Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines.
BACKGROUND
Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors.


METHODS
Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining.


RESULTS
Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole.


CONCLUSIONS
The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance.
Mensah-Osman
Edith J
EJ
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Thomas
Dafydd G
DG
Tabb
Michelle M
MM
Larios
Jose M
JM
Hughes
Dennis P
DP
Giordano
Thomas J
TJ
Lizyness
Michelle L
ML
Rae
James M
JM
Blumberg
Bruce
B
Hollenberg
Paul F
PF
Baker
Laurence H
LH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism*; D000077297:Pregnane X Receptor; D020033:Protein Isoforms / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering; D011987:Receptors, Steroid / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D000077297:Pregnane X Receptor; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D005047:Etoposide; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D012293:Rifampin