Aplidine, a new anticancer agent of marine origin, inhibits vascular endothelial growth factor (VEGF) secretion and blocks VEGF-VEGFR-1 (flt-1) autocrine loop in human leukemia cells MOLT-4

Abstract

The mechanism by which aplidine, a marine natural product in early clinical development as an anticancer agent, induces cell growth inhibition and apoptosis has been investigated in the human leukemia cell line MOLT-4. This cell line is characterized not only by the ability to secrete VEGF, but also for the presence on its surface of the VEGF receptor-1 (VEGFR-1). Previous studies from our laboratory concerned with evaluating early changes in gene expression induced by aplidine in MOLT-4 cells have shown that the drug decreases the expression of VEGFR-1 (Marchini et al. Proc Am Assoc Cancer Res 2000; 41: 833). Here, we report the ability of aplidine to block the VEGF/VEGFR-1 loop. We found that aplidine blocked VEGF secretion that was temporally followed by a decrease in both VEGF and VEGFR-1 production. Aplidine did not directly affect either VEGF transcription or stabilization of its mRNA. Transfection of MOLT-4 cells with an antisense VEGF cDNA construct, resulted in inhibition of colony formations. One clone, transfected with sense VEGF cDNA, secreting 8-10 times more VEGF than parental cells, was less sensitive to aplidineinduced cytotoxicity and apoptosis than control cells. Moreover, addition of VEGF in the medium decreased the activity of aplidine in MOLT-4 cells. These data demonstrate that aplidine inhibits the growth and induces apoptosis in MOLT-4 cells through the inhibition of VEGF secretion which blocks the VEGF/VEGFR-1 autocrine loop necessary for the growth of these cells.

Effect of Aplidin in acute lymphoblastic leukaemia cells

“…The present study also shows that Aplidin is a strong inducer of apoptosis, a finding in keeping with previous data obtained on another human leukaemia Molt-4 cell line (Erba et al, 2002;Broggini et al, 2003). In all the cell lines investigated, the Aplidininduced cell death was clearly related to the induction of apoptosis, even if in Reh cells the apoptosis was found only when the cells were exposed to high concentrations of Aplidin.…”

“…We have recently reported that in Molt-4 cells Aplidin causes an inhibition of VEGF secretion and a downregulation of flt-1 (Broggini et al, 2003). The apoptosis induced by Aplidin in Molt-4 cell could be observed also by exposing the leukaemic cells to anti-VEGF antisense oligonucleotide (Gerber et al, 1998;Nor et al, 1999;Broggini et al, 2001Broggini et al, , 2003.…”

“…The apoptosis induced by Aplidin in Molt-4 cell could be observed also by exposing the leukaemic cells to anti-VEGF antisense oligonucleotide (Gerber et al, 1998;Nor et al, 1999;Broggini et al, 2001Broggini et al, , 2003. In these cells, the addition of VEGF could antagonise the apoptotic process induced by Aplidin.…”

“…By using ELISA assay, we tested the level of VEGF secretion in the four leukaemia cell lines used and found that only ALL-PO secrete detectable amounts of the growth factor. Therefore, we evaluated whether a block in VEGF secretion in ALL-PO cells occurs as previously seen in Molt-4 cells (Broggini et al, 2003). We treated ALL-PO cells with 20 nM Aplidin for 1 h and tested the medium of the cells by means of an ELISA assay used to measure VEGF-A levels of secretion at 0, 6 and 24 h after drug-washout.…”

“…It has been reported by our group that Aplidin causes a strong block of VEGF secretion in Molt-4 cells with a subsequent downregulation of the transcription of VEGF and of its receptor VEGFR1 (Broggini et al, 2003). To test the hypothesis that Aplidin could exert its activity in ALL cell lines by inhibiting the VEGF/ VEGFR-1 autocrine loop, we used an RNAse protection assay in each cell line.…”