Time staggered treatment of EGFR inhibitors and genotoxic drugs result in enhanced cancer cell death. Synergistic relation between the drug can be studied by varying the time gap between their administrations followed by assays for the quantification of pro apoptotic response(s) .
When you treat your cell by some drugs, which may cause apoptotic gene up-regulated, which means the drug can cause the cell death by apoptosis, the drug works.
Dear Srivatsava, in general, Caspases are the enzymes activated during apoptosis. Thus, Caspase-activity assays can be used as functional readout in order to estimate the extend of apoptotic response.
One is a Caspase-III assay, we used this in one of our recent publications:
"Stabilization of Peptides for Intracellular Applications by Phosphoramidate-Linked Polyethylene Glycol Chains"
supporting information p. 16 http://onlinelibrary.wiley.com/doi/10.1002/anie.201303467/suppinfo
I hope this is of help to you.
I think the interaction of phosphatidylinositol and phosphatidylcholine on the cell membrane due to flip flop reaction will lead you to asses the early apoptosis if your drug causes that. The best way to test this interaction is to use PI and annexin V staining. There are several kits for this purpose.
Good Luck
Apoptosis is triggered through two main pathways: the FAS-mediated extrinsic pathway and the intrinsic pathway. Both pathways converge to destroy the mitochondria, activate the caspases, destroy the cell membrane and the nucleus. Trimerization of FAS ligands, ceramide formation, caspase activation and membrane destabilization can be measured through flow cytometry (FACS) with specific antibody targeting every molecule (eg. FAS, ceramide, caspase-3, caspase-8). depolarization of mitochondria can also be measured by FACS. Phosphorylation of pro-apoptotic proteins and cytochromes can be measured with western blots. Damage of the nucleus can either be measured by activation of the nuclease enzymes with blots or checking the degradation of the DNA with BrdU/PI or 7AA staining by FACS or fluorescent microscopy. review articles in Cell Death and Differentiation and Apoptosis can help you a lot.
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