J Gynecol Oncol.  2012 Jul;23(3):182-189. 10.3802/jgo.2012.23.3.182.

The relationship between cisplatin resistance and histone deacetylase isoform overexpression in epithelial ovarian cancer cell lines

• Kim MG
• Pak JH
• Choi WH
• Park JY
• Nam JH
• Kim JH

• KMID: 2245177
• DOI: http://doi.org/10.3802/jgo.2012.23.3.182

Abstract

OBJECTIVE
To investigate the relationship between cisplatin resistance and histone deacetylase (HDAC) isoform overexpression in ovarian cancer cell lines.
METHODS
Expression of four HDAC isoforms (HDAC 1, 2, 3, and 4) in two ovarian cancer cell lines, SKOV3 and OVCAR3, exposed to various concentrations of cisplatin was examined by western blot analyses. Cells were transfected with plasmid DNA of each HDAC. The overexpression of protein and mRNA of each HDAC was confirmed by western blot and reverse transcriptase-polymerase chain reaction analyses, respectively. The cell viability of the SKOV3 and OVCAR3 cells transfected with HDAC plasmid DNA was measured using the cell counting kit-8 assay after treatment with cisplatin.
RESULTS
The 50% inhibitory concentration of the SKOV3 and OVCAR3 cells can be determined 15-24 hours after treatment with 15 microg/mL cisplatin. The expression level of acetylated histone 3 protein in SKOV3 cells increased after exposure to cisplatin. Compared with control cells at 24 hours after cisplatin exposure, the viability of SKOV3 cells overexpressing HDAC 1 and 3 increased by 15% and 13% (p<0.05), respectively. On the other hand, OVCAR3 cells that overexpressed HDAC 2 and 4 exhibited increased cell viability by 23% and 20% (p<0.05), respectively, compared with control cells 24 hours after exposure to cisplatin.
CONCLUSION
In SKOV3 and OVCAR3 epithelial ovarian cancer cell lines, the correlation between HDAC overexpression and cisplatin resistance was confirmed. However, the specific HDAC isoform associated with resistance to cisplatin varied depending on the ovarian cancer cell line. These results may suggest that each HDAC isoform conveys cisplatin resistance via different mechanisms.

Keyword

Cisplatin resistance; Epithelial ovarian cancer cell lines; Histone deacetylase

MeSH Terms

Blotting, Western
Cell Count
Cell Line
Cell Survival
Cisplatin
DNA
Hand
Histone Deacetylases
Histones
Neoplasms, Glandular and Epithelial
Ovarian Neoplasms
Plasmids
Protein Isoforms
RNA, Messenger
Cisplatin
DNA
Histone Deacetylases
Histones
Neoplasms, Glandular and Epithelial
Ovarian Neoplasms
Protein Isoforms
RNA, Messenger

Figure

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  Fig. 1 A comparison of cisplatin resistance in SKOV3 and OVCAR3 cells treated with 15 µg/mL cisplatin. The number of viable cells was measured by CCK-8 assay. *p<0.05.

• 

  Fig. 2 The expression of acetylated histone 3 in SKOV3 cells treated with cisplatin. Cells were treated with 15 µg/mL cisplatin and harvested between 9 and 49 hours, followed by western blot analysis. Western blot for β-actin is shown as a control for protein loading.

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  Fig. 3 Expression levels of histone deacetylase (HDAC) isoforms in SKOV3 cells treated with various concentrations of cisplatin. 30 µg of total soluble protein from each lysate was immunoblotted with anti-HDAC 1, 2, 3, and 4 polyclonal antibodies. Western blot for β-actin is shown as a control for protein loading.

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  Fig. 4 Expression levels of histone deacetylase (HDAC) mRNA in SKOV3 cells transfected with each HDAC isoform.

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  Fig. 5 Protein expression levels of histone deacetylase (HDAC) isoforms in SKOV3 and OVCAR3 cells transfected with HDAC isoform expression vectors or not transfected as a control. Expression of β-actin was used as a loading control. endo, endogenous HDAC 1; exo, exogenous HDAC 1 which was transfected.

• 

  Fig. 6 Effect of overexpressed histone deacetylase (HDAC) isoform on cisplatin resistance in SKOV3 cells. SKOV3 cells transfected with a unique HDAC isoform and nontransfected control cells were treated with 15 µg/mL cisplatin for 24 hours. Cell survival rate was measured by cell counting kit-8 (CCK-8) assay. The values were normalized to time 0 as a starting point and the survival rate was calculated by dividing the average number of treated cells by the average number of untreated cells. Each data set represents a relative percentage of nontransfected control values. Values are expressed as mean±SE for three independent experiments. *p<0.05 compared with control.

• 

  Fig. 7 Effect of overexpressed histone deacetylase (HDAC) isoforms on cisplatin resistance in OVCAR3 cells. OVCAR3 cells transfected with a unique HDAC isoform and nontransfected control cells were treated with 15 µg/mL cisplatin for 24 hours. Cell survival rate was measured by cell counting kit-8 (CCK-8) assay. The values were normalized to time 0 as a starting point and the survival rate was calculated by dividing the average number of treated cells by the average number of untreated cells. Each data set represents a relative percentage of nontransfected control values. Values are presented as mean±SE for three independent experiments. *p<0.05 compared with control.

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