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1.
Nat Commun ; 11(1): 2810, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499572

RESUMO

The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK.


Assuntos
Proteína Rica em Cisteína 61/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neovascularização Patológica , Pericitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Aorta Torácica/patologia , Carcinoma Pulmonar de Lewis/metabolismo , Adesão Celular , Proliferação de Células , Feminino , Quinase 1 de Adesão Focal/genética , Humanos , Linfocinas/metabolismo , Masculino , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/patologia , Fator de Crescimento Placentário/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Transdução de Sinais , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Biochem Biophys Res Commun ; 521(4): 947-951, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31718795

RESUMO

α-Tocopheryl succinate (TS) is a tocopherol derivative and has multifaceted anti-cancer effects; TS not only causes cancer cell-specific apoptosis but also inhibits tumor angiogenesis. Although TS has the potential to be used as a well-tolerated anti-angiogenic drug, it is still unclear which step of the angiogenic process is inhibited by TS. Here, we show that TS inhibits the expression of angiopoietin (Ang)-2, which induces destabilization of vascular structure in the initial steps of the angiogenic process. In mouse melanoma cells, TS treatment decreased mRNA and extracellular protein levels of Ang-2; however, the mRNA level of Ang-1, which stabilizes the vascular structure, remained unchanged. Furthermore, aorta ring and Matrigel plug angiogenesis assays indicated that the conditioned medium from TS-treated cells (CM-TS) inhibited neovascularization and blood leakage from the existing blood vessels, respectively. Following immunohistochemical staining of the vessels treated with CM-TS, imaging studies showed that the vascular endothelial cells were highly packed with pericytes. In conclusion, we found that TS inhibits Ang-2 expression and, consequently, stabilizes the vascular structure during the initial step of tumor angiogenesis.


Assuntos
Inibidores da Angiogênese/farmacologia , Angiopoietina-2/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , alfa-Tocoferol/farmacologia , Inibidores da Angiogênese/química , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Aorta/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Humanos , Masculino , Melanoma/irrigação sanguínea , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/patologia , Relação Estrutura-Atividade , alfa-Tocoferol/química
3.
Eur J Ophthalmol ; 30(4): 723-729, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31210061

RESUMO

PURPOSE: To describe the imaging features of choroidal nevus and melanoma using optical coherence tomography angiography, and evaluate the ability of this technique to establish the differential diagnosis based on the display of the tumor's intrinsic vasculature. METHODS: Comparative analysis of optical coherence tomography angiography findings in consecutive patients diagnosed with choroidal nevus or choroidal melanoma following a complete ophthalmic evaluation, including best-corrected visual acuity and several imaging studies: color fundus photography, B-scan ultrasound, spectral-domain optical coherence tomography, and optical coherence tomography angiography. Optical coherence tomography angiography was used to investigate qualitative differences in the tumor vasculature. RESULTS: Thirty-six eyes (18 cases of choroidal nevus and 18 cases of choroidal melanoma) from 36 consecutive patients were included in the study. Only cases located posterior to equator were included to enable performance of all tests. On optical coherence tomography angiography, choroidal nevus showed well-delimited margins (78%), hyperreflective choroid capillary vasculature (83%), fewer avascular areas (17%), and neovascular membrane in one case (6%). Choroidal melanoma showed imprecise margins (72%), hyporeflective choroidal capillary vasculature (72%), multiple avascular areas (78%), and choroidal vascular changes (e.g. thick vascular networks or vascular loops; 45%). CONCLUSION: Optical coherence tomography angiography can provide useful information for assessing and differentiating between choroidal nevi and small melanomas. Significant differences between these conditions were found for the pattern of reflectivity, and presence/absence of avascular zones and vascular anomalies, which could be helpful for supporting the diagnosis.


