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
Introduction
It has frequently been suggested that antisense constructs assembled against known mRNA sequences might offer a potential means to treat genetic and virus-mediated diseases [12], [15], [31]. Indeed, some antisense drugs have already been tested in clinical trials [31]. Various kinds of antisense structures including oligonucleotides, phosphorothioates, and methylphosphonates have been employed, but perhaps the most promising are those employing peptide nucleic acids (PNAs).
PNAs are comprised of charge–neutral nucleic acid analogs containing a polyamide, pseudopeptide backbone instead of the usual deoxyribose phosphate structure and are more enzymatically stable than antisense oligonucleotides [12], [16]. They can also bind to complementary DNA/RNA resulting in hybrid PNA/DNA or PNA/RNA duplexes which are more thermodynamically stable than the homoduplexes. In addition, PNAs can be conveniently synthesized by regular solid-phase techniques commonly used for normal peptides. Due to these advantages, PNAs have been used as an alternative approach for antisense gene therapy and it has been demonstrated that they have enhanced specificity for target sequences and can inhibit protein expression [5], [29]. Thus, in recent years, PNAs have been promoted as offering a more promising therapeutic approach [12].
As with oligonucleotides, one of the most intractable problems with PNAs is their poor efficiency of passage across cellular membranes [19]. This has been overcome to some extent by conjugating PNAs to short peptide vectors such as transportan or penetratin-1 whereupon cellular uptake is increased [19], [21]. In the present study, we have employed a different, more tumor specific approach using an analog of the hypothalamic peptide, somatostatin (SST). SST is a small tetradecapeptide and, after binding to its type 2 receptor, SST and its octapeptide agonist analogs are rapidly internalized [24] and may even translocate to the cell nucleus [34]. In addition, SSTR2 are widely distributed in various major tumor types including those of the lung, breast, prostate and GI tract [26]. Denzier and Reubi [3] also reported that SSTRs were over-expressed in peritumoral veins in a majority of human epithelial and mesenchymal tumors and that these SSTRs could be considered as novel targets for tumor treatment with SST analogs. Thus, by conjugating PNAs to high-affinity SST analogs, not only should it be possible to effect internalization of the construct, but also specifically target it to tumor cells and epithelial cells in angiogenic blood vessels [34].
In order to evaluate the PNA–SST conjugation approach, we chose the important oncogene target, n-myc, which belongs to the same oncogene family as c-myc, l-myc and v-myc. All these encoding nuclear phosphoproteins are believed to play critical roles in the control of normal cellular proliferation, differentiation, and genesis or progression of diverse tumors [25], [27]. Thus, antisense blockade of these genes might well be a possible means of controlling tumor formation and growth. In fact, there are already several reports indicating that antisense constructs can reduce myc gene expression and inhibit relevant cell growth [32], [35]. Whereas c-myc expression is more generalized, n-myc is only amplified in a restricted set of tissues, especially neuroblastoma [6], rhabdomyosarcoma [8] and small cell lung carcinoma [14].
Based on the above reasoning, in the present study, PNAs complimentary to different regions of the n-myc mRNA sequence were conjugated to a SST agonist analog with retention of high agonist activity. These PNA conjugates were incubated with human neuroblastoma IMR32 cells which could express SSTR2 and amplify the n-myc oncogene. These conjugates were also tested in four other control cell lines lacking expression of either SSTR2 or n-myc, or both.
Section snippets
Synthesis of PNA–SSA conjugates
The designed sequences of synthetic PNAs were complementary to the published mRNA sequence of the n-myc oncogene taken from the GenBank Database (Accession No.: X03293, X03294, M13228). 4-Methylbenzhydrylamine hydrochloride resin was obtained from Advanced ChemTech Inc., Louisville, KY. Na-tert-Butyloxycarbonyl (Boc) protected amino acids were purchased from Bachem Inc., Torrance, CA, Advanced ChemTech Inc., or Synthetech Inc., Albany, OR. The reactive side-chains of the amino acids were
Synthesis
Several PNAs complementary to different regions of n-myc mRNA were chosen for investigation (shown in Fig. 1 and Table 1). These PNAs were assembled directly onto the N-terminus of a highly potent short SST analog (Fig. 2C) using an enzymatically resistant pentapeptide linking motif (d-Lys-d-Tyr-Lys-d-Tyr-d-Lys-) which we have found to be extremely effective for the attachment of large N-terminal groups with retention of good receptor affinity [7]. PNA–SSA conjugates were synthesized in
Discussion
Antisense oligonucleotides have drawn much interest as potential therapeutic agents because of their ability to specifically bind mRNA and inhibit gene expression. In recent years, this approach has been extended to the antisense PNAs based on PNA nucleoside mimetics [12], [15], [17]. However, as with the oligonucleotides, PNAs are unable to effectively penetrate cellular membranes and various new strategies are currently being developed to overcome this and principle among these has been
Acknowledgements
The authors would like to thank Dr. Yiping Chen and his laboratory (Tulane University) for their technical advice, Dr. Eric Wickstrom (Thomas Jefferson University) for his advice on n-myc protein hybridization, as well as Dr. Catherine Anthony (Louisiana State University) for kindly providing IMR32 cells. We also thank Dr. David Hurley (Tulane University) for training in cell culture techniques and our other laboratory colleagues for their support.
References (36)
- et al.
Antitumor and antiangiogenic effects of somatostatin receptor-targeted in situ radiation with -DTPA-JIC2DL
J. Surg. Res.
(2001) - et al.
A targeted cytotoxic somatostatin (SST) analogue, AN-238, inhibits the growth of H-69 small-cell lung carcinoma (SCLC) and H-157 non-SCLC in nude mice
Eur. J. Cancer
(2001) Peptide nucleic acids as therapeutic agents
Curr. Opin. Struct. Biol.
(1999)- et al.
Somatostatin receptors
TEM
(1997) - et al.
Degradation of bradykinin by peritoneal and alveolar macrophages of the guinea pig
Peptides
(2000) - et al.
A new human highly tumorigenic neuroblastoma cell line with undetectable expression of n-myc
Pediatr. Res.
(1990) - et al.
Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal
Nat. Biotechnol.
(2000) - et al.
Expression of somatostatin receptors in peritumoral veins in human tumors
Cancer
(1999) - et al.
Inhibition of proliferation by L-myc antisense DNA for the translational initiation site in human small cell lung cancer
Cancer Res.
(1995) - et al.
Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length
Biochemistry
(2001)
Detection of n-myc oncogene expression in human neuroblastoma by in situ hybridization and blot analysis: relationship to clinical outcome
Cancer Res.
n-myc gene amplification in Rhabdomyosarcoma detected by fluorescence in situ hybridization: its correlation with histologic features
Mod. Pathol.
Replication of proto-oncogenes early during the S phase in mammalian cell lines
Nucleic Acids Res.
Antisense delivery using protamine-oligonucleotide particles
Nucleic Acids Res.
Application of peptide nucleic acid in cancer therapy
Anticancer Drugs
Preparation and evaluation of tumor-targeting peptide–oligonucleotide conjugates
Bioconjugate Chem.
Human small-cell lung cancers show amplification and expression of the n-myc gene
Proc. Natl. Acad. Sci. USA
Peptide nucleic acids: on the road to new gene therapeutic drugs
Pharmacol. Toxicol.
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