RESUMO
Bone metastasis occurs frequently in cancer patients. Conventional therapies have limited therapeutic outcomes, and thus, exploring the mechanisms of cancer progression in bone metastasis is important to develop new effective therapies. In the bone microenvironment, adipocytes are the major stromal cells that interact with cancer cells during bone metastasis. However, the comprehensive functions of bone marrow adipocytes in cancer progression are not yet fully understood. To address this, we investigated the role of bone marrow adipocytes on cancer cells, by focusing on an invasive front that reflects the direct effects of stromal cells on cancer. In comprehensive histopathological and genetic analysis using bone metastasis specimens, we examined invasive fronts in bone metastasis and compared invasive fronts with adipocyte-rich bone marrow (adipo-BM) to those with hematopoietic cell-rich bone marrow (hemato-BM) as a normal counterpart of adipocytes. We found morphological complexity of the invasive front with adipo-BM was significantly higher than that with hemato-BM. Based on immunohistochemistry, the invasive front with adipo-BM comparatively had a significantly increased cancer-associated fibroblast (CAF) marker-positive area and lower density of CD8+ lymphocytes compared to that with hemato-BM. RNA sequencing analysis of primary and bone metastasis cancer revealed that bone metastasized cancer cells acquired drug resistance-related gene expression phenotypes. Clearly, these findings indicate that bone marrow adipocytes provide a favorable tumor microenvironment for cancer invasion and therapeutic resistance of bone metastasized cancers through CAF induction and immune evasion, providing a potential target for the treatment of bone metastasis.
Assuntos
Neoplasias Ósseas , Fibroblastos Associados a Câncer , Humanos , Medula Óssea/metabolismo , Evasão da Resposta Imune , Células Estromais , Células da Medula Óssea/metabolismo , Neoplasias Ósseas/patologia , Adipócitos/patologia , Microambiente TumoralRESUMO
Cancer tissues generally have molecular oxygen and serum component deficiencies because of poor vascularization. Recently, we revealed that ICAM1 is strongly activated through lipophagy in ovarian clear cell carcinoma (CCC) cells in response to starvation of long-chain fatty acids and oxygen and confers resistance to apoptosis caused by these harsh conditions. CD69 is a glycoprotein that is synthesized in immune cells and is associated with their activation through cellular signaling pathways. However, the expression and function of CD69 in nonhematological cells is unclear. Here, we report that CD69 is induced in CCC cells as in ICAM1. Mass spectrometry analysis of phosphorylated peptides followed by pathway analysis revealed that CD69 augments CCC cell binding to fibronectin (FN) in association with the phosphorylation of multiple cellular signaling molecules including the focal adhesion pathway. Furthermore, CD69 synthesized in CCC cells could facilitate cell survival because the CD69-FN axis can induce epithelial-mesenchymal transition. Experiments with surgically removed tumor samples revealed that CD69 is predominantly expressed in CCC tumor cells compared with other histological subtypes of epithelial ovarian cancer. Overall, our data suggest that cancer cell-derived CD69 can contribute to CCC progression through FN.
Assuntos
Fibronectinas , Neoplasias Ovarianas , Humanos , Feminino , Oxigênio , Neoplasias Ovarianas/patologia , Transdução de Sinais , Lipídeos , Linhagem Celular TumoralRESUMO
BACKGROUND: Serum starvation and hypoxia (SSH) mimics a stress condition in tumours. We have shown that intercellular adhesion molecule-1 (ICAM-1) protein is synergistically expressed in ovarian clear cell carcinoma (CCC) cells under SSH in response to an insufficient supply of fatty acids (FAs). This ICAM-1 expression is responsible for resistance against the lethal condition, thereby promoting tumour growth. However, the underlying mechanisms that link SSH-driven ICAM1 gene expression to impaired FA supply and its clinical relevance are unclear. METHODS: The underlying mechanisms of how FA deficiency induces ICAM-1 expression in cooperation with hypoxia were analysed in vitro and in vivo. Clinical significance of CCC cell-derived ICAM-1 and the mechanism associated with the transcriptional synergism were also investigated. RESULTS: ICAM-1 expression was mediated through lipophagy-driven lipid droplet degradation, followed by impaired FA-lipid droplet flow. Lipophagy induced ICAM1 expression through stabilisation of NFκB binding to the promoter region via Sam68 and hTERT. Analyses of clinical specimens revealed that expression of ICAM-1 and LC3B, an autophagy marker associated with lipophagy, significantly correlated with poor prognoses of CCC. CONCLUSIONS: The lipophagy-ICAM-1 pathway induced under a tumour-like stress conditions contributes to CCC progression and is a potential therapeutic target for this aggressive cancer type.
