RESUMO
ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Luz , Retina/efeitos da radiação , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/genética , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Apirase/genética , Apirase/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Retina/fisiologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismoRESUMO
BACKGROUND: Many malignant tumours have increased TSPO expression, which has been related to a poor prognosis. TSPO-PET tracers have not comprehensively been evaluated in peripherally located tumours. This study aimed to evaluate whether N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([18F]F-DPA) can reflect radiotherapy (RT)-induced changes in TSPO activity in head and neck squamous cell carcinoma (HNSCC). METHODS: RT was used to induce inflammatory responses in HNSCC xenografts and cells. [18F]F-DPA uptake was measured in vivo in non-irradiated and irradiated tumours, followed by ex vivo biodistribution, autoradiography, and radiometabolite analysis. In vitro studies were performed in parental and TSPO-silenced (TSPO siRNA) cells. TSPO protein and mRNA expression, as well as tumour-associated macrophages (TAMs), were also assessed. RESULTS: In vivo imaging and ex vivo measurement revealed significantly higher [18F]F-DPA uptake in irradiated, compared to non-irradiated tumours. In vitro labelling studies with cells confirmed this finding, whereas no effect of RT on [18F]F-DPA uptake was detected in TSPO siRNA cells. Radiometabolite analysis showed that the amount of unchanged [18F]F-DPA in tumours was 95%, also after irradiation. PK11195 pre-treatment reduced the tumour-to-blood ratio of [18F]F-DPA by 73% in xenografts and by 88% in cells. TSPO protein and mRNA levels increased after RT, but were highly variable. The proportion of M1/M2 TAMs decreased after RT, whereas the proportion of monocytes and migratory monocytes/macrophages increased. CONCLUSIONS: [18F]F-DPA can detect changes in TSPO expression levels after RT in HNSCC, which does not seem to reflect inflammation. Further studies are however needed to clarify the physiological mechanisms regulated by TSPO after RT.
Assuntos
Radioisótopos de Flúor , Neoplasias de Cabeça e Pescoço , Animais , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/radioterapia , Tomografia por Emissão de Pósitrons , Pirazóis , Pirimidinas , Distribuição TecidualRESUMO
Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate used for the treatment of bone diseases and calcium metabolism. Anticancer activity of ZOL has been established, but its extraskeletal effects are limited due to its rapid uptake and accumulation to bone hydroxyapatite. In this work, we report on the development of tethered lipid bilayer-gated mesoporous silica nanocarriers (MSNs) for the incorporation, retention, and intracellular delivery of ZOL. The in vitro anticancer activity of ZOL-loaded nanocarriers was evaluated by cell viability assay and live-cell imaging. For in vivo delivery, the nanocarriers were tagged with folic acid to boost the affinity for breast cancer cells. Histological examination of the liver revealed no adverse off-target effects stemming from the nanocarriers. Importantly, nonspecific accumulation of ZOL within bone was not observed, which indicated in vivo stability of the tethered lipid bilayers. Further, the intravenously administered ZOL-loaded nanocarriers showed tumor growth suppression in breast cancer xenograft-bearing mice.
Assuntos
Difosfonatos/administração & dosagem , Difosfonatos/química , Imidazóis/administração & dosagem , Imidazóis/química , Bicamadas Lipídicas/química , Nanopartículas/química , Dióxido de Silício/química , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Porosidade , Ácido ZoledrônicoRESUMO
Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Invasividade Neoplásica/genética , Telômero/genética , Receptor Toll-Like 9/genética , Linhagem Celular Tumoral , Fragmentação do DNA , DNA de Neoplasias/genética , Feminino , Quadruplex G , Humanos , Ligantes , Invasividade Neoplásica/patologia , RNA Mensageiro/genética , CatelicidinasRESUMO
INTRODUCTION: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. METHODS: Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). RESULTS: Clinically, we found that high numbers of CD163(+) M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. CONCLUSIONS: This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.
Assuntos
Neoplasias da Mama/patologia , Leucócitos Mononucleares/patologia , Macrófagos/patologia , Neoplasias da Mama/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Citocinas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple-negative, human MDA-MB-231 breast cancer cells stably expressing control, or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , DNA/metabolismo , Inflamação/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Inflamação/genética , Inflamação/imunologia , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Interferência de RNA , Receptor Toll-Like 9/genética , Carga TumoralRESUMO
Introduction: Bisphosphonates (BPs) are bone-protecting osteoclast inhibitors, typically used in the treatment of osteoporosis and skeletal complications of malignancies. When given in the adjuvant setting, these drugs may also prevent relapses and prolong overall survival in early breast cancer (EBC), specifically among postmenopausal patients. Because of these findings, adjuvant nitrogen-containing BPs (N-BPs), such as zoledronate (ZOL), are now the standard of care for high-risk EBC patients, but there are no benefit-associated biomarkers, and the efficacy remains low. BPs have been demonstrated to possess anti-tumor activities, but the mechanisms by which they provide the beneficial effects in EBC are not known. Methods: We used stably transfected 4T1 breast cancer cells together with suppression of CD73 (sh-CD73) or control cells (sh-NT). We compared ZOL effects on tumor growth and infiltrating lymphocytes (TILs) into tumors and lung metastases using two mouse models. B cell depletion was performed using anti-CD20 antibody. Results: Sh-CD73 4T1 cells were significantly more sensitive to the growth inhibitory effects of n-BPs in vitro. However, while ZOL-induced growth inhibition was similar between the tumor groups in vivo, ZOL enhanced B and T lymphocyte infiltration into the orthotopic tumors with down-regulated CD73. A similar trend was detected in lung metastases. ZOL-induced tumor growth inhibition was found to be augmented with B cell depletion in sh-NT tumors, but not in sh-CD73 tumors. As an internal control, ZOL effects on bone were similar in mice bearing both tumor groups. Discussion: Taken together, these results indicate that ZOL modifies TILs in breast cancer, both in primary tumors and metastases. Our results further demonstrate that B cells may counteract the growth inhibitory effects of ZOL. However, all ZOL-induced TIL effects may be influenced by immunomodulatory characteristics of the tumor.
Assuntos
Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Ácido Zoledrônico/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Linfócitos TRESUMO
Toll-like receptor 9 (TLR9) is a cellular DNA-receptor, which is widely expressed in cancer. Synthetic TLR9-ligands induce cancer cell invasion in vitro, but the role of TLR9 in cancer pathophysiology remains unclear. Increased TLR9 expression has been, however, detected in estrogen receptor negative (ER-) breast cancers. In this study, we investigated the effects of ERα expression and sex steroid hormones on TLR9 expression in human ER+ (MCF-7, T47-D) and ER- (MDA-MB-231) breast cancer cell lines in vitro. We also studied TLR9 mRNA expression in archival breast cancer specimens (n = 12) with qRT-PCR, using primer sets that detect only the TLR9A isoform or the isoforms A and B (TLR9A/B). The TLR9 mRNA expression was detected in 10/12 specimens with both primer sets, and in 1/12 with only the TLR9A or the TLR9A/B primer sets. The basal TLR9 mRNA expression levels were significantly lower in the ER+ cell lines as compared with the ER- MDA-MB-231 cells. The transfection of ERα cDNA into MDA-MB-231 cells also resulted in down-regulation of TLR9 expression. While sex steroids had no effect on TLR9 expression in MCF-7 cells, testosterone (10(-8) M) induced TLR9 expression in MDA-MB-231 and T47-D cells. Although bicalutamide blocked this testosterone effect in MDA-MB-231 cells, in T47-D cells bicalutamide increased TLR9 expression and only partially blocked the testosterone effects. Estradiol (10(-8) M) induced TLR9 expression in T47-D cells. The invasive effects of synthetic TLR9-ligands were augmented by testosterone in vitro. This effect was lost in TLR9 siRNA MDA-MB-231 cells and also decreased by over-expression of ERα, which also inhibited NF-κB activation by TLR9-ligands. In conclusion, expression of TLR9 isoforms A and B can be detected in clinical breast cancer specimens. The ERα and sex steroid hormones regulate TLR9 expression and invasive effects in the breast cancer cells. Also, the commonly used hormonal cancer therapy bicalutamide affects TLR9 expression.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Receptor alfa de Estrogênio/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Receptor Toll-Like 9/metabolismo , Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Nitrilas/farmacologia , Isoformas de Proteínas , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Testosterona/análogos & derivados , Testosterona/metabolismo , Receptor Toll-Like 9/genética , Compostos de Tosil/farmacologia , TransfecçãoRESUMO
Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.
