Cytochrome P450 CYP1B1 activity in renal cell carcinoma

Abstract

Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n=11). Measurable levels of active P450R were determined in all normal (n=11) and tumour samples (n=26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.

Figure 1

Graphs illustrating the percentage of CYP1 activity demonstrating the difference in CYP1B1 and CYP1A1 activity (production of resorufin from the O-deethylation of ethoxyresorufin) following the addition of alphanaphthoflavone (0, 0.1, 0.5, 5, 10, 50 and 100 nM final concentration). All assays were performed in triplicate with CYP1A1 (1 pmol) and CYP1B1 (5 pmol) containing microsomes prepared from either CYP1A1- or CYP1B1-expressing human lymphoblastoid cells, respectively.

Figure 2

Efficiency of CYP1B1 activity demonstrated by (A) Michaelis–Menten and (B) Linneweaver–Burke plots illustrating the K_m and V_max for CYP1B1 O-deethylation of ethoxyresorufin.

Figure 3

Distribution of CYP1B1 activity in normal kidney and RCC. CYP1B1 activity according to the change in resorufin production ΔR=R_ni−R_i (ΔR is the change in CYP1B1 activity, R_ni is the resorufin production in the absence of the inhibitor alpha-naphthoflavone and R_i is the resorufin production in the presence of 10 nM alpha-naphthoflavone. Boxes delimit the first and third quartiles with the median inside. Circle (○) represents outlier, defined as individual values 1.5–3 times greater than the interquartile range. No box is present for the normal kidney due to the absence of CYP1B1 activity.

Figure 4

Distribution of P450R activity in normal kidney and RCC, according to cytochrome c production. Boxes delimit the first and third quartiles, with the median inside and bars representing the range of values that fall within 1.5-fold the interquartile range. Circle (○) represents outlier, defined as individual values 1.5–3 times greater than the interquartile range. Triangle (Δ) represents extreme, defined as individual values greater than three times the interquartile range.