RESUMO
Glioblastoma (GBM) is a highly angiogenic malignancy of the central nervous system that resists standard antiangiogenic therapy, in part because of an alternative process to angiogenesis termed vasculogenic mimicry. Intricately linked to GBM, dysregulation of the Hippo signaling pathway leads to overexpression of YAP/TEAD and several downstream effectors involved in therapy resistance. Little is known about whether vasculogenic mimicry and the Hippo pathway intersect in the GBM chemoresistance phenotype. This study seeks to investigate the expression patterns of Hippo pathway regulators within clinically annotated GBM samples, examining their involvement in vitro regarding vasculogenic mimicry. In addition, it aims to assess the potential for pharmacological targeting of this pathway. In-silico analysis of the Hippo signaling members YAP1 , TEAD1 , AXL , NF2 , CTGF , and CYR61 transcript levels in low-grade GBM and GBM tumor tissues was done by Gene Expression Profiling Interactive Analysis. Gene expression was analyzed by real-time quantitative PCR from human U87, U118, U138, and U251 brain cancer cell lines and in clinically annotated brain tumor cDNA arrays. Transient gene silencing was performed with specific small interfering RNA. Vasculogenic mimicry was assessed using a Cultrex matrix, and three-dimensional capillary-like structures were analyzed with Wimasis. CYR61 and CTGF transcript levels were elevated in GBM tissues and were further induced when in-vitro vasculogenic mimicry was assessed. Silencing of CYR61 and CTGF , or treatment with a small-molecule TEAD inhibitor LM98 derived from flufenamic acid, inhibited vasculogenic mimicry. Silencing of SNAI1 and FOXC2 also altered vasculogenic mimicry and reduced CYR61 / CTGF levels. Pharmacological targeting of the Hippo pathway inhibits in-vitro vasculogenic mimicry. Unraveling the connections between the Hippo pathway and vasculogenic mimicry may pave the way for innovative therapeutic strategies.
Assuntos
Neoplasias Encefálicas , Fator de Crescimento do Tecido Conjuntivo , Proteína Rica em Cisteína 61 , Glioblastoma , Fatores de Transcrição , Humanos , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/genética , Neovascularização Patológica/tratamento farmacológico , Fatores de Transcrição de Domínio TEA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
OBJECTIVE: Colorectal cancer (CRC) is the third most diagnosed cancer, and requires surgical resection and reconnection, or anastomosis, of the remaining bowel to re-establish intestinal continuity. Anastomotic leak (AL) is a major complication that increases mortality and cancer recurrence. Our objective is to assess the causal role of gut microbiota in anastomotic healing. DESIGN: The causal role of gut microbiota was assessed in a murine AL model receiving faecal microbiota transplantation (FMT) from patients with CRC collected before surgery and who later developed or not, AL. Anastomotic healing and gut barrier integrity were assessed after surgery. Bacterial candidates implicated in anastomotic healing were identified using 16S rRNA gene sequencing and were isolated from faecal samples to be tested both in vitro and in vivo. RESULTS: Mice receiving FMT from patients that developed AL displayed poor anastomotic healing. Profiling of gut microbiota of patients and mice after FMT revealed correlations between healing parameters and the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementation with A. onderdonkii resulted in a higher rate of leaks in mice, while gavage with P. goldsteinii improved healing by exerting an anti-inflammatory effect. Patients with AL and mice receiving FMT from AL patients presented upregulation of mucosal MIP-1α, MIP-2, MCP-1 and IL-17A/F before surgery. Retrospective analysis revealed that patients with AL present higher circulating neutrophil and monocyte counts before surgery. CONCLUSION: Gut microbiota plays an important role in surgical colonic healing in patients with CRC. The impact of these findings may extend to a vast array of invasive gastrointestinal procedures.
Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Camundongos , Animais , Citocinas , Microbioma Gastrointestinal/fisiologia , Estudos Retrospectivos , RNA Ribossômico 16S , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica/microbiologia , Neoplasias Colorretais/cirurgiaRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) cells' secretome can induce a pro-inflammatory phenotype in human adipose-derived mesenchymal stem cells (hADMSC). This can be prevented by the green tea polyphenol epigallocatechin-3-gallate (EGCG). The impact of EGCG on the paracrine regulation that the extracellular vesicles (EVs) specifically exert within the TNBC secretome remains unknown. METHODS: EVs were obtained from a TNBC-derived serum-starved MDA-MB-231 cell model treated or not with EGCG under normoxic or hypoxic (< 1% O2) culture conditions. RNA-Seq analysis was used to assess the EVs' genetic content. The modulation of inflammatory and senescence markers in hADMSC was evaluated by RT-qPCR using cDNA arrays and validated by immunoblotting. A protein profiler phospho-kinase array was used to explore signaling pathways. RESULTS: While hypoxic culture conditions did not significantly alter the genetic content of MDA-MB-231-secreted EVs, the addition of EGCG significantly modified EVs genetic material at low oxygen tension. Gene expression of cancer-associated adipocyte pro-inflammatory markers CXCL8, CCL2 and IL-1ß was increased in hADMSC treated with EVs. Concomitantly, EVs isolated from MDA-MB-231 treated with EGCG (EGCG-EVs) downregulated CCL2 and IL-1ß, while inducing higher expression of CXCL8 and IL-6 levels. EVs activated CHK-2, c-Jun, AKT and GSK-3ß signaling pathways in hADMSC, whereas EGCG-EVs specifically reduced the latter two as well as the serum starvation-induced senescence markers p21 and ß-galactosidase. Finally, the mitochondrial content within the TNBC cells-derived EVs was found reduced upon EGCG treatment. CONCLUSION: This proof of concept study demonstrates that the chemopreventive properties of diet-derived polyphenols may efficiently target the paracrine regulation that TNBC cells could exert upon their surrounding adipose tissue microenvironment.
RESUMO
Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.
Assuntos
Amina Oxidase (contendo Cobre) , Produtos Biológicos , Animais , Camundongos , Histamina/farmacologia , Histamina/metabolismo , Cimetidina/farmacologia , Catalase , Peróxido de Hidrogênio/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Receptores Histamínicos/genética , Antagonistas dos Receptores Histamínicos H1/farmacologia , Neurônios/metabolismo , Produtos Biológicos/farmacologiaRESUMO
The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.
Assuntos
Ácido Flufenâmico , Neoplasias , Humanos , Ácido Flufenâmico/farmacologia , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Via de Sinalização Hippo , Neoplasias/genéticaRESUMO
Modulations in cell surface receptor ectodomain proteolytic shedding impact on receptor function and cancer biomarker expression. As such, heavily pursued therapeutic avenues have exploited LDL receptor-related protein-1 (LRP-1)-mediated capacity in internalizing Angiopep-2 (An2), a brain-penetrating peptide that allows An2-drug conjugates to cross the blood-brain tumor barrier (BBTB). Given that LRP-1 is proteolytically shed from the cell surface through matrix metalloproteinase (MMP) activity, the balance between MMP expression/function and LRP-1-mediated An2 internalization is unknown. In this study, we found that membrane type-1 (MT1)-MMP expression increased from grade 1 to 4 brain tumors, while that of LRP-1 decreased inversely. MMP pharmacological inhibitors such as Ilomastat, Doxycycline and Actinonin increased in vitro An2 internalization by up to 2.5 fold within a human grade IV-derived U87 glioblastoma cell model. Transient siRNA-mediated MT1-MMP gene silencing resulted in increased basal An2 cell surface binding and intracellular uptake, while recombinant MT1-MMP overexpression reduced both cell surface LRP-1 expression as well as An2 internalization. The addition of Ilomastat to cells overexpressing recombinant MT1-MMP restored LRP-1 expression at the cell surface and An2 uptake to levels comparable to those observed in control cells. Collectively, our data suggest that MT1-MMP expression status dictates An2-mediated internalization processes in part by regulating cell surface LRP-1 functions. Such evidence prompts preclinical evaluations of combined MMP inhibitors/An2-drug conjugate administration to potentially increase the treatment of high-MT1-MMP-expressing brain tumors.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Metaloproteinase 14 da Matriz , Humanos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Ligantes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologiaRESUMO
Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of cancers which lacks the expression and/or amplification of targetable biomarkers (ie, estrogen receptor, progestrogen receptor, and human epidermal growth factor receptor 2), and is often associated with the worse disease-specific outcomes than other breast cancer subtypes. Here, we report that high expression of the sortilin (SORT1) receptor correlates with the decreased survival in TNBC patients, and more importantly in those bearing lymph node metastases. By exploiting SORT1 function in ligand internalization, a new anticancer treatment strategy was designed to target SORT1-positive TNBC-derived cells both in vitro and in two in vivo tumor xenografts models. A peptide (TH19P01), which requires SORT1 for internalization and to which many anticancer drugs could be conjugated, was developed. In vitro, while the TH19P01 peptide itself did not exert any antiproliferative or apoptotic effects, the docetaxel-TH19P01 conjugate (TH1902) exerted potent antiproliferative and antimigratory activities when tested on TNBC-derived MDA-MB-231 cells. TH1902 triggered faster and more potent apoptotic cell death than did unconjugated docetaxel. The apoptotic and antimigratory effects of TH1902 were both reversed by two SORT1 ligands, neurotensin and progranulin, and on siRNA-mediated silencing of SORT1. TH1902 also altered microtubule polymerization and triggered the downregulation of the anti-apoptotic Bcl-xL biomarker. In vivo, both i.p. and i.v. administrations of TH1902 led to greater tumor regression in two MDA-MB-231 and HCC-70 murine xenograft models than did docetaxel, without inducing neutropenia. Altogether, the data demonstrates the high in vivo efficacy and safety of TH1902 against TNBC through a SORT1 receptor-mediated mechanism. This property allows for selective treatment of SORT1-positive TNBC and makes TH1902 a promising avenue for personalized therapy with the potential of improving the therapeutic window of cytotoxic anticancer drugs such as docetaxel.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Combinação de Medicamentos , Descoberta de Drogas , Feminino , Inativação Gênica , Xenoenxertos , Humanos , Metástase Linfática , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Transplante de Neoplasias , Neurotensina/farmacologia , Progranulinas/farmacologia , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , Proteína bcl-X/metabolismoRESUMO
Background: Transforming growth factor (TGF)-ß triggers ovarian cancer metastasis through epithelial-mesenchymal transition (EMT). Whereas drug design strategies targeting the TGF-ß signaling pathway have been envisioned, the anti-TGF structure:function aspect of chemopreventive diet-derived catechins remains unexplored.Aim: We assessed the effects of eight catechins on TGF-ß-mediated cell migration and induction of EMT biomarkers, as well as on In Vitro vasculogenic mimicry (VM), a process partly regulated by EMT-related transcription factors.Results: TGF-ß-mediated phosphorylation of Smad-3 and p38 signaling intermediates was more effective in a chemosensitive ES-2 ovarian cancer cell line but was inoperative in cis-platinum- and adriamycin-chemoresistant SKOV-3 ovarian cancer cells. Increases in cell migration and in gene/protein expression of EMT biomarkers Fibronectin, Snail, and Slug were observed in ES-2 cells. When VM was assessed in ES-2 cells, 3D capillary-like structures were formed and increases in EMT biomarkers found. Catechins bearing the galloyl moiety (CG, ECG, GCG, and EGCG) exerted potent inhibition of TGF-ß-induced cell migration as well as EMT, and inhibited VM, in part through inhibition of Snail and matrix metalloproteinase-2 secretion.Conclusions: Our data suggest that diet-derived catechins exhibit chemopreventive properties that circumvent the TGF-ß-mediated signaling which contributes to the ovarian cancer metastatic phenotype.
Assuntos
Catequina , Transição Epitelial-Mesenquimal , Neoplasias Ovarianas , Catequina/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Dieta , Feminino , Humanos , Metaloproteinase 2 da Matriz , Neoplasias Ovarianas/prevenção & controle , Fator de Crescimento Transformador betaRESUMO
Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-ß signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-ß signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-ß and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-ß triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-ß receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-ß-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-ß-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.
Assuntos
Neoplasias Encefálicas/patologia , Transição Epitelial-Mesenquimal/fisiologia , Glioblastoma/patologia , Metaloproteinase 14 da Matriz/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Concanavalina A , Fibronectinas/biossíntese , Humanos , Metaloproteinase 14 da Matriz/genética , Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Tirfostinas/farmacologiaRESUMO
Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.
