RESUMEN
The success of chemotherapy regimens in patients with non-small cell lung cancer (NSCLC) could be restricted at least in part by cancer stem cells (CSC) niches within the tumor microenvironment (TME). CSC express CD133, CD44, CD47, and SOX2, among other markers and factors. Analysis of public data revealed that high expression of hyaluronan (HA), the main glycosaminoglycan of TME, correlated positively with CSC phenotype and decreased disease-free interval in NSCLC patients. We aimed to cross-validate these findings on human and murine lung cancer cells and observed that CD133 + CSC differentially expressed higher levels of HA, HAS3, ABCC5, SOX2, and CD47 (p < 0.01). We modulated HA expression with 4-methylumbelliferone (4Mu) and detected an increase in sensitivity to paclitaxel (Pa). We evaluated the effect of 4Mu + chemotherapy on survival, HA metabolism, and CSC profile. The combination of 4Mu with Pa reduced the clonogenic and tumor-forming ability of CSC. Pa-induced HAS3, ABCC5, SOX2, and CD47 expression was mitigated by 4Mu. Pa + 4Mu combination significantly reduced in vivo tumor growth, enhancing animal survival and restoring the CSC profile in the TME to basal levels. Our results suggest that HA is involved in lung CSC phenotype and chemosensitivity, and its modulation by 4Mu improves treatment efficacy to inhibit tumor progression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Ácido Hialurónico , Himecromona , Neoplasias Pulmonares , Células Madre Neoplásicas , Paclitaxel , Microambiente Tumoral , Ácido Hialurónico/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Animales , Ratones , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Himecromona/farmacología , Línea Celular Tumoral , Microambiente Tumoral/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patologíaRESUMEN
Glioblastoma (GBM) is the most frequent malignant primary tumor of the CNS in adults, with a median survival of 14.6 months after diagnosis. The effectiveness of GBM therapies remains poor, highlighting the need for new therapeutic alternatives. In this work, we evaluated the effect of 4-methylumbelliferone (4MU), a coumarin derivative without adverse effects reported, in combination with temozolomide (TMZ) or vincristine (VCR) on U251, LN229, U251-TMZ resistant (U251-R) and LN229-TMZ resistant (LN229-R) human GBM cells. We determined cell proliferation by BrdU incorporation, migration through wound healing assay, metabolic and MMP activity by XTT and zymography assays, respectively, and cell death by PI staining and flow cytometry. 4MU sensitizes GBM cell lines to the effect of TMZ and VCR and inhibits metabolic activity and cell proliferation on U251-R cells. Interestingly, the lowest doses of TMZ enhance U251-R and LN229-R cell proliferation, while 4MU reverts this and even sensitizes both cell lines to TMZ and VCR effects. We showed a marked antitumor effect of 4MU on GBM cells alone and in combination with chemotherapy and proved, for the first time, the effect of 4MU on TMZ-resistant models, demonstrating that 4MU would be a potential therapeutic alternative for improving GBM therapy even on TMZ-refractory patients.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/uso terapéutico , Glioblastoma/patología , Himecromona/farmacología , Resistencia a Antineoplásicos , Línea Celular Tumoral , Proliferación Celular , Neoplasias Encefálicas/metabolismo , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Apoptosis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Hialuronano Sintasas , Himecromona/farmacología , Himecromona/uso terapéutico , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , RatonesRESUMEN
Hepatocellular carcinoma (HCC) arises in the setting of advanced liver fibrosis, a dynamic and complex inflammatory disease. The tumor microenvironment (TME) is a mixture of cellular components including cancer cells, cancer stem cells (CSCs), tumor-associated macrophages (TAM), and dendritic cells (DCs), which might drive to tumor progression and resistance to therapies. In this work, we study the effects of 4-methylumbelliferone (4Mu) on TME and how this change could be exploited to promote a potent immune response against HCC. First, we observed that 4Mu therapy induced a switch of hepatic macrophages (MÏ) towards an M1 type profile, and HCC cells (Hepa129 cells) exposed to conditioned medium (CM) derived from MÏ treated with 4Mu showed reduced expression of several CSCs markers and aggressiveness. HCC cells incubated with CM derived from MÏ treated with 4Mu grew in immunosuppressed mice while presented delayed tumor progression in immunocompetent mice. HCC cells treated with 4Mu were more susceptible to phagocytosis by DCs, and when DCs were pulsed with HCC cells previously treated with 4Mu displayed a potent antitumoral effect in therapeutic vaccination protocols. In conclusion, 4Mu has the ability to modulate TME into a less hostile milieu and to potentiate immunotherapeutic strategies against HCC.