Assuntos
Doenças da Coroide/diagnóstico , Neoplasias da Coroide/diagnóstico , Melanoma/diagnóstico , Nevo Pigmentado/diagnóstico , Adulto , Idoso , Neoplasias da Coroide/irrigação sanguínea , Diagnóstico Diferencial , Feminino , Angiofluoresceinografia/métodos , Humanos , Masculino , Melanoma/irrigação sanguínea , Pessoa de Meia-Idade , Nevo Pigmentado/irrigação sanguínea , Estudos Retrospectivos , Tomografia de Coerência Óptica/métodos
4.
Pol J Pathol ; 70(3): 217-222, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820866

RESUMO

The study was aimed to evaluate the number of TAMs and to investigate whether they have association with microvessels density and patients' survival times. 46 cases of melanomas, divided into four groups according to the Breslow scale, were tested immunohistochemically with antibodies anti-CD68, CD163, iNOS to vizualized macrophages and anti-CD34 antibody to stain microvessels. The number of macrophages and the microvessels density were counted by hotspot analysis using an image analysis system. The study revealed increased numbers of CD68 and CD163 positive macrophages in successive stages of Breslow scale, but statistically significant differences were observed only between I and IV group for CD68 positive macrophages, and between I and III, IV group for CD163 positive macrophages. The mean number of the microvessels was significantly increased in group II, III, IV compared to group I. The correlative study showed significant positive correlations between the mean number of CD68 and CD163 positive macrophages and microvessels density. Moreover, the number of CD163 positive macrophages was associated inversely with patient's survival time. The results of our study may indicate that higher infiltration of macrophages, especially CD163 positive cells, is associated with more advanced melanomas, microvessels density and worse patient's prognosis.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Macrófagos/citologia , Melanoma/patologia , Receptores de Superfície Celular/metabolismo , Neoplasias Cutâneas/patologia , Humanos , Macrófagos/metabolismo , Melanoma/irrigação sanguínea , Microvasos , Prognóstico , Neoplasias Cutâneas/irrigação sanguínea
5.
BMC Cancer ; 19(1): 1134, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752759

RESUMO

BACKGROUND: Vasculogenic mimicry (VM) a microvascular system consisting of non-endothelial cells that is newly formed by aggressive tumors, has been proposed as an important therapeutic target in malignant melanoma (MM). We performed a systematic literature review to evaluate the diagnostic and prognostic accuracy of VM status for overall survival of MM patients. METHODS: The quality of the included studies was evaluated using the QUADAS-2 tool. Diagnostic capacity of VM variables, including sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and the area under summary receiver operating characteristic (SROC), were pooled using Meta-DiSc software. RESULTS: A retrospective observational study was conducted based on twelve clinical studies including 978 clinically confirmed melanoma patients with proportion (P). VM+ melanoma cells were associated with poor prognosis in 38% of MM group (P = 0.35, 95% confidence intervals (CI): 0.27-0.42, p < 0.001). The pooled sensitivity and specificity were 0.82 (95% CI: 0.79-0.84) and 0.69 (95% CI: 0.66-0.71), respectively. Furthermore, the pooled PLR, NLR, and DOR were 2.56 (95% CI: 1.94-3.93), 0.17 (95% CI: 0.07-0.42), and 17.75 (95% CI: 5.30-59.44), respectively. Furthermore, the AUC of SROC was 0.63, indicating high reliability of VM status as a biomarker. Importantly, subgroup results suggested that VM+ status is a significantly accurate prognostic biomarker when diagnosed by the CD31-/PAS+ staining methods in Asian MM samples (p < 0.001). CONCLUSIONS: Our findings support the potential of VM status of tumors as a promising prognostic biomarker and emphasize an effective adjuvant therapeutic strategy in the prognosis of Asian MM patients.


Assuntos
Melanoma/irrigação sanguínea , Melanoma/diagnóstico , Neovascularização Patológica/diagnóstico , Feminino , Humanos , Masculino , Melanoma/patologia , Neovascularização Patológica/patologia , Estudos Observacionais como Assunto , Razão de Chances , Prognóstico , Curva ROC , Sensibilidade e Especificidade
6.
Eur J Pharmacol ; 864: 172719, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31586634

RESUMO

Gambogic acid (GA) is a potential anti-cancer compound that is extracted from the resin of Garciania hanburyi. The present study was designed to evaluate the anti-metastatic effect of GA on melanoma cell lines in vitro and to explore the underlying mechanism. The anti-proliferative activity of GA on melanoma cells was assessed by CCK-8 assay. The Wound-healing, transwell, adhesion, and tube formation assays were performed to examine the inhibition of GA on the cell's migration, invasion, adhesion, and angiogenesis capacities, respectively. Enzymatic activity of MMP-2 and MMP-9 were detected by gelatin zymography assay. Protein expressions regulated by GA treatment were tested by Western blot assay. The present results showed that GA significantly inhibited the proliferation of highly metastatic melanoma A375, B16-F10 cells and human umbilical vein endothelial cells (HUVECs) in time- and doses-dependent manners. Furthermore, GA significantly inhibited the migratory, invasive and adhesive properties of A375 and B16-F10 cells, and tube-forming potential of HUVECs at sub-IC50 concentrations, where no significant cytotoxicity was observed. Mechanistically, GA treatment suppressed the EMT and angiogenesis processes and reduced the enzymatic activity of MMP-2 and MMP-9. Moreover, abnormal PI3K/Akt and ERK signaling pathways in A375 and B16-F10 cells and HUVECs were notably suppressed by GA treatment. Collectively, our results suggest that GA exerts anti-metastasis activity in melanoma cells by suppressing the EMT and angiogenesis through the PI3K/Akt and ERK signaling pathways, and might be used as a phytomedicine against metastatic melanoma.


Assuntos
Antineoplásicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Xantonas/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/irrigação sanguínea , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neoplasias Cutâneas/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Xantonas/uso terapêutico
7.
Mol Vis ; 25: 502-516, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588174

RESUMO

Purpose: Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM). We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions. Methods: Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain. Retrospective data on gene expression class and patient survival were retrieved. Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy. Results: The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively. PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02). The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004). Forty percent of the tumors had heterogeneous BAP-1 expression. Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01). Conclusions: PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM. Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Macrófagos/patologia , Melanoma/irrigação sanguínea , Melanoma/genética , Neovascularização Patológica/genética , Reação do Ácido Periódico de Schiff , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Neoplasias Uveais/irrigação sanguínea , Neoplasias Uveais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Corioide/irrigação sanguínea , Corioide/patologia , Corioide/ultraestrutura , Intervalo Livre de Doença , Enucleação Ocular , Feminino , Humanos , Estimativa de Kaplan-Meier , Modelos Logísticos , Macrófagos/metabolismo , Masculino , Melanoma/patologia , Melanoma/ultraestrutura , Pessoa de Meia-Idade , Metástase Neoplásica , Neovascularização Patológica/patologia , Reconhecimento Automatizado de Padrão , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Neoplasias Uveais/patologia , Neoplasias Uveais/ultraestrutura , Adulto Jovem
8.
J Vis Exp ; (149)2019 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-31380841

RESUMO

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc.


Assuntos
Melanoma/irrigação sanguínea , Neovascularização Patológica/patologia , Neutrófilos/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Interferon Tipo I , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Transdução de Sinais
9.
J Cell Biochem ; 120(12): 19721-19729, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31270843

RESUMO

In this study, we used endostatin (ES)-induced apoptosis of endothelial cells to study the role of Hexokinase2 (HK2) in the control of angiogenesis in melanoma. Real-time polymerase chain reaction and Western blot analysis were performed to explore the effect of HK2, lactate, and ES on the levels of caspase-9/3, ATP, and p38/MAPK activation. ES increased the levels of caspase-9/3 while decreasing the level of ATP, whereas ES + HK2 and lactate both restored the normal levels of caspase-9/3 and ATP. In addition, cells transfected with HK2 short hairpin RNA1 (HK2shRNA1) and HK2shRNA2 showed an evident decrease in the levels of caspase-9/3 along with an obvious increase in the level of ATP. Knockdown of HK2 also increased O2 consumption while decreasing the extracellular level of lactate and the phosphorylation of p38-mitogen-activated protein kinase (MAPK). On the other hand, the lactate treatment elevated the phosphorylation of p38-MAPK under time- and concentration-dependent manner. In the study, we clarified the role of HK2 in the control of apoptosis of ECs, which plays an important role in the angiogenesis of melanoma by promoting aerobic glycolysis and activating the p38-MAPK signaling.


Assuntos
Células Endoteliais/metabolismo , Glicólise/fisiologia , Hexoquinase/metabolismo , Melanoma/irrigação sanguínea , Neovascularização Patológica/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Endostatinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Hexoquinase/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Cancer Immunol Immunother ; 68(8): 1287-1301, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31253998

RESUMO

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.


Assuntos
Vasos Sanguíneos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Movimento Celular , Citotoxicidade Imunológica , Humanos , Melanoma/irrigação sanguínea , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Neovascularização Patológica , Fosforilação Oxidativa , Perforina/metabolismo , Neoplasias Cutâneas/irrigação sanguínea
11.
Eur J Pharmacol ; 858: 172463, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31211986

RESUMO

Malignant melanoma is the leading cause of death from skin disease, due in large part to its propensity to metastasize. Scutellarin is an active flavone extracted from traditional Chinese herb Erigeron breviscapus (Vant.) Hand. Mazz. Recent studies have reported that scutellarin can be utilized to treat various types of tumors. In this study, we investigated the effects of scutellarin on melanoma cancer cell invasive potential and the molecular mechanisms underlying these effects using A375 melanoma cells lines. The in-vitro antitumor activity of scutellarin was evaluated by CCK-8 assay, wound-healing assay, transwell assays, adhesion assays, and tube formation assays to assess the cell viability, migration, invasion, adhesion, and angiogenesis, respectively. Also, western blotting assay was used to assess the level of PI3K/Akt/mTOR signaling pathway proteins in A375 cells. We found that scutellarin significantly inhibited melanoma cell lines and HUVECs viability in a time- and concentration-dependent manners. Additionally, scutellarin effectively suppressed tumor cell migration, invasion, adhesion through the suppression of EMT and angiogenesis by inhibiting the PI3K/Akt/mTOR signaling pathway. These results indicated that scutellarin could markedly inhibit the invasive potential of melanoma cell lines by suppressing the EMT and angiogenesis through the PI3K/Akt/mTOR signaling pathway. It suggests that scutellarin might be a potential compound in malignant melanoma treatment.


Assuntos
Apigenina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucuronatos/farmacologia , Melanoma/patologia , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Apigenina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glucuronatos/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/irrigação sanguínea , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Serina-Treonina Quinases TOR/metabolismo
12.
Cancer Lett ; 459: 268-276, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31128215

RESUMO

The incidence of melanoma is increasing faster than any other cancer. In recent years, treatment of melanoma and a range of other deadly cancers has involved immunotherapy with programmed cell death protein-1 (PD-1)/PD-1 ligand (PD-L1) checkpoint blockade which has improved survival. However, many patients do not respond or have partial response, survival benefit is in the order of months and all available PD-1/PD-L1 strategies are antibodies requiring intravenous infusion. There are no clinically approved small molecule pharmacologic inhibitors of the PD-1/PD-L1 system. The benzimidazole derivative flubendazole is a widely used anthelmintic available over the counter in Europe. Here we demonstrate the ability of flubendazole to inhibit human melanoma growth and spread in mice. Flubendazole's ability to block tumor growth and spread was comparable to paclitaxel. Anti-tumor effects were observed when flubendazole was delivered systemically not locally. Flubendazole inhibited CD31/PECAM-1 staining indicating suppression of tumor angiogenesis. Most surprisingly, flubendazole inhibited PD-1 levels within the tumors, but not PD-L1. Western blotting and flow cytometry revealed that flubendazole inhibits PD-1 expression in cultured melanoma cells. Flubendazole also reduced myeloid-derived suppressor cell (MDSC) levels in tumor tissue. Further we found that flubendazole inhibited active (phospho-Tyr705) signal transducer and activator of transcription (STAT3), an upstream regulator of PD-1 expression. These findings uncover that flubendazole is a novel small molecule inhibitor of not only melanoma growth and spread but also of PD-1 and MDSC.


