Specific anti-tumour effects of 6-diazo-5-oxo-L-norleucine (DON) on carcinoid tumour cells

University dissertation from Uppsala : Acta Universitatis Upsaliensis

Abstract: Glutamine is essential for cell growth. 6-diazo-5-oxo-L-norleucine (DON) is a rather old anti-tumour drug developed as a glutamine antagonist. Previous clinical trials with DON, not including carcinoid tumours, did not prove convincing. Hence the drug was not further used.In the present study a human pancreatic carcinoid cell line (BON), which produces serotonin and chromogranin A, was used as a model to evaluate the effect of DON on carcinoid tumours and the mechanisms behind this effect. Parameters used to follow the in vitro effect of DON included (a) aggregate size, (b) amino-acid uptake, (c) DNA and RNA synthesis, and (d) chromogranin A secretion. The results showed that DON (<10μM) had specific inhibitory effect on BON as compared with other tumour cell lines.DON also inhibited the tumour growth in vivo in a model with heterotransplanted BON cells into athymic nude mice.Amidotransferase inhibition has been suggested previously as being the main anti-tumour mechanism of DON. However, experiments done in this work showed that other mechanisms of action contribute alongside the enzyme inhibition. By using (1) dehydrogenase activity (MTT) assay, (2) positron emission tomography, (3) ATP determination and (4) electron microscopy, it was observed that mitochondrial functions were disrupted when BON cells were incubated with a low dose of DON for 5h, and that mitochondrial structure could be damaged if treatment was prolonged. Hence it is bring postulated that the most prominent target of DON in BON cells is the mitochondria.DON induced apoptosis as observed by electron microscopy and TUNEL staining,especially in BON cells as compared to neuroblastoma (LAN) cells according to ELISA analysis. It seems to be consistent with Bcl-2 expression only shown in LAN cells. Results from Western blot suggested that the pathway: mitochondria→cytochrome c→ caspase-3→PARP, participated in DON-induced apoptosis. Apoptosis is another possible anti-tumour mechanism of DON.In conclusion, DON seems to have a specific effect on carcinoid tumour cells, with a primary target being the mitochondria. The fact that this effect is observed at rather low concentrations and persists for a considerable length of time, as shown both in cell culture and inan animal model, motivates clinical trials on patients with carcinoid tumours.

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