Effect of Aplidin in acute lymphoblastic leukaemia cells

Abstract

The cytotoxic effect of Aplidin was investigated on fresh leukaemia cells derived from children with B-cell-precursor (BCP) acute lymphoblastic leukaemia (ALL) by using stromal-layer culture system and on four cell lines, ALL-PO, Reh, ALL/MIK and TOM-1, derived from patients with ALL with different molecular genetic abnormalities. In ALL cell lines Aplidin was cytotoxic at nanomolar concentrations. In the ALL cell lines the drug-induced cell death was clearly related to the induction of apoptosis and appeared to be p53-independent. Only in ALL-PO 20 nM Aplidin treatment caused a block of vascular endothelial growth factor (VEGF) secretion and downregulation of VEGF-mRNA, but Aplidin cytotoxicity does not seem to be related to VEGF inhibition since the sensitivity of ALL-PO cells to Aplidin is comparable to that observed for the other cells used. Aplidin induced a G(1) and a G(2) M block in ALL cell lines. In patient-derived leukaemia cells, Aplidin induced a strong cytotoxicity evidenced in a stroma-supported immunocytometric assay. Cells from children with genetic abnormalities such as t(9;22) and t(4;11) translocations, associated with an inferior treatment outcome, were sensitive to Aplidin to the same extent as that observed in other BCP-ALL cases. Aplidin exerted a strong cell killing effect (>88%) against primary culture cells from five relapsed ALL cases, at concentrations much lower than those reported to be achieved in plasma of patients receiving Aplidin at recommended doses. Taken together these data suggest that Aplidin could be a new anticancer drug to be investigated in ALL patients resistant to available therapy.

Figure 1

Effect of 1 h (A) or 15, 60 min 24 h (B) Aplidin exposure on cell growth evaluated at different time intervals after treatment and drug-washout on ALL-PO, Reh, ALL/MIK and TOM-1. Each point is the mean of three replicates; bars respresent the standard deviation.

Figure 2

Cell cycle phase perturbations induced on (A) ALL-PO, (B) Reh, (C) ALL/MIK and (D) TOM-1 cells treated for 1 h with 0, 5, 10 or 20 nM Aplidin. Monoparametric DNA flow cytometric analysis were performed at different time intervals after drug-washout.

Figure 3

Biparametric BrdU/DNA analysis performed in Reh cells treated for 1 h with 10 nM Aplidin. During the last 15 min of drug treatment 20 μM BrdU was added to the cells, then the cells were washed with PBS and drug-free medium was provided. The flow cytometric analysis were performed at different time intervals after drug-washout. (A) DNA histograms of BrdU-negative control cells; (B) DNA histograms of BrdU-negative Aplidin-treated cells; (C) DNA histograms of BrdU-positive control cells; (D) DNA histograms of BrdU-positive Aplidin-treated cells.

Figure 4

(A) Detection of apoptosis in ALL cells by TdT-dUTP flow cytometric analysis. Cells were treated with different concentrations of Aplidin and the biparametric FSC/TdT-dUTP analysis were performed after 72 h after drug-washout. (B) Percentage of apoptotic cells evaluated by TdT-dUTP flow cytometric analysis. Cells were treated with different concentrations of Aplidin and biparametric FSC/TdT-dUTP analysis were performed at different time intervals after drug-washout.

Figure 5

Detection of active caspase-3 in ALL cells by flow cytometric analysis. Cells were treated with different concentrations of Aplidin and the biparametric FSC/caspase-3 analysis was performed at different times after drug-washout. In the figure are reported the analysis performed at 24 h after drug-washout. (A) control cells; (B) 20 nM Aplidin-treated cells.

Figure 6

Autoradiography of a typical RNAse protection assay on the four different human leukaemic cell lines. Human ovarian cancer A2780 cells were used as an internal control.

Figure 7

Autoradiography of RNAse protection assay on ALL-PO cells treated with 20 nM Aplidin performed at different time intervals after drug-washout.

Figure 8

Vascular endothelial growth factor mRNA levels in human leukaemic ALL-PO, ALL-MIK and Reh cell lines treated with 20 nM Aplidin and performed at different time intervals after drug-washout. Data have been obtained by densitometric analysis and expressend as % of control untreated cells. Each column represents the mean of three independent replicates. The bars represent s.d. ^*=P<0.05 (Duncan test).

Figure 9

Vascular endothelial growth factor-A concentration in the medium of ALL-PO cells treated for 1 h with 20 nM Aplidin and evaluated at 0, 6 and 24 h after drug-washout. The values express the % of VEGF concentration in the medium of treated cells with regard to control cells.

Figure 10

Example of light-scatter dot-plot analysis of blast cells growing on stroma feeder layer evaluated by flow cytometry. Forward and side scatter analysis were evaluated at the beginning of culture (A), after 7 days of culture (B, control cells) and after 7 days with 5 nM Aplidin (C, treated cells).