J Gynecol Oncol.  2010 Dec;21(4):262-268. 10.3802/jgo.2010.21.4.262.

The effect of combined treatment with cisplatin and histone deacetylase inhibitors on HeLa cells

• Jin KL
• Park JY
• Noh EJ
• Hoe KL
• Lee JH
• Kim JH
• Nam JH

• KMID: 2173551
• DOI: http://doi.org/10.3802/jgo.2010.21.4.262

Abstract

OBJECTIVE
To investigate the combined effects of cisplatin and the histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) or sirtinol on HeLa cells and assess the mechanism underlying HDAC inhibitor-cisplatin synergy.
METHODS
The antineoplastic actions of cisplatin, SAHA and sirtinol, alone and in combination, were evaluated using the tetrazolium dye-based MTT cell proliferation assay, DAPI nuclear staining and cytotoxicity analysis.
RESULTS
Exposure to cisplatin, SAHA or sirtinol alone induced a dose-dependent reduction in HeLa cell viability. Combined treatment with cisplatin and SAHA or sirtinol was significantly more cytotoxic than cisplatin alone. Individually, cisplatin, SAHA and sirtinol activated caspase-3 and induced apoptosis, but the effects of combined treatment were greater. Importantly, both HDAC inhibitors dose-dependently inhibited the expression of the antiapoptotic proteins Bcl-2 and x-linked inhibitor of apoptosis protein (XIAP).
CONCLUSION
The combination of cisplatin and SAHA or sirtinol had synergistic effect on the HeLa cell viability. This potentiation of cisplatin activity was associated with HDAC inhibitor-mediated down-regulation of Bcl-2 and XIAP. These may result from the relaxation of chromatin by these HDAC inhibitors that increase cisplatin sensitivity by enhancing the accessibility of DNA to cisplatin and transcriptional regulators.

Keyword

Cervical cancer; Apoptosis; Cisplatin; Suberoylanilide hydroxamic acid; Sirtinol

MeSH Terms

Apoptosis
Benzamides
Caspase 3
Cell Proliferation
Chromatin
Cisplatin
DNA
Down-Regulation
HeLa Cells
Histone Deacetylase Inhibitors
Histone Deacetylases
Histones
Humans
Hydroxamic Acids
Indoles
Naphthols
Proteins
Relaxation
Uterine Cervical Neoplasms
X-Linked Inhibitor of Apoptosis Protein
Benzamides
Caspase 3
Chromatin
Cisplatin
DNA
Histone Deacetylase Inhibitors
Histone Deacetylases
Histones
Hydroxamic Acids
Indoles
Naphthols
Proteins
X-Linked Inhibitor of Apoptosis Protein

Figure

• 

  Fig. 1 Inhibition of HeLa cell growth by suberoylanilide hydroxamic acid (SAHA), sirtinol or cisplatin, alone and in combination. Cells were assayed for viability using MTT assays after treating for 48 hr with increasing doses of SAHA (A), sirtinol (B), or cisplatin (C or D black column) alone, or with a combination of cisplatin and SAHA (C) or cisplatin and sirtinol (D). Results are expressed as the means±SEs of three independent experiments. *p<0.05.

• 

  Fig. 2 Induction of apoptosis in HeLa cells by suberoylanilide hydroxamic acid (SAHA), sirtinol or cisplatin, alone and in combination. Cells were incubated for 48 hr with 2.5 µm SAHA (B), 25 µm sirtinol (F) or 10 µm cisplatin (C and G) alone, or with 2.5 µm SAHA/10 µm cisplatin (D) or 25 µm sirtinol/10 µm cisplatin (H) combinations. (A) and (E) are controls. The cells were sampled, fixed and stained with DAPI; stained nuclei were then observed under a fluorescence microscope using a blue filter.

• 

  Fig. 3 Down-regulation of XIAP and Bcl-2 levels and activation of caspase-3. HeLa cells were treated with the indicated concentrations of SAHA, sirtinol or cisplatin, alone or as SAHA/cisplatin or sirtinol/cisplatin combinations, for 24 hours and then analyzed by immunoblotting. (A, B, D, and E) SAHA (0, 2.5, 5, or 10 µm) or sirtinol (0, 12.5, 25, or 50 µm) induced a dose-dependent reduction in XIAP and Bcl-2 levels. (C) Induction of the cleaved (activated) form of caspase-3 by SAHA (2.5 µm) and cisplatin (20 µm) alone, and by combined SAHA (2.5 µm)/cisplatin (20 µm) treatment. (F) Induction of the cleaved (activated) form of caspase-3 by sirtinol (25 µm) and cisplatin (20 µm) alone, and by combined sirtinol (25 µm)/cisplatin (20 µm) treatment.

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