All-trans retinoic acid and genistein induce cell apoptosis in OVCAR-3 cells by increasing the P14 tumor suppressor gene

Abstract

In this study, we evaluated the effects of all-trans retinoic acid (ATRA) alone or in combination with genistein (GEN) in p14 tumor suppressor gene and subsequent apoptosis of human ovarian carcinoma cells (OVCAR-3). The cells were treated with ATRA or GEN at concentrations of 50 and 25 μM respectively, either alone or in combination for 24 and 48 h. The cell viability was evaluated using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The percentage of cell apoptosis was determined using flow cytometry and p14 gene expression was measured using real time PCR. The MTT results showed that in both ATRA and GEN treated groups, the cell viabilityviability in group treated for 48 h was significantly lower than group treated for 24 h. The flow cytometry results showed that the percentage of apoptotic cells in groups that treated with ATRA and GEN in combination for 24 h and 48 h was significantly more than all other tested groups. The real time results showed that the mRNA level of p14 in cells treated with both drugs for 48 h was significantly higher than all other groups. In conclusion, we confirm that GEN in combination with ATRA is an effective strategy to up regulate the p14 tumor suppressor gene and induce cell apoptosis in OVCAR-3 cell line.

Keywords: ATRA; Genistein; OVCAR-3; p14.

Fig. 1

IC₅₀ assay of ATRA and GEN in OVCAR-3 cancer cell lines. Cells incubated with/without the drug in various concentrations and the relative amount of viable cells determined by measuring the absorbance of MTT solution. Graph of viability versus drug concentration used to calculate IC50 values.

Fig. 2

MTT assay at IC₅₀ concentrations of ATRA and GEN alone and in combination at 24 and 48 h after the treatment. *, Significant difference vs control group (P < 0.05). †, Significant difference of ATRA24 vs ATRA48 and AG48. #, Significant difference of GEN24 vs GEN48 and AG48.

Fig. 3

(A) the apoptosis inducing effects of ATRA and GEN investigated by flow cytometric analysis on OVCAR-3 cells stained with Annexin V-FITC and/or Propidium Iodide. (B) effects of ATRA and GEN on apoptosis of OVCAR-3 cells at 24 and 48 h. *, Significant difference versus control group (P < 0.05). †, Significant difference of GEN48 vs ATRA48. #, Significant difference of AG24 vs all other groups. ×, Significant difference of AG48 vs all other groups.

Fig. 4

Effects of ATRA and GEN on the levels of p14 expression in OVCAR-3 cells at 24 and 48 h after the treatment. *, Significant difference vs control group (P < 0.05). †, Significant diference of AG24 vs AG48 and ATRA24. #, Significant difference of AG48 vs other groups. ×, Significant difference of GEN24 vs ATRA24.