One of our drugs is showing positive Annexin V -Pi and good MTT result that showing cytotoxic activity but there is no immunofluorescent detection of p53 and p21 was found. We have also found that the drug is causing microtubule depolymerisation and cell shrinkage. Again from FACS cell cycle analysis we got most of the cells were at G1-G0 phase.
From these data we are suspecting nonapoptotic cell death.
Can anybody suggest any protocol to analyse the mechanism of a drug causing this type of non-apoptotic cell death?
Thank you Tomasz.Your information is very useful.Still we need a easiest way to discriminate this cell death process from apoptosis.Any experiment that can give initial proof of this necroptosis? then we will proceed for signaling path way .
Caspase-3 activity can be measured to check if the cell death is due to apoptotic pathway or not. Ideally, if the mechanism of cell death is via necrotic pathway then caspase-3 activity should be lower.
Thank you Priyanka. But In our cancer cell line there is no report of Caspase 3 activation.
Even if there is no report of caspase 3 activation by these cells, it does not hurt to check first and see if caspase 3 is being cleaved. If you cannot check for caspase 3 cleavage for any reason, you may then want to to use a kit that detects apoptotic and necrotic cells at the same time: Here is a suggestion, I have not used this kit before but I believe it could be useful for your question. It is a kit made by ENZO Life Sciences: GFP-Certified™ Apoptosis/Necrosis detection kit for microscopy and flow cytometry: http://www.enzolifesciences.com/ENZ-51002-100/gfp-certified-apoptosis-necrosis-detection-kit-for-microscopy-and-flow-cytometry.
As an alternative, you may want to use Real time PCR looking at gene expression for RIP1 and RIP3 in your cells to get an idea of necroptosis activation. You can at the same time check for caspase 8 for extrinsec apoptosis or TRADD and Cleaved caspase 9 or cytochrome c for the intrinsec apotosis activation.
How to interpret the data from this gene expression, I would at least look at protein expression by western to make sure that transcription is followed by translation.
This is not an easy topic but you can make it happen.
GOOD LUCK
Dear saswat, all your data shows that the drug is causing apoptosis. Why are you saying it as non-apoptotic cell death. Annexin V positivity is a celar indication of early apoptosis, more over cell cycle arrest at G0-G1 also pointsout towards apoptotic cell death.
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