Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes.
PDF (299K)
Journal: 2004/April - Antimicrobial Agents and Chemotherapy
ISSN: 0066-4804
PUBMED: 14982775
Abstract:
Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to Leishmania donovani. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of Leishmania donovani HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill L. donovani promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors, z-Val-Ala-DL-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.
Open in
PUBMED | PMC | Google Scholar | Wikipedia
Relations:
Content
Citations
(78)
References
(66)
Chemicals
(6)
Organisms
(2)
Processes
(3)
Similar articles
Articles by the same authors
Discussion board
Antimicrob Agents Chemother 48(3): 852-859
PMID: 14982775

Miltefosine Induces Apoptosis-Like Death in <em>Leishmania donovani</em> Promastigotes

UMR 8076 CNRS, Chimiothérapie Antiparasitaire, INSERM U461, Récepteurs et Signalisation des Interleukines, Faculté de Pharmacie Paris-Sud, 92290 ChÂtenay-Malabry, France2
Corresponding author. Mailing address for Philippe M. Loiseau: UMR 8076 CNRS, Faculté de Pharmacie Paris-Sud, 5 rue Jean-Baptiste Clément, 92290 ChÂtenay-Malabry, France. Phone: (33) 1 46 83 55 53. Fax: (33) 1 46 83 55 57. E-mail: rf.dusp-u.pec@uaesiol.eppilihp. Mailing address for Jacqueline Bréard: INSERM U461, Faculté de Pharmacie Paris-Sud, 5 rue Jean-Baptiste Clément, 92290 ChÂtenay-Malabry, France. Phone: (33) 1 46 83 57 81. Fax: (33) 1 46 83 54 96. E-mail: rf.dusp-u.pec@draerb.enileuqcaj.
Received 2003 Jul 21; Revised 2003 Oct 24; Accepted 2003 Oct 31.
Copyright © 2004, American Society for Microbiology

Abstract

Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to Leishmania donovani. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of Leishmania donovani HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill L. donovani promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors, z-Val-Ala-dl-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.

Abstract

The protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a complex of parasitic diseases that affect 12 million people worldwide and that threaten 350 million people worldwide. The pathology is manifested in visceral, mucocutaneous, or cutaneous forms. Visceral leishmaniasis (VL), the most severe form (which is usually fatal if patients are untreated), which is due to Leishmania donovani, is common in less developed countries, with an estimated 500,000 new cases each year (11, 20). Moreover, cases of Leishmania and human immunodeficiency virus coinfection are expected to rise in many countries, especially in sub-Saharan Africa and Asia, where highly active antiretroviral therapy is not available (39, 27). In the Indian subcontinent, a major area of endemicity of L. donovani VL, there is a worrying increment of resistance to pentavalent antimonial drugs, with more than 50% of infected people being unresponsive (52). In addition, the drugs recommended for the treatment of VL (pentavalent antimonials, amphotericin B, lipid formulations of amphotericin B) have limitations, including parenteral administration, long courses of treatment, toxic side effects, and high costs (20). Recently, miltefosine (hexadecylphosphocholine [HePC]), an alkylphosphocholine originally developed as an anticancer drug and currently used for topical treatment (Miltex) of skin metastases of breast cancer (59), has been proved to be the first effective and safe oral treatment (Impavido) for Indian VL, with cure rates of about 98% (25, 54). Moreover, HePC has been successfully used to treat patients with antimony-resistant VL (53, 55) as well as cutaneous leishmaniasis (51).

Numerous in vitro and in vivo experimental studies have shown that HePC is cytotoxic for both the promastigote and the amastigote stages of various species of Leishmania (7, 8, 15, 16, 29, 31, 58) as well as other protozoan parasites, including Trypanosoma cruzi (8, 32, 46, 47), Trypanosoma brucei (8, 28), Entamoeba histolytica (48), and Acanthamoeba spp. (64). Although the mechanism of the antiprotozoal activity of HePC is poorly understood, several hypotheses have been formulated, such as damage to the flagellar membrane (46), perturbation of alkyl-lipid metabolism and glycosylphosphatidylinositol anchor biosynthesis (33), interference with ether-lipid remodeling through the inhibition of the alkyl-lyso-phosphatidylcholine specific acyl coenzyme A acyltransferase (34), and inhibition of the de novo synthesis of phosphatidylcholine (32).

The antineoplastic activity of HePC has been linked to its capacity to induce apoptosis in numerous tumor cell lines (14, 22, 43, 45). HePC accumulates at the plasma membrane, due to its amphiphilic properties, thus affecting membrane fluidity (18, 61); but the molecular mechanisms implicated in its initiation of apoptotic signaling remain undefined. Suggested pathways include inhibition of phosphatidylcholine biosynthesis by depression of the translocation of CTP:choline phosphate cytidylyltransferase to membranes (5, 19, 21), a sustained increase in Ca levels via calcium channels (23, 65), inhibition of sphingomyelin biosynthesis leading to increased levels of proapoptotic ceramide (67), mitogenic MAPK/ERK signaling inhibition, and proapoptotic SAPK/JNK pathway activation (44).

Apoptosis, a form of programmed cell death (PCD), is a genetically regulated physiological process of cell suicide that is central to the development, homeostasis, and integrity of multicellular organisms (2). Initially, it was assumed that PCD emerged with multicellularity and would have been counterselected in unicellular organisms (41). Recently, several studies have demonstrated that a process of PCD also operates in 10 different single-celled eukaryotic organisms whose phylogenetic origins arose 1 billion to 2 billion years ago (1, 6, 10, 42, 62, 66), including various species of Leishmania (4, 24, 35, 50, 69), among which is L. donovani (9, 30, 37). These studies have also shown that this type of cell death in kinetoplastids, slime mold, ciliates, and dinoflagellates induces some of the structural alterations characteristic of apoptosis in multicellular organisms.

The aim of the present work was to assess more precisely the cell death process induced by HePC in L. donovani promastigotes. We report that dying parasites share numerous cytoplasmic, nuclear, and membrane features with apoptotic metazoan cells, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. Our findings also suggest that proteases are involved, at least in part, in the death machinery operating in L. donovani promastigotes in response to HePC.

Acknowledgments

This work was supported by EC grant QLRT-2000-01404.

We are grateful to Zentaris for providing the HePC used in this study and to Simon L. Croft for providing the promastigote forms of L. donovani WT (MHOM/ET/67/L82) and the derivative HePC-resistant line (HePC-R40).

