Pharmacological Inhibition of p38 MAPK by SB203580 Increases Resistance to Carboplatin in A2780cp Cells and Promotes Growth in Primary Ovarian Cancer Cells

Abstract

Chemoresistance renders current chemotherapy regimens ineffective against advanced epithelial ovarian cancer (EOC). Carboplatin (the first-line chemotherapeutic agent to treat EOC) induces cell death by regulating multiple signaling pathways. The objective of this study is to identify the signaling pathways that contribute to carboplatin resistance in EOC. To this end, we performed a proteome profiler human phospho-kinase array experiment and compared the phosphorylation profiles between the cisplatin-sensitive A2780s versus its derivative cisplatin-resistant A2780cp cells. The phospho-kinase array revealed that A2780s and A2780cp cells displayed different profiles in basal and carboplatin-induced phosphorylation. Phosphorylation of p38 MAPK was increased by carboplatin more markedly in A2780s cells compared to A2780cp cells. Inhibition of p38 MAPK activity by its specific inhibitor SB203580 increased resistance to carboplatin in A2780cp cells, but not in A2780s cells or in ascites-derived high-grade serous EOC cells. Interestingly, SB203580 increased the number of viable cells in the primary EOC cells, which was concomitant with an increase in survivin expression. In conclusion, inhibition of p38 MAPK by SB203580 increases resistance to carboplatin in A2780cp cells and the number of viable cells in the primary EOC cells, suggesting that pharmacological inhibition of p38 MAPK might not be an effective therapeutic strategy for EOC.

Keywords: carboplatin; chemoresistance; epithelial ovarian cancer; p38 MAPK; primary EOC cells; surviving.

Figure 1

Validation of the kinase array data by Western blotting. A2780s and A2780cp cells were left untreated or treated with 50 μM carboplatin for 24 h, and the whole cell lysates were collected for Western blotting to measure total and phosphorylated p38 (A), Chk-2 (B), and p53 (C). β-actin was used as the loading control. (A,B) Phosphorylated p38 (p-p38) and Chk-2 (pChk-2) were quantified using Odyssey imaging software and normalized against total p38 and Chk-2. Relative p-p38/total p38 and pChk-2/total Chk-2 ratio (fold change) were shown as mean ± SE of four and three independent experiments, respectively, with that in untreated A2780s cells designated as 1. (C) Total p53 was quantified using Odyssey imaging software and normalized against β-actin and expressed as relative fold change with that in carboplatin-treated A2780s cells designated as 1. The relative p53 level was shown as mean ± SE of three independent experiments. * Significantly different (p < 0.05).

Figure 2

Effect of p38 MAPK inhibition by SB203580 on carboplatin sensitivity in A2780s and A2780cp cells. (A) A2780s and A2780cp cells were treated with increasing concentrations of carboplatin for 48 h. Phosphorylation of p38 and cleavage of Poly(ADP-ribose) polymerase (PARP) were analyzed by Western blotting. Two antibodies were used to examine the cleaved PARP: an antibody that only recognizes the cleaved PARP (top panel) and an antibody that recognizes both full-length and cleaved PARP (the lower panel). Both antibodies showed the same results. β-actin was used as the loading control. Two independent experiments showed the same results. (B) A2780s and A2780cp cells were treated with increasing concentrations of carboplatin in the presence of SB203580 (10 μM) or an equal volume of DMSO (the vehicle control) for 72 h. Cell viability was determined by the neutral red uptake assay. Data are shown as mean ± SE of seven independent experiments. * Significantly different (p < 0.05).

Figure 3

Inhibition of p38 MAPK by SB203580 has minimal or no effect on carboplatin sensitivity in primary epithelial ovarian cancer (EOC) cells. Primary EOC cells isolated from ascites of six patients with high-grade serous EOC were cultured in adherent condition for one day and then treated with increasing concentrations of carboplatin in the presence of SB203580 (10 μM) or an equal volume of DMSO (the vehicle control) for 72 h. Cell viability was determined by the neutral red uptake assay. (A) Data are shown as mean ± SE of three to five independent experiments. * Significantly different (p < 0.05). (B) The IC₅₀ for carboplatin in these primary EOC cells in the presence of SB203580 or DMSO is shown.

Figure 4

Inhibition of p38 MAPK by SB203580 increases the number of viable cells in primary EOC cells. Primary EOC cells were cultured in adherent condition and treated with 10 μM SB203580 or an equal volume of DMSO (the vehicle control). Viable cells were measured at 48 and 72 h using the neutral red uptake assay and expressed as fold changes (SB203580 versus DMSO). Data are shown as mean ± SE of three independent experiments. * Significantly different (p < 0.05).

Figure 5

Inhibition of p38 MAPK by SB203580 increases survivin expression in primary EOC cells. Primary EOC cells were left untreated or treated with 200 μM carboplatin in the presence of SB203580 (10 μM) or an equal volume of DMSO (the vehicle control) for 48 h. (A) Phosphorylation of p38 MAPK and MAPKAPK2 (a direct substrate target of p38 MAPK), as well as survivin were examined by Western blotting. β-actin was used as the loading control. (B) The Western blotting results of survivin were quantified using Odyssey imaging software. The density of the survivin bands was normalized to that of β-actin. The density of the bands in the DMSO/UT cells was designated as 1. The relative level (fold change) of survivin was shown as mean ± SE of three to six independent experiments. * Significantly different (p < 0.05).

Figure 6

Inhibition of p38 MAPK by SB203580 increases the number of viable cells in the primary EOC cells cultured in spheroids. Primary EOC cells were cultured in spheroids in 24-well low attachment plates (50,000 cells/well) and treated with 10 μM SB203580 or an equal volume of DMSO (the vehicle control) for three days. The number of viable cells was determined using the neutral red uptake assay and shown as absorbance at 540 nm. Data are shown as mean ± SD of three spheroid cultures. * Significantly different (p < 0.05).