THE DNA INTERCALATING ALKALOID CRYPTOLEPINE INTERFERES WITH TOPOISOMERASE-II AND INHIBITS PRIMARILY DNA-SYNTHESIS IN B16 MELANOMA-CELLS

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated fro m the roots of Clyptolepis sanguinolenta. It is characterized by a mul tiplicity of host-mediated biological activities, including antibacter ial, antiviral, and antimalarial properties. To date, the molecular ba sis for its diverse biological effects remains largely uncertain. Seve ral lines of evidence strongly suggest that DNA might correspond to it s principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluore scence, circular, and linear dichroism, as well as by a relaxation ass ay using DNA topoisomerases. The results of various optical and gel el ectrophoresis techniques converge to reveal that the alkaloid binds ti ghtly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts prefere ntially with CC-rich sequences and discriminates against homo-oligomer ic runs of A and T. This study has also led to the discovery that cryp tolepine is a potent topoisomerase II inhibitor and a promising antitu mor agent. It stabilizes topoisomerase II-DNA covalent complexes and s timulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloq uinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes an d accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Fe ulgen reaction and image cytometry reveal that the drug blocks the cel l cycle in G(2)/M phases. It is also shown that the alkaloid is more p otent at inhibiting DNA synthesis rather than RNA and protein synthesi s. Altogether the results provide direct evidence that DNA is the prim ary target of cryptolepine and suggest that this alkaloid is a valid c andidate for the development of tumor active compounds.