I am currently working on antiangiogenic activity of natural bioactive compound. From my MTT result, IC50 is determined at 2ug/ml. Apoptosis occurred at 20 ug/ml but not at the IC50 level. However, when doing cell cycle analysis, no significant difference was observed between negative and treatment group (0.2, 2 and 20 ug/ml).
1. Could I say that the compound is anti-proliferative?
2. Is my cell cycle analysis making any sense?
Many thanks in advance for your help.
Are you distinguishing between apoptosis and necrosis somehow? Maybe it is a completely different cell death pathway.
Furthermore, yes it is possible, that the cells die without interfering in the cell cycle if there is an acute toxicity, there are certain pathways which lead to immediate cell death i would however expect at least a certain degree of decrease in cell proliferation.
Another possibility is a inhomogeneous cell culture. Even with certain cell lines, no all the cells are heterogeneous and if cultured for multiple passages they certainly become inhomogeneous. It is possible, that you are observing different cell populations.
Another possibility is that the substance is interfering with the MTT in a certain way. MTT test is also not the most reliable and not really linear... i determined that myself, a while back.
There are several mechanism of cell death instead of apoptosis. Probably your extract induced cell death through other mechanism except of apoptosis, may be necrosis, ferroptosis or any others. You may consider to them.
About cell cycle arrest, may be you should synchronize your cells to certain cell cycle phase in order to know whether your extract work in that phase.
Dear colleagues,
Just want to add on more information regarding my question:
Proliferation assay = MTT; IC50 = 2 ug/ml
Apoptosis analysis = Annexin V-FITC; 0.2 & 2 ug/ml showed similar viability compared to negative control (~95%), 20 ug/ml showed apoptosis (~60% early apoptosis with no obvious necrosis)
Cell cycle analysis = Flowcytometry; No significant difference (~70% at G1) between negative control and all treatment groups (0.2, 2 & 20 ug/ml), except that 20 ug/ml had significant increased in sub G0.
1. Is it logic to say that the compound used could reduce proliferation at IC50 or lower concentration, induce apoptosis at concentration higher than IC50?
2. The insignificant difference of cell cycle distribution among negative and all treatment groups are possible for the explanation above?
Many thanks!