Acknowledgments

REFERENCES

REFERENCES

References

  • 1. Ameisen, JC. 1995. Apoptosis in a unicellular eukaryotes (Trypanosoma cruzi): implications for the evolutionary origin and role of programmed cell in the control of cell proliferation, differentiation and survival. Cell Death Differ. 2:285-300. [[PubMed][Google Scholar]
  • 2. Ameisen, JC. 2002. On the origin, evolution, and nature of programmed cell death: a timeline of four billion years. Cell Death Differ.9:367-393. [[PubMed][Google Scholar]
  • 3. Aravind, L., V. M. Dixit, and E. V. Koonin. 2001. Apoptotic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons. Science291:1279-1284. [[PubMed]
  • 4. Arnoult, D., K. Akarid, A. Grodet, P. X. Petit, J. Estaquier, and J. C. Ameisen. 2002. On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization. Cell Death Differ.9:65-81. [[PubMed]
  • 5. Boggs, K., C. O. Rock, and S. Jackowski. 1998. The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine. Biochim. Biophys. Acta1389:1-12. [[PubMed]
  • 6. Cornillon, S., C. Foa, J. Davoust, N. Buonavista, J. D. Gross, and P. Golstein. 1994. Programmed cell death in Dictyostelium. J. Cell Sci.107:2691-2704. [[PubMed]
  • 7. Croft, S. L., R. A. Neal, W. Pendergast, and J. H. Chan. 1987. The activity of alkylphosphocholines and related derivatives against Leishmania donovani. Biochem. Pharmacol.36:2633-2636. [[PubMed]
  • 8. Croft, S. L., D. Snowdon, and V. Yardley. 1996. The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. J. Antimicrob. Chemother.38:1041-1047. [[PubMed]
  • 9. Das, M., S. B. Mukherjee, and C. Shaha. 2001. Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes. J. Cell Sci.114:2461-2469. [[PubMed]
  • 10. Davis, M. C., J. G. Ward, G. Herrick, and C. D. Allis. 1992. Programmed nuclear death: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena. Dev. Biol.154:419-432. [[PubMed]
  • 11. Desjeux, P. 1996. Leishmaniasis. Public health aspects and control. Clin. Dermatol.14:417-423. [[PubMed]
  • 12. D'Mello, S. R., F. Aglieco, M. R. Roberts, K. Borodezt, and J. W. Haycock. 1988. A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K deprivation. J. Neurochem.70:1809-1818. [[PubMed]
  • 13. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, and S. Nagata. 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature391:43-50. [[PubMed]
  • 14. Engelmann, J., J. Henke, W. Willker, B. Kutscher, G. Nossner, J. Engel, and D. Leibfritz. 1996. Early stage monitoring of miltefosine induced apoptosis in KB cells by multinuclear NMR spectroscopy. Anticancer Res.16:1429-1439. [[PubMed]
  • 15. Escobar, P., S. Matu, C. Marques, and S. L. Croft. 2002. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH3 (edelfosine) and amphotericin B. Acta Trop.81:151-157. [[PubMed]
  • 16. Escobar, P., V. Yardley, and S. L. Croft. 2001. Activities of hexadecylphosphocholine (miltefosine), AmBisome, and sodium stibogluconate (Pentostam) against Leishmania donovani in immunodeficient scid mice. Antimicrob. Agents Chemother.45:1872-1875.
  • 17. Garcia-Calvo, M., E. P. Peterson, B. Leiting, R. Ruel, D. W. Nicholson, and N. A. Thornberry. 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem.273:32608-32613. [[PubMed]
  • 18. Geilen, C. C., T. Wieder, A. Haase, W. Reutter, D. M. Morre, and D. J. Morre. 1994. Uptake, subcellular distribution and metabolism of the phospholipid analogue hexadecylphosphocholine in MDCK cells. Biochim. Biophys. Acta1211:14-22. [[PubMed]
  • 19. Geilen, C. C., T. Wieder, and W. Reutter. 1992. Hexadecylphosphocholine inhibits translocation of CTP:choline-phosphate cytidylyltransferase in Madin-Darby canine kidney cells. J. Biol. Chem.267:6719-6724. [[PubMed]
  • 20. Guerin, P. J., P. Olliaro, S. Sundar, M. Boelaert, S. L. Croft, P. Desjeux, P., M. K. Wasunna, and A. D. Bryceson. 2002. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infect. Dis.2:494-501. [[PubMed]
  • 21. Haase, R., T. Wieder, C. C. Geilen, and W. Reutter. 1991. The phospholipid analogue hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis in Madin-Darby canine kidney cells. FEBS Lett.288:129-132. [[PubMed]
  • 22. Henke, J., J. Engelmann, U. Flögel, J. Pfeuffer, B. Kutscher, G. Nobner, J. Engel, R. Voegeli, and D. Leibfritz. 1999. Apoptotic effects of hexadecylphosphocholine on resistant and non-resistant cells monitored by NMR spectroscopy. Drugs Today34:37-50. [PubMed]
  • 23. Henke, J., J. Engelmann, B. Kutscher, G. Nobner, J. Engel, R. Voegeli, and D. Leibfritz. 1999. Changes of intracellular calcium, fatty acids and phospholipids during miltefosine-induced apoptosis monitored by fluorescence- and C NMR-spectroscopy. Anticancer Res.19:4027-4032. [[PubMed]
