Screening of cyanobacterial extracts for apoptotic inducers: a combined approach of caspase-3/7 homogeneous assay and time-lapse microscopy

Abstract

Cyanobacteria are well known to produce valuable secondary metabolites, including compounds with anticancer activity. An advantageous feature of candidate anticancer compounds is their ability to induce apoptosis. One possible approach to screen for apoptosis inducers is via the detection of activities of caspases 3 and 7 (Casp3/7), the key apoptotic enzymes. Pilot screening of natural samples may be intricate due to sample complexity. We tested cyanobacterial extracts for their ability to enhance Casp3/7 activity, inhibit proliferation, and cell metabolism in human pancreatic tumor cells PaTu 8902. The majority of extracts inhibited cell division, but this was only partly reflected by a concurrent MTT viability measurement. The time elapsed by the end of the measurement affects the cell number differently in treated and control cells. The resulting cell counts greatly influence the evaluation of the Casp3/7 assay, since we obtained substantially different results when evaluating primary luminescence data (3 hits) as opposed to when the actual cell number was taken into account (23 hits). Based on the fact that crude extracts manifest miscellaneous effect including cytostatic activity, it is necessary to couple Casp3/7 assay with cell count measurement. The crude extract of Nostoc sp. CCAP1453/38 and its active fraction (proapoptotic metabolite nocuolin A) were used to demonstrate the validity of our approach since its effects was detectable only when normalized to cell number. We demonstrate that the Casp3/7 luminescence assay is useful for apoptotic inducer screening from cyanobacterial extracts and present amendments which help deal with the drawbacks of the method.