During my master's dissertation work, studying the biological effects of new potential aromatase inhibitors in hormone-dependent breast cancer (MCF-7aro cells), I observed that the compounds induced activation of caspase-9. However, when performing mitochondrial membrane potential assay by flow cytometry, I see no changes (this has to be confirmed). What's the most likely explanation for this activation of caspase-9 without having changes in mitochondrial membrane potential and therefore cytochrome c release?
Thank you so much for you help!
check all the caspase levels at tat point . Also confirmed the caspase 9 activation with ints specific inhibitor
Thank you for your input!
By the way, I get statistical significance for caspase-9 and no statistical significance for caspase-8 nor -7.
Apoptosis is one hell of a phenomenon to understand. Every pathway is likely backed by at least 4 other pathways to ensure that the cell end up dying. MCF-7 cells lack caspase-3, so other pathways must compensate. If I was you, I'd look at other apoptotic pathways that could be implicated in this compensation, such as the ER stress. Here's an article that could suggest an answer. : http://www.jbc.org/content/277/37/34287.full.
Thank you everyone so much for your feedback!
Bruno, thank you for your input! That paper seems quite interesting and it might, in fact, hold some answers. By the way, following a quick look into the paper and assuming that same pathway is happening in my case, since MCF-7aro cells lack caspase-3 and the pathway itself includes this one, that could explain why I don't have activation of caspase-7, right?
Caspase-7 can be activated in MCF-7. It even has a greater role in those cells because of the absence of caspase-3. Other than looking at caspases activation, what methods have you used to analyse the induction of apoptosis following your treatments ?
Concerning caspases activation, I have no significant activation of caspase-7 nor -8, only caspase-9. Besides that, I did ROS assay, with no production of ROS, mitochondrial membrane potential by flow cytometry, for which I have no alterations (still to be confirmed and this was the main issue linked to my question above). I also did Giemsa and Hoechst staining and I observed cromatin condensation and fragmentation.
Seems like the cells did not die (by apoptosis, at least). How do you know that Caspase-9 was activated?
Flow assays for mitochondrial membrane potential can be tricky. Did you see a change when adding CCCP or FCCP? Why not confirm by fluorescence imaging with TMRE?
Thank you for your answer, Don Newmeyer!
There were features of apoptosis seen by Giemsa and Hoechst staining (chromatin condensation and fragmentation). I did a luminescent caspase-9 assay (kit) and I got significant differences for all my compounds.
As for the mitochondrial membrane potential by flow cytometry, CCCP didn't produce any changes, which is in fact the tricky part. We use DiOC6(3) for mitochondria labeling. The protocol itself has been previously optimized, so I assume there's nothing wrong with this aspect.
I assume that this caspase activity kit uses a fluorogenic peptide substrate. The problem is that hydrolysis of such a substrate does not prove that the activity is due to the caspase it's supposed to measure. A more reliable approach is to immunoprecipitate the caspase with a specific antibody before assaying activity.
If CCCP doesn't do anything (in untreated, living cells), then you have to assume that the assay didn't properly measure the membrane potential. The easiest assay is fluorescence microscopy using a dye like TMRE. CCCP should give a clear difference with healthy cells. Immunostaining for cytochrome c, Smac or Omi is also not difficult.
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