Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines

Abstract

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.CONCLUSION.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. Cancer 2007. © 2007 American Cancer Society.

Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells

“…In osteosarcoma, MDR1 [46] or P-gp [47] expression could be used as a prognostic marker for sensitivity to chemotherapy, allowing the selection of patients for whom alternative treatments may be considered. Recently, other prognostic factors have been described, such as the expression level of clusterin/apolipoprotein J [48] or expression of a pregnane xenobiotic receptor (PXR), a major inducer of cytochrome P450 3A4 [49]. Therefore, these factors may also represent predictive markers correlating with the response of cancer cells to chemotherapy.…”

A microtiter-plate-based cytochrome P450 3A activity assay in fish cell lines

“…We first wanted to know whether this human CYP3A substrate is also metabolized by CYP3A enzymes of different fish. For this reason, we adapted the protocol from Mensah-Osman et al [29] to evaluate whether the human-specific CYP3A substrate BFC [30][31][32] can also be catalyzed through fish CYP3A enzymes into the fluorescent product, as in carp [24]. As shown in Figure 1, fish CYP3A enzymes indeed can catalyze BFC.…”

“…Approximately 2.5 million cells of each cell line were transferred to an Eppendorf tube and incubated in phenol-red-free medium for 5 h. Phenolred-free medium was used to prevent the color of phenol red from interfering with spectrophotometric and fluorescence measurements in assays containing 50 mM BFC (SigmaAldrich) or ACN, respectively. 7-Benzyloxy-4-trifluoromethylcoumarin is metabolized by human CYP3A4 to give highly fluorescent 7-hydroxy-4-(trifluoromethyl)coumarin that can be detected readily spectrofluorometrically [29]. After 5 h, the cells were centrifuged for 4 min at 2,000 g at 4uC.…”

“…In the first set of experiments, we determined the CYP3A activity in vitro according to the protocol of Mensah-Osman et al [29]. Our aim was to evaluate whether the human CYP3A substrate (BFC) is also a substrate for fish CYP3A enzymes.…”

“…Shown are results of at least three independent experiments 6 standard deviation for each cell line. The CYP3A activity assays were performed in 1.5-ml tubes as described by MensahOsman et al [29]. incubated with CYP3A substrate, and the enzyme reaction was stopped at different time points.…”