Assuntos
Catequina/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adiponectina/metabolismo , Animais , Catequina/metabolismo , Catequina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados , Feminino , Humanos , Leptina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Chá/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Epidemiological studies have convincingly demonstrated that diets rich in fruits and vegetables play an important role in preventing cancer due to their polyphenol content. Among polyphenols, the anthocyanidins are known to possess anti-inflammatory, cardioprotective, anti-angiogenic, and anti-carcinogenic properties. Despite the well-known role of transforming growth factor-ß (TGF-ß) in high grade gliomas, the impact of anthocyanidins on TGF-ß-induced epithelial-mesenchymal transition (EMT), a process that allows benign tumor cells to infiltrate surrounding tissues, remains poorly understood. The objective of this study is to investigate the impact of anthocyanidins such as cyanidin (Cy), delphinidin (Dp), malvidin (Mv), pelargonidin (Pg), and petunidin (Pt) on TGF-ß-induced EMT and to determine the mechanism(s) underlying such action. Human U-87 glioblastoma (U-87 MG) cells were treated with anthocyanidins prior to, along with or following the addition of TGF-ß. We found that anthocyanidins differently affected TGF-ß-induced EMT, depending on the treatment conditions. Dp was the most potent EMT inhibitor through its inhibitory effect on the TGF-ß Smad and non-Smad signaling pathways. These effects altered expression of the EMT mesenchymal markers fibronectin and Snail, as well as markedly reducing U-87 MG cell migration. Our study highlights a new action of anthocyanidins against EMT that supports their beneficial health and chemopreventive effects in dietary-based strategies against cancer. © 2016 Wiley Periodicals, Inc.
Assuntos
Antocianinas/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Success in developing therapeutic approaches to target brain tumor-associated inflammation in patients has been limited. Given that the inflammatory microenvironment is a hallmark signature of solid tumor development, anti-inflammatory targeting strategies have been envisioned as preventing glioblastoma initiation or progression. Consumption of foods from plant origin is associated with reduced risk of developing cancers, a chemopreventive effect that is, in part, attributed to their high content of phytochemicals with potent anti-inflammatory properties. We explored whether luteolin, a common flavonoid in many types of plants, may inhibit interleukin (IL)-1ß function induction of the inflammation biomarker cyclooxygenase (COX)-2. We found that IL-1ß triggered COX-2 expression in U-87 glioblastoma cells and synergized with luteolin to potentiate or inhibit that induction in a biphasic manner. Luteolin pretreatment of cells inhibited IL-1ß-mediated phosphorylation of inhibitor of κB, nuclear transcription factor-κB (NF-κB) p65, extracellular signal-regulated kinase-1/2, and c-Jun amino-terminal kinase in a concentration-dependent manner. Luteolin also inhibited AKT phosphorylation and survivin expression, while it triggered both caspase-3 cleavage and expression of glucose-regulated protein 78. These effects were all potentiated by IL-1ß, in part through increased nuclear translocation of NF-κB p65. Finally, luteolin was able to reduce IL-1 receptor gene expression, and treatment with IL-1 receptor antagonist or gene silencing of IL-1 receptor prevented IL-1ß/luteolin-induced COX-2 expression. Our results document a novel adaptive cellular response to luteolin, which triggers anti-survival and anti-inflammatory mechanisms that contribute to the chemopreventive properties of this diet-derived molecule.
Assuntos
Ciclo-Oxigenase 2/genética , Glioblastoma/enzimologia , Interleucina-1beta/farmacologia , Luteolina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores de Interleucina-1/fisiologia , Fator de Transcrição RelA/metabolismoRESUMO
Progression of astrocytic tumors is, in part, related to their dysregulated autophagy capacity. Recent evidence indicates that upstream autophagy signaling events can be triggered by MT1-MMP, a membrane-bound matrix metalloproteinase that contributes to the invasive phenotype of brain cancer cells. The signaling functions of MT1-MMP require its intracellular domain, and recent identification of MTCBP-1, a cytoplasmic 19 kDa protein involved in the inhibition of MT1-MMP-mediated cell migration, suggests that modulation of MT1-MMP cytoplasmic domain-mediated signaling may affect other carcinogenic processes. Using qPCR and screening of cDNA generated from brain tumor tissues of grades I, II, III, and IV, MT1-MMP gene expression was found to correlate with increased grade of tumors. Inversely, MTCBP-1 expression decreased with increasing grade of brain tumor. Confocal microscopy and fluorescence resonance energy transfer (FRET) analysis revealed that overexpressing a cytoplasmic-deleted MT1-MMP recombinant protein mutant prevented MTCBP-1 recruitment to the intracellular leaf of plasma membrane in U87 glioblastoma cells. The interaction between MTCBP-1 and the 20 amino acids peptide representing the MT1-MMP cytoplasmic domain was confirmed by surface plasmon resonance. Overexpression of a full-length Wt-MT1-MMP triggered acidic autophagy vesicle formation and autophagic puncta formation for green fluorescent microtubule-associated protein 1 light chain 3 (GFP-LC3). Autophagic vesicles and GFP-LC3 puncta formation were abrogated in the presence of MTCBP-1. Our data elucidate a new role for MTCBP-1 regulating the intracellular function of MT1-MMP-mediated autophagy. The inverse correlation between MTCBP-1 and MT1-MMP expression with brain tumor grades could also contribute to the decreased autophagic index observed in high-grade tumors.
