Cer and So are involved in regulation of apoptosis and cell cycle arrest while on the other hand S1P promotes cell growth and inhibits apoptosis. The antagonistic effects of these metabolites are regulated by enzymes that interconvert Cer, So, and S1P. In this work two of these enzymes were studied: sphingosine phosphate lyase and acid ceramidase. First several methods to determine the activity of these enzymes were developed and optimized, resulting in the publication of sensitive fluorogenic and chromatographic methods for enzyme activity.
Particularly the assay optimized for acid ceramidase activity was used in the finding and identification of new inhibitors in several compound libraries. As a result compounds RBM2-1B, RBM2-1D and RBM2-1E were identified as acid ceramidase and dihydroceramide desaturase inhibitors. Furthermore compounds RBM1-12, RBM1-13 and SABRAC were also described as acid ceramidase inhibitors. Since several publications described the upregulation of acid ceramidase in advanced prostate cancer, we decided to investigate the particular effect of the acid ceramidase inhibition in a cellular model of advanced prostate cancer. Using cells PC-3/Mc we inhibited acid ceramidase through two different approaches: first silencing the gen ASAH1 and comparing the effects with chemical inhibition of the enzyme using compounds RBM1-12, RBM1-13 and SABRAC.
We evaluate the effect of acid ceramidase inhibition in cell growth, invasivity and 3D growth in vitro, finding a diminished growth and 3D growth in both cells those knockdown for ASAH1 and treated with inhibitors. Finally, the effect of ASAH1 silencing in in vivo tumor growth and lung colonization was also determined. To this end male NOD-SCID mice were used for xenotransplants with cells PC-3/Mc_ASAH1_KD or control and the tumor growth or lung colonization was followed by luminometry. We found that the silencing of ASAH1 in PC-3/Mc cells delayed the growth and also the lung colonization, highlighting the potential of acid ceramidase inhibition as adjuvant in the treatment of prostate cancer and also in the prevention of metastases formation.
Identifer | oai:union.ndltd.org:TDX_UB/oai:www.tdx.cat:10803/21624 |
Date | 31 March 2011 |
Creators | Camacho Castillo, Luz del Carmen |
Contributors | Fabriàs Domingo, Gemma, Marfany i Nadal, Gemma, Universitat de Barcelona. Departament de Genètica |
Publisher | Universitat de Barcelona |
Source Sets | Universitat de Barcelona |
Language | English |
Detected Language | English |
Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
Format | 193 p., application/pdf |
Source | TDX (Tesis Doctorals en Xarxa) |
Rights | ADVERTIMENT. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs., info:eu-repo/semantics/openAccess |
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