No abstract

No abstract

Miltefosine causes leishmanial death, but the possible mechanism(s) of action is not known. The mode of action of miltefosine was investigated in vitro in Leishmania donovani promastigotes as well as in extra-and intracellular amastigotes. Here, we demonstrate that miltefosine induces apoptosis-like death in L. donovani based on observed phenomena such as nuclear DNA condensation, DNA fragmentation with accompanying ladder formation, and in situ labeling of DNA fragments by the terminal deoxyribonucleotidyltransferasemediated dUTP-biotin nick end labeling method. Understanding of miltefosine-mediated death will facilitate the design of new therapeutic strategies against Leishmania parasites.Miltefosine (1-O-hexadecylphosphocholine), an alkylphosphocholine and a membrane-active synthetic ether-lipid analogue originally developed for the treatment of cutaneous metastasis from mammary carcinomas (21, 32), has proved to be an effective treatment for human visceral leishmaniasis (25,38,(52)(53)(54)(55). It has been hailed as potentially the first oral treatment of human leishmaniasis (8,15,16,20,25,31,47,50). The leishmaniacidal activities of miltefosine have been associated with perturbation of the alkyl-phospholipid metabolism and the biosynthesis of alkyl-anchored glycolipids and glycoproteins (33,34). Although potential antitumor cell mechanisms of action of miltefosine have been elaborated in mammalian cells (13,44), its exact mode(s) of cytotoxicity has not been determined in Leishmania spp. (9,25,39,46,48,55). It has been known to induce apoptotic death in various cancer cell lines (13,28,32,43,60). However, it is not yet established whether miltefosine can bring about apoptosis-like death in all the forms of Leishmania parasite.There are now increasing numbers of reports regarding unicellular organisms undergoing apoptosis-like death, whose induction is not obligatory but activated under threatening circumstances (1,36,49). Cell death resembling metazoan apoptosis has been reported in several parasitic protozoans (4,10,30,36,49,59,61). Apoptosis greatly affects the host-parasite relationship, since the survival of the parasite inside the vector as well as in the macrophage requires strict control of the population of the parasite (12, 58). Apoptosis could be a useful mechanism to avoid killing of the entire population (36) and thus influence the chemotherapeutic strategies to limit the parasite (11).In the present study we sought to determine the mode of action of miltefosine in Leishmania donovani promastigotes as well as extra-and intracellular amastigotes. We have demonstrated that miltefosine causes apoptosis-like death in L. donovani. Our data set the stage for future development of this class of drug for better treatment of leishmaniasis. MATERIALS AND METHODSReagents. Miltefosine was a kind gift from Zentaris (Frankfurt, Germany). RPMI 1640 culture medium was from Gibco BRL. Fetal calf serum was obtained from Biological Industries (Kibbutz Beit Haemek, Israel). ApoAlert DNA fragmentation assay kit was ...

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells di erentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of di erentiation of the C2C12 myotubes. Insulinstimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to signi®cant levels (P50.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were una ected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin-and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the e ect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.

No abstract