  • 24. Holzmuller, P., D. Sereno, M. Cavaleyrz, I. Mangot, S. Daulouede, P. Vincendeau, and J. L. Lemesre. 2002. Nitric oxide-mediated proteasome-dependent oligonucleosomal DNA fragmentation in Leishmania amazonensis amastigotes. Infect. Immun.70:3727-3735.
  • 25. Jha, T. K., S. Sundar, C. P. Thakur, P. Bachmann, J. Karbwang, C. Fischer, A. Voss, and J. Berman. 1999. Miltefosine, an oral agent, for the treatment of Indian visceral leishmaniasis. N. Engl. J. Med.341:1795-1800. [[PubMed]
  • 26. Johnson, DE. 2000. Noncaspase proteases in apoptosis. Leukemia14:1695-1703. [[PubMed][Google Scholar]
  • 27. The Joint United Nations Programme on HIV/AIDS. December 2002. AIDS epidemic update. [Online.] .[PubMed]
  • 28. Konstantinov, S. M., R. Kaminsky, R. Brun, M. R. Berger, and U. Zillmann. 1997. Efficacy of anticancer alkylphosphocholines in Trypanosoma brucei subspecies. Acta Trop.64:145-154. [[PubMed]
  • 29. Kuhlencord, A., T. Maniera, H. Eibl, and C. Unger. 1992. Hexadecylphosphocholine: oral treatment of visceral leishmaniasis in mice. Antimicrob. Agents Chemother.36:1630-1634.
  • 30. Lee, N., S. Bertholet, A. Debradant, J. Muller, R. Duncan, and H. L. Nakhasi. 2002. Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ.9:53-64. [[PubMed]
  • 31. Le Fichoux, Y., D. Rousseau, B. Ferrua, S. Ruette, A. Lelievre, D. Grousson, and J. Kubar. 1998. Short- and long term-term efficacy of hexadecylphosphocholine against established Leishmania infantum infection in BALB/c mice. Antimicrob. Agents Chemother.42:654-658.
  • 32. Lira, R., L. M. Contreras, R. M. Santa-Rita, and J. A. Urbina. 2001. 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.47:537-546. [[PubMed]
  • 33. Lux, H., D. T. Hart, P. J. Parker, and T. Klenner. 1996. Ether lipid metabolism, GPI anchor biosynthesis, and signal transduction are putative targets for anti-leishmanial alkyl phospholipid analogues. Adv. Exp. Med. Biol.416:201-211. [[PubMed]
  • 34. Lux, H., N. Heise, T. Klenner, D. Hart, and F. R. Opperdoes. 2000. Ether-lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether-lipid analogues in Leishmania. Mol. Biochem. Parasitol.111:1-14. [[PubMed]
  • 35. Moreira, M. E., H. A. Del Portillo, R. V. Milder, J. M. Balanco, and M. A. Barcinski. 1996. Heat shock induction of apoptosis in promastigotes of the unicellular organism Leishmania (Leishmania) amazonensis. J. Cell. Physiol.167:305-313. [[PubMed]
  • 36. Mottram, J. C., M. J. Helms, G. H. Coombs, and M. Sajid. 2003. Clan CD cysteine peptidases of parasitic protozoa. Trends Parasitol.19:182-187. [[PubMed]
  • 37. Mukherjee, S. B., M. Das, G. Sudhandiran, and C. Shaha. 2002. Increase in cytosolic Ca 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. [[PubMed]
  • 38. Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, and C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods139:271-279. [[PubMed]
  • 39. Paredes, R., J. Munoz, I. Diaz, P. Domingo, M. Gurgui, and B. Clotet. 2003. Leismaniasis in HIV infection. J. Postgrad. Med.49:39-49. [[PubMed]
  • 40. Paugam, A., A. L. Bulteau, J. Dupouy-Camet, C. Creuzet, and B. Friguet. 2003. Characterization and role of protozoan parasite proteasomes. Trends Parasitol.19:55-59. [[PubMed]
  • 41. Raff, MC. 1992. Social controls on cell survival and cell death. Nature356:397-400. [[PubMed][Google Scholar]
  • 42. Ridgley, E. L., Z. H. Xiong, and L. Ruben. 1999. Reactive oxygen species activate a Ca-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei. Biochem. J.340:33-40.
  • 43. Ruiter, G. A., M. Verheij, S. F. Zerp, and W. J. Van Blitterswijk. 2001. Alkyl-lysophospholipids as anticancer agents and enhancers of radiation-induced apoptosis. Int. J. Radiat. Oncol. Biol. Phys.49:415-419. [[PubMed]
  • 44. Ruiter, G. A., S. F. Zerp, H. Bartelink, W. J. Van Blitterswijk, and M. Verheij. 1999. Alkyl-lysophospholipids activate the SAPK/ JNK pathway and enhance radiation-induced apoptosis. Cancer Res.59:2457-2463. [[PubMed]
  • 45. Rybczynska, M., M. Spitaler, N. G. Knebel, G. Boeck, H. Grunicke, and J. Hofmann. 2001. Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities. Biochem. Pharmacol.62:765-772. [[PubMed]
  • 46. Santa-Rita, R. M., H. Santos Barbosa, M. N. Meirelles, and S. L. De Castro. 2000. Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi. Acta Trop.75:219-228. [[PubMed]
  • 47. Saraiva, V. B., D. Gibaldi, J. O. Previato, L. Mendonça-Previato, M. T. Bozza, C. G. Freire-De-Lima, and N. Heise. 2002. Proinflammatory and cytotoxic effects of hexadecylphosphocholine (miltefosine) against drug-resistant strains of Trypanosoma cruzi. Antimicrob. Agents Chemother.46:3472-3477.
  • 48. Seifert, K., M. Duchene, W. H. Wernsdorfer, H. Kollaritsch, O. Scheiner, G. Wiedermann, T. Hottkowitz, and H. Eibl. 2001. Effects of miltefosine and other alkylphosphocholines on human intestinal parasite Entamoeba histolytica. Antimicrob. Agents Chemother.45:1505-1510.
  • 49. Seifert, K., S. Matu, F. J. Pérez-Victoria, S. Castanys, F. Gamarro, and S. L. Croft. 2003. Characterization of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int. J. Antimicrob. Agents22:380-387. [[PubMed]
  • 50. Sereno, D., P. Holzmuller, I. Mangot, G. Cuny, A. Ouaissi, and J. L. Lemesre. 2001. Antimonial-mediated DNA fragmentation in Leishmania infantum amastigotes. Antimicrob. Agents Chemother.45:2064-2069.
  • 51. Soto, J., J. Toledo, P. Gutierrez, R. S. Nicholls, J. Padilla, J. Engel, C. Fischer, A. Voss, and J. Berman. 2001. Treatment of American cutaneous leishmaniasis with miltefosine, an oral agent. Clin. Infect. Dis.33:E57-E61. [[PubMed]
  • 52. Sundar, S. 2001. Drug resistance in Indian visceral leishmaniasis. Trop. Med. Int. Health6:849-854. [[PubMed]
  • 53. Sundar, S., L. B. Gupta, M. K. Makharia, M. K. Singh, A. Voss, F. Rosenkaimer, J. Engel, and H. W. Murray. 1999. Oral treatment of visceral leishmaniasis with miltefosine. Ann. Trop. Med. Parasitol.93:589-597. [[PubMed]
  • 54. Sundar, S., A. Makharia, D. K. More, G. Agrawal, A. Voss, C. Fischer, P. Bachmann, and H. W. Murray. 2000. Short-course of oral miltefosine for treatment of visceral leishmaniasis. Clin. Infect. Dis.31:1110-1113. [[PubMed]
  • 55. Sundar, S., F. Rosenkaimer, M. K. Makharia, A. K. Goyal, A. K. Mandal, A. Voss, P. Hilgard, and H. W. Murray. 1998. Trial of oral miltefosine for visceral leishmaniasis. Lancet352:1821-1823. [[PubMed]
  • 56. Szallies, A., B. K. Kubata, and M. Duszenko. 2002. A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae. FEBS Lett.517:144-150. [[PubMed]
  • 57. Thornberry, N. A., and Y. Lazebnik. 1998. Caspases: enemies within. Science281:1312-1316. [[PubMed]
  • 58. Unger, C., T. Maniera, P. Kaufmann-Kolle, and H. Eibl. 1998. In vivo antileishmanial activity of hexadecylphosphocholine and other alkylphosphocholines. Drugs Today34:133-140. [PubMed]
  • 59. Unger, C., M. Peukert, H. Sindermann, P. Hilgard, G. Nagel, and H. Eibl. 1990. Hexadecylphosphocholine in the topical treatment of skin metastases in breast cancer patients. Cancer Treat. Rev.17:243-246. [[PubMed]
  • 60. Uren, A. G., K. O'Rourke, L. A. Aravind, M. T. Pisabarro, S. Seshagiri, E. V. Koonin, and V. M. Dixit. 2000. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol. Cell6:961-967. [[PubMed]
  • 61. Van Blitterswijk, W. J., H. Hilkmann, and G. A. Storme. 1987. Accumulation of an alkyl lysophospholipid in tumor cell membranes affects membrane fluidity and tumor cell invasion. Lipids22:820-823. [[PubMed]
  • 62. Vardi, A., I. Berman-Frank, T. Rozenberg, O. Hadas, A. Kaplan, and A. Levine. 1999. Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO2 limitation and oxidative stress. Curr. Biol.9:1061-1064. [[PubMed]
  • 63. Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger. 1995. A novel assay for apoptosis. Flow cytometry detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled annexin V. J. Immunol. Methods184:39-51. [[PubMed]
  • 64. Walochnik, J., M. Duchene, K. Seifert, A. Obwaller, T. Hottkowitz, G. Wiedermann, H. Eibl, and H. Aspock. 2002. Cytotoxic activities of alkylphosphocholines against clinical isolates of Acanthamoeba spp. Antimicrob. Agents Chemother.46:695-701.
  • 65. Wang, Y. Z., Y. B. Chang, C. Xing, and D. Fu. 1998. The interference effects of hexadecylphosphocholine on proliferation and membrane phospholipid metabolism in human myeloid leukaemia cell lines. Int. J. Tissue React.20:101-107. [[PubMed]
  • 66. Welburn, S. C., S. Lillico, and N. B. Murphy. 1999. Programmed cell death in procyclic form Trypanosoma brucei rhodesiense: identification of differentially expressed genes during Con A induced death. Mem. Inst. Oswaldo Cruz94:229-234. [[PubMed]
  • 67. Wieder, T., C. E. Orfanos, and C. C. Geilen. 1998. Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog, hexadecylphosphocholine. J. Biol. Chem.273:11025-11031. [[PubMed]
  • 68. Wyllie, AH. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature284:555-556. [[PubMed][Google Scholar]
  • 69. Zangger, H., J. C. Mottram, and N. Fasel. 2002. Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis? Cell Death Differ.9:1126-1139. [[PubMed]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.