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

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 20μM – 100μM, 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 500μl 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 1μM 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 60μM 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 100μM 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.