• 1.

    Olivier M, Gregory DJ, Forget G, 2005. Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view. Clin Microbiol Rev 18: 293305.

    • Search Google Scholar
    • Export Citation
  • 2.

    Kaye PM, Aebischer T, 2011. Visceral leishmaniasis: immunology and prospects for vaccine. Clin Microbiol Infect 17: 14621470.

  • 3.

    Selvapandiyan A, Duncan R, Debrabant A, Lee N, Sreenivas G, Salotra P, Nakhasi HL, 2006. Genetically modified live attenuated parasites as vaccines for leishmaniasis. Indian J Med Res 123: 455466.

    • Search Google Scholar
    • Export Citation
  • 4.

    Solomon M, Pavlotsky F, Leshem E, 2011. Liposomal amphotericin B treatment of cutaneous leishmaniasis due to Leishmania tropica. J Eur Acad Dermatol Venereol 25: 973977.

    • Search Google Scholar
    • Export Citation
  • 5.

    Wortmann G, Zapor M, Ressner R, Fraser S, Hartzell J, Pierson J, Weintrob A, Magill A, 2010. Lipsosomal amphotericin B for treatment of cutaneous leishmaniasis. Am J Trop Med Hyg 83: 10281033.

    • Search Google Scholar
    • Export Citation
  • 6.

    Mishra M, Biswas UK, Jha DN, Khan AB, 1992. Amphotericin versus pentamidine in antimony-unresponsive kala-azar. Lancet 340: 12561257.

  • 7.

    Sindermann H, Engel J, 2006. Development of miltefosine as an oral treatment for leishmaniasis. Trans R Soc Trop Med Hyg 100: S17S20.

  • 8.

    Seifert K, Perez-Victoria FJ, Stettler M, Sánchez-Cañete MP, Castanys S, Gamarro F, Croft SL, 2007. Inactivation of the miltefosine transporter, LdMT, causes miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani and persists in vivo. Int J Antimicrob Agents 30: 229235.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lillig CH, Holmgren A, 2007. Thioredoxin and related molecules–from biology to health and disease. Antioxid Redox Signal 9: 2547.

  • 10.

    Krauth-Siegel RL, Leroux AE, 2012. Low-molecular-mass antioxidants in parasites. Antioxid Redox Signal 17: 583607.

  • 11.

    Krauth-Siegel RL, Comini MA, 2008. Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim Biophys Acta 1780: 12361248.

    • Search Google Scholar
    • Export Citation
  • 12.

    Dormeyer M, Reckenfelderbaumer N, Ludemann H, Krauth-Siegel RL, 2001. Trypanothione-dependent synthesis of deoxyribonucleotides by Trypanosoma brucei ribonucleotide reductase. J Biol Chem 276: 1060210606.

    • Search Google Scholar
    • Export Citation
  • 13.

    Krauth-Siegel RL, Lüdemann H, 1996. Reduction of dehydroascorbate by trypanothione. Mol Biochem Parasitol 80: 203208.

  • 14.

    Flohe L, Hecht HJ, Steinert P, 1999. Glutathione and trypanothione in parasitic hydroperoxide metabolism. Free Radic Biol Med 27: 966984.

  • 15.

    Comini MA, Guerrero SA, Haile S, Menge U, Lünsdorf H, Flohé L, 2004. Validation of Trypanosoma brucei trypanothione synthetase as drug target. Free Radic Biol Med 36: 12891302.

    • Search Google Scholar
    • Export Citation
  • 16.

    Ariyanayagam MR, Oza SL, Guther ML, Fairlamb AH, 2005. Phenotypic analysis of trypanothione synthetase knockdown in the African trypanosome. Biochem J 39: 425432.

    • Search Google Scholar
    • Export Citation
  • 17.

    Saudagar P, Dubey VK, 2011. Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani. Biol Chem 392: 11131122.

    • Search Google Scholar
    • Export Citation
  • 18.

