We would like to find a typical, preferably clinically used drug for in vitro study of apoptosis in cancer cells. It seems that most in vitro studies reporting apoptotic response in cancer cell lines used a dose much higher than the reported plasma concentration than in patients in other literature. I did a dose response curve of HeLa cells treated with cisplatin and found that the percentage of inhibition of the plasma concentration (2.0ug/ml) was only ~10-20%. Should I incubate the cells with drug in plasma concentration and refresh the drug and medium every day for a longer period like 5 days or should I simply switch to doses like IC50, IC90 that can lead to a high proportion of apoptotic cells within 24h?
Hi Karen,
As i know Hela cells require higher dose to kill. I would like to suggest for you is to start a dose response curve following take the IC50 value for your following experiments. Cheers
Regards
Boon Shing
Agree to Boon Shing. In addition to that you might want to think about some possibility that may differ your finding with the literature :
1) passage number of your HeLa cells ; would it be possible your cells could no longer sensitive to cisplatin ?
2) Cisplatin treatment period ? 24hr ? 48 hrs? 72 hrs?
3) solvent for dissolving cisplatin ? Cisplatin tends to decompose and changed to its metabolite in some solvents; also the solubility of cisplatin will affect your drug's efficiency
Thanks for the kind comments from Boon Shing and Chun Wai.
I am not so familiar with the relationship between passage number and sensitivity. Could you please provide more information? Thanks for pointing it out.
I seeded 4000 Hela cells in 96 well plate, replaced with medium containing cisplatin and incubated for 72h. IC50 is around 18uM. I dissolved the cisplatin in 0.9%NaCl before diluting with medium.
The reason why I am so concerned with plasma concentration is that we want to mimic in vivo situation as much as possible. Some scientists questioned the significance of in vitro apoptosis study using high doses.
Cell line might undergone morphological changes after long passage numbers (many rounds of sub-culture) and thus alter the gene expression in the cells. And thus it might have developed resistance to some treatment in "in vitro" but it doesnt resemble the true behavior of the cells.
It depends on your objectives when u perform the in vivo and the in vitro test. I believe as long as you could justify the reason of a dose is selected for your in vivo / in vitro study, no one could possible question on your designing approach. Usually in vivo dose is slightly higher than in vitro due to the role of metabolic pathways present in the in vivo model, which tends to be neglected during in vitro (cell-based) study
My humble suggestion: If we goal is to develop such a drug eventually into a therapeutic, first confirm it is active at concentrations that can be achieved at mg/kg doses. Therefore, first check micromolar concentrations (0, 1, 3, 10, 30, 100). If the drug is inactive at these concentrations, make a "NO GO" decision. If the drug is active at low micromolar concetrations, broaden the concentration range to nanomolar concentrations.
choose the peak plasma concentration (X) use that as well as dose escalation up 10X
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