Implicación de células madre tumorales de la side population e hipoxia en la resistencia a fármacos en líneas celulares de astrocitomas

Resumen

Astrocytomas, the most common type of gliomas are frequently incurable, due to chemoresistance. Temozolomide is the first line of treatment for gliomas and its response in tumors has been associated with MGMT and the mismatch repair pathway (MMR). Hypoxia is a frequent event in solid tumors, conferring drug resistance. This thesis work is focused on the isolation of tumor stem cells of the Side Population (SP cells) and determination of its chemoresistance. The SP phenotype is conferred by the ABCG2 membrane transporter. The role of cell culture in hypoxia and drug resistance, and the study of expression and inhibition of the Sonic Hedgehog signaling pathway (HH) as a potential chemotherapeutic target was performed. We identified SP cells in all astrocytoma cell lines. The percentage of SP cells was higher in the highest tumor grades (III and IV). The cells (parental cells and their SP cells, in normoxia and hypoxia), were treated with temozolomide alone and in combination with O6-benzylguanine, to inhibit MGMT. We found that some parental lines were sensitive to temozolomide, but their SP cells and the same parental line cultured in hypoxia were resistant to temozolomide. The combined treatment increased the cytotoxicity of temozolomide, but not in SP cells. The resistance to temozolomide of SP cells appears to be independent of MGMT and MMR in astrocytoma cell lines, but not in normal astrocytes, where the it is possibly due to a deficiency of MMR. In addition, treatment with temozolomide increased the levels of expression of two genes related to drug resistance: ABCG2 and BCL-2 (anti-apoptotic protein), suggesting that chemotherapy can induce acquired resistance during treatment. Astrocytoma cell lines showed expression of almost all components of the HH pathway, , while astrocytes presented an inactive HH pathway. A combination of cyclopamine (to inhibit HH) and temozolomide, increased cytotoxicity, except in SP cells, that were resistant. SP cells must have mechanisms for self-renewal and proliferation capacity different to the HH pathway. Furthermore, the combined treatment of cyclopamine and temozolomide, seems to have no further effects on ABCG2 and BCL-2 gene expression to those found with temozolomide only, suggesting that cyclopamine does not seem to act, possibly because it is a substrate of ABCG2, and might be extruded out of the cell.