    Reutrakul V, Anantachoke N, Pohmakotr M, Jaipetch T, Yoosook C, Kasisit J, Napaswa C, Panthong A, Santisuk T, Prabpai S, Kongsaeree P, Tuchinda P, 2010. Anti-HIV-1 and anti-inflammatory lupanes from the leaves, twigs, and resin of Garcinia hanburyi. Planta Med 76: 368371.

    • Search Google Scholar
    • Export Citation
  • 19.

    Steele JC, Warhurst DC, Kirby GC, Simmonds MS, 1999. In vitro and in vivo evaluation of betulinic acid as an antimalarial. Phytother Res 13: 115119.

    • Search Google Scholar
    • Export Citation
  • 20.

    Laszczyk MN, 2009. Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy. Planta Med 75: 15491560.

    • Search Google Scholar
    • Export Citation
  • 21.

    Li Y, He K, Huang Y, Zheng D, Gao C, Cui L, Jin YH, 2010. Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells. Mol Carcinog 49: 630640.

    • Search Google Scholar
    • Export Citation
  • 22.

    Chowdhury S, Mukherjee T, Sengupta S, Chowdhury SR, Mukhopadhyay S, Majumder HK, 2011. Novel Betulin derivatives as antileishmanial agents with mode of action targeting type Ib DNA topoisomerase. Mol Pharmacol 80: 694703.

    • Search Google Scholar
    • Export Citation
  • 23.

    Mosmann T, 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 5563.

    • Search Google Scholar
    • Export Citation
  • 24.

    Shukla AK, Patra S, Dubey VK, 2012. Iridoid glucosides from Nyctanthes arbortristis result in increased reactive oxygen species and cellular redox homeostasis imbalance in Leishmania parasite. Eur J Med Chem 54: 4958.

    • Search Google Scholar
    • Export Citation
  • 25.

    Shukla AK, Patra S, Dubey VK, 2011. Evaluation of selected antitumor agents as subversive substrate and potential inhibitor of trypanothione reductase: an alternative approach for chemotherapy of leishmaniasis. Mol Cell Biochem 352: 261270.

    • Search Google Scholar
    • Export Citation
  • 26.

    Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A Laboratory Manual. Second edition. New York: Cold Spring Harbor Laboratory, 5.615.64.

    • Search Google Scholar
    • Export Citation
  • 27.

    Boonstra J, Post JA, 2004. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 337: 113.

    • Search Google Scholar
    • Export Citation
  • 28.

    Sen N, Das BB, Gangulyet A, Mukherjee T, Tripathi G, Bandyopadhyay S, Rakshit S, Sen T, Majumder HK, 2004. Camptothecin-induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani. Cell Death Differ 11: 924936.

    • Search Google Scholar
    • Export Citation
  • 29.

    Roy A, Ganguly A, Dasgupta SB, Das BB, Pal C, Jaisankar P, Majumder HK, 2008. Mitochondria dependent reactive oxygen species mediated programmed cell death induced by 3,3-di indolylmethane through inhibition of F0F1-ATP synthase in unicellular protozoan parasite Leishmania donovani. Mol Pharm 74: 12921307.

    • Search Google Scholar
    • Export Citation
  • 30.

    Kupchan SM, Anderson WK, Bollinger P, Doskotch RW, Smith RM, Renauld JA, Schnoes HK, Burlingame AL, Smith DH, 1969. Tumor inhibitors. XXXIX. Active principles of Acnistus arborescens. Isolation and structural and spectral studies of with aferin A and with acristin. J Org Chem 34: 38583866.

    • Search Google Scholar
    • Export Citation
  • 31.

    Zou H, Henzel WJ, Liu X, Lutschg A, Wang X, 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90: 405413.

    • Search Google Scholar
    • Export Citation
  • 32.

