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Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma.

Ren J, Chen GG, Liu Y, Su X, Hu B, Leung BC, Wang Y, Ho RL, Yang S, Lu G, Lee CG, Lai PB - PLoS ONE (2016)

Bottom Line: The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME.The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib.The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong; New Territories, Hong Kong, China.

ABSTRACT
Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.

No MeSH data available.


Related in: MedlinePlus

Effects of gene overexpression on E2-mediated inhibition of cell proliferation.(A) Growth rate of the transfected cells. Hep3B cells were transfected with vector control or ERα, ERβ, GPR30, CYP1A2, CYP3A4, COMT plasmid DNAs, respectively, and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours, at which time MTT was performed with one set of the cells (Day0). MTT for another set of the cells was performed 48 hours later (Day2). The growth ratios of the cells are expressed as the value of Day2 cells versus that of the Day0 cells of the same transfection (= 100%). (B) Hep3B cells were transfected and reseeded as mentioned in (A). Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (C) CYP1A2 mediates E2-inhibition of cell proliferation in 293T cells. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%) Consistent results were obtained in three independent experiments. (D) CYP1A2 stable Hep3B cells migrated slowly under E2 treatment. Hep3B stable cells overexpressing CYP1A2 or empty vector were grown to confluence and a 2mm-thin wound gap was generated by scratching with a rubber policeman (Day0). The cells were then grown in medium supplemented with or without 1 μM E2 for 10 days (Day10) before microscopic images were taken. The lines indicate the wound edges. Consistent results were obtained in three independent experiments. (E) Hep3B cells were co-transfected with siRNA against CYP1A2 (siCYP1A2) or COMT (siCOMT) and CYP1A2-expressing plasmids and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours. Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (E) Hep3B cells were transfected with CYP1A2 and grown for 24 hours. Then the cells were grown in 10μM E2 supplemented medium for another 24 hours. Afterwards the cells were harvested and lysed in PBS by sonication. Concentrations of E2 and 2-ME were measured with respective EIA kits mentioned in Materials and Methods. The values represent the relative concentrations normalized against the protein concentration in the same sample. Each column represents mean±s.d. of data obtained from three independent assays.
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pone.0153863.g003: Effects of gene overexpression on E2-mediated inhibition of cell proliferation.(A) Growth rate of the transfected cells. Hep3B cells were transfected with vector control or ERα, ERβ, GPR30, CYP1A2, CYP3A4, COMT plasmid DNAs, respectively, and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours, at which time MTT was performed with one set of the cells (Day0). MTT for another set of the cells was performed 48 hours later (Day2). The growth ratios of the cells are expressed as the value of Day2 cells versus that of the Day0 cells of the same transfection (= 100%). (B) Hep3B cells were transfected and reseeded as mentioned in (A). Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (C) CYP1A2 mediates E2-inhibition of cell proliferation in 293T cells. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%) Consistent results were obtained in three independent experiments. (D) CYP1A2 stable Hep3B cells migrated slowly under E2 treatment. Hep3B stable cells overexpressing CYP1A2 or empty vector were grown to confluence and a 2mm-thin wound gap was generated by scratching with a rubber policeman (Day0). The cells were then grown in medium supplemented with or without 1 μM E2 for 10 days (Day10) before microscopic images were taken. The lines indicate the wound edges. Consistent results were obtained in three independent experiments. (E) Hep3B cells were co-transfected with siRNA against CYP1A2 (siCYP1A2) or COMT (siCOMT) and CYP1A2-expressing plasmids and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours. Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (E) Hep3B cells were transfected with CYP1A2 and grown for 24 hours. Then the cells were grown in 10μM E2 supplemented medium for another 24 hours. Afterwards the cells were harvested and lysed in PBS by sonication. Concentrations of E2 and 2-ME were measured with respective EIA kits mentioned in Materials and Methods. The values represent the relative concentrations normalized against the protein concentration in the same sample. Each column represents mean±s.d. of data obtained from three independent assays.

Mentions: To further elucidate how E2 exerts its suppressive effect on HCC cells, the cells were transfected ERα, ERβ, GPR30, CYP1A2, CYP3A4, or COMT DNA and overexpression of these genes alone did not significantly influence HCC cell proliferation “Fig 3A”. The inhibitory effect of E2 was significantly enhanced in CYP1A2- and GPR30-overexpressing cells but not in CYP3A4, COMT, ERα or ERβ-overexpressing cells “Fig 3B”, indicating the possible involvement of CYP1A2 and GPR30 in the E2-induced inhibition. Considering the liver-specific expression of CYP1A2 and the fact that the levels of CYP1A2 but not GPR30 were significantly reduced in HCC “Fig 2C and 2D”, we found it more interesting to further investigate the functions of CYP1A2 rather than that of GPR30 in mediating the E2-induced inhibition. In addition, the consistent result regarding the effect of CYP1A2 was obtained with 293T cells, excluding the possibility that above CYP1A2 effect was cell line specific. Moreover, due to higher transfection efficiency in 293T cells, a more robust effect of CYP1A2 was observed “Fig 3C”.


Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma.

