| 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.
Methods: Cells grown to 90% confluence in 25cm3 flasks were incubated with
concentrations of CLOM from 20muM-100muM, 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 500mul 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 1muM 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.
Results: Results show that, of the five malignant gliomas tested, the two
established cell lines had the lower apoptotic threshold, with a
significant percentage of apoptotic cells present at 60muM and above when
compared to the control sample. The three early passage cultures,
developed 'in house' from biopsy, had higher apoptotic thresholds,
withstanding up to 100muM CLOM incubation for six hours. Normal human
astrocytes were assayed in parallel, and show that CLOM does not cause
cell death at the concentrations tested.
Conclusions: It may be possible, in a larger study, to predict individual
patient response to CLOM using the annexin-V assay, alongside Bcl-2
analysis and CYP gene testing, on the individual patient's tumour cells.
The difference in sensitivities between glioma, in this small study,
indicates the importance of analysing early passage cultures, which retain
original morphology and characteristics to a greater extent, alongside
established cell lines.
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