Cyclophosphamide is supposed to be activated by Cytochrome P450. I've treated Huh7 and PLC liver cancer cell lines with Cytochrome P450, but there is no sensitivity whatsoever, even at 500uM. Quite literally, nothing happens. What am I doing wrong? What cell lines are naturally sensitive to cyclophosphamide without pre-activation?
Hi Florian,
Did you add cytochrome P450 cofactor, NADH or NADPH? without cofactor, enzyme do not work. These cofactors act as the source of electrons.
I hope this helps
NADPH should be present in Huh7 cells...I think what you are refering to are isolated microsomes added to the cells with NADPH. I understand that this is done for testing cyclophosphamide with say leukemia cells, which do not express Cytocrhome P450 but for Hepatocellular carcinoma cell lines like Huh7 and Plc, which should express plenty of Cyt P450, adding microsomes should not be nescessary......or am I wrong about that?
Dear Florian,
Sorry I got your question wrong. I thought you really treated your cell lines with pure Cytochrome p450 enzyme. So what exactly did you mean "treating Huh7 and PLC by cytp450" then? Did you mean "treating cells with p450 inducers"? What are you trying to do? If you explain little more, it will be useful.
By the way, I searched the literature and I realized that HepG2 cells have been used for biotransformation of drugs.
I typed a bit too fast and didn't get my point accross correctly. Huh7 and PLC are supposed to be liver cancer cell lines...unless I am totally mistaken, they should express a decent amount of cytochrome P450's. Since cyclophosphamide is activated by cytochrome P450, I should observe some toxicity, especially at higher doses (500 uM).
Actually you should induce CYP2B6 in these cell lines first by phenobarbital or other inducer. After that these cell lines transform cyclophosphamide to its metabolites. Phenobarbital is a inducer, cyclophosphamide is a substrate of the enzyme.
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm
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