Background Survivin (BIRC5), the smallest member of the Inhibitor of Apoptosis (IAP) gene family, is a multifunctional protein which participates in the control of mitosis, apoptosis regulation and cellular stress response [1]. Survivin is undetectable in most healthy adult tissues while it is overexpressed in all human cancers; it is associated with resistance to therapy and with reduced survival in patients with solid and hematologic cancers [1]. Therefore, it is considered a therapeutic target and a marker with prognostic significance in the evolution of different tumors including melanoma. Among the wide spectrum of survivin inhibitors which may be used in therapy, molecules silencing survivin expression are promising agents. Actually a antisense oligonucleotide (LY2181308, gataparsen sodium) is undergoing to advanced phase II trial in different tumors [2]. The use of an oligonucleotide which functions as molecular beacon, able to generate a fluorescent signal when it hybridizes with the survivin mRNA, may offer a new strategy conjugating imaging with the pharmacological silencing activity [3]. Aim of the study In this study we evaluated a lipofectamine-delivered molecular beacon-oligodeoxynucleotide (MB-ODN), targeting survivin mRNA, to reveal its expression and its pharmacological activity in living melanoma A375 cells. Materials and methods Human malignant melanoma cells (A375) were maintained in culture according to the suppliers recommendations (DMEM added with 10% FBS). Human monocyte cells, used as a negative control (not expressing survivin) were isolated from the buffy coats of blood donors and maintained in the same medium as A375 cells. Survivin expression was studied by Reverse Transcriptase-PCR (RT-PCR) using GAPDH as housekeeping gene. MB targeting survivin mRNA (5'ATTO 647-GAGAAAGGGCTGCCATTCTC-3'BBQ) was synthesized by IBA Nucleic Acids Synthesis (Göttingen, Germany). Confocal microscopy evaluation of living cells was carried out plating the cells in Ibidi dishes 24 hours before transfection. The trasfection complex was obtained in Opti-MEM with survivin MB-ODN 100 nmol/L and Lipofectamine 2000 (Monza, Life Technologies Italia). The fluorescence was evaluated in vivo by confocal microscopy at different time points. Results Fluorescence was observed in the cytoplasm of A375 cells at 1,2,3 and 6 h after transfection, with an increasing time-dependent signal. On the contrary, no fluorescent signal was observed in the extracellular milieu and in not expressing survivin cells (monocytes). After 3 and 6 h exposure,in A375 cells, MB-ODN transfection induced cytoplasmic blebbing typical of the apoptotic process revealing the ability of the nucleic acid molecule to inhibit the survivin-mediated antiapoptotic activity. Conclusion Our data indicate that the MB-ODN is a specific and sensitive molecular probe for survivin detection in melanoma living cells revealing, at the same time, its activity as pharmacological agent. Further pharmacological investigations to elucidate the chemosensitizing activity of MB-ODN in melanoma cells are in progress. References [1] D.C. Altieri, Survivin, cancer networks and pathway-directed drug discovery, Nat. Rev. Cancer 8 (2008) 61–70. [2] clinicaltrials.gov [3] F. Baldini , M. Ballestri , S. Carpi, S.G. Conticello, G. Giambastiani, A. Giannetti , A. Guerrini, R. Mercatelli, P. Nieri , F. Quercioli, F. Severi, G. Sotgiu, S. Tombelli, C. Trono, G. Tuci ,G. Varchi Molecular beacon-coated PMMA nanoparticles for the intracellular detection of tumourassociated mRNA, Nanotechitaly2012, in press
Survivin mRNA detection and silencing by a molecular beacon oligodeoxynucleotide in living melanoma cells
Carpi S;
2012-01-01
Abstract
Background Survivin (BIRC5), the smallest member of the Inhibitor of Apoptosis (IAP) gene family, is a multifunctional protein which participates in the control of mitosis, apoptosis regulation and cellular stress response [1]. Survivin is undetectable in most healthy adult tissues while it is overexpressed in all human cancers; it is associated with resistance to therapy and with reduced survival in patients with solid and hematologic cancers [1]. Therefore, it is considered a therapeutic target and a marker with prognostic significance in the evolution of different tumors including melanoma. Among the wide spectrum of survivin inhibitors which may be used in therapy, molecules silencing survivin expression are promising agents. Actually a antisense oligonucleotide (LY2181308, gataparsen sodium) is undergoing to advanced phase II trial in different tumors [2]. The use of an oligonucleotide which functions as molecular beacon, able to generate a fluorescent signal when it hybridizes with the survivin mRNA, may offer a new strategy conjugating imaging with the pharmacological silencing activity [3]. Aim of the study In this study we evaluated a lipofectamine-delivered molecular beacon-oligodeoxynucleotide (MB-ODN), targeting survivin mRNA, to reveal its expression and its pharmacological activity in living melanoma A375 cells. Materials and methods Human malignant melanoma cells (A375) were maintained in culture according to the suppliers recommendations (DMEM added with 10% FBS). Human monocyte cells, used as a negative control (not expressing survivin) were isolated from the buffy coats of blood donors and maintained in the same medium as A375 cells. Survivin expression was studied by Reverse Transcriptase-PCR (RT-PCR) using GAPDH as housekeeping gene. MB targeting survivin mRNA (5'ATTO 647-GAGAAAGGGCTGCCATTCTC-3'BBQ) was synthesized by IBA Nucleic Acids Synthesis (Göttingen, Germany). Confocal microscopy evaluation of living cells was carried out plating the cells in Ibidi dishes 24 hours before transfection. The trasfection complex was obtained in Opti-MEM with survivin MB-ODN 100 nmol/L and Lipofectamine 2000 (Monza, Life Technologies Italia). The fluorescence was evaluated in vivo by confocal microscopy at different time points. Results Fluorescence was observed in the cytoplasm of A375 cells at 1,2,3 and 6 h after transfection, with an increasing time-dependent signal. On the contrary, no fluorescent signal was observed in the extracellular milieu and in not expressing survivin cells (monocytes). After 3 and 6 h exposure,in A375 cells, MB-ODN transfection induced cytoplasmic blebbing typical of the apoptotic process revealing the ability of the nucleic acid molecule to inhibit the survivin-mediated antiapoptotic activity. Conclusion Our data indicate that the MB-ODN is a specific and sensitive molecular probe for survivin detection in melanoma living cells revealing, at the same time, its activity as pharmacological agent. Further pharmacological investigations to elucidate the chemosensitizing activity of MB-ODN in melanoma cells are in progress. References [1] D.C. Altieri, Survivin, cancer networks and pathway-directed drug discovery, Nat. Rev. Cancer 8 (2008) 61–70. [2] clinicaltrials.gov [3] F. Baldini , M. Ballestri , S. Carpi, S.G. Conticello, G. Giambastiani, A. Giannetti , A. Guerrini, R. Mercatelli, P. Nieri , F. Quercioli, F. Severi, G. Sotgiu, S. Tombelli, C. Trono, G. Tuci ,G. Varchi Molecular beacon-coated PMMA nanoparticles for the intracellular detection of tumourassociated mRNA, Nanotechitaly2012, in pressI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