Assuntos
Adenocarcinoma de Células Claras , Molécula 1 de Adesão Intercelular , Neoplasias Ovarianas , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/metabolismo , Autofagia/genética , Ácidos Graxos/metabolismo , Feminino , Humanos , Hipóxia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , PrognósticoRESUMO
Exosomes are small endosome-derived vesicles containing a wide range of functional proteins, mRNA, and miRNA. Exosomal miRNA from cancer cells helps modulate the microenvironment. In multiple myeloma (MM), the massive proliferation of malignant plasma cells causes hypoxia. To date, the majority of in vitro hypoxia studies of cancer cells have used acute hypoxic exposure (3-24 hours). Thus, we attempted to clarify the role of MM-derived exosomes in hypoxic bone marrow by using MM cells grown continuously in vitro under chronic hypoxia (hypoxia-resistant MM [HR-MM] cells). The HR-MM cells produced more exosomes than the parental cells under normoxia or acute hypoxia conditions, and miR-135b was significantly upregulated in exosomes from HR-MM cells. Exosomal miR-135b directly suppressed its target factor-inhibiting hypoxia-inducible factor 1 (FIH-1) in endothelial cells. Finally, exosomal miR-135b from HR-MM cells enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway. This in vitro HR myeloma cell model will be useful for investigating MM cell-endothelial cell interactions under hypoxic conditions, which may mimic the in vivo bone marrow microenvironment. Although tumor angiogenesis is regulated by various factors, exosomal miR-135b may be a target for controlling MM angiogenesis.
Assuntos
Exossomos/genética , MicroRNAs/genética , Oxigenases de Função Mista/genética , Neovascularização Patológica/genética , Proteínas Repressoras/genética , Regiões 3' não Traduzidas/genética , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , Células Cultivadas , Exossomos/ultraestrutura , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Microscopia Eletrônica de Transmissão , Oxigenases de Função Mista/metabolismo , Mieloma Múltiplo/irrigação sanguínea , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/metabolismoRESUMO
Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.
Assuntos
Hipóxia Celular , Células Endoteliais/metabolismo , Exossomos/metabolismo , Leucemia/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Células K562 , Leucemia/patologia , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Extracellular vesicles (EVs) in the tumor microenvironment facilitate intercellular communication. Cancer cell-derived EVs act as an immunosuppressor by transporting cargos and presenting transmembrane proteins. By contrast, CD8+ cytotoxic T-lymphocytes (CTLs) exert anti-cancer cytotoxicity via the pore-forming protein perforin. Here, we hypothesize that although EVs are destroyed by perforin, cancer cell-derived EVs might possess mechanisms that enable them to avoid this destruction. We used a breast cancer cell line, MDA-MB-231-luc-D3H2LN (D3H2LN), to generate EVs. Destruction of the EVs by perforin was demonstrated visually using atomic force microscopy. To investigate immunosuppressive metabolites within cancer cell-derived EVs, we performed metabolomic profiling of EVs from D3H2LN cells cultured for 48 h with or without IFN-γ, which induces metabolic changes in the cells. We found that both types of EV from IFN-γ treated D3H2LN cells and non-treated D3H2LN cells contained adenosine, which has immunosuppressive effects. When we exposed cancer cell-derived EVs to CTLs, perforin secretion by CTLs fell significantly. In addition, the decreases in perforin secretion were ameliorated by treatment with adenosine deaminase, which degrades extracellular adenosine. Taken together, these results suggest that after perforin secreted by CTLs disrupts the membrane of EVs, adenosine released from the EVs acts as an immunosuppressive metabolite by binding to the adenosine receptor on the CTL membrane. This mechanism provides a novel survival strategy using cancer cell-derived EVs.
