The result is interesting.

As your approach is majorly inhibitor dependent its hard to comment in specific way. because chemical inhibitor s has diverse effects on cell. If apoptososme inhibitor protects cell then theoretically pan caspase inhibitor should also work. However, in this case its not working.

I will suggest to KD cyt C as its a precursor of apoptosome formation or KD apaf1 and try check what happens. This will be more specific.

Also try to check autophagic cell death process.

There is some report where cell dies only by the activation of PARP but independent of caspase. May be apoptososme could have induce PARP dependent cell death also.

Good luck.

Omer

Are any caspases activated in cells treated with your compound? Secondly, 20 mM is an extremely high concentration of zVAD. It should be uM, right?

Regards

Magnus

My guess is that your compound induces intrinsic apoptosis, which shifts to necrosis upon caspase inhibition.

Have a look at the morphology of the cells +/- zVAD, and verify if caspases are activated or not. you can also check PARP-1 cleavage and DNA fragmentation.

Mostly if you interfer with cell death pathways with inhibitors, they shift to another pathway.

I have, as similarly pointed out by others the feeling that your compound triggers mitochondrial apoptosis.

Here some suggestions

i) I guess that you intended 20 uM of Z-VAD. In this case, if you have a robust apoptotic response and depending from the cell line that you are using, you could try to increase the concentration up to 100uM to see eventually an inhibition.

ii) You should use DKO cells for Bax and Bak to switch-off mitochondrial apoptosis.

iii) Use a couple of caspase substrates (there are hundreds PARP, beta-catenin, HDAC4,... ) to monitor in immunoblot the activation of caspases and the efficiency of z-VAD inhibition.

Once proved these aspects, You are ready to design experiments to characterise a caspase-independent cell death.

What Tom, Magnus, and Claudio said (Hi guys).

Also, which cells/cell lines are you using (e.g. are they type I or II).

I cannot recommend knocking down cyt-c. Firstly, there typically is massive cyt-c in cells so that you will hardly be able to get it down to rate limiting amounts.

In addition: Excluding necroptosis, zVAD is always more potent in preventing apoptosis throught the extrinsic pathway than through the intrinsic pathway. This is due to zVAD easily blocking the rather modest amounts of slowly activated caspase-8. In contrast, during intrinsic apptosis, you have full blown cyt-c/Smac release and caspase-9 activation within 1-3 min in individual cells, whcih triggers amplifying feedbacks through caspase-3 as well. Take for example HeLa cells. With 20-40 uM zVAD you block extrinsic apoptosis completely, but you need up to 200 uM to maximally block intrinsic apoptotic caspase activation. As a first step, you may therefore just like to try out a higher amount of zVAD?

I never worked with the Apaf-1 inhibitor you used, and I would say that it is not widely in use in the field (correct me if I'm worng, Tom, Magnus). If you want to knock something down, try caspase-9. In molar amounts, this would typically be the protein with the lowest expression in the downstream pathway.

I can also offer you a plasmid from which you can express fluorescently labelled XIAP. If you overexpress that in your cells, it could serve as a protein based control rather than relying only on your apoptosome inhibitor or zVAD alone.

I suspect, might be your compound has some ROS inducing property.

Following suggestions:-

1) Elevated intracellular ROS may induce caspase independent cell death as well, NAC can be used to confirm the role of ROS.

2) Confirm the possibility of necrotic cell death with the inhibitor necrosatain.

3) Confirm the mitochondrial integrity upon treatment with your compound by JC-1 or TMRE assay.

Hope these suggestions would be helpful for your experimentation!!

Good Luck

Note also that in some cases zVAD can upregulates caspase-9 cleavage and activity

see http://www.ncbi.nlm.nih.gov/pubmed/22613767

Best

First of all, thanks for all the suggestions and let me give some more information.

I am using UACC 903 melanoma cells in most of the experiments. However, I used MEF Bax-/- and wt cells to investigate the involvement of Bax in cell death.

The concentration of z-VAD fmk was 20 uM and cells were treated 30 min before the drug treatment.

Based on the cells morphology, it does not like that they are undergoing to necrosis. Also I used Necrostatin-1 and it was not able to protect cells. However I did not have a

positive control for this experiment. What can I use as a positive control that will trigger necrosis and that I can supress through Necrostatin-1?

Caspase 3 activity was up-regulated based on Caspase 3 assay. However, I thought that this may be a secondary effect and not the primary cause of cell death...

PARP-1 cleavage was observed at 12-24 hour. and there was no significant DNA fragmentation.

3 hr treatment of compound diminishes mitochondrial membrane potential in 15-20 % of the cells (When I gate both living and apoptotic cells from the FSCH- SSCH histogram)

I am going to get shRNA constructs for Bad, Bim and Bmf from Addgene. I will try to generate cell lines and test them also.

Now,based on suggestions,

First, I will try increasing the concentration of z-Vad fmk to 100-200 uM...

I will try to do a Caspase activity assay in conjugation with z-VAD fmk treatments.

Thanks for all suggestions and I will inform you back soon...

All of the preceding comments are useful. You can also consider the possibility of a non-apoptotic programmed cell death (paraptotic). One example of such a mechanism is the spontaneous elimination of dopaminergic neurons during development in mice with the weaver mutation (J Neurosci. 1996 Mar 1;16(5):1819-26). A similar mechanism can be triggered in vitro by excitatory stimulation of AMPA receptors, and is mediated by NFkB translocation and transcriptional activation of p53 (Mol Pharmacol. 2003 Apr;63(4):784-90).. You may want to look into this pathway as an alterate explanation.