Assuntos
Mebendazol/análogos & derivados , Melanoma/tratamento farmacológico , Células Supressoras Mieloides/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Antinematódeos/farmacologia , Antineoplásicos/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Mebendazol/farmacologia , Melanoma/irrigação sanguínea , Melanoma/patologia , Camundongos , Camundongos SCID , Células Supressoras Mieloides/patologia , Metástase Neoplásica , Neovascularização Patológica/tratamento farmacológico , Distribuição Aleatória , Fator de Transcrição STAT3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Sci Rep ; 9(1): 8052, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142788

RESUMO

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.


Assuntos
Biomarcadores Tumorais/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Neovascularização Patológica/genética , Neoplasias Cutâneas/genética , Apoptose/genética , Biomarcadores Tumorais/análise , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Cocultura , Biologia Computacional , Conjuntos de Dados como Assunto , Endoglina/análise , Endoglina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Melanócitos , Melanoma/irrigação sanguínea , Melanoma/patologia , Neovascularização Patológica/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Cultura Primária de Células , Domínios Proteicos/genética , Proteômica , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética
14.
Biochimie ; 162: 198-207, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075281

RESUMO

Malignant melanoma remains a challenge for clinical practice and novel therapeutic strategies are urgently needed. Herbacetin, a natural flavonoid compound that has multiple pharmacological activities, exerts anticancer effects on several human tumors. In this study, the anti-angiogenesis effect of Herbacetin in human malignant melanoma was investigated. The results indicated that Herbacetin treatment significantly suppressed tumor growth and angiogenesis of malignant melanoma both in vitro and in vivo. In melanoma A375 and Hs294T cells, Herbacetin treatment suppressed both EGF-induced and constitutive phosphorylation of EGFR, accelerated the internalization and degradation of EGFR, and subsequently suppressed the activation of the downstream kinases (AKT and ERK). Moreover, MMP9 was determined as a key angiogenic factor in Herbacetin treated melanoma cells. Knockdown of MMP9 suppressed the in vitro angiogenesis while overexpression of MMP9 in Herbacetin treated melanoma cells restored the angiogenesis ability. We concluded that Herbacetin suppressed melanoma angiogenesis through blocking EGFR-ERK/AKT-MMP9 signaling pathway and Herbacetin may be developed as a potential drug for melanoma treatment.


Assuntos
Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/irrigação sanguínea , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Flavonoides/uso terapêutico , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
15.
Eur J Pharmacol ; 853: 316-324, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30954563

