For a certain anticancer agent it was found a good cytotoxic action at 24 hr, even better at 48 hr, but a significant reduction in cytotoxicity at 72 hr when analysed on a cell line with MTT assay. Could it be something with the half life of the agent? if so how could you administer in vivo.
The first possibility that comes to my mind is that many cancer cell lines express multi-drug resistance proteins that scavenge such small molecules to impart resistance to therapy. From the information you provided, this would be my best guess.
Dear Manu,
In your experimental setup, do you start from the same cell number, add the compound and measure the MTT assay after the given time points? Or do seed the cells at day one and add the compounds at different time points to reach the incubation times you mentioned? In order to compare, it might be good to have the cells more or less confluent in the untreated control wells for all setups (Especially as in case of the MTT assay, there are steps where you change the medium, and in less confluent wells there might be a higher probability of losing cells in that step). Maybe it would be worth to compare both methods…
Regarding the multidrug resistance protein theory, I think the kinetics you observe are a bit unusual for efflux proteins.
Good luck, Veronika
Dear Veronika and Manu, possible explanation for unusual appearance of effect could be selection of cells in the presence of cytotoxic agent.
Dear Dmitry,
Thats true. Although to me that effect seems a bit fast for that expaination - after 3d? But of course it is possible, maybe the cell line Manu uses reacts that fast... In that case I would check the literature, if there is any information about the used substance to be a substrate for a transporter. If there is no information, it would be interesting to investigate the expression of MDR proteins after 72h treatment with a not yet toxic concentration.
Unfortunately, we do not know, how much cells were resistant to the moment of the start of culture. As a rule, many of cultured cell lines are heterogeneous after many passages. Frequently, I force my students to perform recloning of them. This is especially required after retroviral transduction and subsequent incubation with selecting reagent. Up to now, this approach helped very much.
Dear Veronika and Dmitry, thanks for the active discussion.
As Veronica suggested , the experiment was performed where the drugs were added at the same time and incubated for the respective time periods. So all the incubation periods have same cell number. May be as you mentioned resistance could have emerged after prolong exposure of drug... may be so right ?
Dear Manu, during 72 h period cells should be divided 3 times as minimum. Cell concentration in control wells should be increased in 8 times. So, unlikely the statement about the same cell number during incubation period is correct. I can see two reasons for obtained effects: 1) selection and subsequent growth of cytotoxic drug resistant cells in experimental wells, and 2) overgrowing and subsequent death of cells in control wells.
Thank you Dmitry, the second aspect ie, overgrowing of cells in the control is the one i never thought off.. I t could be a potential reason..Since 48 hr incubation itself shows almost confluence cells in control. It is a great piece of information.thank you so much.
Exactly! this is why I asked for the exact assay conditions. Because if you say that you add the compounds to the same cell number at the same time, you will not have the same cell number in the control wells after different time points. You will see the difference when you look at your raw data of the untreated controls. But if you say the wells were confluent after 48h, it is quite probable, that this is the explanation for your observation.
In any case: how much is the difference in toxicity between the 48h and 72h measurements? I mean, if you build the ratio of IC50 72h/48h? Is this a reproducible and significant difference?
If it is, and if you still think about the resistance theory, repeat the experiment in the other way I suggested yesterday, but before: adjust the cell number ;-)
And another approach: you could select the cells in a low concentration which is not toxic, yet (maybe IC10 or below), for a longer time (in your cell culture), and repeat the experiment with cells treated for different times.
The MTT assay is a measure of the metabolic activity of cells (NAD(P)H-dependent oxidoreductases). It is NOT a direct measure of cytotoxicity, and would need to be validated, particularly at 24h using a dye exclusion assay. For example, your compound could be cytostatic rather than cytotoxic in which case very little MTT would be reduced at 24h and perhaps even at 48h due to low metabolic activity and low NAD(P)H flux. Loss of compound over these time periods could then result in resumption of cell division without cytotoxicity, and therefore reversal of loss of MTT responsiveness. I suggest you check for cytotoxicity at each timepoint (% viable cells and total viable cells) and be careful to use the MTT assay appropriately.
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