Author/Editor		Parker, Katherine A; Pilkington, Geoffrey J
Title		Apoptosis of human malignant glioma-derived cell cultures treated with clomipramine hydrochloride, as detected by Annexin-V assay
Translated title		Klomipramin hidroklorid in ugotavljanje apoptoze na celičnih kulturah humanih malignih gliomov s pomočjo pretočne citometrije ob uporabi Annexina-V
Type		članek
Source		Radiol Oncol
Vol. and No.		Letnik 40, št. 2
Publication year		2006
Volume		str. 87-93
Language		eng
Abstract		Background. Previous research in our laboratories has shown that Clomipramine Hydrochloride (CLOM), a tricyclic antidepressant in use for over thirty years, selectively kills neoplastic glial cells in vitro whilst leaving normal brain cells unaffected. The purpose of this study was to evaluate whether a range of early passage cell cultures and established cell lines, derived from a number of patients with malignant glioma, would display different sensitivities when exposed to CLOM. The particular assay of interest, following our discovery that CLOM targets the mitochondria of tumour cells and triggers Caspase 3 mitochondrially-mediated apoptosis, was Annexin-V flow cytometry. This assay was used to determine the mechanism of cell death, either necrosis or apoptosis, according to drug concentration and period of incubation. Method. Cells grown to 90% confluence in 25cm3 flasks were incubated with concentrations of CLOM from 20miM - 100miM, for up to 6 hours. Cells were harvested and resuspended in calcium binding buffer, which triggers translocation of calcium-regulated phosphatidylserine residues to the nuclear envelope, before removing 500mil of the single cell suspension to a Facs tube. Controls used in the analysis were performed by omission of the drug incubation in one flask, and addition of 1miM staurosporine to one flask. These served as negative and positive controls respectively. Annexin-V FITC and propidium iodide were added to all tubes and incubated for 15 minutes at room temperature, in the dark. Subsequent to this, binding buffer was added to each tube and analysed using a BD FACScalibur. (Abstract truncated at 2000 characters)
Summary		Izhodišča. Predhodne raziskave v našem laboratoriju so pokazale, da klomipramin hidroklorid (CLOM), triciklični antidepresiv, ki ga uporabljamo že 30 let, in vitro selektivno ubija neoplastične glialne celice in pri tem ne prizadene normalnih možganskih celic. Namen naše raziskave je bil oceniti celične kulture malignega glioma, ki smo jih odvzeli različnim bolnikom. Želeli smo ugotoviti, ali so različno občutljive na CLOM. Posebno nas je zanimala apoptoza, saj CLOM deluje na mitohondrije tumorskih celic in na ta način sproži apoptozo. Pri tem smo uporabljali pretočno citometrijo in Annexin-V. Glede na koncentracijo zdravila in čas inkubacije smo želeli ugotaviti mehanizem celične smrti, ali ta nastane predvsem zaradi nekroze ali zaradi apoptoze. Metode. Celice smo inkubirali do 6 ur z različno koncentracijo CLOM-a (20miM - 100miM). Sledila je priprava celic za pretočno citometrijo, kjer smo uporabili tudi Annexin-V FITC in propidium iodid. Rezultati. Preiskavo smo naredili s petimi malignimi gliomi. Pri dveh so imele celice manj apoptoze, koncentracija CLOM-a je bila 60miM ali več. Pri treh, kjer smo uporabili zgodnje celične linije, pa smo opazili zelo izrazito apoptozo, koncentacija CLOM-a je bila do 100miM, inkubacija pa 6 ur. Vzporedno smo preiskovali normalne humane astrocite in ugotovili, da CLOM v omenjenih koncentracijah ni povzročil njihove smrti. Zaključki. Preiskava z Annexinom-V bi lahko služila testiranju posamičnih bolnikov - ob analizi Bcl-2 in genskem CYP preiskovanju - ugotavljali bi lahko, ali so njihove tumorske celice občutljive na CLOM.
Descriptors		BRAIN NEOPLASMS GLIOBLASTOMA TUMOR CELLS, CULTURED APOPTOSIS CLOMIPRAMINE ASTROCYTOMA ANNEXIN V