Antiproliferative and proapoptotic activities of pyranoxanthenones, pyranothioxanthenones and their pyrazole-fused derivatives in HL-60 cells.

Background: Synthetic pyranoxanthenones, pyranothioxanthenones and their pyrazole-fused derivatives, which bind to DNA, block the G2 + M-phases of the cell cycle and inhibit the proliferation of ascitic and solid tumor cell lines in vitro, were tested for their ability to induce apoptosis in the HL-60 cell system.

Materials And Methods: Various markers of tumor cell metabolism, apoptosis induction and mitochondrial permeability transition (MPT) were assayed in vitro to evaluate drug cytotoxicity.

Results: All these compounds, and especially the pyrazole-fused pyranoxanthenones 7, 8 and 10, which were effective in the 3-5 microM range and were more potent than the pyranoxanthenones, reduced the proliferation of HL-60 cells at 2 and 4 days. These antitumor drugs inhibited DNA synthesis at 2 h in relation to their ability to block the cellular uptake of purine and pyrimidine nucleosides within 15 min. Internmucleosomal DNA fragmentation, a late marker of apoptosis, was induced in a concentration-dependent manner by 7 and 10 at 24 h. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, was detected within 12 h in HL-60 cells treated with 7 and 10. In accord with the fact that the caspase cascade is responsible for PARP-1 cleavage, 7 and 10 induced the activities of initiator caspases-2 and -9 and effector caspase-3 within 9 h in HL-60 cells. The release of mitochondrial cytochrome c (Cyt c) was also detected within 9 h in HL-60 cells treated with 7 and 10, consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. However, 7 and 10 neither caused the rapid collapse of mitochondrial transmembrane potential, nor the mitochondrial swelling linked to MPT.

Conclusion: Pyrazole-fused pyranoxanthenones are DNA-interacting antiproliferative drugs that do not directly target mitochondria in cell and cell-free systems to induce the intrinsic pathway of apoptosis.