I am treating drug which causes cell death (according to mtt assay), in dose dependent manner, but in western blot same ratio of drug does not show increased activity of cleaved- Caspase3 and cleaved -Parp but the expression is found decreasing what would be the reason?
If I understand the question correctly, treatment of cells with a drug caused cell death (as determined by the MTT assay), however, "cell death" was not accompanied by detection of cleavage of both caspase-3 and PARP proteins in immunoblotting. Further, levels of uncleaved forms of both the proteins decreased. Several factors could account for your above observations. The simplest explanation would be that you do not have cell death by apoptosis and levels of all proteins decrease upon treatment. If you suspect apoptosis following treatment of your cells with the drug, you need a control to inhibit caspase-3 in your assay. Further, include positive control lysates from cells that are known to undergo apoptosis following a treatment, resulting in cleavage of both caspase-3 and PARP.
I agree with Mr. Choubey. The MTT assay tells you the viability and metabolism of the cells. This might very well be the case that that your drug is causing reduced metabolism or cell death via necrosis. Try positive controls to activation of caspase 3. I have been using CD95L with cycloheximide that induce apoptosis via intrisic pathway.
Hi, the question does not addressed where author measured apoptosis or not. you need to select the drug dose at different concentration to see clear picture. make sure all your measurements are of cells undergoing death, not in the floaters. check cell cycle arrest.
Are lower concentrations of the drug generating apoptotic markers in WB? If so, a solution to the problem might be that higher doses cause an alternative death response, such as necrosis. Alternatively, cells might under these conditions generate a fast apoptotic response after which cells enter a secondary necrotic state. In this situation, some cytosolic proteins, cleaved casp-3 included, will diffuse to the extracellular environment, thereby becoming undetectable in WB. Apart from what already been suggested, I would increase the time window of your experiment and in parallel to WB perform LDH-release analysis.
Agree with above and would add one further note. In my readings of papers that look at parp cleavage, the loss of full length is an indicator of cleavage, as is the appearance of cleaved parp. However, the quantitative amount of either of these (or the ratio) is not a quantitative indicator of apoptosis. This is in part because immunoblot is not a quantitative technique (no matter what they tell you).
Probably there is a difference in stability of the cleaved form, such that once formed it is degraded faster than the full length protein. Depending on the cell line we have seen marked reduction of total parp with a slight increase in the cleaved form or slight reduction in full length and marked increase in cleaved form- with the same apoptotic stimulus.
Take-home message is to use a secondary assay for apoptosis, always. MTT is a way to estimate cell number (or more carefully, mitochondrial activity). It can indicate the number of remaining cells and is then an indirect measure of death, but it is never an apoptosis assay.
This is simply due to protein degradation after cleavage and all along the dismantling of the cells
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