I pre-treat my cells with 100mM reduced L-gluthatione and then I treat my cells with my anticancer drug that induces apoptosis, and instead of the normal apoptosis I get, I get all of my cells with permeabilization of the plasma membrane, but no change in shape of the cells, nor do I get condensed chromatin.
100 mM GSH is very high. We only use 5- 10 mM to inhibit apoptosis induced by ROS. Maybe your dose of GSH is killing them?
I'm confused? Are you asking why they aren't apoptotic? Heavy quantities of a strong antioxidant would slow down the respiratory burst, right? Add some vitamin e and they'd probably survive completely. Are you using physiologic Oxygen?
I guess it depends on how strong your drug is and what conditions the cells were grown in.
The concentration used is too high. L-gluthatione inhibit or limit ROS but in lower concentrations. Also you should check if your antibiotic concentration is too high.
Yeah, I wasn't sure the concentration was appropriate, but I read a few papers using up to 100mM L-glut to inhibit apoptosis. I suppose VitE would be a good add-on to the experiment with N-acetyl-L-cysteine. Thanks!
This is well known and NAC (N -acetyl cysteine) does the same thing. Try BHA (butylated hydroxyanisole). It worked well for us.
what concentration of BHA have you been using? I think I might go and purchase some, along with vitamin E.
There is an extensive literature on the topic. I think the concentration of GSH that you used is too high and this may have created some unintended side effects (cell growth arrest and cell death). In my experience, 2 or 5 mM of N-acetylcysteine are enough to inhibit apoptosis, but the efficiency of such protection depends strongly on many factors, including cell histotype.
GSH inhibits caspases (http://www.pnas.org/content/95/6/2956.full.pdf). Caspase inhibition in the presence of apoptotic stimuli induces caspase independent cell death/necrosis (eg. caspase inhibitor z-VAD-fmk induced cell death with TNF family members). See
http://www.nature.com/onc/journal/v15/n13/abs/1201324a.html
Your target here is RIP1/3. Good luck.
If you use a so elevated concentration, you may be cytotoxic for the cells.
This may also explain your results.
Anyhow your concentration is to hig (at least an orderto high), 1 to 5 mM wil be OK, and 10 mM being quite high...
hi! yes, it´s possible you drive them into different cell death modalities, including necrosis or even pyroptosis (they are fast than apoptosis)..see Goodwin Jinesh´s answer!....playing around with the redox-balance of the cells can have different outcomes...easy way how you can follow (document) non-apoptotic-cell death is to add DAPI for 5min directly to the cells in culture, then put PFA into the medium to fix....DAPI will penetrate into cells with a damaged cell membran....BHT is not tolerated by all cell-types! is an aromatic compound, so it will induce NRF2/KEAP dep. upregulation e.g. antioxidative enzymes/proteins....basic question: does your anticancer-drug induce ROS-formation? and can you compensate for it with GSH?, is it mitochondrial pathway of apoptosis you want to investigate? ...can you block drug-induced apoptosis with z-VAD-fmk?
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