Estudio sinérgico de los fármacos APR-246, DZ-Nep, panobinostat y temozolamida como tratamiento farmacológico en gliobastoma
- Autores: Francisco Javier de la Rosa Fernández-Pacheco
- Directores de la Tesis: Francisco Javier Sáez Castresana (dir. tes.), Miguel-Ángel Idoate (codir. tes.)
- Lectura: En la Universidad de Navarra ( España ) en 2016
- Idioma: español
- Tribunal Calificador de la Tesis: María Jesús López Zabalza (presid.), Beatriz Pelacho (secret.), José Angel García García (voc.), Miguel Martorell Cebollada (voc.), Cristina Montiel Duarte (voc.)
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- Resumen
Glioblastoma Multiforme (GBM) is the most malignant brain tumour accounting for 60-70% of all gliomas. Current treatment against this tumour consists in maximal surgical resection without threatening the patients life, followed by a treatment with temozolomide, an alkylating agent, with or without combined radiotherapy. Nevertheless, current achieved median survival ranges only 12-15 months. In addition, this malignancy is able to acquire resistance to therapeutic drugs. Numerous pathways can be attacked in combination using molecularly targeted therapeutics, improving the response to the therapy. The p53 signalling pathway plays a key role in apoptosis and senescence programs. Many gliomas have mutations in this gene, featuring a high resistance to apoptosis. APR-246, a new promising drug, can bind to mutant p53 restoring its wild-type activity, leading the tumour cell to an apoptotic induced cell death. The inhibition of EZH2, a histone methylase which is overexpressed in cancer cells with related functions in angiogenesis and metastasis, is also a new strategy tested nowadays. HDACs inhibitors induce a high cytotoxic effect in cancer cells. In this work, the drugs temozolomide, APR-246, DZ-Nep (an EZH2 inhibitor) and panobinostat (a HDACi) were tested in regular glioblastoma cells, and in TMZ resistant cells in order to analyse and compare the effects of these compounds. Moreover, the four drugs were evaluated individually or in combination of pairs or trios, to confirm if these drugs could behave in a synergic, additive or antagonistic way. For this purpose, commercial cell lines used were T98G, A172, A172-TMZR (a temozolomide resistant variant of A172), U87MG, MOG-G-CCM, LN405, LN405TZMR (a temozolomide resistant variant of LN405), and GOS-3, together with PE8 and PE9, primary cultures derived from GBM samples. Cell lines were exposed to single treatments of the drugs and to all possible combinations among them. Colony formation, scratching, and synergistic assays, together with western blots and qRT-PCR, were performed in order to show the cellular and molecular changes induced by all possible combinations of the drugs on the cell lines. Cells treated with double combinations had a significant decrease in the number of produced colonies. Those cell lines in which a double treatment was synergic, increased their levels of pro-apoptotic genes. Migration seemed to not be affected by the treatment. Lastly, combinatorial studies showed the additive or synergic effect of some of the dual combinations. A high synergic effect was obtained when the drugs were supplied in trios. Curiously, despite of the high effect produced by APR-246, we found that APR-26 could not reactivate p53 in the p53 mutant glioblastoma cell lines. We can conclude that the therapeutic potential of the combination of these four drugs in GBM is clear. However, further studies are required in order to understand the mechanisms of the additive or synergic effect followed by these compounds, as well as the impact of these compounds on the cells, depending on the expression of relevant genes as MGMT or PTEN.
