Tesis doctoral de Jessica Castro Gallegos
Ribonucleases are promising agents for use in anticancer therapy. In contrast to many chemotherapeutic agents, which act by interfering with dna synthesis and cell division, cytotoxic ribonucleases are non-mutagenic agents that exert their effects by interfering with rna functions such as protein synthesis or gene regulation and are able to kill non-dividing cells. The mechanism of cytotoxicity of pe5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase, a ribonuclease with antitumor activity that is currently in phase iiib clinical trials in patients with malignant mesothelioma. The results obtained in this study show that pe5 does not require the pro-apoptotic activity of p53 to trigger cell death. In addition, the cytotoxic effect is not prevented by a multiple drug resistance phenotype. Using different complementary techniques, it is demonstrated that the cytotoxicity of pe5 is produced through apoptosis. The data also show that in vitro pe5 is selective for tumor cells and that both ribonucleases induce cell death to the same extent although onconase also arrest the cell growth. The cytotoxic effects of both ribonucleases and the cytostatic effect of onconase have been compared by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. Pe5 increases the number of cells in s and g2/m cell cycle phases, which is accompanied by the increased expression of cyclin e and p21waf1/cip1 together with the underphosphorylation of p46 forms of jnk. At 5 ¿m, onconase is highly cytotoxic. While this effect is accompanied by the reduced expression of xiap and bcl-2, the overphosphorylation of jnk is not produced. On the other hand, cell growth arrest induced by onconase produces a prolongation of the overall cell cycle, which is accompanied by overphosphorylation of p46 forms of jnk and reduced expression of cyclins d1 and e. The present results show that pe5 and onconase kill the cells through mechanisms with significant differences. in addition, pe5 cytotoxicity is synergic with doxorubicin on the doxorubicin-resistant nci/adr-res cell line but not on the mcf-7 sensitive cell line. Since this result could be explained by a reduction in the level of p-glycoprotein induced by pe5, this thesis investigates whether pe5 could specifically inhibit the accumulation of p-glycoprotein in multidrug-resistant cells. The results show that pe5, but not onconase, is able to reduce the amount of p-glycoprotein in two different multidrug-resistant cell lines -nci-h460/r and nci/adr-res- while glutathione s-transferase-¿ is not affected. The reduction in p-glycoprotein accumulation, which has been functionally confirmed by flow cytometry analysis, may be caused by the previously reported underphosphorylation of jnk induced by pe5.
Datos académicos de la tesis doctoral «A human pancreatic ribonuclease variant kills cancer cells by apoptosis and reduces the expression of p-glycoprotein in mdr cell lines«
- Título de la tesis: A human pancreatic ribonuclease variant kills cancer cells by apoptosis and reduces the expression of p-glycoprotein in mdr cell lines
- Autor: Jessica Castro Gallegos
- Universidad: Girona
- Fecha de lectura de la tesis: 26/10/2010
Dirección y tribunal
- Director de la tesis
- María Vilanova Brugués
- Tribunal
- Presidente del tribunal: claudi Cuchillo foix
- Antonio Villaverde corrales (vocal)
- francesc Canals surís (vocal)
- ramon Colomer bosch (vocal)