Assuntos
Adenosina/metabolismo , Vesículas Extracelulares/metabolismo , Perforina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Vesículas Extracelulares/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Perforina/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
AIMS: The effects of vitamin K (VK) derivatives VK3 and VK5 on human immune cells have not been extensively investigated. We examined the effects of VK3 and VK5 on proliferation, apoptosis, cytokine production, and CD4+CD25+Foxp3+ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. MAIN METHODS: Anti-proliferative effects of VK3 and VK5 on T-cell mitogen activated PBMCs were assessed by WST assay procedures. Apoptotic cells were determined as Annexin V positive/propidium iodide (PI) negative cells. Cytokine concentrations in the supernatant of the culture medium were measured with bead-array procedures followed by analysis with flow cytometry. The CD4+CD25+Foxp3+Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. KEY FINDINGS: VK3 and VK5 suppressed the mitogen-activated proliferation of PBMCs significantly at 10-100µM (p<0.05). The data also suggest that VK3 and VK5 promote apoptosis in the mitogen-activated T cells. VK3 and VK5 significantly inhibited the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 10-100µM (p<0.05). In contrast, VK3 and VK5 significantly increased Treg cell-frequency in the activated PBMCs at concentrations more than 10µM (p<0.001). SIGNIFICANCE: Our data suggest that VK3 and VK5 attenuate T cell mediated immunity by inhibiting the proliferative response and inducing apoptosis in activated T cells.
Assuntos
Citocinas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Vitamina K 3/análogos & derivados , Vitamina K 3/farmacologia , Adulto , Apoptose , Proliferação de Células/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/citologia , Masculino , Padrões de Referência , Linfócitos T Reguladores/citologiaRESUMO
The aim of this study was to assess the suitability of decellularized porcine aorta as a vascular graft material by measuring its permeability to protein. Aorta samples were decellularized by treatment with either high hydrostatic pressurization (HHP) or sodium dodecyl sulfate (SDS). Histological evaluation showed that the structure of an HHP-treated sample was similar to that of an untreated sample, while the structure of an SDS-treated sample was surfactant-damaged. A two-chamber diffusion system was used to measure permeability to lysozyme and bovine serum albumin. The lysozyme and bovine serum albumin mass transfer coefficients calculated for an SDS-treated sample were significantly larger than those calculated for an untreated sample, while the mass transfer coefficients for an HHP-treated sample were similar to those for an untreated sample. The mass transfer coefficients showed very good agreement with the tissue structure characterization, which means that differences in permeability reflected differences in tissue structure.
Assuntos
Aorta/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Aorta/citologia , Eletroforese em Gel de Poliacrilamida , Pressão Hidrostática , Microscopia Eletrônica de Varredura , Permeabilidade , SuínosRESUMO
OBJECTIVES: For patients with advanced melanoma, no treatment options are available at present that provide either sufficient response rates or a significant prolongation of overall survival. The present study examines the effects of two inorganic and six organic arsenic compounds on cell proliferation and cell invasion of melanoma cells in vitro. METHODS: The effects of arsenic compounds on proliferation of human melanoma A375 cells and murine melanoma B16F10 cells were examined by MTT assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and the effects of the compounds on cell invasion were examined by the Boyden chamber invasion assay. The amounts of active matrix metalloproteinase (MMP)-2 and pro-MMP-2 in the culture supernatant of A375 cells were determined by an MMP-2 activity assay system. KEY FINDINGS: Arsenate and arsenic trioxide (As(2) O(3) ) inhibited the proliferation of A375 and B16F10 cells significantly at concentration ranges of 0.1-20µg/ml (P<0.001), while the organic compounds arsenobetaine, arsenocholine, dimethylarsinic acid, methylarsonic acid, tetramethylarsonium and trimethylarsine oxide did not show any inhibitory effects even at 20µg/ml. Cell invasion of A375 and B16F10 cells through a layer of collagen IV was significantly inhibited by 0.1-20 µg/ml of arsenate or As(2) O(3) (P<0.05), while the organic compounds did not inhibit cell invasion. Arsenate or As(2) O(3) at 0.2-10µg/ml significantly inhibited the amount of active MMP-2 and pro-MMP-2 secreted into the A375 cell culture supernatant (P<0.05). CONCLUSIONS: Our findings show that the inorganic arsenic compounds arsenate and As(2) O(3) inhibit cell proliferation and prevent the invasive properties of melanoma cells, possibly by decreasing MMP-2 production from the cells.