Assuntos
Neoplasias da Mama/metabolismo , Expressão Gênica , Receptor Toll-Like 9/metabolismo , Animais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estimativa de Kaplan-Meier , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz Secretadas/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Receptor Toll-Like 9/genética , Carga Tumoral , Regulação para CimaRESUMO
We recently reported N4-substituted 3-methylcytidine-5'-α,ß-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by â¼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Nucleotídeos de Citosina/farmacologia , Difosfonatos/farmacologia , Inibidores Enzimáticos/farmacologia , 5'-Nucleotidase/metabolismo , Adulto , Nucleotídeos de Citosina/síntese química , Nucleotídeos de Citosina/metabolismo , Difosfonatos/síntese química , Difosfonatos/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Neoplasias/enzimologia , Tonsila Palatina/enzimologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Astrocytes and microglia are able to degrade potentially neurotoxic ß-amyloid (Aß) deposits typical for Alzheimer's disease (AD) pathology. Contrary to microglia, astrocytes degrade human Aß from tissue sections in vitro without any additional stimulation, but it has remained unclear whether transplanted astrocytes are able to clear deposited human Aß in vivo. We transplanted adult mouse astrocytes into the hippocampi of transgenic mice mimicking AD and observed their fate, effects on microglial responses, and Aß clearance. After 2-months follow-up time, we discovered a significant reduction in Aß burden compared with AD mice infused with PBS only. The remaining Aß deposits were fragmented and most of the Aß immunoreactivity was seen within the transplanted astrocytes. Concomitant to Aß reduction, both CD68 and CD45 immunoreactivities were significantly upregulated but phagocytic microglia were often surrounding and engulfing Aß burdened, TUNEL-positive astrocytes rather than co-localizing with Aß alone. Astrocytes are known to degrade Aß also by secreting proteases involved in Aß catabolism. To study the contribution of neprilysin (NEP), angiotensin-converting enzyme-1 (ACE-1), and endothelin-converting enzyme-2 (ECE-2) in human Aß clearance, we utilized an ex vivo assay to demonstrate that adult astrocytes respond to human Aß by upregulating NEP expression. Further, incubation of adult astrocytes with known inhibitors of NEP, ACE-1, or ECE-2 significantly inhibited the removal of human Aß from the tissue suggesting an important role for these proteases in Aß clearance by adult astrocytes ex vivo.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Astrócitos/transplante , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Apoptose/fisiologia , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA/metabolismo , Enzimas Conversoras de Endotelina , Feminino , Gliose/patologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Antígenos Comuns de Leucócito/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Microglia/metabolismo , Neprilisina/metabolismo , Peptidil Dipeptidase A/metabolismo , Inibidores de Proteases/farmacologiaRESUMO
CD73 is a cell surface ecto-5'-nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5'-(α, ß-methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial-mesenchymal transition.
Assuntos
5'-Nucleotidase/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/enzimologia , Neoplasias Mamárias Animais/enzimologia , Proteínas de Neoplasias/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , 5'-Nucleotidase/genética , Animais , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.
RESUMO
BACKGROUND: Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members. RESULTS: We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. CONCLUSIONS: Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Humanos , Camundongos , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genéticaRESUMO
BACKGROUND: Prostate tumours are commonly poorly oxygenated which is associated with tumour progression and development of resistance to chemotherapeutic drugs and radiotherapy. Fibroblast growth factor 8b (FGF8b) is a mitogenic and angiogenic factor, which is expressed at an increased level in human prostate tumours and is associated with a poor prognosis. We studied the effect of FGF8b on tumour oxygenation and growth parameters in xenografts in comparison with vascular endothelial growth factor (VEGF)-expressing xenografts, representing another fast growing and angiogenic tumour model. METHODS: Subcutaneous tumours of PC-3 cells transfected with FGF8b, VEGF or empty (mock) vectors were produced and studied for vascularity, cell proliferation, glucose metabolism and oxygenation. Tumours were evaluated by immunohistochemistry (IHC), flow cytometry, use of radiolabelled markers of energy metabolism ([18F]FDG) and hypoxia ([18F]EF5), and intratumoral polarographic measurements of pO2. RESULTS: Both FGF8b and VEGF tumours grew rapidly in nude mice and showed highly vascularised morphology. Perfusion studies, pO2 measurements, [18F]EF5 and [18F]FDG uptake as well as IHC staining for glucose transport protein (GLUT1) and hypoxia inducible factor (HIF) 1 showed that VEGF xenografts were well-perfused and oxygenised, as expected, whereas FGF8b tumours were as hypoxic as mock tumours. These results suggest that FGF8b-induced tumour capillaries are defective. Nevertheless, the growth rate of hypoxic FGF8b tumours was highly increased, as that of well-oxygenised VEGF tumours, when compared with hypoxic mock tumour controls. CONCLUSION: FGF8b is able to induce fast growth in strongly hypoxic tumour microenvironment whereas VEGF-stimulated growth advantage is associated with improved perfusion and oxygenation of prostate tumour xenografts.
Assuntos
Fator 8 de Crescimento de Fibroblasto/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/metabolismo , Animais , Humanos , Hipóxia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Oxigênio/química , Oxigênio/metabolismo , Prognóstico , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC-3 prostate cancer cells. This was done by using FTI-277, GGTI-298 or NE-10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)-I and -II, respectively. Treatment of PC-3 cells with GGTI-298 and FTI-277 inhibited migration and invasion in a time- and dose-dependent manner. This was associated with disruption of F-actin organization and decreased recovery of GFP-actin. Immunoblot analysis of various cytoskeleton-associated proteins showed that the most striking change in GGTI-298- and FTI-277-treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC-3 cells with GGTI-298 also affected the dynamics of GFP-paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p-21-associated kinase)-2 were also lowered by GGTI-298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI-298 had a minor effect on the activity of MMP-9. RNAi knockdown of GGTase-Ibeta inhibited invasion, disrupted F-actin organization and decreased the level of cofilin in PC-3 cells. NE-10790 did not have any effect on PC-3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase-I- and FTase-catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Alquil e Aril Transferases/antagonistas & inibidores , Neoplasias da Próstata/enzimologia , Citoesqueleto de Actina/ultraestrutura , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Alquil e Aril Transferases/genética , Benzamidas/farmacologia , Difosfonatos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Masculino , Metionina/análogos & derivados , Metionina/farmacologia , Paxilina/metabolismo , Fosforilação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/ultraestrutura , Prenilação de Proteína/efeitos dos fármacos , Piridinas/farmacologia , Interferência de RNA , Células Tumorais Cultivadas , Quinases Ativadas por p21/metabolismoRESUMO
Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.
RESUMO
Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.
RESUMO
Clear signaling roles for ATP and adenosine have been established in all tissues, including the eye. The magnitude of signaling responses is governed by networks of enzymes; however, little is known about the regulatory mechanisms of purinergic signaling in the eye. By employing thin-layer chromatographic assays with 3H-labeled substrates, this study aimed to evaluate the role of nucleotide homeostasis in the pathogenesis of vitreoretinal diseases in humans. We have identified soluble enzymes ecto-5'-nucleotidase/CD73, adenylate kinase-1, and nucleoside diphosphate kinase in the vitreous fluid that control active cycling between pro-inflammatory ATP and anti-inflammatory adenosine. Strikingly, patients with proliferative form of diabetic retinopathy (DR) had higher adenylate kinase activity and ATP concentration, when compared to non-proliferative DR eyes and non-diabetic controls operated for rhegmatogenous retinal detachment, macular hole, and pucker. The non-parametric correlation analysis revealed positive correlations between intravitreal adenylate kinase and concentrations of ATP, ADP, and other angiogenic (angiopoietins-1 and -2), profibrotic (transforming growth factor-ß1), and proteolytic (matrix metalloproteinase-9) factors but not erythropoietin and VEGF. Immunohistochemical staining of postmortem human retina additionally revealed selective expression of ecto-5'-nucleotidase/CD73 on the rod-and-cone-containing photoreceptor cells. Collectively, these findings provide novel insights into the regulatory mechanisms that influence purinergic signaling in diseased eye and open up new possibilities in the development of enzyme-targeted therapeutic approaches for prevention and treatment of DR. KEY MESSAGE: Ecto-5'-nucleotidase/CD73 and adenylate kinase-1 circulate in human vitreous fluid. Adenylate kinase activity is high in diabetic eyes with proliferative retinopathy. Diabetic eyes display higher intravitreal ATP/ADP ratio than non-diabetic controls. Soluble adenylate kinase maintains resynthesis of inflammatory ATP in diabetic eyes.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Retinopatia Diabética/metabolismo , Retina/metabolismo , 5'-Nucleotidase/metabolismo , Adenilato Quinase/metabolismo , Adulto , Idoso , Cromatografia em Camada Fina , Eritropoetina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Núcleosídeo-Difosfato Quinase/metabolismo , Estatísticas não Paramétricas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/enzimologiaRESUMO
BACKGROUND: The pim family genes encode oncogenic serine/threonine kinases which in hematopoietic cells have been implicated in cytokine-dependent signaling as well as in lymphomagenesis, especially in cooperation with other oncogenes such as myc, bcl-2 or Runx family genes. The Runx genes encode alpha-subunits of heterodimeric transcription factors which regulate cell proliferation and differentiation in various tissues during development and which can become leukemogenic upon aberrant expression. RESULTS: Here we have identified novel protein-protein interactions between the Pim-1 kinase and the RUNX family transcription factors. Using the yeast two-hybrid system, we were able to show that the C-terminal part of human RUNX3 associates with Pim-1. This result was confirmed in cell culture, where full-length murine Runx1 and Runx3 both coprecipitated and colocalized with Pim-1. Furthermore, catalytically active Pim-1 kinase was able to phosphorylate Runx1 and Runx3 proteins and enhance the transactivation activity of Runx1 in a dose-dependent fashion. CONCLUSION: Altogether, our results suggest that mammalian RUNX family transcription factors are novel binding partners and substrates for the Pim-1 kinase, which may be able to regulate their activities during normal hematopoiesis as well as in leukemogenesis.