Cryptolepine and
neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two
alkaloids, which only differ by the respective orientation of their
indole and
quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and
antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into
DNA and interfere with the catalytic activity of human
topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human
leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry.
Cryptolepine, and to a lesser extent
neocryptolepine, provoke a massive accumulation of P388 murine
leukemia cells in the G2/M phase. With HL-60 human
leukemia cells, the treatment with
cryptolepine leads to the appearance of a hypo-diploid
DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells,
cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer
drug mitoxantrone suggests that
topoisomerase II may not represent the essential cellular target for the
alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human
leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that
cryptolepine, but not
neocryptolepine, induces cleavage of
poly(ADP-ribose) polymerase but both
alkaloids induce the release of
cytochrome c from the mitochondria. The cleavage of
poly(ADP-ribose) polymerase observed with
cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of
Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-
caspases was found to be enhanced much more strongly with
cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the
caspase cascade, we did not detect internucleosomal cleavage of
DNA in the HL-60 cells treated with the
alkaloids. Altogether, the results shed light on the mechanism of action of these two
plant alkaloids.