    Ariyanayagam MR, Fairlamb AH, 2001. Ovothiol and trypanothione as antioxidants in trypanosomatids. Mol Biochem Parasitol 115: 189198.

  • 33.

    Steenkamp DJ, 2002. Trypanosomal antioxidants and emerging aspects of redox regulation in the trypanosomatids. Antioxid Redox Signal 4: 105121.

    • Search Google Scholar
    • Export Citation
  • 34.

    Bocedi A, Dawood KF, Fabrini R, Federici G, Gradoni L, Pedersen JZ, Ricci G, 2010. Trypanothione efficiently intercepts nitric oxide as a harmless iron complex in trypanosomatid parasites. FASEB J 24: 10351042.

    • Search Google Scholar
    • Export Citation
  • 35.

    Tovar J, Cunningham ML, Smith AC, Croft SL, Fairlamb AH, 1998. Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival. Proc Natl Acad Sci USA 95: 53115316.

    • Search Google Scholar
    • Export Citation
  • 36.

    Das M, Mukherjee SB, Shaha C, 2001. Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes. J Cell Sci 114: 24612469.

    • Search Google Scholar
    • Export Citation
  • 37.

    Das R, Roy A, Dutta N, Majumder HK, 2008. Reactive oxygen species and imbalance of calcium homeostasis contributes to curcumin induced programmed cell death in Leishmania donovani. Apoptosis 13: 867882.

    • Search Google Scholar
    • Export Citation
  • 38.

    Lee N, Bertholet S, Debrabant A, Muller J, Duncan R, Nakhasi HL, 2002. Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ 9: 5364.

    • Search Google Scholar
    • Export Citation
  • 39.

    Castanys-Munoz E, Brown E, Coombs GH, Mottram JV, 2012. Leishmania mexicana metacaspase is a negative regulator of amastigote proliferation in mammalian cells. Cell Death Dis 3: e385.

    • Search Google Scholar
    • Export Citation
  • 40.

    Moss CX, Westrop GD, Juliano L, Coombs GH, Mottram JC, 2007. Metacaspase 2 of Trypanosoma brucei is a calcium-dependent cysteine peptidase active without processing. FEBS Lett 581: 56355639.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 
 

 

 

 

 

 

 

Molecular Mechanisms of In vitro Betulin-Induced Apoptosis of Leishmania donovani

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  • Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India

Although leishmanial infections of humans occur globally, the major health impact lies in developing nations, thus, leishmaniases remain “neglected” diseases for new drugs development. Multidrug resistance has been documented in most countries where leishmaniases is endemic. Betulin is a widely available and affordable natural product exerting leishmanicidal activity at micromolar concentration. In this study, the molecular mechanisms of death that contribute to the anti-leishmanial activity of betulin are investigated. In promastigotes, betulin stimulated reactive oxygen species generation at micromolar concentrations in Leishmania. Apoptosis was observed in betulin-treated promastigotes using flow cytometric analysis of treated cells stained with annexin V-FITC and propidium iodide. Furthermore, betulin treatment of promastigotes led to mitochondrial membrane damage, activation of caspase-like proteases, and DNA fragmentation in Leishmania donovani promastigotes. Betulin treatment of amastigotes cultured within macrophages, resulted in a reduced number of amastigotes, with no substantive cytotoxic damage to the host macrophage cells at leishmanicidal drug concentrations.

Author Notes

* Address correspondence to Vikash K. Dubey, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India 781039. E-mail: vdubey@iitg.ernet.in

Financial support: Research fellowship to PS by IIT Guwahati is acknowledged. Financial support by Department of Biotechnology, Government of India in the form of research grant (Project no: BT/PR3409/MED/29/326/2011) to VKD is also acknowledged.

Authors' addresses: Prakash Saudagar and Vikash Kumar Dubey, Indian Institute of Technology Guwahati – Biotechnology, Guwahati, Assam, India, E-mails: vdubey@iitg.ernet.in and saudagar@iitg.ernet.in.

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