J Gynecol Oncol.  2009 Mar;20(1):48-54. 10.3802/jgo.2009.20.1.48.

Synergistic growth inhibition by combination of adenovirus mediated p53 transfer and cisplatin in ovarian cancer cell lines

• Ryu SY
• Kim K
• Lee WS
• Kwon HC
• Lee KH
• Kim CM
• Kang SB

• KMID: 2055649
• DOI: http://doi.org/10.3802/jgo.2009.20.1.48

Abstract

OBJECTIVE
This study was to investigate the synergistic growth inhibitory effect by combination of adenovirus mediated p53 gene transfer and cisplatin in ovarian cancer cell lines with different p53 gene mutation patterns.
METHODS
Three ovarian cancer cell lines, p53 deleted SKOV3, p53 mutated OVCAR-3, and PA-1 with wild-type p53 were transduced with human adenovirus vectors carrying p53 gene (Ad-p53) and treated with a sublethal concentration of cisplatin before and after Ad-p53. The cell number was counted daily for 5 days after Ad-p53 transduction. Western blotting was used to identify p53 and p21 protein expressions, and flow cytometric analysis was performed to investigate any change of DNA ploidy after Ad-p53 transfer.
RESULTS
Ad-p53 transduced cells successfully expressed p53 and p21 proteins after 48 hours of Ad-p53 transduction. Synergistic growth inhibition by combination of Ad-p53 and cisplatin was detected only in SKOV3 and OVCAR-3 cells, but not in PA-1 cells. In p53 deleted SKOV3 cells, cisplatin treatment after Ad-p53 showed higher growth inhibition than the treatment before Ad-p53 transduction, and reverse relationship was observed in p53 mutated OVCAR-3 cells. In SKOV3 cells, the fraction of cells at G2/M phase increased after cisplatin treatment, however, it decreased dramatically with Ad-p53 transduction.
CONCLUSION
The synergistic growth inhibition by combination of Ad-p53 and cisplatin may depend on the p53 status and the temporal sequence of cisplatin treatment, suggesting judicious selective application of this strategy in clinical trials.

Keyword

p53; Adenoviruses; Gene therapy; Cisplatin

MeSH Terms

Adenoviridae
Adenoviruses, Human
Blotting, Western
Cell Count
Cell Line
Cisplatin
DNA
Genes, p53
Genetic Therapy
Lifting
Ovarian Neoplasms
Ploidies
Proteins
Cisplatin
DNA
Proteins

Figure

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  Fig. 1 PCR amplification of p53 target sequences in recombinant Ad-p53 shows 240 bp product of p53 exon 4 (a), and 850 bp product of adenovirus E2B (b). M: size marker, c) positive control for p53, d) Ad-Luc, e) negative control.

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  Fig. 2 Transduction efficiency of adenovirus in ovarian cancer cell lines. MOIs of adenovirus with 70-80% transduction efficiency were 20 in SKOV3, 5 in OVCAR-3, and 50 in PA-1 cells.

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  Fig. 3 Sublethal concentration of cisplatin in ovarian cancer cells. Concentration of cisplatin with growth inhibition below 10% of the control was 0.05 µg/ml in SKOV3, OVCAR-3 and PA-1 cells.

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  Fig. 4 Western blotting for p53 and p21 protein expressions after Ad-p53 shows effective p53 and p21 protein expressions in SKOV3 cells. OVCAR-3 and PA-1 cells already expressed p53 and p21 proteins.

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  Fig. 5 The growth curves of ovarian cancer cells show growth inhibition with Ad-p53 (p53 group) (A, B, C). The synergistic tumor growth suppression effect with combination of Ad-p53 and cisplatin was observed only in p53+P group of SKOV3 (A) and P+p53 group of OVCAR-3 cells (B). Growth curve of PA-1 cells did not show a synergy with combination of Ad-p53 and cisplatin (C). Points: mean; bars: standard error.

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  Fig. 6 Flow cytometric analysis show that cell fraction at G2/M phase increased after cisplatin treatment in p53 deleted SKOV3 cells, however, G2/M phase fraction decreased dramatically after Ad-p53 transfer (A). The changes of G2/M phase fraction of OVCAR-3 and PA-1 cells were not definitive after cisplatin treatment, but G1/S phase fraction were slightly increased after Ad-p53 transfer (B, C). Legends are the same as described in Fig. 5.

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