural Wiskott-Aldrich Syndrome protein (N-WASP) in HT-29 cells. We tested the potential role of various tyrosine kinases involved in N-WASP activation and uncovered a previously unappreciated role for Bruton's tyrosine kinase (Btk) in actin tail formation in intestinal cells. We showed that Btk depletion led to a decrease in N-WASP phosphorylation which affected N-WASP recruitment to the bacterial surface, decreased the number of bacteria displaying actin-based motility, and ultimately affected the efficiency of spread from cell to cell. Finally, we showed that the levels of N-WASP phosphorylation and Btk expression were increased in response to infection, which suggests that S. flexneri infection not only triggers the production of proinflammatory factors as previously described but also manipulates cellular processes required for dissemination in intestinal cells.
Assuntos
Disenteria Bacilar/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Proteínas Tirosina Quinases/metabolismo , Shigella flexneri/metabolismo , Actinas/metabolismo , Tirosina Quinase da Agamaglobulinemia , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/microbiologia , Disenteria Bacilar/enzimologia , Disenteria Bacilar/microbiologia , Células HT29 , Humanos , Mucosa Intestinal/enzimologia , Fosforilação , Shigella flexneri/patogenicidade , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
The tumor microenvironment (TME) is the source of important cues that govern epithelial-to-mesenchymal transition (EMT) and facilitate the acquisition of aggressive traits by cancer cells. It is now recognized that EMT is not a binary program, and cancer cells rarely switch to a fully mesenchymal phenotype. Rather, cancer cells exist in multiple hybrid epithelial/mesenchymal (E/M) states responsible for cell population heterogeneity, which is advantageous for the ever-changing environment during tumor development and metastasis. How are these intermediate states generated and maintained is not fully understood. Here, we show that direct interaction between small cell lung carcinoma cells and lung fibroblasts induces a hybrid EMT phenotype in cancer cells in which several mesenchymal genes involved in receptor interaction with the extracellular matrix (ECM) and ECM remodeling are upregulated while epithelial genes such as E-cadherin remain unchanged or slightly increase. We also demonstrate that several core EMT-regulating transcription factors (EMT-TFs) are upregulated in cancer cells during direct contact with fibroblasts, as is Yes-associated protein (YAP1), a major regulator of the Hippo pathway. Further, we show that these changes are transient and reverse to the initial state once the interaction is disrupted. Altogether, our results provide evidence that tumor cells' direct contact with the fibroblasts in the TME initiates a signaling cascade responsible for hybrid E/M states of cancer cells. These hybrid states are maintained during the interaction and possibly contribute to therapy resistance and immune evasion, while interference with direct contact will result in slow recovery and switch to the initial states.
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Host membranes are inherently critical for niche homeostasis of vacuolar pathogens. Thus, intracellular bacteria frequently encode the capacity to regulate host lipogenesis as well as to modulate the lipid composition of host membranes. One membrane component that is often subverted by vacuolar bacteria is cholesterol - an abundant lipid that mammalian cells produce de novo at the endoplasmic reticulum (ER) or acquire exogenously from serum-derived lipoprotein carriers. Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires' disease. From within a unique ER-derived vacuole L. pneumophila promotes host lipogenesis and experimental evidence indicates that cholesterol production might be one facet of this response. Here we investigated the link between cellular cholesterol and L. pneumophila intracellular replication and discovered that disruption of cholesterol biosynthesis or cholesterol trafficking lowered bacterial replication in infected cells. These growth defects were rescued by addition of exogenous cholesterol. Conversely, bacterial growth within cholesterol-leaden macrophages was enhanced. Importantly, the growth benefit of cholesterol was observed strictly in cellular infections and L. pneumophila growth kinetics in axenic cultures did not change in the presence of cholesterol. Microscopy analyses indicate that cholesterol regulates a step in L. pneumophila intracellular lifecycle that occurs after bacteria begin to replicate within an established intracellular niche. Collectively, we provide experimental evidence that cellular cholesterol promotes L. pneumophila replication within a membrane bound organelle in infected macrophages.
