Liposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis.
OBJECTIVE
The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODN's).


METHODS
Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODN's using a polycationic liposome preparation. Specific hybridization of antisense ODN's was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis.


RESULTS
Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis.


CONCLUSIONS
Polycationic liposome-mediated transfection of bcl-2 antisense ODN's causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).
Department of Nuclear Medicine, University of Ulm Robert-Koch-Str. 8, D-89070 Ulm. andreas.buck@medizin.uni-ulm.de
Andreas C
AC
Buck
Changxian
C
Shen
Holger
H
Schirrmeister
Alexandra
A
Schmid-Kotsas
Gerd
G
Munzert
Albrecht
A
Guhlmann
Gerhard
G
Mehrke
Nicole
N
Klug
Hans-Jürgen
HJ
Gross
Max
M
Bachem
Sven N
SN
Reske
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D016503:Drug Delivery Systems; D024202:Electrophoretic Mobility Shift Assay; D019254:Genes, bcl-2 / Q000235:genetics*; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D008081:Liposomes; D008223:Lymphoma / Q000188:drug therapy* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017304:Annexin A5; D017931:DNA Primers; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger