Trypanocidal activity of the proteasome inhibitor and anti-cancer drug bortezomib
© Steverding and Wang; licensee BioMed Central Ltd. 2009
Received: 21 June 2009
Accepted: 07 July 2009
Published: 07 July 2009
The proteasome inhibitor and anti-cancer drug bortezomib was tested for in vitro activity against bloodstream forms of Trypanosoma brucei. The concentrations of bortezomib required to reduce the growth rate by 50% and to kill all trypanosomes were 3.3 nM and 10 nM, respectively. In addition, bortezomib was 10 times more toxic to trypanosomes than to human HL-60 cells. Moreover, exposure of trypanosomes to 10 nM bortezomib for 16 h was enough to kill 90% of the parasites following incubation in fresh medium. However, proteasomal peptidase activities of trypanosomes exposed to bortezomib were only inhibited by 10% and 30% indicating that the proteasome is not the main target of the drug. The results suggest that bortezomib may be useful as drug for the treatment of human African trypanosomiasis.
Human African trypanosomiasis or sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei. The parasites live and multiply extracellularly in the blood and tissue fluids in the human host and are transmitted by the bite of infected tsetse flies (Glossina spp.). Millions of people living in 36 sub-Saharan countries are threatened with the disease and the estimated number of infected people is currently between 50,000 and 70,000 . There are only four drugs available for chemotherapy of sleeping sickness and all show some degree of toxic side effects . In addition, drug resistance in T. brucei is an increasing problem [3, 4]. Moreover, at the turn of the millennium, the production of anti-sleeping sickness drugs was under threat as their manufacture was not profitable . Thus, new strategies for the development of new drugs for treatment of sleeping sickness are urgently needed.
The trypanocidal activity of bortezomib was determined with T. brucei bloodstream forms 427-221a while the general cytotoxicity of the drug was evaluated with human myeloid leukaemia HL-60 cells. The tests were analysed by the Alamar Blue assay as described previously . In brief, cells were seeded in 24-well plates in a final volume of 1 ml of appropriate culture medium (trypanosomes: Baltz medium plus 16.7% heat-inactivated foetal bovine serum ; HL-60 cells: RPMI 1640 plus 16.7% heat-inactivated foetal bovine serum) containing various concentration of bortezomib and 1% DMSO. Wells containing medium and 1% DMSO served as controls. The initial densities were 104/ml for trypanosomes and 105/ml for HL-60 cells. After 24 h incubation, 100 μl Alamar Blue (11.11 mg resazurin sodium salt in 100 ml PBS) was added and the cells were incubated for a further 48 h so that the total incubation time was 72 h. Then, the plates were read on a microplate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. The 50% growth inhibition (GI50) values, i.e. the concentration of the drug necessary to reduce the growth rate of trypanosomes and HL-60 cells by 50% to that of controls, was determined by linear interpolation according to the method described in . The minimum inhibitory concentration (MIC) values, i.e. the concentration of the drug at which all trypanosomes and HL-60 cells were killed, was determined microscopically.
To check whether the trypanocidal activity of bortezomib is due to inhibition of the proteasome, the activity of this multi-enzyme complex in trypanosomes exposed to the drug was determined. In this experiment, 107 trypanosomes/ml were incubated with or without 100 μM bortezomib for 2 h. After washing three times with PBS/1% glucose, the trypanosomes were lysed in 10 mM Tris, 2 mM ATP, 0.1 mM EDTA, pH 7, 1 mM DTT, 0.2% NP-40 and centrifuged at 16000 × g. The chymotrypsin-like activity and the trypsin-like activity of the clarified supernatant were assayed in 50 mM HEPES, pH 7.5 with 10 μM Suc-LLVY-AMC and 10 μM Z-GGR-AMC, respectively. Surprisingly, bortezomib inhibited the chymotrypsin-like activity and the trypsin-like activity of the proteasome in trypanosomes only by 30% and 10%, respectively. Under the same experimental conditions, the drug inhibited the proteasomal chymotrypsin-like and trypsin-like activity in HL-60 cells by 100% and 90%, respectively. These findings indicate that the trypanocidal action of bortezomib is most likely not the result from inactivation of the proteasome.
In conclusion, bortezomib has been shown to display substantial trypanocidal activity. The current therapy regime of bortezomib to treat cancer is probably not applicable for treatment of sleeping sickness. The results of this study indicate that a shorter treatment regime with a higher dosage may be appropriate. Importantly, bortezomib can be also administered subcutaneously  whereas most of the current anti-sleeping sickness drugs have to be given intraveneously . Before developing bortezomib as an anti-sleeping sickness drug, animal experiments would need to be performed to establish the in vivo efficacy of this proteasome inhibitor. However, a selectivity index of 10 may be regarded as insufficient for proceeding to animal experiments. The Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR) recommends a selectivity index of >100 to pursue such animal studies . Compared with normal cells, however, the cytotoxicity of bortezomib determined for HL-60 cells may be overestimated. In addition, bortezomib may serve as a lead for the development of analogues with improved selectivity. Another possibility would be to use bortezomib in combination with the current drugs to treat sleeping sickness. Such drug combination regimes may lead to synergistic effects, in which lower amounts of drugs sufficient to kill the parasites would lead to a reduction in toxicity.
