MGMT- and P450 3A-inhibitors do not sensitize glioblastoma cell cultures against nitrosoureas.
AIM, MATERIAL AND METHODS: Using RT-PCR and immunohistochemistry the expression of cytochrome P450 was evaluated in a series of 22 glioblastomas and 4 anaplastic astrocytomas (WHO grade III). Since rat liver P450 can catalyze the denitrosation of the nitrosourea compound BCNU in vitro, cell culture experiments were performed to test a possible sensitizing effect of P450 3A inhibitors (tiamulin and ketoconazole) in BCNU treatment of glial tumor cells. O6-benzylguanine (BG), an inhibitor of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT), was used in parallel experiments, since MGMT is discussed as a main mechanism in nitrosourea resistance.


RESULTS
RT-PCR reactions with primers designed according to the sequences for CYP1A1 and CYP2E1 were always positive, while those for CYP1A2 and CYP2D6 were negative. The strongest PCR products were detected with CYP3A primers, but CYP3A expression was heterogeneous within the tumor samples. Antibodies to human liver CYP3A4 stained a subfraction of tumor cells (18% of the cells in glioblastomas and 14% in grade III astrocytomas) and to some extend neurons in normal brain areas, while astrocytes were negative. For cell culture experiments with P450 3A and MGMT inhibitors, early passages of 3 glioblastomas, a late passage of an immortalized cell line derived from a reoccurring glioblastoma, and the human glioblastoma line LN405 were used. The sensitivity of the tumor cells for both nitrosourea compounds was very low, when low concentrations were applied (comparable to the achievable blood concentrations in glioma patients). Strong effects did only occur when the concentrations were raised 9-fold or 27-fold.


CONCLUSIONS
In no case could a significant sensitizing effect of P450 3A- and MGMT inhibitors be demonstrated.
Kirches
E
E
Institute for Neuropathology, Otto-von-Guericke University, Magdeburg, Germany.
Scherlach
C
C
von Bossanyi
P
P
Schneider
T
T
Szibor
R
R
Kutz
E
E
Warich-Kirches
M
M
Dietzmann
K
K
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001189:Aryl Hydrocarbon Hydroxylases; D002330:Carmustine / Q000627:therapeutic use; D002470:Cell Survival / Q000187:drug effects; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D004224:Diterpenes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005909:Glioblastoma / Q000188:drug therapy* / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007654:Ketoconazole / Q000494:pharmacology; D015376:Nimustine / Q000627:therapeutic use; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors*; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured