PRIMA-1MET induces apoptosis through accumulation of intracellular reactive oxygen species irrespective of p53 status and chemo-sensitivity in epithelial ovarian cancer cells

Abstract

There is an intensive need for the development of novel drugs for the treatment of epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy due to the high recurrence rate. TP53 mutation is a common event in EOC, particularly in high-grade serous ovarian cancer, where it occurs in more than 90% of cases. Recently, PRIMA-1 and PRIMA‑1MET (p53 reactivation and induction of massive apoptosis and its methylated form) were shown to have an antitumor effect on several types of cancer. Despite that PRIMA-1MET is the first compound evaluated in clinical trials, the antitumor effects of PRIMA-1MET on EOC remain unclear. In this study, we investigated the therapeutic potential of PRIMA-1MET for the treatment of EOC cells. PRIMA-1MET treatment of EOC cell lines (n=13) resulted in rapid apoptosis at various concentrations (24 h IC50 2.6-20.1 µM). The apoptotic response was independent of the p53 status and chemo-sensitivity. PRIMA‑1MET treatment increased intracellular reactive oxygen species (ROS), and PRIMA-1MET-induced apoptosis was rescued by an ROS scavenger. Furthermore, RNA expression analysis revealed that the mechanism of action of PRIMA‑1MET may be due to inhibition of antioxidant enzymes, such as Prx3 and GPx-1. In conclusion, our results suggest that PRIMA-1MET represents a novel therapeutic strategy for the treatment of ovarian cancer irrespective of p53 status and chemo-sensitivity.

Figure 1

p53 protein expression in the EOC cells. EOC cells were culture in 6-well plates, and then lysed with lysis buffer. Immunoblot analysis revealed the p53 protein expression of EOC cells. The samples were loaded and separated by 10% SDS-PAGE with running buffer. The separated proteins were transferred to a PVDF membrane. After blocking with 1% skim milk, the membrane was incubated with anti-p53 or anti-β-actin at 4°C for 1 day. The proteins were visualized using enhanced chemiluminescence. β-actin was used as a loading control.

Figure 2

The effects of PRIMA-1^MET on tumor cell growth in ovarian cancer cells. (a) PRIMA-1^MET-induced growth suppressive dose response curves of EOC cells following 48 h of PRIMA-1^MET treatment. The IC₅₀ values of PRIMA-1^MET were determined in a total of 9 EOC cell lines by cell viability assay. Data represent the mean ± standard deviation (SD) from triplicate reactions. (b) Correlation between PRIMA-1^MET 48 h IC₅₀ values and TP53 status. EOC cell lines with wild-type p53 were slightly more sensitive than those with mutant p53 (not significant). (c) PRIMA-1^MET-induced morphological changes in EOC cells. ES-2, OVCAR-3, and SKOV-3 cells were cultured in a 6-well plate, and then treated with 0, 50, 100 µM PRIMA-1^MET for 24 h. PRIMA-1^MET-induced morphological changes were observed using microscopy (scale bar, 200 µm). (d) PRIMA-1^MET-induced growth suppressive dose response curves of NOS2, NOS3, and their chemo-resistant cells. The IC₅₀ values of 48 h PRIMA-1^MET were determined by cell viability assay. Data displays mean ± standard deviation (SD) from triplicate reactions.

Figure 3

PRIMA-1^MET induces apoptosis in a dose-dependent manner in EOC cell lines. (a) TOV21G and A2780 cells were treated with 0, 10, 20 µM of PRIMA-1^MET for 16 h, and then stained with Annexin V-FITC and PI. The Annexin V-positive and PI-negative cells were defined as early apoptotic, and the Annexin V-positive and PI-positive cells were defined as late apoptotic (dead cells). (b) Viable, early apoptotic, and late apoptotic cells were quantified from triplicate samples. Asterisk indicates statistical significance (P<0.001). Error bars represent standard deviations. (c) Representative images of Hoechst 33342 staining of NOS3TR cells after 24 h of PRIMA-1^MET treatment. Apoptotic cells were defined by their condensed and fragmented nuclei with fluorescence microscopy (scale bar, 200 µm). (d) Apoptosis levels of NOS2 and NOS3 cells, and their chemo-resistant cells after 0, 10, 25, 50 µM 20 h PRIMA-1^MET treatment. Bars represent the percentages of apoptotic nuclei counted in each treatment group and are expressed as the mean ± SD.

Figure 4

PRIMA-1^MET induces PARP cleavage in EOC cells. NOS2 and NOS3 cells, and their chemo-resistant cells were treated with 0, 25 and 50 µM PRIMA-1^MET for 24 h, and then lysed with lysis buffer. Cell lysates were subjected to western blot analysis. β-actin was used as a loading control.

Figure 5

PRIMA-1^MET induces intracellular ROS accumulation in EOC cells. (a) Intracellular ROS accumulation after 16 h of PRIMA-1^MET treatment in the NOS2 and NOS3 cells, and their chemo-resistant cells. Intracellular ROS levels were detected by CM-H₂DCFDA, resulting in fluorescence positivity under fluorescence microscopy. (b) Dose-dependent intracellular ROS accumulation in TOV21G cells. TOV21G cells were maintained in medium with the indicated concentrations of PRIMA-1^MET, labeled with 5 µM CM-H₂DCFDA, and then subjected to flow cytometry. The percentage of cells with fluorescence intensity above the level of 1,000 FL was measured. (c) Bars represent the mean percentage of fluorescence-positive cells. Error bars represent standard deviations.

Figure 6

N-acetyl cysteine (NAC) inhibits the biological effect of PRIMA-1^MET. (a) NAC completely blocked the cytotoxic effect of 16 h of PRIMA-1^MET treatment in TOV21G cells, as shown by Annexin V-FITC and PI staining analysis. After 20 µM of PRIMA-1^MET treatment with or without 10 mM NAC for 16 h, the fraction of viable, early apoptotic, and late apoptotic cells were examined. Bars represents the mean percentages. Error bars represent standard deviations. Asterisk indicates statistical significance (P<0.05). (b) Treatment with NAC prevented the PRIMA-1^MET-induced growth suppressive effect in the TOV21G and A2780 cells according to the cell viability assay. After treatment with several concentrations of PRIMA-1^MET with or without 10 mM NAC, viable cells were evaluated. Bars represents the mean percentages of viable cells. Error bars represent standard deviations. Asterisk indicates statistical significance (P<0.05). (c) Inhibition of antioxidant enzymes by PRIMA-1^MET. TOV21G and A2780 cells were treated with PRIMA-1^MET for 20 h and thereafter the mRNA levels of Prx3 and GPx-1 were evaluated by real-time RT-PCR. Data represent the mean ± SD from triplicate reactions.