The importance of RHAMM in the normal brain and gliomas: physiological and pathological roles.

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.

The Effects of Cuprizone on Murine Subventricular Zone-Derived Neural Stem Cells and Progenitor Cells Grown as Neurospheres.

Despite the extensive use of the cuprizone (CPZ) demyelination animal model, there is little evidence regarding the effects of CPZ on a cellular level. Initial studies have suggested that oligodendrocytes (OL) are the main cell targets for CPZ toxicity. However, recent data have revealed additional effects on neural stem cells and progenitor cells (NSC/NPC), which constitute a reservoir for OL regeneration during brain remyelination. We cultured NSC/NPC as neurospheres to investigate CPZ effects on cell mechanisms which are thought to be involved in demyelination and remyelination processes in vivo. Proliferating NSC/NPC cultures exposed to CPZ showed overproduction of intracellular reactive oxygen species and increased progenitor migration at the expense of a significant inhibition of cell proliferation. Although NSC/NPC survival was not affected by CPZ in proliferative conditions, we found that CPZ-treated cultures undergoing cell differentiation were more prone to cell death than controls. The commitment and cell differentiation towards neural lineages did not seem to be affected by CPZ, as shown by the conserved proportions of OL, astrocytes, and neurons. Nevertheless, when CPZ treatment was performed after cell differentiation, we detected a significant reduction in the number and the morphological complexity of OL, astrogliosis, and neuronal damage. We conclude that, in addition to damaging mature OL, CPZ also reduces NSC/NPC proliferation and activates progenitor migration. These results shed light on CPZ direct effects on NSC proliferation and the progression of in vitro differentiation.

Antitumor effect of 4MU on glioblastoma cells is mediated by senescence induction and CD44, RHAMM and p-ERK modulation.

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.

4-Methylumbelliferone as a potent and selective antitumor drug on a glioblastoma model.

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.