| pA2 online © Copyright 2004 The British Pharmacological Society |
045P
University of Newcastle Winter Meeting December 2004 |
Cannabinoid induced apoptosis in human medulloblastoma cells is caspase dependent but caspase independent in rat glioma cells Storer LCD1, Walker DA2, Coyle B3 and Parker TL1. 1Institute of Neuroscience, School of Biomedical Sciences, 2Department of Child Health, 3Institute of Genetics, School of Biology, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH (Introduced by David A. Kendall). |
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Print abstractCannabinoids have been shown to induce apoptosis, decrease tumour growth and inhibit tumour angiogenesis in a number of different transformed cell lines and primary cells ( Sanchez et al., 1998; Blazquez et al., 2003; Guzman, 2003). In this study the effect of the cannabinoid 1 (CB1) and 2 (CB2) receptor agonist, WIN-55,212-2, was investigated on human medulloblastoma (Daoy) tumour cells and rat glioma (C6) cells.
Cells were seeded onto poly-D-lysine coated coverslips at a density of 75,000cells/ml. The presence of CB1 receptors was determined by fluorescent immunocytochemistry. The apoptotic effect of cannabinoids was studied by replacing the media after 24 hours with either WIN-55,212-2, the CB1 antagonist, AM281 or WIN-55,212-2 and AM281 in combination. Positive controls were incubated with 5ng/ml TNF
or 10 µM MG132, known apoptotic inducers. Cells grown in the presence of DMSO (carrier) provided the baseline level of apoptosis. Cells were stained with Hoechst 33258 or terminal deoxynucleotidyl transferase nick end labelling (TUNEL). Apoptotic and normal cells were uniformly random sampled and data analysed using ANOVA and Tukey’s multiple correction test (n = 10). Western blot analysis using antibodies against caspase-3 and caspase-9 was performed to identify cleavage of proforms and hence activation of the caspase cascade.
CB1 receptors were identified on both Daoy and C6 cells. The morphological and biochemical changes induced by WIN-55,212-2 were consistent with the apoptotic changes observed in positive controls. DNA fragmentation and apoptotic bodies were observed. Drug dependent, apoptosis was observed with WIN-55,212-2 concentrations of 10-4M, 5x10-5M, 2x10-5M and 10-5M [apoptotic index of 84.3%. 72.4%, 63.8%, (p<0.001) and 34.9% (p>0.05) respectively] in Daoy cells. Drug dependent, apoptosis was observed with WIN-55,212-2 concentrations of 10-4M, 5x10-5M and 2x10-5M [apoptotic index of 73.6% (p<0.001), 48.2% and 28.0% (p>0.05) respectively] in C6 cells. Antagonist treatment alone had no effect on apoptosis, and apoptosis was inhibited in both cell types when the combination was administered. For western blotting, cells were incubated with either 5x10-5M WIN-55,212-2 or 5x10-5M WIN-55,212-2 and 10-4M AM281 in combination for 3 and 7 days. Loss of full length (proform) caspase-3 and caspase-9 was observed in Daoy cells following incubation with WIN-55,212-2. No loss of either proform was detected following incubation with the combination, Unexpectedly, no loss of caspase-3 or caspase-9 proforms were observed in C6 cells following incubation with WIN-55,212-2 or the combination.
These results indicate that the apoptotic effect of WIN-55,212-2 on Daoy and C6 cells is mediated by the CB1 receptor, with different apoptotic mechanisms for Daoy and C6 cells: Daoy cells being caspase dependent and occurring via mitochondria peturbation, and C6 cells being caspase independent. We intend to further confirm this using the caspase inhibitor, ZVAD. These results provide evidence to justify further investigation of cannabinoids as therapeutic agents in the treatment of medulloblastoma and glioma brain tumours.
Blazquez et al. (2003) The FASEB Journal 17 :529-531.
Guzman, M. (2003) Nature Reviews 3: 745-755.
Sanchez et al. (1998) FEBS Letters 472: 39-44.