RESUMO

Tivantinib (TivB) was reported previously to target MET and microtubule assembly in different cells resulting in cytotoxicity. However, its other cellular targets remain unknown, especially the proteins involved in focal adhesion and cytoskeletal organization. We studied the effect of TivB on vinculin a focal adhesion protein, and RhoC, a GTPase which promote the reorganization of cytoskeleton. Biomolecules involved in vasculogenic mimicry (VM) previously not reported in melanoma, and their susceptibility to TivB was also evaluated. TivB affects the viability and apoptosis of human melanoma cells depending on the cell type. Vinculin and RhoC were increased in the presence of TivB and affected the integrity of actin filaments and altered the cellular morphology. TivB disrupts the VM exhibited by melanoma cells in 3D matrix. Roundabout Guidance Receptor 4 (Robo4), a receptor protein implicated in axonal guidance and angiogenesis and its ligand Slit2 are expressed in human C8161 and WM793 melanoma cells, but absent in other melanoma cells including normal melanocytes. VM is more prominent in C8161 cells and could be blocked by siRNA mediated silencing of Robo4 mRNA, but TivB does not affect Robo4 in C8161 cells. Immunoblot analysis indicated no changes in Robo4 and Slit2 protein expression, however, both vinculin and RhoC protein increased in TivB treated melanoma cells. These results suggest that TivB affects cell cytoskeleton and morphology by altering proteins such as vinculin and RhoC. Our studies indicate TivB could target molecules other than MET in melanoma cells, which may provide insight into its alternate mechanism of action.


Assuntos
Melanoma/patologia , Terapia de Alvo Molecular , Neovascularização Patológica , Pirrolidinonas/farmacologia , Quinolinas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inativação Gênica , Humanos , Melanoma/irrigação sanguínea , Melanoma/tratamento farmacológico , Pirrolidinonas/uso terapêutico , Quinolinas/uso terapêutico , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
16.
Int J Hyperthermia ; 36(1): 511-515, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30987478

RESUMO

BACKGROUND: Isolated limb perfusion (ILP) is a treatment option for unresectable in-transit melanoma metastases of the extremities. Approximately two-thirds of the patients have a complete response, and known predictive factors mainly regard tumor burden. In an attempt to identify subgroups with higher response rates, we retrospectively analyzed the predictive value of the BRAF V600E/K mutation for response at our institution. METHODS: Between January 2012 and December 2017, 98 consecutive patients underwent first-time ILP with melphalan for melanoma in-transit metastases and were included in the study. Data was retrieved from our prospectively kept database. Tumor burden was assessed preoperatively as number of lesions and largest tumor diameter. BRAF status was determined according to clinical routine. Response rates were classified according to WHO criteria. RESULTS: Of the 98 patients included in the analysis, 32 patients had a BRAF V600E/K mutation (33%) and 66 patients were BRAF wild type (wt). There was no difference in age, sex or tumor burden between the groups. Comparing response between BRAF V600E/K mutation and BRAF wt, the overall response rate was 69% vs. 77% (p=.36) and the complete response rate was 47% vs. 52% (p=.67). There was no difference in survival, with a median survival of 47 months. CONCLUSION: In this consecutive series of patients, BRAF V600E/K mutation was not found to be a significant factor for response or survival following ILP.


Assuntos
Extremidades/irrigação sanguínea , Melanoma/irrigação sanguínea , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
17.
Biomed Microdevices ; 21(2): 33, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30906958

RESUMO

We couple a tumor growth model embedded in a microenvironment, with a bio distribution model able to simulate a whole organ. The growth model yields the evolution of tumor cell population, of the differential pressure between cell populations, of porosity of ECM, of consumption of nutrients due to tumor growth, of angiogenesis, and related growth factors as function of the locally available nutrient. The bio distribution model on the other hand operates on a frozen geometry but yields a much refined distribution of nutrient and other molecules. The combination of both models will enable simulating the growth of a tumor in a whole organ, including a realistic distribution of therapeutic agents and allow hence to evaluate the efficacy of these agents.


