Cationic liposomes loaded with proapoptotic peptide D-(KLAKLAK)(2) and Bcl-2 antisense oligodeoxynucleotide G3139 for enhanced anticancer therapy - PubMed (original) (raw)

Cationic liposomes loaded with proapoptotic peptide D-(KLAKLAK)(2) and Bcl-2 antisense oligodeoxynucleotide G3139 for enhanced anticancer therapy

Young Tag Ko et al. Mol Pharm. 2009 May-Jun.

Abstract

The treatment of cancer using macromolecular therapeutics such as oligonucleotides or peptides requires efficient delivery systems capable of intracellular penetration and may also benefit from use of a combination of therapeutics with different mechanisms of action. With this possibility in mind, we constructed cationic liposome loaded with the proapoptotic peptide, d-(KLAKLAK)(2) and the Bcl-2 antisense oligodeoxynucleotide, G3139, and determined whether the combination of the proapoptotic macromolecules in a single cationic liposome can enhance antitumor efficacy. Advantage was taken of alternating charge interaction to entrap macromolecules of opposite charge. The polycationic peptide d-(KLAKLAK)(2) was first condensed with the polyanionic oligodeoxynucleotide G3139 to obtain overall negatively charged peptide/oligodeoxynucleotide complexes. The complexes were then entrapped into DOTAP/DOPE cationic liposomes (CL). This sequential charge interaction ensured efficient entrapment of d-(KLAKLAK)(2) and G3139 with a high loading efficiency (50%) and capacity (7.5 wt %). In vitro treatment of mouse melanoma B16(F10) with CL loaded with d-(KLAKLAK)(2)/G3139 led to significantly enhanced antitumor efficacy, mediated by stimulated induction of apoptotic (caspase 3/7) activity, when compared to CL loaded with G3139 alone. Intratumoral injection of CL loaded with d-(KLAKLAK)(2)/G3139 in B16(F10) mice xenograft also led to suppressed tumor growth associated with enhanced apoptotic activity. Thus, the combination of proapoptotic peptide d-(KLAKLAK)(2) and antisense oligonucleotide G3139 in a cationic liposome led to enhanced apoptotic/antitumor efficacy and may provide a promising tool for cancer treatment.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Preparation of cationic liposome (CL) loaded with D-(KLAKLAK)2 peptide and G3139 ODN. (a) Agarose gel electrophoresis confirmed the complex formation between peptide and ODN and subsequent entrapment of the complex with cationic liposome. Lane1: naked peptide/ODN complexes, nontreated; Lane2: naked peptide/ODN complexes, treated with heparin; Lane 3: CL loaded with peptide/ODN complexes, nontreated; Lane 4: CL loaded with peptide/ODN complexes, treated with heparin and Triton X-100; (b) Size exclusion chromatography (SEC) using FAM-D-(KLAKLAK)2 tracer revealed that about 54 % of peptide was loaded in CL. Free peptide was separated from CL-loaded peptide on a Sepharose CL4B column. Entrapped D-(KLAKLAK)2 eluted with CL at void volume. Without pre-complexation, the loading of peptide into CL was insignificant.

Figure 2

Figure 2

In vitro anti-tumor activity of CL loaded with D-(KLAKLAK)2/G3139 toward B16(F10) melanoma cells. The cells were treated for 24 hours before detection of redox enzymatic activity. CL loaded with G3139 only, complexes of the D-(KLAKLAK)2 peptide with a reverse sequence ODN G3622, and G3622 only were used as controls. Relative cell viability was expressed as a percentage of control cells treated with the medium. Reduced cell viability was observed in the cells treated with CL loaded with the D-(KLAKLAK)2 peptide and G3139 ODN at a relatively low ODN concentration, demonstrating the advantage of the combined apoptotic therapeutic.

Figure 3

Figure 3

Caspase 3/7 activity in B16(F10) melanoma cells treated with CL loaded with D-(KLAKLAK)2/G3139. Cells were treated for 4 hours followed by 24 hours incubation before detection of the enzymatic activity. CL loaded with G3139 only, complexes of D-(KLAKLAK)2 peptide with a control ODN G3622, and G3622 only served as controls. Significantly increased Caspase 3/7 activity was observed in the cells treated with CL loaded with D-(KLAKLAK)2 and G3139, demonstrating the advantages of combining the apoptotic therapeutics for enhanced anti-tumor efficacy through increased apoptotic activity.

Figure 4

Figure 4

In vivo anti-tumor efficacy of CL loaded with D-(KLAKLAK)2/G3139 in the tumor-bearing mice. The mice bearing B16(F10) tumors were administered via intratumoral injection with CL loaded with D-(KLAKLAK)2/G3139 or G3139 alone, CL(peptide/G3139) and CL(G3139) respectively. After 4 days post-injection, (a) tumor volumes were measured and (b) relative inhibition of tumor growth was accessed by comparing tumor volumes of the treated animals to saline-treated controls. Intra-tumoral administration of CL(peptide/G3139) led to significantly decreased tumor volumes whereas CL(G3139) alone showed no significant decrease (n=4, one-way ANOVA followed by Tukey post-hoc test).

Figure 5

Figure 5

In vivo induction of apoptosis by CL loaded with D-(KLAKLAK)2/G3139 in the tumor-bearing mice. The mice bearing B16(F10) tumors were administered CL loaded with the peptide/G3139 via intra-tumoral injection. At 24 hrs post-injection, apoptotic activity in tumors was accessed by DNA fragmentation with TUNEL staining. Dual channel fluorescence microscopy of frozen tumor sections from in vivo grown-B16(F10) tumors is shown. (a) Tumor section from a non-treated animal (background pattern); (b) Tumor section from an animal injected with CL loaded with D-(KLAKLAK)2/G3139. Intra-tumoral injection of CL loaded with D-(KLAKLAK)2/G3139 led to bright green staining of apoptotic nuclei.

References

    1. Green DR, Kroemer G. Pharmacological manipulation of cell death: clinical applications in sight? J Clin Invest. 2005;115(10):2610–7. - PMC - PubMed
    1. Bouchier-Hayes L, Lartigue L, Newmeyer DD. Mitochondria: pharmacological manipulation of cell death. J Clin Invest. 2005;115(10):2640–7. - PMC - PubMed
    1. Jacotot E, Deniaud A, Borgne-Sanchez A, Touat Z, Briand JP, Le Bras M, Brenner C. Therapeutic peptides: Targeting the mitochondrion to modulate apoptosis. Biochim Biophys Acta. 2006;1757(910):1312–23. - PubMed
    1. Javadpour MM, Juban MM, Lo WC, Bishop SM, Alberty JB, Cowell SM, Becker CL, McLaughlin ML. De novo antimicrobial peptides with low mammalian cell toxicity. J Med Chem. 1996;39(16):3107–13. - PubMed
    1. Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Rio GD, Krajewski S, Lombardo CR, Rao R, Ruoslahti E, Bredesen DE, Pasqualini R. Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med. 1999;5(9):1032–8. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources