Cisplatin-induced expression of Gb3 enables verotoxin-1 treatment of cisplatin resistance in malignant pleural mesothelioma cells

Abstract

Background: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the membrane glycolipid globotriasosylceramide (Gb3), a molecule associated with drug resistance. Cisplatin- and VT-1-induced apoptosis involves mitogen-activated protein kinase (MAPK) activation, and deactivation of MAPKs is associated with cisplatin resistance. This study aimed to investigate whether a sub-toxic concentration of VT-1 could enhance cisplatin-induced apoptosis and overcome acquired-cisplatin resistance in cultured cancer cell lines.

Method: P31 and H1299 cells with corresponding cisplatin-resistant sub-lines (P31res/H1299res) were incubated with VT-1 and/or cisplatin followed by determination of Gb3 expression, cell viability, apoptosis, and signalling pathways.

Results: Cells from the resistant sub-lines had elevated Gb3 expression compared with the parental cell lines, and cisplatin further increased Gb3 expression, whereas VT-1 reduced the percentage of Gb3-expressing cells. Combination of cisplatin and sub-toxic concentrations of VT-1 led to a super-additive increase of cytotoxicity and TUNEL staining, especially in the cisplatin-resistant sub-lines. Blockade of Gb3 synthesis by a Gb3 synthesis inhibitor not only led to eradicated TUNEL staining of P31 cells, but also sensitised P31res cells to the induction of apoptosis by cisplatin alone. Cisplatin- and VT-1-induced apoptosis involved the MAPK pathways with increased C-Jun N-terminal kinase and MAPK kinase-3 and -6 phosphorylation.

Conclusions: We show the presence of Gb3 in acquired-cisplatin resistance in P31res and H1299res cells. Cisplatin up-regulated Gb3 expression in all cells and thus sensitised the cells to VT-1-induced cytotoxicity. A strong super-additive effect of combined cisplatin and a sub-toxic concentration of VT-1 in cisplatin-resistant malignant pleural mesothelioma cells were observed, indicating a new potential clinical-treatment approach.

Figure 1

Flow cytometry analysis of Gb3 expression in P31 and H1299 cells. Gb3 expression in cells not incubated with and cells incubated for 72 h with 5 mg l^–1 cisplatin or 0.1 μg l^–1 VT-1, respectively. The percentage of Gb3-expressing cells is noted in the right quadrant in each dot plot. Blank shows unspecific secondary anti-body binding, whereas control shows cell not incubated with either cisplatin or VT-1. Representative results out of at least three independent experiments are shown.

Figure 2

Cell viability (FMCA assay) after exposure to increasing concentrations of VT-1 for 72 h. (A) P31 (straight line) and P31res (dotted line) cells exposed to 0.1–5.0 μg l^–1 VT-1. (B) H1299 (straight line) and H1299res (dotted line) cells exposed to 0.001–1 μg l^–1 VT-1. Significant differences (P<0.05) between cell lines is indicated (^*). Mean±s.d. (n =3).

Figure 3

Cell viability (FMCA assay) after exposure of MPM and NSCLC cells to 0.1–10 mg l^–1 cisplatin alone (filled line) or in combination with 0.1 μg l^–1 (P31 sub-lines) or 0.001 μg l^–1 (H1299 sub-lines) of VT-1 (dotted line) for 72 h. (A) P31cells, (B) P31res cells, (C) H1299t cells, and (D) H1299res cells. Significant differences (P<0.05) between cell lines with cisplatin alone and when combined with VT-1 is indicated (^*). Mean±s.d. (n=3).

Figure 4

Flow cytometry analysis of Gb3- and MDR1/Pgp expression of cisplatin-resistant cell sub-lines. (A) Cell surface expression of MDR1/PgP (ordinate) and Gb3 (abscissa) of P31res cells. (B) Cell surface expression of MDR1/PgP (ordinate) and Gb3 (abscissa) of H1299res cells. Representative results are shown and the percentage mean (n=3) of stained cells is noted in each dot plot.

Figure 5

Flow cytometry analysis of Gb3- and MDR1/PgP expression of P31 and H1299 cells and their cisplatin-resistant sub-lines incubated with 10 μmol l^–1 cyclosporin A or 2 μmol l^–1 PPMP for 72 h. Representative results are shown and the percentage mean (n=3) of stained cells is noted in each dot plot.

Figure 6

(A) Flow cytometry analysis of TUNEL staining in P31 cells after 72 h incubation with 5 mg l^–1 cisplatin and 0.1 μg l^–1 VT-1, alone or in combination. Green dots indicate unstained cells and red TUNEL-stained cells. The percentage mean±s.d. (n=3) of TUNEL-stained cells is noted in the dot plots. (B) Flow cytometry analysis of Gb3 expression in P31 and P31res cells after 72 h incubation with 2 μmol l^–1 PPMP. The percentage mean±s.d. (n=3) of Gb3-stained cells is noted in each dot plot. (C) Flow cytometry analysis of TUNEL staining in P31and P31res cells pre-incubated with 2 μmol l^–1 PPMP, and after 72 h incubation with 5 mg l^–1 cisplatin and 0.1 μg l^–1 VT-1, alone or in combination. Green dots indicate unstained cells and red TUNEL-stained cells. The percentage mean±s.d. (n=3) of TUNEL-stained cells is noted in each dot plot. Representative results are shown in A–C.

Figure 7

(A) Caspase-3, (B) -8, and (C) -9 enzyme activity in P31 (black bars) and P31res cells (grey bars) after 24 h incubation with 5 mg l^–1 cisplatin and 0.1 μg l^–1 VT-1, alone or in combination. Significant enzyme activity differences (P<0.05) compared with untreated control is indicated (^*). Mean±s.d. (n=3).

Figure 8

Western blot analysis of total and phosphorylated JNK1/2 and MKK3/6 protein expression of P31and P31res cells after 24 h incubation with 5 mg l^–1 cisplatin and 0.1 μg l^–1 VT-1, alone or in combination. Representative blot (n=3).