Assuntos
Neoplasias Encefálicas/patologia , Dioxigenases/metabolismo , Glioblastoma/patologia , Metaloproteinase 14 da Matriz/metabolismo , Autofagia , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Dioxigenases/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/genética , Prognóstico , Estrutura Terciária de ProteínaRESUMO
RATIONALE: An isotopic labeling strategy based on derivatizing amine-containing metabolites has been developed using light ((12) C6 ) and heavy ((13) C6 ) N-benzoyloxysuccinimide reagents for semi-targeted metabolomic applications. METHODS: Differentially labeled samples were combined and analyzed simultaneously by liquid chromatography/high-resolution tandem mass spectrometry (LC/HR-MS/MS) to compare relative amounts of amine-containing metabolites. The selectivity of the reaction was determined with model metabolites and was shown to also be applicable to thiol and phenol moieties. The potential for relative quantitation was evaluated in cell extracts and the method was then applied to quantify metabolic perturbations occurring in human cultured cells under normal vs. oxidative stress conditions. RESULTS: A total of 279 derivatized features were detected in HL60 cell extracts, 77 of which yielded significant concentration changes upon oxidative stress treatment. Based on accurate mass measurements and MS/MS spectral matching with reference standard solutions, 10 metabolites were clearly identified. Derivatized compounds were found to have diagnostic fragment ions from the reagent itself, as well as structurally informative ions useful for metabolite identification. CONCLUSIONS: This simple derivatization reaction can be applied to the relative quantitation of amine-, thiol- and phenol-containing compounds, with improved sensitivity and chromatographic peak shapes due to the increased hydrophobicity of polar metabolites not readily amenable to reversed-phase LC/MS analysis.
Assuntos
Cromatografia Líquida/métodos , Metabolômica/métodos , Succinimidas/química , Espectrometria de Massas em Tandem/métodos , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Células HL-60 , Humanos , Marcação por IsótopoRESUMO
BACKGROUND: CSF-2 and CSF-3 confer proangiogenic and immunomodulatory properties to mesenchymal stromal cells (MSCs). RESULTS: Transcriptional regulation of CSF-2 and CSF-3 in concanavalin A-activated MSCs requires MT1-MMP signaling and is inhibited by EGCG. CONCLUSION: The chemopreventive properties of diet-derived EGCG alter MT1-MMP-mediated intracellular signaling. SIGNIFICANCE: Pharmacological targeting of MSCs proangiogenic functions may prevent their contribution to tumor development. Epigallocatechin gallate (EGCG), a major form of tea catechins, possesses immunomodulatory and antiangiogenic effects, both of which contribute to its chemopreventive properties. In this study, we evaluated the impact of EGCG treatment on the expression of colony-stimulating factors (CSF) secreted from human bone marrow-derived mesenchymal stromal cells (MSCs), all of which also contribute to the immunomodulatory and angiogenic properties of these cells. MSCs were activated with concanavalin A (ConA), a Toll-like receptor (TLR)-2 and TLR-6 agonist as well as a membrane type 1 matrix metalloproteinase (MT1-MMP) inducer, which increased granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP gene expression. EGCG antagonized the ConA-induced CSF-2 and CSF-3 gene expression, and this process required an MT1-MMP-mediated sequential activation of the Src and JAK/STAT pathways. Gene silencing of MT1-MMP expression further demonstrated its requirement in the phosphorylation of Src and STAT3, whereas overexpression of a nonphosphorylatable MT1-MMP mutant (Y573F) abrogated CSF-2 and CSF-3 transcriptional increases. Given that MSCs are recruited within vascularizing tumors and are believed to contribute to tumor angiogenesis, possibly through secretion of CSF-2 and CSF-3, our study suggests that diet-derived polyphenols such as EGCG may exert chemopreventive action through pharmacological targeting of the MT1-MMP intracellular signaling.
