BACKGROUND. Therapeutic options for metastatic melanoma are still very limited. Survivin, a multifunctional protein that regulates multiple pathways required for tumour phenotype maintenance, may also have a role in melanoma development and progression (1). Noteworthy, survivin is overexpressed in cancer cells while it is usually undetectable in most healthy tissues, making it an important molecular target for novel therapeutic interventions with minimal side effects. The use of an antisense oligonucleotide (which acts as molecular beacon) able to generate a fluorescent signal when it hybridizes with the survivin mRNA, may represent an innovative option that conjugates the ability of imaging with the pharmacological, silencing activity (2). This study was aimed at investigating a lipofectamine-delivered molecular beacon-oligodeoxynucleotide (MB- ODN) targeting survivin mRNA, as potential anticancer strategy against human melanoma. MATERIALS AND METHODS. Experiments were performed on the human melanoma A375 cell line, using human monocytes as negative control. RT-PCR and western blot were used to analyse survivin mRNA and protein expression, respectively. The fluorescence signal by MB-ODN in living cells was evaluated by confocal microscopy at 1, 2, 3 and 6 h after lipofectamine-delivered transfection. Cell viability was determined by colorimetric assays (WST-1 and neutral red reagents). All experiments were performed in triplicate. RESULTS. A time-dependent increase in the fluorescence signal was observed in the cytoplasm of A375 cells after lipofectamine-delivered MB-ODN transfection, while no signal was observed in human monocytes. Cytoplasmic blebbing typical of the apoptotic process became evident after 3 and 6 h exposure to MB-ODN. Western blot analysis revealed that survivin expression was decreased by 35%, as compared to untreated cells, thus confirming the pharmacological silencing activity of MB-ODN. Cytotoxicity induced by the nucleic acid was also observed in MB-ODN- transfected cells. CONCLUSIONS. Our data indicate that MB-ODN may represent a specific and sensitive molecular probe for survivin detection in melanoma living cells and a potential novel pharmacological strategy for preventing, overcoming or reversing drug resistance in human melanoma.

Survivin mRNA detection and silencing by a Molecular Beacon Oligodeoxynucleotide in living cutaneous melanoma cells

ADINOLFI, BARBARA;CARPI, SARA
Secondo
;
FOGLI, STEFANO;PELLEGRINO, MARIO;MARTINOTTI, ENRICA;BRESCHI, MARIA CRISTINA
Penultimo
;
NIERI, PAOLA
Ultimo
2012

Abstract

BACKGROUND. Therapeutic options for metastatic melanoma are still very limited. Survivin, a multifunctional protein that regulates multiple pathways required for tumour phenotype maintenance, may also have a role in melanoma development and progression (1). Noteworthy, survivin is overexpressed in cancer cells while it is usually undetectable in most healthy tissues, making it an important molecular target for novel therapeutic interventions with minimal side effects. The use of an antisense oligonucleotide (which acts as molecular beacon) able to generate a fluorescent signal when it hybridizes with the survivin mRNA, may represent an innovative option that conjugates the ability of imaging with the pharmacological, silencing activity (2). This study was aimed at investigating a lipofectamine-delivered molecular beacon-oligodeoxynucleotide (MB- ODN) targeting survivin mRNA, as potential anticancer strategy against human melanoma. MATERIALS AND METHODS. Experiments were performed on the human melanoma A375 cell line, using human monocytes as negative control. RT-PCR and western blot were used to analyse survivin mRNA and protein expression, respectively. The fluorescence signal by MB-ODN in living cells was evaluated by confocal microscopy at 1, 2, 3 and 6 h after lipofectamine-delivered transfection. Cell viability was determined by colorimetric assays (WST-1 and neutral red reagents). All experiments were performed in triplicate. RESULTS. A time-dependent increase in the fluorescence signal was observed in the cytoplasm of A375 cells after lipofectamine-delivered MB-ODN transfection, while no signal was observed in human monocytes. Cytoplasmic blebbing typical of the apoptotic process became evident after 3 and 6 h exposure to MB-ODN. Western blot analysis revealed that survivin expression was decreased by 35%, as compared to untreated cells, thus confirming the pharmacological silencing activity of MB-ODN. Cytotoxicity induced by the nucleic acid was also observed in MB-ODN- transfected cells. CONCLUSIONS. Our data indicate that MB-ODN may represent a specific and sensitive molecular probe for survivin detection in melanoma living cells and a potential novel pharmacological strategy for preventing, overcoming or reversing drug resistance in human melanoma.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/223532
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