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 XenoenxertoRESUMO
Chemotherapy mistreatment is partially due to a lack of rapid and reliable tools to discriminate between sensitive and resistant phenotypes. In many cases, the resistance mechanism is not fully understood, contributing to the diagnostic tools' absence. This work aims to determine the capacity of MALDI-TOF-MS profiling to discriminate between chemotherapy-resistant and sensitive phenotypes in leukemia and glioblastoma cells. A multivariate analysis of two therapy-resistant leukemia cell lines (Ki562 and Kv562) and two TMZ-resistant glioblastoma cell lines (U251-R and LN229-R) and their sensitive counterparts was performed. In this work, we first show MALDI-TOF-MS patterns analysis ability to differentiate these cancer cell lines by their chemotherapy-resistant status. We present a rapid and inexpensive tool that would guide and complement the therapeutic decision.
Assuntos
Glioblastoma , Leucemia , Humanos , Glioblastoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linhagem Celular Tumoral , FenótipoRESUMO
Although the literature about the functions of hyaluronan and the CD44 receptor in the brain and brain tumours is extensive, the role of the receptor for hyaluronan-mediated motility (RHAMM) in neural stem cells and gliomas remain poorly explored. RHAMM is considered a multifunctional receptor which performs various biological functions in several normal tissues and plays a significant role in cancer development and progression. RHAMM was first identified for its ability to bind to hyaluronate, the extracellular matrix component associated with cell motility control. Nevertheless, additional functions of this protein imply the interaction with different partners or cell structures to regulate other biological processes, such as mitotic-spindle assembly, gene expression regulation, cell-cycle control and proliferation. In this review, we summarise the role of RHAMM in normal brain development and the adult brain, focusing on the neural stem and progenitor cells, and discuss the current knowledge on RHAMM involvement in glioblastoma progression, the most aggressive glioma of the central nervous system. Understanding the implications of RHAMM in the brain could be useful to design new therapeutic approaches to improve the prognosis and quality of life of glioblastoma patients.
Assuntos
Glioblastoma , Glioma , Humanos , Glioblastoma/genética , Ácido Hialurônico , Qualidade de Vida , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glioma/genética , Encéfalo/metabolismo , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismoRESUMO
Cancer is one of the leading causes of death worldwide and has been associated with ageing. Although there are numerous reports that have demonstrated the dual role of hyaluronic acid and senescence induction in cancer prevention and promotion, both players have been linked to ageing in opposite ways. Hyaluronan is recognized for its antiaging role, whereas senescence is associated with ageing. In this review we address these dual roles, showing their interrelation, hypothesizing that the downregulation of senescence mediated by HA would be a key factor in the ambivalent effects described. Likewise, the deforestation allegory aims to explain, through the use of a metaphor, the contradictory yet valid results found in the literature. Considering this background, we propose new strategies for improving tumor therapy. Understanding the biology of these complex diseases and the temporal implication of the different players in dissimilar contexts could bring us closer to the therapeutic improvements needed in the field of oncology.
Assuntos
Ácido Hialurônico , Neoplasias , Senescência Celular/fisiologia , Humanos , Receptores de HialuronatosRESUMO
The extracellular matrix plays a key role in cancer progression. Hyaluronan, the main glycosaminoglycan of the extracellular matrix, has been related to several tumor processes. Hyaluronan acts through the interaction with cell membrane receptors as CD44 and RHAMM and triggers signaling pathways as MEK/ERK. 4-methylumbelliferone (4MU), a well-known hyaluronan synthesis inhibitor, is a promising alternative for cancer therapy. 4MU is a coumarin derivative without adverse effects that has been studied in several tumors. However, little is known about its use in glioblastoma (GBM), the most malignant primary brain tumor in adults. Glioblastoma is characterized by fast growth, migration and tissue invasiveness, and a poor median survival of the patients after treatment. Several reports linked glioblastoma progression with HA levels and even with CD44 and RHAMM expression, as well as MEK/ERK activation. Previously, we showed on a murine GBM cell line that HA enhances GBM migration, while 4MU markedly inhibits it. In this work we showed for the first time, that 4MU decreases cell migration and induces senescence in U251 and LN229 human GBM cell lines. Furthermore, we observed that HA promotes GBM cell migration on both cell lines and that such effects depend on CD44 and RHAMM, as well as MEK/ERK signaling pathway. Interestingly, we observed that the exogenous HA failed to counteract the effects of 4MU, indicating that 4MU effects are independent of HA synthesis inhibition. We found that 4MU decreases total CD44 and RHAMM membrane expression, which could explain the effect of 4MU on cell migration. Furthermore, we observed that 4MU increases the levels of RHAMM inside the cell while decreases the nucleus/cytoplasm relation of p-ERK, associated with 4MU effects on cell proliferation and senescence induction. Overall, 4MU should be considered as a promising therapeutic alternative to improve the outcome of patients with GBM.
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 CultivadasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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/metabolismoRESUMO
Hyaluronan (HA) is the major glycosaminoglycan present in the extracellular matrix. It is produced by some tumours and promotes proliferation, differentiation and migration among others cellular processes. Gestational trophoblastic disease (GTD) is composed by non-tumour entities, such as hydatidiform mole (HM), which is the most common type of GTD and also malignant entities such as choriocarcinoma (CC) and placental site trophoblastic tumour (PSTT), being CC the most aggressive tumour. Although there is a growing understanding of GTD biology, the role of HA in the pathogenesis of this group of diseases remains largely unknown. The aim of this work was to study the role of HA in the pathogenesis of GTD by defining the expression pattern of HA and its receptors CD44 and RHAMM, as well as to determine if HA can modulate proliferation, differentiation and migration of CC cells. Receptors and signalling pathways involved were also analyzed. We demonstrated that HA and RHAMM are differently expressed among GTD entities and even among trophoblast subtypes. We also showed that HA is able to enhance the expression of extravillous trophoblast markers and also to induce migration of JEG-3 cells, the latter mediated by RHAMM as well as PI3K and MAPK pathways. These findings indicate a novel regulatory mechanism for CC cell biology and also contribute to the understanding of GTD pathophysiology.
Assuntos
Movimento Celular/efeitos dos fármacos , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Peso Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.