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Himecromona/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Himecromona/efectos adversos , Inmunidad/efectos de los fármacos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Macrófagos Asociados a Tumores/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma (GBM), the most frequent primary tumor of the central nervous system, has a median survival of 14.6 months. 4-Methylumbelliferone (4MU) is a coumarin derivative widely used as a hyaluronan synthesis inhibitor with proven antitumor activity and without toxic effects reported. We aim to evaluate the antitumor effect of 4MU alone or combined with temozolomide (TMZ) on a GBM cell line, its absence of toxicity on brain cells and its selectivity for tumor cells. The antitumor effect of 4MU alone or combined with TMZ was evaluated on GL26 cells by assessing the metabolic activity through the XTT assay, cell proliferation by BrdU incorporation assay, migration by the wound healing assay, cell death by fluorescein diacetate/propidium iodide (FDA/PI) staining, apoptosis by membrane asymmetry and DNA fragmentation and metalloproteinase activity by zymography. The levels of hyaluronan and its capacity to counteract the effects of 4MU and the expression of RHAMM and CD44 were also determined. The toxicity and selectivity of 4MU were determined by XTT assay and PI staining on normal brain primary cell culture (NBPC-GFP) and GL26/NBPC-GFP cocultures. The GL26 cells expressed RHAMM but not CD44 while synthetized hyaluronan. 4MU decreased hyaluronan synthesis, diminished proliferation and induced apoptosis while reducing cell migration and the activity of metalloproteinases, which was restored by addition of hyaluronic acid. Furthermore, 4MU sensitized GL26 cells to the TMZ effect and showed selective toxicity on tumor cells without exhibiting neurotoxic effects. We demonstrated for the first time the cytotoxic effect of 4MU on GBM cells, highlighting its potential usefulness to improve GBM treatment.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Himecromona/farmacología , Animales , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/metabolismo , Glioblastoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Tumorales CultivadasRESUMEN
Hyaluronan (HA) is the main glycosaminoglycan of the extracellular matrix. CD44 is the most important HA receptor, and both have been associated with poor prognosis in cancer. Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively activated tyrosine kinase (Breakpoint Cluster Region - Abelson murine leukemia viral oncogene homolog1, BCR-ABL). It is mainly treated with BCR-ABL inhibitors, such as imatinib. However, the selection of resistant cells leads to treatment failure. The aim of this work was to determine the capacity of HA (high molecular weight) to counteract the effect of imatinib in human CML cell lines (K562 and Kv562). We demonstrated that imatinib decreased HA levels and the surface expression of CD44 in both cell lines. Furthermore, HA abrogated the anti-proliferative and pro-senescent effect of Imatinib without modifying the imatinib-induced apoptosis. Moreover, the inhibition of HA synthesis with 4-methylumbelliferone enhanced the anti-proliferative effect of imatinib. These results suggest that Imatinib-induced senescence would depend on the reduction in HA levels, describing, for the first time, the role of HA in the development of resistance to imatinib. These findings show that low levels of HA are crucial for an effective therapy with imatinib in CML.
Asunto(s)
Antineoplásicos/farmacología , Ácido Hialurónico/metabolismo , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Senescencia Celular/efectos de los fármacos , Senescencia Celular/fisiología , Resistencia a Antineoplásicos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Himecromona/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The tumor microenvironment (TME) represents a complex interplay between different cellular components, including tumor cells and cancer stem cells (CSCs), with the associated stroma; such interaction promotes tumor immune escape and sustains tumor growth. Several experimental approaches for cancer therapy are focused on TME remodeling, resulting in increased antitumor effects. We previously demonstrated that the hyaluronan synthesis inhibitor 4-methylumbelliferone (4Mu) decreases liver fibrosis and induces antitumor activity in hepatocellular carcinoma (HCC). In this work, 4Mu, in combination with an adenovirus encoding interleukin-12 genes (AdIL-12), elicited a potent antitumor effect and significantly prolonged animal survival (p < 0.05) in an orthotopic HCC model established in fibrotic livers. In assessing the presence of CSCs, we found reduced mRNA levels of CD133+, CD90+, EpCAM+, CD44+, and CD13+ CSC markers within HCC tumors (p < 0.01). Additionally, 4Mu downregulated the expression of the CSC marker CD47+ on HCC cells, promoted phagocytosis by antigen-presenting cells, and, combined with Ad-IL12, elicited a potent cytotoxic-specific T cell response. Finally, animal survival was increased when CD133low HCC cells, generated upon 4Mu treatment, were injected in a metastatic HCC model. In conclusion, the combined strategy ameliorates HCC aggressiveness by targeting CSCs and as a result of the induction of anticancer immunity.
Asunto(s)
Antígeno CD47/genética , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Interleucina-12/metabolismo , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Biomarcadores , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Himecromona/farmacología , Interleucina-12/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fagocitosis/inmunología , Linfocitos T/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To establish a successful infection, microorganisms have developed strategies to invade host cells. One of the most important human pathogens and the greatest cause of urinary tract infections, Escherichia coli, still do not have its invasion mechanisms fully understood. This work aims to present a new approach for detecting bacterial invasion of lineage cells, based on an enzymatic-fluorogenic method. The focus of this technique is the detection of E. coli invasion of HeLa cells, exploring ß-glucuronidase, a specific constitutive enzyme of this bacterium. This enzyme hydrolyses the key substrate of this work, 4-methylumbelliferyl-ß-d-glucuronide (MUG), resulting in a fluorogenic molecule, 4-methylumbelliferone. The fluorescence curve created by this method, analyzed by Tukey statistical test, demonstrated that this detection can be efficiently performed after 5â¯h incubation with MUG. When testing uropathogenic E. coli and E. coli isolated from human gastrointestinal microbiota, the proposed method presented similar results to those exhibited by plate counting invasion detection. Data examination by Duncan statistical test allowed the creation of an intensity range of bacterial invasion, which is part of the process of results interpretation. Detection by this enzymatic-fluorogenic method, compared to other existing bacterial invasion detection techniques, is less burdensome, more sensitive and allows fast achievement of reliable results.
Asunto(s)
Técnicas Bacteriológicas/métodos , Escherichia coli/aislamiento & purificación , Células HeLa/microbiología , Infecciones Urinarias/diagnóstico , Escherichia coli Uropatógena/aislamiento & purificación , Técnicas de Cultivo de Célula , Recuento de Colonia Microbiana , Colorantes Fluorescentes , Microbioma Gastrointestinal , Glucuronidasa , Humanos , Himecromona/análogos & derivados , Reproducibilidad de los Resultados , Especificidad por Sustrato , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/patogenicidadRESUMEN
The IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safeguards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress, and that has also emerged as a key regulator of dendritic cell (DC) homeostasis. However, in the context of DC activation, the regulation of the IRE1α/XBP1s axis is not fully understood. In this work, we report that cell lysates generated from melanoma cell lines markedly induce XBP1s and certain members of the UPR such as the chaperone BiP in bone marrow derived DCs (BMDCs). Activation of IRE1α endonuclease upon innate recognition of melanoma cell lysates was required for amplification of proinflammatory cytokine production and was necessary for efficient cross-presentation of melanoma-associated antigens without modulating the MHC-II antigen presentation machinery. Altogether, this work provides evidence indicating that ex-vivo activation of the IRE1α/XBP1 pathway in BMDCs enhances CD8+ T cell specific responses against tumor antigens.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Endorribonucleasas/metabolismo , Melanoma/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Reactividad Cruzada/efectos de los fármacos , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Estrés del Retículo Endoplásmico/inmunología , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Humanos , Himecromona/análogos & derivados , Himecromona/farmacología , Activación de Linfocitos/efectos de los fármacos , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/inmunología , Proteína 1 de Unión a la X-Box/inmunología , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative syndrome characterized by the presence of the Philadelphia chromosome which encodes a constitutively activated tyrosine kinase (BCR-ABL). The first line treatment for CML consists on BCR-ABL inhibitors such as Imatinib. Nevertheless, such treatment may lead to the selection of resistant cells. Therefore, it is of great value to find molecules that enhance the anti-proliferative effect of first-line drugs. Hyaluronan is the main glycosaminglican of the extracellular matrix which is involved in tumor progression and multidrug resistance. We have previously demonstrated that the inhibition of hyaluronan synthesis by 4-methylumbelliferone (4MU) induces senescence and can revert Vincristine resistance in CML cell lines. However, the effect of 4MU on Imatinib therapy remains unknown. The aim of this work was to determine whether the combination of 4MU with Imatinib is able to modulate the proliferation as well as apoptosis and senescence induction in human CML cell lines. For this purpose the ATCC cell line K562, and its multidrug resistant derivate, Kv562 were used. Cells were exposed to 4MU, Imatinib or a combination of both. We demonstrated that 4MU and Imatinib co-treatment abrogated the proliferation of both cell lines. However, such co-treatment did not increase the levels of apoptosis when compared with the treatment with Imatinib alone. For both cell lines the mechanisms of tumor suppression involved was senescence, since the combination of 4MU and Imatinib arrested the cell cycle and increased senescence associated ß-galactosidase activity and senescence associated heterochromatin foci presence when compared to each drug alone. Moreover, 4MU, Imatinib and 4MU + Imatinib decreased pAkt/Akt ratio in both cell lines and reduced the pERK/ERK ratio only in K562 cells. These findings highlight the potential use of 4MU together with Imatinib for CML therapy.
Asunto(s)
Antineoplásicos/farmacología , Himecromona/farmacología , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
BACKGROUND: The development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA) through its receptor CD44 has been described to be involved in the process of angiogenesis. OBJECTIVE: To study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU) on endometriosis-related angiogenesis. MATERIALS AND METHODS: The cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6), 20 mg/kg 4-MU (n = 8) or 80 mg/kg 4-MU (n = 7) throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry. MAIN RESULTS: Non-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions. CONCLUSIONS: The present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the treatment of endometriosis.
Asunto(s)
Endometriosis/etiología , Ácido Hialurónico/antagonistas & inhibidores , Himecromona/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Animales , Aorta/metabolismo , Aorta/patología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Endometrio/irrigación sanguínea , Endometrio/metabolismo , Endometrio/trasplante , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Microvasos/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously demonstrated that a low dose of cyclophosphamide (Cy) combined with gene therapy of interleukin-12 (AdIL-12) has a synergistic, although limited, antitumoral effect in mice with colorectal carcinoma. The main mechanism involved in the efficacy of Cy+AdIL-12 was the induction of a specific immune response mediated by cytotoxic T lymphocytes. Our current aims were to evaluate the effects of 4-methylumbelliferone (4Mu), a selective inhibitor of hyaluronan (HA) synthesis, on tumor microenvironment (TME) and to investigate how 4Mu affects the therapeutic efficacy of Cy+AdIL-12. The results showed that 4Mu significantly reduced the amount of tumoral HA leading to a significant decrease in tumor interstitial pressure (TIP). As a consequence, tumor perfusion was improved allowing an increased adenoviral transgene expression. In addition, treatment with 4Mu boosted the number of cytotoxic T lymphocytes that reach the tumor after adoptive transfer resulting in a potent inhibition of tumor growth. Importantly, we observed complete tumor regression in 75% of mice when 4Mu was administrated in combination with Cy+AdIL-12. The triple combination 4Mu+Cy+AdIL-12 also induced a shift toward antiangiogenic factors production in tumor milieu. Our results showed that TME remodeling is an interesting strategy to increase the efficacy of anticancer immunotherapies based on gene and/or cell therapy.
