CYP1B1 enhances the resistance of epithelial ovarian cancer cells to paclitaxel in vivo and in vitro

Abstract

Ovarian cancer (OC) is the most frequent cause of mortality among gynecological malignancies, with a 5-year survival rate of approximately 30%. The standard regimen for OC therapy includes a platinum agent combined with a taxane, to which the patients frequently acquire resistance. Resistance arises from the oxidation of anticancer drugs by CYP1B1, a cytochrome P450 enzyme overexpressed in malignant OC. The aim of the present study was to determine the role of CYP1B1 expression in the drug resistance of OC to the taxane, paclitaxel (PTX). Immunohistochemical staining was used to assess CYP1B1 expression in a panel of ovarian samples (53 primary cancer samples, 14 samples of metastastic cancer, 30 benign tumor samples and 19 normal tissue samples). Semi-quantitative RT-PCR was also performed to determine CYP1B1 expression in several OC cell lines. Finally, we used proliferation and toxicity assays, as well as a mouse xenograft model using nude mice to determine whether α-naphthoflavone (ANF), a CYP1B1 specific inhibitor, reduces resistance to PTX. CYP1B1 was overexpressed in the samples from primary and metastatic loci of epithelial ovarian cancers. In some cell lines, PTX induced CYP1B1 expression, which resulted in drug resistance. Exposure to ANF reduced drug resistance and enhanced the sensitivity of OC cells to PTX in vitro and in vivo. The expression profile of CYP1B1 suggests that it has the potential to be a useful diagnostic marker and prognostic factor for malignant OC. The inhibition of CYP1B1 expression by specific agents may provide a novel therapeutic strategy for the treatment of patients resistant to PTX and may improve the prognosis of these patients.

Figure 1

Expression of CYP1B1 in ovarian cancer, benign ovarian tumor and normal ovarian tissue samples. (A) Negative expression of CYP1B1 in normal ovarian tissue. (B) Expression of CYP1B1 in benign ovarian tumors. (C) Positive expression of CYP1B1 in ovarian cancer. (D) Positive expression of CYP1B1 in metastatic ovarian cancer.

Figure 2

Paclitaxel (PTX)-induced CYP1B1 expression in ovarian cancer cell lines. (A) RT-PCR of total mRNA or mRNA levels of CYP1B1, MDR-1 and β-actin (as a loading control) in epithelial ovarian cancer cell lines (COC1, HO-8910, HO-8910PM, A2780 and A2780TS) as indicated. (B) The COC1, HO-8910, HO-8910PM, A2780 and A2780TS ovarian cancer cell lines were cultured and treated with various concentrations (0, 2.5, 5, 10 and 20 μg/ml) of PTX for 24 h before harvesting. The leftmost column in each group represents the lowest concentration of PTX (0 μg/ml), while the rightmost column in each group represents the highest concentration of PTX (20 μg/ml). From left to right, the intervening columns represent 2.5, 5 and 10 μg/ml of PTX. The mRNA expression level of CYP1B1 was measured by RT-PCR. The relative expression was calculated by normalization to the mRNA level of β-actin. The data are presented as the means ± standard deviation.

Figure 3

Treatment with α-naphthoflavone (ANF) reverses resistance to paclitaxel (PTX) in A2780TS cells. (A) Growth inhibition of A2780 and A2780TS cells following treatment with <1–50 mg/ml (PTX) for 72 h. (B) Growth inhibitory effects on A2780TS cells in the presence of titrated concentrations (1, 10 or 100 μM) of ANF. (C and D) Effects of combined treatment with 20 μg/ml PTX and 1, 10 or 100 μM ANF on A2780TS cells. (C) Effects of combined PTX and ANF treatment on the growth (IC50) of A2780TS cells. (D) Effects of combined treatment with PTX and ANF or 100 μM ANF alone on the mRNA and protein expression of CYP1B1, as assessed by RT-PCR and western blot analysis (WB), respectively.

Figure 4

Analysis of the cell cycle in A2780TS cells treated with paclitaxel (PTX), α-naphthoflavone (ANF) or PTX and ANF. (A) Flow cytometric analysis of untreated A2780TS cells or A2780TS cells treated with PTX, ANF or a combination of PTX and ANF. (a) Untreated cells; (b) cells treated with 20 μg/ml PTX; (c) cells treated with 100 μM ANF; (d) cells treated with 20 μg/ml PTX + 1 μM ANF; (e) cells treated with 20 μg/ml PTX + 10 μM ANF; (f) cells treated with 20 μg/ml PTX + 100 μM ANF. (B) Analysis of apoptosis and the cell cycle (G0/G1, S and G2/M phase) in untreated A2780TS cells and A2780TS cells treated with PTX (20 μg/ml), ANF (100 μM) or a combination of PTX and ANF (20 μg/ml PTX + 1, 10 or 100 μM ANF).

Figure 5

Treatment with α-naphthoflavone (ANF) antagonizes the resistance of ovarian cancer cells to paclitaxel (PTX) in vivo. (A) Images of representative tumors resulting from the subcutaneous injetion of A2780TS cells into nude BALB/c mice that were treated with saline (control, top row), 3 mg/kg PTX (middle row) or 3 mg/kg PTX and 20 mg/kg ANF (bottom row) when the tumors reached 10 mm³. (B) Tumor growth curves in nude mice with xenograft tumors treated with saline (control), PTX or PTX and ANF. (C) Images of hematoxylin and eosin-stained sections from representative tumors resulting from tumor xenografts in nude mice treated with (a) saline; (b) PTX; (c) PTX and ANF. (D) Representative images of hematoxylin and eosin-stained sections of (a) kidney and (b) liver from nude mice with xenograft tumors treated as described in (A) with PTX and ANF.