RESUMO
Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires' disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)-derived organelle within which bacterial replication takes place. Bacteria-derived proteins are deposited in the host cytosol and in the lumen of the pathogen-occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella -containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes - such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis - and trans - isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid - a C16:1 monounsaturated fatty acid - that promotes membrane disorder when enriched in GPLs significantly increased bacterial replication within human and murine macrophages but not in axenic growth assays. Lipidome analysis of infected macrophages showed that treatment with exogenous palmitoleic acid resulted in membrane acyl chain reprogramming in a manner that promotes membrane disorder and live-cell imaging revealed that the consequences of increasing membrane disorder impinge on several LCV homeostasis parameters. Collectively, we provide experimental evidence that L. pneumophila replication within its intracellular niche is a function of the lipid bilayer disorder and hydrophobic thickness.
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Fusarochromanone is an experimental drug with unique and potent anti-cancer activity. Current cancer therapies often incorporate a combination of drugs to increase efficacy and decrease the development of drug resistance. In this study, we used drug combinations and cellular phenotypic screens to address important questions about FC101's mode of action and its potential therapeutic synergies in triple negative breast cancer (TNBC). We hypothesized that FC101's activity against TNBC is similar to the mTOR inhibitor, everolimus, because FC101 downregulates the phosphorylation of two mTOR substrates, S6K and S6. Since everolimus synergistically enhances the anti-cancer activities of two known EGFR inhibitors (erlotinib or lapatinib) in TNBC, we performed analogous studies with FC101. Phenotypic cellular assays helped assess whether FC101 acts similarly to everolimus, in both single and combination treatments with the two inhibitors. FC101 outperformed all other single treatments in both cell proliferation and viability assays. However, unlike everolimus, FC101 produced a sustained decrease in cell viability in drug washout studies. None of the other drugs were able to maintain comparable effects upon removal. Although we observed slightly additive effects when the TNBC cells were treated with FC101 and the two EGFR inhibitors, those effects were not truly synergistic in the manner displayed with everolimus.
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Vitamin E TPGS is a tocopherol (α-T) based nonionic surfactant that was used in the formulation of the Tocosol™ paclitaxel nanoemulsion, which was withdrawn from phase III clinical trials. Unlike tocopherols, however, the tocotrienol (T3) isomers of vitamin E were found to have innate anticancer activity and were shown to potentiate the antitumor activity of paclitaxel. The primary objective of the present study was therefore to develop a paclitaxel nanoemulsions by substituting α-T oil core of Tocosol™ with γ-T3 in, and vitamin E TPGS with PEGylated γ-T3 as the shell, and test the nanoemulsions against Bx-PC-3 and PANC-1 pancreatic tumor cells. A secondary objective was to test the activity of paclitaxel when directly conjugated with the γ-T3 isomer of vitamin E. The synthesis of the conjugates was confirmed by NMR and mass spectroscopy. Developed nanoemulsions were loaded with free or lipid conjugated paclitaxel. Nanoemulsions droplets were <300 nm with fastest release observed with formulations loaded with free paclitaxel when γ-T3 was used as the core. Substituting α-T with γ-T3 was also found to potentiate the anticancer activity of the nanoemulsions. Although marginal increase in activity was observed when nanoemulsions were loaded with free paclitaxel, a significant increase in activity was observed when lipid conjugates were used. The results from this study suggest that the developed paclitaxel nanoemulsions with either γ-T3, PEGylated γ-T3, or paclitaxel lipid conjugates may represent a more promising option for paclitaxel delivery in cancer chemotherapy.
Assuntos
Emulsões/química , Lipídeos/química , Nanopartículas/química , Paclitaxel/química , Tocotrienóis/química , Antineoplásicos/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Humanos , Polietilenoglicóis/química , Vitamina E/químicaRESUMO
Thymosin alpha 1 (Talpha1) has therapeutic potential in the treatment of infectious diseases and cancer. However, the exact molecular pathways for Talpha1 action are not fully understood. We found that Talpha1 induces the production of interleukin-6 (IL-6), IL-10, and IL-12 in murine bone marrow-derived macrophages (BMDMs) through IKK and MAPK pathways. Talpha1 triggers the activation of AP-1 and the phosphorylation of JNK and p38. Inhibition of p38 impairs IL-6 production in response to Talpha1. Further, TRAF6 is involved in the activation of JNK and IRAK4 is involved for the activation of IKK and PKCzeta in a Talpha1-induced system. Loss of IRAK4 largely blocked induction of IL-6. Thus, our studies define early signal events that are critical for the Talpha1-induced immune responses.
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Quinase I-kappa B/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/fisiologia , Células Dendríticas/imunologia , Ativação Enzimática , Humanos , Interleucina-12/metabolismo , Proteína Quinase C/metabolismo , Timalfasina , Timosina/análogos & derivados , Timosina/fisiologiaRESUMO
The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.
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Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Antifúngicos/química , Candida albicans/genética , Candida albicans/fisiologia , Linhagem Celular , Corantes , Cumarínicos , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Pigmentação/genética , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Vacúolos/fisiologia , Vacúolos/ultraestruturaRESUMO
The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) signaling axis has gained considerable attention as an attractive molecular target for therapeutic blockade of cancer. Inspired by the chemical structure of S (-)-oleocanthal, a natural secoiridoid from extra-virgin olive oil with documented anticancer activity against c-Met-dependent malignancies, the research presented herein reports on the discovery of the novel olive-derived homovanillyl sinapate (HVS) as a promising c-Met inhibitor. HVS was distinguished for its remarkable potency against wild-type c-Met and its oncogenic variant in cell-free assays and confirmed by in silico docking studies. Furthermore, HVS substantially impaired the c-Met-mediated growth across a broad spectrum of breast cancer cells, while similar treatment doses had no effect on the non-tumorigenic mammary epithelial cell growth. In addition, HVS caused a dose-dependent inhibition of HGF-induced, but not epidermal growth factor (EGF)-induced, cell scattering in addition to HGF-mediated migration, invasion, and 3-dimensional (3D) proliferation of tumor cell spheroids. HVS treatment effects were mediated via inhibition of ligand-mediated c-Met activation and its downstream mitogenic signaling and blocking molecular mediators involved in cellular motility across different cellular contexts. An interesting feature of HVS is its good selectivity for c-Met and Abelson murine leukemia viral oncogene homolog 1 (ABL1) when profiled against a panel of kinases. Docking studies revealed interactions likely to impart high dual affinity for both ABL1 and c-Met kinases. HVS markedly reduced tumor growth, showed excellent pharmacodynamics, and suppressed cell proliferation and microvessel density in an orthotopic model of triple negative breast cancer. Collectively, the present findings suggested that the oleocanthal-based HVS is a promising c-Met inhibitor lead entity with excellent therapeutic potential to control malignancies with aberrant c-Met activity.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ácidos Cumáricos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catálise , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos Nus , Simulação de Acoplamento Molecular , Estrutura Molecular , Invasividade Neoplásica , Fosforilação , Mutação Puntual , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Relação Estrutura-Atividade , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dysregulated receptor tyrosine kinase c-Met and its ligand HGF is valid and attractive molecular target for therapeutic blockade in cancer. Inspired by the chemical structure of the naturally occurring olive secoiridoid (-)-oleocanthal (1) and its documented anticancer activity against c-Met-dependent malignancies, a previous study reported tyrosol sinapate (4) as a c-Met inhibitor hit. This study reports additional semisynthetic optimization and SAR of 4 to improve its selective activity against c-Met-dependent breast cancer by increasing its capacity to inhibit c-Met phosphorylation. Forty-three compounds (5-47) were synthesized, among which the novel analog homovanillyl sinapate (HVS-16) was distinguished for its remarkable activity. HVS-16 substantially impaired c-Met-mediated proliferation, migration, and invasion across human breast cancer cell lines in two- and three-dimensional culture systems, while similar treatment doses were found to have effect neither on the non-tumorigenic human mammary epithelial cell growth nor on the c-Met independent breast cancer cell viability. HVS-16 showed a dose-dependent inhibition of ligand-mediated c-Met activation in human breast cancer cells. Docking studies revealed that HVS-16 fits very well inside c-Met crystal structures, satisfying critical interactions at the ATP binding site. This study identified important structural pharmacophoric features in HVS-16 and correlated its postulated binding pose with c-Met kinase assay data that would guide future olive secoiridoid bioisostere lead design. Results presented herein suggest HVS-16 as a promising c-Met inhibitor validated hit with potential to control invasive breast malignancies with aberrant c-Met activity.
RESUMO
Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell.