Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis

Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolat ed 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 ce lls and present antibacterial and antiparasitic properties. Our previous mo lecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. He re we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxici ty of the drugs and their effects on the cell cycle were measured by flow c ytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a m assive accumulation of P388 murine leukemia cells in the G2/M phase. With H L-60 human leukemia cells, the treatment with cryptolepine leads to the app earance of a hypo-diploid DNA content peak (sub-G1) characteristic of the a poptotic cell population. With both P388 and HL-60 cells, cryptolepine prov ed about four times more toxic than its isomer. But the use of the HL-60/MX 2 cell line resistant to the anticancer drug mitoxantrone suggests that top oisomerase II may not represent the essential cellular target for the alkal oids, which are both only two times less toxic to the resistant HL-60/MX2 c ells compared to the parental cells. The capacity of the drugs to induce ap optosis of HL-60 human leukemia cells was examined by complementary biochem ical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but bo th alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlat es with the appearance of a marked sub-G1 peak in the cell cycle experiment s. 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 it s isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal clea vage 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. (C) 2000 Elsevier Science B.V. All rights reserved.