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Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin.

Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, van Reet N, Amin DN, Oberg B, Andersson B, Kristensson K, Wigzell H, Rottenberg ME - PLoS Negl Trop Dis (2009)

Bottom Line: Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment.Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites.Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/principal findings: Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/significance: Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

No MeSH data available.


Related in: MedlinePlus

Cordycepin induces programmed cell death of trypanosomes.T.b. brucei were treated for the indicated times with 1 µM cordycepin and staining with annexin V, propidium iodide (PI), and Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) was evaluated by FACS analysis. A T.b. brucei were incubated for the indicated time with 1 µM cordycepin, fixed with digitonin and stained with PI. Note presence of subG1 peaks indicative of DNA fragmentation at 1 h after treatment and extensive DNA degradation at 3 h after incubation with cordycepin. B T.b. brucei were incubated with 1 µM cordycepin, incubated at 37°C and stained with Annexin V at the indicated time points. C DNA fragmentation in individual fixed T.b. brucei before and after incubation with 1 µM cordycepin was visualized by detection of biotinylated nucleotides incorporated onto the free 3′–hydroxyl residues of DNA fragments. Biotinylated nucleotides were then stained with streptavidin-FITC. As a positive control, T.b. brucei were fixed, treated with DNAse I and the fragmented DNA was labeled biotinylated nucleotides. Negative controls included cordycepin untreated labeled and unlabeled parasites. D To study necrosis after treatment of T.b. brucei with 1 µM cordycepin, nuclei were stained with propidium iodide (5 µg/ml) in the absence of a cell permeant and analyzed by FACS.
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pntd-0000495-g007: Cordycepin induces programmed cell death of trypanosomes.T.b. brucei were treated for the indicated times with 1 µM cordycepin and staining with annexin V, propidium iodide (PI), and Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) was evaluated by FACS analysis. A T.b. brucei were incubated for the indicated time with 1 µM cordycepin, fixed with digitonin and stained with PI. Note presence of subG1 peaks indicative of DNA fragmentation at 1 h after treatment and extensive DNA degradation at 3 h after incubation with cordycepin. B T.b. brucei were incubated with 1 µM cordycepin, incubated at 37°C and stained with Annexin V at the indicated time points. C DNA fragmentation in individual fixed T.b. brucei before and after incubation with 1 µM cordycepin was visualized by detection of biotinylated nucleotides incorporated onto the free 3′–hydroxyl residues of DNA fragments. Biotinylated nucleotides were then stained with streptavidin-FITC. As a positive control, T.b. brucei were fixed, treated with DNAse I and the fragmented DNA was labeled biotinylated nucleotides. Negative controls included cordycepin untreated labeled and unlabeled parasites. D To study necrosis after treatment of T.b. brucei with 1 µM cordycepin, nuclei were stained with propidium iodide (5 µg/ml) in the absence of a cell permeant and analyzed by FACS.

Mentions: The mechanisms accounting for the trypanocidal effect of cordycepin were subsequently explored. Several trypanocidal drugs have been shown to activate a programmed cell death of T. brucei [26]. We found that incubation with 1 µM cordycepin induced degradation of DNA by measured by propidium iodide (PI) staining of permeabilized parasites. DNA degradation was detected 1 h after cordycepin treatment and it increased with time (Figure 7A). DNA fragments were also detected by the TUNEL assay in cordycepin-treated parasites (Figure 7C). One of the earliest indications of apoptosis is the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Parasites treated with 1 µM cordycepin showed translocation of phosphatidylserine as indicated by the binding of annexin V as early as 1 h after treatment (Figure 7B). On the other hand, no alterations in mitochondrial redox potential of cordycepin-treated parasites were detected (Figure S1; Text S1). DNA degradation occurred in the absence of cell membrane disruption, which could only be detected 7 h after cordycepin treatment (Figure 7D), indicating that cordycepin induced programmed cell death of T.b. brucei which was followed by a secondary necrosis.


Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin.

Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, van Reet N, Amin DN, Oberg B, Andersson B, Kristensson K, Wigzell H, Rottenberg ME - PLoS Negl Trop Dis (2009)

Cordycepin induces programmed cell death of trypanosomes.T.b. brucei were treated for the indicated times with 1 µM cordycepin and staining with annexin V, propidium iodide (PI), and Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) was evaluated by FACS analysis. A T.b. brucei were incubated for the indicated time with 1 µM cordycepin, fixed with digitonin and stained with PI. Note presence of subG1 peaks indicative of DNA fragmentation at 1 h after treatment and extensive DNA degradation at 3 h after incubation with cordycepin. B T.b. brucei were incubated with 1 µM cordycepin, incubated at 37°C and stained with Annexin V at the indicated time points. C DNA fragmentation in individual fixed T.b. brucei before and after incubation with 1 µM cordycepin was visualized by detection of biotinylated nucleotides incorporated onto the free 3′–hydroxyl residues of DNA fragments. Biotinylated nucleotides were then stained with streptavidin-FITC. As a positive control, T.b. brucei were fixed, treated with DNAse I and the fragmented DNA was labeled biotinylated nucleotides. Negative controls included cordycepin untreated labeled and unlabeled parasites. D To study necrosis after treatment of T.b. brucei with 1 µM cordycepin, nuclei were stained with propidium iodide (5 µg/ml) in the absence of a cell permeant and analyzed by FACS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713411&req=5

pntd-0000495-g007: Cordycepin induces programmed cell death of trypanosomes.T.b. brucei were treated for the indicated times with 1 µM cordycepin and staining with annexin V, propidium iodide (PI), and Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) was evaluated by FACS analysis. A T.b. brucei were incubated for the indicated time with 1 µM cordycepin, fixed with digitonin and stained with PI. Note presence of subG1 peaks indicative of DNA fragmentation at 1 h after treatment and extensive DNA degradation at 3 h after incubation with cordycepin. B T.b. brucei were incubated with 1 µM cordycepin, incubated at 37°C and stained with Annexin V at the indicated time points. C DNA fragmentation in individual fixed T.b. brucei before and after incubation with 1 µM cordycepin was visualized by detection of biotinylated nucleotides incorporated onto the free 3′–hydroxyl residues of DNA fragments. Biotinylated nucleotides were then stained with streptavidin-FITC. As a positive control, T.b. brucei were fixed, treated with DNAse I and the fragmented DNA was labeled biotinylated nucleotides. Negative controls included cordycepin untreated labeled and unlabeled parasites. D To study necrosis after treatment of T.b. brucei with 1 µM cordycepin, nuclei were stained with propidium iodide (5 µg/ml) in the absence of a cell permeant and analyzed by FACS.
Mentions: The mechanisms accounting for the trypanocidal effect of cordycepin were subsequently explored. Several trypanocidal drugs have been shown to activate a programmed cell death of T. brucei [26]. We found that incubation with 1 µM cordycepin induced degradation of DNA by measured by propidium iodide (PI) staining of permeabilized parasites. DNA degradation was detected 1 h after cordycepin treatment and it increased with time (Figure 7A). DNA fragments were also detected by the TUNEL assay in cordycepin-treated parasites (Figure 7C). One of the earliest indications of apoptosis is the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Parasites treated with 1 µM cordycepin showed translocation of phosphatidylserine as indicated by the binding of annexin V as early as 1 h after treatment (Figure 7B). On the other hand, no alterations in mitochondrial redox potential of cordycepin-treated parasites were detected (Figure S1; Text S1). DNA degradation occurred in the absence of cell membrane disruption, which could only be detected 7 h after cordycepin treatment (Figure 7D), indicating that cordycepin induced programmed cell death of T.b. brucei which was followed by a secondary necrosis.

Bottom Line: Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment.Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites.Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/principal findings: Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/significance: Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

No MeSH data available.


Related in: MedlinePlus