Asunto(s)
Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Himecromona/farmacología , Inmunoterapia , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Adenoviridae/genética , Traslado Adoptivo , Animales , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/terapia , Terapia Combinada , Ciclofosfamida/farmacología , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Inmunoterapia/métodos , Interleucina-12/genética , Interleucina-12/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/terapia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transducción Genética , Transgenes , Carga Tumoral/genética , Carga Tumoral/inmunologíaRESUMEN
Cirrhosis is characterized by an excessive accumulation of extracellular matrix components including hyaluronic acid (HA) and is widely considered a preneoplastic condition for hepatocellular carcinoma (HCC). 4-Methylumbelliferone (4MU) is an inhibitor of HA synthesis and has anticancer activity in an orthotopic HCC model with underlying fibrosis. Our aim was to explore the effects of HA inhibition by 4MU orally administered on tumor microenvironment. Hepa129 tumor cells were inoculated orthotopically in C3H/HeJ male mice with fibrosis induced by thioacetamide. Mice were orally treated with 4MU. The effects of 4MU on angiogenesis were evaluated by immunostaining of CD31 and quantification of proangiogenic factors (vascular endothelial growth factor, VEGF, interleukin-6, IL-6 and C-X-C motif chemokine 12, CXCL12). IL-6 was also quantified in Hepa129 cells in vitro after treatment with 4MU. Migration of endothelial cells and tube formation were also analyzed. As a result, 4MU treatment decreases tumor growth and increased animal survival. Systemic levels of VEGF were significantly inhibited in 4MU-treated mice. Expression of CD31 was reduced after 4MU therapy in liver parenchyma in comparison with control group. In addition, mRNA expression and protein levels of IL-6 and VEGF were inhibited both in tumor tissue and in nontumoral liver parenchyma. Interestingly, IL-6 production was dramatically reduced in Kupffer cells isolated from 4MU-treated mice, and in Hepa129 cells in vitro. Besides, 4MU was able to inhibit endothelial cell migration and tube formation. In conclusion, 4MU has antitumor activity in vivo and its mechanisms of action involve an inhibition of angiogenesis and IL-6 production. 4MU is an orally available molecule with potential for HCC treatment.
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Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Himecromona/farmacología , Cirrosis Hepática/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Administración Oral , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Cirrosis Hepática/mortalidad , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C3H , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Transducción de Señal , Análisis de Supervivencia , Tioacetamida , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control. METHODOLOGY/PRINCIPAL FINDINGS: We conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production. CONCLUSIONS/SIGNIFICANCE: The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.
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Aedes/enzimología , Genoma de los Insectos , Oviposición/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Vitelogeninas/genética , Aedes/efectos de los fármacos , Aedes/genética , Secuencia de Aminoácidos , Animales , Cuerpo Adiposo/efectos de los fármacos , Cuerpo Adiposo/enzimología , Femenino , Regulación de la Expresión Génica , Himecromona/análogos & derivados , Himecromona/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Ovario/efectos de los fármacos , Ovario/enzimología , Oviposición/efectos de los fármacos , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vanadatos/farmacología , Vitelogeninas/antagonistas & inhibidores , Vitelogeninas/biosíntesisRESUMEN
Hyaluronan (HA) is one of the major components of the extracellular matrix. Several solid tumors produce high levels of HA, which promotes survival and multidrug resistance (MDR). HA oligomers (oHAs) can block HA effects. However, little is known about the role of HA in hematological malignancies. The aim of this work was to determine whether HA or its oligomers can modulate the proliferation of leukemia cells as well as their effect on MDR. Receptors and signaling pathways involved were also analyzed. For this purpose, the human leukemic cell lines K562 and Kv562, which are sensitive and resistant to Vincristine (VCR), respectively, were used. We demonstrated that HA induced cell proliferation in both cell lines. On K562 cells, this effect was mediated by cluster differentiation 44 (CD44) and activation of both phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways, whereas on Kv562 cells, the effect was mediated by receptor for hyaluronan-mediated motility (RHAMM) and PI3K/Akt activation. The inhibition of HA synthesis by 4-methylumbelliferone (4MU) decreased cell line proliferation and sensitized Kv562 to the effect of VCR through P-glycoprotein (Pgp) inhibition, in both cases with senescence induction. Moreover, oHAs inhibited K562 proliferation mediated by CD44 as well as Akt and ERK down-regulation. Furthermore, oHAs sensitized Kv562 cells to VCR by Pgp inhibition inducing senescence. We postulate that the synthesis of HA would promote leukemia progression mediated by the triggering of the above-mentioned proliferative signals. These findings highlight the potential use of oHAs and 4MU as coadjuvant for drug-resistant leukemia.
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Senescencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/farmacología , Leucemia/tratamiento farmacológico , Leucemia/patología , Vincristina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Humanos , Ácido Hialurónico/antagonistas & inhibidores , Himecromona/farmacología , Células K562 , Leucemia/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Vincristina/uso terapéuticoRESUMEN
Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-ß-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na](+) ions correlated with the theoretical monoisotopic mass (calcd [M+Na](+): 507.44 m/z; obs. [M+Na](+): 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-ß-glucosidases by zymographic analysis.
