Skip Navigation


JAC Advance Access originally published online on July 10, 2007
Journal of Antimicrobial Chemotherapy 2007 60(3):521-525; doi:10.1093/jac/dkm245
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
60/3/521    most recent
dkm245v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by McBride, J.
Right arrow Articles by Roberts, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McBride, J.
Right arrow Articles by Roberts, C. W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Differential cytotoxicity of phospholipid analogues to pathogenic Acanthamoeba species and mammalian cells

James McBride, Alexander B. Mullen, K. Christine Carter and Craig W. Roberts*

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, Scotland, UK

* Corresponding author. Tel/Fax: +44-141-548-4823; E-mail: c.w.roberts{at}strath.ac.uk

Received 6 February 2007; returned 18 March 2007; revised 1 June 2007; accepted 12 June 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Objectives: Previous studies have reported the ability of several phospholipid analogues to successfully inhibit the growth of Acanthamoeba species in vitro. This study tests further phospholipid analogues, either as free drug or in liposomal formulations, and unlike previous studies, examines their comparative toxicities to mammalian cells.

Methods: The relative cytotoxic activities of the phospholipid derivatives hexadecyl-PC, octadecyl-PC, elaidyl-PC, erucyl-PC and edelfosine, against Acanthamoeba castellanii, Acanthamoeba polyphaga and a rabbit corneal epithelial (RCE) cell line, was determined by the alamarBlueTMTM assay. Free and liposomal formulations were compared for hexadecyl-PC and elaidyl-PC.

Results: Both hexadecyl-PC and octadecyl-PC (IC50 values between 3.9 and 7.8 µM) demonstrated considerable activity against A. castellanii, as did elaidyl-PC (IC50 values between 15.6 and 31.25 µM). Both hexadecyl-PC and elaidyl-PC also proved effective against A. polyphaga (IC50 values between 15.6 and 31.25 and between 31.25 and 62.5 µM, respectively). In contrast, neither erucyl-PC nor edelfosine was inhibitory against either Acanthamoeba species. The growth of RCE cells was inhibited by octadecyl-PC, erucyl-PC and edelfosine (octadecyl-PC and erucyl-PC IC50 values between 7.8 and 15.6 µM and edelfosine IC50 values between 31.25 and 62.5 µM). Liposomal formulations of hexadecyl-PC and elaidyl-PC were less effective than free drug against both Acanthamoeba species.

Conclusions: These results demonstrate that hexadecyl-PC has the highest therapeutic index and is the most promising for the treatment of acanthamoebiasis.

Keywords: APC , liposome , keratitis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Acanthamoeba species are free-living protozoan organisms and opportunistic human pathogens that have a ubiquitous distribution in the environment.1–5 They are responsible for both granulomatous amoebic encephalitis and Acanthamoeba keratitis in humans.1–5 Curative therapy for systemic infection is extremely rare, although treatment of ocular disease is arduous and subject to frequent complications, such as recrudescence of disease when trophic amoebae re-emerge from dormant cysts. There is a requirement for more effective antimicrobials to treat Acanthamoeba infection.

Phospholipid analogues demonstrate varying degrees of efficacy against several protozoan organisms including Leishmania spp., Trypanosoma cruzi, Trypanosoma brucei subspecies, Entamoeba histolytica and Acanthamoeba spp.6–16 As phospholipid analogues were originally developed as antineoplastics, their inherent toxicity to mammalian cells also needs to be taken into account. Herein, the alamarBlueTM assay method was used to compare the activity of hexadecyl-PC, octadecyl-PC, elaidyl-PC and erucyl-PC and the alkylglycerophosphocholine (AGPC) edelfosine, against Acanthamoeba castellanii, Acanthamoeba polyphaga and rabbit corneal epithelial (RCE) cells. For both hexadecyl-PC and elaidyl-PC, liposomal formulations were produced and their activities against Acanthamoeba and RCE cells were compared with those of the free drugs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Transparency declarations
 References
 
Acanthamoeba

A. castellanii (Neff strain) and A. polyphaga (strain 1501/18) were maintained as previously described.13

RCE cells

The RCE cell line (ECACC no. 95081046) was cultured in Dulbecco's modified Eagle's medium Ham's F-12 (1:1) containing 15 mM HEPES and L-glutamine (2 mM), supplemented with 10% heat-inactivated fetal calf serum (Harlan Sera Lab, Loughborough, UK), 10 ng/mL murine epidermal growth factor (Invitrogen Life Technologies, UK) and 5 mg/L bovine insulin (Biowhittaker Europe, UK), penicillin (100 U/mL), streptomycin (100 mg/L) and amphotericin B (250 ng/mL).

Comparison of the relative susceptibilities of A. castellanii, A. polyphaga and RCE cells to phospholipid analogues

Conditions for assessing the susceptibility of Acanthamoeba to compounds have been described previously.13 RCE cells were seeded in triplicate, at 280 cells per well in 50 µL of medium, in 96-well tissue culture plates and allowed to adhere for 3 h at 37°C, 5% CO2. A medium containing hexadecyl-PC, octadecyl-PC, elaidyl-PC or erucyl-PC, each at a concentration range of 500 to 3.9 µM, and edelfosine (all Alexis Biochemicals, Nottingham, UK) at a concentration of 1000 to 7.8 µM, was freshly prepared and 50 µL volumes added to all wells except untreated control cultures, which were instead supplemented with a further 50 µL medium. Phospholipid analogues were dissolved in ethanol and diluted approximately 200–225-fold in culture medium. Plates were incubated for 96 h, either at room temperature for Acanthamoeba or at 37°C for RCE cells, in the dark, with addition of 10 µL of alamarBlueTMTM reagent to control and test wells performed 6 h prior to the end of incubation. Percentage inhibition of alamarBlueTMTM reduction was calculated, as described previously.13 Results are expressed as mean values for each triplicate culture ± SE.

Comparison of the relative susceptibilities of A. castellanii, A. polyphaga and RCE cells to liposomal formulations of hexadecyl-PC and elaidyl-PC

Briefly, 5000 µM of vesicle constituents, comprising 3:3:1 molar ratio of alkylphosphocholine (APC; either hexadecyl-PC or elaidyl-PC), cholesterol and dicetyl phosphate, was melted by heating at 130°C for 2 min. The molten mixture was cooled to 70°C prior to hydration with 5 mL preheated (70°C) tissue culture media to allow vesicle formation. Size reduction was afforded by homogenizing the samples at 8000 ± 100 rpm for 15 min at 70°C, using a Silverson mixer, Model L4R SU (Silverson Machines, UK), fitted with a 5/8'' tubular work head. A. castellanii, A. polyphaga and RCE cells were seeded as before and a medium containing liposomal formulations of hexadecyl-PC and elaidyl-PC, each at a concentration range of 250 to 3.9 µM, was freshly prepared and 50 µL volumes added to all wells except untreated control cultures, which were instead supplemented with a further 50 µL of medium. Incubation of plates with alamarBlueTM TM reagent was performed as before.

Statistics and experimental design

Experiments were performed at least twice, with similar findings. Comparisons were performed by the Mann–Whitney U-test. Tests were performed to establish the range of concentrations at which statistical significance was achieved.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Transparency declarations
 References
 
Susceptibilities of A. castellanii and A. polyphaga to phospholipid analogues

A. castellanii was significantly (P < 0.05) more susceptible to hexadecyl-PC than was A. polyphaga from 1.95 to 15.6 µM. Furthermore, treatment with 15.6 µM was sufficient to almost completely inhibit A. castellanii proliferation, whereas treatment with 31.25 µM was required to inhibit A. polyphaga growth (Figure 1a). Similarly, A. castellanii was more susceptible to octadecyl-PC than was A. polyphaga (P < 0.05) from 7.8 to 125 µM (Figure 1b). A. castellanii was also more susceptible to elaidyl-PC than was A. polyphaga (P < 0.05) from 7.8 to 62.5 µM. Complete inhibition of A. castellanii proliferation occurred at 62.5 µM, whereas A. polyphaga required 125 µM (Figure 1c). Neither A. castellanii nor A. polyphaga was susceptible to erucyl-PC or edelfosine, with significant inhibition of the growth of the amoebae only obtained at the highest doses tested, of 250 and 500 µM, respectively (P < 0.05, Figure 1d and e).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Relative susceptibilities of A. castellanii (closed squares), A. polyphaga (closed diamonds) and RCE cells (open circles), to the following over 96 h: (a) hexadecyl-PC (IC50 values were between 3.9 and 7.8 µM, 15.6 and 31.25 µM and 62.5 and 125 µM, for A. castellanii, A. polyphaga and RCE cells, respectively); (b) octadecyl-PC (IC50 values were between 3.9 and 7.8 µM, 31.25 and 62.5 µM and 7.8 and 15.6 µM, for A. castellanii, A. polyphaga and RCE cells, respectively); (c) elaidyl-PC (IC50 values were between 15.6 and 31.25 µM, 31.25 and 62.5 µM and 62.5 and 125 µM, for A. castellanii, A. polyphaga and RCE cells, respectively); (d) erucyl-PC (IC50 values were between 125 and 250 µM for both A. castellanii and A. polyphaga and between 7.8 and 15.6 µM for RCE cells); (e) edelfosine (IC50 values were between 250 and 500 µM, in excess of 500 µM and between 31.25 and 62.5 µM, for A. castellanii, A. polyphaga and RCE cells, respectively); (f) hexadecyl-PC vesicles (IC50 values were in excess of 125 µM for both A. castellanii and A. polyphaga and between 7.8 and 15.6 µM for RCE cells); (g) elaidyl-PC vesicles (IC50 values were between 62.5 and 125 µM, in excess of 125 µM, and between 15.6 and 31.25 µM, for A. castellanii, A. polyphaga and RCE cells, respectively). Acanthamoeba and RCE cell numbers were assessed by measuring the percentage of alamarBlueTM reduction relative to untreated control cultures over 96 h. Results are expressed as mean values for triplicate cultures ± SEs.

 
Susceptibilities of RCE cells to phospholipid analogues

RCE cells were significantly less susceptible to hexadecyl-PC and elaidyl-PC when compared with either species of Acanthamoeba from 15.6 to 62.5 µM (P < 0.05, Figure 1a and c). RCE cells were more susceptible to octadecyl-PC treatment from 15.6 to 125 µM than was A. polyphaga (P < 0.05), but less susceptible than A. castellanii (P < 0.05). However, RCE cells were more susceptible to edelfosine than either Acanthamoeba spp. from 62.5 to 500 µM (P < 0.05, Figure 1e).

Comparison of the relative susceptibilities of A. castellanii, A. polyphaga and RCE cells to liposomal formulations of hexadecyl-PC and elaidyl-PC

Neither A. castellanii nor A. polyphaga was susceptible to liposomal formulations of hexadecyl-PC at any concentration tested, from 1.95 to 125 µM (Figure 1f). A. castellanii was more susceptible to liposomal elaidyl-PC when compared with A. polyphaga from 31.25 to 125 µM. Nevertheless, the liposomal formulation of elaidyl-PC was less effective than free drug (P < 0.05, Figure 1c and g). RCE cells were more susceptible to liposomal hexadecyl-PC than either species of Acanthamoeba from concentrations of 3.9 to 125 µM. Moreover, RCE cells were more susceptible to the liposomal formulation when compared with free drug treatment from concentrations of 7.8–62.5 µM (P < 0.05). Similarly, RCE cells were more sensitive to liposomal elaidyl-PC than were either Acanthamoeba spp., and the liposomal formulation was again more effective than free drug treatment from 15.6 to 62.5 µM (P < 0.05).

The relative susceptibilities of each Acanthamoeba species and RCE cells to the various phospholipids analogues and liposomal formulations are summarized in Table 1.


View this table:
[in this window]
[in a new window]

  		Table 1. Phospholipid derivatives and their respective IC50 values (µM) for A. castellanii, A. polyphaga and RCE cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Transparency declarations
 References
 
Hexadecyl-PC and elaidyl-PC demonstrated efficacy against both A. castellanii and A. polyphaga at concentrations that were non-toxic to RCE cells. Although octadecyl-PC was effective against A. castellanii, higher concentrations more readily inhibited proliferation of RCE cells than A. polyphaga. Other derivatives, such as erucyl-PC and edelfosine, failed to reduce Acanthamoeba proliferation. Although the reason for their ineffectiveness is not known, it is tempting to speculate that their comparatively longer chain lengths preclude their internalization by trophic amoebae.

The results of the present study partly concur with those of a previous study, which reported 40 µM hexadecyl-PC (16-carbon chain length) to be completely effective against both A. castellanii and A. polyphaga (minor differences may reflect differences in the incubation times used in each study).16 The previous study also reported a moderate efficacy for octadecyl-PC (18-carbon chain length) in the inhibition of A. polyphaga growth.16 Walochnik et al.16 also assessed alkylphospholipids of longer chain lengths with various degrees of saturation and, with the exception of (Z,Z)-6,12-eicosadienylphosphocholine, reported little success. Therefore, the effects of erucyl-PC, an APC with a 22-carbon chain length, and edelfosine, an AGPC with an 18-carbon chain length, were assessed and both found to be poorly effective against either species of Acanthamoeba. Interestingly, previous studies have reported the efficacy of edelfosine both against Leishmania donovani and a number of other trypanosomatids at low micromolar levels.7 Thus, Acanthamoeba species appear to be susceptible to a more limited range of available phospholipid analogues when compared with other protozoa. The antiprotozoan activity of phospholipid derivatives requires that they be successfully internalized. Interestingly, this process is reported not to occur in L. donovani cells with a 15-fold reduced sensitivity to HPC.17 Thus, one possible mechanism for the lack of efficacy demonstrated by both erucyl-PC and edelfosine might be due to these analogues not being readily internalized by Acanthamoeba. This hypothesis would need to be addressed in future studies.

Various mechanisms have been suggested to explain how phospholipid analogues exert their antiprotozoan activities, which include inhibition of lipid remodelling, phospholipid biosynthesis and intracellular signalling, as well as targeting of cellular membranes and induction of apoptosis.7,12,16 However, the precise relevance of each is yet to be elucidated. Further, there are no comparable data available regarding the inhibition of amoebae, although targeting of the cell membrane has been suggested.7 Consistent with this, we observed that hexadecyl-PC, octadecyl-PC and elaidyl-PC induce lysis of Acanthamoeba at high concentrations (data not shown).

As phospholipid analogues were originally used as antineoplastics, they exhibit toxicity against mammalian cells and parallel toxicity assays using mammalian cell lines are necessary to understand the specificity of their actions. RCE cells proved more susceptible than Acanthamoeba to octadecyl-PC, erucyl-PC and edelfosine, rendering them unsuitable for the treatment of Acanthamoeba infection. In contrast, both hexadecyl-PC and elaidyl-PC had reduced toxicity to RCE cells, compared with either Acanthamoeba species, indicating their potential for the treatment of Acanthamoeba infection. As previously observed for E. histolytica, liposomal alkylphosphocholine formulations had reduced efficacy against Acanthamoeba when compared with free drug.15

Taken together, these data demonstrate that hexadecyl-PC has the highest therapeutic index of the phospholipid analogues investigated and warrants testing in animal models.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
References
 
None to declare.


   Acknowledgements
 
This work was funded by the William Ross Foundation and the University of Strathclyde Research and Development Fund.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
1 Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev (2003) 16:273–307.[Abstract/Free Full Text]

2 Martinez AJ, Visvesvara GS. Free-living, amphizoic and opportunistic amoebas. Brain Pathol (1997) 7:583–98.[Web of Science][Medline]

3 Niederkorn JY, Alizadeh H, Leher H, et al. The pathogenesis of Acanthamoeba keratitis. Microbes Infect (1999) 1:437–43.[CrossRef][Web of Science][Medline]

4 Schuster FL, Visvesvara GS. Opportunistic amoebae: challenges in prophylaxis and treatment. Drug Resist Updat (2004) 7:41–51.[CrossRef][Web of Science][Medline]

5 Schuster FL, Visvesvara GS. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol (2004) 34:1001–27.[CrossRef][Web of Science][Medline]

6 Azzouz S, Maache M, Garcia RG, et al. Leishmanicidal activity of edelfosine, miltefosine, and ilmofosine. Basic Clin Pharmacol Toxicol (2005) 96:60–5.[CrossRef][Web of Science][Medline]

7 Croft SL, Seifert K, Duchene M. Antiprotozoal activities of phospholipid analogues. Mol Biochem Parasitol (2003) 126:165–72.[CrossRef][Web of Science][Medline]

8 Croft SL, Snowdon D, Yardley V. The activities of four anticancer allkylphospholipids against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei. J Antimicrob Chemother (1996) 38:1041–7.[Abstract/Free Full Text]

9 Escobar P, Yardley V, Croft SL. Activities of hexadecylphosphocholine (miltefosine), ambisome, and sodium stibogluconate (pentostam) against Leishmania donovani in immunodeficient scid mice. Antimicrob Agents Chemother (2001) 45:1872–5.[Abstract/Free Full Text]

10 Escobar P, Matu S, Marques C, et al. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH3 (edelfosine) and amphotericin B. Acta Trop (2002) 81:151–7.[CrossRef][Web of Science][Medline]

11 Konstantinov SM, Kaminsky R, Brun R, et al. Efficacy of anticancer alkylphosphocholines in Trypanosoma brucei subspecies. Acta Trop (1997) 64:145–54.[CrossRef][Web of Science][Medline]

12 Lira R, Contreas LM, Santa-Rita RM, et al. Mechanism of action of anti-proliferative lysophospholipid analogues against the protozoan parasite Trypanosoma cruzi: potentiation of in vitro activity by the sterol biosynthesis inhibitor ketoconazole. J Antimicrob Chemother (2001) 47:537–46.[Abstract/Free Full Text]

13 McBride J, Ingram PR, Henriquez FL, et al. Development of a colorimetric microtitre plate assay for assessment of antimicrobials against Acanthamoeba. J Clin Microbiol (2005) 43:629–34.[Abstract/Free Full Text]

14 Santa-Rita RM, Santos Barbosa H, Meirelles M, et al. Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi. Acta Trop (2000) 75:219–28.[CrossRef][Web of Science][Medline]

15 Seifert K, Duchene M, Wernsclofer WH, et al. Effects of miltefosine and other alkylphosphocholines on the human intestinal parasite Entamoeba histolytica. Antimicrob Agents Chemother (2001) 45:1505–10.[Abstract/Free Full Text]

16 Walochnik J, Duchene M, Seifert K, et al. Cytotoxic activities of alkylphosphocholines against clinical isolates of Acanthamoeba spp. Antimicrob Agents Chemother (2002) 46:695.[Abstract/Free Full Text]

17 Perez-Victoria FJ, Castanys S, Gamarro F. Leishmania donovani resistance to miltefosine involves a defective inward translocation of the drug. Antimicrob Agents Chemother (2003) 47:2397–403.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J Antimicrob ChemotherHome page
J. Walochnik, A. Obwaller, F. Gruber, M. Mildner, E. Tschachler, M. Suchomel, M. Duchene, and H. Auer
Anti-Acanthamoeba efficacy and toxicity of miltefosine in an organotypic skin equivalent
J. Antimicrob. Chemother., September 1, 2009; 64(3): 539 - 545.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
60/3/521    most recent
dkm245v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by McBride, J.
Right arrow Articles by Roberts, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McBride, J.
Right arrow Articles by Roberts, C. W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?