Anticancer efficacy of cisplatin and trichostatin A or 5-aza-2'-deoxycytidine on ovarian cancer

Abstract

Background: To evaluate the anticancer efficacy of the combination of epigenetic modifiers and cisplatin in human ovarian cancer.

Methods: The effect of trichostatin A (TSA) and 5-aza-2'-deoxycytidine alone or in combination with low-dose cisplatin was evaluated on human ovarian cancer cell lines in vitro. We measured drug interaction by MTS assay, migration by transwell assay, expression of epithelial to mesenchymal transition (EMT) markers (Twist, Snail, Slug, E-cadherin, and N-cadherin), pluripotency markers (Oct4, Sox2, and Nanog), and epigenetic markers (DNMT3A, LSD1 and H3K4me2, H3K4me3, H3K9me2, and H3K9me3) by western blot, and the impact on and characteristics of spheroid growth when exposed to these drugs. Mouse xenografts were used to evaluate the anticancer effect of sequential drug treatment.

Results: Combination treatment had greater efficacy than single drugs and significantly suppressed cell viability, migration, and spheroid formation and growth. Sequential treatment of cisplatin (1 mg kg(-1)) followed by TSA (0.3 mg kg(-1)) significantly suppressed tumorigenicity of HEY xenografts through inhibition of EMT and decreased pluripotency of ovarian cancer cells.

Conclusion: Epigenetic modifiers potentiate the anticancer efficacy of low-dose cisplatin in ovarian cancer through regulation of EMT and pluripotency, and may provide a promising treatment for ovarian cancer patients.

Figure 1

Effect of TSA, 5-aza-CdR, and cisplatin alone or in combination on the cell viability of ovarian cancer cell lines. (A) The chemical structure of TSA, 5-aza-CdR, and cisplatin. (B) Dose–response curve of SKOV3 cells in the presence of increasing doses of drugs for 48 h in vitro. The experiment was performed in triplicate and the data were presented as mean±s.e.m. Synergism was investigated by exposing SKOV3 cells to a fixed ratio of (C) TSA and cisplatin (0.3 : 1) or (D) 5-aza-CdR and cisplatin (10 : 1) and cell viability were detected by MTS assay. The numbers labelled indicate the fraction (0.5) and multiples (1.0, 2.0, and 3.0) of the IC₅₀ concentration in a fixed ratio (TSA/cisplatin=0.3/1; 5-aza-CdR/cisplatin=10/1). Combination index <1 indicates synergistic effect. All dosages had a CI<1.

Figure 2

Effect of TSA, 5-aza-CdR, and cisplatin alone or in combination on cell migration, chemosensitivity, and the expression of important proteins associated with EMT and pluripotency. (A) Quantitative analysis of migration of ovarian cancer cells (HEY, SKOV3, and A2780) when treated with TSA, 5-aza-CdR, and cisplatin alone or in combination expression as fold change compared with the untreated cells, mean±s.e.m., n=5 (B) Chemotherapy sensitisation assay with HEY cells treated with a fixed dose of cisplatin (1 μℳ) with increasing doses of TSA or 5-aza-CdR measured by cell viability. Each point represents mean±s.e.m. (C) HEY and SKOV3 cells were exposed to the same drugs as the migration assay and the expression levels of proteins were detected by western blot, β-actin is a loading control. Quantitation was done using density of the blots and was a percentage compared with the control cells.

Figure 3

Epigenetic regulation of ovarian cancer cells in the presence of different drugs. (A) Effect of treatment on the expression of epigenetic regulation enzymes DNMT3A and LSD1 in SKOV3 and A2780 cells. Both cells lines were treated for 48 h in vitro and the expression of DNMT3A and LSD1 were detected by western blot. (B) Histone methylation markers H3K4me2/me3 and H3K9me2/me3 were also measured in SKOV3 and A2780 cells. Histone H3 is the loading control.

Figure 4

Inhibitory effect of TSA, 5-aza-CdR, and cisplatin alone or in combination on the formation and growth of spheroids. (A) Representative images of normally occurring spheroids in malignant ascites and spheroids grown in vitro from ovarian cancer cell lines HEY, SKOV3, A2780, and normal epithelium IOSE at × 100. (B) The expression of EMT markers of spheroid and parental cells were assessed by western blot. (C) Effect of different concentration of TSA, 5-aza-CdR, and cisplatin alone or in combination on spheroid number in SKOV3 cells.

Figure 5

Effect of sequential drug administration in tumour xenograft mice model (A) The scheme of sequential treatment strategies. All drugs were administrated at the same time point on each treatment day. (B) Tumour weights were evaluated at day 26, the results presented as mean±s.e.m., P<0.05 denotes statistical significance. (C) Tumour volume was measured and calculated for each treatment group. (D) The expression of EMT markers and pluripotency markers from tumour were detected by western blot. β-actin is a loading control. (E) The mice were weighed at day 26 and compared between treatment groups. n=6.