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An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Published on: September 18, 2013
G3139 (oblimersen) may inhibit prostate cancer cell growth in a partially bis-CpG-dependent non-antisense manner
Jonathan C Lai1, Luba Benimetskaya, Regina M Santella
1Department of Medicine, Columbia University, New York, NY, USA.
Abstract:
G3139 is an 18-mer phosphorothioate oligodeoxyribonucleotide, which is targeted to the initiation codon region of the bcl-2mRNA. Although treatment of PC3 prostate cancer cells with G3139, which contains two CpG motifs, causes a dramatic decrease in bcl-2 protein expression after 3 days, it did not result in significant cellular apoptosis, as it does in many other cell lines. The absence of apoptosis was demonstrated by the absence of pro-caspase 3 cleavage products and of Annexin V cell surface expression. In addition, ATP production and the mitochondrial membrane potential DeltaPsim were preserved. Despite this, G3139 significantly inhibited the rate of cellular proliferation in complete media and blocked cloning in soft agar. G4232, a variant of G3139 that down-regulates bcl-2 expression to the same extent but has both CpG cytidines C5 methylated, was only minimally antiproliferative. A series of mismatched G3139-related oligomers were synthesized that could also substantially down-regulate bcl-2 protein expression, but only if the CpG motifs were preserved, demonstrating the presence of additional non-antisense mechanisms. G3139 caused production of reactive oxygen species in growth-arrested cells and oxidation of nuclear guanosine to 8-hydroxy-2'-deoxyguanosine, as determined by 1F7 monoclonal antibody staining. Bromodeoxyuridine incorporation studies demonstrated that G3139 induced a G1-S entry block and an intra-S-phase block in PC3 cells that persisted as long as 3 days. This finding coincides with the observation that expression of several proteins encoded by S-phase genes, including c-myb and poly(ADP-ribose) polymerase, were significantly reduced. These results illustrate the complexity of the mechanism of action of G3139 in PC3 cells.
Insights
G3139 oligodeoxynucleotide reduces bcl-2 expression in PC3 prostate cancer cells, inhibiting proliferation and causing cell cycle arrest without inducing apoptosis. CpG motifs are crucial for its non-antisense effects.
Area of Science:
- Molecular Biology
- Cancer Research
- Oligonucleotide Therapeutics
Background:
- The bcl-2 gene plays a critical role in cell survival and is often overexpressed in various cancers.
- Oligodeoxynucleotides, such as G3139, are being investigated as potential therapeutic agents targeting specific mRNA sequences.
- PC3 prostate cancer cells exhibit unique responses to G3139, differing from other cell lines.
Purpose of the Study:
- To investigate the mechanism of action of G3139 in PC3 prostate cancer cells.
- To determine if G3139 induces apoptosis in PC3 cells.
- To explore the role of CpG motifs and non-antisense mechanisms in G3139's effects.
Main Methods:
- Treatment of PC3 cells with G3139 and its variants (G4232, mismatched oligomers).
- Assessment of bcl-2 protein expression, apoptosis markers (caspase 3, Annexin V), mitochondrial membrane potential, and ATP production.
- Analysis of cellular proliferation, colony formation in soft agar, reactive oxygen species production, and DNA damage.
- Bromodeoxyuridine incorporation studies to evaluate cell cycle progression.
Main Results:
- G3139 significantly decreased bcl-2 protein expression but did not induce apoptosis in PC3 cells.
- G3139 inhibited cellular proliferation and colony formation, while a methylated variant (G4232) showed minimal antiproliferative effects.
- CpG motifs were essential for G3139's non-antisense mediated effects.
- G3139 induced reactive oxygen species, DNA oxidation, and cell cycle arrest at G1-S and intra-S phases, reducing S-phase gene expression.
Conclusions:
- G3139 exerts complex anti-cancer effects in PC3 cells, primarily through non-antisense mechanisms involving cell cycle arrest.
- The presence of unmethylated CpG motifs is critical for G3139's antiproliferative activity.
- Further research is warranted to fully elucidate G3139's therapeutic potential and mechanisms in prostate cancer.
