Expression profile of CYP1A1 and CYP1B1 enzymes in colon and bladder tumors

Abstract

Background: The cytochrome P450 CYP1A1 and CYP1B1 enzymes are involved in carcinogenesis via activation of pro-carcinogenic compounds to carcinogenic metabolites. CYP1A1 and CYP1B1 have shown elevated levels in human tumors as determined by qRT-PCR and immunohistochemical studies. However studies that have examined CYP1 expression by enzyme activity assays are limited.

Results: In the current study the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of bladder and colorectal origin by qRT-PCR and enzyme activity assays. The results demonstrated that 35% (7/20) of bladder tumors and 35% (7/20) of colon tumors overexpressed active CYP1 enzymes. CYP1B1 mRNA was overexpressed in 65% and 60% of bladder and colon tumors respectively, whereas CYP1A1 was overexpressed in 65% and 80% of bladder and colon tumors. Mean mRNA levels of CYP1B1 and CYP1A1 along with mean CYP1 activity were higher in bladder and colon tumors compared to normal tissues (p<0.05). Statistical analysis revealed CYP1 expression levels to be independent of TNM status. Moreover, incubation of tumor microsomal protein in 4 bladder and 3 colon samples with a CYP1B1 specific antibody revealed a large reduction (72.5 ± 5.5 % for bladder and 71.8 ± 7.2% for colon) in catalytic activity, indicating that the activity was mainly attributed to CYP1B1 expression.

Conclusions: The study reveals active CYP1 overexpression in human tumors and uncovers the potential use of CYP1 enzymes and mainly CYP1B1 as targets for cancer therapy.

Figure 1. Expression profiling of CYP1A1 and CYP1B1 mRNA in bladder samples.

qRT-PCR analysis of CYP1B1 and CYP1A1 in 20 matched normal and tumor pairs derived from bladder tissue. Each bar represents an average of triplicate reactions. The numbers in the X axis correspond to patient numbers. Ta/T1 and T2/T3 represent the different stages of tumors according to TNM classification. ns not statistically significant, * statistically different p< 0.05.

Figure 2. Expression profiling of CYP1A1 and CYP1B1 mRNA in colon samples.

qRT-PCR analysis of CYP1B1 and CYP1A1 in 20 matched normal and tumor pairs derived from colon tissue. Each bar represents an average of triplicate reactions. The numbers in the X axis correspond to patient numbers. Ta/T1 and T2/T3 represent the different stages of tumors according to TNM classification. ns not statistically significant, * statistically different p< 0.05.

Figure 3. Mean mRNA levels of CYP1A1 and CYP1B1 transcripts in human tumors.

Box plots indicate mean ± STDEV for (A) bladder and (B) colon tumor and normal samples. Statistical analysis was conducted using paired T test and Wilcoxon ranks test. Statistical differences were obtained for bladder (n=20) and colon tumors (n=20) vs normals (p< 0.05 for CYP1A1 and CYP1B1).

Figure 4. The metabolism of 4´ methoxy 3´,5,7 trihydroxy flavone (diosmetin) by recombinant CYP1A1 and CYP1B1.

Diosmetin (10 μM) was incubated with recombinant CYP1A1 (1mg/ml) and CYP1B1 (1mg/ml) at various time points in the presence of NADPH (5mM) and MgCl2 (0.5mM) as described in Materials and Methods. (A) LC-MS analysis of diosmetin metabolism by CYP1A1. Mass spectrometric trace indicated two positively charged ions (287, 301) and one negatively charged ion (315) that correspond to diosmetin, 3´,4´,5,7 tetrahydroxy flavone (luteolin) and hydroxy - 4´ methoxy tetrahydroxy flavone (hydroxy diosmetin) respectively. (B) Enzyme profile of diosmetin metabolism by CYP1A1. Diosmetin was incubated for 20 min with increasing enzyme concentrations corresponding to 0.5, 0.8, 1.2 and 3,5 mg/ml. Green line to orange line: increasing concentrations of CYP1A1 recombinant enzyme. (C) Enzyme profile of diosmetin metabolism by CYP1B1. Diosmetin was incubated for 20 min with increasing enzyme concentrations corresponding to 0.5, 0.8, 1.2 and 3,5 mg/ml. Orange line to black line: increasing concentrations of CYP1B1 recombinant enzyme. (D) Time dependence analysis of diosmetin (10 μM) metabolism by CYP1A1 at 1, 5, 10, 15, 30, 60 min. Black to blue line: 1-60 min. (E) Time dependence analysis of diosmetin metabolism by CYP1B1 (1mg/ml) at 1, 5, 10, 15, 30, 60 min time intervals. Black to blue line: 1-60 min. (F) Michaelis-Menten kinetics of luteolin production by CYP1A1 and CYP1B1-mediated metabolism of 4´ methoxy 3´,5,7 trihydroxy flavone indicating maximum activity (Vmax).

Figure 5. Determination of CYP1 expression by enzyme activity assay.

The X axis corresponds to patient numbers while the Y axis to CYP1 activity levels. Activity was calculated from production of the metabolite luteolin per time per amount of microsomal protein. Each bar represents an average of triplicate reactions. Ta-T1 and T2-T3 correspond to different stages of tumors according to TNM. (A) Bladder samples (n=10). Colon samples (n=8). ns not statistically significant, * statistically different p< 0.05. (B) Mean CYP1 activity levels in human tumors. Bar charts indicate mean ± STDEV for bladder (n=10) and colon (n=8) tumors and normal samples. Statistical analysis was conducted using paired T test and Wilcoxon ranks test. Statistical differences were obtained for bladder and colon tumors vs normals (p<0.05).

Figure 6. Correlation of CYP1 mRNA expression levels with tumor stage.

Group pairs were compared using Mann-U-Whitney test. Bars depict the mean values. Scatterplot depicting mRNA levels of CYP1A1 and CYP1B1 genes in tumor samples of different TNM status and normal samples of (A) bladder and (B) colorectal origin. Statistical significance was set at p < 0.05. Arrows and horizontal lines indicate groups compared with statistical tests.

Figure 7. Correlation of CYP1 enzyme activity levels with tumor stage.

Group pairs were compared using Mann-U-Whitney test. Bars depict the mean values. Scatterplot depicting CYP1 activity levels in tumor samples of different TNM status and normal samples of bladder and colorectal origin. Statistical significance was set at p < 0.05. Arrows and horizontal lines indicate groups compared with statistical tests.

Figure 8. CYP1 activity is mainly indicative of active CYP1B1 in human tumors.

Correlation of CYP1A1 and CYP1B1 mRNA T/N expression ratio with CYP1 activity T/N expression ratio in (A) bladder and (B) colon tumors using linear regression analysis.

Figure 9. Reduction of CYP1 activity in colon (n=3) and bladder tumors (n=4) by (A) CYP1B1 antibody (1:500) (B) CYP1A1 antibody (1:300).

Figure 10. Reduction of CYP1 activity in colon (n=3) and bladder tumors (n=4) by α-napthoflavone (0.5 μM).