Ren J, Chen GG, Liu Y, Su X, Hu B, Leung BC, Wang Y, Ho RL, Yang S, Lu G, Lee CG, Lai PB - PLoS ONE (2016)

Effects of gene overexpression on E2-mediated inhibition of cell proliferation.(A) Growth rate of the transfected cells. Hep3B cells were transfected with vector control or ERα, ERβ, GPR30, CYP1A2, CYP3A4, COMT plasmid DNAs, respectively, and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours, at which time MTT was performed with one set of the cells (Day0). MTT for another set of the cells was performed 48 hours later (Day2). The growth ratios of the cells are expressed as the value of Day2 cells versus that of the Day0 cells of the same transfection (= 100%). (B) Hep3B cells were transfected and reseeded as mentioned in (A). Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (C) CYP1A2 mediates E2-inhibition of cell proliferation in 293T cells. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%) Consistent results were obtained in three independent experiments. (D) CYP1A2 stable Hep3B cells migrated slowly under E2 treatment. Hep3B stable cells overexpressing CYP1A2 or empty vector were grown to confluence and a 2mm-thin wound gap was generated by scratching with a rubber policeman (Day0). The cells were then grown in medium supplemented with or without 1 μM E2 for 10 days (Day10) before microscopic images were taken. The lines indicate the wound edges. Consistent results were obtained in three independent experiments. (E) Hep3B cells were co-transfected with siRNA against CYP1A2 (siCYP1A2) or COMT (siCOMT) and CYP1A2-expressing plasmids and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours. Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (E) Hep3B cells were transfected with CYP1A2 and grown for 24 hours. Then the cells were grown in 10μM E2 supplemented medium for another 24 hours. Afterwards the cells were harvested and lysed in PBS by sonication. Concentrations of E2 and 2-ME were measured with respective EIA kits mentioned in Materials and Methods. The values represent the relative concentrations normalized against the protein concentration in the same sample. Each column represents mean±s.d. of data obtained from three independent assays.
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Related In: Results  -  Collection

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pone.0153863.g003: Effects of gene overexpression on E2-mediated inhibition of cell proliferation.(A) Growth rate of the transfected cells. Hep3B cells were transfected with vector control or ERα, ERβ, GPR30, CYP1A2, CYP3A4, COMT plasmid DNAs, respectively, and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours, at which time MTT was performed with one set of the cells (Day0). MTT for another set of the cells was performed 48 hours later (Day2). The growth ratios of the cells are expressed as the value of Day2 cells versus that of the Day0 cells of the same transfection (= 100%). (B) Hep3B cells were transfected and reseeded as mentioned in (A). Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (C) CYP1A2 mediates E2-inhibition of cell proliferation in 293T cells. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%) Consistent results were obtained in three independent experiments. (D) CYP1A2 stable Hep3B cells migrated slowly under E2 treatment. Hep3B stable cells overexpressing CYP1A2 or empty vector were grown to confluence and a 2mm-thin wound gap was generated by scratching with a rubber policeman (Day0). The cells were then grown in medium supplemented with or without 1 μM E2 for 10 days (Day10) before microscopic images were taken. The lines indicate the wound edges. Consistent results were obtained in three independent experiments. (E) Hep3B cells were co-transfected with siRNA against CYP1A2 (siCYP1A2) or COMT (siCOMT) and CYP1A2-expressing plasmids and grown for 24 hours. Then the cells were seeded at 5x103 cells/per well of 96-well plates and grown for 24 hours. Afterwards the cells were treated as indicated for 48 hours and analyzed for proliferation rate by MTT. The proliferation rate is expressed as the value of treated cells versus that of the untreated cells of same transfection (= 100%). Each column represents mean±s.d. of data obtained from four replicate wells. Consistent results were obtained in three independent experiments. (E) Hep3B cells were transfected with CYP1A2 and grown for 24 hours. Then the cells were grown in 10μM E2 supplemented medium for another 24 hours. Afterwards the cells were harvested and lysed in PBS by sonication. Concentrations of E2 and 2-ME were measured with respective EIA kits mentioned in Materials and Methods. The values represent the relative concentrations normalized against the protein concentration in the same sample. Each column represents mean±s.d. of data obtained from three independent assays.
Mentions: To further elucidate how E2 exerts its suppressive effect on HCC cells, the cells were transfected ERα, ERβ, GPR30, CYP1A2, CYP3A4, or COMT DNA and overexpression of these genes alone did not significantly influence HCC cell proliferation “Fig 3A”. The inhibitory effect of E2 was significantly enhanced in CYP1A2- and GPR30-overexpressing cells but not in CYP3A4, COMT, ERα or ERβ-overexpressing cells “Fig 3B”, indicating the possible involvement of CYP1A2 and GPR30 in the E2-induced inhibition. Considering the liver-specific expression of CYP1A2 and the fact that the levels of CYP1A2 but not GPR30 were significantly reduced in HCC “Fig 2C and 2D”, we found it more interesting to further investigate the functions of CYP1A2 rather than that of GPR30 in mediating the E2-induced inhibition. In addition, the consistent result regarding the effect of CYP1A2 was obtained with 293T cells, excluding the possibility that above CYP1A2 effect was cell line specific. Moreover, due to higher transfection efficiency in 293T cells, a more robust effect of CYP1A2 was observed “Fig 3C”.

Bottom Line: The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME.The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib.The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong; New Territories, Hong Kong, China.

ABSTRACT
Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.

No MeSH data available.


Related in: MedlinePlus