Short communicationApoptosis-like death in Leishmania donovani promastigotes induced by diospyrin and its ethanolamine derivative
Introduction
Leishmaniasis, a protozoan parasitic disease, is currently a global health problem occurring as visceral, cutaneous and mucocutaneous manifestations. Visceral leishmaniasis (VL), or kala-azar, caused by Leishmania donovani, is often associated with marked suppression of the host's cell-mediated immune response, leading to severe morbidity, even mortality, if left untreated [1]. Recently, VL is appearing as an opportunistic infection among immunocompromised patients around the world [2]. Treatment of VL mainly relies on pentavalent antimonials and second-line drugs such as amphotericin B and pentamidine. However, resistance to antimonial chemotherapy, in addition to the limitations of high cost, toxicity and difficult route of administration, is causing concern in endemic regions such as Bihar in India [3].
In view of the ongoing challenge to introduce new antileishmanial drugs, traditional medicinal plants are expected to provide valuable ‘leads’. In fact, quinonoid phytochemicals isolated from plants growing in Leishmania-endemic regions around Amazonian forests have shown promise against diseases caused by Trypanosomatidae parasites, including Leishmania spp. [4]. In our laboratory, a tumour-inhibitory naphthoquinonoid, namely diospyrin (1), isolated from Diospyros montana Roxb. (Ebenaceae) was found to inhibit the growth of L. donovani promastigotes [5]. Thereafter, 1 was structurally modified through synthesis of derivatives that, in comparison with 1, exhibited enhanced antiproliferative activity against human cancer cell lines [6], [7]. Incidentally, the cytotoxicity of 1 and analogous quinonoids, isolated from another Diospyros sp., against L. donovani amastigotes has been reported recently [8].
The present study was therefore undertaken on the antileishmanial activity of dimeric naphthoquinonoids comprising alkyl ether, amino, mercapto and epoxide analogues of diospyrin (1). The study revealed a pronounced enhancement in cytotoxicity of the ethanolamino derivative against L. donovani promastigotes in comparison with 1. This prompted us to elucidate for the first time the underlying mechanism of cell death induced by this derivative, as well as its precursor, with regard to the leishmanicidal activity of a quinonoid natural product in vitro.
Section snippets
Drugs
Diospyrin (1), isolated from the stem bark of D. montana Roxb. (Ebenaceae), was converted into its dimethyl ether (2), diethyl ether (3), diamine (4), mono-amine (5), p-chloroaniline (6), β-naphthylamine (7), benzylamine (8), ethyl glycinate (9), ethanolamine (10), aminoacetyl (11), diepoxide (12) and mercaptoethanol (13) derivatives, as described previously [6], [7]. Stock solutions were prepared aseptically in dimethyl sulphoxide (DMSO) and diluted in culture medium so that the DMSO
Cytotoxic effect of diospyrin (1) and its derivatives (2–13) on Leishmania donovani promastigotes in vitro
The cytotoxicity of test compounds against AG83 promastigotes was determined by MTT assay. Amphotericin B, a second-line antileishmanial drug, exhibited an IC50 value of 0.4 ± 0.1 μM. Tested over a concentration range of 0.1–25 μM, six of the twelve diospyrin derivatives (3, 5, 7, 8, 9 and 13) could not inhibit 50% of the parasite cells at 25 μM. However, three other derivatives (2, 10 and 12) showed significant enhancement of activity, with IC50 values of 5.5 ± 0.6 μM (P < 0.05), 2.9 ± 0.5 μM (P < 0.01) and
Discussion
In this study, it was found that the ethanolamine-substituted derivative (10) of diospyrin exhibited significant enhancement of activity against L. donovani promastigotes in comparison with its natural precursor (1). Therefore, the mechanism of cell death was investigated, since to the best of our knowledge no such study on quinonoids has been reported.
Protozoan parasites such as Leishmania can undergo programmed cell death (PCD) in response to stress [11] and drug application [9], [11], [12].
Acknowledgments
The authors are grateful to Prof. A.C. Ghose, Emeritus Scientist, Indian Council of Medical Research (ICMR), at the National Institute of Cholera and Enteric Diseases, Kolkata, India, for guidance and advice throughout the study. Diospyrin derivatives were prepared with the help of Ms. Madhushree Das Sarma (SRF), Council of Scientific & Industrial Research (CSIR).
Funding: Part of this work was supported by research grants from ICMR and Defence Research and Development Organisation (DRDO), New
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Present address: Molecular Oncology Program, H. Lee Moffitt Cancer Centre & Research Institute, Tampa, FL 33613, USA.