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1.
Sci Rep ; 14(1): 16803, 2024 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-39039104

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Ácido Hialurônico , Himecromona , Neoplasias Pulmonares , Células-Tronco Neoplásicas , Paclitaxel , Microambiente Tumoral , Ácido Hialurônico/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Animais , Camundongos , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Himecromona/farmacologia , Linhagem Celular Tumoral , Microambiente Tumoral/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia
2.
Sci Rep ; 13(1): 9356, 2023 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-37291120

RESUMO

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.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/uso terapêutico , Glioblastoma/patologia , Himecromona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Encefálicas/metabolismo , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Apoptose , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Liver Int ; 42(2): 444-457, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34800352

RESUMO

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.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Hialuronan Sintases , Himecromona/farmacologia , Himecromona/uso terapêutico , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Camundongos
4.
Sci Rep ; 11(1): 6310, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33737571

RESUMO

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 (Mϕ) towards an M1 type profile, and HCC cells (Hepa129 cells) exposed to conditioned medium (CM) derived from Mϕ treated with 4Mu showed reduced expression of several CSCs markers and aggressiveness. HCC cells incubated with CM derived from Mϕ 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.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Himecromona/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Himecromona/efeitos adversos , Imunidade/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Macrófagos Associados a Tumor/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Glycobiology ; 31(1): 29-43, 2021 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-32472122

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Himecromona/farmacologia , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Glioblastoma/metabolismo , Glioblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais Cultivadas
6.
Sci Rep ; 9(1): 10930, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358779

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Ácido Hialurônico/metabolismo , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Himecromona/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
7.
Mol Ther ; 26(12): 2738-2750, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30301668

RESUMO

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.


Assuntos
Antígeno CD47/genética , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Interleucina-12/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Biomarcadores , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Himecromona/farmacologia , Interleucina-12/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fagocitose/imunologia , Linfócitos T/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Front Immunol ; 9: 3050, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30687308

RESUMO

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.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Endorribonucleases/metabolismo , Melanoma/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Apresentação Cruzada/efeitos dos fármacos , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Endorribonucleases/imunologia , Humanos , Himecromona/análogos & derivados , Himecromona/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/imunologia , Proteína 1 de Ligação a X-Box/imunologia , Proteína 1 de Ligação a X-Box/metabolismo
9.
Invest New Drugs ; 35(1): 1-10, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27718039

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Himecromona/farmacologia , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo
10.
PLoS One ; 11(3): e0152302, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27018976

RESUMO

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.


Assuntos
Endometriose/etiologia , Ácido Hialurônico/antagonistas & inibidores , Himecromona/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Animais , Aorta/metabolismo , Aorta/patologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Endométrio/irrigação sanguínea , Endométrio/metabolismo , Endométrio/transplante , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microvasos/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley
11.
Mol Ther ; 23(9): 1444-55, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26105158

RESUMO

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.


Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Himecromona/farmacologia , Imunoterapia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Adenoviridae/genética , Transferência Adotiva , Animais , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Terapia Combinada , Ciclofosfamida/farmacologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Imunoterapia/métodos , Interleucina-12/genética , Interleucina-12/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/terapia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética , Transgenes , Carga Tumoral/genética , Carga Tumoral/imunologia
12.
Glycobiology ; 25(8): 825-35, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25882295

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Himecromona/farmacologia , Cirrose Hepática/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Administração Oral , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/mortalidade , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Tioacetamida , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Glycobiology ; 23(12): 1463-76, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24013961

RESUMO

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.


Assuntos
Senescência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ácido Hialurônico/biossíntese , Ácido Hialurônico/farmacologia , Leucemia/tratamento farmacológico , Leucemia/patologia , Vincristina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Ácido Hialurônico/antagonistas & inibidores , Himecromona/farmacologia , Células K562 , Leucemia/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Vincristina/uso terapêutico
14.
Glycobiology ; 22(3): 400-10, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22038477

RESUMO

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.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Glucuronosiltransferase/antagonistas & inibidores , Himecromona/análogos & derivados , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases , Ácido Hialurônico/biossíntese , Ácido Hialurônico/metabolismo , Himecromona/farmacologia , Himecromona/uso terapêutico , Himecromona/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/tratamento farmacológico , Cirrose Hepática Experimental/patologia , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Tioacetamida , Carga Tumoral/efeitos dos fármacos
15.
Mol Hum Reprod ; 6(8): 699-706, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10908279

RESUMO

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.


Assuntos
Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Animais , Células CHO , Cricetinae , Feminino , Humanos , Concentração de Íons de Hidrogênio , Himecromona/análogos & derivados , Himecromona/metabolismo , Himecromona/farmacologia , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/farmacologia
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