Apoptosis of human malignant glioma-derived cell cultures treated with clomipramine hydrochloride, as detected by annexin-V assay

english

2006

volume 40, issue 2

Analysis   Annexin V   Apoptosis   Astrocytoma   Brain Neoplasms   Clomipramine   Drug Effects   therapy   tumor   Tumor Cells, Cultured

Društvo radiologije in onkologije
(Obvezni izvod spletne publikacije)

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 andtriggers 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 triggerstranslocation 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 minutesat 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)

16.09.2009