Figure 1.
Negative effects of cytostatic drugs on vinca alkaloid-induced apoptosis in tumor cell lines and primary tumor cells. (A) SHEP neuroblastoma cells were stimulated with doxorubicin (doxo; 100 ng/ml), 4-hydro-peroxy-cyclophosphamide (cyclo; 3 μM), etoposide (VP-16; 1 μM), 5-fluorouracil (5-FU; 10 μM) or methotrexate (MTX; 30 μM) and vincristine (VCR, 300 ng/ml) as indicated for 48 h. (B) SHEP cells from Figure 1A were analyzed for cell-cycle distribution using the recently described flow cytometric staining against cyclin-D1 to discriminate cells in G0 and G1 phase and against p-Histone H3 to separate cells in G2 and M-phase in combination with propidium iodide staining (5, 6). (C) SHEP cells were stimulated with betulinic acid (BA, 1 μg/ml), dacarbacine (1 ng/ml) or the death-inducing ligand TRAIL (3 ng/ml) and VCR, as in Figure 1A. (D) 12 different primary samples were simultaneously stimulated with doxo (300 ng/ml), cyclo (1 μM), MTX (30 μM) or VP-16 (0,1 μM) plus VCR (300 ng/ml). Detailed analyses using the Fractional Product method to discriminate synergistic and antagonistic effects are presented in Figure 5E-H. Primary samples were separated by the effects of the combinatorial stimulation in comparison to the independent application. Fractions are presented as percentages with antagonistic (black bars), unchanged (grey bars) or synergistic (white bars) effects when both drugs were applied simultaneously. For all cell line experiments, apoptosis was determined by PI staining of fragmented DNA and FACscan analysis and data are presented as mean±SEM of at least three independent experiments if not stated differently. Statistical analysis was performed using ANOVA, *p<0.05. NS, Not significant.