Assuntos
Inibidores da Angiogênese/farmacologia , Catequina/análogos & derivados , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Metaloproteinase 14 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transcrição Gênica , Catequina/farmacologia , Células Cultivadas , Concanavalina A/farmacologia , Inativação Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Janus Quinases/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Metaloproteinase 14 da Matriz/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Fosforilação , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/agonistas , Receptor 6 Toll-Like/genética , Receptor 6 Toll-Like/metabolismo , Quinases da Família src/metabolismoRESUMO
In the original publication [...].
RESUMO
The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10 melanoma syngeneic tumor model demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes.
Assuntos
Antígeno B7-H1 , Nucleotidiltransferases , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Morte CelularRESUMO
[This corrects the article DOI: 10.3389/fimmu.2024.1355945.].
RESUMO
Human bone marrow-derived mesenchymal stromal cells (MSCs) express Toll-like receptors (TLRs) and produce cytokines and chemokines, all of which contribute to these cells' immunomodulatory and proangiogenic properties. Among the secreted cytokines, colony-stimulating factors (CSFs) regulate angiogenesis through activation of endothelial cell proliferation and migration. Since MSC are recruited within hypoxic tumors where they signal paracrine-regulated angiogenesis, the aim of this study was to evaluate which CSF members are expressed and are inducible in activated MSC. Furthermore, we investigated the JAK/STAT signal transducing pathway that may impact on CSF transcription. MSC were activated with Concanavalin-A (ConA), a TLR-2/6 agonist as well as a membrane type-1 matrix metalloproteinase (MT1-MMP) inducer, and we found increased transcription of granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP. Gene silencing of either STAT3 or MT1-MMP prevented ConA-induced phosphorylation of STAT3, and reversed ConA effects on CSF-2 and CSF-3. Treatment with the Janus Kinase (JAK)2 inhibitor AG490 antagonized the ConA induction of MT1-MMP and CSF-2, while the pan-JAK inhibitor Tofacitinib reversed ConA-induced CSF-2 and -3 gene expression. Silencing of JAK2 prevented the ConA-mediated increase of CSF-2, while silencing of JAK1, JAK3 and TYK2 prevented the increase in CSF-3. Given that combined TLR-activation and locally-produced CSF-2 and CSF-3 could regulate immunomodulation and neovascularization, pharmacological targeting of TLR-2/6-induced MT1-MMP/JAK/STAT3 signalling pathway may prevent MSC contribution to tumor development.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Janus Quinases/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Transcrição STAT3/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Concanavalina A , Inibidores Enzimáticos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Fator Estimulador de Colônias de Macrófagos/genética , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Neovascularização Fisiológica , Fosforilação , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição STAT3/genética , Transdução de Sinais , TYK2 Quinase/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Transcrição Gênica , Tirfostinas/farmacologiaRESUMO
Pharmacological targeting of inflammation through STAT3 and NF-κB signaling pathways is, among other inflammatory biomarkers, associated with cyclooxygenase (COX)-2 inhibition and is believed to play a crucial role in prevention and therapy of cancer. Recently, inflammatory factors were found to impact on mesenchymal stromal cells (MSC) contribution to tumor angiogenesis. Given MSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in transducing NF-κB intracellular signaling pathways, we tested whether STAT3 regulation by MT1-MMP may also contribute to the expression balance of COX-2 in MSC. We demonstrate that STAT3 phosphorylation was triggered in MSC treated with the MT1-MMP inducer lectin Concanavalin-A (ConA), and that this phosphorylation was abrogated by the JAK2 inhibitor AG490. MT1-MMP gene silencing significantly inhibited ConA-induced STAT3 phosphorylation and this was correlated with reduced proMMP-2 activation and COX-2 expression. On the other hand, STAT3 gene silencing potentiated ConA-induced COX-2 expression, providing evidence for a new MT1-MMP/JAK/STAT3 signaling axis that may, in part, explain how MT1-MMP contributes to proinflammatory intracellular signaling. Given that MSC are avidly recruited within inflammatory microenvironments and within experimental vascularizing tumors, these mechanistic observations support a possible dual control of cell adaptation to inflammation by MT1-MMP and that may enable MSC to be active participants within inflamed tissues.