Luteolin, an Abundant Dietary Component is a Potent Anti-leishmanial Agent that Acts by Inducing Topoisomerase II-mediated Kinetoplast DNA Cleavage Leading to Apoptosis

Molecular Medicine 6(6): 527–541, 2000 Molecular Medicine © 2000 The Picower Institute Press Luteolin, an Abundant Dietary Component is a Potent Anti-leishmanial Agent that Acts by Inducing Topoisomerase II-mediated Kinetoplast DNA Cleavage Leading to Apoptosis Bidyottam Mittra,1 Asim Saha,2 Arnab Roy Chowdhury,1 Chiranjib Pal,2 Suparna Mandal,3 Sibabrata Mukhopadhyay,3 Santu Bandyopadhyay,2 and Hemanta K. Majumder1 1 Molecular Parasitology Laboratory, 2Cellular Immunology Division, 3Medicinal Chemistry Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta, India Accepted March 24, 2000. Abstract Background: Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. Materials and Methods: Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apop- tosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)labeled Annexin V. Results: Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-IImediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 M and 45.5 M, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. Conclusion: Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design. Introduction stituents of a number of herbal and traditional medicines (2). Quercetin and luteolin, important members of the flavonoid family, are present in fairly large amounts in fruits, vegetables, olive oil, red wine, tea, and the propolis of beehive (3). The average human daily intake of major flavonoids, such as quercetin, myricetin, luteolin, etc., is in the range of 16 mg per day (4). In addition to having many therapeutic uses, quercetin was found to inhibit the growth of leukemic cells, Ehrlich ascites tumor cells, and other ascites tumor cells (5–8). Quercetin also was shown to The flavonoids are a large and complex group of compounds that occur throughout the plant kingdom, providing color and flavor. They exhibit a wide spectrum of pharmacological properties (1). Flavonoids form active con- Address correspondence and reprint requests to: Dr. Hemanta K. Majumder, Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta - 700 032, India. Fax: 91-33-473-5197/0284; E-mail: hkmajum@cal2. vsnl.net.in 528 Molecular Medicine, Volume 6, Number 6, June 2000 potentiate the cytotoxicity of DNA-damaging anticancer drugs, such as cis-platin (9–11). Several flavonoid compounds have been shown to exert their action by interacting with DNA topoisomerases, the key enzymes that govern important cellular processes like replication, transcription, recombination, integration, and chromosome segregation (12). The nonintercalating flavonoids, genistein and orobol, and the DNA-intercalating flavonoids, quercetin, myricetin, and biacelin, promote topoisomerasemediated, site-specific DNA cleavage in mammalian cells in vitro (13,14). Recently, it was reported that quercetin and the related flavones, acacetin, apigenin, kaempherol, and morin, inhibited eukaryotic topoisomerase I-catalyzed DNA religation. These compounds do not act directly on the catalytic intermediate and also do not interfere with DNA cleavage (15). As stabilization of topoisomerase II DNA “cleavable complex” is a known cytotoxic lesion in vivo, these flavonoids are potential candidates in designing antineoplastic, antibacterial, or antiparasitic drugs. Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions, through the disfiguring mucocutaneous forms, to the often fatal visceralizing form (16). With the spread of human immunodeficiency virus (HIV) and general low standards of public hygiene, the parasites pose a much greater threat than before, particularly in developing countries. To make the situation even worse, some parasite strains also have developed resistance against the classical antimonial drugs, like sodium stibogluconate or megalumine antimonate. The second line of drugs, amphotericin B and pentamidines, although used clinically, are very toxic (17). Therefore, improved therapy of leishmanial infections is still desirable and the need for newer molecular targets on which to base the future treatment strategies clearly is justified. In search for such strategies, the DNA topoisomerases of Leishmania offer most attractive targets. Leishmania parasites contain a unique DNA structure, the kinetoplast DNA (kDNA) within their mitochondria. kDNA is a network of thousands of topologically interlocked minicircles (~1 kb) and several thousands of maxicircles (~25 kb). Replication of this massive intercatenated network structure involves numerous topological interconversions. The re- lease and reattachment of individual minicircles from the network and the final splitting of the double-sized parent network into daughter networks all involve kinetoplast specific topoII-mediated manipulation of DNA topology (18). Several important anti-trypanosomal and anti-leishmanial drugs, like pentamidine, berenil or samorin, target the kinetoplast-specific topoisomerase II (19). These drugs and the antitumor drug, etoposide and 4-[9-Acridinylamino]N-[methanesulfonyl]-m-anisidine (mAMSA) promote the formation of a “cleavable complex” between kinetoplast minicircle DNA and mitochondrial type II topoisomerase (20). Both type I and type II topoisomerases have been characterized in Leishmania (21,22). A plant-derived bisnaphthoquinone, diospyrin, that induces topomediated DNA cleavage recently was reported from our laboratory (23). Identification of indigenous plant-derived compounds that induce topoisomerase-II-mediated cleavage of kDNA minicircles and have potentiality to act as lead compounds for drug design against l (...truncated)