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. 2016;2:016.
doi: 10.24947/2380-5552/2/1/120. Epub 2016 May 3.

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque

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Free PMC article

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque

Minlu Hu et al. BAOJ Pharm Sci. .
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Abstract

This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in macaque tissues were usually comparable to those of human tissues. In addition, menopause did not significantly alter the ectocervical mRNA levels of CYP1A1, CYP1B1, or NRs.

Keywords: CYP1A1; CYP1B1; Cancer; Colorectal Tissue; Female Genital Tract; Nuclear Receptor; Pigtailed Macaque.

Figures

Figure 1
Figure 1
The mRNA levels of CYP1A1 (A) and CYP1B1 (B) in the genital tract and colorectal tissue of premenopausal women and pigtailed macaques. The tissues for endocervix, ectocervix, vagina, sigmoid colon and liver were from human donors (endocervix: n= 4; ectocervix: n= 6; vagina: n= 5; sigmoid colon: n= 5; liver: n= 6). For each type of tissue, 50-200 mg of tissue was collected from each donor and used for RNA extraction and subsequent RT-PCR analysis. The threshold cycle numbers (Ct) of enzymes and GAPDH of each sample were measured in triplicates, and the average Ct was used to reflect the Ct of a tested gene. All tested gene levels were generated using 2-ΔCt method and normalized to GAPDH and multiplied by 106. The data shown represent mean ± standard deviation of all samples. (□): endocervix, ( formula image): ectocervix, ( formula image): vagina, ( formula image): colorectal tissue, ( formula image): liver. *, p<0.05; ***, p<0.001
Figure 2
Figure 2
Localization of CYP1A1 protein in the genital tract and colorectal tissue of premenopausal women and pigtailed macaques. A-E: human uterus, endocervix, ectocervix, vagina and colorectal tissue. F-J: macaque uterus, endocervix, ectocervix, vagina and colorectal tissue. Black arrow: basal layers of the squamous epithelium, white arrows: vascular endothelial cells. Scale bar: 50μm for all.
Figure 3
Figure 3
Localization of CYP1B1 protein in the genital tract and colorectal tissue of premenopausal women and pigtailed macaques. A-E: human uterus, endocervix, ectocervix, vagina and colorectal tissue. F-J: macaque uterus, endocervix, ectocervix, vagina and colorectal tissue. White arrows: vascular endothelial cells. Scale bar is 50 μm for A, B, D, E, F, G, I, H, and J; 100 μm for C. K: Western blot analysis of CYP1B1 in cytoplasmic and nuclear fractions from human premenopausal ectocervix. Upper panel: CYP1B1; lower panel: GAPDH as internal reference; Cyto: cytoplasmic fraction; Nu: nuclear fraction.
Figure 4
Figure 4
The mRNA levels of 17 nuclear receptors in the genital tract and colorectal tissue of premenopausal women (A) and pigtailed macaques (B). For human tissues, endocervix: n= 4; ectocervix: n= 6; vagina: n= 5; sigmoid colon: n= 5; liver: n= 6. For macaque tissues, n=3 for all tissue types. For each type of tissue, 50-200 mg of tissue was collected from each donor and used for RNA extraction and subsequent RT-PCR analysis. The threshold cycle numbers (Ct) of enzymes and GAPDH of each sample were measured in triplicate, and the average Ct was used to reflect the Ct of a tested gene. All tested gene levels were generated using 2-ΔCt method and normalized to GAPDH and multiplied by 106. The data shown represent mean ± standard deviation of all samples.
Figure 5
Figure 5
The effect of menopause on the expression of CYP1A1, CYP1B1, and NRs in human ectocervix. A: fold change in mRNA levels of CYP1A1 and CYP1B1 in human ectocervix collected from pre- (n=6) and post-menopausal women (n=6). B and C: protein localization of CYP1A1 (B) and CYP1B1 (C) in postmenopausal human ectocervix. Black arrow: basal layers of the squamous epithelium, white arrows: vascular endothelial cells. Scale bar is 100 μm for B and C. D: fold changes in mRNA expression of 17 NRs in human ectocervix (n=6). For A and D: The threshold cycle numbers (Ct) of enzymes and GAPDH of each sample were measured in triplicate, and the average Ct was used to reflect the Ct of a tested gene. All tested gene levels were generated using 2-ΔCt method and normalized to GAPDH and multiplied by 106. The data shown represent mean ± standard deviation of all samples. (□): endocervix, ( formula image): ectocervix, ( formula image): vagina, ( formula image): colorectal tissue, ( formula image): liver.

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