Assuntos
Melanoma/metabolismo , Melanoma/patologia , Modelos Biológicos , Proliferação de Células , Matriz Extracelular/metabolismo , Melanoma/irrigação sanguínea , Neovascularização Patológica , Nutrientes/farmacocinética , Distribuição Tecidual , Microambiente Tumoral
18.
J Biophotonics ; 12(6): e201800421, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30734505

RESUMO

Optical-resolution photoacoustic microscopy (OR-PAM) has been shown to be an excellent imaging modality for monitoring and study of tumor microvasculature. However, previous studies focused mainly on the normal tissues and did not quantify the tumor microvasculature. In this study, we present an in vivo OR-PAM imaging of the melanomas and hepatoma implanted in the mouse ear. We quantify the vessel growth by extracting the skeletons of both dense and thin branches of the tumor microvasculature obtained by Hessian matrix enhancement followed by improved two-step multistencils fast marching method. Compared with the previous methods of using OR-PAM for normal tissues, our method was more effective in extracting the binary vascular network in the tumor images and in obtaining the complete and continuous microvascular skeleton maps. Our demonstration of using OR-PAM in improving microvasculature of tumors and quantification of tumor growth would push deep this technology for the early diagnosis and treatment of cancers.


Assuntos
Carcinoma Hepatocelular/irrigação sanguínea , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Hepáticas/irrigação sanguínea , Melanoma/irrigação sanguínea , Microvasos/diagnóstico por imagem , Fenômenos Ópticos , Técnicas Fotoacústicas , Animais , Fractais , Camundongos
19.
PLoS One ; 14(1): e0210399, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30640942

RESUMO

Angiogenesis is important for the progression of cutaneous melanoma. Here, we analyzed the prognostic impact of the angiogenic factor urokinase plasminogen activator resecptor (uPAR), vascular proliferation index (VPI) and tumor necrosis as a measure of hypoxia in a patient series of nodular melanomas (n = 255) and matched loco-regional metastases (n = 78). Expression of uPAR was determined by immunohistochemistry and VPI was assessed by dual immunohistochemistry using Factor-VIII/Ki67 staining. Necrosis was recorded based on HE-slides. As novel findings, high uPAR expression and high VPI were associated with each other, and with increased tumor thickness, presence of tumor necrosis, tumor ulceration, increased mitotic count and reduced cancer specific survival in primary melanoma. In matched cases, VPI was decreased in metastases, whereas the frequency of necrosis was increased. Our findings demonstrate for the first time the impact on melanoma specific survival of uPAR expression and VPI in primary tumors, and of increased necrosis as an indicator of tumor hypoxia in loco-regional metastases. These findings support the importance of tumor angiogenesis in melanoma aggressiveness, and suggest uPAR as an indicator of vascular proliferation and a potential biomarker in melanoma.


Assuntos
Melanoma/irrigação sanguínea , Melanoma/metabolismo , Neovascularização Patológica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Índice Mitótico , Necrose , Prognóstico , Neoplasias Cutâneas/patologia , Adulto Jovem
20.
Biochimie ; 158: 165-171, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30639360

RESUMO

The potent cytotoxicity of reactive oxygen species (ROS) can cause various diseases, however, it may also serve as a powerful chemotherapeutic strategy capable of killing cancer cells. Oxalomalate (OMA, α-hydroxy-ß-oxalosuccinic acid), a tricarboxylic acid intermediate, is a well-known competitive inhibitor of two classes of NADP+-dependent isocitrate dehydrogenase (IDH) isoenzymes, which serve as the major antioxidants and redox regulators in the mitochondria and cytosol. In this study, we investigated the therapeutic effects of OMA in melanoma and elucidated the associated underlying mechanisms of action using in vitro and in vivo models. OMA targeting IDH enzymes suppressed melanoma growth through activation of apoptosis and inhibition of angiogenesis. Mechanistically, our findings showed that OMA activated p53-mediated apoptosis through ROS-dependent ATM-Chk2 signaling and reduced the expression of vascular endothelial growth factor through ROS-dependent E2F1-mediated hypoxia inducible factor-1α degradation. In particular, OMA-induced suppression of IDH activity resulted in induction of ROS stress response, ultimately leading to apoptotic cell death and antiangiogenic effects in melanoma cells. Thus, OMA might be a potential candidate drug for melanoma skin cancer therapy.


Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Oxalatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/metabolismo , Isocitrato Desidrogenase/metabolismo , Masculino , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/metabolismo
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