Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis

• DOI: 10.1158/1535-7163.mct-04-0213
• Publication Date: 2005-09-22T20:50:40Z
• Authors: Roberto Tonelli; Stefania Purgato; Consuelo Camerin; Raffaele Fronza; Fabrizio Bologna; Simone Alboresi; Monica Franzoni; Roberto Corradini; Stefano Sforza; Andrea Faccini; Jason M. Shohet; Rosangela Marchelli; Andrea Pession
• Keywords: Gene Expression Regulation, Neoplastic; Peptide Nucleic Acids; Proto-Oncogene Proteins c-myc; 0301 basic medicine; Neuroblastoma; 03 medical and health sciences; Tumor Cells, Cultured; Humans; Apoptosis

Abstract We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH2 terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G1-phase accumulation (39–53%) in IMR-32 and apoptosis (56% annexin V–positive cells at 24 hours in IMR-32 and 22% annexin V–positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA–based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.