Antisense peptide nucleic acids conjugated to somatostatin analogs and targeted at the n-myc oncogene display enhanced cytotoxity to human neuroblastoma IMR32 cells expressing somatostatin receptors.
Peptide nucleic acid (PNA) sequences are synthetic versions of naturally occurring oligonucleotides which display improved binding properties to DNA and RNA, but are still poorly internalized across cell membranes. In an effort to employ the rapid binding/internalization properties of somatostatin agonist analogs and the over-expression of somatostatin receptors on many types of tumor cells, PNAs complementary to target sites throughout 5'-UTR, translation start site and coding region of the n-myc oncogene were conjugated to a somatostatin analog (SSA) with retention of high somatostatin biological potency. IMR32 cells, which over-express somatostatin receptor type 2 (SSTR2) and contain the n-myc oncogene, were treated with these PNA-SSA conjugates. The results show that PNA conjugates targeted to the 5'-UTR terminus and to regions at or close to the translation start site could effectively inhibit n-myc gene expression and cell growth, whereas the non-conjugate PNAs were without effect at similar doses. The most potent inhibition of cell growth was achieved with PNAs binding to the translation start site, but those complementary to the middle coding region or middle upstream site between 5'-UTR and translation start site displayed no inhibition of gene expression. These observations were extended to four other cell lines: GH3 cells which express SSTRs with the n-myc gene, SKNSH cells containing a silent n-myc gene without SSTR2, HT-29 cells carrying the c-myc but no n-myc gene, and CHO-K1 cells lacking SSTR2 with n-myc gene. The results show that there was almost no effect on these four cell lines. Our study indicates that PNAs conjugated to SSA exhibited improved inhibition of gene expression possibly due to facilitated cellular uptake of the PNAs. These conjugates were mRNA sequence- and SSTR2-specific suggesting that many other genes associated with tumor growth could be targeted using this approach and that SSA could be a novel and effective transportation vector for the PNA antisense strategy.
Sun
Lichun
L
Department of Medicine, Peptide Research Laboratories, Tulane Health Sciences Center, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112-2699, USA.
Fuselier
Joseph A
JA
Murphy
William A
WA
Coy
David H
DH
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D004396:Coloring Agents / Q000494:pharmacology; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D020135:Peptide Nucleic Acids / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017481:Receptors, Somatostatin / Q000378:metabolism*; D013004:Somatostatin / Q000031:analogs & derivatives*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D004396:Coloring Agents; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D013778:Tetrazolium Salts; D013844:Thiazoles; D013004:Somatostatin; D013006:Growth Hormone; C022616:thiazolyl blue