Journal Article

Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan <i>in vitro</i> and <i>in vivo</i>

Marzia Pennati, Mara Binda, Michelandrea De Cesare, Graziella Pratesi, Marco Folini, Lorenzo Citti, Maria Grazia Daidone, Franco Zunino and Nadia Zaffaroni

in Carcinogenesis

Volume 25, issue 7, pages 1129-1136
Published in print July 2004 | ISSN: 0143-3334
Published online July 2004 | e-ISSN: 1460-2180 | DOI: https://dx.doi.org/10.1093/carcin/bgh107
Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo

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The ability of melanoma cells to evade engagement of apoptosis plays a significant role in their resistance to chemotherapy. In an attempt to lower the apoptotic threshold of melanoma cells as a possible strategy to increase their drug sensitivity, we generated a hammerhead ribozyme to down-regulate the expression of the anti-apoptotic protein survivin. The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv (targeting the 3′ end of the GUC294 triplet in the exon 3 of the survivin mRNA) or the catalytically inactive ribozyme mutRZsurv (carrying a mutation in the catalytic core of RZsurv). Two polyclonal cell populations expressing the active (JR8/RZsurv) or the mutant (JR8/mutRZsurv) ribozyme were selected for the study. JR8/RZsurv cells were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction in survivin expression was observed in JR8/mutRZsurv cells. JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) compared with JR8/mutRZsurv cells. Moreover, the extent of drug-induced apoptosis (in terms of percentage of apoptotic nuclei and level of caspase-9 and caspase-3 catalytic activity) was significantly greater in JR8/RZsurv than in JR8/mutRZsurv cells. Finally, an increased antitumor activity of oral topotecan was observed in JR8/RZsurv cells grown as xenograft tumors in athymic nude mice compared with JR8/mutRZsurv cells. These results demonstrate that attenuation of survivin expression renders human melanoma cells more susceptible to topotecan-induced apoptosis and more responsive to in vivo treatment, and support the concept that survivin is an attractive target for new therapeutic interventions in melanoma.

Keywords: DOTAP, N-(1-(2,3 dioleoyloxy)propyl)-N,N,N-trimethylammoniummethyl sulfate; IAP, inhibitors of apoptosis protein

Journal Article.  5790 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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