We thank Dr Penelope Powell for critical reading of the manuscript.
- Steverding D: The history of African trypanosomiasis. Parasit Vectors. 2008, 1: 3-10.1186/1756-3305-1-3.PubMed CentralView ArticlePubMedGoogle Scholar
- Fairlamb AH: Chemotherapy of human African trypanosomiasis: current and future prospects. Trends Parasitol. 2003, 19: 488-94. 10.1016/j.pt.2003.09.002.View ArticlePubMedGoogle Scholar
- Matovu E, Seebeck T, Enyaru JCK, Kaminsky R: Drug resistance in Trypanosoma brucei spp., the causative agents of sleeping sickness in man and nagana in cattle. Microbes Infect. 2001, 3: 763-70. 10.1016/S1286-4579(01)01432-0.View ArticlePubMedGoogle Scholar
- Delespaux V, de Koning HP: Drugs and drug resistance in African trypanosomiasis. Drug Resist Updat. 2007, 10: 30-50. 10.1016/j.drup.2007.02.004.View ArticlePubMedGoogle Scholar
- Wickware P: Resurrecting the resurrection drug. Nat Med. 2002, 8: 908-9. 10.1038/nm0902-908b.View ArticlePubMedGoogle Scholar
- Caffrey CR, Steverding D: Recent initiatives and strategies to developing new drugs for tropical parasitic diseases. Expert Opin Drug Discov. 2008, 3: 173-86. 10.1517/174604126.96.36.199.View ArticlePubMedGoogle Scholar
- Barrett SV, Barrett MP: Anti-sleeping sickness drugs and cancer chemotherapy. Parasitol Today. 2000, 16: 7-9. 10.1016/S0169-4758(99)01560-4.View ArticlePubMedGoogle Scholar
- Nkemgu-Njinkeng J, Rosenkranz V, Wink M, Steverding D: Antitrypanosomal activities of proteasome inhibitors. Antimicrob Agents Chemother. 2002, 46: 2038-40. 10.1128/AAC.46.6.2038-2040.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Glenn RJ, Pemberton AJ, Royle HJ, Spackman RW, Smith E, Rivett AJ, Steverding D: Trypanocidal effect of α', β'-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity. Int J Antimicrob Agents. 2004, 24: 286-9. 10.1016/j.ijantimicag.2004.02.023.View ArticlePubMedGoogle Scholar
- Steverding D, Spackman RW, Royle HJ, Glenn RJ: Trypanocidal activities of trileucine methyl vinyl sulfone proteasome inhibitors. Parasitol Res. 2005, 95: 73-6. 10.1007/s00436-004-1253-y.View ArticlePubMedGoogle Scholar
- Steverding D, Pemberton AJ, Royle H, Spackman RW, Rivett AJ: Evaluation of the anti-trypanosomal activity of tyropeptin A. Planta Med. 2006, 72: 761-3. 10.1055/s-2006-931600.View ArticlePubMedGoogle Scholar
- Steverding D: The proteasome as a potential target for chemotherapy of African trypanosomiasis. Drug Dev Res. 2007, 68: 205-12. 10.1002/ddr.20188.View ArticleGoogle Scholar
- Utecht KN, Kolesar J: Bortezomib: a novel chemotherapeutic agent for hematologic malignancies. Am J Health Syst Pharm. 2008, 65: 1221-31. 10.2146/ajhp070272.View ArticlePubMedGoogle Scholar
- Merschjohann K, Sporer F, Steverding D, Wink M: In vitro effect of alkaloids on bloodstream forms of Trypanosoma brucei and T. congolense. Planta Med. 2001, 67: 623-7. 10.1055/s-2001-17351.View ArticlePubMedGoogle Scholar
- Baltz T, Baltz D, Giroud C, Crockett J: Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 1985, 4: 1273-7.PubMed CentralPubMedGoogle Scholar
- Huber W, Koella JC: A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop. 1993, 55: 257-61. 10.1016/0001-706X(93)90083-N.View ArticlePubMedGoogle Scholar
- Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliot PJ: Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 1999, 59: 2615-22.PubMedGoogle Scholar
- Millennium Pharmaceuticals Inc: Velcade® (bortezomib) for injection. Full prescribing information. Cambridge, MA, USA; issued June 2008. [http://velcade.com/full_prescrib_velcade.pdf]
- Moreau P, Coiteux V, Hulin C, Leleu X, van de Velde H, Acharya M, Harousseau JL: Prospective comparison of subcutaneous versus intravenous administration of bortezomib in patients with multiple myeloma. Haematologica. 2008, 93: 1908-11. 10.3324/haematol.13285.View ArticlePubMedGoogle Scholar
- Hesse F, Selzer PM, Mühlstädt K, Duszenko M: A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro. Mol Biochem Parasitol. 1995, 70: 157-66. 10.1016/0166-6851(95)00027-X.View ArticlePubMedGoogle Scholar
- Kuzoe FA: Current situation of African trypanosomiasis. Acta Trop. 1993, 54: 153-62. 10.1016/0001-706X(93)90089-T.View ArticlePubMedGoogle Scholar
- Nwaka S, Hudson A: Innovative lead discovery strategies for tropical diseases. Nat Rev Drug Discov. 2006, 5: 941-55. 10.1038/nrd2144.View ArticlePubMedGoogle Scholar
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