Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo.
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.
Pennati
Marzia
M
Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, I-20133 Milan and Istituto di Fisiologia Clinica-CNR, Pisa, Italy.
Binda
Mara
M
De Cesare
Michelandrea
M
Pratesi
Graziella
G
Folini
Marco
M
Citti
Lorenzo
L
Daidone
Maria Grazia
MG
Zunino
Franco
F
Zaffaroni
Nadia
N
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D066298:In Vitro Techniques; D051034:Inhibitor of Apoptosis Proteins; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics*; D009363:Neoplasm Proteins; D016337:RNA, Catalytic / Q000378:metabolism*; D000077022:Survivin; D059004:Topoisomerase I Inhibitors; D019772:Topotecan / Q000494:pharmacology*; D014183:Transplantation, Heterologous
D000970:Antineoplastic Agents; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016337:RNA, Catalytic; D000077022:Survivin; D059004:Topoisomerase I Inhibitors; D019772:Topotecan; C506433:CASP9 protein, human; C506434:Casp9 protein, mouse; D053453:Caspase 9; D020169:Caspases