I have 2 cell lines ( cancer cell lines) and a compound. First, I perform the MTT assay to identify the IC50 of 2 cell lines (~5 micro molar). Then the flow cytometry was perform to see that this compound can induced apoptosis or not. The result I got show that it can induced the apoptosis. Then, I perform WB, the result I get is the cytochrome c is increase, bcl2 and survivin is also increase. Can anyone help me explain this situation
As you mentioned that you treated your cells with IC50 dose, which means 50% cells live or we can say resist the apoptotic effect of your drug. Bcl2 and survivin are two proteins well established as reason behind drug resistance in various cancers. So one explanation is the Bcl2 and survivin is upregulated in some cells. A better way to visualize this is Confocal microscopy with Annexin-V FITC staining coupled with either Bcl2 or survivin to see whether Annexin-V FITC and Bcl2/survivin signal overlap or not.
Good luck
Induction of apoptosis is about the disruption between the balance of pro- and anti- apoptotic molecules. In your case, it may be possible that BCL2 and survivin are induced despite an apoptotic response. I suggests you to use other assays to further quantify the apoptotic response (Ie. Caspase cleavages, annexin/PI apoptotic assay etc..) Also, measuring the levels of cytochrome C is not conclusive on the intrinsic apoptotic induction. I suggest you to look at the localisation of cytochrome c instead..
To verify cytochrome c release, I suggest to look for other pro-apoptotic Bcl2 members downstream of cytochrome c, especially Bak and Bax (alternatively look for Puma, Bad, Bim or Noxa). Furthermore, I would check for caspase-9 levels in WB, as this is the characteristic caspase for the intrinsic apoptosis pathway.
Bcl-2 is the protein that is involved in the crosstalk between apoptosis and autophagy. Bcl-2 can bind to beclin-1 and thereby block omegasome formation, which leads to the inhibition of autophagy. If the situation remains unclear, you should consider investigating LC3-II levels (with and without lysosomal protease inhibitors like PSA or E-64d) for autophagic flux analysis.
Good luck,
CL
- Badges
- Science method