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Combination anti-gene therapy targeting c-myc and p53 in ovarian cancer cell lines
M F Janicek1, B U Sevin, H N Nguyen
1Department of Gynecology and Obstetrics, University of Miami School of Medicine, Florida 33136, USA.
Abstract:
Gene therapy clinical trials targeting p53 and other genes are underway in nongynecologic cancer systems. To explore the potential for antigene therapy in gynecologic oncology, we examined the in vitro effects of oligonucleotides targeting c-myc and p53 in the ovarian cancer cell lines CAOV-3, SKOV-3, and BG-1. The ATP cell viability assay was used to measure growth effects after 6-day treatments with 27-mer antisense phosphorothioate oligodeoxyribonucleotides (oligos) targeting the Puf/nm23 binding region of c-myc and promoter/ATG region of p53. A random sequence of the p53 27-mer was used as a control, and an untransformed fibroblast cell line was used for comparison. IC50 was defined as the oligo concentration required for 50% growth reduction compared to untreated controls. Synergistic vs antagonistic effects of oligo combinations were quantitated by combination indexes (CI) as calculated from median effect parameters by the methods of Chou and Talalay. Mean +/- SE IC50's of c-myc and p53 antisense oligos in CAOV-3 and SKOV-3 ranged from 1.0 +/- 0.2 to 9.7 +/- 1.3 microM. The IC50's of c-myc oligos were consistently lower than corresponding p53 oligos in all cell lines (P < 0.034, t test). The fibroblast cell line was sensitive to anti-c-myc and combination anti-c-myc/p53 oligos (IC50 = 1.5 +/- 0.6 and 1.4 +/- 0.2 microM, respectively), but not to anti-p53 oligos alone (IC50 > 16 microM). Nonspecific toxicity was observed at concentrations of 16 microM for all cell lines except in BG-1, where maximal growth stimulation occurred at this concentration with anti-p53 oligos. Growth stimulation was also observed in BG-1 with anti-c-myc and anti-c-myc/p53 combinations at intermediate doses, with inhibition at higher doses. While c-myc/p53 combinations in CAOV-3 were synergistic (CI < 0.8), they were antagonistic in SKOV-3 (CI > 3.2). Phosphorothioate oligos directed against c-myc and p53 in different cell lines were shown to have both antiproliferative and stimulatory activity, as single agents and in combination, at concentrations that are achievable in vivo. Because of the complex patterns of effects, further in vitro studies are warranted before considering clinical trials with these agents in gynecologic cancers.
Insights
Antisense oligonucleotides targeting c-myc and p53 genes show varied effects in ovarian cancer cells. While some combinations are synergistic, others are antagonistic, warranting further in vitro research for gynecologic cancer gene therapy.
Area of Science:
- Molecular Oncology
- Gene Therapy
- Oligonucleotide Therapeutics
Background:
- Gene therapy targeting p53 and other genes is being explored in non-gynecologic cancers.
- The potential of antisense oligonucleotide therapy for gynecologic oncology remains to be fully elucidated.
- Ovarian cancer cell lines provide a model system to investigate novel therapeutic strategies.
Purpose of the Study:
- To investigate the in vitro effects of antisense oligonucleotides targeting c-myc and p53 in human ovarian cancer cell lines.
- To evaluate the antiproliferative and synergistic/antagonistic effects of these oligonucleotides, alone and in combination.
- To assess the potential for developing oligonucleotide-based gene therapy for gynecologic malignancies.
Main Methods:
- Utilized ATP cell viability assays to measure growth effects after 6-day treatment with 27-mer antisense phosphorothioate oligodeoxyribonucleotides (oligos).
- Targeted specific regions of c-myc and p53 genes, employing a random p53 sequence as a control.
- Quantified synergistic and antagonistic effects using combination indexes (CI) based on median effect parameters.
Main Results:
- Antisense oligos targeting c-myc and p53 demonstrated both antiproliferative and growth-stimulatory activities across different ovarian cancer cell lines (CAOV-3, SKOV-3, BG-1).
- c-myc antisense oligos generally showed lower IC50 values (higher potency) than p53 antisense oligos.
- Combination therapy exhibited synergistic effects in CAOV-3 cells (CI < 0.8) but antagonistic effects in SKOV-3 cells (CI > 3.2).
- A control fibroblast cell line showed sensitivity to anti-c-myc and combination oligos but not to anti-p53 alone.
- Nonspecific toxicity was observed at higher concentrations, with notable exceptions of growth stimulation in BG-1 cells.
Conclusions:
- Phosphorothioate oligonucleotides targeting c-myc and p53 exhibit complex, cell-line-specific effects, including both inhibition and stimulation at achievable in vivo concentrations.
- The observed synergistic and antagonistic interactions highlight the need for careful consideration of combination strategies.
- Further in vitro studies are essential to understand these intricate patterns before advancing to clinical trials in gynecologic cancers.