I am trying to see the effect of my treatment on both pathways of apoptosis. So far treatment has affected both pathways but I want to figure out which pathway is mostly affected by blocking each pathway. So, I need some suggestions on the potent inhibitors that I can use to block each pathway. I have seen many papers using z-ietd-fmk as Caspase 8 inhibitor. So, I think I can use z-ietd-fmk as extrinsic pathway inhibitor. But what I should use for intrinsic pathway inhibitor?
Usually Cyclosphorin A is used as MPTP inhibitor. But couple of papers showed Cys A to redirect the cell to the necrosis pathway. So, need some suggestion. Thank you.
there are a few inhibitors you can try :
For Casp8
http://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=9563391
For Casp 9
http://www.sigmaaldrich.com/catalog/product/sigma/scp0113?lang=en®ion=US
http://www.biovision.com/caspase-9-inhibitor-z-lehd-fmk-2979.html
you can complement this with other assays such as Annexin and measure if apoptosis was blocked by the inhibitor, of course there are many different approaches but these are pretty simple and straight forward with well documented references.
Am curious about what you mentioned regarding Cys A. It can block just cylcophiln D of MPTP, so I don't think using just that to block intrinsic pathway is sufficient to begin with. Looks like you need either blockers of other mPTP components too or sth that targets TRAP1 . Am not aware of specific intrinsic blockers though!
All these artificial inhibitors are generally very poor in their specificity. Further, as you induce both pathways, inhibition of one is likely to enhance death through the other pathway, unless you can detect extrinsic and intrinsic apoptosis very early . If so, with the caspase-8 inhibitor at hand (which admittedly is one of the more specific ones), my suggestion would be to just observe caspase-3 cleavage (by Western) with and without the inhibitor present. My guess would be that if extrinsic apoptosis dominates you should not see much cleavage of caspase-3 then. Alternatively, if caspase-3 cleavage is just as pronunced as without the inhibitor then caspase-8 mediated apoptosis might just be a "by-product".
Thank you very much @Chris, Rt P, Gerhard..
I think the activation of extrinsic pathway could also be to truncate BID protein. Do you agree with that?@Gerhard Krumschnabel
Sure, you my see tBID if the extrinsic pathway is activted (and you might see if you detect this with and without caspase-8 inhibitor). But I think you need to see this early on or else you may see BID truncation as a secondary event. BID may also be cleaved by other proteases and by caspase-2 and the role of this protease is still very elusive. Later on in intrinsic apoptosis, when caspase-3 activity is high, you may see all sort of not really specific cleavage of apparent substrates. So I believe thatpure forms of exterinsic or intrisic can only be discerned at early times.
- Badges
- Science method