İnvestigation of drug effects on apoptosis in promastigotes of leishmania tropica

Leishmania infection continues to be a serious public health problem. After the existence of programmed cell death in multicellular organisms discovered in unicellular organisms has sparked interest on leishmania protozoan that is the causative agent of the leishmaniasis. Elucidation of the mechanisms of apoptosis is important in terms of Leishmania species to develop drugs for the treatment of leishmaniasis. For this reason in this study the effects of the drugs which are Glukantim® and miltefosin aganist the programmed cell death markers was investigated on L. tropica promastigotes. İn the study, L. tropica promastigotes which were produced in NNN were incubated in cell culture medium by addition of 10% fetal calf serum for grow that 24-26ºC. When the promastigotes growth reached logaritmic phase the drugs that were miltefosine and Glucantim® were administrated and they were taken to the analysis for three apoptotic markers that were DNA fragmentation by electrophoresis method, DNA fragmentation by TUNEL method and bcl-2 concentration by ELİSA method. Staurosporine, the drug was administered as a positive control to compare the two drugs. The application dose of miltefosin and staurosporin was adjusted as 100 µmol, 50 µmol, 25 µmol, 12.5 µmol and 6.25 µmol then promastigotes were incubated 24 hours .When comes to Glucantime® the application dose was adjusted as 15mg/ml, 7.5mg/ml, 3.75mg/ml, 1.875 mg/ml and 0.9375 mg/ml then promastigotes were incubated 72 hours. In the determination of DNA fragmentation by electrophoresis method in promastigotes with the drug staurosporine as a positive control yielded DNA fragmentation image in each dose which were 50 µmol, 25 µmol, 12.5 µmol and 6.25 µmol.At the same doses, apoptotic DNA fragmentation was observed also by TUNEL assay analysis in promastigotes.In the drug which was miltefosine displayed oligonucleosomal DNA fragmentation in 25 µmol dose. The dose which was 12.5 µmol,ıt was not displayed a full clear fragmentation. It was observed DNA fragmentation at 25 µmol and 12.5 µmol doses in also TUNEL assay analysis in promastigotes with miltefosin. Correspond to this two drugs, ıt was not observed oligo nucleosomal DNA fragmentation in promastigotes with the drug which was Glucantime®. Only at 0.9375 mg / mL dose, a vague image that was similar to DNA fragmentation was exposed. In TUNNEL analysis Glucantime® again at 0.9375mg / ml dose occured a very light color was due to a vague image which was similar to DNA fragmentation. Apoptosis was not observed in the other dose finding. Thus, an effective oral medication of miltefosine for the treatment of Leishmania can be said to be superior compared to Glukantim® on the apoptotic effect that induced by drug in promastigotes of Leishmania tropica. There was not a statistically significant difference between the concentration values for each drug that was administered at five different doses, when we looked at the bcl-2 concentration which was the other apoptotic marker. In contrast, when each drug chart values compared with each other from the highest dose, to the lowest dose of bcl-2 concentration Glucantime® displayed a high value according to the other two drugs in five doses. The lowest value occurred in staurosporine. The high bcl-2 concentration of Glukantim® drug may be associated with that any finding of apoptosis does not occur . A high concentration of bcl-2 is thought to inhibit apoptosis. Efforts to induce programmed cell death in various Leishmania species of Leishmania in the treatment of drug effect is still ongoing. Induction of programmed cell death can be an effective way to treat Leishmania. But diversity and the development of resistance to drugs against parasites of Leishmania species makes it difficult to gain clarity of apoptosis to be observed in Leishmania protozoa. The findings of this study is thought to contribute to the drug treatment for Leishmania infection and to shed light on the work planned. Keywords: Leishmania tropica, Programmed cell death, Glucantime®, Miltefosine.