Most of the successful anticancer drugs trigger apoptotic cell death in target cells primarily through mitochondrial permeabilization followed by cytochrome c release. Hence mitochondrial permeabilization or upstream signaling that contributes to mitochondrial permeabilization could be a sensitive readout for anticancer activity. One such early change that contributes for the mitochondrial permeabilization is Bax conformational activation and its translocation to mitochondria.
To engineer, several cancer cell lines expressing Bax EGFP and mitochondrial with DsRed to identify compounds that induce Bax conformational activation and its translocation to Mitochondria as a read-out of anticancer activity.
Different cell lines like HeLa, Si Ha, MEF, HCT-116 were maintained and transfected with expression vectors as per our standard protocol. Transgene expressing cells were sorted by FACS based on EGFP fluorescence to enrich probe expressing cells. Several clones were selected, expanded and studied for functional competence live cell imaging after treating with standard anticancer drugs.
Automated well plate imaging followed by segmentation and granularity index calculation in drug treated cells reliably identified positive hits in a compound library-screening test. Additionally automated co-localization index within cytoplasm in the segmented cells confirmed the Bax activation status. Using this assay we could systematically analyze the dependence of trans-membrane potential loss during Bax activation in selected anticancer drugs. Since Bax overexpression sensitized few cancer cell lines because of its inherent pro-apoptotic activity, we have also utilized a Bax knock out colon cancer cell line HCT116 for stable expression of Bax EGFP that served as a best cell based assay system to screen Bax activating compounds.
This user friendly, sensitive assay system developed can be used for monitoring apoptosis in live cells and for preliminary screening of plant derived bioactive compounds.