• BCRP
• blood-brain barrier
• chemoresistance
• doxorubicin
• miR-21
• P-gp
• pediatric glioblastoma multiforme

Various signal transduction pathways, and various proteins of the blood-brain barrier (BBB) seem to be involved in chemoresistance of pediatric glioblastoma multiforme (GBM). MicroRNA-21 (miR-21) is an important oncogenic microRNA (miRNA) which modulates drug resistance of tumor cells.
Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are ATP-binding cassette (ABC) transporters that also remove drugs through BBB, limiting brain penetration and accumulation. These proteins are also expressed by cancer cells, but their role in chemoresistance is unclear.
In GBM and in other brain tumors, miR-21 seems regulate the expression levels of BCRP and P-gp with an indirect regulation mechanism.
It has already been shown that doxorubicin (DOX) is an effective anti-glioma agent with antitumor activity also against GBM stem cells. However clinical trials disappointed these data. Preclinical studies suggest that chemoresistance is a multi-levels, dynamic reality, that can be modulated. In particular, in animal models, Morphine (Morph) temporarily alters the permeability of BBB for large molecules, such as DOX. However, its role is unknown in cellular resistance. Moreover, prolonged exposure to DOX showed a significant induction of apoptosis in GBM stem cells while the commonly used etoposide and temozolomide were not quite effective.
We analyzed the expression of 5 miRNAs, previously found to be dysregulated in high grade gliomas (HGG), in 9 pediatric GBM samples. Furthermore, we selected a DOX resistant GBM cell line with similar miRNA expression profile, in order to study mechanisms of chemoresistance (miRNAs and ABC trasporters).
Analysis on pediatric GBM samples showed an overexpression of miR-21 and a reduced or lack of expression of miR-7, miR-124, miR-128 and miR-137.
Analysis of sensitivity to DOX in 3 GBM cell lines (A172, U87MG and T98G) showed a cytotoxic effect after 48h of treatment in A172 and U87MG, but not in T98G cells. TUNEL assay verified also that DOX induced apoptosis in A172 and U87MG but not in T98G. About miRNA expression profile in this cell line, miR-21 in T98G cells was over-expressed likely pediatric GBM samples.
We evaluated both the role of Morph and prolonged exposure to DOX, on chemoresistance of T98G. In our experience, treatment with Morph plus DOX did not interfere with the cytotoxic effect of chemotherapy that was observed only after prolonged exposure to anthracycline (96h).
Moreover we evaluated the role of miR-21 and ABC transporters in DOX chemoresistance of T98G cells.
To validate the possible association of miR-21 with drug resistance of T98G cells, we transfected anti-miR-21 inhibitor into the cells. The expression level of miR-21 was significantly lower in T98G transfected cells (than in the parental control ones). Transfected cells showed a high apoptotic rate compared to control after DOX treatment by TUNEL assay, suggesting a key role of miR-21 in DOX chemoresistance.
Finally, we evaluated the cellular expression (basal and after treatment) of P-gp and BCRP in T98G cells. T98G cells constitutively express BCRP, but not P-gp. After treatment with DOX or DOX plus Morph we have seen that both DOX and Morph down-regulate BCRP expression.
In conclusion, we have demonstrated that miR-21 was over-expressed in T98G GBM cells and in our pediatric GBM samples. miR-21 could play an important role in DOX chemoresistance of T98G cells. However, DOX chemoresistance seems to be independent from BCRP expression and Morph co-treatment. Only prolonged exposure (96h) to DOX makes T98G cells sensible to DOX.