The DNA Intercalating Alkaloid Cryptolepine Interferes with Topoisomerase II and Inhibits Primarily DNA Synthesis in B16 Melanoma Cells

Abstract

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds.

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

“…Extracts of the roots are used in decoction to treat fevers due to malaria and stomach disorders [4]. Cryptolepine presents a large spectrum of biological properties, including hypotensive and antipyretic, antimuscarinic, antibacterial and antiinflammatory effects [5]. It also possesses potent activity in vitro against Plasmodium falciparum, which is one of the main parasite species responsible for malaria.…”

“…The mechanism of action of this antimalarial product remains unclear; at least two independent effects may together lead to a potent activity. First, it behaves like a DNA intercalator with a preference for GC-rich sequences [5]. Second, it may act like chloroquin by inhibiting the detoxification of haeme in red blood cells [6].…”

“…Second, it may act like chloroquin by inhibiting the detoxification of haeme in red blood cells [6]. It has been proposed that crytolepine exerts its cytotoxic action via the inhibition of DNA synthesis and stabilization of topoisomerase II-DNA covalent complexes [5]. Neocryptolepine differs from the parent isomer only in terms of the orientation of the indole unit with respect to the quinoline moiety (see Fig.…”

Qualitative and Quantitative Phytochemicalanalysis and DPPH Radical Scavenging Activity of Different Extractsof Flower of Bauhinia Acuminata Linn

“…Alkaloids are a diverse group of secondary metabolites found to have antimicrobial activity by inhibiting DNA topoisomerase [22]. Alkaloid constituents in flower showed that the chloroform extract had higher amounts (0.72±0.03) atropine/g of the extract followed by (0.66±0.02) atropine/g of the ethyl acetate extract.…”

Economic Maximization of Alfalfa Antimicrobial Efficacy Using Stressful Factors

“…For alkaloids, they were intensively reported to possess dignified antimicrobial due to their potency for DNA intercalation and/or topoisomerase inhibition [13]. The functional antimicrobial activity of flavonoids may be because of their capability of complexing with soluble extracellular proteins and with the microbial cell walls [14].…”