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Acremonium/enzimología , Disacáridos/química , Disacáridos/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Glucosidasas/metabolismo , Glicósidos/química , Glicósidos/síntesis química , Glicosilación , Himecromona/síntesis química , Himecromona/química , Solubilidad , Solventes/química , Especificidad por Sustrato , Agua/químicaRESUMEN
Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.
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Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Glucuronosiltransferasa/antagonistas & inhibidores , Himecromona/análogos & derivados , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/metabolismo , Himecromona/farmacología , Himecromona/uso terapéutico , Himecromona/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/tratamiento farmacológico , Cirrosis Hepática Experimental/patología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Tioacetamida , Carga Tumoral/efectos de los fármacosRESUMEN
OBJECTIVE: This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. METHODS: The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. RESULTS: Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. CONCLUSION: These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.
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Fosfatasa Ácida/metabolismo , Candida albicans/enzimología , Seronegatividad para VIH , Seropositividad para VIH/microbiología , Fosfatasa Ácida/antagonistas & inhibidores , Animales , Recuento de Linfocito CD4 , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular , Niño , Inhibidores Enzimáticos/farmacología , Células Epiteliales/microbiología , VIH/aislamiento & purificación , Seropositividad para VIH/virología , Humanos , Concentración de Iones de Hidrógeno , Himecromona/análogos & derivados , Indicadores y Reactivos , Mucosa Bucal/microbiología , Mucosa Bucal/patología , Vanadatos/farmacología , Carga ViralRESUMEN
Tail regeneration in Xenopus tadpoles is a favorable model system to understand the molecular and cellular basis of tissue regeneration. Although turnover of the extracellular matrix (ECM) is a key event during tissue injury and repair, no functional studies to evaluate its role in appendage regeneration have been performed. Studying the role of Hyaluronan (HA), an ECM component, is particularly attractive because it can activate intracellular signaling cascades after tissue injury. Here we studied the function of HA and components of the HA pathway in Xenopus tadpole tail regeneration. We found that transcripts for components of this pathway, including Hyaluronan synthase2 (HAS2), Hyaluronidase2 and its receptors CD44 and RHAMM, were transiently upregulated in the regenerative bud after tail amputation. Concomitantly, an increase in HA levels was observed. Functional experiments using 4-methylumbelliferone, a specific HAS inhibitor that blocked the increase in HA levels after tail amputation, and transgenesis demonstrated that the HA pathway is required during the early phases of tail regeneration. Proper levels of HA are required to sustain proliferation of mesenchymal cells in the regenerative bud. Pharmacological and genetic inhibition of GSK3beta was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3beta is downstream of the HA pathway. We have demonstrated that HA is an early component of the regenerative pathway and is required for cell proliferation during the early phases of Xenopus tail regeneration. In addition, a crosstalk between HA and GSK3beta signaling during tail regeneration was demonstrated.
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Ácido Hialurónico/metabolismo , Larva , Regeneración/fisiología , Cola (estructura animal)/fisiología , Xenopus laevis , Animales , Animales Modificados Genéticamente , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Glucuronosiltransferasa/antagonistas & inhibidores , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas , Ácido Hialurónico/genética , Himecromona/análogos & derivados , Himecromona/metabolismo , Larva/anatomía & histología , Larva/fisiología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Transducción de Señal/fisiología , Cola (estructura animal)/anatomía & histología , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/anatomía & histología , Xenopus laevis/fisiologíaRESUMEN
We compared the accuracy and reliability of three amplification systems for enzyme immunoassays in the detection of specific IgG antibodies for the diagnosis of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in patients from an endemic area in Rio de Janeiro, Brazil. Partially soluble antigens obtained from the promastigote forms of L. (V.) braziliensis were used. For development of the reaction, two chromogens, 1,2-orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB), and a fluorogen, 4-methylumbelliferylphosphate (MUP), were tested. The performance of each system was compared using the following parameters: accuracy, intraclass correlation coefficient (ICC), and area under the receiver operating characteristic (ROC) curve. Sensitivity was the same (97.4%) for all systems. The reliability was excellent (ICC = 98.6, 98.7, and 99.1%) and specificity was 93.7, 95.8, and 97.4% for OPD, MUP, and TMB, respectively, showing no statistical significance. Despite the absence of differences in the performance of the three systems, the use of TMB is suggested because of its operational advantages, such as low cost compared with fluorogens, easy manipulation, greater stability, and lower toxicity.