Treatment of MCF7 cells with a carcinogenic agent increased ki-67 expression as well as annexin v marker by flow cytometry. what is the reason behind high expression of annexin in treated cells ?
Is that a compensatory mechanism of apoptotic cells to proliferate more ?
Can anyone help plz?
Cancer cells tend to turn off any redundant or "unnecessary" (from their point-of-view) signaling pathways so that they can allocate resources towards growing. I think what you're seeing here is that this carcinogenic agent is toxic to malignantly transformed cells like MCF7.
Since these cells probably don't have any intact systems for dealing with the toxic effects of the drug anymore, exposure is probably just causing them to die off. Hence, the annexin V signal.
Although Ki67 is used as a proliferation marker in IH staining, for in vitro studies using MCF7 cells other methods might be more suitable as KI67 is expressed in all cell cycle stages except resting cells (G0). Assay like BrDu incorporation, cell counting, cell viability (Cell titer Blue/Glo) over time can be used to measure proliferation. Annexin V is a good marker for early apoptosis due to translocation of PS to outer leaflet of PM. It could be that the drug has some toxicity and you detect apoptosis at early time point in a few cells but overtime the majority of the cells proliferate.
Maybe you can check this : https://pubmed.ncbi.nlm.nih.gov/12427252/
They claim that there is a correlation between KI-67 expression and TNFa IFNg expression (experiment on MCF7).
About the flow cytometry itself, when you do apoptosis experiment, your cells have to be in exponential phase of cell groth to avoid apoptosis-related-confluence.
Hope you'll find some answers,
Best
@ Govinda thanks for your explanation but if the cells die off and this agent is cytotoxic, what is the cause of proliferation ?
@ Sima thanks a lot, does this agent considered to be carcinogenic or not ? as one of hallmarks of carcinogenesis evades apoptosis or this principle isn't applied in cancer cell lines ?
@ Gaetan thanks a lot for sharing your opinion
Annexin V is a very early marker of apoptosis and, depending on the turnover of Ki67 protein in cells, it would not be surprising if there was temporal overlap between Annexin V and Ki67 in cells that are slowing their proliferation and initiating apoptosis.
I Marwa
Just an advice. What don't kill you makes you stronger. A dose-response experiment coupled with annexin V testing should help you to clarify your carcinogen effect on proliferation and maybe choose the right dose to study your compound in ''controled'' (not cytotoxic) metabolic condition
Good luck
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