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Screening for pro-apoptotic agents

The M30-Apoptosense® assay is a versatile tool to for screen for pro-apoptotic drugs (Hägg et al. 2002).

In the FIGURE below, MDA-MB-231 cells (cytokeratin 18 positive human breast carcinoma) were seeded in 96 well microtiter plates at a density of 10 000 cells/well (in 200 µl medium). Treatment with the pro-apoptotic candidates was initiated after 6 hours. After 40 hours, NP-40 was added to 0,5% final concentration to the medium and the plates were frozen. By this procedure, it was possible to assay total caspase-cleaved CK18 produced (cell associated + antigen released into the medium). 25 µl of the medium/extract was used for the M30-Apoptosense® assay.

A number of pro-apoptotic drugs were identified from a chemical library. The exact number of drugs that can be identified depends on the nature and size of the library used, the drug concentrations chosen and their time course kinetics.

The M30-Apoptosense® assay can be used to screen for drugs that induce apoptosis of tumor cells under specific conditions. One example is the dependence on the p53 gene product. By screening drug libraries for induction of caspase cleavage of CK18 in p53wt and p53null cells, interesting drug candidates can be identified.

In the FIGURE below, HCT116 cells with functional p53 were assayed for apoptosis induction parallel to HCT116 cells deficient in p53. As expected, many compounds induce a more efficient response in p53wt cells. However, a large number of compounds induce apoptosis also in p53null cells.

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