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

    loading  Checking for direct PDF access through Ovid


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.

Related Topics

    loading  Loading Related Articles