Elsevier

Peptides

Volume 23, Issue 9, September 2002, Pages 1557-1565
Peptides

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

https://doi.org/10.1016/S0196-9781(02)00096-7Get rights and content

Abstract

Peptide nucleic acid (PNA) sequences are synthetic versions of naturally ocurring 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.

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)

  • E.F Grady-Leopardi et al.

    Detection of n-myc oncogene expression in human neuroblastoma by in situ hybridization and blot analysis: relationship to clinical outcome

    Cancer Res.

    (1986)
  • Y Hachitanda et al.

    n-myc gene amplification in Rhabdomyosarcoma detected by fluorescence in situ hybridization: its correlation with histologic features

    Mod. Pathol.

    (1998)
  • M.A Iqbal et al.

    Replication of proto-oncogenes early during the S phase in mammalian cell lines

    Nucleic Acids Res.

    (1987)
  • M Junghans et al.

    Antisense delivery using protamine-oligonucleotide particles

    Nucleic Acids Res.

    (2000)
  • H Knudsen et al.

    Application of peptide nucleic acid in cancer therapy

    Anticancer Drugs

    (1997)
  • W Mier et al.

    Preparation and evaluation of tumor-targeting peptide–oligonucleotide conjugates

    Bioconjugate Chem.

    (2000)
  • M.M Nau et al.

    Human small-cell lung cancers show amplification and expression of the n-myc gene

    Proc. Natl. Acad. Sci. USA

    (1986)
  • P.E Nielsen

    Peptide nucleic acids: on the road to new gene therapeutic drugs

    Pharmacol. Toxicol.

    (2000)
  • Cited by (36)

    • Copper-64-labeled anti-bcl-2 PNA-peptide conjugates selectively localize to bcl-2-positive tumors in mouse models of B-cell lymphoma

      2015, Nuclear Medicine and Biology
      Citation Excerpt :

      However, these studies were performed in rat AR42J rat pancreatic tumor cells, which are bcl-2-negative, as shown in Fig. 1. Sun et al. used a somatostatin analogue conjugated to an anti-n-myc PNA to enhance cytotoxicity in IMR32 neuroblastoma cells [30]. Wickstrom and colleagues reported a series of insulin growth factor receptor-targeted PNA-peptide chimeras labeled with 99mTc or 64Cu [31,32] for imaging oncogene mRNAs, such as CCND1, in mouse models of breast cancer.

    • In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder

      2010, Blood
      Citation Excerpt :

      Examples of tumor-specific peptides include somatostatin4 and bombesin/gastrin-releasing peptide,5 which exhibit high affinities for the cognate cell-surface receptors. Peptides also facilitate the transport of cytotoxic compounds and stretches of nucleic acid into specific tumor tissues.6 Random peptide libraries (RPLs) allow the selection of therapeutic peptides for tumor cell-surface receptors.7

    • MYC in Oncogenesis and as a Target for Cancer Therapies

      2010, Advances in Cancer Research
      Citation Excerpt :

      One MYCN targeting approach still awaiting clinical trials is the employment of peptide nucleic acids (PNAs), DNA analogs modified for a higher stability and longer duration of activity (reviewed in Morgenstern and Anderson, 2006). An antisense MYCN PNA conjugated to a somatostatin analog was demonstrated to be rapidly internalized and significantly inhibited cell growth of neuroblastoma cells (Sun et al., 2002). An even better outcome was observed by the use of antigene PNAs, designed to be complementary to the coding DNA strand (Tonelli et al., 2005).

    • A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids

      2007, Molecular Therapy
      Citation Excerpt :

      The most frequently used delivery vehicles today are formulas based on cationic liposomes that are highly efficient in vitro and easy to use but are unable to transport uncharged PNAs. Therefore, various peptides, such as receptor ligands and nuclear localization signal sequences, have been conjugated to PNA to promote cellular uptake.8,9 Another class of molecules that suffer from low bioavailability owing to limited cellular uptake are proteins, and, unlike in the case of ONs, few vectors are available for protein transduction.

    View all citing articles on Scopus
    View full text