Our combination treatments cause a reduction in cell number and a 2 fold increase in ROS but our other experiments do not suggest apoptosis: no cleavage of Parp, or increase in Bad expression by immunoblotting, no Annexin staining increase, etc. Are there other forms of cell death that is induced by an increase in ROS?
A two fold increase in ROS does not necessarily mean that there is cell death.
You can achieve cell death with no change in ROS by high doses of metformin associated with quercetin. While complex I inhibition increases lactic acid quercetin impedes it's extrusion. Cells die through acidic stress without increasing ROS. The pathway requires intact Tp53.
Other forms of death induced by ROS are necrosis, necroptosis. ROS is also implicated in autophagic death. Markers of these cell death mechanisms can be checked for confirmation.
Increased ROS levels do not necessarily indicate apoptosis induction. In fact, you clearly proove that with your own experimental results. Cells are equipped with a very effective antioxidative machinery (e.g. catalase, SOD, GPx, GSH), which is able to prevent apoptosis after high ROS levels occurred (How did you measure ROS levels anyway?). On the other hand, a form of programmed cell death called ferroptosis is characterized by enhanced lipid peroxidation (LPO) after failure of GSH-related enzymes, such as GPx4. You could check for LPO with BODIPY-dyes to verify that. Maybe your high ROS levels are somehow related to ferroptosis.
Good luck - CL
You could look into oncosis vs apoptosis. There are reports where PS is not externalized during oncosis. ATP loss and mito.membrane potential could also help
@Mrinalini can you describe the markers of necrosis, necroptosis and autophagy that I could use? Thank you
Necrosis can be confirmed by loss of membrane integrity, e.g. via LDH or ATP release assay.
Autophagy can be investigated by LC3-II levels (in presence and absence of lysosmal protease inhibitors like pepstatin A and E-64d for full autophagic flux analysis) and/or p62 (sequestosome-1) in western blot analysis.
@Carsten I used the ROS-glo assay from Promega normalized to cell number to determine the increase in ROS. Can you recommend an LDH or ATP release assay? I'll also try the p62 immunoblot. Thank you!
If you have access to a flow cytometer, measurement of ROS levels with H2DCF-DA could be an easy and quick additional method to confirm your findings.
For the LDH and ATP assays you will find multiple commercial kits available. I only worked with the LDH release assay and performed it with the help of a publication by Chan et al. (doi:10.1007/978-1-62703-290-2_7).
For autophagy experiments, I suggest to look for LC3-II turnover instead of p62 alone. Use p62 only to confirm autophagy. I highly recommend to check the latest version of Klionsky's guidelines for monitoring of autophagy (doi: 10.1080/15548627.2015.1100356).
Good luck - CL
- Badges
- Science method