RESUMEN
Cryptolepine and neocryptolepine are two indoloquinoline alkaloids isolated from the roots of the African plant Cryptolepis sanguinolenta. Both drugs have revealed antibacterial and antiparasitic activities and are strongly cytotoxic to tumour cells. We have recently shown that cryptolepine can intercalate into DNA and stimulates DNA cleavage by human topoisomerase II. In this study, we have investigated the mechanism of action and cytotoxicity of neocryptolepine, which differs from the parent isomer only by the orientation of the indole unit with respect to the quinoline moiety. The biochemical and physicochemical results presented here indicate that neocryptolepine also intercalates into DNA, preferentially at GC-rich sequences, but exhibits a reduced affinity for DNA compared with cryptolepine. The two alkaloids interfere with the catalytic activity of human topoisomerase II but the poisoning activity is slightly more pronounced with cryptolepine than with its isomer. The data provide a molecular basis to account for the reduced cytotoxicity of neocryptolepine compared with the parent drug.
Asunto(s)
Alcaloides/toxicidad , Antineoplásicos Fitogénicos/toxicidad , Indoles , Sustancias Intercalantes/toxicidad , Quinolinas , Inhibidores de Topoisomerasa II , Alcaloides/metabolismo , Animales , Antineoplásicos Fitogénicos/metabolismo , Bovinos , Dicroismo Circular , ADN/efectos de los fármacos , ADN/metabolismo , Daño del ADN , Huella de ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Alcaloides Indólicos , Sustancias Intercalantes/metabolismo , Células KB , Melanoma Experimental/tratamiento farmacológico , Ratones , Ratones Endogámicos , Raíces de Plantas/química , Plantas Medicinales/química , Especificidad por SustratoRESUMEN
Cryptolepine, matadine, and serpentine are three indoloquinoline alkaloids isolated from the roots of African plants: Cryptolepis sanguinolenta, Strychnos gossweileri, and Rauwolfia serpentina, respectively. For a long time, these alkaloids have been used in African folk medicine in the form of plant extracts for the treatment of multiple diseases, in particular as antimalarial drugs. To date, the molecular basis for their diverse biological effects remains poorly understood. To elucidate their mechanism of action, we studied their interaction with DNA and their effects on topoisomerase II. The strength and mode of binding to DNA of the three alkaloids were investigated by spectroscopy. The alkaloids bind tightly to DNA and behave as typical intercalating agents. All three compounds stabilize the topoisomerase II-DNA covalent complex and stimulate the cutting of DNA by topoisomerase II. The poisoning effect is more pronounced with cryptolepine than with matadine and serpentine, but none of the drugs exhibit a preference for cutting at a specific base. Cryptolepine which binds 10-fold more tightly to DNA than the two related alkaloids proves to be much more cytotoxic toward B16 melanoma cells than matadine and serpentine. The cellular consequences of the inhibition of topoisomerase II by cryptolepine were investigated using the HL60 leukemia cell line. The flow cytometry analysis shows that the drug alters the cell cycle distribution, but no sign of drug-induced apoptosis was detected when evaluating the internucleosomal fragmentation of DNA in cells. Cryptolepine-treated cells probably die via necrosis rather than via apoptosis. The results provide evidence that DNA and topoisomerase II are the primary targets of cryptolepine, matadine, and serpentine.
Asunto(s)
Alcaloides/farmacología , ADN-Topoisomerasas de Tipo II/química , ADN/química , ADN/efectos de los fármacos , Indoles , Sustancias Intercalantes/farmacología , Quinolinas , Alcaloides/química , Alcaloides/metabolismo , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión/efectos de los fármacos , Muerte Celular/efectos de los fármacos , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Electroquímica , Inhibidores Enzimáticos/farmacología , Polarización de Fluorescencia , Células HL-60 , Humanos , Alcaloides Indólicos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Alcaloides de Triptamina Secologanina/química , Alcaloides de Triptamina Secologanina/metabolismo , Alcaloides de Triptamina Secologanina/farmacología , Análisis Espectral , Inhibidores de Topoisomerasa IIRESUMEN
The properties of certain hybrids 3 and 5 bearing a photoactivatable psoralen group attached to DNA sequence recognizing lexitropsin carriers have been examined. The hybrids bind to poly(dA-dT) with Kapp of 2.8 and 0.9 x 10(7) M-1, i.e. greater than or equal to that of netropsin (Kapp = 1.0 x 10(7) M-1), indicating that the psoralen moiety may contribute to binding in the case of 5. Photoinduced cross-linking of DNA by 3 and 5, while efficient, is less so than that of individual psoralens and reaches a maximum at a ligand to DNA base pair ratio (r) of 0.2. Complementary strand methidium-propyl-EDTA (MPE).Fe(II) footprinting demonstrated that, in the dark, the sequence preferential recognition of hybrids 3 and 5 is dominated by the lexitropsin moiety. Examination of 360 nm photoinduced DNA cross-linking by the hybrids 3 and 5 was carried out using an exonuclease III stop assay. This revealed that > 95% of the DNA remained double stranded, indicating that 3 and 5 generate primarily biadducts at AT-rich sequences. This assay also located individual monoadduct sites, some of which are remote from the dominant cross-linked sites. When the samples were exposed to 254 nm UV light before loading onto the gel to reverse the photoproducts, the pattern of the exonuclease III stop bands was not altered significantly compared with the experiment without 254 nm irradiation. It is concluded that these termination sites include both mono- and biadducts. Electric linear dichroism examination of the DNA complexes of hybrids 3 and 5 (without light activation) provides evidence that the lexitropsin portion binds in the minor groove, while the psoralen portion intercalates in a suitably located site for subsequent photoinduced cross-linking.