Synergistic cytotoxic effect of sulindac and pyrrolidine dithiocarbamate against ovarian cancer cells

Abstract

Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac) in vitro compounds affected the growth of tumor cells to a similar extent and synergized in cytotoxicity with PDTC. Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.

Figure 1

Effect of PDTC and sulindac (A) or sulindac sulfide (B) on viability of OVA-14 ovarian cancer cells. OVA-14 cells were incubated for 24 h with 1, 2, 4, 8 or 16 μM of PDTC in combination with 50, 100 or 200 μM of sulindac or sulindac sulfide. Cell viability was measured by MTT assay. The data show the mean ± SD (n=3). Strong synergistic effect is exhibited especially in cultures incubated with 200 μM sulindac or sulindac sulfide with 16 μM PDTC (CI<0.01 and CI<0.4, respectively).

Figure 2

Effect of PDTC and sulindac on the cell viability of different ovarian cancer cell lines: (A) OVP-10, (B) SKOV-3, (C) CAOV-1 and (D) MDAH 2774. The cells were incubated with different concentrations of sulindac and/or PDTC for 24 h and cell viability was measured by MTT assay. The data show the mean ± SD (n=3).

Figure 3

Effect of sulindac and PDTC on the cell cycle in OVA-14 cells. The cells were incubated with 100 μM sulindac and/or 16 μM PDTC for 24 h, fixed in 70% ethanol, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry.

Figure 4

Annexin-V/PI dual staining of OVA-14 cells incubated with 100 μM sulindac and/or 16 μM PDTC for 24 h. Numbers express the percentage of cells that were double-stained, single-stained or unstained with Annexin-V and propidium iodide. The lower right quadrant represent early apoptosis and the upper right quadrant (double-stained cells) late apoptosis cells.

Figure 5

Effect of sulindac and PDTC on expression of Bcl-2, Bax, procaspase-9, NF-κB p50 and NF-κB p65 in OVA-14 cells incubated with 100 μM sulindac and/or 16 μM PDTC for 1 or 4 h. Cytoplasmic cell lysates were immunoblotted with antibodies against each of the above-mentioned proteins, as described in Materials and methods. GAPDH was used as control. C1, C2, controls; lanes 1 and 4, cells incubated with sulindac alone; lanes 2, 5, cells incubated with PDTC alone; lanes 3 and 6, cells incubated with both sulindac and PDTC.