Good luck

Gabriel

Apoptosome formation usually leads to casp-3 activation as a downstream process which is why you might be observing increased casp-3 activity. Moreover it is also due to the same reason NS3694 is able to protect the cells. i would encourage you to do a basic check up of the following:

1. if NS3694 is able to inhibit apoptosome mediated cell death (as you already found), then it might reduce casp-3 activation as well (also check casp-9 activity which is also a linked phenomenon).

2. Since the property of this inhibitor notifies "NS3694 exhibits no effect on apoptosome-independent caspase activation and cell death induced by FasL. Inhibits neither cytochrome c release nor the enzymatic activity of caspases" which i found from the following link "http://www.emdmillipore.com/chemicals/apoptosis-inhibitor-ii-ns3694/EMD_BIO-178494/p_iLqb.s1LOdkAAAEWwWEfVhTm" it demonstrates that apoptosome formation is primarily the main mediator of cell death induced by your compound and caspase are really a collateral molecules (a you pointed out already)

3. Now most importantly, since cyt-c is a prerequisite in apoptosome formation and thus mitochondrial membrane perturbations are thus involved to release cyt-c you can try using cyclosporine-A and ruthenium red to understand the role of mitochondria. if they are able to protect against your compound, then there is a possibility of the involvement of AIF and ENDO-G which also induces caspase independent cell death. since both AIF and ENDO-G translocates to nucleus and incude DNA damage (may be this is why you are getting PARP-cleavage on a later time) you can isolate the nuclear fraction of treated cells and simply do western blotting to understand their involvement.

I hope that these may help you to at least identify the pathway that you have to follow for your further work.

All the best.

with regards,

Sumonto Mitra

Hey,

Just a warning, I'm still very new to this field and graduate school in general, but I've been messing around with inducing necroptosis in MOVAS for a few months, and maybe some of my observations might help you out.

1) What confluency are you at when treating your cells? I've come to realize anything over 70-80% confluency ruins the amount of necrosis I see when sorting with 7-AAD and Annexin V. The higher confluency tends to cause a shift to apoptosis.

2) Are you using Necrostatin-1 or Necrostatin-1s the more stable and specific RIP-1 inhibitor? I would suggest using Nec-1s just because it's more robust and specifiic ( I use at 10uM concentration)

3) The controls I use is TNFa (50ng/ul) + zVADfmk (40uM) which induces necroptosis. DMSO as the blank, essentially. And, then I block that necroptosis by adding TNFa+zVADfmk+ Nec1s.

4) So, I work on RIP3 which is a part of the necrosome downstream of RIP1, and we have noticed that upregulation of RIP3 can actually activate the same amount of necrosis seen with TNFa+ zVAD, without zVAD. which means there might be a RIP3 activated necroptosis that completely bipasses inhibiting caspase 8? possibly. Maybe see what happens with your inhibitor when you use a cell line that doesn't naturally express RIP3? I have no idea if your cell line falls into that category or not, but it might be an interesting aspect to look into.

5) And, if it happens to be downstream you can test NSA ( necrosulfamide) which is an inhibitor for MLKL, another part of the necrosome.

And, thanks for the thread, it's been really interesting to follow. Also if my logic is unsound in some way, please point it out to me. Again, I'm really new to this and any discussion on the topic really helps me out.

Cheers,

Kirsti Walker

Point 1-4 of Kirsti are correct.

Concerning point 5, NSA only works in human cells.

Hi..

You need to more focus on the mitochondrial mediated death. As you said earlier Necrosatain-1 unable to protect cell death and within 3h mitochondrial integrity compromised with your compound, suggests that the cell death you are getting due to the unhealthy mitochondria.

1. Any change in the expression of Bcl2?

2. Just confirm the level of mtROS once.

3. If possible, perform BN-PAGE assay for in-depth understanding of mitochondrial functioning.

Good luck

Kishu

I have new updates!

1- I tried upto 200 uM of zVADfmk and it did not protected cells from cell death.

2- Beside Necrostatin 5 (upto 50 uM) I also tried another necrosis inhibitor IM54 (up to 20 uM) and their combination. None of them was able to protect cells.

3- In Annexin V - PI staining there is an increase in early apoptotic cells (PI- Annexin V +) (5-10 % increase) and in PI + Annexin V- (10-15 %) and in late apoptotic/necrotic cells (40-50%).

As note, I treat my cells at 870 to 80 % confluency, I did not check Bcl2 expression. I will check and inform you about the results. I had checked ROS levels with DCFDA but I did not see a significant change.

Hi..

you can refer following links to measure mtROS:-

http://www.sciencedirect.com/science/article/pii/S1046202302000397

http://online.liebertpub.com/doi/abs/10.1089/ars.2012.4886

https://www.lifetechnologies.com/order/catalog/product/M36008

It is possible that your compound is triggering two modes of cell death simultaneously. I agree with Kishu Ranjan, as necrostatin and caspase inhibitor both are not effective it should be mitochodria that is prime in this case. It is very likely that your compound disturbs mitochondria releasing caspase dependent (Smac/Diablo, cytochrome C...) and independent (AIF endonuclease G,....) factors and so in this case if you inhibit one pathway of cell death cells would take up alternative pathway to die. You can try checking what kind of DNA fragmentation (ladder, large scale or shearing) you are getting with different treatments that you are using this will give a clearer picture. Also you can check the energy status (pyridine nucleotides and ATP/ADP) of the cells.