Skip to main content
SpringerLink
Log in

Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Swaminath CS, Shortt HE, Anderson LAP (2006) Transmission of Indian kala-azar to man by the bites of Phlebotomus argentipes, ann and brun. Indian J Med Res 123:473–477

    CAS  PubMed  Google Scholar 

  2. Desjeux P (1996) Leishmaniasis public health aspects and control. Clin Dermatol 14:417–423

    Article  CAS  PubMed  Google Scholar 

  3. Alvar J, Vélez ID, Bern C et al (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7:e35671. doi:10.1371/journal.pone.0035671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. NVBDCP National Vector Borne Disease Control Programme. http://nvbdcp.gov.in/ka-cd.html. Accessed 29 Dec 2015

  5. Singh N (2006) Drug resistance mechanisms in clinical isolates of Leishmania donovani. Indian J Med Res 123:411–422

    CAS  PubMed  Google Scholar 

  6. Verma RK, Prajapati VK, Verma GK et al (2012) Molecular docking and in vitro antileishmanial evaluation of chromene-2-thione analogues. ACS Med Chem Lett 3:243–247. doi:10.1021/ml200280r

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 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 352:261–270. doi:10.1007/s11010-011-0762-0

    Article  CAS  PubMed  Google Scholar 

  8. Kawiak A, Zawacka-Pankau J, Lojkowska E (2012) Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway. J Nat Prod 75:747–751. doi:10.1021/np3000409

    Article  CAS  PubMed  Google Scholar 

  9. Seshadri P, Rajaram A, Rajaram R (2011) Plumbagin and juglone induce caspase-3-dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes. Free Radic Biol Med 51:2090–2107. doi:10.1016/j.freeradbiomed.2011.09.009

    Article  CAS  PubMed  Google Scholar 

  10. Srinivas P, Gopinath G, Banerji A et al (2004) Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells. Mol Carcinog 40:201–211. doi:10.1002/mc.20031

    Article  CAS  PubMed  Google Scholar 

  11. Lorsuwannarat N, Saowakon N, Ramasoota P et al (2013) The anthelmintic effect of plumbagin on Schistosoma mansoni. Exp Parasitol 133:18–27. doi:10.1016/j.exppara.2012.10.003

    Article  CAS  PubMed  Google Scholar 

  12. Sumsakul W, Plengsuriyakarn T, Chaijaroenkul W et al (2014) Antimalarial activity of plumbagin in vitro and in animal models. BMC Complement Altern Med 14:15. doi:10.1186/1472-6882-14-15

    Article  PubMed  PubMed Central  Google Scholar 

  13. Croft SL, Evans AT, Neal RA (1985) The activity of plumbagin and other electron carriers against Leishmania donovani and Leishmania mexicana amazonensis. Ann Trop Med Parasitol 79:651–653

    CAS  PubMed  Google Scholar 

  14. Hazra B, Sarkar R, Bhattacharyya S et al (2002) Synthesis of plumbagin derivatives and their inhibitory activities against Ehrlich ascites carcinoma in vivo and Leishmania donovani promastigotes in vitro. Phytother Res 16:133–137

    Article  CAS  PubMed  Google Scholar 

  15. Sharma N, Shukla AK, Das M, Dubey VK (2012) Evaluation of plumbagin and its derivative as potential modulators of redox thiol metabolism of Leishmania parasite. Parasitol Res 110:341–348. doi:10.1007/s00436-011-2498-x

    Article  PubMed  Google Scholar 

  16. Khan MOF (2007) Trypanothione reductase: a viable chemotherapeutic target for antitrypanosomal and antileishmanial drug design. Drug Target Insights 2:129–146

    PubMed  PubMed Central  Google Scholar 

  17. Houghton P, Fang R, Techatanawat I et al (2007) The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 42:377–387. doi:10.1016/j.ymeth.2007.01.003

    Article  CAS  PubMed  Google Scholar 

  18. Gupta S, Tiwari S, Bhaduri AP, Jain GK (2002) Anilino-(2-bromophenyl) acetonitrile: a promising orally effective antileishmanial agent. Acta Trop 84:165–173

    Article  CAS  PubMed  Google Scholar 

  19. Kathuria M, Bhattacharjee A, Sashidhara KV et al (2014) Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by orally active clerodane diterpene. Antimicrob Agents Chemother 58:5916–5928. doi:10.1128/AAC.02459-14

    Article  PubMed  PubMed Central  Google Scholar 

  20. McGuire SO, James-Kracke MR, Sun GY, Fritsche KL (1997) An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells. Lipids 32:219–226

    Article  CAS  PubMed  Google Scholar 

  21. Dolai S, Pal S, Yadav RK, Adak S (2011) Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism. J Biol Chem 286:13638–13646. doi:10.1074/jbc.M110.201889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fairlamb A, Blackburn P, Ulrich P et al (1985) Trypanothione: a novel bis (glutathionyl) spermidine cofactor for glutathione reductase in trypanosomatids. Science 80:1485–1487

    Article  Google Scholar 

  23. Carvalho L, Luque-Ortega JR, López-Martín C et al (2011) The 8-aminoquinoline analogue sitamaquine causes oxidative stress in Leishmania donovani promastigotes by targeting succinate dehydrogenase. Antimicrob Agents Chemother 55:4204–4210. doi:10.1128/AAC.00520-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Das M, Mukherjee SB, Shaha C (2001) Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes. J Cell Sci 114:2461–2469

    CAS  PubMed  Google Scholar 

  25. BoseDasgupta S, Das BB, Sengupta S et al (2008) The caspase-independent algorithm of programmed cell death in Leishmania induced by baicalein: the role of LdEndoG, LdFEN-1 and LdTatD as a DNA “degradesome”. Cell Death Differ 15:1629–1640. doi:10.1038/cdd.2008.85

    Article  CAS  PubMed  Google Scholar 

  26. Roy A, Ganguly A, BoseDasgupta S et al (2008) Mitochondria-dependent reactive oxygen species-mediated programmed cell death induced by 3,3′-diindolylmethane through inhibition of F0F1-ATP synthase in unicellular protozoan parasite Leishmania donovani. Mol Pharmacol 74:1292–1307. doi:10.1124/mol.108.050161

    Article  CAS  PubMed  Google Scholar 

  27. Inbaraj JJ, Chignell CF (2004) Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chem Res Toxicol 17:55–62. doi:10.1021/tx034132s

    Article  CAS  PubMed  Google Scholar 

  28. Mukherjee SB, Das M, Sudhandiran G, Shaha C (2002) Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes. J Biol Chem 277:24717–24727. doi:10.1074/jbc.M201961200

    Article  CAS  PubMed  Google Scholar 

  29. Sen N, Das BB, Ganguly A et al (2004) Camptothecin-induced imbalance in intracellular cation homeostasis regulates programmed cell death in unicellular hemoflagellate Leishmania donovani. J Biol Chem 279:52366–52375. doi:10.1074/jbc.M406705200

    Article  CAS  PubMed  Google Scholar 

  30. Irigoín F, Inada NM, Fernandes MP et al (2009) Mitochondrial calcium overload triggers complement-dependent superoxide-mediated programmed cell death in Trypanosoma cruzi. Biochem J 418:595–604. doi:10.1042/BJ20081981

    Article  PubMed  Google Scholar 

  31. López-Arencibia A, García-Velázquez D, Martín-Navarro CM et al (2015) In vitro activities of hexaazatrinaphthylenes against Leishmania spp. Antimicrob Agents Chemother 59:2867–2874. doi:10.1128/AAC.00226-15

    Article  PubMed  PubMed Central  Google Scholar 

  32. Meslin B, Zalila H, Fasel N et al (2011) Are protozoan metacaspases potential parasite killers? Parasit Vectors 4:26. doi:10.1186/1756-3305-4-26

    Article  PubMed  PubMed Central  Google Scholar 

  33. Britta EA, Scariot DB, Falzirolli H et al (2014) Cell death and ultrastructural alterations in Leishmania amazonensis caused by new compound 4-Nitrobenzaldehyde thiosemicarbazone derived from S-limonene. BMC Microbiol 14:236. doi:10.1186/s12866-014-0236-0

    Article  PubMed  PubMed Central  Google Scholar 

  34. Mehta A, Shaha C (2004) Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J Biol Chem 279:11798–11813. doi:10.1074/jbc.M309341200

    Article  CAS  PubMed  Google Scholar 

  35. De Souza W, Rodrigues JCF (2009) Sterol biosynthesis pathway as target for anti-trypanosomatid drugs. Interdiscip Perspect Infect Dis 2009:642502. doi:10.1155/2009/642502

    PubMed  PubMed Central  Google Scholar 

  36. Fridberg A, Buchanan KT, Engman DM (2007) Flagellar membrane trafficking in kinetoplastids. Parasitol Res 100:205–212. doi:10.1007/s00436-006-0329-2

    Article  PubMed  Google Scholar 

  37. Légaré D, Richard D, Mukhopadhyay R et al (2001) The Leishmania ATP-binding Cassette Protein PGPA is an Intracellular Metal-Thiol Transporter ATPase. J Biol Chem 276:26301–26307. doi:10.1074/jbc.M102351200

    Article  PubMed  Google Scholar 

  38. Lazardi K, Urbina JA, de Souza W (1990) Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob Agents Chemother 34:2097–2105. doi:10.1128/AAC.34.11.2097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Funding from CSIR network project HOPE (BSC0114) is acknowledged. BA and MK are recipients of ICMR and CSIR Senior Research fellowships. We thank Dr. A.A. Sahasrabuddhe, Dr. Neena Goyal and Dr. Susanta Kar for generously sharing MHOM/IN/80/DD8, luciferase transfected Leishmania promastigotes and J774.1 macrophages. We thank Mr. A.L. Vishwakarma for assistance in flow cytometry experiments. We also thank Director, CDRI for supporting and providing facilities for this work. This is CDRI communication number 9252.

Author information

Authors and Affiliations

  1. Electron Microscopy Unit, Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, India

    Bhanu Priya Awasthi, Manoj Kathuria, Garima Pant, Neema Kumari & Kalyan Mitra

  2. Academy of Scientific and Innovative Research, Chennai, 600113, India

    Bhanu Priya Awasthi & Kalyan Mitra

Authors
  1. Bhanu Priya Awasthi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manoj Kathuria
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Garima Pant
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Neema Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kalyan Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kalyan Mitra.

Ethics declarations

Conflict of Interest

None

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Awasthi, B.P., Kathuria, M., Pant, G. et al. Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study. Apoptosis 21, 941–953 (2016). https://doi.org/10.1007/s10495-016-1259-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-016-1259-9

Keywords

Search

Navigation